A photoaffinity scan maps regions of the p85 SH2 domain involved in phosphoprotein binding.

PubMed

Williams, K P; Shoelson, S E

1993-03-15

Src homology 2 (SH2) domains are modular phosphotyrosine binding pockets found within a wide variety of cytoplasmic signaling molecules. Here we develop a new approach to analyzing protein-protein interfaces termed photoaffinity scanning, and apply the method to map regions of the phosphatidylinositol 3-kinase p85 SH2 domain that participate in phospho-protein binding. Each residue except phosphotyrosine (pY) within a tightly binding, IRS-1-derived phosphopeptide (GNGDpYMPMSPKS) was substituted with the photoactive amino acid, benzoylphenylalanine (Bpa). Whereas most substitutions had little effect on binding affinity, Bpa substitution of either Met (+1 and +3 with respect to pY) reduced affinity 50-100-fold to confirm their importance in the pYMXM recognition motif. In three cases photolysis of SH2 domain/Bpa phosphopeptide complexes led to cross-linking of > 50% of the SH2 domain; cross-link positions were identified by microsequence, amino acid composition, and electrospray mass spectrometric analyses. Bpa-1 cross-links within alpha-helix I, whereas Bpa+1 and Bpa+4 cross-link the SH2 domain within the flexible loop C-terminal to alpha-helix II. Moreover, cross-linking at any position prevents SH2 domain cleavage at a trypsin-sensitive site within the flexible loop between beta-strands 1 and 2. Therefore, at least three distinct SH2 regions in addition to the beta-sheet participate in phosphoprotein binding; the loop cross-linked by phosphopeptide residues C-terminal to pY appears to confer specificity to the phosphoprotein/SH2 domain interaction.

SH2/SH3 signaling proteins.

PubMed

Schlessinger, J

1994-02-01

SH2 and SH3 domains are small protein modules that mediate protein-protein interactions in signal transduction pathways that are activated by protein tyrosine kinases. SH2 domains bind to short phosphotyrosine-containing sequences in growth factor receptors and other phosphoproteins. SH3 domains bind to target proteins through sequences containing proline and hydrophobic amino acids. SH2 and SH3 domain containing proteins, such as Grb2 and phospholipase C gamma, utilize these modules in order to link receptor and cytoplasmic protein tyrosine kinases to the Ras signaling pathway and to phosphatidylinositol hydrolysis, respectively. The three-dimensional structures of several SH2 and SH3 domains have been determined by NMR and X-ray crystallography, and the molecular basis of their specificity is beginning to be unveiled.

Two amino acid residues confer different binding affinities of Abelson family kinase SRC homology 2 domains for phosphorylated cortactin.

PubMed

Gifford, Stacey M; Liu, Weizhi; Mader, Christopher C; Halo, Tiffany L; Machida, Kazuya; Boggon, Titus J; Koleske, Anthony J

2014-07-11

The closely related Abl family kinases, Arg and Abl, play important non-redundant roles in the regulation of cell morphogenesis and motility. Despite similar N-terminal sequences, Arg and Abl interact with different substrates and binding partners with varying affinities. This selectivity may be due to slight differences in amino acid sequence leading to differential interactions with target proteins. We report that the Arg Src homology (SH) 2 domain binds two specific phosphotyrosines on cortactin, a known Abl/Arg substrate, with over 10-fold higher affinity than the Abl SH2 domain. We show that this significant affinity difference is due to the substitution of arginine 161 and serine 187 in Abl to leucine 207 and threonine 233 in Arg, respectively. We constructed Abl SH2 domains with R161L and S187T mutations alone and in combination and find that these substitutions are sufficient to convert the low affinity Abl SH2 domain to a higher affinity "Arg-like" SH2 domain in binding to a phospho-cortactin peptide. We crystallized the Arg SH2 domain for structural comparison to existing crystal structures of the Abl SH2 domain. We show that these two residues are important determinants of Arg and Abl SH2 domain binding specificity. Finally, we expressed Arg containing an "Abl-like" low affinity mutant Arg SH2 domain (L207R/T233S) and find that this mutant, although properly localized to the cell periphery, does not support wild type levels of cell edge protrusion. Together, these observations indicate that these two amino acid positions confer different binding affinities and cellular functions on the distinct Abl family kinases. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Distinct peptide binding specificities of Src homology 3 (SH3) protein domains can be determined by modulation of local energetics across the binding interface.

PubMed

Gorelik, Maryna; Davidson, Alan R

2012-03-16

The yeast Nbp2p SH3 and Bem1p SH3b domains bind certain target peptides with similar high affinities, yet display vastly different affinities for other targets. To investigate this unusual behavior, we have solved the structure of the Nbp2p SH3-Ste20 peptide complex and compared it with the previously determined structure of the Bem1p SH3b bound to the same peptide. Although the Ste20 peptide interacts with both domains in a structurally similar manner, extensive in vitro studies with domain and peptide mutants revealed large variations in interaction strength across the binding interface of the two complexes. Whereas the Nbp2p SH3 made stronger contacts with the peptide core RXXPXXP motif, the Bem1p SH3b domain made stronger contacts with residues flanking the core motif. Remarkably, this modulation of local binding energetics can explain the distinct and highly nuanced binding specificities of these two domains.

The C-terminal SH2 domain of p85 accounts for the high affinity and specificity of the binding of phosphatidylinositol 3-kinase to phosphorylated platelet-derived growth factor beta receptor.

PubMed Central

Klippel, A; Escobedo, J A; Fantl, W J; Williams, L T

1992-01-01

Upon stimulation by its ligand, the platelet-derived growth factor (PDGF) receptor associates with the 85-kDa subunit of phosphatidylinositol (PI) 3-kinase. The 85-kDa protein (p85) contains two Src homology 2 (SH2) domains and one SH3 domain. To define the part of p85 that interacts with the PDGF receptor, a series of truncated p85 mutants was analyzed for association with immobilized PDGF receptor in vitro. We found that a fragment of p85 that contains a single Src homology domain, the C-terminal SH2 domain (SH2-C), was sufficient for directing the high-affinity interaction with the receptor. Half-maximal binding of SH2-C to the receptor was observed at an SH2-C concentration of 0.06 nM. SH2-C, like full-length p85, was able to distinguish between wild-type PDGF receptor and a mutant receptor lacking the PI 3-kinase binding site. An excess of SH2-C blocked binding of full-length p85 and PI 3-kinase to the receptor but did not interfere with the binding of two other SH2-containing proteins, phospholipase C-gamma and GTPase-activating protein. These results demonstrate that a region of p85 containing a single SH2 domain accounts both for the high affinity and specificity of binding of PI 3-kinase to the PDGF receptor. Images PMID:1312663

Specific phosphopeptide binding regulates a conformational change in the PI 3-kinase SH2 domain associated with enzyme activation.

PubMed Central

Shoelson, S E; Sivaraja, M; Williams, K P; Hu, P; Schlessinger, J; Weiss, M A

1993-01-01

SH2 (src-homology 2) domains define a newly recognized binding motif that mediates the physical association of target phosphotyrosyl proteins with downstream effector enzymes. An example of such phosphoprotein-effector coupling is provided by the association of phosphatidylinositol 3-kinase (PI 3-kinase) with specific phosphorylation sites within the PDGF receptor, the c-Src/polyoma virus middle T antigen complex and the insulin receptor substrate IRS-1. Notably, phosphoprotein association with the SH2 domains of p85 also stimulates an increase in catalytic activity of the PI 3-kinase p110 subunit, which can be mimicked by phosphopeptides corresponding to targeted phosphoprotein phosphorylation sites. To investigate how phosphoprotein binding to the p85 SH2 domain stimulates p110 catalytic activation, we have examined the differential effects of phosphotyrosine and PDGF receptor-, IRS-1- and c-Src-derived phosphopeptides on the conformation of an isolated SH2 domain of PI 3-kinase. Although phosphotyrosine and both activating and non-activating phosphopeptides bind to the SH2 domain, activating phosphopeptides bind with higher affinity and induce a qualitatively distinct conformational change as monitored by CD and NMR spectroscopy. Amide proton exchange and protease protection assays further show that high affinity, specific phosphopeptide binding induces non-local dynamic SH2 domain stabilization. Based on these findings we propose that specific phosphoprotein binding to the p85 subunit induces a change in SH2 domain structure which is transmitted to the p110 subunit and regulates enzymatic activity by an allosteric mechanism. Images PMID:8382612

Structural insights into the specific binding of huntingtin proline-rich region with the SH3 and WW domains.

PubMed

Gao, Yong-Guang; Yan, Xian-Zhong; Song, Ai-Xin; Chang, Yong-Gang; Gao, Xue-Chao; Jiang, Nan; Zhang, Qi; Hu, Hong-Yu

2006-12-01

The interactions of huntingtin (Htt) with the SH3 domain- or WW domain-containing proteins have been implicated in the pathogenesis of Huntington's disease (HD). We report the specific interactions of Htt proline-rich region (PRR) with the SH3GL3-SH3 domain and HYPA-WW1-2 domain pair by NMR. The results show that Htt PRR binds with the SH3 domain through nearly its entire chain, and that the binding region on the domain includes the canonical PxxP-binding site and the specificity pocket. The C terminus of PRR orients to the specificity pocket, whereas the N terminus orients to the PxxP-binding site. Htt PRR can also specifically bind to WW1-2; the N-terminal portion preferentially binds to WW1, while the C-terminal portion binds to WW2. This study provides structural insights into the specific interactions between Htt PRR and its binding partners as well as the alteration of these interactions that involve PRR, which may have implications for the understanding of HD.

Identification and specificity studies of small-molecule ligands for SH3 protein domains.

PubMed

Inglis, Steven R; Stojkoski, Cvetan; Branson, Kim M; Cawthray, Jacquie F; Fritz, Daniel; Wiadrowski, Emma; Pyke, Simon M; Booker, Grant W

2004-10-21

The Src Homology 3 (SH3) domains are small protein-protein interaction domains that bind proline-rich sequences and mediate a wide range of cell-signaling and other important biological processes. Since deregulated signaling pathways form the basis of many human diseases, the SH3 domains have been attractive targets for novel therapeutics. High-affinity ligands for SH3 domains have been designed; however, these have all been peptide-based and no examples of entirely nonpeptide SH3 ligands have previously been reported. Using the mouse Tec Kinase SH3 domain as a model system for structure-based ligand design, we have identified several simple heterocyclic compounds that selectively bind to the Tec SH3 domain. Using a combination of nuclear magnetic resonance chemical shift perturbation, structure-activity relationships, and site-directed mutagenesis, the binding of these compounds at the proline-rich peptide-binding site has been characterized. The most potent of these, 2-aminoquinoline, bound with Kd = 125 microM and was able to compete for binding with a proline-rich peptide. Synthesis of 6-substituted-2-aminoquinolines resulted in ligands with up to 6-fold improved affinity over 2-aminoquinoline and enhanced specificity for the Tec SH3 domain. Therefore, 2-aminoquinolines may potentially be useful for the development of high affinity small molecule ligands for SH3 domains.

The Src SH2 domain interacts dynamically with the focal adhesion kinase binding site as demonstrated by paramagnetic NMR spectroscopy.

PubMed

Lindfors, Hanna E; Drijfhout, Jan Wouter; Ubbink, Marcellus

2012-06-01

The interaction between the tyrosine kinases Src and focal adhesion kinase (FAK) is a key step in signaling processes from focal adhesions. The phosphorylated tyrosine residue 397 in FAK is able to bind the Src SH2 domain. To establish the extent of the FAK binding motif, the binding affinity of the SH2 domain for phosphorylated and unphosphorylated FAK-derived peptides of increasing length was determined and compared with that of the internal Src SH2 binding site. It is shown that the FAK peptides have higher affinity than the internal binding site and that seven negative residues adjacent to the core SH2 binding motif increase the binding constant 30-fold. A rigid spin-label incorporated in the FAK peptides was used to establish on the basis of paramagnetic relaxation enhancement whether the peptide-protein complex is well defined. A large spread of the paramagnetic effects on the surface of the SH2 domain suggests that the peptide-protein complex exhibits dynamics, despite the high affinity of the peptide. The strong electrostatic interaction between the positive side of the SH2 domain and the negative peptide results in a high affinity but may also favor a dynamic interaction. Copyright © 2012 Wiley Periodicals, Inc.

Distinct mechanisms of a phosphotyrosyl peptide binding to two SH2 domains.

PubMed

Pang, Xiaodong; Zhou, Huan-Xiang

2014-05-01

Protein phosphorylation is very common post-translational modification, catalyzed by kinases, for signaling and regulation. Phosphotyrosines frequently target SH2 domains. The spleen tyrosine kinase (Syk) is critical for tyrosine phosphorylation of multiple proteins and for regulation of important pathways. Phosphorylation of both Y342 and Y346 in Syk linker B is required for optimal signaling. The SH2 domains of Vav1 and PLC-γ both bind this doubly phosphorylated motif. Here we used a recently developed method to calculate the effects of Y342 and Y346 phosphorylation on the rate constants of a peptide from Syk linker B binding to the SH2 domains of Vav1 and PLC-γ. The predicted effects agree well with experimental observations. Moreover, we found that the same doubly phosphorylated peptide binds the two SH2 domains via distinct mechanisms, with apparent rigid docking for Vav1 SH2 and dock-and-coalesce for PLC-γ SH2.

SH3 domain-mediated binding of the Drk protein to Dos is an important step in signaling of Drosophila receptor tyrosine kinases.

PubMed

Feller, Stephan M; Wecklein, Heike; Lewitzky, Marc; Kibler, Eike; Raabe, Thomas

2002-08-01

Activation of the Sevenless (Sev) receptor tyrosine kinase (RTK) in the developing Drosophila eye is required for the specification of the R7 photoreceptor cell fate. Daughter of Sevenless (Dos), a putative multi-site adaptor protein, is a substrate of the Sev kinase and is known to associate with the tyrosine phosphatase Corkscrew (Csw). Binding of Csw to Dos depends on the Csw Src homology 2 (SH2) domains and is an essential step for signaling by the Sev RTK. Dos, however, lacks a recognizable phosphotyrosine interaction domain and it was previously unclear how it is recruited to the Sev receptor. Here it is shown that the SH2/SH3 domain adaptor protein Drk can provide this link. Drk binds with its SH2 domain to the autophosphorylated Sev receptor while the C-terminal SH3 domain is able to associate with Dos. The Drk SH3 domain binding motifs on Dos were mapped to two sites which do not conform the known Drk SH3 domain binding motif (PxxPxR) but instead have the consensus PxxxRxxKP. Mutational analysis in vitro and in vivo provided evidence that both Drk binding sites fulfil an important function in the context of Sev and Drosophila epidermal growth factor receptor mediated signaling processes.

Characterizing SH2 Domain Specificity and Network Interactions Using SPOT Peptide Arrays.

PubMed

Liu, Bernard A

2017-01-01

Src Homology 2 (SH2) domains are protein interaction modules that recognize and bind tyrosine phosphorylated ligands. Their ability to distinguish binding to over thousands of potential phosphotyrosine (pTyr) ligands within the cell is critical for the fidelity of receptor tyrosine kinase (RTK) signaling. Within humans there are over a hundred SH2 domains with more than several thousand potential ligands across many cell types and cell states. Therefore, defining the specificity of individual SH2 domains is critical for predicting and identifying their physiological ligands. Here, in this chapter, I describe the broad use of SPOT peptide arrays for examining SH2 domain specificity. An orientated peptide array library (OPAL) approach can uncover both favorable and non-favorable residues, thus providing an in-depth analysis to SH2 specificity. Moreover, I discuss the application of SPOT arrays for paneling SH2 ligand binding with physiological peptides.

SH2 Domains Recognize Contextual Peptide Sequence Information to Determine Selectivity*

PubMed Central

Liu, Bernard A.; Jablonowski, Karl; Shah, Eshana E.; Engelmann, Brett W.; Jones, Richard B.; Nash, Piers D.

2010-01-01

Selective ligand recognition by modular protein interaction domains is a primary determinant of specificity in signaling pathways. Src homology 2 (SH2) domains fulfill this capacity immediately downstream of tyrosine kinases, acting to recruit their host polypeptides to ligand proteins harboring phosphorylated tyrosine residues. The degree to which SH2 domains are selective and the mechanisms underlying selectivity are fundamental to understanding phosphotyrosine signaling networks. An examination of interactions between 50 SH2 domains and a set of 192 phosphotyrosine peptides corresponding to physiological motifs within FGF, insulin, and IGF-1 receptor pathways indicates that individual SH2 domains have distinct recognition properties and exhibit a remarkable degree of selectivity beyond that predicted by previously described binding motifs. The underlying basis for such selectivity is the ability of SH2 domains to recognize both permissive amino acid residues that enhance binding and non-permissive amino acid residues that oppose binding in the vicinity of the essential phosphotyrosine. Neighboring positions affect one another so local sequence context matters to SH2 domains. This complex linguistics allows SH2 domains to distinguish subtle differences in peptide ligands. This newly appreciated contextual dependence substantially increases the accessible information content embedded in the peptide ligands that can be effectively integrated to determine binding. This concept may serve more broadly as a paradigm for subtle recognition of physiological ligands by protein interaction domains. PMID:20627867

Simultaneous Binding of Two Peptidyl Ligands by a Src Homology 2 Domain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Yanyan; Zhang, Jinjin; Yuan, Chunhua

Src homology 2 (SH2) domains mediate protein-protein interactions by recognizing phosphotyrosine (pY)-containing sequences of target proteins. In all of the SH2 domain-pY peptide interactions described to date, the SH2 domain binds to a single pY peptide. Here, determination of the cocrystal structure of the N-terminal SH2 domain of phosphatase SHP-2 bound to a class IV peptide (VIpYFVP) revealed a noncanonical 1:2 (protein-peptide) complex. The first peptide binds in a canonical manner with its pY side chain inserted in the usual binding pocket, while the second pairs up with the first to form two antiparallel {beta}-strands that extend the central {beta}-sheetmore » of the SH2 domain. This unprecedented binding mode was confirmed in the solution phase by NMR experiments and shown to be adopted by pY peptides derived from cellular proteins. Site-directed mutagenesis and surface plasmon resonance studies revealed that the binding of the first peptide is pY-dependent, but phosphorylation is not required for the second peptide. Our findings suggest a potential new function for the SH2 domain as a molecular clamp to promote dimerization of signaling proteins.« less

Lipid binding by the Unique and SH3 domains of c-Src suggests a new regulatory mechanism

PubMed Central

Pérez, Yolanda; Maffei, Mariano; Igea, Ana; Amata, Irene; Gairí, Margarida; Nebreda, Angel R.; Bernadó, Pau; Pons, Miquel

2013-01-01

c-Src is a non-receptor tyrosine kinase involved in numerous signal transduction pathways. The kinase, SH3 and SH2 domains of c-Src are attached to the membrane-anchoring SH4 domain through the flexible Unique domain. Here we show intra- and intermolecular interactions involving the Unique and SH3 domains suggesting the presence of a previously unrecognized additional regulation layer in c-Src. We have characterized lipid binding by the Unique and SH3 domains, their intramolecular interaction and its allosteric modulation by a SH3-binding peptide or by Calcium-loaded calmodulin binding to the Unique domain. We also show reduced lipid binding following phosphorylation at conserved sites of the Unique domain. Finally, we show that injection of full-length c-Src with mutations that abolish lipid binding by the Unique domain causes a strong in vivo phenotype distinct from that of wild-type c-Src in a Xenopus oocyte model system, confirming the functional role of the Unique domain in c-Src regulation. PMID:23416516

Quantitative relation between intermolecular and intramolecular binding of pro-rich peptides to SH3 domains.

PubMed

Zhou, Huan-Xiang

2006-11-01

Flexible linkers are often found to tether binding sequence motifs or connect protein domains. Here we analyze three usages of flexible linkers: 1), intramolecular binding of proline-rich peptides (PRPs) to SH3 domains for kinase regulation; 2), intramolecular binding of PRP for increasing the folding stability of SH3 domains; and 3), covalent linking of PRPs and other ligands for high-affinity bivalent binding. The basis of these analyses is a quantitative relation between intermolecular and intramolecular binding constants. This relation has the form K(i) = K(e0)p for intramolecular binding and K(e) = K(e01)K(e02)p for bivalent binding. The effective concentration p depends on the length of the linker and the distance between the linker attachment points in the bound state. Several applications illustrate the usefulness of the quantitative relation. These include intramolecular binding to the Itk SH3 domain by an internal PRP and to a circular permutant of the alpha-spectrin SH3 domain by a designed PRP, and bivalent binding to the two SH3 domains of Grb2 by two linked PRPs. These and other examples suggest that flexible linkers and sequence motifs tethered to them, like folded protein domains, are also subject to tight control during evolution.

SH2 domains of the p85 alpha subunit of phosphatidylinositol 3-kinase regulate binding to growth factor receptors.

PubMed Central

McGlade, C J; Ellis, C; Reedijk, M; Anderson, D; Mbamalu, G; Reith, A D; Panayotou, G; End, P; Bernstein, A; Kazlauskas, A

1992-01-01

The binding of cytoplasmic signaling proteins such as phospholipase C-gamma 1 and Ras GTPase-activating protein to autophosphorylated growth factor receptors is directed by their noncatalytic Src homology region 2 (SH2) domains. The p85 alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase, which associates with several receptor protein-tyrosine kinases, also contains two SH2 domains. Both p85 alpha SH2 domains, when expressed individually as fusion proteins in bacteria, bound stably to the activated beta receptor for platelet-derived growth factor (PDGF). Complex formation required PDGF stimulation and was dependent on receptor tyrosine kinase activity. The bacterial p85 alpha SH2 domains recognized activated beta PDGF receptor which had been immobilized on a filter, indicating that SH2 domains contact autophosphorylated receptors directly. Several receptor tyrosine kinases within the PDGF receptor subfamily, including the colony-stimulating factor 1 receptor and the Steel factor receptor (Kit), also associate with PI 3-kinase in vivo. Bacterially expressed SH2 domains derived from the p85 alpha subunit of PI 3-kinase bound in vitro to the activated colony-stimulating factor 1 receptor and to Kit. We infer that the SH2 domains of p85 alpha bind to high-affinity sites on these receptors, whose creation is dependent on receptor autophosphorylation. The SH2 domains of p85 are therefore primarily responsible for the binding of PI 3-kinase to activated growth factor receptors. Images PMID:1372092

The Abl SH2-kinase linker naturally adopts a conformation competent for SH3 domain binding.

PubMed

Chen, Shugui; Brier, Sébastien; Smithgall, Thomas E; Engen, John R

2007-04-01

The core of the Abelson tyrosine kinase (c-Abl) is structurally similar to Src-family kinases where SH3 and SH2 domains pack against the backside of the kinase domain in the down-regulated conformation. Both kinase families depend upon intramolecular association of SH3 with the linker joining the SH2 and kinase domains for suppression of kinase activity. Hydrogen deuterium exchange (HX) and mass spectrometry (MS) were used to probe intramolecular interaction of the c-Abl SH3 domain with the linker in recombinant constructs lacking the kinase domain. Under physiological conditions, the c-Abl SH3 domain undergoes partial unfolding, which is stabilized by ligand binding, providing a unique assay for SH3:linker interaction in solution. Using this approach, we observed dynamic association of the SH3 domain with the linker in the absence of the kinase domain. Truncation of the linker before W254 completely prevented cis-interaction with SH3, while constructs containing amino acids past this point showed SH3:linker interactions. The observation that the Abl linker sequence exhibits SH3-binding activity in the absence of the kinase domain is unique to Abl and was not observed with Src-family kinases. These results suggest that SH3:linker interactions may have a more prominent role in Abl regulation than in Src kinases, where the down-regulated conformation is further stabilized by a second intramolecular interaction between the C-terminal tail and the SH2 domain.

Conformational change of Sos-derived proline-rich peptide upon binding Grb2 N-terminal SH3 domain probed by NMR

NASA Astrophysics Data System (ADS)

Ogura, Kenji; Okamura, Hideyasu

2013-10-01

Growth factor receptor-bound protein 2 (Grb2) is a small adapter protein composed of a single SH2 domain flanked by two SH3 domains. The N-terminal SH3 (nSH3) domain of Grb2 binds a proline-rich region present in the guanine nucleotide releasing factor, son of sevenless (Sos). Using NMR relaxation dispersion and chemical shift analysis methods, we investigated the conformational change of the Sos-derived proline-rich peptide during the transition between the free and Grb2 nSH3-bound states. The chemical shift analysis revealed that the peptide does not present a fully random conformation but has a relatively rigid structure. The relaxation dispersion analysis detected conformational exchange of several residues of the peptide upon binding to Grb2 nSH3.

Structural and biophysical investigation of the interaction of a mutant Grb2 SH2 domain (W121G) with its cognate phosphopeptide.

PubMed

Papaioannou, Danai; Geibel, Sebastian; Kunze, Micha B A; Kay, Christopher W M; Waksman, Gabriel

2016-03-01

The adaptor protein Grb2 is a key element of mitogenetically important signaling pathways. With its SH2 domain it binds to upstream targets while its SH3 domains bind to downstream proteins thereby relaying signals from the cell membranes to the nucleus. The Grb2 SH2 domain binds to its targets by recognizing a phosphotyrosine (pY) in a pYxNx peptide motif, requiring an Asn at the +2 position C-terminal to the pY with the residue either side of this Asn being hydrophobic. Structural analysis of the Grb2 SH2 domain in complex with its cognate peptide has shown that the peptide adopts a unique β-turn conformation, unlike the extended conformation that phosphopeptides adopt when bound to other SH2 domains. TrpEF1 (W121) is believed to force the peptide into this unusual conformation conferring this unique specificity to the Grb2 SH2 domain. Using X-ray crystallography, electron paramagnetic resonance (EPR) spectroscopy, and isothermal titration calorimetry (ITC), we describe here a series of experiments that explore the role of TrpEF1 in determining the specificity of the Grb2 SH2 domain. Our results demonstrate that the ligand does not adopt a pre-organized structure before binding to the SH2 domain, rather it is the interaction between the two that imposes the hairpin loop to the peptide. Furthermore, we find that the peptide adopts a similar structure when bound to both the wild-type Grb2 SH2 domain and a TrpEF1Gly mutant. This suggests that TrpEF1 is not the determining factor for the conformation of the phosphopeptide. © 2015 The Protein Society.

Purification of SOCS (Suppressor of Cytokine Signaling) SH2 Domains for Structural and Functional Studies.

PubMed

Liau, Nicholas P D; Laktyushin, Artem; Babon, Jeffrey J

2017-01-01

Src Homology 2 (SH2) domains are protein domains which have a high binding affinity for specific amino acid sequences containing a phosphorylated tyrosine residue. The Suppressors of Cytokine Signaling (SOCS) proteins use an SH2 domain to bind to components of certain cytokine signaling pathways to downregulate the signaling cascade. The recombinantly produced SH2 domains of various SOCS proteins have been used to undertake structural and functional studies elucidating the method of how such targeting occurs. Here, we describe the protocol for the recombinant production and purification of SOCS SH2 domains, with an emphasis on SOCS3.

SH2 Ligand Prediction-Guidance for In-Silico Screening.

PubMed

Li, Shawn S C; Li, Lei

2017-01-01

Systematic identification of binding partners for SH2 domains is important for understanding the biological function of the corresponding SH2 domain-containing proteins. Here, we describe two different web-accessible computer programs, SMALI and DomPep, for predicting binding ligands for SH2 domains. The former was developed using a Scoring Matrix method and the latter based on the Support Vector Machine model.

Dissection of the BCR-ABL signaling network using highly specific monobody inhibitors to the SHP2 SH2 domains.

PubMed

Sha, Fern; Gencer, Emel Basak; Georgeon, Sandrine; Koide, Akiko; Yasui, Norihisa; Koide, Shohei; Hantschel, Oliver

2013-09-10

The dysregulated tyrosine kinase BCR-ABL causes chronic myelogenous leukemia in humans and forms a large multiprotein complex that includes the Src-homology 2 (SH2) domain-containing phosphatase 2 (SHP2). The expression of SHP2 is necessary for BCR-ABL-dependent oncogenic transformation, but the precise signaling mechanisms of SHP2 are not well understood. We have developed binding proteins, termed monobodies, for the N- and C-terminal SH2 domains of SHP2. Intracellular expression followed by interactome analysis showed that the monobodies are essentially monospecific to SHP2. Two crystal structures revealed that the monobodies occupy the phosphopeptide-binding sites of the SH2 domains and thus can serve as competitors of SH2-phosphotyrosine interactions. Surprisingly, the segments of both monobodies that bind to the peptide-binding grooves run in the opposite direction to that of canonical phosphotyrosine peptides, which may contribute to their exquisite specificity. When expressed in cells, monobodies targeting the N-SH2 domain disrupted the interaction of SHP2 with its upstream activator, the Grb2-associated binder 2 adaptor protein, suggesting decoupling of SHP2 from the BCR-ABL protein complex. Inhibition of either N-SH2 or C-SH2 was sufficient to inhibit two tyrosine phosphorylation events that are critical for SHP2 catalytic activity and to block ERK activation. In contrast, targeting the N-SH2 or C-SH2 revealed distinct roles of the two SH2 domains in downstream signaling, such as the phosphorylation of paxillin and signal transducer and activator of transcription 5. Our results delineate a hierarchy of function for the SH2 domains of SHP2 and validate monobodies as potent and specific antagonists of protein-protein interactions in cancer cells.

Changes in signal transducer and activator of transcription 3 (STAT3) dynamics induced by complexation with pharmacological inhibitors of Src homology 2 (SH2) domain dimerization.

PubMed

Resetca, Diana; Haftchenary, Sina; Gunning, Patrick T; Wilson, Derek J

2014-11-21

The activity of the transcription factor signal transducer and activator of transcription 3 (STAT3) is dysregulated in a number of hematological and solid malignancies. Development of pharmacological STAT3 Src homology 2 (SH2) domain interaction inhibitors holds great promise for cancer therapy, and a novel class of salicylic acid-based STAT3 dimerization inhibitors that includes orally bioavailable drug candidates has been recently developed. The compounds SF-1-066 and BP-1-102 are predicted to bind to the STAT3 SH2 domain. However, given the highly unstructured and dynamic nature of the SH2 domain, experimental confirmation of this prediction was elusive. We have interrogated the protein-ligand interaction of STAT3 with these small molecule inhibitors by means of time-resolved electrospray ionization hydrogen-deuterium exchange mass spectrometry. Analysis of site-specific evolution of deuterium uptake induced by the complexation of STAT3 with SF-1-066 or BP-1-102 under physiological conditions enabled the mapping of the in silico predicted inhibitor binding site to the STAT3 SH2 domain. The binding of both inhibitors to the SH2 domain resulted in significant local decreases in dynamics, consistent with solvent exclusion at the inhibitor binding site and increased rigidity of the inhibitor-complexed SH2 domain. Interestingly, inhibitor binding induced hot spots of allosteric perturbations outside of the SH2 domain, manifesting mainly as increased deuterium uptake, in regions of STAT3 important for DNA binding and nuclear localization. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

The development and application of a quantitative peptide microarray platform to SH2 domain specificity space

NASA Astrophysics Data System (ADS)

Engelmann, Brett Warren

The Src homology 2 (SH2) domains evolved alongside protein tyrosine kinases (PTKs) and phosphatases (PTPs) in metazoans to recognize the phosphotyrosine (pY) post-translational modification. The human genome encodes 121 SH2 domains within 111 SH2 domain containing proteins that represent the primary mechanism for cellular signal transduction immediately downstream of PTKs. Despite pY recognition contributing to roughly half of the binding energy, SH2 domains possess substantial binding specificity, or affinity discrimination between phosphopeptide ligands. This specificity is largely imparted by amino acids (AAs) adjacent to the pY, typically from positions +1 to +4 C-terminal to the pY. Much experimental effort has been undertaken to construct preferred binding motifs for many SH2 domains. However, due to limitations in previous experimental methodologies these motifs do not account for the interplay between AAs. It was therefore not known how AAs within the context of individual peptides function to impart SH2 domain specificity. In this work we identified the critical role context plays in defining SH2 domain specificity for physiological ligands. We also constructed a high quality interactome using 50 SH2 domains and 192 physiological ligands. We next developed a quantitative high-throughput (Q-HTP) peptide microarray platform to assess the affinities four SH2 domains have for 124 physiological ligands. We demonstrated the superior characteristics of our platform relative to preceding approaches and validated our results using established biophysical techniques, literature corroboration, and predictive algorithms. The quantitative information provided by the arrays was leveraged to investigate SH2 domain binding distributions and identify points of binding overlap. Our microarray derived affinity estimates were integrated to produce quantitative interaction motifs capable of predicting interactions. Furthermore, our microarrays proved capable of resolving subtle contextual differences within motifs that modulate interaction affinities. We conclude that contextually informed specificity profiling of protein interaction domains using the methodologies developed in this study can inform efforts to understand the interconnectivity of signaling networks in normal and aberrant states. Three supplementary tables containing detailed lists of peptides, interactions, and sources of corroborative information are provided.

An Efficient Semi-supervised Learning Approach to Predict SH2 Domain Mediated Interactions.

PubMed

Kundu, Kousik; Backofen, Rolf

2017-01-01

Src homology 2 (SH2) domain is an important subclass of modular protein domains that plays an indispensable role in several biological processes in eukaryotes. SH2 domains specifically bind to the phosphotyrosine residue of their binding peptides to facilitate various molecular functions. For determining the subtle binding specificities of SH2 domains, it is very important to understand the intriguing mechanisms by which these domains recognize their target peptides in a complex cellular environment. There are several attempts have been made to predict SH2-peptide interactions using high-throughput data. However, these high-throughput data are often affected by a low signal to noise ratio. Furthermore, the prediction methods have several additional shortcomings, such as linearity problem, high computational complexity, etc. Thus, computational identification of SH2-peptide interactions using high-throughput data remains challenging. Here, we propose a machine learning approach based on an efficient semi-supervised learning technique for the prediction of 51 SH2 domain mediated interactions in the human proteome. In our study, we have successfully employed several strategies to tackle the major problems in computational identification of SH2-peptide interactions.

SH2-catalytic domain linker heterogeneity influences allosteric coupling across the SFK family.

PubMed

Register, A C; Leonard, Stephen E; Maly, Dustin J

2014-11-11

Src-family kinases (SFKs) make up a family of nine homologous multidomain tyrosine kinases whose misregulation is responsible for human disease (cancer, diabetes, inflammation, etc.). Despite overall sequence homology and identical domain architecture, differences in SH3 and SH2 regulatory domain accessibility and ability to allosterically autoinhibit the ATP-binding site have been observed for the prototypical SFKs Src and Hck. Biochemical and structural studies indicate that the SH2-catalytic domain (SH2-CD) linker, the intramolecular binding epitope for SFK SH3 domains, is responsible for allosterically coupling SH3 domain engagement to autoinhibition of the ATP-binding site through the conformation of the αC helix. As a relatively unconserved region between SFK family members, SH2-CD linker sequence variability across the SFK family is likely a source of nonredundant cellular functions between individual SFKs via its effect on the availability of SH3 and SH2 domains for intermolecular interactions and post-translational modification. Using a combination of SFKs engineered with enhanced or weakened regulatory domain intramolecular interactions and conformation-selective inhibitors that report αC helix conformation, this study explores how SH2-CD sequence heterogeneity affects allosteric coupling across the SFK family by examining Lyn, Fyn1, and Fyn2. Analyses of Fyn1 and Fyn2, isoforms that are identical but for a 50-residue sequence spanning the SH2-CD linker, demonstrate that SH2-CD linker sequence differences can have profound effects on allosteric coupling between otherwise identical kinases. Most notably, a dampened allosteric connection between the SH3 domain and αC helix leads to greater autoinhibitory phosphorylation by Csk, illustrating the complex effects of SH2-CD linker sequence on cellular function.

Targeting Self-Binding Peptides as a Novel Strategy To Regulate Protein Activity and Function: A Case Study on the Proto-oncogene Tyrosine Protein Kinase c-Src.

PubMed

Bai, Zhengya; Hou, Shasha; Zhang, Shilei; Li, Zhongyan; Zhou, Peng

2017-04-24

Previously, we have reported a new biomolecular phenomenon spanning between protein folding and binding, termed as self-binding peptides (SBPs), where a short peptide segment in monomeric protein functions as a molecular switch by dynamically binding to/unbinding from its cognate domain in the monomer (Yang et al. J. Chem. Inf. 2015, 55, 329-342). Here, we attempt to raise the SBP as a new class of druggable targets to regulate the biological activity and function of proteins. A case study was performed on the proto-oncogene nonreceptor tyrosine kinase, c-Src, which contains two SBPs that bind separately to SH3 and SH2 domains of the kinase. State-of-the-art molecular dynamics (MD) simulations and post binding energetics analysis revealed that disrupting the kinase-intramolecular interactions of SH3 and SH2 domains with their cognate SBP ligands can result in totally different effects on the structural dynamics of c-Src kinase architecture; targeting the SH2 domain unlocks the autoinhibitory form of the kinase-this is very similar to the pTyr527 dephosphorylation that functionally activates the kinase, whereas targeting the SH3 domain can only release the domain from the tightly packed kinase but has a moderate effect on the kinase activity. Subsequently, based on the cognate SBP sequence we computationally designed a number of SH2-binding phosphopeptides using a motif grafting strategy. Fluorescence polarization (FP) assay observed that most of the designed phosphopeptides have higher binding affinity to SH2 domain as compared to the native SBP segment (K d = 53 nM). Kinase assay identified a typical dose-response relationship of phosphopeptides against kinase activation, substantiating that disruption of SH2-SBP interaction can mimic c-Src dephosphorylation and activate the kinase. Two rationally designed phosphopeptides, namely EPQpYEEIEN and EPQpYEELEN, were determined as strong binders of SH2 domain (K d = 8.3 and 15 nM, respectively) and potent activators of c-Src kinase (EC 50 = 3.2 and 41 μM, respectively).

Binding Assays Using Recombinant SH2 Domains: Far-Western, Pull-Down, and Fluorescence Polarization.

PubMed

Machida, Kazuya; Liu, Bernard

2017-01-01

Recognition of phosphotyrosine-containing sequences by SH2 domains confers specificity in tyrosine kinase pathways. By assessing interactions between isolated SH2 domains and their binding proteins, it is possible to gain insight into otherwise inaccessible complex cellular systems. Far-Western, pull-down, and fluorescence polarization (FP) have been frequently used for characterization of phosphotyrosine signaling. Here, we outline standard protocols for these established assays using recombinant SH2 domain, emphasizing the importance of appropriate sample preparation and assay controls.

Insight into the Selectivity of the G7-18NATE Inhibitor Peptide for the Grb7-SH2 Domain Target.

PubMed

Watson, Gabrielle M; Lucas, William A H; Gunzburg, Menachem J; Wilce, Jacqueline A

2017-01-01

Growth factor receptor bound protein 7 (Grb7) is an adaptor protein with established roles in the progression of both breast and pancreatic cancers. Through its C-terminal SH2 domain, Grb7 binds to phosphorylated tyrosine kinases to promote proliferative and migratory signaling. Here, we investigated the molecular basis for the specificity of a Grb7 SH2-domain targeted peptide inhibitor. We identified that arginine 462 in the BC loop is unique to Grb7 compared to Grb2, another SH2 domain bearing protein that shares the same consensus binding motif as Grb7. Using surface plasmon resonance we demonstrated that Grb7-SH2 binding to G7-18NATE is reduced 3.3-fold when the arginine is mutated to the corresponding Grb2 amino acid. The reverse mutation in Grb2-SH2 (serine to arginine), however, was insufficient to restore binding of G7-18NATE to Grb2-SH2. Further, using a microarray, we confirmed that G7-18NATE is specific for Grb7 over a panel of 79 SH2 domains, and identified that leucine at the βD6 position may also be a requirement for Grb7-SH2 binding. This study provides insight into the specificity defining features of Grb7 for the inhibitor molecule G7-18NATE, that will assist in the development of improved Grb7 targeted inhibitors.

Insight into the Selectivity of the G7-18NATE Inhibitor Peptide for the Grb7-SH2 Domain Target

PubMed Central

Watson, Gabrielle M.; Lucas, William A. H.; Gunzburg, Menachem J.; Wilce, Jacqueline A.

2017-01-01

Growth factor receptor bound protein 7 (Grb7) is an adaptor protein with established roles in the progression of both breast and pancreatic cancers. Through its C-terminal SH2 domain, Grb7 binds to phosphorylated tyrosine kinases to promote proliferative and migratory signaling. Here, we investigated the molecular basis for the specificity of a Grb7 SH2-domain targeted peptide inhibitor. We identified that arginine 462 in the BC loop is unique to Grb7 compared to Grb2, another SH2 domain bearing protein that shares the same consensus binding motif as Grb7. Using surface plasmon resonance we demonstrated that Grb7-SH2 binding to G7-18NATE is reduced 3.3-fold when the arginine is mutated to the corresponding Grb2 amino acid. The reverse mutation in Grb2-SH2 (serine to arginine), however, was insufficient to restore binding of G7-18NATE to Grb2-SH2. Further, using a microarray, we confirmed that G7-18NATE is specific for Grb7 over a panel of 79 SH2 domains, and identified that leucine at the βD6 position may also be a requirement for Grb7-SH2 binding. This study provides insight into the specificity defining features of Grb7 for the inhibitor molecule G7-18NATE, that will assist in the development of improved Grb7 targeted inhibitors. PMID:29018805

SH2 and SH3 domains: elements that control interactions of cytoplasmic signaling proteins.

PubMed

Koch, C A; Anderson, D; Moran, M F; Ellis, C; Pawson, T

1991-05-03

Src homology (SH) regions 2 and 3 are noncatalytic domains that are conserved among a series of cytoplasmic signaling proteins regulated by receptor protein-tyrosine kinases, including phospholipase C-gamma, Ras GTPase (guanosine triphosphatase)-activating protein, and Src-like tyrosine kinases. The SH2 domains of these signaling proteins bind tyrosine phosphorylated polypeptides, implicated in normal signaling and cellular transformation. Tyrosine phosphorylation acts as a switch to induce the binding of SH2 domains, thereby mediating the formation of heteromeric protein complexes at or near the plasma membrane. The formation of these complexes is likely to control the activation of signal transduction pathways by tyrosine kinases. The SH3 domain is a distinct motif that, together with SH2, may modulate interactions with the cytoskeleton and membrane. Some signaling and transforming proteins contain SH2 and SH3 domains unattached to any known catalytic element. These noncatalytic proteins may serve as adaptors to link tyrosine kinases to specific target proteins. These observations suggest that SH2 and SH3 domains participate in the control of intracellular responses to growth factor stimulation.

Dimer formation through domain swapping in the crystal structure of the Grb2-SH2-Ac-pYVNV complex.

PubMed

Schiering, N; Casale, E; Caccia, P; Giordano, P; Battistini, C

2000-11-07

Src homology 2 (SH2) domains are key modules in intracellular signal transduction. They link activated cell surface receptors to downstream targets by binding to phosphotyrosine-containing sequence motifs. The crystal structure of a Grb2-SH2 domain-phosphopeptide complex was determined at 2.4 A resolution. The asymmetric unit contains four polypeptide chains. There is an unexpected domain swap so that individual chains do not adopt a closed SH2 fold. Instead, reorganization of the EF loop leads to an open, nonglobular fold, which associates with an equivalent partner to generate an intertwined dimer. As in previously reported crystal structures of canonical Grb2-SH2 domain-peptide complexes, each of the four hybrid SH2 domains in the two domain-swapped dimers binds the phosphopeptide in a type I beta-turn conformation. This report is the first to describe domain swapping for an SH2 domain. While in vivo evidence of dimerization of Grb2 exists, our SH2 dimer is metastable and a physiological role of this new form of dimer formation remains to be demonstrated.

The N-methyl-D-aspartate receptor subunits NR2A and NR2B bind to the SH2 domains of phospholipase C-gamma.

PubMed

Gurd, J W; Bissoon, N

1997-08-01

The NMDA receptor has recently been found to be phosphorylated on tyrosine. To assess the possible connection between tyrosine phosphorylation of the NMDA receptor and signaling pathways in the postsynaptic cell, we have investigated the relationship between tyrosine phosphorylation and the binding of NMDA receptor subunits to the SH2 domains of phospholipase C-gamma (PLC-gamma). A glutathione S-transferase (GST) fusion protein containing both the N- and the C-proximal SH2 domains of PLC-gamma was bound to glutathione-agarose and reacted with synaptic junctional proteins and glycoproteins. Tyrosine-phosphorylated PSD-GP180, which has been identified as the NR2B subunit of the NMDA receptor, bound to the SH2-agarose beads in a phosphorylation-dependent fashion. Immunoblot analysis with antibodies specific for individual NMDA receptor subunits showed that both NR2A and NR2B subunits bound to the SH2-agarose. No binding occurred to GST-agarose lacking an associated SH2 domain, indicating that binding was specific for the SH2 domains. The binding of receptor subunits increased after the incubation of synaptic junctions with ATP and decreased after treatment of synaptic junctions with exogenous protein tyrosine phosphatase. Immunoprecipitation experiments confirmed that NR2A and NR2B were phosphorylated on tyrosine and further that tyrosine phosphorylation of each of the subunits was increased after incubation with ATP. The results demonstrate that NMDA receptor subunits NR2A and NR2B will bind to the SH2 domains of PLC-gamma and that isolated synaptic junctions contain endogenous protein tyrosine kinase(s) that can phosphorylate both NR2A and NR2B receptor subunits, and suggest that interaction of the tyrosine-phosphorylated NMDA receptor with proteins that contain SH2 domains may serve to link it to signaling pathways in the postsynaptic cell.

Linker length dependent binding of a focal adhesion kinase derived peptide to the Src SH3-SH2 domains.

PubMed

Lindfors, Hanna E; Venkata, Bharat Somireddy; Drijfhout, Jan W; Ubbink, Marcellus

2011-02-18

The interaction between a peptide encompassing the SH3 and SH2 binding motifs of focal adhesion kinase (FAK) and the Src SH3-SH2 domains has been investigated with NMR spectroscopy and calorimetry. The binding to both motifs is anti-cooperative. Reduction of the long linker connecting the motifs does not lead to cooperativity. Short linkers that do not allow simultaneous intramolecular binding of the peptide to both motifs cause peptide-mediated dimerisation, even with a linker of only three amino acids. The role of the SH3 binding motif is discussed in view of the independent nature of the SH interactions. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Differential Recognition Preferences of the Three Src Homology 3 (SH3) Domains from the Adaptor CD2-associated Protein (CD2AP) and Direct Association with Ras and Rab Interactor 3 (RIN3)*

PubMed Central

Rouka, Evgenia; Simister, Philip C.; Janning, Melanie; Kumbrink, Joerg; Konstantinou, Tassos; Muniz, João R. C.; Joshi, Dhira; O'Reilly, Nicola; Volkmer, Rudolf; Ritter, Brigitte; Knapp, Stefan; von Delft, Frank; Kirsch, Kathrin H.; Feller, Stephan M.

2015-01-01

CD2AP is an adaptor protein involved in membrane trafficking, with essential roles in maintaining podocyte function within the kidney glomerulus. CD2AP contains three Src homology 3 (SH3) domains that mediate multiple protein-protein interactions. However, a detailed comparison of the molecular binding preferences of each SH3 remained unexplored, as well as the discovery of novel interactors. Thus, we studied the binding properties of each SH3 domain to the known interactor Casitas B-lineage lymphoma protein (c-CBL), conducted a peptide array screen based on the recognition motif PxPxPR and identified 40 known or novel candidate binding proteins, such as RIN3, a RAB5-activating guanine nucleotide exchange factor. CD2AP SH3 domains 1 and 2 generally bound with similar characteristics and specificities, whereas the SH3-3 domain bound more weakly to most peptide ligands tested yet recognized an unusually extended sequence in ALG-2-interacting protein X (ALIX). RIN3 peptide scanning arrays revealed two CD2AP binding sites, recognized by all three SH3 domains, but SH3-3 appeared non-functional in precipitation experiments. RIN3 recruited CD2AP to RAB5a-positive early endosomes via these interaction sites. Permutation arrays and isothermal titration calorimetry data showed that the preferred binding motif is Px(P/A)xPR. Two high-resolution crystal structures (1.65 and 1.11 Å) of CD2AP SH3-1 and SH3-2 solved in complex with RIN3 epitopes 1 and 2, respectively, indicated that another extended motif is relevant in epitope 2. In conclusion, we have discovered novel interaction candidates for CD2AP and characterized subtle yet significant differences in the recognition preferences of its three SH3 domains for c-CBL, ALIX, and RIN3. PMID:26296892

Diversity in peptide recognition by the SH2 domain of SH2B1.

PubMed

McKercher, Marissa A; Guan, Xiaoyang; Tan, Zhongping; Wuttke, Deborah S

2018-02-01

SH2B1 is a multidomain protein that serves as a key adaptor to regulate numerous cellular events, such as insulin, leptin, and growth hormone signaling pathways. Many of these protein-protein interactions are mediated by the SH2 domain of SH2B1, which recognizes ligands containing a phosphorylated tyrosine (pY), including peptides derived from janus kinase 2, insulin receptor, and insulin receptor substrate-1 and -2. Specificity for the SH2 domain of SH2B1 is conferred in these ligands either by a hydrophobic or an acidic side chain at the +3 position C-terminal to the pY. This specificity for chemically disparate species suggests that SH2B1 relies on distinct thermodynamic or structural mechanisms to bind to peptides. Using binding and structural strategies, we have identified unique thermodynamic signatures for each peptide binding mode, and several SH2B1 residues, including K575 and R578, that play distinct roles in peptide binding. The high-resolution structure of the SH2 domain of SH2B1 further reveals conformationally plastic protein loops that may contribute to the ability of the protein to recognize dissimilar ligands. Together, numerous hydrophobic and electrostatic interactions, in addition to backbone conformational flexibility, permit the recognition of diverse peptides by SH2B1. An understanding of this expanded peptide recognition will allow for the identification of novel physiologically relevant SH2B1/peptide interactions, which can contribute to the design of obesity and diabetes pharmaceuticals to target the ligand-binding interface of SH2B1 with high specificity. © 2017 Wiley Periodicals, Inc.

Computational dissection of allosteric inhibition of the SH2 domain of Bcr-Abl kinase by the monobody inhibitor AS25.

PubMed

Ji, Mingfei; Zheng, Guodong; Li, Xiaolong; Zhang, Zhongqin; Jv, Guanqun; Wang, Xiaowei; Wang, Jialin

2017-06-01

The deregulated breakpoint cluster region (Bcr)-Abelson tyrosine kinase (Abl) fusion protein represents an attractive pharmacological target for the treatment of chronic myeloid leukemia (CML). The high affinity of monobody AS25 was designed to target the Src homology 2 (SH2) domain of Bcr-Abl, leading to allosteric inhibition of Bcr-Abl through formation of protein-protein interactions. An I164E mutation in the SH2 domain disrupts AS25 binding to the SH2 domain of Bcr-Abl. The detailed mechanisms, however, remain to be unresolved. Here, molecular dynamics (MD) simulations and binding free energy calculations were performed to explore the conformational and energetic differences between the wild-type (WT) complexes of Bcr-Abl SH2 domain and AS25 (SH2 WT -AS25) as well as the mutated complexes (SH2 I164E -AS25). The results revealed that I164E mutation not only caused an increase in the conformational flexibility of SH2-AS25 complexes, but also weakened the binding affinity of AS25 to SH2. The comparative binding modes of SH2-AS25 complexes between WT and the I164E mutant were comprehensively analyzed to unravel the disruption of hydrophobic and hydrogen bonding interactions in the interface of the SH2-AS25 complex triggered by the I164E mutation. The results obtained may help to design the next generation of higher affinity Bcr-Abl SH2-specific peptide inhibitors.

Functional diversity of Csk, Chk, and Src SH2 domains due to a single residue variation.

PubMed

Ayrapetov, Marina K; Nam, Nguyen Hai; Ye, Guofeng; Kumar, Anil; Parang, Keykavous; Sun, Gongqin

2005-07-08

The C-terminal Src kinase (Csk) family of protein tyrosine kinases contains two members: Csk and Csk homologous kinase (Chk). Both phosphorylate and inactivate Src family kinases. Recent reports suggest that the Src homology (SH) 2 domains of Csk and Chk may bind to different phosphoproteins, which provides a basis for different cellular functions for Csk and Chk. To verify and characterize such a functional divergence, we compared the binding properties of the Csk, Chk, and Src SH2 domains and investigated the structural basis for the functional divergence. First, the study demonstrated striking functional differences between the Csk and Chk SH2 domains and revealed functional similarities between the Chk and Src SH2 domains. Second, structural analysis and mutagenic studies revealed that the functional differences among the three SH2 domains were largely controlled by one residue, Glu127 in Csk, Ile167 in Chk, and Lys200 in Src. Mutating these residues in the Csk or Chk SH2 domain to the Src counterpart resulted in dramatic gain of function similar to Src SH2 domain, whereas mutating Lys200 in Src SH2 domain to Glu (the Csk counterpart) resulted in loss of Src SH2 function. Third, a single point mutation of E127K rendered Csk responsive to activation by a Src SH2 domain ligand. Finally, the optimal phosphopeptide sequence for the Chk SH2 domain was determined. These results provide a compelling explanation for the functional differences between two homologous protein tyrosine kinases and reveal a new structure-function relationship for the SH2 domains.

The ShcA SH2 domain engages a 14-3-3/PI3'K signaling complex and promotes breast cancer cell survival.

PubMed

Ursini-Siegel, J; Hardy, W R; Zheng, Y; Ling, C; Zuo, D; Zhang, C; Podmore, L; Pawson, T; Muller, W J

2012-11-29

The ShcA adapter protein transmits activating signals downstream of receptor and cytoplasmic tyrosine kinases through the establishment of phosphotyrosine-dependent complexes. In this regard, ShcA possesses both a phosphotyrosine-binding domain (PTB) and Src homology 2 domain (SH2), which bind phosphotyrosine residues in a sequence-specific manner. Although the majority of receptor tyrosine kinases expressed in breast cancer cells bind the PTB domain, very little is known regarding the biological importance of SH2-driven ShcA signaling during mammary tumorigenesis. To address this, we employed transgenic mice expressing a mutant ShcA allele harboring a non-functional SH2 domain (ShcR397K) under the transcriptional control of the endogenous ShcA promoter. Using transplantation approaches, we demonstrate that SH2-dependent ShcA signaling within the mammary epithelial compartment is essential for breast tumor outgrowth, survival and the development of lung metastases. We further show that the ShcA SH2 domain activates the AKT pathway, potentially through a novel SH2-mediated complex between ShcA, 14-3-3ζ and the p85 regulatory subunit of phosphatidylinositol 3 (PI3') kinase. This study is the first to demonstrate that the SH2 domain of ShcA is critical for tumor survival during mammary tumorigenesis.

Differential recognition of syk-binding sites by each of the two phosphotyrosine-binding pockets of the Vav SH2 domain.

PubMed

Chen, Chih-Hong; Piraner, Dan; Gorenstein, Nina M; Geahlen, Robert L; Beth Post, Carol

2013-11-01

The association of spleen tyrosine kinase (Syk), a central tyrosine kinase in B cell signaling, with Vav SH2 domain is controlled by phosphorylation of two closely spaced tyrosines in Syk linker B: Y342 and Y346. Previous studies established both singly phosphorylated and doubly phosphorylated forms play a role in signaling. The structure of the doubly phosphorylated form identified a new recognition of phosphotyrosine whereby two phosphotyrosines bind simultaneously to the Vav SH2 domain, one in the canonical pTyr pocket and one in the specificity pocket on the opposite side of the central β-sheet. It is unknown if the specificity pocket can bind phosphotyrosine independent of phosphotyrosine binding the pTyr pocket. To address this gap in knowledge, we determined the structure of the complex between Vav1 SH2 and a peptide (SykLB-YpY) modeling the singly phosphorylated-Y346 form of Syk with unphosphorylated Y342. The nuclear magnetic resonance (NMR) data conclusively establish that recognition of phosphotyrosine is swapped between the two pockets; phosphorylated pY346 binds the specificity pocket of Vav1 SH2, and unphosphorylated Y342 occupies what is normally the pTyr binding pocket. Nearly identical changes in chemical shifts occurred upon binding all three forms of singly and doubly phosphorylated peptides; however, somewhat smaller shift perturbations for SykLB-YpY from residues in regions of high internal mobility suggest that internal motions are coupled to binding affinity. The differential recognition that includes this swapped binding of phosphotyrosine to the specificity pocket of Vav SH2 increases the repertoire of possible phosphotyrosine binding by SH2 domains in regulating protein-protein interactions in cellular signaling. Copyright © 2013 Wiley Periodicals, Inc.

SH2-PLA: a sensitive in-solution approach for quantification of modular domain binding by proximity ligation and real-time PCR.

PubMed

Thompson, Christopher M; Bloom, Lee R; Ogiue-Ikeda, Mari; Machida, Kazuya

2015-06-26

There is a great interest in studying phosphotyrosine dependent protein-protein interactions in tyrosine kinase pathways that play a critical role in many aspects of cellular function. We previously established SH2 profiling, a phosphoproteomic approach based on membrane binding assays that utilizes purified Src Homology 2 (SH2) domains as a molecular tool to profile the global tyrosine phosphorylation state of cells. However, in order to use this method to investigate SH2 binding sites on a specific target in cell lysate, additional procedures such as pull-down or immunoprecipitation which consume large amounts of sample are required. We have developed PLA-SH2, an alternative in-solution modular domain binding assay that takes advantage of Proximity Ligation Assay and real-time PCR. The SH2-PLA assay utilizes oligonucleotide-conjugated anti-GST and anti-EGFR antibodies recognizing a GST-SH2 probe and cellular EGFR, respectively. If the GST-SH2 and EGFR are in close proximity as a result of SH2-phosphotyrosine interactions, the two oligonucleotides are brought within a suitable distance for ligation to occur, allowing for efficient complex amplification via real-time PCR. The assay detected signal across at least 3 orders of magnitude of lysate input with a linear range spanning 1-2 orders and a low femtomole limit of detection for EGFR phosphotyrosine. SH2 binding kinetics determined by PLA-SH2 showed good agreement with established far-Western analyses for A431 and Cos1 cells stimulated with EGF at various times and doses. Further, we showed that PLA-SH2 can survey lung cancer tissues using 1 μl lysate without requiring phospho-enrichment. We showed for the first time that interactions between SH2 domain probes and EGFR in cell lysate can be determined in a microliter-scale assay using SH2-PLA. The obvious benefit of this method is that the low sample requirement allows detection of SH2 binding in samples which are difficult to analyze using traditional protein interaction assays. This feature along with short assay runtime makes this method a useful platform for the development of high throughput assays to determine modular domain-ligand interactions which could have wide-ranging applications in both basic and translational cancer research.

Evolution of Src Homology 2 (SH2) Domain to Recognize Sulfotyrosine.

PubMed

Ju, Tong; Niu, Wei; Guo, Jiantao

2016-09-16

Protein tyrosine O-sulfation is considered as the most common type of post-translational tyrosine modification in nature and plays important roles in extracellular biomolecular interactions. To facilitate the mapping, biological study, and medicinal application of this type of post-translational modification, we seek to evolve a small protein scaffold that recognizes sulfotyrosine with high affinity. We focused our efforts on the engineering of the Src Homology 2 (SH2) domain, which represents the largest class of known phosphotyrosine-recognition domain in nature and has a highly evolvable binding pocket. By using phage display, we successfully engineered the SH2 domain to recognize sulfotyrosine with high affinity. The best mutant, SH2-60.1, displayed more than 1700 fold higher sulfotyrosine-binding affinity than that of the wild-type SH2 domain. We also demonstrated that the evolved SH2 domain mutants could be used to detect sulfoprotein levels on the cell surface. These evolved SH2 domain mutants can be potentially applied to the study of protein tyrosine O-sulfation with proper experimental designs.

Coupled motions in the SH2 and kinase domains of Csk control Src phosphorylation.

PubMed

Wong, Lilly; Lieser, Scot A; Miyashita, Osamu; Miller, Meghan; Tasken, Kjetil; Onuchic, Josè N; Adams, Joseph A; Woods, Virgil L; Jennings, Patricia A

2005-08-05

The C-terminal Src kinase (Csk) phosphorylates and down-regulates Src family tyrosine kinases. The Csk-binding protein (Cbp) localizes Csk close to its substrates at the plasma membrane, and increases the specific activity of the kinase. To investigate this long-range catalytic effect, the phosphorylation of Src and the conformation of Csk were investigated in the presence of a high-affinity phosphopeptide derived from Cbp. This peptide binds tightly to the SH2 domain and enhances Src recognition (lowers K(m)) by increasing the apparent phosphoryl transfer rate in the Csk active site, a phenomenon detected in rapid quench flow experiments. Previous studies demonstrated that the regulation of Csk activity is linked to conformational changes in the enzyme that can be probed with hydrogen-deuterium exchange methods. We show that the Cbp peptide impacts deuterium incorporation into its binding partner (the SH2 domain), and into the SH2-kinase linker and several sequences in the kinase domain, including the glycine-rich loop in the active site. These findings, along with computational data from normal mode analyses, suggest that the SH2 domain moves in a cantilever fashion with respect to the small lobe of the kinase domain, ordering the active site for catalysis. The binding of a small Cbp-derived peptide to the SH2 domain of Csk modifies these motions, enhancing Src recognition.

Structural basis of nSH2 regulation and lipid binding in PI3Kα.

PubMed

Miller, Michelle S; Schmidt-Kittler, Oleg; Bolduc, David M; Brower, Evan T; Chaves-Moreira, Daniele; Allaire, Marc; Kinzler, Kenneth W; Jennings, Ian G; Thompson, Philip E; Cole, Philip A; Amzel, L Mario; Vogelstein, Bert; Gabelli, Sandra B

2014-07-30

We report two crystal structures of the wild-type phosphatidylinositol 3-kinase α (PI3Kα) heterodimer refined to 2.9 Å and 3.4 Å resolution: the first as the free enzyme, the second in complex with the lipid substrate, diC4-PIP₂, respectively. The first structure shows key interactions of the N-terminal SH2 domain (nSH2) and iSH2 with the activation loop that suggest a mechanism by which the enzyme is inhibited in its basal state. In the second structure, the lipid substrate binds in a positively charged pocket adjacent to the ATP-binding site, bordered by the P-loop, the activation loop and the iSH2 domain. An additional lipid-binding site was identified at the interface of the ABD, iSH2 and kinase domains. The ability of PI3Kα to bind an additional PIP₂ molecule was confirmed in vitro by fluorescence quenching experiments. The crystal structures reveal key differences in the way the nSH2 domain interacts with wild-type p110α and with the oncogenic mutant p110αH1047R. Increased buried surface area and two unique salt-bridges observed only in the wild-type structure suggest tighter inhibition in the wild-type PI3Kα than in the oncogenic mutant. These differences may be partially responsible for the increased basal lipid kinase activity and increased membrane binding of the oncogenic mutant.

Presence of an SH2 domain in the actin-binding protein tensin.

PubMed

Davis, S; Lu, M L; Lo, S H; Lin, S; Butler, J A; Druker, B J; Roberts, T M; An, Q; Chen, L B

1991-05-03

The molecular cloning of the complementary DNA coding for a 90-kilodalton fragment of tensin, an actin-binding component of focal contacts and other submembraneous cytoskeletal structures, is reported. The derived amino acid sequence revealed the presence of a Src homology 2 (SH2) domain. This domain is shared by a number of signal transduction proteins including nonreceptor tyrosine kinases such as Abl, Fps, Src, and Src family members, the transforming protein Crk, phospholipase C-gamma 1, PI-3 (phosphatidylinositol) kinase, and guanosine triphosphatase-activating protein (GAP). Like the SH2 domain found in Src, Crk, and Abl, the SH2 domain of tensin bound specifically to a number of phosphotyrosine-containing proteins from v-src-transformed cells. Tensin was also found to be phosphorylated on tyrosine residues. These findings suggest that by possessing both actin-binding and phosphotyrosine-binding activities and being itself a target for tyrosine kinases, tensin may link signal transduction pathways with the cytoskeleton.

Tyr721 regulates specific binding of the CSF-1 receptor kinase insert to PI 3'-kinase SH2 domains: a model for SH2-mediated receptor-target interactions.

PubMed Central

Reedijk, M; Liu, X; van der Geer, P; Letwin, K; Waterfield, M D; Hunter, T; Pawson, T

1992-01-01

Efficient binding of active phosphatidylinositol (PI) 3'-kinase to the autophosphorylated macrophage colony stimulating factor receptor (CSF-1R) requires the noncatalytic kinase insert (KI) region of the receptor. To test whether this region could function independently to bind PI 3'-kinase, the isolated CSF-1R KI was expressed in Escherichia coli, and was inducibly phosphorylated on tyrosine. The tyrosine phosphorylated form of the CSF-1R KI bound PI 3'-kinase in vitro, whereas the unphosphorylated form had no binding activity. The p85 alpha subunit of PI 3'-kinase contains two Src homology (SH)2 domains, which are implicated in the interactions of signalling proteins with activated receptors. Bacterially expressed p85 alpha SH2 domains complexed in vitro with the tyrosine phosphorylated CSF-1R KI. Binding of the CSF-1R KI to PI 3'-kinase activity, and to the p85 alpha SH2 domains, required phosphorylation of Tyr721 within the KI domain, but was independent of phosphorylation at Tyr697 and Tyr706. Tyr721 was also critical for the association of activated CSF-1R with PI 3'-kinase in mammalian cells. Complex formation between the CSF-1R and PI 3'-kinase can therefore be reconstructed in vitro in a specific interaction involving the phosphorylated receptor KI and the SH2 domains of p85 alpha. Images PMID:1314163

Systematic characterization of the specificity of the SH2 domains of cytoplasmic tyrosine kinases.

PubMed

Zhao, Bing; Tan, Pauline H; Li, Shawn S C; Pei, Dehua

2013-04-09

Cytoplasmic tyrosine kinases (CTK) generally contain a Src-homology 2 (SH2) domain, whose role in the CTK family is not fully understood. Here we report the determination of the specificity of 25 CTK SH2 domains by screening one-bead-one-compound (OBOC) peptide libraries. Based on the peptide sequences selected by the SH2 domains, we built Support Vector Machine (SVM) models for the prediction of binding ligands for the SH2 domains. These models yielded support for the progressive phosphorylation model for CTKs in which the overlapping specificity of the CTK SH2 and kinase domains has been proposed to facilitate targeting of the CTK substrates with at least two potential phosphotyrosine (pTyr) sites. We curated 93 CTK substrates with at least two pTyr sites catalyzed by the same CTK, and showed that 71% of these substrates had at least two pTyr sites predicted to bind a common CTK SH2 domain. More importantly, we found 34 instances where there was at least one pTyr site predicted to be recognized by the SH2 domain of the same CTK, suggesting that the SH2 and kinase domains of the CTKs may cooperate to achieve progressive phosphorylation of a protein substrate. This article is part of a Special Issue entitled: From protein structures to clinical applications. Copyright © 2012 Elsevier B.V. All rights reserved.

Identification of a New Interaction Mode between the Src Homology 2 Domain of C-terminal Src Kinase (Csk) and Csk-binding Protein/Phosphoprotein Associated with Glycosphingolipid Microdomains♦

PubMed Central

Tanaka, Hiroaki; Akagi, Ken-ichi; Oneyama, Chitose; Tanaka, Masakazu; Sasaki, Yuichi; Kanou, Takashi; Lee, Young-Ho; Yokogawa, Daisuke; Dobenecker, Marc-Werner; Nakagawa, Atsushi; Okada, Masato; Ikegami, Takahisa

2013-01-01

Proteins with Src homology 2 (SH2) domains play major roles in tyrosine kinase signaling. Structures of many SH2 domains have been studied, and the regions involved in their interactions with ligands have been elucidated. However, these analyses have been performed using short peptides consisting of phosphotyrosine followed by a few amino acids, which are described as the canonical recognition sites. Here, we report the solution structure of the SH2 domain of C-terminal Src kinase (Csk) in complex with a longer phosphopeptide from the Csk-binding protein (Cbp). This structure, together with biochemical experiments, revealed the existence of a novel binding region in addition to the canonical phosphotyrosine 314-binding site of Cbp. Mutational analysis of this second region in cells showed that both canonical and novel binding sites are required for tumor suppression through the Cbp-Csk interaction. Furthermore, the data indicate an allosteric connection between Cbp binding and Csk activation that arises from residues in the βB/βC loop of the SH2 domain. PMID:23548896

Mutational analysis of the SRC homology 2 domain protein-tyrosine phosphatase Corkscrew.

PubMed

Allard, J D; Herbst, R; Carroll, P M; Simon, M A

1998-05-22

The SRC homology 2 (SH2) domain protein-tyrosine phosphatase, Corkscrew (CSW) is required for signaling by receptor tyrosine kinases, including the Sevenless receptor tyrosine kinase (SEV), which directs Drosophila R7 photoreceptor cell development. To investigate the role of the different domains of CSW, we constructed domain-specific csw mutations and assayed their effects on CSW function. Our results indicate that CSW SH2 domain function is essential, but either CSW SH2 domain can fulfill this requirement. We also found that CSW and activated SEV are associated in vivo in a manner that does not require either CSW SH2 domain function or tyrosine phosphorylation of SEV. In contrast, the interaction between CSW and Daughter of Sevenless, a CSW substrate, is dependent on SH2 domain function. These results suggest that the role of the CSW SH2 domains during SEV signaling is to bind Daughter of Sevenless rather than activated SEV. We also found that although CSW protein-tyrosine phosphatase activity is required for full CSW function, a catalytically inactive CSW is capable of providing partial function. In addition, we found that deletion of either the CSW protein- tyrosine phosphatase insert or the entire CSW carboxyl terminus, which includes a conserved DRK/GRB2 SH2 domain binding sequence, does not abolish CSW function.

Introduction: History of SH2 Domains and Their Applications.

PubMed

Liu, Bernard A; Machida, Kazuya

2017-01-01

The Src Homology 2 (SH2) domain is the prototypical protein interaction module that lies at the heart of phosphotyrosine signaling. Since its serendipitous discovery, there has been a tremendous advancement in technologies and an array of techniques available for studying SH2 domains and phosphotyrosine signaling. In this chapter, we provide a glimpse of the history of SH2 domains and describe many of the tools and techniques that have been developed along the way and discuss future directions for SH2 domain studies. We highlight the gist of each chapter in this volume in the context of: the structural biology and phosphotyrosine binding; characterizing SH2 specificity and generating prediction models; systems biology and proteomics; SH2 domains in signal transduction; and SH2 domains in disease, diagnostics, and therapeutics. Many of the individual chapters provide an in-depth approach that will allow scientists to interrogate the function and role of SH2 domains.

IRS-1 activates phosphatidylinositol 3'-kinase by associating with src homology 2 domains of p85.

PubMed Central

Myers, M G; Backer, J M; Sun, X J; Shoelson, S; Hu, P; Schlessinger, J; Yoakim, M; Schaffhausen, B; White, M F

1992-01-01

IRS-1 is an insulin receptor substrate that undergoes tyrosine phosphorylation and associates with the phosphatidylinositol (PtdIns) 3'-kinase immediately after insulin stimulation. Recombinant IRS-1 protein was tyrosine phosphorylated by the insulin receptor in vitro and associated with the PtdIns 3'-kinase from lysates of quiescent 3T3 fibroblasts. Bacterial fusion proteins containing the src homology 2 domains (SH2 domains) of the 85-kDa subunit (p85) of the PtdIns 3'-kinase bound quantitatively to tyrosine phosphorylated, but not unphosphorylated, IRS-1, and this association was blocked by phosphotyrosine-containing synthetic peptides. Moreover, the phosphorylated peptides and the SH2 domains each inhibited binding of PtdIns 3'-kinase to IRS-1. Phosphorylated IRS-1 activated PtdIns 3'-kinase in anti-p85 immunoprecipitates in vitro, and this activation was blocked by SH2 domain fusion proteins. These data suggest that the interaction between PtdIns 3'-kinase and IRS-1 is mediated by tyrosine phosphorylated motifs on IRS-1 and the SH2 domains of p85, and IRS-1 activates PtdIns 3'-kinase by binding to the SH2 domains of p85. Thus, IRS-1 likely serves to transmit the insulin signal by binding and regulating intracellular enzymes containing SH2 domains. Images PMID:1332046

The heat-shock protein Apg-2 binds to the tight junction protein ZO-1 and regulates transcriptional activity of ZONAB.

PubMed

Tsapara, Anna; Matter, Karl; Balda, Maria S

2006-03-01

The tight junction adaptor protein ZO-1 regulates intracellular signaling and cell proliferation. Its Src homology 3 (SH3) domain is required for the regulation of proliferation and binds to the Y-box transcription factor ZO-1-associated nucleic acid binding protein (ZONAB). Binding of ZO-1 to ZONAB results in cytoplasmic sequestration and hence inhibition of ZONAB's transcriptional activity. Here, we identify a new binding partner of the SH3 domain that modulates ZO-1-ZONAB signaling. Expression screening of a cDNA library with a fusion protein containing the SH3 domain yielded a cDNA coding for Apg-2, a member of the heat-shock protein 110 (Hsp 110) subfamily of Hsp70 heat-shock proteins, which is overexpressed in carcinomas. Regulated depletion of Apg-2 in Madin-Darby canine kidney cells inhibits G(1)/S phase progression. Apg-2 coimmunoprecipitates with ZO-1 and partially localizes to intercellular junctions. Junctional recruitment and coimmunoprecipitation with ZO-1 are stimulated by heat shock. Apg-2 competes with ZONAB for binding to the SH3 domain in vitro and regulates ZONAB's transcriptional activity in reporter gene assays. Our data hence support a model in which Apg-2 regulates ZONAB function by competing for binding to the SH3 domain of ZO-1 and suggest that Apg-2 functions as a regulator of ZO-1-ZONAB signaling in epithelial cells in response to cellular stress.

The Heat-Shock Protein Apg-2 Binds to the Tight Junction Protein ZO-1 and Regulates Transcriptional Activity of ZONAB

PubMed Central

Tsapara, Anna; Matter, Karl; Balda, Maria S.

2006-01-01

The tight junction adaptor protein ZO-1 regulates intracellular signaling and cell proliferation. Its Src homology 3 (SH3) domain is required for the regulation of proliferation and binds to the Y-box transcription factor ZO-1-associated nucleic acid binding protein (ZONAB). Binding of ZO-1 to ZONAB results in cytoplasmic sequestration and hence inhibition of ZONAB's transcriptional activity. Here, we identify a new binding partner of the SH3 domain that modulates ZO-1–ZONAB signaling. Expression screening of a cDNA library with a fusion protein containing the SH3 domain yielded a cDNA coding for Apg-2, a member of the heat-shock protein 110 (Hsp 110) subfamily of Hsp70 heat-shock proteins, which is overexpressed in carcinomas. Regulated depletion of Apg-2 in Madin-Darby canine kidney cells inhibits G1/S phase progression. Apg-2 coimmunoprecipitates with ZO-1 and partially localizes to intercellular junctions. Junctional recruitment and coimmunoprecipitation with ZO-1 are stimulated by heat shock. Apg-2 competes with ZONAB for binding to the SH3 domain in vitro and regulates ZONAB's transcriptional activity in reporter gene assays. Our data hence support a model in which Apg-2 regulates ZONAB function by competing for binding to the SH3 domain of ZO-1 and suggest that Apg-2 functions as a regulator of ZO-1–ZONAB signaling in epithelial cells in response to cellular stress. PMID:16407410

Global transformation of erythrocyte properties via engagement of an SH2-like sequence in band 3

PubMed Central

Turrini, Francesco M.; Li, Yen-Hsing; Low, Philip S.

2016-01-01

Src homology 2 (SH2) domains are composed of weakly conserved sequences of ∼100 aa that bind phosphotyrosines in signaling proteins and thereby mediate intra- and intermolecular protein–protein interactions. In exploring the mechanism whereby tyrosine phosphorylation of the erythrocyte anion transporter, band 3, triggers membrane destabilization, vesiculation, and fragmentation, we discovered a SH2 signature motif positioned between membrane-spanning helices 4 and 5. Evidence that this exposed cytoplasmic sequence contributes to a functional SH2-like domain is provided by observations that: (i) it contains the most conserved sequence of SH2 domains, GSFLVR; (ii) it binds the tyrosine phosphorylated cytoplasmic domain of band 3 (cdb3-PO4) with Kd = 14 nM; (iii) binding of cdb3-PO4 to erythrocyte membranes is inhibited both by antibodies against the SH2 signature sequence and dephosphorylation of cdb3-PO4; (iv) label transfer experiments demonstrate the covalent transfer of photoactivatable biotin from isolated cdb3-PO4 (but not cdb3) to band 3 in erythrocyte membranes; and (v) phosphorylation-induced binding of cdb3-PO4 to the membrane-spanning domain of band 3 in intact cells causes global changes in membrane properties, including (i) displacement of a glycolytic enzyme complex from the membrane, (ii) inhibition of anion transport, and (iii) rupture of the band 3–ankyrin bridge connecting the spectrin-based cytoskeleton to the membrane. Because SH2-like motifs are not retrieved by normal homology searches for SH2 domains, but can be found in many tyrosine kinase-regulated transport proteins using modified search programs, we suggest that related cases of membrane transport proteins containing similar motifs are widespread in nature where they participate in regulation of cell properties. PMID:27856737

Global transformation of erythrocyte properties via engagement of an SH2-like sequence in band 3.

PubMed

Puchulu-Campanella, Estela; Turrini, Francesco M; Li, Yen-Hsing; Low, Philip S

2016-11-29

Src homology 2 (SH2) domains are composed of weakly conserved sequences of ∼100 aa that bind phosphotyrosines in signaling proteins and thereby mediate intra- and intermolecular protein-protein interactions. In exploring the mechanism whereby tyrosine phosphorylation of the erythrocyte anion transporter, band 3, triggers membrane destabilization, vesiculation, and fragmentation, we discovered a SH2 signature motif positioned between membrane-spanning helices 4 and 5. Evidence that this exposed cytoplasmic sequence contributes to a functional SH2-like domain is provided by observations that: (i) it contains the most conserved sequence of SH2 domains, GSFLVR; (ii) it binds the tyrosine phosphorylated cytoplasmic domain of band 3 (cdb3-PO 4 ) with K d = 14 nM; (iii) binding of cdb3-PO 4 to erythrocyte membranes is inhibited both by antibodies against the SH2 signature sequence and dephosphorylation of cdb3-PO 4 ; (iv) label transfer experiments demonstrate the covalent transfer of photoactivatable biotin from isolated cdb3-PO 4 (but not cdb3) to band 3 in erythrocyte membranes; and (v) phosphorylation-induced binding of cdb3-PO 4 to the membrane-spanning domain of band 3 in intact cells causes global changes in membrane properties, including (i) displacement of a glycolytic enzyme complex from the membrane, (ii) inhibition of anion transport, and (iii) rupture of the band 3-ankyrin bridge connecting the spectrin-based cytoskeleton to the membrane. Because SH2-like motifs are not retrieved by normal homology searches for SH2 domains, but can be found in many tyrosine kinase-regulated transport proteins using modified search programs, we suggest that related cases of membrane transport proteins containing similar motifs are widespread in nature where they participate in regulation of cell properties.

The SH2 and SH3 domains of mammalian Grb2 couple the EGF receptor to the Ras activator mSos1.

PubMed

Rozakis-Adcock, M; Fernley, R; Wade, J; Pawson, T; Bowtell, D

1993-05-06

Many tyrosine kinases, including the receptors for hormones such as epidermal growth factor (EGF), nerve growth factor and insulin, transmit intracellular signals through Ras proteins. Ligand binding to such receptors stimulates Ras guanine-nucleotide-exchange activity and increases the level of GTP-bound Ras, suggesting that these tyrosine kinases may activate a guanine-nucleotide releasing protein (GNRP). In Caenorhabditis elegans and Drosophila, genetic studies have shown that Ras activation by tyrosine kinases requires the protein Sem-5/drk, which contains a single Src-homology (SH) 2 domain and two flanking SH3 domains. Sem-5 is homologous to the mammalian protein Grb2, which binds the autophosphorylated EGF receptor and other phosphotyrosine-containing proteins such as Shc through its SH2 domain. Here we show that in rodent fibroblasts, the SH3 domains of Grb2 are bound to the proline-rich carboxy-terminal tail of mSos1, a protein homologous to Drosophila Sos. Sos is required for Ras signalling and contains a central domain related to known Ras-GNRPs. EGF stimulation induces binding of the Grb2-mSos1 complex to the autophosphorylated EGF receptor, and mSos1 phosphorylation. Grb2 therefore appears to link tyrosine kinases to a Ras-GNRP in mammalian cells.

Disabled is a putative adaptor protein that functions during signaling by the sevenless receptor tyrosine kinase.

PubMed

Le, N; Simon, M A

1998-08-01

DRK, the Drosophila homolog of the SH2-SH3 domain adaptor protein Grb2, is required during signaling by the sevenless receptor tyrosine kinase (SEV). One role of DRK is to provide a link between activated SEV and the Ras1 activator SOS. We have investigated the possibility that DRK performs other functions by identifying additional DRK-binding proteins. We show that the phosphotyrosine-binding (PTB) domain-containing protein Disabled (DAB) binds to the DRK SH3 domains. DAB is expressed in the ommatidial clusters, and loss of DAB function disrupts ommatidial development. Moreover, reduction of DAB function attenuates signaling by a constitutively activated SEV. Our biochemical analysis suggests that DAB binds SEV directly via its PTB domain, becomes tyrosine phosphorylated upon SEV activation, and then serves as an adaptor protein for SH2 domain-containing proteins. Taken together, these results indicate that DAB is a novel component of the SEV signaling pathway.

SH2 domains: modulators of nonreceptor tyrosine kinase activity.

PubMed

Filippakopoulos, Panagis; Müller, Susanne; Knapp, Stefan

2009-12-01

The Src homology 2 (SH2) domain is a sequence-specific phosphotyrosine-binding module present in many signaling molecules. In cytoplasmic tyrosine kinases, the SH2 domain is located N-terminally to the catalytic kinase domain (SH1) where it mediates cellular localization, substrate recruitment, and regulation of kinase activity. Initially, structural studies established a role of the SH2 domain stabilizing the inactive state of Src family members. However, biochemical characterization showed that the presence of the SH2 domain is frequently required for catalytic activity, suggesting a crucial function stabilizing the active state of many nonreceptor tyrosine kinases. Recently, the structure of the SH2-kinase domain of Fes revealed that the SH2 domain stabilizes the active kinase conformation by direct interactions with the regulatory helix alphaC. Stabilizing interactions between the SH2 and the kinase domains have also been observed in the structures of active Csk and Abl. Interestingly, mutations in the SH2 domain found in human disease can be explained by SH2 domain destabilization or incorrect positioning of the SH2. Here we summarize our understanding of mechanisms that lead to tyrosine kinase activation by direct interactions mediated by the SH2 domain and discuss how mutations in the SH2 domain trigger kinase inactivation.

Biochemical and genetic analysis of the Drk SH2/SH3 adaptor protein of Drosophila.

PubMed

Raabe, T; Olivier, J P; Dickson, B; Liu, X; Gish, G D; Pawson, T; Hafen, E

1995-06-01

The Drk SH3-SH2-SH3 adaptor protein has been genetically identified in a screen for rate-limiting components acting downstream of the Sevenless (Sev) receptor tyrosine kinase in the developing eye of Drosophila. It provides a link between the activated Sev receptor and Sos, a guanine nucleotide release factor that activates Ras1. We have used a combined biochemical and genetic approach to study the interactions between Sev, Drk and Sos. We show that Tyr2546 in the cytoplasmic tail of Sev is required for Drk binding, probably because it provides a recognition site for the Drk SH2 domain. Interestingly, a mutation at this site does not completely block Sev function in vivo. This may suggest that Sev can signal in a Drk-independent, parallel pathway or that Drk can also bind to an intermediate docking protein. Analysis of the Drk-Sos interaction has identified a high affinity binding site for Drk SH3 domains in the Sos tail. We show that the N-terminal Drk SH3 domain is primarily responsible for binding to the tail of Sos in vitro, and for signalling to Ras in vivo.

Structure of lipid kinase p110β/p85β elucidates an unusual SH2-domain-mediated inhibitory mechanism.

PubMed

Zhang, Xuxiao; Vadas, Oscar; Perisic, Olga; Anderson, Karen E; Clark, Jonathan; Hawkins, Phillip T; Stephens, Len R; Williams, Roger L

2011-03-04

Phosphoinositide 3-kinases (PI3Ks) are essential for cell growth, migration, and survival. The structure of a p110β/p85β complex identifies an inhibitory function for the C-terminal SH2 domain (cSH2) of the p85 regulatory subunit. Mutagenesis of a cSH2 contact residue activates downstream signaling in cells. This inhibitory contact ties up the C-terminal region of the p110β catalytic subunit, which is essential for lipid kinase activity. In vitro, p110β basal activity is tightly restrained by contacts with three p85 domains: the cSH2, nSH2, and iSH2. RTK phosphopeptides relieve inhibition by nSH2 and cSH2 using completely different mechanisms. The binding site for the RTK's pYXXM motif is exposed on the cSH2, requiring an extended RTK motif to reach and disrupt the inhibitory contact with p110β. This contrasts with the nSH2 where the pY-binding site itself forms the inhibitory contact. This establishes an unusual mechanism by which p85 SH2 domains contribute to RTK signaling specificities. Copyright © 2011 Elsevier Inc. All rights reserved.

Probing SH2-domains using Inhibitor Affinity Purification (IAP).

PubMed

Höfener, Michael; Heinzlmeir, Stephanie; Kuster, Bernhard; Sewald, Norbert

2014-01-01

Many human diseases are correlated with the dysregulation of signal transduction processes. One of the most important protein interaction domains in the context of signal transduction is the Src homology 2 (SH2) domain that binds phosphotyrosine residues. Hence, appropriate methods for the investigation of SH2 proteins are indispensable in diagnostics and medicinal chemistry. Therefore, an affinity resin for the enrichment of all SH2 proteins in one experiment would be desirable. However, current methods are unable to address all SH2 proteins simultaneously with a single compound or a small array of compounds. In order to overcome these limitations for the investigation of this particular protein family in future experiments, a dipeptide-derived probe has been designed, synthesized and evaluated. This probe successfully enriched 22 SH2 proteins from mixed cell lysates which contained 50 SH2 proteins. Further characterization of the SH2 binding properties of the probe using depletion and competition experiments indicated its ability to enrich complexes consisting of SH2 domain bearing regulatory PI3K subunits and catalytic phosphoinositide 3-kinase (PI3K) subunits that have no SH2 domain. The results make this probe a promising starting point for the development of a mixed affinity resin with complete SH2 protein coverage. Moreover, the additional findings render it a valuable tool for the evaluation of PI3K complex interrupting inhibitors.

Rosette Assay: Highly Customizable Dot-Blot for SH2 Domain Screening.

PubMed

Ng, Khong Y; Machida, Kazuya

2017-01-01

With a growing number of high-throughput studies, structural analyses, and availability of protein-protein interaction databases, it is now possible to apply web-based prediction tools to SH2 domain-interactions. However, in silico prediction is not always reliable and requires experimental validation. Rosette assay is a dot blot-based reverse-phase assay developed for the assessment of binding between SH2 domains and their ligands. It is conveniently customizable, allowing for low- to high-throughput analysis of interactions between various numbers of SH2 domains and their ligands, e.g., short peptides, purified proteins, and cell lysates. The binding assay is performed in a 96-well plate (MBA or MWA apparatus) in which a sample spotted membrane is incubated with up to 96 labeled SH2 domains. Bound domains are detected and quantified using a chemiluminescence or near-infrared fluorescence (IR) imaging system. In this chapter, we describe a practical protocol for rosette assay to assess interactions between synthesized tyrosine phosphorylated peptides and a library of GST-tagged SH2 domains. Since the methodology is not confined to assessment of SH2-pTyr interactions, rosette assay can be broadly utilized for ligand and drug screening using different protein interaction domains or antibodies.

Using peptide array to identify binding motifs and interaction networks for modular domains.

PubMed

Li, Shawn S-C; Wu, Chenggang

2009-01-01

Specific protein-protein interactions underlie all essential biological processes and form the basis of cellular signal transduction. The recognition of a short, linear peptide sequence in one protein by a modular domain in another represents a common theme of macromolecular recognition in cells, and the importance of this mode of protein-protein interaction is highlighted by the large number of peptide-binding domains encoded by the human genome. This phenomenon also provides a unique opportunity to identify protein-protein binding events using peptide arrays and complementary biochemical assays. Accordingly, high-density peptide array has emerged as a useful tool by which to map domain-mediated protein-protein interaction networks at the proteome level. Using the Src-homology 2 (SH2) and 3 (SH3) domains as examples, we describe the application of oriented peptide array libraries in uncovering specific motifs recognized by an SH2 domain and the use of high-density peptide arrays in identifying interaction networks mediated by the SH3 domain. Methods reviewed here could also be applied to other modular domains, including catalytic domains, that recognize linear peptide sequences.

Expression and Production of SH2 Domain Proteins.

PubMed

Liu, Bernard A; Ogiue-Ikeda, Mari; Machida, Kazuya

2017-01-01

The Src Homology 2 (SH2) domain lies at the heart of phosphotyrosine signaling, coordinating signaling events downstream of receptor tyrosine kinases (RTKs), adaptors, and scaffolds. Over a hundred SH2 domains are present in mammals, each having a unique specificity which determines its interactions with multiple binding partners. One of the essential tools necessary for studying and determining the role of SH2 domains in phosphotyrosine signaling is a set of soluble recombinant SH2 proteins. Here we describe methods, based on a broad experience with purification of all SH2 domains, for the production of SH2 domain proteins needed for proteomic and biochemical-based studies such as peptide arrays, mass-spectrometry, protein microarrays, reverse-phase microarrays, and high-throughput fluorescence polarization (HTP-FP). We describe stepwise protocols for expression and purification of SH2 domains using GST or poly His-tags, two widely adopted affinity tags. In addition, we address alternative approaches, challenges, and validation studies for assessing protein quality and provide general characteristics of purified human SH2 domains.

Itk tyrosine kinase substrate docking is mediated by a nonclassical SH2 domain surface of PLCgamma1.

PubMed

Min, Lie; Joseph, Raji E; Fulton, D Bruce; Andreotti, Amy H

2009-12-15

Interleukin-2 tyrosine kinase (Itk) is a Tec family tyrosine kinase that mediates signaling processes after T cell receptor engagement. Activation of Itk requires recruitment to the membrane via its pleckstrin homology domain, phosphorylation of Itk by the Src kinase, Lck, and binding of Itk to the SLP-76/LAT adapter complex. After activation, Itk phosphorylates and activates phospholipase C-gamma1 (PLC-gamma1), leading to production of two second messengers, DAG and IP(3). We have previously shown that phosphorylation of PLC-gamma1 by Itk requires a direct, phosphotyrosine-independent interaction between the Src homology 2 (SH2) domain of PLC-gamma1 and the kinase domain of Itk. We now define this docking interface using a combination of mutagenesis and NMR spectroscopy and show that disruption of the Itk/PLCgamma1 docking interaction attenuates T cell signaling. The binding surface on PLCgamma1 that mediates recognition by Itk highlights a nonclassical binding activity of the well-studied SH2 domain providing further evidence that SH2 domains participate in important signaling interactions beyond recognition of phosphotyrosine.

The ASPP interaction network: electrostatic differentiation between pro- and anti-apoptotic proteins.

PubMed

Benyamini, Hadar; Friedler, Assaf

2011-01-01

The ASPP proteins are apoptosis regulators: ASPP1 and ASPP2 promote, while iASPP inhibits, apoptosis. The mechanism by which these different outcomes are achieved is still unknown. The C-terminal ankyrin repeats and SH3 domain (ANK-SH3) mediate the interactions of the ASPP proteins with major apoptosis regulators such as p53, Bcl-2, and NFκB. The structure of the complex between ASPP2(ANK-SH3) and the core domain of p53 (p53CD) was previously determined. We have recently characterized the individual interactions of ASPP2(ANK-SH3) with Bcl-2 and NFκB, as well as a regulatory intramolecular interaction with the proline rich domain of ASPP2. Here we compared the ASPP interactions at two levels: ASPP2(ANK-SH3) with different proteins, and different ASPP family members with each protein partner. We found that the binding sites of ASPP2 to p53CD, Bcl-2, and NFκB are different, yet lie on the same face of ASPP2(ANK-SH3) . The intramolecular binding site to the proline rich domain overlaps the three intermolecular binding sites. To reveal the basis of functional diversity in the ASPP family, we compared their protein-binding domains. A subset of surface-exposed residues differentiates ASPP1 and ASPP2 from iASPP: ASPP1/2 are more negatively charged in specific residues that contact positively charged residues of p53CD, Bcl-2, and NFκB. We also found a gain of positive charge at the non-protein binding face of ASPP1/2, suggesting a role in electrostatic direction towards the negatively charged protein binding face. The electrostatic differences in binding interfaces between the ASPP proteins may be one of the causes for their different function. Copyright © 2010 John Wiley & Sons, Ltd.

Binding of the cSH3 Domain of Grb2 Adaptor to Two Distinct RXXK Motifs within Gab1 Docker Employs Differential Mechanisms

PubMed Central

McDonald, Caleb B.; Seldeen, Kenneth L.; Deegan, Brian J.; Bhat, Vikas; Farooq, Amjad

2010-01-01

A ubiquitous component of cellular signaling machinery, Gab1 docker plays a pivotal role in routing extracellular information in the form of growth factors and cytokines to downstream targets such as transcription factors within the nucleus. Here, using isothermal titration calorimetry (ITC) in combination with macromolecular modeling (MM), we show that although Gab1 contains four distinct RXXK motifs, designated G1, G2, G3 and G4, only G1 and G2 motifs bind to the cSH3 domain of Grb2 adaptor and do so with distinct mechanisms. Thus, while the G1 motif strictly requires the PPRPPKP consensus sequence for high-affinity binding to the cSH3 domain, the G2 motif displays preference for the PXVXRXLKPXR consensus. Such sequential differences in the binding of G1 and G2 motifs arise from their ability to adopt distinct polyproline type II (PPII)- and 310-helical conformations upon binding to the cSH3 domain, respectively. Collectively, our study provides detailed biophysical insights into a key protein-protein interaction involved in a diverse array of signaling cascades central to health and disease. PMID:21472810

Fluorescence Polarization Screening Assays for Small Molecule Allosteric Modulators of ABL Kinase Function

PubMed Central

Grover, Prerna; Shi, Haibin; Baumgartner, Matthew; Camacho, Carlos J.; Smithgall, Thomas E.

2015-01-01

The ABL protein-tyrosine kinase regulates intracellular signaling pathways controlling diverse cellular processes and contributes to several forms of cancer. The kinase activity of ABL is repressed by intramolecular interactions involving its regulatory Ncap, SH3 and SH2 domains. Small molecules that allosterically regulate ABL kinase activity through its non-catalytic domains may represent selective probes of ABL function. Here we report a screening assay for chemical modulators of ABL kinase activity that target the regulatory interaction of the SH3 domain with the SH2-kinase linker. This fluorescence polarization (FP) assay is based on a purified recombinant ABL protein consisting of the N-cap, SH3 and SH2 domains plus the SH2-kinase linker (N32L protein) and a short fluorescein-labeled probe peptide that binds to the SH3 domain. In assay development experiments, we found that the probe peptide binds to the recombinant ABL N32L protein in vitro, producing a robust FP signal that can be competed with an excess of unlabeled peptide. The FP signal is not observed with control N32L proteins bearing either an inactivating mutation in the SH3 domain or enhanced SH3:linker interaction. A pilot screen of 1200 FDA-approved drugs identified four compounds that specifically reduced the FP signal by at least three standard deviations from the untreated controls. Secondary assays showed that one of these hit compounds, the antithrombotic drug dipyridamole, enhances ABL kinase activity in vitro to a greater extent than the previously described ABL agonist, DPH. Docking studies predicted that this compound binds to a pocket formed at the interface of the SH3 domain and the linker, suggesting that it activates ABL by disrupting this regulatory interaction. These results show that screening assays based on the non-catalytic domains of ABL can identify allosteric small molecule regulators of kinase function, providing a new approach to selective drug discovery for this important kinase system. PMID:26222440

Fluorescence Polarization Screening Assays for Small Molecule Allosteric Modulators of ABL Kinase Function.

PubMed

Grover, Prerna; Shi, Haibin; Baumgartner, Matthew; Camacho, Carlos J; Smithgall, Thomas E

2015-01-01

The ABL protein-tyrosine kinase regulates intracellular signaling pathways controlling diverse cellular processes and contributes to several forms of cancer. The kinase activity of ABL is repressed by intramolecular interactions involving its regulatory Ncap, SH3 and SH2 domains. Small molecules that allosterically regulate ABL kinase activity through its non-catalytic domains may represent selective probes of ABL function. Here we report a screening assay for chemical modulators of ABL kinase activity that target the regulatory interaction of the SH3 domain with the SH2-kinase linker. This fluorescence polarization (FP) assay is based on a purified recombinant ABL protein consisting of the N-cap, SH3 and SH2 domains plus the SH2-kinase linker (N32L protein) and a short fluorescein-labeled probe peptide that binds to the SH3 domain. In assay development experiments, we found that the probe peptide binds to the recombinant ABL N32L protein in vitro, producing a robust FP signal that can be competed with an excess of unlabeled peptide. The FP signal is not observed with control N32L proteins bearing either an inactivating mutation in the SH3 domain or enhanced SH3:linker interaction. A pilot screen of 1200 FDA-approved drugs identified four compounds that specifically reduced the FP signal by at least three standard deviations from the untreated controls. Secondary assays showed that one of these hit compounds, the antithrombotic drug dipyridamole, enhances ABL kinase activity in vitro to a greater extent than the previously described ABL agonist, DPH. Docking studies predicted that this compound binds to a pocket formed at the interface of the SH3 domain and the linker, suggesting that it activates ABL by disrupting this regulatory interaction. These results show that screening assays based on the non-catalytic domains of ABL can identify allosteric small molecule regulators of kinase function, providing a new approach to selective drug discovery for this important kinase system.

High resolution crystal structure of the Grb2 SH2 domain with a phosphopeptide derived from CD28.

PubMed

Higo, Kunitake; Ikura, Teikichi; Oda, Masayuki; Morii, Hisayuki; Takahashi, Jun; Abe, Ryo; Ito, Nobutoshi

2013-01-01

Src homology 2 (SH2) domains play a critical role in cellular signal transduction. They bind to peptides containing phosphotyrosine (pY) with various specificities that depend on the flanking amino-acid residues. The SH2 domain of growth-factor receptor-bound protein 2 (Grb2) specifically recognizes pY-X-N-X, whereas the SH2 domains in phosphatidylinositol 3-kinase (PI3K) recognize pY-X-X-M. Binding of the pY site in CD28 (pY-M-N-M) by PI3K and Grb2 through their SH2 domains is a key step that triggers the CD28 signal transduction for T cell activation and differentiation. In this study, we determined the crystal structure of the Grb2 SH2 domain in complex with a pY-containing peptide derived from CD28 at 1.35 Å resolution. The peptide was found to adopt a twisted U-type conformation, similar to, but distinct from type-I β-turn. In all previously reported crystal structures, the peptide bound to the Grb2 SH2 domains adopts a type-I β-turn conformation, except those with a proline residue at the pY+3 position. Molecular modeling also suggests that the same peptide bound to PI3K might adopt a very different conformation.

Progress towards the development of SH2 domain inhibitors.

PubMed

Kraskouskaya, Dziyana; Duodu, Eugenia; Arpin, Carolynn C; Gunning, Patrick T

2013-04-21

Src homology 2 (SH2) domains are 100 amino acid modular units, which recognize and bind to tyrosyl-phosphorylated peptide sequences on their target proteins, and thereby mediate intracellular protein-protein interactions. This review summarizes the progress towards the development of synthetic agents that disrupt the function of the SH2 domains in different proteins as well as the clinical relevance of targeting a specific SH2 domain. Since 1986, SH2 domains have been identified in over 110 human proteins, including kinases, transcription factors, and adaptor proteins. A number of these proteins are over-activated in many diseases, including cancer, and their function is highly dependent on their SH2 domain. Thus, inhibition of a protein's function through disrupting that of its SH2 domain has emerged as a promising approach towards the development of novel therapeutic modalities. Although targeting the SH2 domain is a challenging task in molecular recognition, the progress reported here demonstrates the feasibility of such an approach.

Two signaling molecules share a phosphotyrosine-containing binding site in the platelet-derived growth factor receptor.

PubMed

Nishimura, R; Li, W; Kashishian, A; Mondino, A; Zhou, M; Cooper, J; Schlessinger, J

1993-11-01

Autophosphorylation sites of growth factor receptors with tyrosine kinase activity function as specific binding sites for Src homology 2 (SH2) domains of signaling molecules. This interaction appears to be a crucial step in a mechanism by which receptor tyrosine kinases relay signals to downstream signaling pathways. Nck is a widely expressed protein consisting exclusively of SH2 and SH3 domains, the overexpression of which causes cell transformation. It has been shown that various growth factors stimulate the phosphorylation of Nck and its association with autophosphorylated growth factor receptors. A panel of platelet-derived growth factor (PDGF) receptor mutations at tyrosine residues has been used to identify the Nck binding site. Here we show that mutation at Tyr-751 of the PDGF beta-receptor eliminates Nck binding both in vitro and in living cells. Moreover, the Y751F PDGF receptor mutant failed to mediate PDGF-stimulated phosphorylation of Nck in intact cells. A phosphorylated Tyr-751 is also required for binding of phosphatidylinositol-3 kinase to the PDGF receptor. Hence, the SH2 domains of p85 and Nck share a binding site in the PDGF receptor. Competition experiments with different phosphopeptides derived from the PDGF receptor suggest that binding of Nck and p85 is influenced by different residues around Tyr-751. Thus, a single tyrosine autophosphorylation site is able to link the PDGF receptor to two distinct SH2 domain-containing signaling molecules.

Characterization of breakpoint cluster region kinase and SH2-binding activities.

PubMed

Afar, D E; Witte, O N

1995-01-01

BCR is an interesting signaling protein, whose cellular function is currently unknown. Its biochemical properties include serine kinase activity, SH2-binding activity, and a GTPase-activating activity. The SH2-binding activity is particularly interesting because it may link BCR to signaling pathways involving SH2-containing molecules. Since tyrosine phosphorylation of BCR has been detected in CML-derived cell lines and since tyrosine-phosphorylated BCR shows increased affinity toward certain SH2 domains, it seems particularly important to further characterize this activity. This chapter described a simple purification scheme for partial purification of BCR, which can be used to assess in vitro kinase and SH2-binding activities.

Cloning and characterization of a novel human STAR domain containing cDNA KHDRBS2.

PubMed

Wang, Liu; Xu, Jian; Zeng, Li; Ye, Xin; Wu, Qihan; Dai, Jianfeng; Ji, Chaoneng; Gu, Shaohua; Zhao, Chunhua; Xie, Yi; Mao, Yumin

2002-12-01

KHDRBS2, KH domain containing, RNA binding, signal transduction associated 2, is an RNA-binding protein that is tyrosine phosphorylated by Src during mitosis. It contains a KH domain,which is embedded in a larger conserved domain called the STAR domain. This protein has a 99% sequence identity with rat SLM-1 (the Sam68-like mammalian protein 1) and 98% sequence identity with mouse SLM-1 in its STAR domain. KHDRBS2 has the characteristic Sam68 SH2 and SH3 domain binding sites. RT-PCR analysis showed its transcript is ubiquitously expressed. The characterization of KHDRBS2 indicates it may link tyrosine kinase signaling cascades with some aspect of RNA metabolism.

Crystal Structure of the FERM-SH2 Module of Human Jak2.

PubMed

McNally, Randall; Toms, Angela V; Eck, Michael J

2016-01-01

Jak-family tyrosine kinases mediate signaling from diverse cytokine receptors. Binding of Jaks to their cognate receptors is mediated by their N-terminal region, which contains FERM and SH2 domains. Here we describe the crystal structure of the FERM-SH2 region of Jak2 at 3.0Å resolution. The structure reveals that these domains and their flanking linker segments interact intimately to form an integrated structural module. The Jak2 FERM-SH2 structure closely resembles that recently described for Tyk2, another member of the Jak family. While the overall architecture and interdomain orientations are preserved between Jak2 and Tyk2, we identify residues in the putative receptor-binding groove that differ between the two and may contribute to the specificity of receptor recognition. Analysis of Jak mutations that are reported to disrupt receptor binding reveals that they lie in the hydrophobic core of the FERM domain, and are thus expected to compromise the structural integrity of the FERM-SH2 unit. Similarly, analysis of mutations in Jak3 that are associated with severe combined immunodeficiency suggests that they compromise Jak3 function by destabilizing the FERM-SH2 structure.

Structural coupling of SH2-kinase domains links Fes and Abl substrate recognition and kinase activation.

PubMed

Filippakopoulos, Panagis; Kofler, Michael; Hantschel, Oliver; Gish, Gerald D; Grebien, Florian; Salah, Eidarus; Neudecker, Philipp; Kay, Lewis E; Turk, Benjamin E; Superti-Furga, Giulio; Pawson, Tony; Knapp, Stefan

2008-09-05

The SH2 domain of cytoplasmic tyrosine kinases can enhance catalytic activity and substrate recognition, but the molecular mechanisms by which this is achieved are poorly understood. We have solved the structure of the prototypic SH2-kinase unit of the human Fes tyrosine kinase, which appears specialized for positive signaling. In its active conformation, the SH2 domain tightly interacts with the kinase N-terminal lobe and positions the kinase alphaC helix in an active configuration through essential packing and electrostatic interactions. This interaction is stabilized by ligand binding to the SH2 domain. Our data indicate that Fes kinase activation is closely coupled to substrate recognition through cooperative SH2-kinase-substrate interactions. Similarly, we find that the SH2 domain of the active Abl kinase stimulates catalytic activity and substrate phosphorylation through a distinct SH2-kinase interface. Thus, the SH2 and catalytic domains of active Fes and Abl pro-oncogenic kinases form integrated structures essential for effective tyrosine kinase signaling.

Modification by covalent reaction or oxidation of cysteine residues in the tandem-SH2 domains of ZAP-70 and Syk can block phosphopeptide binding.

PubMed

Visperas, Patrick R; Winger, Jonathan A; Horton, Timothy M; Shah, Neel H; Aum, Diane J; Tao, Alyssa; Barros, Tiago; Yan, Qingrong; Wilson, Christopher G; Arkin, Michelle R; Weiss, Arthur; Kuriyan, John

2015-01-01

Zeta-chain associated protein of 70 kDa (ZAP-70) and spleen tyrosine kinase (Syk) are non-receptor tyrosine kinases that are essential for T-cell and B-cell antigen receptor signalling respectively. They are recruited, via their tandem-SH2 (Src-homology domain 2) domains, to doubly phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) on invariant chains of immune antigen receptors. Because of their critical roles in immune signalling, ZAP-70 and Syk are targets for the development of drugs for autoimmune diseases. We show that three thiol-reactive small molecules can prevent the tandem-SH2 domains of ZAP-70 and Syk from binding to phosphorylated ITAMs. We identify a specific cysteine residue in the phosphotyrosine-binding pocket of each protein (Cys39 in ZAP-70, Cys206 in Syk) that is necessary for inhibition by two of these compounds. We also find that ITAM binding to ZAP-70 and Syk is sensitive to the presence of H2O2 and these two cysteine residues are also necessary for inhibition by H2O2. Our findings suggest a mechanism by which the reactive oxygen species generated during responses to antigen could attenuate signalling through these kinases and may also inform the development of ZAP-70 and Syk inhibitors that bind covalently to their SH2 domains.

Binding of influenza A virus NS1 protein to the inter-SH2 domain of p85 suggests a novel mechanism for phosphoinositide 3-kinase activation.

PubMed

Hale, Benjamin G; Batty, Ian H; Downes, C Peter; Randall, Richard E

2008-01-18

Influenza A virus NS1 protein stimulates host-cell phosphoinositide 3-kinase (PI3K) signaling by binding to the p85beta regulatory subunit of PI3K. Here, in an attempt to establish a mechanism for this activation, we report further on the functional interaction between NS1 and p85beta. Complex formation was found to be independent of NS1 RNA binding activity and is mediated by the C-terminal effector domain of NS1. Intriguingly, the primary direct binding site for NS1 on p85beta is the inter-SH2 domain, a coiled-coil structure that acts as a scaffold for the p110 catalytic subunit of PI3K. In vitro kinase activity assays, together with protein binding competition studies, reveal that NS1 does not displace p110 from the inter-SH2 domain, and indicate that NS1 can form an active heterotrimeric complex with PI3K. In addition, it was established that residues at the C terminus of the inter-SH2 domain are essential for mediating the interaction between p85beta and NS1. Equivalent residues in p85alpha have previously been implicated in the basal inhibition of p110. However, such p85alpha residues were unable to substitute for those in p85beta with regards NS1 binding. Overall, these data suggest a model by which NS1 activates PI3K catalytic activity by masking a normal regulatory element specific to the p85beta inter-SH2 domain.

SH2-dependent autophosphorylation within the Tec family kinase Itk.

PubMed

Joseph, Raji E; Severin, Andrew; Min, Lie; Fulton, D Bruce; Andreotti, Amy H

2009-08-07

The Tec family kinase, Itk (interleukin-2 tyrosine kinase), undergoes an in cis autophosphorylation on Y180 within its Src homology 3 (SH3) domain. Autophosphorylation of the Itk SH3 domain by the Itk kinase domain is strictly dependent on the presence of the intervening Src homology 2 (SH2) domain. A direct docking interaction between the Itk kinase and SH2 domains brings the Itk SH3 domain into the active site where Y180 is then phosphorylated. We now identify the residues on the surface of the Itk SH2 domain responsible for substrate docking and show that this SH2 surface mediates autophosphorylation in the full-length Itk molecule. The canonical phospholigand binding site on the SH2 domain is not involved in substrate docking, instead the docking site consists of side chains from three loop regions (AB, EF and BG) and part of the betaD strand. These results are extended into Btk (Bruton's tyrosine kinase), a Tec family kinase linked to the B-cell deficiency X-linked agammaglobulinemia (XLA). Our results suggest that some XLA-causing mutations might impair Btk phosphorylation.

Development of binding assays for the SH2 domain of Grb7 and Grb2 using fluorescence polarization.

PubMed

Luzy, Jean-Philippe; Chen, Huixiong; Gril, Brunilde; Liu, Wang-Qing; Vidal, Michel; Perdereau, Dominique; Burnol, Anne-Françoise; Garbay, Christiane

2008-02-01

Adaptor proteins Grb7 and Grb2 have been implicated as being 2 potential therapeutic targets in several human cancers, especially those that overexpress ErbB2. These 2 proteins contain both a SH2 domain (Src homology 2) that binds to phosphorylated tyrosine residues contained within ErbB2 and other specific protein targets. Two assays based on enzyme-linked immunosorbent assay and fluorescence polarization methods have been developed and validated to find and rank inhibitors for both proteins binding to the pY(1139). Fluorescence polarization assays allowed the authors to determine quickly and reproducibly affinities of peptides from low nanomolar to high micromolar range and to compare them directly for Grb7 and Grb2. As a result, the assays have identified a known peptidomimetic Grb2 SH2 inhibitor (mAZ-pTyr-(alphaMe)pTyr-Asn-NH(2)) that exhibits the most potent affinity for the Grb7 SH2 domain described to date.

Fusion protein based on Grb2-SH2 domain for cancer therapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Saito, Yuriko; Graduate School of Pharmaceutical Sciences, Chiba University; Furukawa, Takako, E-mail: tfuru@nirs.go.jp

2010-08-20

Research highlights: {yields} Grb2 mediates EGFR signaling through binding to phosphorylate EGFR with SH2 domain. {yields} We generated fusion proteins containing 1 or 2 SH2 domains of Grb2 added with TAT. {yields} The one with 2 SH2 domains (TSSF) interfered ERK phosphorylation. {yields} TSSF significantly delayed the growth of EGFR overexpressing tumor in a mouse model. -- Abstract: Epidermal growth factor receptor (EGFR) is one of the very attractive targets for cancer therapy. In this study, we generated fusion proteins containing one or two Src-homology 2 (SH2) domains of growth factor receptor bound protein 2 (Grb2), which bind to phosphorylatedmore » EGFR, added with HIV-1 transactivating transcription for cell membrane penetration (termed TSF and TSSF, respectively). We examined if they can interfere Grb2-mediated signaling pathway and suppress tumor growth as expected from the lack of SH3 domain, which is necessary to intermediate EGFR-Grb2 cell signaling, in the fusion proteins. The transduction efficiency of TSSF was similar to that of TSF, but the binding activity of TSSF to EGFR was higher than that of TSF. Treatment of EGFR-overexpressing cells showed that TSSF decreased p42-ERK phosphorylation, while TSF did not. Both the proteins delayed cell growth but did not induce cell death in culture. TSSF also significantly suppressed tumor growth in vivo under consecutive administration. In conclusion, TSSF showed an ability to inhibit EGFR-Grb2 signaling and could have a potential to treat EGFR-activated cancer.« less

SH2 dependent autophosphorylation within the Tec family kinase Itk

PubMed Central

Joseph, Raji E.; Severin, Andrew; Min, Lie; Fulton, D. Bruce; Andreotti, Amy H.

2009-01-01

The Tec family kinase, Itk, undergoes an in cis autophosphorylation on Y180 within its SH3 domain. Autophosphorylation of the Itk SH3 domain by the Itk kinase domain is strictly dependent on the presence of the intervening SH2 domain. A direct docking interaction between the Itk kinase and SH2 domains brings the Itk SH3 domain into the active site where Y180 is then phosphorylated. We now identify the residues on the surface of the Itk SH2 domain responsible for substrate docking and show that this SH2 surface mediates autophosphorylation in the full length Itk molecule. The canonical phospholigand binding site on the SH2 domain is not involved in substrate docking, instead the docking site consists of side chains from three loop regions (AB, EF and BG) and part of the βD strand. These results are extended into Btk, a Tec family kinase linked to the B cell deficiency X-linked agammaglobulinemia (XLA). Our results suggest that some XLA causing mutations might impair Btk phosphorylation. PMID:19523959

Binding of the cSH3 domain of Grb2 adaptor to two distinct RXXK motifs within Gab1 docker employs differential mechanisms.

PubMed

McDonald, Caleb B; Seldeen, Kenneth L; Deegan, Brian J; Bhat, Vikas; Farooq, Amjad

2011-01-01

A ubiquitous component of cellular signaling machinery, Gab1 docker plays a pivotal role in routing extracellular information in the form of growth factors and cytokines to downstream targets such as transcription factors within the nucleus. Here, using isothermal titration calorimetry (ITC) in combination with macromolecular modeling (MM), we show that although Gab1 contains four distinct RXXK motifs, designated G1, G2, G3, and G4, only G1 and G2 motifs bind to the cSH3 domain of Grb2 adaptor and do so with distinct mechanisms. Thus, while the G1 motif strictly requires the PPRPPKP consensus sequence for high-affinity binding to the cSH3 domain, the G2 motif displays preference for the PXVXRXLKPXR consensus. Such sequential differences in the binding of G1 and G2 motifs arise from their ability to adopt distinct polyproline type II (PPII)- and 3(10) -helical conformations upon binding to the cSH3 domain, respectively. Collectively, our study provides detailed biophysical insights into a key protein-protein interaction involved in a diverse array of signaling cascades central to health and disease. Copyright © 2010 John Wiley & Sons, Ltd.

Semi-supervised prediction of SH2-peptide interactions from imbalanced high-throughput data.

PubMed

Kundu, Kousik; Costa, Fabrizio; Huber, Michael; Reth, Michael; Backofen, Rolf

2013-01-01

Src homology 2 (SH2) domains are the largest family of the peptide-recognition modules (PRMs) that bind to phosphotyrosine containing peptides. Knowledge about binding partners of SH2-domains is key for a deeper understanding of different cellular processes. Given the high binding specificity of SH2, in-silico ligand peptide prediction is of great interest. Currently however, only a few approaches have been published for the prediction of SH2-peptide interactions. Their main shortcomings range from limited coverage, to restrictive modeling assumptions (they are mainly based on position specific scoring matrices and do not take into consideration complex amino acids inter-dependencies) and high computational complexity. We propose a simple yet effective machine learning approach for a large set of known human SH2 domains. We used comprehensive data from micro-array and peptide-array experiments on 51 human SH2 domains. In order to deal with the high data imbalance problem and the high signal-to-noise ration, we casted the problem in a semi-supervised setting. We report competitive predictive performance w.r.t. state-of-the-art. Specifically we obtain 0.83 AUC ROC and 0.93 AUC PR in comparison to 0.71 AUC ROC and 0.87 AUC PR previously achieved by the position specific scoring matrices (PSSMs) based SMALI approach. Our work provides three main contributions. First, we showed that better models can be obtained when the information on the non-interacting peptides (negative examples) is also used. Second, we improve performance when considering high order correlations between the ligand positions employing regularization techniques to effectively avoid overfitting issues. Third, we developed an approach to tackle the data imbalance problem using a semi-supervised strategy. Finally, we performed a genome-wide prediction of human SH2-peptide binding, uncovering several findings of biological relevance. We make our models and genome-wide predictions, for all the 51 SH2-domains, freely available to the scientific community under the following URLs: http://www.bioinf.uni-freiburg.de/Software/SH2PepInt/SH2PepInt.tar.gz and http://www.bioinf.uni-freiburg.de/Software/SH2PepInt/Genome-wide-predictions.tar.gz, respectively.

Identification of a Src kinase SH3 binding site in the C-terminal domain of the human ErbB2 receptor tyrosine kinase.

PubMed

Bornet, Olivier; Nouailler, Matthieu; Feracci, Michaël; Sebban-Kreuzer, Corinne; Byrne, Deborah; Halimi, Hubert; Morelli, Xavier; Badache, Ali; Guerlesquin, Françoise

2014-06-05

Overexpression of the ErbB2 receptor tyrosine kinase is associated with most aggressive tumors in breast cancer patients and is thus one of the main investigated therapeutic targets. Human ErbB2 C-terminal domain is an unstructured anchor that recruits specific adaptors for signaling cascades resulting in cell growth, differentiation and migration. Herein, we report the presence of a SH3 binding motif in the proline rich unfolded ErbB2 C-terminal region. NMR analysis of this motif supports a PPII helix conformation and the binding to Fyn-SH3 domain. The interaction of a kinase of the Src family with ErbB2 C-terminal domain could contribute to synergistic intracellular signaling and enhanced oncogenesis. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Coordinated autoinhibition of F-BAR domain membrane binding and WASp activation by Nervous Wreck.

PubMed

Stanishneva-Konovalova, Tatiana B; Kelley, Charlotte F; Eskin, Tania L; Messelaar, Emily M; Wasserman, Steven A; Sokolova, Olga S; Rodal, Avital A

2016-09-20

Membrane remodeling by Fes/Cip4 homology-Bin/Amphiphysin/Rvs167 (F-BAR) proteins is regulated by autoinhibitory interactions between their SRC homology 3 (SH3) and F-BAR domains. The structural basis of autoregulation, and whether it affects interactions of SH3 domains with other cellular ligands, remain unclear. Here we used single-particle electron microscopy to determine the structure of the F-BAR protein Nervous Wreck (Nwk) in both soluble and membrane-bound states. On membrane binding, Nwk SH3 domains do not completely dissociate from the F-BAR dimer, but instead shift from its concave surface to positions on either side of the dimer. Unexpectedly, along with controlling membrane binding, these autoregulatory interactions inhibit the ability of Nwk-SH3a to activate Wiskott-Aldrich syndrome protein (WASp)/actin related protein (Arp) 2/3-dependent actin filament assembly. In Drosophila neurons, Nwk autoregulation restricts SH3a domain-dependent synaptopod formation, synaptic growth, and actin organization. Our results define structural rearrangements in Nwk that control F-BAR-membrane interactions as well as SH3 domain activities, and suggest that these two functions are tightly coordinated in vitro and in vivo.

Allosteric Inhibition of Bcr-Abl Kinase by High Affinity Monobody Inhibitors Directed to the Src Homology 2 (SH2)-Kinase Interface*

PubMed Central

Wojcik, John; Lamontanara, Allan Joaquim; Grabe, Grzegorz; Koide, Akiko; Akin, Louesa; Gerig, Barbara; Hantschel, Oliver; Koide, Shohei

2016-01-01

Bcr-Abl is a constitutively active kinase that causes chronic myelogenous leukemia. We have shown that a tandem fusion of two designed binding proteins, termed monobodies, directed to the interaction interface between the Src homology 2 (SH2) and kinase domains and to the phosphotyrosine-binding site of the SH2 domain, respectively, inhibits the Bcr-Abl kinase activity. Because the latter monobody inhibits processive phosphorylation by Bcr-Abl and the SH2-kinase interface is occluded in the active kinase, it remained undetermined whether targeting the SH2-kinase interface alone was sufficient for Bcr-Abl inhibition. To address this question, we generated new, higher affinity monobodies with single nanomolar KD values targeting the kinase-binding surface of SH2. Structural and mutagenesis studies revealed the molecular underpinnings of the monobody-SH2 interactions. Importantly, the new monobodies inhibited Bcr-Abl kinase activity in vitro and in cells, and they potently induced cell death in chronic myelogenous leukemia cell lines. This work provides strong evidence for the SH2-kinase interface as a pharmacologically tractable site for allosteric inhibition of Bcr-Abl. PMID:26912659

Direct association between the Ret receptor tyrosine kinase and the Src homology 2-containing adapter protein Grb7.

PubMed

Pandey, A; Liu, X; Dixon, J E; Di Fiore, P P; Dixit, V M

1996-05-03

Adapter proteins containing Src homology 2 (SH2) domains link transmembrane receptor protein-tyrosine kinases to downstream signal transducing molecules. A family of SH2 containing adapter proteins including Grb7 and Grb10 has been recently identified. We had previously shown that Grb10 associates with Ret via its SH2 domain in an activation-dependent manner (Pandey, A., Duan, H., Di Fiore, P.P., and Dixit, V.M. (1995) J. Biol, Chem. 270, 21461-21463). We now demonstrate that the related adapter molecule Grb7 also associates with Ret in vitro and in vivo, and that the binding of the SH2 domain of Grb7 to Ret is direct. This binding is dependent upon Ret autophosphorylation since Grb7 is incapable of binding a kinase-defective mutant of Ret. Thus two members of the Grb family, Grb7 and Grb10, likely relay signals emanating from Ret to other, as yet, unidentified targets within the cell.

Intramolecular interactions regulate SAP97 binding to GKAP

PubMed Central

Wu, Hongju; Reissner, Carsten; Kuhlendahl, Sven; Coblentz, Blake; Reuver, Susanne; Kindler, Stefan; Gundelfinger, Eckart D.; Garner, Craig C.

2000-01-01

Membrane-associated guanylate kinase homologs (MAGUKs) are multidomain proteins found to be central organizers of cellular junctions. In this study, we examined the molecular mechanisms that regulate the interaction of the MAGUK SAP97 with its GUK domain binding partner GKAP (GUK-associated protein). The GKAP–GUK interaction is regulated by a series of intramolecular interactions. Specifically, the association of the Src homology 3 (SH3) domain and sequences situated between the SH3 and GUK domains with the GUK domain was found to interfere with GKAP binding. In contrast, N-terminal sequences that precede the first PDZ domain in SAP97, facilitated GKAP binding via its association with the SH3 domain. Utilizing crystal structure data available for PDZ, SH3 and GUK domains, molecular models of SAP97 were generated. These models revealed that SAP97 can exist in a compact U-shaped conformation in which the N-terminal domain folds back and interacts with the SH3 and GUK domains. These models support the biochemical data and provide new insights into how intramolecular interactions may regulate the association of SAP97 with its binding partners. PMID:11060025

In silico simulations of STAT1 and STAT3 inhibitors predict SH2 domain cross-binding specificity.

PubMed

Szelag, Malgorzata; Sikorski, Krzysztof; Czerwoniec, Anna; Szatkowska, Katarzyna; Wesoly, Joanna; Bluyssen, Hans A R

2013-11-15

Signal transducers and activators of transcription (STATs) comprise a family of transcription factors that are structurally related and which participate in signaling pathways activated by cytokines, growth factors and pathogens. Activation of STAT proteins is mediated by the highly conserved Src homology 2 (SH2) domain, which interacts with phosphotyrosine motifs for specific contacts between STATs and receptors and for STAT dimerization. By generating new models for human (h)STAT1, hSTAT2 and hSTAT3 we applied comparative in silico docking to determine SH2-binding specificity of the STAT3 inhibitor stattic, and of fludarabine (STAT1 inhibitor). Thus, we provide evidence that by primarily targeting the highly conserved phosphotyrosine (pY+0) SH2 binding pocket stattic is not a specific hSTAT3 inhibitor, but is equally effective towards hSTAT1 and hSTAT2. This was confirmed in Human Micro-vascular Endothelial Cells (HMECs) in vitro, in which stattic inhibited interferon-α-induced phosphorylation of all three STATs. Likewise, fludarabine inhibits both hSTAT1 and hSTAT3 phosphorylation, but not hSTAT2, by competing with the highly conserved pY+0 and pY-X binding sites, which are less well-preserved in hSTAT2. Moreover we observed that in HMECs in vitro fludarabine inhibits cytokine and lipopolysaccharide-induced phosphorylation of hSTAT1 and hSTAT3 but does not affect hSTAT2. Finally, multiple sequence alignment of STAT-SH2 domain sequences confirmed high conservation between hSTAT1 and hSTAT3, but not hSTAT2, with respect to stattic and fludarabine binding sites. Together our data offer a molecular basis that explains STAT cross-binding specificity of stattic and fludarabine, thereby questioning the present selection strategies of SH2 domain-based competitive small inhibitors. © 2013 Elsevier B.V. All rights reserved.

The SH2 domain interaction landscape.

PubMed

Tinti, Michele; Kiemer, Lars; Costa, Stefano; Miller, Martin L; Sacco, Francesca; Olsen, Jesper V; Carducci, Martina; Paoluzi, Serena; Langone, Francesca; Workman, Christopher T; Blom, Nikolaj; Machida, Kazuya; Thompson, Christopher M; Schutkowski, Mike; Brunak, Søren; Mann, Matthias; Mayer, Bruce J; Castagnoli, Luisa; Cesareni, Gianni

2013-04-25

Members of the SH2 domain family modulate signal transduction by binding to short peptides containing phosphorylated tyrosines. Each domain displays a distinct preference for the sequence context of the phosphorylated residue. We have developed a high-density peptide chip technology that allows for probing of the affinity of most SH2 domains for a large fraction of the entire complement of tyrosine phosphopeptides in the human proteome. Using this technique, we have experimentally identified thousands of putative SH2-peptide interactions for more than 70 different SH2 domains. By integrating this rich data set with orthogonal context-specific information, we have assembled an SH2-mediated probabilistic interaction network, which we make available as a community resource in the PepspotDB database. A predicted dynamic interaction between the SH2 domains of the tyrosine phosphatase SHP2 and the phosphorylated tyrosine in the extracellular signal-regulated kinase activation loop was validated by experiments in living cells. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Integrated In Silico-In Vitro Identification and Characterization of the SH3-Mediated Interaction between Human PTTG and its Cognate Partners in Medulloblastoma.

PubMed

Liu, Jiangang; Wang, Dapeng; Li, Yanyan; Yao, Hui; Zhang, Nan; Zhang, Xuewen; Zhong, Fangping; Huang, Yulun

2018-06-01

The human pituitary tumor-transforming gene is an oncogenic protein which serves as a central hub in the cellular signaling network of medulloblastoma. The protein contains two vicinal PxxP motifs at its C terminus that are potential binding sites of peptide-recognition SH3 domains. Here, a synthetic protocol that integrated in silico analysis and in vitro assay was described to identify the SH3-binding partners of pituitary tumor-transforming gene in the gene expression profile of medulloblastoma. In the procedure, a variety of structurally diverse, non-redundant SH3 domains with high gene expression in medulloblastoma were compiled, and their three-dimensional structures were either manually retrieved from the protein data bank database or computationally modeled through bioinformatics technique. The binding capability of these domains towards the two PxxP-containing peptides m1p: 161 LGPPSPVK 168 and m2p: 168 KMPSPPWE 175 of pituitary tumor-transforming gene were ranked by structure-based scoring and fluorescence-based assay. Consequently, a number of SH3 domains, including MAP3K and PI3K, were found to have moderate or high affinity for m1p and/or m2p. Interestingly, the two overlapping peptides exhibits a distinct binding profile to these identified domain partners, suggesting that the binding selectivity of m1p and m2p is optimized across the medulloblastoma expression spectrum by competing for domain candidates. In addition, two redesigned versions of m1p peptide ware obtained via a structure-based rational mutation approach, which exhibited an increased affinity for the domain as compared to native peptide.

Abl N-terminal Cap stabilization of SH3 domain dynamics†

PubMed Central

Chen, Shugui; Dumitrescu, Teodora Pene; Smithgall, Thomas E.; Engen, John R.

2008-01-01

Crystal structures and other biochemical data indicate that the N-terminal cap (NCap) region of the Abelson tyrosine kinase (c-Abl) is important for maintaining the downregulated conformation of the kinase domain. The exact contributions that NCap makes in stabilizing the various intramolecular interactions within c-Abl are less clear. While the NCap appears important for locking the SH3/SH2 domains to the back of the kinase domain, there may be other more subtle elements of regulation. Hydrogen exchange (HX) and mass spectrometry (MS) were used to determine if the NCap contributes to intramolecular interactions involving the Abl SH3 domain. Under physiological conditions, the Abl SH3 domain underwent partial unfolding and its unfolding half-life was slowed during binding to the SH2-kinase linker, providing a unique assay to test NCap-induced stabilization of the SH3 domain in various constructs. The results showed that NCap stabilizes the dynamics of the SH3 domain in certain constructs but does not increase the relative affinity of the SH3 domain for the native SH2-kinase linker. The stabilization effect was absent in constructs of just NCap + SH3 but was obvious when the SH2 domain and the SH2-kinase linker were present. These results suggest that interactions between NCap and the SH3 domain can contribute to c-Abl stabilization in constructs that contain at least the SH2 domain, an effect that may partially compensate for the absence of the negative regulatory C-terminal tail found in the related Src family of kinases. PMID:18452309

Abl N-terminal cap stabilization of SH3 domain dynamics.

PubMed

Chen, Shugui; Dumitrescu, Teodora Pene; Smithgall, Thomas E; Engen, John R

2008-05-27

Crystal structures and other biochemical data indicate that the N-terminal cap (NCap) region of the Abelson tyrosine kinase (c-Abl) is important for maintaining the downregulated conformation of the kinase domain. The exact contributions that the NCap makes in stabilizing the various intramolecular interactions within c-Abl are less clear. While the NCap appears to be important for locking the SH3 and SH2 domains to the back of the kinase domain, there may be other more subtle elements of regulation. Hydrogen exchange (HX) and mass spectrometry (MS) were used to determine if the NCap contributes to intramolecular interactions involving the Abl SH3 domain. Under physiological conditions, the Abl SH3 domain underwent partial unfolding and its unfolding half-life was slowed during binding to the SH2 kinase linker, providing a unique assay for testing NCap-induced stabilization of the SH3 domain in various constructs. The results showed that the NCap stabilizes the dynamics of the SH3 domain in certain constructs but does not increase the relative affinity of the SH3 domain for the native SH2 kinase linker. The stabilization effect was absent in constructs of just the NCap and SH3 but was obvious when the SH2 domain and the SH2 kinase linker were present. These results suggest that interactions between the NCap and the SH3 domain can contribute to c-Abl stabilization in constructs that contain at least the SH2 domain, an effect that may partially compensate for the absence of the negative regulatory C-terminal tail found in the related Src family of kinases.

Integrin-mediated signal transduction linked to Ras pathway by GRB2 binding to focal adhesion kinase.

PubMed

Schlaepfer, D D; Hanks, S K; Hunter, T; van der Geer, P

The cytoplasmic focal adhesion protein-tyrosine kinase (FAK) localizes with surface integrin receptors at sites where cells attach to the extracellular matrix. Increased FAK tyrosine phosphorylation occurs upon integrin engagement with fibronectin. Here we show that adhesion of murine NIH3T3 fibroblasts to fibronectin promotes SH2-domain-mediated association of the GRB2 adaptor protein and the c-Src protein-tyrosine kinase (PTK) with FAK in vivo, and also results in activation of mitogen-activated protein kinase (MAPK). In v-Src-transformed NIH3T3, the association of v-Src, GRB2 and Sos with FAK is independent of cell adhesion to fibronectin. The GRB2 SH2 domain binds directly to tyrosine-phosphorylated FAK. Mutation of tyrosine residue 925 of FAK (YENV motif) to phenylalanine blocks GRB2 SH2-domain binding to FAK in vitro. Our results show that fibronectin binding to integrins on NIH3T3 fibroblasts promotes c-Src and FAK association and formation of an integrin-activated signalling complex. Phosphorylation of FAK at Tyr 925 upon fibronectin stimulation creates an SH2-binding site for GRB2 which may link integrin engagement to the activation of the Ras/MAPK signal transduction pathway.

Identification of a human src homology 2-containing protein-tyrosine-phosphatase: a putative homolog of Drosophila corkscrew.

PubMed Central

Freeman, R M; Plutzky, J; Neel, B G

1992-01-01

src homology 2 (SH2) domains direct binding to specific phosphotyrosyl proteins. Recently, SH2-containing protein-tyrosine-phosphatases (PTPs) were identified. Using degenerate oligonucleotides and the PCR, we have cloned a cDNA for an additional PTP, SH-PTP2, which contains two SH2 domains and is expressed ubiquitously. When expressed in Escherichia coli, SH-PTP2 displays tyrosine-specific phosphatase activity. Strong sequence similarity between SH-PTP2 and the Drosophila gene corkscrew (csw) and their similar patterns of expression suggest that SH-PTP2 is the human corkscrew homolog. Sequence comparisons between SH-PTP2, SH-PTP1, corkscrew, and other SH2-containing proteins suggest the existence of a subfamily of SH2 domains found specifically in PTPs, whereas comparison of the PTP domains of the SH2-containing PTPs with other tyrosine phosphatases suggests the existence of a subfamily of PTPs containing SH2 domains. Since corkscrew, a member of the terminal class signal transduction pathway, acts in concert with D-raf to positively transduce the signal generated by the receptor tyrosine kinase torso, these findings suggest several mechanisms by which SH-PTP2 may participate in mammalian signal transduction. Images PMID:1280823

Potent blockade of hepatocyte growth factor-stimulated cell motility, matrix invasion and branching morphogenesis by antagonists of Grb2 Src homology 2 domain interactions.

PubMed

Atabey, N; Gao, Y; Yao, Z J; Breckenridge, D; Soon, L; Soriano, J V; Burke, T R; Bottaro, D P

2001-04-27

Hepatocyte growth factor (HGF) stimulates mitogenesis, motogenesis, and morphogenesis in a wide range of cellular targets during development, homeostasis and tissue regeneration. Inappropriate HGF signaling occurs in several human cancers, and the ability of HGF to initiate a program of protease production, cell dissociation, and motility has been shown to promote cellular invasion and is strongly linked to tumor metastasis. Upon HGF binding, several tyrosines within the intracellular domain of its receptor, c-Met, become phosphorylated and mediate the binding of effector proteins, such as Grb2. Grb2 binding through its SH2 domain is thought to link c-Met with downstream mediators of cell proliferation, shape change, and motility. We analyzed the effects of Grb2 SH2 domain antagonists on HGF signaling and observed potent blockade of cell motility, matrix invasion, and branching morphogenesis, with ED(50) values of 30 nm or less, but only modest inhibition of mitogenesis. These compounds are 1000-10,000-fold more potent anti-motility agents than any previously characterized Grb2 SH2 domain antagonists. Our results suggest that SH2 domain-mediated c-Met-Grb2 interaction contributes primarily to the motogenic and morphogenic responses to HGF, and that these compounds may have therapeutic application as anti-metastatic agents for tumors where the HGF signaling pathway is active.

Structural Basis of the High Affinity Interaction between the Alphavirus Nonstructural Protein-3 (nsP3) and the SH3 Domain of Amphiphysin-2.

PubMed

Tossavainen, Helena; Aitio, Olli; Hellman, Maarit; Saksela, Kalle; Permi, Perttu

2016-07-29

We show that a peptide from Chikungunya virus nsP3 protein spanning residues 1728-1744 binds the amphiphysin-2 (BIN1) Src homology-3 (SH3) domain with an unusually high affinity (Kd 24 nm). Our NMR solution complex structure together with isothermal titration calorimetry data on several related viral and cellular peptide ligands reveal that this exceptional affinity originates from interactions between multiple basic residues in the target peptide and the extensive negatively charged binding surface of amphiphysin-2 SH3. Remarkably, these arginines show no fixed conformation in the complex structure, indicating that a transient or fluctuating polyelectrostatic interaction accounts for this affinity. Thus, via optimization of such dynamic electrostatic forces, viral peptides have evolved a superior binding affinity for amphiphysin-2 SH3 compared with typical cellular ligands, such as dynamin, thereby enabling hijacking of amphiphysin-2 SH3-regulated host cell processes by these viruses. Moreover, our data show that the previously described consensus sequence PXRPXR for amphiphysin SH3 ligands is inaccurate and instead define it as an extended Class II binding motif PXXPXRpXR, where additional positive charges between the two constant arginine residues can give rise to extraordinary high SH3 binding affinity. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Efficient T-cell receptor signaling requires a high-affinity interaction between the Gads C-SH3 domain and the SLP-76 RxxK motif.

PubMed

Seet, Bruce T; Berry, Donna M; Maltzman, Jonathan S; Shabason, Jacob; Raina, Monica; Koretzky, Gary A; McGlade, C Jane; Pawson, Tony

2007-02-07

The relationship between the binding affinity and specificity of modular interaction domains is potentially important in determining biological signaling responses. In signaling from the T-cell receptor (TCR), the Gads C-terminal SH3 domain binds a core RxxK sequence motif in the SLP-76 scaffold. We show that residues surrounding this motif are largely optimized for binding the Gads C-SH3 domain resulting in a high-affinity interaction (K(D)=8-20 nM) that is essential for efficient TCR signaling in Jurkat T cells, since Gads-mediated signaling declines with decreasing affinity. Furthermore, the SLP-76 RxxK motif has evolved a very high specificity for the Gads C-SH3 domain. However, TCR signaling in Jurkat cells is tolerant of potential SLP-76 crossreactivity, provided that very high-affinity binding to the Gads C-SH3 domain is maintained. These data provide a quantitative argument that the affinity of the Gads C-SH3 domain for SLP-76 is physiologically important and suggest that the integrity of TCR signaling in vivo is sustained both by strong selection of SLP-76 for the Gads C-SH3 domain and by a capacity to buffer intrinsic crossreactivity.

Allosteric Inhibition of Bcr-Abl Kinase by High Affinity Monobody Inhibitors Directed to the Src Homology 2 (SH2)-Kinase Interface.

PubMed

Wojcik, John; Lamontanara, Allan Joaquim; Grabe, Grzegorz; Koide, Akiko; Akin, Louesa; Gerig, Barbara; Hantschel, Oliver; Koide, Shohei

2016-04-15

Bcr-Abl is a constitutively active kinase that causes chronic myelogenous leukemia. We have shown that a tandem fusion of two designed binding proteins, termed monobodies, directed to the interaction interface between the Src homology 2 (SH2) and kinase domains and to the phosphotyrosine-binding site of the SH2 domain, respectively, inhibits the Bcr-Abl kinase activity. Because the latter monobody inhibits processive phosphorylation by Bcr-Abl and the SH2-kinase interface is occluded in the active kinase, it remained undetermined whether targeting the SH2-kinase interface alone was sufficient for Bcr-Abl inhibition. To address this question, we generated new, higher affinity monobodies with single nanomolar KD values targeting the kinase-binding surface of SH2. Structural and mutagenesis studies revealed the molecular underpinnings of the monobody-SH2 interactions. Importantly, the new monobodies inhibited Bcr-Abl kinase activity in vitro and in cells, and they potently induced cell death in chronic myelogenous leukemia cell lines. This work provides strong evidence for the SH2-kinase interface as a pharmacologically tractable site for allosteric inhibition of Bcr-Abl. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Critical Role of the Src Homology 2 (SH2) Domain of Neuronal SH2B1 in the Regulation of Body Weight and Glucose Homeostasis in Mice

PubMed Central

Morris, David L.; Cho, Kae Won; Rui, Liangyou

2010-01-01

SH2B1 is an SH2 domain-containing adaptor protein that plays a key role in the regulation of energy and glucose metabolism in both rodents and humans. Genetic deletion of SH2B1 in mice results in obesity and type 2 diabetes. Single-nucleotide polymorphisms in the SH2B1 loci and chromosomal deletions of the SH2B1 loci associate with obesity and insulin resistance in humans. In cultured cells, SH2B1 promotes leptin and insulin signaling by binding via its SH2 domain to phosphorylated tyrosines in Janus kinase 2 and the insulin receptor, respectively. Here we generated three lines of mice to analyze the role of the SH2 domain of SH2B1 in the central nervous system. Transgenic mice expressing wild-type, SH2 domain-defective (R555E), or SH2 domain-alone (ΔN503) forms of SH2B1 specifically in neurons were crossed with SH2B1 knockout mice to generate KO/SH2B1, KO/R555E, or KO/ΔN503 compound mutant mice. R555E had a replacement of Arg555 with Glu within the SH2 domain. ΔN503 contained an intact SH2 domain but lacked amino acids 1-503. Neuron-specific expression of recombinant SH2B1, but not R555E or ΔN503, corrected hyperphagia, obesity, glucose intolerance, and insulin resistance in SH2B1 null mice. Neuron-specific expression of R555E in wild-type mice promoted obesity and insulin resistance. These results indicate that in addition to the SH2 domain, N-terminal regions of neuronal SH2B1 are also required for the maintenance of normal body weight and glucose metabolism. Additionally, mutations in the SH2 domain of SH2B1 may increase the susceptibility to obesity and type 2 diabetes in a dominant-negative manner. PMID:20484460

Critical role of the Src homology 2 (SH2) domain of neuronal SH2B1 in the regulation of body weight and glucose homeostasis in mice.

PubMed

Morris, David L; Cho, Kae Won; Rui, Liangyou

2010-08-01

SH2B1 is an SH2 domain-containing adaptor protein that plays a key role in the regulation of energy and glucose metabolism in both rodents and humans. Genetic deletion of SH2B1 in mice results in obesity and type 2 diabetes. Single-nucleotide polymorphisms in the SH2B1 loci and chromosomal deletions of the SH2B1 loci associate with obesity and insulin resistance in humans. In cultured cells, SH2B1 promotes leptin and insulin signaling by binding via its SH2 domain to phosphorylated tyrosines in Janus kinase 2 and the insulin receptor, respectively. Here we generated three lines of mice to analyze the role of the SH2 domain of SH2B1 in the central nervous system. Transgenic mice expressing wild-type, SH2 domain-defective (R555E), or SH2 domain-alone (DeltaN503) forms of SH2B1 specifically in neurons were crossed with SH2B1 knockout mice to generate KO/SH2B1, KO/R555E, or KO/DeltaN503 compound mutant mice. R555E had a replacement of Arg(555) with Glu within the SH2 domain. DeltaN503 contained an intact SH2 domain but lacked amino acids 1-503. Neuron-specific expression of recombinant SH2B1, but not R555E or DeltaN503, corrected hyperphagia, obesity, glucose intolerance, and insulin resistance in SH2B1 null mice. Neuron-specific expression of R555E in wild-type mice promoted obesity and insulin resistance. These results indicate that in addition to the SH2 domain, N-terminal regions of neuronal SH2B1 are also required for the maintenance of normal body weight and glucose metabolism. Additionally, mutations in the SH2 domain of SH2B1 may increase the susceptibility to obesity and type 2 diabetes in a dominant-negative manner.

Mechanism and function of Vav1 localisation in TCR signalling

PubMed Central

Ksionda, Olga; Saveliev, Alexander; Köchl, Robert; Rapley, Jonathan; Faroudi, Mustapha; Smith-Garvin, Jennifer E.; Wülfing, Christoph; Rittinger, Katrin; Carter, Tom; Tybulewicz, Victor L. J.

2012-01-01

Summary The antigen-specific binding of T cells to antigen presenting cells results in recruitment of signalling proteins to microclusters at the cell-cell interface known as the immunological synapse (IS). The Vav1 guanine nucleotide exchange factor plays a critical role in T cell antigen receptor (TCR) signalling, leading to the activation of multiple pathways. We now show that it is recruited to microclusters and to the IS in primary CD4+ and CD8+ T cells. Furthermore, we show that this recruitment depends on the SH2 and C-terminal SH3 (SH3B) domains of Vav1, and on phosphotyrosines 112 and 128 of the SLP76 adaptor protein. Biophysical measurements show that Vav1 binds directly to these residues on SLP76 and that efficient binding depends on the SH2 and SH3B domains of Vav1. Finally, we show that the same two domains are critical for the phosphorylation of Vav1 and its signalling function in TCR-induced calcium flux. We propose that Vav1 is recruited to the IS by binding to SLP76 and that this interaction is critical for the transduction of signals leading to calcium flux. PMID:22956543

Mechanism and function of Vav1 localisation in TCR signalling.

PubMed

Ksionda, Olga; Saveliev, Alexander; Köchl, Robert; Rapley, Jonathan; Faroudi, Mustapha; Smith-Garvin, Jennifer E; Wülfing, Christoph; Rittinger, Katrin; Carter, Tom; Tybulewicz, Victor L J

2012-11-15

The antigen-specific binding of T cells to antigen presenting cells results in recruitment of signalling proteins to microclusters at the cell-cell interface known as the immunological synapse (IS). The Vav1 guanine nucleotide exchange factor plays a critical role in T cell antigen receptor (TCR) signalling, leading to the activation of multiple pathways. We now show that it is recruited to microclusters and to the IS in primary CD4(+) and CD8(+) T cells. Furthermore, we show that this recruitment depends on the SH2 and C-terminal SH3 (SH3(B)) domains of Vav1, and on phosphotyrosines 112 and 128 of the SLP76 adaptor protein. Biophysical measurements show that Vav1 binds directly to these residues on SLP76 and that efficient binding depends on the SH2 and SH3(B) domains of Vav1. Finally, we show that the same two domains are critical for the phosphorylation of Vav1 and its signalling function in TCR-induced calcium flux. We propose that Vav1 is recruited to the IS by binding to SLP76 and that this interaction is critical for the transduction of signals leading to calcium flux.

Analysis of the thermodynamics of binding of an SH3 domain to proline-rich peptides using a chimeric fusion protein.

PubMed

Candel, Adela M; van Nuland, Nico A J; Martin-Sierra, Francisco M; Martinez, Jose C; Conejero-Lara, Francisco

2008-03-14

A complete understanding of the thermodynamic determinants of binding between SH3 domains and proline-rich peptides is crucial to the development of rational strategies for designing ligands for these important domains. Recently we engineered a single-chain chimeric protein by fusing the alpha-spectrin Src homology region 3 (SH3) domain to the decapeptide APSYSPPPPP (p41). This chimera mimics the structural and energetic features of the interaction between SH3 domains and proline-rich peptides. Here we show that analysing the unfolding thermodynamics of single-point mutants of this chimeric fusion protein constitutes a very useful approach to deciphering the thermodynamics of SH3-ligand interactions. To this end, we investigated the contribution of each proline residue of the ligand sequence to the SH3-peptide interaction by producing six single Pro-Ala mutants of the chimeric protein and analysing their unfolding thermodynamics by differential scanning calorimetry (DSC). Structural analyses of the mutant chimeras by circular dichroism, fluorescence and NMR together with NMR-relaxation measurements indicate conformational flexibility at the binding interface, which is strongly affected by the different Pro-Ala mutations. An analysis of the DSC thermograms on the basis of a three-state unfolding model has allowed us to distinguish and separate the thermodynamic magnitudes of the interaction at the binding interface. The model assumes equilibrium between the "unbound" and "bound" states at the SH3-peptide binding interface. The resulting thermodynamic magnitudes classify the different proline residues according to their importance in the interaction as P2 approximately P7 approximately P10>P9 approximately P6>P8, which agrees well with Lim's model for the interaction between SH3 domains and proline-rich peptides. In addition, the thermodynamic signature of the interaction is the same as that usually found for this type of binding, with a strong enthalpy-entropy compensation for all the mutants. This compensation appears to derive from an increase in conformational flexibility concomitant to the weakening of the interactions at the binding interface. We conclude that our approach, based on DSC and site-directed mutagenesis analysis of chimeric fusion proteins, may serve as a suitable tool to analyse the energetics of weak biomolecular interactions such as those involving SH3 domains.

Modification by covalent reaction or oxidation of cysteine residues in the Tandem-SH2 Domains of ZAP-70 and Syk Can Block Phosphopeptide Binding

PubMed Central

Visperas, Patrick R.; Winger, Jonathan A.; Horton, Timothy M.; Shah, Neel H.; Aum, Diane J.; Tao, Alyssa; Barros, Tiago; Yan, Qingrong; Wilson, Christopher G.; Arkin, Michelle R.; Weiss, Arthur; Kuriyan, John

2015-01-01

Zeta-chain Associated Protein of 70kDa (ZAP-70) and Spleen tyrosine kinase (Syk) are non-receptor tyrosine kinases that are essential for T-cell and B-cell antigen receptor signaling, respectively. They are recruited, via their tandem-SH2 domains, to doubly-phosphorylated Immunoreceptor Tyrosine-based Activation Motifs (ITAMs) on invariant chains of immune antigen receptors. Because of their critical roles in immune signaling, ZAP-70 and Syk are targets for the development of drugs for autoimmune diseases. We show that three thiol-reactive small molecules can prevent the tandem-SH2 domains of ZAP-70 and Syk from binding to phosphorylated ITAMs. We identify a specific cysteine residue in the phosphotyrosine-binding pocket of each protein (Cys 39 in ZAP-70, Cys 206 in Syk) that is necessary for inhibition by two of these compounds. We also find that ITAM binding to ZAP-70 and Syk is sensitive to the presence of hydrogen peroxide, and these two cysteine residues are also necessary for inhibition by hydrogen peroxide. Our findings suggest a mechanism by which the generation of reactive oxygen species generated during responses to antigen could attenuate signaling through these kinases, and may also inform the development of ZAP-70 and Syk inhibitors that bind covalently to their SH2 domains. PMID:25287889

HIV-1 Tat binds to SH3 domains: cellular and viral outcome of Tat/Grb2 interaction

PubMed Central

Rom, Slava; Pacifici, Marco; Passiatore, Giovanni; Aprea, Susanna; Waligorska, Agnieszka; Valle, Luis Del; Peruzzi, Francesca

2011-01-01

The Src-homology 3 (SH3) domain is one of the most frequent protein recognition modules (PRMs), being represented in signal transduction pathways and in several pathologies such as cancer and AIDS. Grb2 (growth factor receptor-bound protein 2) is an adaptor protein that contains two SH3 domains and is involved in receptor tyrosine kinase (RTK) signal transduction pathways. The HIV-1 transactivator factor Tat is required for viral replication and it has been shown to bind directly or indirectly to several host proteins, deregulating their functions. In this study, we show interaction between the cellular factor Grb2 and the HIV-1 trans-activating protein Tat. The binding is mediated by the proline-rich sequence of Tat and the SH3 domain of Grb2. As the adaptor protein Grb2 participates in a wide variety of signaling pathways, we characterized at least one of the possible downstream effects of the Tat/Grb2 interaction on the well-known IGF-1R/Raf/MAPK cascade. We show that the binding of Tat to Grb2 impairs activation of the Raf/MAPK pathway, while potentiating the PKA/Raf inhibitory pathway. The Tat/Grb2 interaction affects also viral function by inhibiting the Tat-mediated transactivation of HIV-1 LTR and viral replication in infected primary microglia. PMID:21745501

Crystal Structures and Thermodynamic Analysis Reveal Distinct Mechanisms of CD28 Phosphopeptide Binding to the Src Homology 2 (SH2) Domains of Three Adaptor Proteins*

PubMed Central

Inaba, Satomi; Numoto, Nobutaka; Ogawa, Shuhei; Morii, Hisayuki; Ikura, Teikichi; Abe, Ryo; Ito, Nobutoshi; Oda, Masayuki

2017-01-01

Full activation of T cells and differentiation into effector T cells are essential for many immune responses and require co-stimulatory signaling via the CD28 receptor. Extracellular ligand binding to CD28 recruits protein-tyrosine kinases to its cytoplasmic tail, which contains a YMNM motif. Following phosphorylation of the tyrosine, the proteins growth factor receptor-bound protein 2 (Grb2), Grb2-related adaptor downstream of Shc (Gads), and p85 subunit of phosphoinositide 3-kinase may bind to pYMNM (where pY is phosphotyrosine) via their Src homology 2 (SH2) domains, leading to downstream signaling to distinct immune pathways. These three adaptor proteins bind to the same site on CD28 with variable affinity, and all are important for CD28-mediated co-stimulatory function. However, the mechanism of how these proteins recognize and compete for CD28 is unclear. To visualize their interactions with CD28, we have determined the crystal structures of Gads SH2 and two p85 SH2 domains in complex with a CD28-derived phosphopeptide. The high resolution structures obtained revealed that, whereas the CD28 phosphopeptide bound to Gads SH2 is in a bent conformation similar to that when bound to Grb2 SH2, it adopts a more extended conformation when bound to the N- and C-terminal SH2 domains of p85. These differences observed in the peptide-protein interactions correlated well with the affinity and other thermodynamic parameters for each interaction determined by isothermal titration calorimetry. The detailed insight into these interactions reported here may inform the development of compounds that specifically inhibit the association of CD28 with these adaptor proteins to suppress excessive T cell responses, such as in allergies and autoimmune diseases. PMID:27927989

Crystal Structures and Thermodynamic Analysis Reveal Distinct Mechanisms of CD28 Phosphopeptide Binding to the Src Homology 2 (SH2) Domains of Three Adaptor Proteins.

PubMed

Inaba, Satomi; Numoto, Nobutaka; Ogawa, Shuhei; Morii, Hisayuki; Ikura, Teikichi; Abe, Ryo; Ito, Nobutoshi; Oda, Masayuki

2017-01-20

Full activation of T cells and differentiation into effector T cells are essential for many immune responses and require co-stimulatory signaling via the CD28 receptor. Extracellular ligand binding to CD28 recruits protein-tyrosine kinases to its cytoplasmic tail, which contains a YMNM motif. Following phosphorylation of the tyrosine, the proteins growth factor receptor-bound protein 2 (Grb2), Grb2-related adaptor downstream of Shc (Gads), and p85 subunit of phosphoinositide 3-kinase may bind to pYMNM (where pY is phosphotyrosine) via their Src homology 2 (SH2) domains, leading to downstream signaling to distinct immune pathways. These three adaptor proteins bind to the same site on CD28 with variable affinity, and all are important for CD28-mediated co-stimulatory function. However, the mechanism of how these proteins recognize and compete for CD28 is unclear. To visualize their interactions with CD28, we have determined the crystal structures of Gads SH2 and two p85 SH2 domains in complex with a CD28-derived phosphopeptide. The high resolution structures obtained revealed that, whereas the CD28 phosphopeptide bound to Gads SH2 is in a bent conformation similar to that when bound to Grb2 SH2, it adopts a more extended conformation when bound to the N- and C-terminal SH2 domains of p85. These differences observed in the peptide-protein interactions correlated well with the affinity and other thermodynamic parameters for each interaction determined by isothermal titration calorimetry. The detailed insight into these interactions reported here may inform the development of compounds that specifically inhibit the association of CD28 with these adaptor proteins to suppress excessive T cell responses, such as in allergies and autoimmune diseases. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Characterization of domain-peptide interaction interface: a case study on the amphiphysin-1 SH3 domain.

PubMed

Hou, Tingjun; Zhang, Wei; Case, David A; Wang, Wei

2008-02-29

Many important protein-protein interactions are mediated by peptide recognition modular domains, such as the Src homology 3 (SH3), SH2, PDZ, and WW domains. Characterizing the interaction interface of domain-peptide complexes and predicting binding specificity for modular domains are critical for deciphering protein-protein interaction networks. Here, we propose the use of an energetic decomposition analysis to characterize domain-peptide interactions and the molecular interaction energy components (MIECs), including van der Waals, electrostatic, and desolvation energy between residue pairs on the binding interface. We show a proof-of-concept study on the amphiphysin-1 SH3 domain interacting with its peptide ligands. The structures of the human amphiphysin-1 SH3 domain complexed with 884 peptides were first modeled using virtual mutagenesis and optimized by molecular mechanics (MM) minimization. Next, the MIECs between domain and peptide residues were computed using the MM/generalized Born decomposition analysis. We conducted two types of statistical analyses on the MIECs to demonstrate their usefulness for predicting binding affinities of peptides and for classifying peptides into binder and non-binder categories. First, combining partial least squares analysis and genetic algorithm, we fitted linear regression models between the MIECs and the peptide binding affinities on the training data set. These models were then used to predict binding affinities for peptides in the test data set; the predicted values have a correlation coefficient of 0.81 and an unsigned mean error of 0.39 compared with the experimentally measured ones. The partial least squares-genetic algorithm analysis on the MIECs revealed the critical interactions for the binding specificity of the amphiphysin-1 SH3 domain. Next, a support vector machine (SVM) was employed to build classification models based on the MIECs of peptides in the training set. A rigorous training-validation procedure was used to assess the performances of different kernel functions in SVM and different combinations of the MIECs. The best SVM classifier gave satisfactory predictions for the test set, indicated by average prediction accuracy rates of 78% and 91% for the binding and non-binding peptides, respectively. We also showed that the performance of our approach on both binding affinity prediction and binder/non-binder classification was superior to the performances of the conventional MM/Poisson-Boltzmann solvent-accessible surface area and MM/generalized Born solvent-accessible surface area calculations. Our study demonstrates that the analysis of the MIECs between peptides and the SH3 domain can successfully characterize the binding interface, and it provides a framework to derive integrated prediction models for different domain-peptide systems.

Crystal Structure of a Complex of the Intracellular Domain of Interferon λ Receptor 1 (IFNLR1) and the FERM/SH2 Domains of Human JAK1.

PubMed

Zhang, Di; Wlodawer, Alexander; Lubkowski, Jacek

2016-11-20

The crystal structure of a construct consisting of the FERM and SH2-like domains of the human Janus kinase 1 (JAK1) bound to a fragment of the intracellular domain of the interferon-λ receptor 1 (IFNLR1) has been determined at the nominal resolution of 2.1Å. In this structure, the receptor peptide forms an 85-Å-long extended chain, in which both the previously identified box1 and box2 regions bind simultaneously to the FERM and SH2-like domains of JAK1. Both domains of JAK1 are generally well ordered, with regions not seen in the crystal structure limited to loops located away from the receptor-binding regions. The structure provides a much more complete and accurate picture of the interactions between JAK1 and IFNLR1 than those given in earlier reports, illuminating the molecular basis of the JAK-cytokine receptor association. A glutamate residue adjacent to the box2 region in IFNLR1 mimics the mode of binding of a phosphotyrosine in classical SH2 domains. It was shown here that a deletion of residues within the box1 region of the receptor abolishes stable interactions with JAK1, although it was previously shown that box2 alone is sufficient to stabilize a similar complex of the interferon-α receptor and TYK2. Published by Elsevier Ltd.

Metallofullerenol Gd@C82(OH)22 distracts the proline-rich-motif from putative binding on the SH3 domain

NASA Astrophysics Data System (ADS)

Kang, Seung-Gu; Huynh, Tien; Zhou, Ruhong

2013-03-01

Biocompatibility is often regarded as one important aspect of de novo designed nanomaterials for biosafety. However, the toxicological effect, appearing along with its latency, is much more difficult to address by linearly mapping physicochemical properties of related nanomaterials with biological effects such as immune or cellular regulatory responses due to the complicated protein-protein interactions. Here, we investigate a potential interference of a metallofullerenol, Gd@C82(OH)22, on the function of SH3 domain, a highly promiscuous protein-protein interaction mediator involved in signaling and regulatory pathways through its binding with the proline-rich motif (PRM) peptides, using the atomistic molecular dynamics simulation. Our study shows that when only Gd@C82(OH)22 and the SH3 domain are present (without the PRM ligand), Gd@C82(OH)22 can interact with the SH3 domain by either directly blocking the hydrophobic active site or binding with a hydrophilic off-site with almost equal probability, which can be understood from its intrinsic amphiphilic nature. In a binding competition with the PRM onto the SH3 domain, however, the on-site binding mode is depleted while Gd@C82(OH)22 effectively intercepts the PRM from the putative binding site of the SH3 domain, implying that Gd@C82(OH)22 can disturb protein-protein interactions mediated by the SH3 domain. Despite a successful surface modification in an aqueous biological medium and a more recent demonstration as potential de novo cancer therapeutics, our study indicates that greater attention is needed in assessing the potential cytotoxicity of these nanomaterials.Biocompatibility is often regarded as one important aspect of de novo designed nanomaterials for biosafety. However, the toxicological effect, appearing along with its latency, is much more difficult to address by linearly mapping physicochemical properties of related nanomaterials with biological effects such as immune or cellular regulatory responses due to the complicated protein-protein interactions. Here, we investigate a potential interference of a metallofullerenol, Gd@C82(OH)22, on the function of SH3 domain, a highly promiscuous protein-protein interaction mediator involved in signaling and regulatory pathways through its binding with the proline-rich motif (PRM) peptides, using the atomistic molecular dynamics simulation. Our study shows that when only Gd@C82(OH)22 and the SH3 domain are present (without the PRM ligand), Gd@C82(OH)22 can interact with the SH3 domain by either directly blocking the hydrophobic active site or binding with a hydrophilic off-site with almost equal probability, which can be understood from its intrinsic amphiphilic nature. In a binding competition with the PRM onto the SH3 domain, however, the on-site binding mode is depleted while Gd@C82(OH)22 effectively intercepts the PRM from the putative binding site of the SH3 domain, implying that Gd@C82(OH)22 can disturb protein-protein interactions mediated by the SH3 domain. Despite a successful surface modification in an aqueous biological medium and a more recent demonstration as potential de novo cancer therapeutics, our study indicates that greater attention is needed in assessing the potential cytotoxicity of these nanomaterials. Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr33756a

Hydrophobic interaction between the SH2 domain and the kinase domain is required for the activation of Csk.

PubMed

Mikkola, Esa T; Gahmberg, Carl G

2010-06-18

The protein tyrosine kinase C-terminal Src kinase (Csk) is activated by the engagement of its Src homology (SH) 2 domain. However, the molecular mechanism required for this is not completely understood. The crystal structure of the active Csk indicates that Csk could be activated by contact between the SH2 domain and the beta3-alphaC loop in the N-terminal lobe of the kinase domain. To study the importance of this interaction for the SH2-domain-mediated activation of Csk, we mutated the amino acid residues forming the contacts between the SH2 domain and the beta3-alphaC loop. The mutation of the beta3-alphaC loop Ala228 to glycine and of the SH2 domain Tyr116, Tyr133, Leu138, and Leu149 to alanine resulted in the inability of the SH2 domain ligand to activate Csk. Furthermore, the overexpressed Csk mutants A228G, Y133A/Y116A, L138A, and L149A were unable to efficiently inactivate endogenous Src in human embryonic kidney 293 cells. The results suggest that the SH2-domain-mediated activation of Csk is dependent on the binding of the beta3-alphaC loop Ala228 to the hydrophobic pocket formed by the side chains of Tyr116, Tyr133, Leu138, and Leu149 on the surface of the SH2 domain. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Enhanced SH3/Linker Interaction Overcomes Abl Kinase Activation by Gatekeeper and Myristic Acid Binding Pocket Mutations and Increases Sensitivity to Small Molecule Inhibitors*

PubMed Central

Panjarian, Shoghag; Iacob, Roxana E.; Chen, Shugui; Wales, Thomas E.; Engen, John R.; Smithgall, Thomas E.

2013-01-01

Multidomain kinases such as c-Src and c-Abl are regulated by complex allosteric interactions involving their noncatalytic SH3 and SH2 domains. Here we show that enhancing natural allosteric control of kinase activity by SH3/linker engagement has long-range suppressive effects on the kinase activity of the c-Abl core. Surprisingly, enhanced SH3/linker interaction also dramatically sensitized the Bcr-Abl tyrosine kinase associated with chronic myelogenous leukemia to small molecule inhibitors that target either the active site or the myristic acid binding pocket in the kinase domain C-lobe. Dynamics analyses using hydrogen exchange mass spectrometry revealed a remarkable allosteric network linking the SH3 domain, the myristic acid binding pocket, and the active site of the c-Abl core, providing a structural basis for the biological observations. These results suggest a rational strategy for enhanced drug targeting of Bcr-Abl and other multidomain kinase systems that use multiple small molecules to exploit natural mechanisms of kinase control. PMID:23303187

Investigating MUC1/ICAM-1 Binding Induced Signaling in Breast Cancer Metastasis

DTIC Science & Technology

2011-05-01

expected that covalently linked species would remain intact. Reducing (R, + !-mercaptoethanol) and non-reducing (NR, no !-mercaptoethanol) samples were...binding site, containing both proline and arginine residues. We mutated the SH2 and/or putative SH3 binding domains on the MUC1-CFP-Fv plasmid...Structure and regulation of Src family kinases. Oncogene 2004, 23:7918- 7927. 31. Li SSC: Specificity and versatility of SH3 and other proline -recognition

From Binding-Induced Dynamic Effects in SH3 Structures to Evolutionary Conserved Sectors.

PubMed

Zafra Ruano, Ana; Cilia, Elisa; Couceiro, José R; Ruiz Sanz, Javier; Schymkowitz, Joost; Rousseau, Frederic; Luque, Irene; Lenaerts, Tom

2016-05-01

Src Homology 3 domains are ubiquitous small interaction modules known to act as docking sites and regulatory elements in a wide range of proteins. Prior experimental NMR work on the SH3 domain of Src showed that ligand binding induces long-range dynamic changes consistent with an induced fit mechanism. The identification of the residues that participate in this mechanism produces a chart that allows for the exploration of the regulatory role of such domains in the activity of the encompassing protein. Here we show that a computational approach focusing on the changes in side chain dynamics through ligand binding identifies equivalent long-range effects in the Src SH3 domain. Mutation of a subset of the predicted residues elicits long-range effects on the binding energetics, emphasizing the relevance of these positions in the definition of intramolecular cooperative networks of signal transduction in this domain. We find further support for this mechanism through the analysis of seven other publically available SH3 domain structures of which the sequences represent diverse SH3 classes. By comparing the eight predictions, we find that, in addition to a dynamic pathway that is relatively conserved throughout all SH3 domains, there are dynamic aspects specific to each domain and homologous subgroups. Our work shows for the first time from a structural perspective, which transduction mechanisms are common between a subset of closely related and distal SH3 domains, while at the same time highlighting the differences in signal transduction that make each family member unique. These results resolve the missing link between structural predictions of dynamic changes and the domain sectors recently identified for SH3 domains through sequence analysis.

From Binding-Induced Dynamic Effects in SH3 Structures to Evolutionary Conserved Sectors

PubMed Central

Ruiz Sanz, Javier; Schymkowitz, Joost; Rousseau, Frederic

2016-01-01

Src Homology 3 domains are ubiquitous small interaction modules known to act as docking sites and regulatory elements in a wide range of proteins. Prior experimental NMR work on the SH3 domain of Src showed that ligand binding induces long-range dynamic changes consistent with an induced fit mechanism. The identification of the residues that participate in this mechanism produces a chart that allows for the exploration of the regulatory role of such domains in the activity of the encompassing protein. Here we show that a computational approach focusing on the changes in side chain dynamics through ligand binding identifies equivalent long-range effects in the Src SH3 domain. Mutation of a subset of the predicted residues elicits long-range effects on the binding energetics, emphasizing the relevance of these positions in the definition of intramolecular cooperative networks of signal transduction in this domain. We find further support for this mechanism through the analysis of seven other publically available SH3 domain structures of which the sequences represent diverse SH3 classes. By comparing the eight predictions, we find that, in addition to a dynamic pathway that is relatively conserved throughout all SH3 domains, there are dynamic aspects specific to each domain and homologous subgroups. Our work shows for the first time from a structural perspective, which transduction mechanisms are common between a subset of closely related and distal SH3 domains, while at the same time highlighting the differences in signal transduction that make each family member unique. These results resolve the missing link between structural predictions of dynamic changes and the domain sectors recently identified for SH3 domains through sequence analysis. PMID:27213566

SH2 Ligand-Like Effects of Second Cytosolic Domain of Na/K-ATPase α1 Subunit on Src Kinase.

PubMed

Banerjee, Moumita; Duan, Qiming; Xie, Zijian

2015-01-01

Our previous studies have suggested that the α1 Na/K-ATPase interacts with Src to form a receptor complex. In vitro binding assays indicate an interaction between second cytosolic domain (CD2) of Na/K-ATPase α1 subunit and Src SH2 domain. Since SH2 domain targets Src to specific signaling complexes, we expressed CD2 as a cytosolic protein and studied whether it could act as a Src SH2 ligand in LLC-PK1 cells. Co-immunoprecipitation analyses indicated a direct binding of CD2 to Src, consistent with the in vitro binding data. Functionally, CD2 expression increased basal Src activity, suggesting a Src SH2 ligand-like property of CD2. Consistently, we found that CD2 expression attenuated several signaling pathways where Src plays an important role. For instance, although it increased surface expression of Na/K-ATPase, it decreased ouabain-induced activation of Src and ERK by blocking the formation of Na/K-ATPase/Src complex. Moreover, it also attenuated cell attachment-induced activation of Src/FAK. Consequently, CD2 delayed cell spreading, and inhibited cell proliferation. Furthermore, these effects appear to be Src-specific because CD2 expression had no effect on EGF-induced activation of EGF receptor and ERK. Hence, the new findings indicate the importance of Na/K-ATPase/Src interaction in ouabain-induced signal transduction, and support the proposition that the CD2 peptide may be utilized as a Src SH2 ligand capable of blocking Src-dependent signaling pathways via a different mechanism from a general Src kinase inhibitor.

Dynamics and intramolecular ligand binding of DtxR studied by MD simulations and NMR spectroscopy

NASA Astrophysics Data System (ADS)

Yi, Myunggi; Bhattacharya, Nilakshee; Zhou, Huan-Xiang

2005-11-01

Diphtheria toxin repressor (DtxR) regulates the expression of the diphtheria toxin gene through intramolecular ligand binding (Wylie et al., Biochemistry 2005, 44:40-51). Protein dynamics is essential to the binding process of the Pro-rich (Pr) ligand to the C-terminal SH3 domain. We present MD and NMR results on the dynamics and ligand interactions of a Pr-SH3 construct of DtxR. NMR relaxation data (T1, T2, and NOE) showed that the Pr ligand is very flexible, suggesting that it undergoes binding/unbinding transitions. A 50-ns MD trajectory of the protein was used to calculate T1, T2, and NOE, reproducing the NMR results for the SH3 domain but not for the Pr segment. During the MD simulation, the ligand stayed bound to the SH3 domain; thus the simulation represented the bound state. The NMR data for the Pr-segment could be explained by assuming that they represented the average behavior of a fast binding/unbinding exchange. Though unbinding was not observed in the MD simulation, the simulation did show large fluctuations of a loop which forms part of the wall of the binding pocket. The fluctuations led to opening up of the binding pocket, thus weakening the interaction with the Pr segment and perhaps ultimately leading to ligand unbinding.

Association of 3BP2 with SHP-1 regulates SHP-1-mediated production of TNF-α in RBL-2H3 cells.

PubMed

Chihara, Kazuyasu; Nakashima, Kenji; Takeuchi, Kenji; Sada, Kiyonao

2011-12-01

Adaptor protein 3BP2, a c-Abl Src homology 3 (SH3) domain-binding protein, is tyrosine phosphorylated and positively regulates mast cell signal transduction after the aggregation of the high affinity IgE receptor (FcεRI). Overexpression of the Src homology 2 (SH2) domain of 3BP2 results in the dramatic suppression of antigen-induced degranulation in rat basophilic leukemia RBL-2H3 cells. Previously, a linker for activation of T cells (LAT) was identified as one of the 3BP2 SH2 domain-binding protein. In this report, to further understand the functions of 3BP2 in FcεRI-mediated activation of mast cell, we explored the protein that associates with the SH2 domain of 3BP2 and found that SH2 domain-containing phosphatase-1 (SHP-1) inducibly interacts with the SH2 domain of 3BP2 after the aggregation of FcεRI. The phosphorylation of Tyr(564) in the carboxy (C)-terminal tail region of SHP-1 is required for the direct interaction of SHP-1 to the SH2 domain of 3BP2. The expression of the mutant form of SHP-1 which was unable to interact with 3BP2 resulted in the significant reduction in SHP-1-mediated tumor necrosis factor-α (TNF-α) production without any effects on the degranulation in antigen-stimulated RBL-2H3 cells. These findings suggest that 3BP2 directly interacts with Tyr(564) -phosphorylated form of SHP-1 and positively regulates the function of SHP-1 in FcεRI-mediated signaling in mast cells. © 2011 The Authors. Journal compilation © 2011 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

PLC-γ directly binds activated c-Src, which is necessary for carbachol-mediated inhibition of NHE3 activity in Caco-2/BBe cells

PubMed Central

Lee, Luke J.; Kovbasnjuk, Olga; Li, Xuhang; Donowitz, Mark

2013-01-01

Elevated levels of intracellular Ca2+ ([Ca2+]i) inhibit Na+/H+ exchanger 3 (NHE3) activity in the intact intestine. We previously demonstrated that PLC-γ directly binds NHE3, an interaction that is necessary for [Ca2+]i inhibition of NHE3 activity, and that PLC-γ Src homology 2 (SH2) domains may scaffold Ca2+ signaling proteins necessary for regulation of NHE3 activity. [Ca2+]i regulation of NHE3 activity is also c-Src dependent; however, the mechanism by which c-Src is involved is undetermined. We hypothesized that the SH2 domains of PLC-γ might link c-Src to NHE3-containing complexes to mediate [Ca2+]i inhibition of NHE3 activity. In Caco-2/BBe cells, carbachol (CCh) decreased NHE3 activity by ∼40%, an effect abolished with the c-Src inhibitor PP2. CCh treatment increased the amount of active c-Src as early as 1 min through increased Y416 phosphorylation. Coimmunoprecipitation demonstrated that c-Src associated with PLC-γ, but not NHE3, under basal conditions, an interaction that increased rapidly after CCh treatment and occurred before the dissociation of PLC-γ and NHE3 that occurred 10 min after CCh treatment. Finally, direct binding to c-Src only occurred through the PLC-γ SH2 domains, an interaction that was prevented by blocking the PLC-γ SH2 domain. This study demonstrated that c-Src 1) activity is necessary for [Ca2+]i inhibition of NHE3 activity, 2) activation occurs rapidly (∼1 min) after CCh treatment, 3) directly binds PLC-γ SH2 domains and associates dynamically with PLC-γ under elevated [Ca2+]i conditions, and 4) does not directly bind NHE3. Under elevated [Ca2+]i conditions, PLC-γ scaffolds c-Src into NHE3-containing multiprotein complexes before dissociation of PLC-γ from NHE3 and subsequent endocytosis of NHE3. PMID:23703528

PLC-γ directly binds activated c-Src, which is necessary for carbachol-mediated inhibition of NHE3 activity in Caco-2/BBe cells.

PubMed

Zachos, Nicholas C; Lee, Luke J; Kovbasnjuk, Olga; Li, Xuhang; Donowitz, Mark

2013-08-01

Elevated levels of intracellular Ca(2+) ([Ca(2+)]i) inhibit Na(+)/H(+) exchanger 3 (NHE3) activity in the intact intestine. We previously demonstrated that PLC-γ directly binds NHE3, an interaction that is necessary for [Ca(2+)]i inhibition of NHE3 activity, and that PLC-γ Src homology 2 (SH2) domains may scaffold Ca(2+) signaling proteins necessary for regulation of NHE3 activity. [Ca(2+)]i regulation of NHE3 activity is also c-Src dependent; however, the mechanism by which c-Src is involved is undetermined. We hypothesized that the SH2 domains of PLC-γ might link c-Src to NHE3-containing complexes to mediate [Ca(2+)]i inhibition of NHE3 activity. In Caco-2/BBe cells, carbachol (CCh) decreased NHE3 activity by ∼40%, an effect abolished with the c-Src inhibitor PP2. CCh treatment increased the amount of active c-Src as early as 1 min through increased Y(416) phosphorylation. Coimmunoprecipitation demonstrated that c-Src associated with PLC-γ, but not NHE3, under basal conditions, an interaction that increased rapidly after CCh treatment and occurred before the dissociation of PLC-γ and NHE3 that occurred 10 min after CCh treatment. Finally, direct binding to c-Src only occurred through the PLC-γ SH2 domains, an interaction that was prevented by blocking the PLC-γ SH2 domain. This study demonstrated that c-Src 1) activity is necessary for [Ca(2+)]i inhibition of NHE3 activity, 2) activation occurs rapidly (∼1 min) after CCh treatment, 3) directly binds PLC-γ SH2 domains and associates dynamically with PLC-γ under elevated [Ca(2+)]i conditions, and 4) does not directly bind NHE3. Under elevated [Ca(2+)]i conditions, PLC-γ scaffolds c-Src into NHE3-containing multiprotein complexes before dissociation of PLC-γ from NHE3 and subsequent endocytosis of NHE3.

Enhanced Prediction of Src Homology 2 (SH2) Domain Binding Potentials Using a Fluorescence Polarization-derived c-Met, c-Kit, ErbB, and Androgen Receptor Interactome*

PubMed Central

Leung, Kin K.; Hause, Ronald J.; Barkinge, John L.; Ciaccio, Mark F.; Chuu, Chih-Pin; Jones, Richard B.

2014-01-01

Many human diseases are associated with aberrant regulation of phosphoprotein signaling networks. Src homology 2 (SH2) domains represent the major class of protein domains in metazoans that interact with proteins phosphorylated on the amino acid residue tyrosine. Although current SH2 domain prediction algorithms perform well at predicting the sequences of phosphorylated peptides that are likely to result in the highest possible interaction affinity in the context of random peptide library screens, these algorithms do poorly at predicting the interaction potential of SH2 domains with physiologically derived protein sequences. We employed a high throughput interaction assay system to empirically determine the affinity between 93 human SH2 domains and phosphopeptides abstracted from several receptor tyrosine kinases and signaling proteins. The resulting interaction experiments revealed over 1000 novel peptide-protein interactions and provided a glimpse into the common and specific interaction potentials of c-Met, c-Kit, GAB1, and the human androgen receptor. We used these data to build a permutation-based logistic regression classifier that performed considerably better than existing algorithms for predicting the interaction potential of several SH2 domains. PMID:24728074

Allostery Mediates Ligand Binding to Grb2 Adaptor in a Mutually Exclusive Manner

PubMed Central

McDonald, Caleb B.; El Hokayem, Jimmy; Zafar, Nawal; Balke, Jordan E.; Bhat, Vikas; Mikles, David C.; Deegan, Brian J.; Seldeen, Kenneth L.; Farooq, Amjad

2012-01-01

Allostery plays a key role in dictating the stoichiometry and thermodynamics of multi-protein complexes driving a plethora of cellular processes central to health and disease. Herein, using various biophysical tools, we demonstrate that although Sos1 nucleotide exchange factor and Gab1 docking protein recognize two non-overlapping sites within the Grb2 adaptor, allostery promotes the formation of two distinct pools of Grb2-Sos1 and Grb2-Gab1 binary signaling complexes in concert in lieu of a composite Sos1-Grb2-Gab1 ternary complex. Of particular interest is the observation that the binding of Sos1 to the nSH3 domain within Grb2 sterically blocks the binding of Gab1 to the cSH3 domain and vice versa in a mutually exclusive manner. Importantly, the formation of both the Grb2-Sos1 and Grb2-Gab1 binary complexes is governed by a stoichiometry of 2:1, whereby the respective SH3 domains within Grb2 homodimer bind to Sos1 and Gab1 via multivalent interactions. Collectively, our study sheds new light on the role of allostery in mediating cellular signaling machinery. PMID:23334917

The SH2 Domain Regulates c-Abl Kinase Activation by a Cyclin-Like Mechanism and Remodulation of the Hinge Motion

PubMed Central

Dölker, Nicole; Górna, Maria W.; Sutto, Ludovico; Torralba, Antonio S.; Superti-Furga, Giulio; Gervasio, Francesco L.

2014-01-01

Regulation of the c-Abl (ABL1) tyrosine kinase is important because of its role in cellular signaling, and its relevance in the leukemiogenic counterpart (BCR-ABL). Both auto-inhibition and full activation of c-Abl are regulated by the interaction of the catalytic domain with the Src Homology 2 (SH2) domain. The mechanism by which this interaction enhances catalysis is not known. We combined computational simulations with mutagenesis and functional analysis to find that the SH2 domain conveys both local and global effects on the dynamics of the catalytic domain. Locally, it regulates the flexibility of the αC helix in a fashion reminiscent of cyclins in cyclin-dependent kinases, reorienting catalytically important motifs. At a more global level, SH2 binding redirects the hinge motion of the N and C lobes and changes the conformational equilibrium of the activation loop. The complex network of subtle structural shifts that link the SH2 domain with the activation loop and the active site may be partially conserved with other SH2-domain containing kinases and therefore offer additional parameters for the design of conformation-specific inhibitors. PMID:25299346

The SH2 domain regulates c-Abl kinase activation by a cyclin-like mechanism and remodulation of the hinge motion.

PubMed

Dölker, Nicole; Górna, Maria W; Sutto, Ludovico; Torralba, Antonio S; Superti-Furga, Giulio; Gervasio, Francesco L

2014-10-01

Regulation of the c-Abl (ABL1) tyrosine kinase is important because of its role in cellular signaling, and its relevance in the leukemiogenic counterpart (BCR-ABL). Both auto-inhibition and full activation of c-Abl are regulated by the interaction of the catalytic domain with the Src Homology 2 (SH2) domain. The mechanism by which this interaction enhances catalysis is not known. We combined computational simulations with mutagenesis and functional analysis to find that the SH2 domain conveys both local and global effects on the dynamics of the catalytic domain. Locally, it regulates the flexibility of the αC helix in a fashion reminiscent of cyclins in cyclin-dependent kinases, reorienting catalytically important motifs. At a more global level, SH2 binding redirects the hinge motion of the N and C lobes and changes the conformational equilibrium of the activation loop. The complex network of subtle structural shifts that link the SH2 domain with the activation loop and the active site may be partially conserved with other SH2-domain containing kinases and therefore offer additional parameters for the design of conformation-specific inhibitors.

The Janus Kinase (JAK) FERM and SH2 Domains: Bringing Specificity to JAK-Receptor Interactions.

PubMed

Ferrao, Ryan; Lupardus, Patrick J

2017-01-01

The Janus kinases (JAKs) are non-receptor tyrosine kinases essential for signaling in response to cytokines and interferons and thereby control many essential functions in growth, development, and immune regulation. JAKs are unique among tyrosine kinases for their constitutive yet non-covalent association with class I and II cytokine receptors, which upon cytokine binding bring together two JAKs to create an active signaling complex. JAK association with cytokine receptors is facilitated by N-terminal FERM and SH2 domains, both of which are classical mediators of peptide interactions. Together, the JAK FERM and SH2 domains mediate a bipartite interaction with two distinct receptor peptide motifs, the proline-rich "Box1" and hydrophobic "Box2," which are present in the intracellular domain of cytokine receptors. While the general sidechain chemistry of Box1 and Box2 peptides is conserved between receptors, they share very weak primary sequence homology, making it impossible to posit why certain JAKs preferentially interact with and signal through specific subsets of cytokine receptors. Here, we review the structure and function of the JAK FERM and SH2 domains in light of several recent studies that reveal their atomic structure and elucidate interaction mechanisms with both the Box1 and Box2 receptor motifs. These crystal structures demonstrate how evolution has repurposed the JAK FERM and SH2 domains into a receptor-binding module that facilitates interactions with multiple receptors possessing diverse primary sequences.

SH3-domain binding protein 1 in the tumor microenvironment promotes hepatocellular carcinoma metastasis through WAVE2 pathway

PubMed Central

Tao, Yiming; Hu, Kuan; Tan, Fengbo; Zhang, Sai; Zhou, Ming; Luo, Jia; Wang, Zhiming

2016-01-01

SH3-domain binding protein-1 (SH3BP1) specifically inactivating Rac1 and its target WAVE2 is required for cell motility. The present study shows SH3BP1 expression patterns in human HCC tissues and cell lines were examined. The regulation of SH3BP1 on HCC cell migration and invasion related to Rac1-WAVE2 signaling was characterized using in vitro and in vivo models. SH3BP1 overexpressed in HCC tissues and highly metastatic HCC cells was significantly associated vascular invasion (VI). SH3BP1 promoted VEGF secretion via Rac1-WAVE2 signaling, so as to exert an augmentation on cell invasion and microvessel formation. In three study cohorts with a total of 516 HCC patients, high SH3BP1 expression combined with high microvessel density (MVD) was confirmed as a powerful independent predictor of HCC prognosis in both training cohorts and validation cohort. Being an important angiogenic factor of HCC through Rac1-WAVE2 signaling, SH3BP1 promotes tumor invasion and microvessel formation contributing to HCC metastasis and recurrence. SH3BP1 is a novel WAVE2 regulator, a prognostic marker and a potential therapeutic target of HCC. PMID:26933917

SH3-domain binding protein 1 in the tumor microenvironment promotes hepatocellular carcinoma metastasis through WAVE2 pathway.

PubMed

Tao, Yiming; Hu, Kuan; Tan, Fengbo; Zhang, Sai; Zhou, Ming; Luo, Jia; Wang, Zhiming

2016-04-05

SH3-domain binding protein-1 (SH3BP1) specifically inactivating Rac1 and its target WAVE2 is required for cell motility. The present study shows SH3BP1 expression patterns in human HCC tissues and cell lines were examined. The regulation of SH3BP1 on HCC cell migration and invasion related to Rac1-WAVE2 signaling was characterized using in vitro and in vivo models. SH3BP1 overexpressed in HCC tissues and highly metastatic HCC cells was significantly associated vascular invasion (VI). SH3BP1 promoted VEGF secretion via Rac1-WAVE2 signaling, so as to exert an augmentation on cell invasion and microvessel formation. In three study cohorts with a total of 516 HCC patients, high SH3BP1 expression combined with high microvessel density (MVD) was confirmed as a powerful independent predictor of HCC prognosis in both training cohorts and validation cohort. Being an important angiogenic factor of HCC through Rac1-WAVE2 signaling, SH3BP1 promotes tumor invasion and microvessel formation contributing to HCC metastasis and recurrence. SH3BP1 is a novel WAVE2 regulator, a prognostic marker and a potential therapeutic target of HCC.

Thermodynamic contribution of backbone conformational entropy in the binding between SH3 domain and proline-rich motif.

PubMed

Zeng, Danyun; Shen, Qingliang; Cho, Jae-Hyun

2017-02-26

Biological functions of intrinsically disordered proteins (IDPs), and proteins containing intrinsically disordered regions (IDRs) are often mediated by short linear motifs, like proline-rich motifs (PRMs). Upon binding to their target proteins, IDPs undergo a disorder-to-order transition which is accompanied by a large conformational entropy penalty. Hence, the molecular mechanisms underlying control of conformational entropy are critical for understanding the binding affinity and selectivity of IDPs-mediated protein-protein interactions (PPIs). Here, we investigated the backbone conformational entropy change accompanied by binding of the N-terminal SH3 domain (nSH3) of CrkII and PRM derived from guanine nucleotide exchange factor 1 (C3G). In particular, we focused on the estimation of conformational entropy change of disordered PRM upon binding to the nSH3 domain. Quantitative characterization of conformational dynamics of disordered peptides like PRMs is limited. Hence, we combined various methods, including NMR model-free analysis, δ2D, DynaMine, and structure-based calculation of entropy loss. This study demonstrates that the contribution of backbone conformational entropy change is significant in the PPIs mediated by IDPs/IDRs. Copyright © 2017 Elsevier Inc. All rights reserved.

Dynamically Coupled Residues within the SH2 Domain of FYN Are Key to Unlocking Its Activity.

PubMed

Huculeci, Radu; Cilia, Elisa; Lyczek, Agatha; Buts, Lieven; Houben, Klaartje; Seeliger, Markus A; van Nuland, Nico; Lenaerts, Tom

2016-11-01

Src kinase activity is controlled by various mechanisms involving a coordinated movement of kinase and regulatory domains. Notwithstanding the extensive knowledge related to the backbone dynamics, little is known about the more subtle side-chain dynamics within the regulatory domains and their role in the activation process. Here, we show through experimental methyl dynamic results and predicted changes in side-chain conformational couplings that the SH2 structure of Fyn contains a dynamic network capable of propagating binding information. We reveal that binding the phosphorylated tail of Fyn perturbs a residue cluster near the linker connecting the SH2 and SH3 domains of Fyn, which is known to be relevant in the regulation of the activity of Fyn. Biochemical perturbation experiments validate that those residues are essential for inhibition of Fyn, leading to a gain of function upon mutation. These findings reveal how side-chain dynamics may facilitate the allosteric regulation of the different members of the Src kinase family. Copyright © 2016 Elsevier Ltd. All rights reserved.

Novel Multiplexed Assay for Identifying SH2 Domain Antagonists of STAT Family Proteins

PubMed Central

Takakuma, Kazuyuki; Ogo, Naohisa; Uehara, Yutaka; Takahashi, Susumu; Miyoshi, Nao; Asai, Akira

2013-01-01

Some of the signal transducer and activator of transcription (STAT) family members are constitutively activated in a wide variety of human tumors. The activity of STAT depends on their Src homology 2 (SH2) domain-mediated binding to sequences containing phosphorylated tyrosine. Thus, antagonizing this binding is a feasible approach to inhibiting STAT activation. We have developed a novel multiplexed assay for STAT3- and STAT5b-SH2 binding, based on amplified luminescent proximity homogeneous assay (Alpha) technology. AlphaLISA and AlphaScreen beads were combined in a single-well assay, which allowed the binding of STAT3- and STAT5b-SH2 to phosphotyrosine peptides to be simultaneously monitored. Biotin-labeled recombinant human STAT proteins were obtained as N- and C-terminal deletion mutants. The spacer length of the DIG-labeled peptide, the reaction time, and the concentration of sodium chloride were optimized to establish a HTS system with Z’ values of greater than 0.6 for both STAT3- and STAT5b-SH2 binding. We performed a HTS campaign for chemical libraries using this multiplexed assay and identified hit compounds. A 2-chloro-1,4-naphthalenedione derivative, Compound 1, preferentially inhibited STAT3-SH2 binding in vitro, and the nuclear translocation of STAT3 in HeLa cells. Initial structure activity relationship (SAR) studies using the multiplexed assay showed the 3-substituent effect on both the activity and selectivity of STAT3 and STAT5b inhibition. Therefore, this multiplexed assay is useful for not only searching for potential lead compounds but also obtaining SAR data for developing new STAT3/STAT5b inhibitors. PMID:23977103

Novel multiplexed assay for identifying SH2 domain antagonists of STAT family proteins.

PubMed

Takakuma, Kazuyuki; Ogo, Naohisa; Uehara, Yutaka; Takahashi, Susumu; Miyoshi, Nao; Asai, Akira

2013-01-01

Some of the signal transducer and activator of transcription (STAT) family members are constitutively activated in a wide variety of human tumors. The activity of STAT depends on their Src homology 2 (SH2) domain-mediated binding to sequences containing phosphorylated tyrosine. Thus, antagonizing this binding is a feasible approach to inhibiting STAT activation. We have developed a novel multiplexed assay for STAT3- and STAT5b-SH2 binding, based on amplified luminescent proximity homogeneous assay (Alpha) technology. AlphaLISA and AlphaScreen beads were combined in a single-well assay, which allowed the binding of STAT3- and STAT5b-SH2 to phosphotyrosine peptides to be simultaneously monitored. Biotin-labeled recombinant human STAT proteins were obtained as N- and C-terminal deletion mutants. The spacer length of the DIG-labeled peptide, the reaction time, and the concentration of sodium chloride were optimized to establish a HTS system with Z' values of greater than 0.6 for both STAT3- and STAT5b-SH2 binding. We performed a HTS campaign for chemical libraries using this multiplexed assay and identified hit compounds. A 2-chloro-1,4-naphthalenedione derivative, Compound 1, preferentially inhibited STAT3-SH2 binding in vitro, and the nuclear translocation of STAT3 in HeLa cells. Initial structure activity relationship (SAR) studies using the multiplexed assay showed the 3-substituent effect on both the activity and selectivity of STAT3 and STAT5b inhibition. Therefore, this multiplexed assay is useful for not only searching for potential lead compounds but also obtaining SAR data for developing new STAT3/STAT5b inhibitors.

Evolution of SH2 domains and phosphotyrosine signalling networks

PubMed Central

Liu, Bernard A.; Nash, Piers D.

2012-01-01

Src homology 2 (SH2) domains mediate selective protein–protein interactions with tyrosine phosphorylated proteins, and in doing so define specificity of phosphotyrosine (pTyr) signalling networks. SH2 domains and protein-tyrosine phosphatases expand alongside protein-tyrosine kinases (PTKs) to coordinate cellular and organismal complexity in the evolution of the unikont branch of the eukaryotes. Examination of conserved families of PTKs and SH2 domain proteins provides fiduciary marks that trace the evolutionary landscape for the development of complex cellular systems in the proto-metazoan and metazoan lineages. The evolutionary provenance of conserved SH2 and PTK families reveals the mechanisms by which diversity is achieved through adaptations in tissue-specific gene transcription, altered ligand binding, insertions of linear motifs and the gain or loss of domains following gene duplication. We discuss mechanisms by which pTyr-mediated signalling networks evolve through the development of novel and expanded families of SH2 domain proteins and the elaboration of connections between pTyr-signalling proteins. These changes underlie the variety of general and specific signalling networks that give rise to tissue-specific functions and increasingly complex developmental programmes. Examination of SH2 domains from an evolutionary perspective provides insight into the process by which evolutionary expansion and modification of molecular protein interaction domain proteins permits the development of novel protein-interaction networks and accommodates adaptation of signalling networks. PMID:22889907

Structure of the SH3 Domain of Rat Endophilin A2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Loll,P.; Swain, E.; Chen, Y.

2008-01-01

The crystal structure of the SH3 domain of rat endophilin A2 has been determined by the multiwavelength anomalous dispersion method and refined at a resolution of 1.70 Angstroms to R and Rfree values of 0.196 and 0.217, respectively. The structure adheres to the canonical SH3-domain fold and is highly similar to those of the corresponding domains of endophilins A1 and A3. An intermolecular packing interaction between two molecules in the lattice exploits features that are commonly observed in SH3-domain ligand recognition, including the insertion of a proline side chain into the ligand-binding groove of the protein and the recognition ofmore » a basic residue by a cluster of acidic side chains on the RT loop.« less

GW domains of the Listeria monocytogenes invasion protein InlB are SH3-like and mediate binding to host ligands

PubMed Central

Marino, Michael; Banerjee, Manidipa; Jonquières, Renaud; Cossart, Pascale; Ghosh, Partho

2002-01-01

InlB, a surface-localized protein of Listeria monocytogenes, induces phagocytosis in non-phagocytic mammalian cells by activating Met, a receptor tyrosine kinase. InlB also binds glycosaminoglycans and the protein gC1q-R, two additional host ligands implicated in invasion. We present the structure of InlB, revealing a highly elongated molecule with leucine-rich repeats that bind Met at one end, and GW domains that dissociably bind the bacterial surface at the other. Surprisingly, the GW domains are seen to resemble SH3 domains. Despite this, GW domains are unlikely to act as functional mimics of SH3 domains since their potential proline-binding sites are blocked or destroyed. However, we do show that the GW domains, in addition to binding glycosaminoglycans, bind gC1q-R specifically, and that this binding requires release of InlB from the bacterial surface. Dissociable attachment to the bacterial surface via the GW domains may be responsible for restricting Met activation to a small, localized area of the host cell and for coupling InlB-induced host membrane dynamics with bacterial proximity during invasion. PMID:12411480

A Discovery Strategy for Selective Inhibitors of c-Src in Complex with the Focal Adhesion Kinase SH3/SH2-binding Region.

PubMed

Moroco, Jamie A; Baumgartner, Matthew P; Rust, Heather L; Choi, Hwan Geun; Hur, Wooyoung; Gray, Nathanael S; Camacho, Carlos J; Smithgall, Thomas E

2015-08-01

The c-Src tyrosine kinase co-operates with the focal adhesion kinase to regulate cell adhesion and motility. Focal adhesion kinase engages the regulatory SH3 and SH2 domains of c-Src, resulting in localized kinase activation that contributes to tumor cell metastasis. Using assay conditions where c-Src kinase activity required binding to a tyrosine phosphopeptide based on the focal adhesion kinase SH3-SH2 docking sequence, we screened a kinase-biased library for selective inhibitors of the Src/focal adhesion kinase peptide complex versus c-Src alone. This approach identified an aminopyrimidinyl carbamate compound, WH-4-124-2, with nanomolar inhibitory potency and fivefold selectivity for c-Src when bound to the phospho-focal adhesion kinase peptide. Molecular docking studies indicate that WH-4-124-2 may preferentially inhibit the 'DFG-out' conformation of the kinase active site. These findings suggest that interaction of c-Src with focal adhesion kinase induces a unique kinase domain conformation amenable to selective inhibition. © 2014 John Wiley & Sons A/S.

Evidence for in vivo phosphorylation of the Grb2 SH2-domain binding site on focal adhesion kinase by Src-family protein-tyrosine kinases.

PubMed

Schlaepfer, D D; Hunter, T

1996-10-01

Focal adhesion kinase (FAK) is a nonreceptor protein-tyrosine kinase (PTK) that associates with integrin receptors and participates in extracellular matrix-mediated signal transduction events. We showed previously that the c-Src nonreceptor PTK and the Grb2 SH2/SH3 adaptor protein bound directly to FAK after fibronectin stimulation (D. D. Schlaepfer, S.K. Hanks, T. Hunter, and P. van der Geer, Nature [London] 372:786-791, 1994). Here, we present evidence that c-Src association with FAK is required for Grb2 binding to FAK. Using a tryptic phosphopeptide mapping approach, the in vivo phosphorylation of the Grb2 binding site on FAK (Tyr-925) was detected after fibronectin stimulation of NIH 3T3 cells and was constitutively phosphorylated in v-Src-transformed NIH 3T3 cells. In vitro, c-Src phosphorylated FAK Tyr-925 in a glutathione S-transferase-FAK C-terminal domain fusion protein, whereas FAK did not. Using epitope-tagged FAK constructs, transiently expressed in human 293 cells, we determined the effect of site-directed mutations on c-Src and Grb2 binding to FAK. Mutation of FAK Tyr-925 disrupted Grb2 binding, whereas mutation of the c-Src binding site on FAK (Tyr-397) disrupted both c-Src and Grb2 binding to FAK in vivo. These results support a model whereby Src-family PTKs are recruited to FAK and focal adhesions following integrin-induced autophosphorylation and exposure of FAK Tyr-397. Src-family binding and phosphorylation of FAK at Tyr-925 creates a Grb2 SH2-domain binding site and provides a link to the activation of the Ras signal transduction pathway. In Src-transformed cells, this pathway may be constitutively activated as a result of FAK Tyr-925 phosphorylation in the absence of integrin stimulation.

SH3 interactome conserves general function over specific form

PubMed Central

Xin, Xiaofeng; Gfeller, David; Cheng, Jackie; Tonikian, Raffi; Sun, Lin; Guo, Ailan; Lopez, Lianet; Pavlenco, Alevtina; Akintobi, Adenrele; Zhang, Yingnan; Rual, Jean-François; Currell, Bridget; Seshagiri, Somasekar; Hao, Tong; Yang, Xinping; Shen, Yun A; Salehi-Ashtiani, Kourosh; Li, Jingjing; Cheng, Aaron T; Bouamalay, Dryden; Lugari, Adrien; Hill, David E; Grimes, Mark L; Drubin, David G; Grant, Barth D; Vidal, Marc; Boone, Charles; Sidhu, Sachdev S; Bader, Gary D

2013-01-01

Src homology 3 (SH3) domains bind peptides to mediate protein–protein interactions that assemble and regulate dynamic biological processes. We surveyed the repertoire of SH3 binding specificity using peptide phage display in a metazoan, the worm Caenorhabditis elegans, and discovered that it structurally mirrors that of the budding yeast Saccharomyces cerevisiae. We then mapped the worm SH3 interactome using stringent yeast two-hybrid and compared it with the equivalent map for yeast. We found that the worm SH3 interactome resembles the analogous yeast network because it is significantly enriched for proteins with roles in endocytosis. Nevertheless, orthologous SH3 domain-mediated interactions are highly rewired. Our results suggest a model of network evolution where general function of the SH3 domain network is conserved over its specific form. PMID:23549480

Tyrosine phosphorylation of the orphan receptor ESDN/DCBLD2 serves as a scaffold for the signaling adaptor CrkL.

PubMed

Aten, Tyler M; Redmond, Miranda M; Weaver, Sheila O; Love, Collin C; Joy, Ryan M; Lapp, Aliya S; Rivera, Osvaldo D; Hinkle, Karen L; Ballif, Bryan A

2013-08-02

A quantitative proteomics screen to identify substrates of the Src family of tyrosine kinases (SFKs) whose phosphorylation promotes CrkL-SH2 binding identified the known Crk-associated substrate (Cas) of Src as well as the orphan receptor endothelial and smooth muscle cell-derived neuropilin-like protein (ESDN). Mutagenesis analysis of ESDN's seven intracellular tyrosines in YxxP motifs found several contribute to the binding of ESDN to the SH2 domains of both CrkCT10 regulator of kinase Crk-Like (CrkL) and a representative SFK Fyn. Quantitative mass spectrometry showed that at least three of these (Y565, Y621 and Y750), as well as non-YxxP Y715, are reversibly phosphorylated. SFK activity was shown to be sufficient, but not required for the interaction between ESDN and the CrkL-SH2 domain. Finally, antibody-mediated ESDN clustering induces ESDN tyrosine phosphorylation and CrkL-SH2 binding. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

The auto-inhibitory state of Rho guanine nucleotide exchange factor ARHGEF5/TIM can be relieved by targeting its SH3 domain with rationally designed peptide aptamers.

PubMed

He, Ping; Tan, De-Li; Liu, Hong-Xiang; Lv, Feng-Lin; Wu, Wei

2015-04-01

The short isoform of Rho guanine nucleotide exchange factor ARHGEF5 is known as TIM, which plays diverse roles in, for example, tumorigenesis, neuronal development and Src-induced podosome formation through the activation of its substrates, the Rho family of GTPases. The activation is auto-inhibited by a putative helix N-terminal to the DH domain of TIM, which is stabilized by the intramolecular interaction of C-terminal SH3 domain with a poly-proline sequence between the putative helix and the DH domain. In this study, we systematically investigated the structural basis, energetic landscape and biological implication underlying TIM auto-inhibition by using atomistic molecular dynamics simulations and binding free energy analysis. The computational study revealed that the binding of SH3 domain to poly-proline sequence is the prerequisite for the stabilization of TIM auto-inhibition. Thus, it is suggested that targeting SH3 domain with competitors of the poly-proline sequence would be a promising strategy to relieve the auto-inhibitory state of TIM. In this consideration, we rationally designed a number of peptide aptamers for competitively inhibiting the SH3 domain based on modeled TIM structure and computationally generated data. Peptide binding test and guanine nucleotide exchange analysis solidified that these designed peptides can both bind to the SH3 domain potently and activate TIM-catalyzed RhoA exchange reaction effectively. Interestingly, a positive correlation between the peptide affinity and induced exchange activity was observed. In addition, separate mutation of three conserved residues Pro49, Pro52 and Lys54 - they are required for peptide recognition by SH3 domain -- in a designed peptide to Ala would completely abolish the capability of this peptide activating TIM. All these come together to suggest an intrinsic relationship between peptide binding to SH3 domain and the activation of TIM. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Crystal structure of the Src family kinase Hck SH3-SH2 linker regulatory region supports an SH3-dominant activation mechanism.

PubMed

Alvarado, John J; Betts, Laurie; Moroco, Jamie A; Smithgall, Thomas E; Yeh, Joanne I

2010-11-12

Most mammalian cell types depend on multiple Src family kinases (SFKs) to regulate diverse signaling pathways. Strict control of SFK activity is essential for normal cellular function, and loss of kinase regulation contributes to several forms of cancer and other diseases. Previous x-ray crystal structures of the SFKs c-Src and Hck revealed that intramolecular association of their Src homology (SH) 3 domains and SH2 kinase linker regions has a key role in down-regulation of kinase activity. However, the amino acid sequence of the Hck linker represents a suboptimal ligand for the isolated SH3 domain, suggesting that it may form the polyproline type II helical conformation required for SH3 docking only in the context of the intact structure. To test this hypothesis directly, we determined the crystal structure of a truncated Hck protein consisting of the SH2 and SH3 domains plus the linker. Despite the absence of the kinase domain, the structures and relative orientations of the SH2 and SH3 domains in this shorter protein were very similar to those observed in near full-length, down-regulated Hck. However, the SH2 kinase linker adopted a modified topology and failed to engage the SH3 domain. This new structure supports the idea that these noncatalytic regions work together as a "conformational switch" that modulates kinase activity in a manner unique to the SH3 domain and linker topologies present in the intact Hck protein. Our results also provide fresh structural insight into the facile induction of Hck activity by HIV-1 Nef and other Hck SH3 domain binding proteins and implicate the existence of innate conformational states unique to individual Src family members that "fine-tune" their sensitivities to activation by SH3-based ligands.

Nck-2, a Novel Src Homology2/3-containing Adaptor Protein That Interacts with the LIM-only Protein PINCH and Components of Growth Factor Receptor Kinase-signaling Pathways

PubMed Central

Tu, Yizeng; Li, Fugang; Wu, Chuanyue

1998-01-01

Many of the protein–protein interactions that are essential for eukaryotic intracellular signal transduction are mediated by protein binding modules including SH2, SH3, and LIM domains. Nck is a SH3- and SH2-containing adaptor protein implicated in coordinating various signaling pathways, including those of growth factor receptors and cell adhesion receptors. We report here the identification, cloning, and characterization of a widely expressed, Nck-related adaptor protein termed Nck-2. Nck-2 comprises primarily three N-terminal SH3 domains and one C-terminal SH2 domain. We show that Nck-2 interacts with PINCH, a LIM-only protein implicated in integrin-linked kinase signaling. The PINCH-Nck-2 interaction is mediated by the fourth LIM domain of PINCH and the third SH3 domain of Nck-2. Furthermore, we show that Nck-2 is capable of recognizing several key components of growth factor receptor kinase-signaling pathways including EGF receptors, PDGF receptor-β, and IRS-1. The association of Nck-2 with EGF receptors was regulated by EGF stimulation and involved largely the SH2 domain of Nck-2, although the SH3 domains of Nck-2 also contributed to the complex formation. The association of Nck-2 with PDGF receptor-β was dependent on PDGF activation and was mediated solely by the SH2 domain of Nck-2. Additionally, we have detected a stable association between Nck-2 and IRS-1 that was mediated primarily via the second and third SH3 domain of Nck-2. Thus, Nck-2 associates with PINCH and components of different growth factor receptor-signaling pathways via distinct mechanisms. Finally, we provide evidence indicating that a fraction of the Nck-2 and/or Nck-1 proteins are associated with the cytoskeleton. These results identify a novel Nck-related SH2- and SH3-domain–containing protein and suggest that it may function as an adaptor protein connecting the growth factor receptor-signaling pathways with the integrin-signaling pathways. PMID:9843575

Interactive roles of Ras, insulin receptor substrate-1, and proteins with Src homology-2 domains in insulin signaling in Xenopus oocytes.

PubMed

Chuang, L M; Hausdorff, S F; Myers, M G; White, M F; Birnbaum, M J; Kahn, C R

1994-11-04

Insulin receptor substrate-1 (IRS-1) serves as the major immediate substrate of insulin/insulin-like growth factor (IGF)-1 receptors and following tyrosine phosphorylation binds to specific Src homology-2 (SH2) domain-containing proteins including the p85 subunit of phosphatidylinositol (PI) 3-kinase and GRB2, a molecule believed to link IRS-1 to the Ras pathway. To investigate how these SH2-containing signaling molecules interact to regulate insulin/IGF-1 action, IRS-1, glutathione S-transferase (GST)-SH2 domain fusion proteins and Ras proteins were microinjected into Xenopus oocytes. We found that pleiotropic insulin actions are mediated by IRS-1 through two independent, but convergent, pathways involving PI 3-kinase and GRB2. Thus, microinjection of GST-fusion proteins of either p85 or GRB2 inhibited IRS-1-dependent activation of mitogen-activated protein (MAP) and S6 kinases and oocyte maturation, although only the GST-SH2 of p85 reduced insulin-stimulated PI 3-kinase activation. Co-injection of a dominant negative Ras (S17N) with IRS-1 inhibited insulin-stimulated MAP and S6 kinase activation. Micro-injection of activated [Arg12,Thr59]Ras increased basal MAP and S6 kinase activities and sensitized the oocytes to insulin-stimulated maturation without altering insulin-stimulated PI 3-kinase. The Ras-enhanced oocyte maturation response, but not the elevated basal level of MAP and S6 kinase, was partially blocked by the SH2-p85, but not SH2-GRB2. These data strongly suggest that IRS-1 can mediate many of insulin's actions on cellular enzyme activation and cell cycle progression requires binding and activation of multiple different SH2-domain proteins.

Kinase activation through dimerization by human SH2-B.

PubMed

Nishi, Masahiro; Werner, Eric D; Oh, Byung-Chul; Frantz, J Daniel; Dhe-Paganon, Sirano; Hansen, Lone; Lee, Jongsoon; Shoelson, Steven E

2005-04-01

The isoforms of SH2-B, APS, and Lnk form a family of signaling proteins that have been described as activators, mediators, or inhibitors of cytokine and growth factor signaling. We now show that the three alternatively spliced isoforms of human SH2-B readily homodimerize in yeast two-hybrid and cellular transfections assays, and this is mediated specifically by a unique domain in its amino terminus. Consistent with previous reports, we further show that the SH2 domains of SH2-B and APS bind JAK2 at Tyr813. These findings suggested a model in which two molecules of SH2-B or APS homodimerize with their SH2 domains bound to two JAK2 molecules, creating heterotetrameric JAK2-(SH2-B)2-JAK2 or JAK2-(APS)2-JAK2 complexes. We further show that APS and SH2-B isoforms heterodimerize. At lower levels of SH2-B or APS expression, dimerization approximates two JAK2 molecules to induce transactivation. At higher relative concentrations of SH2-B or APS, kinase activation is blocked. SH2-B or APS homodimerization and SH2-B/APS heterodimerization thus provide direct mechanisms for activating and inhibiting JAK2 and other kinases from the inside of the cell and for potentiating or attenuating cytokine and growth factor receptor signaling when ligands are present.

Novel Autophosphorylation Sites of Src Family Kinases Regulate Kinase Activity and SH2 Domain Binding Capacity

PubMed Central

Weir, Marion E.; Mann, Jacqueline E.; Corwin, Thomas; Fulton, Zachary W.; Hao, Jennifer M.; Maniscalco, Jeanine F.; Kenney, Marie C.; Roque, Kristal M. Roman; Chapdelaine, Elizabeth F.; Stelzl, Ulrich; Deming, Paula B.; Ballif, Bryan A.; Hinkle, Karen L.

2016-01-01

Src family tyrosine kinases (SFKs) are critical players in normal and aberrant biological processes. While phosphorylation importantly-regulates SFKs at two known tyrosines, large-scale phosphoproteomics have revealed four additional tyrosines commonly-phosphorylated in SFKs. We found these novel tyrosines to be autophosphorylation sites. Mimicking phosphorylation at the site C-terminal to the activation loop decreased Fyn activity. Phosphomimetics and direct phosphorylation at the three SH2 domain sites increased Fyn activity while reducing phosphotyrosine-dependent interactions. While 68% of human SH2 domains exhibit conservation of at least one of these tyrosines, few have been found phosphorylated except when found in cis to a kinase domain. PMID:27001024

Thermodynamic dissection of the binding energetics of proline-rich peptides to the Abl-SH3 domain: implications for rational ligand design.

PubMed

Palencia, Andrés; Cobos, Eva S; Mateo, Pedro L; Martínez, Jose C; Luque, Irene

2004-02-13

The inhibition of the interactions between SH3 domains and their targets is emerging as a promising therapeutic strategy. To date, rational design of potent ligands for these domains has been hindered by the lack of understanding of the origins of the binding energy. We present here a complete thermodynamic analysis of the binding energetics of the p41 proline-rich decapeptide (APSYSPPPPP) to the SH3 domain of the c-Abl oncogene. Isothermal titration calorimetry experiments have revealed a thermodynamic signature for this interaction (very favourable enthalpic contributions opposed by an unfavourable binding entropy) inconsistent with the highly hydrophobic nature of the p41 ligand and the Abl-SH3 binding site. Our structural and thermodynamic analyses have led us to the conclusion, having once ruled out any possible ionization events or conformational changes coupled to the association, that the establishment of a complex hydrogen-bond network mediated by water molecules buried at the binding interface is responsible for the observed thermodynamic behaviour. The origin of the binding energetics for proline-rich ligands to the Abl-SH3 domain is further investigated by a comparative calorimetric analysis of a set of p41-related ligands. The striking effects upon the enthalpic and entropic contributions provoked by conservative substitutions at solvent-exposed positions in the ligand confirm the complexity of the interaction. The implications of these results for rational ligand design are discussed.

Unexpected involvement of staple leads to redesign of selective bicyclic peptide inhibitor of Grb7

NASA Astrophysics Data System (ADS)

Gunzburg, Menachem J.; Kulkarni, Ketav; Watson, Gabrielle M.; Ambaye, Nigus D.; Del Borgo, Mark P.; Brandt, Rebecca; Pero, Stephanie C.; Perlmutter, Patrick; Wilce, Matthew C. J.; Wilce, Jacqueline A.

2016-06-01

The design of potent and specific peptide inhibitors to therapeutic targets is of enormous utility for both proof-of-concept studies and for the development of potential new therapeutics. Grb7 is a key signaling molecule in the progression of HER2 positive and triple negative breast cancers. Here we report the crystal structure of a stapled bicyclic peptide inhibitor G7-B1 in complex with the Grb7-SH2 domain. This revealed an unexpected binding mode of the peptide, in which the staple forms an alternative contact with the surface of the target protein. Based on this structural information, we designed a new series of bicyclic G7 peptides that progressively constrain the starting peptide, to arrive at the G7-B4 peptide that binds with an approximately 2-fold enhanced affinity to the Grb7-SH2 domain (KD = 0.83 μM) compared to G7-B1 and shows low affinity binding to Grb2-, Grb10- and Grb14-SH2 domains (KD > 100 μM). Furthermore, we determined the structure of the G7-B4 bicyclic peptide in complex with the Grb7-SH2 domain, both before and after ring closing metathesis to show that the closed staple is essential to the target interaction. The G7-B4 peptide represents an advance in the development of Grb7 inhibitors and is a classical example of structure aided inhibitor development.

Multipoint Binding of the SLP-76 SH2 Domain to ADAP Is Critical for Oligomerization of SLP-76 Signaling Complexes in Stimulated T Cells

PubMed Central

Coussens, Nathan P.; Hayashi, Ryo; Brown, Patrick H.; Balagopalan, Lakshmi; Balbo, Andrea; Akpan, Itoro; Houtman, Jon C. D.; Barr, Valarie A.; Schuck, Peter; Appella, Ettore

2013-01-01

The adapter molecules SLP-76 and LAT play central roles in T cell activation by recruiting enzymes and other adapters into multiprotein complexes that coordinate highly regulated signal transduction pathways. While many of the associated proteins have been characterized, less is known concerning the mechanisms of assembly for these dynamic and potentially heterogeneous signaling complexes. Following T cell receptor (TCR) stimulation, SLP-76 is found in structures called microclusters, which contain many signaling complexes. Previous studies showed that a mutation to the SLP-76 C-terminal SH2 domain nearly abolished SLP-76 microclusters, suggesting that the SH2 domain facilitates incorporation of signaling complexes into microclusters. S. C. Bunnell, A. L. Singer, D. I. Hong, B. H. Jacque, M. S. Jordan, M. C. Seminario, V. A. Barr, G. A. Koretzky, and L. E. Samelson, Mol. Cell. Biol., 26:7155–7166, 2006). Using biophysical methods, we demonstrate that the adapter, ADAP, contains three binding sites for SLP-76, and that multipoint binding to ADAP fragments oligomerizes the SLP-76 SH2 domain in vitro. These results were complemented with confocal imaging and functional studies of cells expressing ADAP with various mutations. Our results demonstrate that all three binding sites are critical for SLP-76 microcluster assembly, but any combination of two sites will partially induce microclusters. These data support a model whereby multipoint binding of SLP-76 to ADAP facilitates the assembly of SLP-76 microclusters. This model has implications for the regulation of SLP-76 and LAT microclusters and, as a result, T cell signaling. PMID:23979596

Multipoint binding of the SLP-76 SH2 domain to ADAP is critical for oligomerization of SLP-76 signaling complexes in stimulated T cells.

PubMed

Coussens, Nathan P; Hayashi, Ryo; Brown, Patrick H; Balagopalan, Lakshmi; Balbo, Andrea; Akpan, Itoro; Houtman, Jon C D; Barr, Valarie A; Schuck, Peter; Appella, Ettore; Samelson, Lawrence E

2013-11-01

The adapter molecules SLP-76 and LAT play central roles in T cell activation by recruiting enzymes and other adapters into multiprotein complexes that coordinate highly regulated signal transduction pathways. While many of the associated proteins have been characterized, less is known concerning the mechanisms of assembly for these dynamic and potentially heterogeneous signaling complexes. Following T cell receptor (TCR) stimulation, SLP-76 is found in structures called microclusters, which contain many signaling complexes. Previous studies showed that a mutation to the SLP-76 C-terminal SH2 domain nearly abolished SLP-76 microclusters, suggesting that the SH2 domain facilitates incorporation of signaling complexes into microclusters. S. C. Bunnell, A. L. Singer, D. I. Hong, B. H. Jacque, M. S. Jordan, M. C. Seminario, V. A. Barr, G. A. Koretzky, and L. E. Samelson, Mol. Cell. Biol., 26:7155-7166, 2006). Using biophysical methods, we demonstrate that the adapter, ADAP, contains three binding sites for SLP-76, and that multipoint binding to ADAP fragments oligomerizes the SLP-76 SH2 domain in vitro. These results were complemented with confocal imaging and functional studies of cells expressing ADAP with various mutations. Our results demonstrate that all three binding sites are critical for SLP-76 microcluster assembly, but any combination of two sites will partially induce microclusters. These data support a model whereby multipoint binding of SLP-76 to ADAP facilitates the assembly of SLP-76 microclusters. This model has implications for the regulation of SLP-76 and LAT microclusters and, as a result, T cell signaling.

Structure-based characterization of the binding of peptide to the human endophilin-1 Src homology 3 domain using position-dependent noncovalent potential analysis.

PubMed

Fu, Chunjiang; Wu, Gang; Lv, Fenglin; Tian, Feifei

2012-05-01

Many protein-protein interactions are mediated by a peptide-recognizing domain, such as WW, PDZ, or SH3. In the present study, we describe a new method called position-dependent noncovalent potential analysis (PDNPA), which can accurately characterize the nonbonding profile between the human endophilin-1 Src homology 3 (hEndo1 SH3) domain and its peptide ligands and quantitatively predict the binding affinity of peptide to hEndo1 SH3. In this procedure, structure models of diverse peptides in complex with the hEndo1 SH3 domain are constructed by molecular dynamics simulation and a virtual mutagenesis protocol. Subsequently, three noncovalent interactions associated with each position of the peptide ligand in the complexed state are analyzed using empirical potential functions, and the resulting potential descriptors are then correlated with the experimentally measured affinity on the basis of 1997 hEndo1 SH3-binding peptides with known activities, using linear partial least squares regression (PLS) and the nonlinear support vector machine (SVM). The results suggest that: (i) the electrostatics appears to be more important than steric properties and hydrophobicity in the formation of the hEndo1 SH3-peptide complex; (ii) P(-4) of the core decapeptide ligand with the sequence pattern P(-6)P(-5)P(-4)P(-3)P(-2)P(-1)P(0)P(1)P(2)P(3) is the most important position in terms of determining both the stability and specificity of the architecture of the complex, and; (iii) nonlinear SVM appears to be more effective than linear PLS for accurately predicting the binding affinity of a peptide ligand to hEndo1 SH3, whereas PLS models are straightforward and easy to interpret as compared to those built by SVM.

Simian Immunodeficiency Virus and Human Immunodeficiency Virus Type 1 Nef Proteins Show Distinct Patterns and Mechanisms of Src Kinase Activation

PubMed Central

Greenway, Alison L.; Dutartre, Hélène; Allen, Kelly; McPhee, Dale A.; Olive, Daniel; Collette, Yves

1999-01-01

The nef gene from human and simian immunodeficiency viruses (HIV and SIV) regulates cell function and viral replication, possibly through binding of the nef product to cellular proteins, including Src family tyrosine kinases. We show here that the Nef protein encoded by SIVmac239 interacts with and also activates the human Src kinases Lck and Hck. This is in direct contrast to the inhibitory effect of HIV type 1 (HIV-1) Nef on Lck catalytic activity. Unexpectedly, however, the interaction of SIV Nef with human Lck or Hck is not mediated via its consensus proline motif, which is known to mediate HIV-1 Nef binding to Src homology 3 (SH3) domains, and various experimental analyses failed to show significant interaction of SIV Nef with the SH3 domain of either kinase. Instead, SIV Nef can bind Lck and Hck SH2 domains, and its N-terminal 50 amino acid residues are sufficient for Src kinase binding and activation. Our results provide evidence for multiple mechanisms by which Nef binds to and regulates Src kinases. PMID:10364375

Microscopic insight into thermodynamics of conformational changes of SAP-SLAM complex in signal transduction cascade

NASA Astrophysics Data System (ADS)

Samanta, Sudipta; Mukherjee, Sanchita

2017-04-01

The signalling lymphocytic activation molecule (SLAM) family of receptors, expressed by an array of immune cells, associate with SLAM-associated protein (SAP)-related molecules, composed of single SH2 domain architecture. SAP activates Src-family kinase Fyn after SLAM ligation, resulting in a SLAM-SAP-Fyn complex, where, SAP binds the Fyn SH3 domain that does not involve canonical SH3 or SH2 interactions. This demands insight into this SAP mediated signalling cascade. Thermodynamics of the conformational changes are extracted from the histograms of dihedral angles obtained from the all-atom molecular dynamics simulations of this structurally well characterized SAP-SLAM complex. The results incorporate the binding induced thermodynamic changes of individual amino acid as well as the secondary structural elements of the protein and the solvent. Stabilization of the peptide partially comes through a strong hydrogen bonding network with the protein, while hydrophobic interactions also play a significant role where the peptide inserts itself into a hydrophobic cavity of the protein. SLAM binding widens SAP's second binding site for Fyn, which is the next step in the signal transduction cascade. The higher stabilization and less fluctuation of specific residues of SAP in the Fyn binding site, induced by SAP-SLAM complexation, emerge as the key structural elements to trigger the recognition of SAP by the SH3 domain of Fyn. The thermodynamic quantification of the protein due to complexation not only throws deeper understanding in the established mode of SAP-SLAM interaction but also assists in the recognition of the relevant residues of the protein responsible for alterations in its activity.

Microscopic insight into thermodynamics of conformational changes of SAP-SLAM complex in signal transduction cascade.

PubMed

Samanta, Sudipta; Mukherjee, Sanchita

2017-04-28

The signalling lymphocytic activation molecule (SLAM) family of receptors, expressed by an array of immune cells, associate with SLAM-associated protein (SAP)-related molecules, composed of single SH2 domain architecture. SAP activates Src-family kinase Fyn after SLAM ligation, resulting in a SLAM-SAP-Fyn complex, where, SAP binds the Fyn SH3 domain that does not involve canonical SH3 or SH2 interactions. This demands insight into this SAP mediated signalling cascade. Thermodynamics of the conformational changes are extracted from the histograms of dihedral angles obtained from the all-atom molecular dynamics simulations of this structurally well characterized SAP-SLAM complex. The results incorporate the binding induced thermodynamic changes of individual amino acid as well as the secondary structural elements of the protein and the solvent. Stabilization of the peptide partially comes through a strong hydrogen bonding network with the protein, while hydrophobic interactions also play a significant role where the peptide inserts itself into a hydrophobic cavity of the protein. SLAM binding widens SAP's second binding site for Fyn, which is the next step in the signal transduction cascade. The higher stabilization and less fluctuation of specific residues of SAP in the Fyn binding site, induced by SAP-SLAM complexation, emerge as the key structural elements to trigger the recognition of SAP by the SH3 domain of Fyn. The thermodynamic quantification of the protein due to complexation not only throws deeper understanding in the established mode of SAP-SLAM interaction but also assists in the recognition of the relevant residues of the protein responsible for alterations in its activity.

Structure-based design of competitive ligands to target Spon2 in gastric cancer: An integration of molecular modeling and in vitro assay.

PubMed

Xu, Zhenglei; Yu, Zhichao; Nai, Shumei; Shi, Ruiyue; Tang, Qinhong; Zhang, Haiyang; Ye, Lijuan; Wang, Lisheng; Hong, Yincai

2017-10-01

Spon2 is a proto-oncogene matrix protein that plays an essential role in the tumorigenesis and metastasis of gastric cancer. The protein has recently been found to function as a guanine nucleotide exchange factor through the activation of RhoGTPase. Here, computational modeling and bioinformatics analysis were employed to investigate the molecular mechanism and biological implication underlying Spon2 autoinhibition. It is revealed that the binding of PxxP motif to SH domain can stabilize the intramolecular interaction between the N-terminal helix and DH domain of Spon2, thus shifting the protein into an autoinhibitory state. Here, we proposed releasing Spon2 autoinhibition by targeting SH domain with competitive peptide ligands. To verify this notion, the PxxP sequence was adopted as the start to derive an array of efficient SH binders by using a structure-based rational design strategy, which were then substantiated with fluorescence spectroscopy analysis and guanine nucleotide exchange test. Consequently, the obtained peptide ligands were determined to have a moderate or high affinity for SH domain; they can also enhance Spon2 exchange activity by 1.2-6.1 folds, exhibiting a significant correlation with their SH-binding affinity (Pearson's coefficient=0.92). In addition, neutral substitution of conserved residues in a high-affinity peptide ligand can largely reduce its Spon2-activating potency, confirming that the designed peptide activates Spon2 by competitively disrupting SH-PxxP interaction. Copyright © 2017 Elsevier Inc. All rights reserved.

Identification of Tyrosine Phosphorylated Proteins by SH2 Domain Affinity Purification and Mass Spectrometry.

PubMed

Buhs, Sophia; Gerull, Helwe; Nollau, Peter

2017-01-01

Phosphotyrosine signaling plays a major role in the control of many important biological functions such as cell proliferation and apoptosis. Deciphering of phosphotyrosine-dependent signaling is therefore of great interest paving the way for the understanding of physiological and pathological processes of signal transduction. On the basis of the specific binding of SH2 domains to phosphotyrosine residues, we here present an experimental workflow for affinity purification and subsequent identification of tyrosine phosphorylated proteins by mass spectrometry. In combination with SH2 profiling, a broadly applicable platform for the characterization of phosphotyrosine profiles in cell extracts, our pull down strategy enables researchers by now to identify proteins in signaling cascades which are differentially phosphorylated and selectively recognized by distinct SH2 domains.

The SLP-76 SH2 domain is required for T cell development and activation

PubMed Central

Burns, Jeremy C.; Corbo, Evann; Degen, Janine; Gohil, Mercy; Anterasian, Christine; Schraven, Burkart; Koretzky, Gary A.; Kliche, Stefanie; Jordan, Martha S.

2011-01-01

The adaptor protein Src homology 2 (SH2) domain containing leukocyte protein of 76 kDa (SLP-76) is critical for multiple aspects of T cell development and function. Through its protein-binding domains, SLP-76 serves as a platform for the assembly of multiple enzymes and adaptor proteins that function together to activate second messengers required for TCR signal propagation. The N-terminus of SLP-76, which contains three tyrosines that serve as docking sites for SH2 domain-containing proteins, and the central proline-rich region of SLP-76 have been well studied and are known to be important for both thymocyte selection and activation of peripheral T cells. Less is known about the function of the C-terminal SH2 domain of SLP-76. This region inducibly associates with the adhesion- and degranulation-promoting adaptor protein (ADAP) and hematopoietic progenitor kinase 1 (HPK1). Combining regulated deletion of endogenous SLP-76 with transgenic expression of a SLP-76 SH2 domain mutant, we demonstrate that the SLP-76 SH2 domain is required for peripheral T cell activation and positive selection of thymocytes, a function not previously attributed to this region. This domain is also important for T cell proliferation, IL-2 production and phosphorylation of protein kinase D (PKD) and IκB. ADAP-deficient T cells display similar, but in some cases less severe, defects despite phosphorylation of a negative regulatory site on SLP-76 by HPK1, a function that is lost in SLP-76 SH2 domain mutant T cells. PMID:21949020

Contributions of F-BAR and SH2 Domains of Fes Protein Tyrosine Kinase for Coupling to the FcɛRI Pathway in Mast Cells▿ †

PubMed Central

McPherson, Victor A.; Everingham, Stephanie; Karisch, Robert; Smith, Julie A.; Udell, Christian M.; Zheng, Jimin; Jia, Zongchao; Craig, Andrew W. B.

2009-01-01

This study investigates the roles of Fer-CIP4 homology (FCH)-Bin/amphiphysin/Rvs (F-BAR) and SH2 domains of Fes protein tyrosine kinase in regulating its activation and signaling downstream of the high-affinity immunoglobulin G (IgE) receptor (FcɛRI) in mast cells. Homology modeling of the Fes F-BAR domain revealed conservation of some basic residues implicated in phosphoinositide binding (R113/K114). The Fes F-BAR can bind phosphoinositides and induce tubulation of liposomes in vitro. Mutation of R113/K114 to uncharged residues (RK/QQ) caused a significant reduction in phosphoinositide binding in vitro and a more diffuse cytoplasmic localization in transfected COS-7 cells. RBL-2H3 mast cells expressing full-length Fes carrying the RK/QQ mutation show defects in FcɛRI-induced Fes tyrosine phosphorylation and degranulation compared to cells expressing wild-type Fes. This correlated with reduced localization to Lyn kinase-containing membrane fractions for the RK/QQ mutant compared to wild-type Fes in mast cells. The Fes SH2 domain also contributes to Fes signaling in mast cells, via interactions with the phosphorylated FcɛRI β chain and the actin regulatory protein HS1. We show that Fes phosphorylates C-terminal tyrosine residues in HS1 implicated in actin stabilization. Thus, coordinated actions of the F-BAR and SH2 domains of Fes allow for coupling to FcɛRI signaling and potential regulation the actin reorganization in mast cells. PMID:19001085

Contributions of F-BAR and SH2 domains of Fes protein tyrosine kinase for coupling to the FcepsilonRI pathway in mast cells.

PubMed

McPherson, Victor A; Everingham, Stephanie; Karisch, Robert; Smith, Julie A; Udell, Christian M; Zheng, Jimin; Jia, Zongchao; Craig, Andrew W B

2009-01-01

This study investigates the roles of Fer-CIP4 homology (FCH)-Bin/amphiphysin/Rvs (F-BAR) and SH2 domains of Fes protein tyrosine kinase in regulating its activation and signaling downstream of the high-affinity immunoglobulin G (IgE) receptor (FcepsilonRI) in mast cells. Homology modeling of the Fes F-BAR domain revealed conservation of some basic residues implicated in phosphoinositide binding (R113/K114). The Fes F-BAR can bind phosphoinositides and induce tubulation of liposomes in vitro. Mutation of R113/K114 to uncharged residues (RK/QQ) caused a significant reduction in phosphoinositide binding in vitro and a more diffuse cytoplasmic localization in transfected COS-7 cells. RBL-2H3 mast cells expressing full-length Fes carrying the RK/QQ mutation show defects in FcepsilonRI-induced Fes tyrosine phosphorylation and degranulation compared to cells expressing wild-type Fes. This correlated with reduced localization to Lyn kinase-containing membrane fractions for the RK/QQ mutant compared to wild-type Fes in mast cells. The Fes SH2 domain also contributes to Fes signaling in mast cells, via interactions with the phosphorylated FcepsilonRI beta chain and the actin regulatory protein HS1. We show that Fes phosphorylates C-terminal tyrosine residues in HS1 implicated in actin stabilization. Thus, coordinated actions of the F-BAR and SH2 domains of Fes allow for coupling to FcepsilonRI signaling and potential regulation the actin reorganization in mast cells.

The Gab1 protein is a docking site for multiple proteins involved in signaling by the B cell antigen receptor.

PubMed

Ingham, R J; Holgado-Madruga, M; Siu, C; Wong, A J; Gold, M R

1998-11-13

Gab1 is a member of the docking/scaffolding protein family which includes IRS-1, IRS-2, c-Cbl, p130(cas), and p62(dok). These proteins contain a variety of protein-protein interaction motifs including multiple tyrosine residues that when phosphorylated can act as binding sites for Src homology 2 (SH2) domain-containing signaling proteins. We show in the RAMOS human B cell line that Gab1 is tyrosine-phosphorylated in response to B cell antigen receptor (BCR) engagement. Moreover, tyrosine phosphorylation of Gab1 correlated with the binding of several SH2-containing signaling proteins to Gab1 including Shc, Grb2, phosphatidylinositol 3-kinase, and the SHP-2 tyrosine phosphatase. Far Western analysis showed that the SH2 domains of Shc, SHP-2, and the p85 subunit of phosphatidylinositol 3-kinase could bind directly to tyrosine-phosphorylated Gab1 isolated from activated RAMOS cells. In contrast, the Grb2 SH2 domain did not bind directly to Gab1 but instead to the Shc and SHP-2 associated with Gab1. We also show that Gab1 is present in the membrane-enriched particulate fraction of RAMOS cells and that Gab1/signaling protein complexes are found in this fraction after BCR engagement. Thus, tyrosine-phosphorylated Gab1 may recruit cytosolic signaling proteins to cellular membranes where they can act on membrane-bound targets. This may be a critical step in the activation of multiple BCR signaling pathways.

Structural basis for recognition of the T cell adaptor protein SLP-76 by the SH3 domain of phospholipase Cgamma1.

PubMed

Deng, Lu; Velikovsky, C Alejandro; Swaminathan, Chittoor P; Cho, Sangwoo; Mariuzza, Roy A

2005-09-09

The enzyme phospholipase Cgamma1 (PLCgamma1) is essential for T cell signaling and activation. Following T cell receptor ligation, PLCgamma1 interacts through its SH2 and SH3 domains with the adaptors LAT and SLP-76, respectively, to form a multiprotein signaling complex that leads to activation of PLCgamma1 by Syk tyrosine kinases. To identify the binding site for PLCgamma1 in SLP-76, we used isothermal titration calorimetry to measure affinities for the interaction of PLCgamma1-SH3 with a set of overlapping peptides spanning the central proline-rich region of SLP-76. PLCgamma1-SH3 bound with high specificity to the SLP-76 motif 186PPVPPQRP193, which represents the minimal binding site. To understand the basis for selective recognition, we determined the crystal structures of PLCgamma1-SH3 in free form, and bound to a 10-mer peptide containing this site, to resolutions of 1.60 A and 1.81 A, respectively. The structures reveal that several key contacting residues of the SH3 shift toward the SLP-76 peptide upon complex formation, optimizing the fit and strengthening hydrophobic interactions. Selectivity results mainly from strict shape complementarity between protein and peptide, rather than sequence-specific hydrogen bonding. In addition, Pro193 of SLP-76 assists in positioning Arg192 into the compass pocket of PLCgamma1-SH3, which coordinates the compass residue through an unusual aspartate. The PLCgamma1-SH3/SLP-76 structure provides insights into ligand binding by SH3 domains related to PLCgamma1-SH3, as well as into recognition by PLCgamma1 of signaling partners other than SLP-76.

The SLP-76 Src homology 2 domain is required for T cell development and activation.

PubMed

Burns, Jeremy C; Corbo, Evann; Degen, Janine; Gohil, Mercy; Anterasian, Christine; Schraven, Burkart; Koretzky, Gary A; Kliche, Stefanie; Jordan, Martha S

2011-11-01

The adapter protein Src homology 2 (SH2) domain-containing leukocyte protein of 76 kDa (SLP-76) is critical for multiple aspects of T cell development and function. Through its protein-binding domains, SLP-76 serves as a platform for the assembly of multiple enzymes and adapter proteins that function together to activate second messengers required for TCR signal propagation. The N terminus of SLP-76, which contains three tyrosines that serve as docking sites for SH2 domain-containing proteins, and the central proline-rich region of SLP-76 have been well studied and are known to be important for both thymocyte selection and activation of peripheral T cells. Less is known about the function of the C-terminal SH2 domain of SLP-76. This region inducibly associates with ADAP and HPK1. Combining regulated deletion of endogenous SLP-76 with transgenic expression of a SLP-76 SH2 domain mutant, we demonstrate that the SLP-76 SH2 domain is required for peripheral T cell activation and positive selection of thymocytes, a function not previously attributed to this region. This domain is also important for T cell proliferation, IL-2 production, and phosphorylation of protein kinase D and IκB. ADAP-deficient T cells display similar, but in some cases less severe, defects despite phosphorylation of a negative regulatory site on SLP-76 by HPK1, a function that is lost in SLP-76 SH2 domain mutant T cells.

Conserved interdomain linker promotes phase separation of the multivalent adaptor protein Nck

PubMed Central

Banjade, Sudeep; Wu, Qiong; Mittal, Anuradha; Peeples, William B.; Pappu, Rohit V.; Rosen, Michael K.

2015-01-01

The organization of membranes, the cytosol, and the nucleus of eukaryotic cells can be controlled through phase separation of lipids, proteins, and nucleic acids. Collective interactions of multivalent molecules mediated by modular binding domains can induce gelation and phase separation in several cytosolic and membrane-associated systems. The adaptor protein Nck has three SRC-homology 3 (SH3) domains that bind multiple proline-rich segments in the actin regulatory protein neuronal Wiskott-Aldrich syndrome protein (N-WASP) and an SH2 domain that binds to multiple phosphotyrosine sites in the adhesion protein nephrin, leading to phase separation. Here, we show that the 50-residue linker between the first two SH3 domains of Nck enhances phase separation of Nck/N-WASP/nephrin assemblies. Two linear motifs within this element, as well as its overall positively charged character, are important for this effect. The linker increases the driving force for self-assembly of Nck, likely through weak interactions with the second SH3 domain, and this effect appears to promote phase separation. The linker sequence is highly conserved, suggesting that the sequence determinants of the driving forces for phase separation may be generally important to Nck functions. Our studies demonstrate that linker regions between modular domains can contribute to the driving forces for self-assembly and phase separation of multivalent proteins. PMID:26553976

Novel autophosphorylation sites of Src family kinases regulate kinase activity and SH2 domain-binding capacity.

PubMed

Weir, Marion E; Mann, Jacqueline E; Corwin, Thomas; Fulton, Zachary W; Hao, Jennifer M; Maniscalco, Jeanine F; Kenney, Marie C; Roman Roque, Kristal M; Chapdelaine, Elizabeth F; Stelzl, Ulrich; Deming, Paula B; Ballif, Bryan A; Hinkle, Karen L

2016-04-01

Src family tyrosine kinases (SFKs) are critical players in normal and aberrant biological processes. While phosphorylation importantly regulates SFKs at two known tyrosines, large-scale phosphoproteomics have revealed four additional tyrosines commonly phosphorylated in SFKs. We found these novel tyrosines to be autophosphorylation sites. Mimicking phosphorylation at the C-terminal site to the activation loop decreased Fyn activity. Phosphomimetics and direct phosphorylation at the three SH2 domain sites increased Fyn activity while reducing phosphotyrosine-dependent interactions. While 68% of human SH2 domains exhibit conservation of at least one of these tyrosines, few have been found phosphorylated except when found in cis to a kinase domain. © 2016 Federation of European Biochemical Societies.

Insights into substrate specificity of NlpC/P60 cell wall hydrolases containing bacterial SH3 domains

DOE PAGES

Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.; ...

2015-09-15

Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. In addition, these enzymes all have γ-d-Glu-A 2pm (A 2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structuremore » consisting of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation.Peptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling enzymes, show that they may have evolved from a common molecular architecture, where the substrate preference is modulated by local changes. These results also suggest that new pathways for recycling PG turnover products, such as tracheal cytotoxin, may have evolved in bacteria in the human gut microbiome that involve NlpC/P60 cell wall hydrolases.« less

Insights into substrate specificity of NlpC/P60 cell wall hydrolases containing bacterial SH3 domains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.

Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. In addition, these enzymes all have γ-d-Glu-A 2pm (A 2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structuremore » consisting of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation.Peptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling enzymes, show that they may have evolved from a common molecular architecture, where the substrate preference is modulated by local changes. These results also suggest that new pathways for recycling PG turnover products, such as tracheal cytotoxin, may have evolved in bacteria in the human gut microbiome that involve NlpC/P60 cell wall hydrolases.« less

Insights into Substrate Specificity of NlpC/P60 Cell Wall Hydrolases Containing Bacterial SH3 Domains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.

ABSTRACT Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. These enzymes all have γ-d-Glu-A 2pm (A 2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structure consistingmore » of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation. IMPORTANCEPeptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling enzymes, show that they may have evolved from a common molecular architecture, where the substrate preference is modulated by local changes. These results also suggest that new pathways for recycling PG turnover products, such as tracheal cytotoxin, may have evolved in bacteria in the human gut microbiome that involve NlpC/P60 cell wall hydrolases.« less

HCV NS5A protein containing potential ligands for both Src homology 2 and 3 domains enhances autophosphorylation of Src family kinase Fyn in B cells.

PubMed

Nakashima, Kenji; Takeuchi, Kenji; Chihara, Kazuyasu; Horiguchi, Tomoko; Sun, Xuedong; Deng, Lin; Shoji, Ikuo; Hotta, Hak; Sada, Kiyonao

2012-01-01

Hepatitis C virus (HCV) infects B lymphocytes and induces mixed cryoglobulinemia and B cell non-Hodgkin's lymphoma. The molecular mechanism for the pathogenesis of HCV infection-mediated B cell disorders remains obscure. To identify the possible role for HCV nonstructural 5A (NS5A) protein in B cells, we generated the stable B cell lines expressing Myc-His tagged NS5A. Immunoprecipitation study in the presence or absence of pervanadate (PV) implied that NS5A was tyrosine phosphorylated by pervanadate (PV) treatment of the cells. Therefore we examined pull-down assay by using glutathione S-transferase (GST)-fusion proteins of various Src homology 2 (SH2) domains, which associates with phosphotyrosine within a specific amino acid sequence. The results showed that NS5A specifically bound to SH2 domain of Fyn from PV-treated B cells in addition to Src homology 3 (SH3) domain. Substitution of Arg(176) to Lys in the SH2 domain of Fyn abrogated this interaction. Deletion mutational analysis demonstrated that N-terminal region of NS5A was not required for the interaction with the SH2 domain of Fyn. Tyr(334) was identified as a tyrosine phosphorylation site in NS5A. Far-western analysis revealed that SH2 domain of Fyn directly bound to NS5A. Fyn and NS5A were colocalized in the lipid raft. These results suggest that NS5A directly binds to the SH2 domain of Fyn in a tyrosine phosphorylation-dependent manner. Lastly, we showed that the expression of NS5A in B cells increased phosphorylation of activation loop tyrosine in the kinase domain of Fyn. NS5A containing ligand for both SH2 and SH3 domains enhances an aberrant autophosphorylation and kinase activity of Fyn in B cells.

Binding preference of carbon nanotube over proline-rich motif ligand on SH3-domain: a comparison with different force fields.

PubMed

Shi, Biyun; Zuo, Guanghong; Xiu, Peng; Zhou, Ruhong

2013-04-04

With the widespread applications of nanomaterials such as carbon nanotubes, there is a growing concern on the biosafety of these engineered nanoparticles, in particular their interactions with proteins. In molecular simulations of nanoparticle-protein interactions, the choice of empirical parameters (force fields) plays a decisive role, and thus is of great importance and should be examined carefully before wider applications. Here we compare three commonly used force fields, CHARMM, OPLSAA, and AMBER in study of the competitive binding of a single wall carbon nanotube (SWCNT) with a native proline-rich motif (PRM) ligand on its target protein SH3 domain, a ubiquitous protein-protein interaction mediator involved in signaling and regulatory pathways. We find that the SWCNT displays a general preference over the PRM in binding with SH3 domain in all the three force fields examined, although the degree of preference can be somewhat different, with the AMBER force field showing the highest preference. The SWCNT prevents the ligand from reaching its native binding pocket by (i) occupying the binding pocket directly, and (ii) binding with the ligand itself and then being trapped together onto some off-sites. The π-π stacking interactions between the SWCNT and aromatic residues are found to play a significant role in its binding to the SH3 domain in all the three force fields. Further analyses show that even the SWCNT-ligand binding can also be relatively more stable than the native ligand-protein binding, indicating a serious potential disruption to the protein SH3 function.

Binding of the Ras activator son of sevenless to insulin receptor substrate-1 signaling complexes.

PubMed

Baltensperger, K; Kozma, L M; Cherniack, A D; Klarlund, J K; Chawla, A; Banerjee, U; Czech, M P

1993-06-25

Signal transmission by insulin involves tyrosine phosphorylation of a major insulin receptor substrate (IRS-1) and exchange of Ras-bound guanosine diphosphate for guanosine triphosphate. Proteins containing Src homology 2 and 3 (SH2 and SH3) domains, such as the p85 regulatory subunit of phosphatidylinositol-3 kinase and growth factor receptor-bound protein 2 (GRB2), bind tyrosine phosphate sites on IRS-1 through their SH2 regions. Such complexes in COS cells were found to contain the heterologously expressed putative guanine nucleotide exchange factor encoded by the Drosophila son of sevenless gene (dSos). Thus, GRB2, p85, or other proteins with SH2-SH3 adapter sequences may link Sos proteins to IRS-1 signaling complexes as part of the mechanism by which insulin activates Ras.

T cell specific adaptor protein (TSAd) promotes interaction of Nck with Lck and SLP-76 in T cells.

PubMed

Hem, Cecilie Dahl; Sundvold-Gjerstad, Vibeke; Granum, Stine; Koll, Lise; Abrahamsen, Greger; Buday, Laszlo; Spurkland, Anne

2015-07-11

The Lck and Src binding adaptor protein TSAd (T cell specific adaptor) regulates actin polymerization in T cells and endothelial cells. The molecular details as to how TSAd regulates this process remain to be elucidated. To identify novel interaction partners for TSAd, we used a scoring matrix-assisted ligand algorithm (SMALI), and found that the Src homology 2 (SH2) domain of the actin regulator Non-catalytic region of tyrosine kinase adaptor protein (Nck) potentially binds to TSAd phosphorylated on Tyr(280) (pTyr(280)) and pTyr(305). These predictions were confirmed by peptide array analysis, showing direct binding of recombinant Nck SH2 to both pTyr(280) and pTyr(305) on TSAd. In addition, the SH3 domains of Nck interacted with the proline rich region (PRR) of TSAd. Pull-down and immunoprecipitation experiments further confirmed the Nck-TSAd interactions through Nck SH2 and SH3 domains. In line with this Nck and TSAd co-localized in Jurkat cells as assessed by confocal microscopy and imaging flow cytometry. Co-immunoprecipitation experiments in Jurkat TAg cells lacking TSAd revealed that TSAd promotes interaction of Nck with Lck and SLP-76, but not Vav1. TSAd expressing Jurkat cells contained more polymerized actin, an effect dependent on TSAd exon 7, which includes interactions sites for both Nck and Lck. TSAd binds to and co-localizes with Nck. Expression of TSAd increases both Nck-Lck and Nck-SLP-76 interaction in T cells. Recruitment of Lck and SLP-76 to Nck by TSAd could be one mechanism by which TSAd promotes actin polymerization in activated T cells.

Quantifying protein-protein interactions in high throughput using protein domain microarrays.

PubMed

Kaushansky, Alexis; Allen, John E; Gordus, Andrew; Stiffler, Michael A; Karp, Ethan S; Chang, Bryan H; MacBeath, Gavin

2010-04-01

Protein microarrays provide an efficient way to identify and quantify protein-protein interactions in high throughput. One drawback of this technique is that proteins show a broad range of physicochemical properties and are often difficult to produce recombinantly. To circumvent these problems, we have focused on families of protein interaction domains. Here we provide protocols for constructing microarrays of protein interaction domains in individual wells of 96-well microtiter plates, and for quantifying domain-peptide interactions in high throughput using fluorescently labeled synthetic peptides. As specific examples, we will describe the construction of microarrays of virtually every human Src homology 2 (SH2) and phosphotyrosine binding (PTB) domain, as well as microarrays of mouse PDZ domains, all produced recombinantly in Escherichia coli. For domains that mediate high-affinity interactions, such as SH2 and PTB domains, equilibrium dissociation constants (K(D)s) for their peptide ligands can be measured directly on arrays by obtaining saturation binding curves. For weaker binding domains, such as PDZ domains, arrays are best used to identify candidate interactions, which are then retested and quantified by fluorescence polarization. Overall, protein domain microarrays provide the ability to rapidly identify and quantify protein-ligand interactions with minimal sample consumption. Because entire domain families can be interrogated simultaneously, they provide a powerful way to assess binding selectivity on a proteome-wide scale and provide an unbiased perspective on the connectivity of protein-protein interaction networks.

An SH2 domain-based tyrosine kinase assay using biotin ligase modified with a terbium(III) complex.

PubMed

Sueda, Shinji; Shinboku, Yuki; Kusaba, Takeshi

2013-01-01

Src homology 2 (SH2) domains are modules of approximately 100 amino acids and are known to bind phosphotyrosine-containing sequences with high affinity and specificity. In the present work, we developed an SH2 domain-based assay for Src tyrosine kinase using a unique biotinylation reaction from archaeon Sulfolobus tokodaii. S. tokodaii biotinylation has a unique property that biotin protein ligase (BPL) forms a stable complex with its biotinylated substrate protein (BCCP). Here, an SH2 domain from lymphocyte-specific tyrosine kinase was genetically fused to a truncated BCCP, and the resulting fusion protein was labeled through biotinylation with BPL carrying multiple copies of a luminescent Tb(3+) complex. The labeled SH2 fusion proteins were employed to detect a phosphorylated peptide immobilized on the surface of the microtiter plate, where the phosphorylated peptide was produced by phosphorylation to the substrate peptide by Src tyrosine kinase. Our assay allows for a reliable determination of the activity of Src kinase lower than 10 pg/μL by a simple procedure.

Nck-Interacting Ste20 Kinase Couples Eph Receptors to c-Jun N-Terminal Kinase and Integrin Activation

PubMed Central

Becker, Elena; Huynh-Do, Uyen; Holland, Sacha; Pawson, Tony; Daniel, Tom O.; Skolnik, Edward Y.

2000-01-01

The mammalian Ste20 kinase Nck-interacting kinase (NIK) specifically activates the c-Jun amino-terminal kinase (JNK) mitogen-activated protein kinase module. NIK also binds the SH3 domains of the SH2/SH3 adapter protein Nck. To determine whether Nck functions as an adapter to couple NIK to a receptor tyrosine kinase signaling pathway, we determined whether NIK is activated by Eph receptors (EphR). EphRs constitute the largest family of receptor tyrosine kinases (RTK), and members of this family play important roles in patterning of the nervous and vascular systems. In this report, we show that NIK kinase activity is specifically increased in cells stimulated by two EphRs, EphB1 and EphB2. EphB1 kinase activity and phosphorylation of a juxtamembrane tyrosine (Y594), conserved in all Eph receptors, are both critical for NIK activation by EphB1. Although pY594 in the EphB1R has previously been shown to bind the SH2 domain of Nck, we found that stimulation of EphB1 and EphB2 led predominantly to a complex between NIK/Nck, p62dok, RasGAP, and an unidentified 145-kDa tyrosine-phosphorylated protein. Tyrosine-phosphorylated p62dok most probably binds directly to the SH2 domain of Nck and RasGAP and indirectly to NIK bound to the SH3 domain of Nck. We found that NIK activation is also critical for coupling EphB1R to biological responses that include the activation of integrins and JNK by EphB1. Taken together, these findings support a model in which the recruitment of the Ste20 kinase NIK to phosphotyrosine-containing proteins by Nck is an important proximal step in the signaling cascade downstream of EphRs. PMID:10669731

Elucidation of the binding preferences of peptide recognition modules: SH3 and PDZ domains.

PubMed

Teyra, Joan; Sidhu, Sachdev S; Kim, Philip M

2012-08-14

Peptide-binding domains play a critical role in regulation of cellular processes by mediating protein interactions involved in signalling. In recent years, the development of large-scale technologies has enabled exhaustive studies on the peptide recognition preferences for a number of peptide-binding domain families. These efforts have provided significant insights into the binding specificities of these modular domains. Many research groups have taken advantage of this unprecedented volume of specificity data and have developed a variety of new algorithms for the prediction of binding specificities of peptide-binding domains and for the prediction of their natural binding targets. This knowledge has also been applied to the design of synthetic peptide-binding domains in order to rewire protein-protein interaction networks. Here, we describe how these experimental technologies have impacted on our understanding of peptide-binding domain specificities and on the elucidation of their natural ligands. We discuss SH3 and PDZ domains as well characterized examples, and we explore the feasibility of expanding high-throughput experiments to other peptide-binding domains. Copyright © 2012. Published by Elsevier B.V.

Molecular Mechanisms of SH2- and PTB-Domain-Containing Proteins in Receptor Tyrosine Kinase Signaling

PubMed Central

Wagner, Melany J.; Stacey, Melissa M.; Liu, Bernard A.; Pawson, Tony

2013-01-01

Intracellular signaling is mediated by reversible posttranslational modifications (PTMs) that include phosphorylation, ubiquitination, and acetylation, among others. In response to extracellular stimuli such as growth factors, receptor tyrosine kinases (RTKs) typically dimerize and initiate signaling through phosphorylation of their cytoplasmic tails and downstream scaffolds. Signaling effectors are recruited to these phosphotyrosine (pTyr) sites primarily through Src homology 2 (SH2) domains and pTyr-binding (PTB) domains. This review describes how these conserved domains specifically recognize pTyr residues and play a major role in mediating precise downstream signaling events. PMID:24296166

Molecular mechanisms of SH2- and PTB-domain-containing proteins in receptor tyrosine kinase signaling.

PubMed

Wagner, Melany J; Stacey, Melissa M; Liu, Bernard A; Pawson, Tony

2013-12-01

Intracellular signaling is mediated by reversible posttranslational modifications (PTMs) that include phosphorylation, ubiquitination, and acetylation, among others. In response to extracellular stimuli such as growth factors, receptor tyrosine kinases (RTKs) typically dimerize and initiate signaling through phosphorylation of their cytoplasmic tails and downstream scaffolds. Signaling effectors are recruited to these phosphotyrosine (pTyr) sites primarily through Src homology 2 (SH2) domains and pTyr-binding (PTB) domains. This review describes how these conserved domains specifically recognize pTyr residues and play a major role in mediating precise downstream signaling events.

Co-clustering of Fcgamma and B cell receptors induces dephosphorylation of the Grb2-associated binder 1 docking protein.

PubMed

Koncz, G; Tóth, G K; Bökönyi, G; Kéri, G; Pecht, I; Medgyesi, D; Gergely, J; Sármay, G

2001-07-01

The immunoreceptor tyrosine-based inhibitory motif (ITIM) of human type IIb Fcgamma receptor (FcgammaRIIb) is phosphorylated on its tyrosine upon co-clustering with the B cell receptor (BCR). The phosphorylated ITIM (p-ITIM) binds to the SH2 domains of polyphosphoinositol 5-phosphatase (SHIP) and the tyrosine phosphatase, SHP-2. We investigated the involvement of the molecular complex composed of the phosphorylated SHIP and FcgammaRIIb in the activation of SHP-2. As a model compound, we synthesized a bisphosphopeptide, combining the sequences of p-ITIM and the N-terminal tyrosine phosphorylated motif of SHIP with a flexible spacer. This compound bound to the recombinant SH2 domains of SHP-2 with high affinity and activated the phosphatase in an in vitro assay. These data suggest that the phosphorylated FcgammaRII-SHIP complexes formed in the intact cells may also activate SHP-2. Grb2-associated binder 1 (Gab1) is a multisite docking protein, which becomes tyrosine-phosphorylated in response to various types of signaling, including BCR. In turn it binds to the SH2 domains of SHP-2, SHIP and the p85 subunit of phosphatidyl inositol 3-kinase (PtdIns3-K) and may regulate their activity. Gab1 is a potential substrate of SHP-2, thus its binding to FcgammaRIIb may modify the Gab1-bound signaling complex. We show here that Gab1 is part of the multiprotein complex assembled by FcgammaRIIb upon its co-clustering with BCR. Gab1 may recruit SH2 domain-containing molecules to the phosphorylated FcgammaRIIb. SHP-2, activated upon the binding to FcgammaRIIb-SHIP complex, partially dephosphorylates Gab1, resulting in the release of PtdIns3-K and ultimately in the inhibition of downstream activation pathways in BCR/FcgammaRIIb co-aggregated cells.

Application of Ring-Closing Metathesis to Grb2 SH3 Domain-Binding Peptides | Center for Cancer Research

Cancer.gov

In silico-generated hypothetical interactions of a ring-closing metathesis-macrocylized peptide bound to the amino terminal SH3 domain of the growth factor receptor bound protein 2 (Grb2). The complex was derived from the NMR solution structure of the bound parent peptide, Ac-V-P-P-P-V-P-P-R-R-R-amide (Protein Data Bank: 3GBQ). The protein surface is shown as electrostatic

SOX10 Regulates Expression of the SH3-Domain Kinase Binding Protein 1 (Sh3kbp1) locus in Schwann Cells via an Alternative Promoter

PubMed Central

Hodonsky, Chani J.; Kleinbrink, Erica L.; Charney, Kira N.; Prasad, Megana; Bessling, Seneca L.; Jones, Erin A.; Srinivasan, Rajini; Svaren, John; McCallion, Andrew S.; Antonellis, Anthony

2011-01-01

The transcription factor SOX10 has essential roles in neural crest-derived cell populations, including myelinating Schwann cells—specialized glial cells responsible for ensheathing axons in the peripheral nervous system. Importantly, SOX10 directly regulates the expression of genes essential for proper myelin function. To date, only a handful of SOX10 target loci have been characterized in Schwann cells. Addressing this lack of knowledge will provide a better understanding of Schwann cell biology and candidate loci for relevant diseases such as demyelinating peripheral neuropathies. We have identified a highly-conserved SOX10 binding site within an alternative promoter at the SH3-domain kinase binding protein 1 (Sh3kbp1) locus. The genomic segment identified at Sh3kbp1 binds to SOX10 and displays strong promoter activity in Schwann cells in vitro and in vivo. Mutation of the SOX10 binding site ablates promoter activity, and ectopic expression of SOX10 in SOX10-negative cells promotes the expression of endogenous Sh3kbp1. Combined, these data reveal Sh3kbp1 as a novel target of SOX10 and raise important questions regarding the function of SH3KBP1 isoforms in Schwann cells. PMID:22037207

Estrogen Receptor Folding Modulates cSrc Kinase SH2 Interaction via a Helical Binding Mode.

PubMed

Nieto, Lidia; Tharun, Inga M; Balk, Mark; Wienk, Hans; Boelens, Rolf; Ottmann, Christian; Milroy, Lech-Gustav; Brunsveld, Luc

2015-11-20

The estrogen receptors (ERs) feature, next to their transcriptional role, important nongenomic signaling actions, with emerging clinical relevance. The Src Homology 2 (SH2) domain mediated interaction between cSrc kinase and ER plays a key role in this; however the molecular determinants of this interaction have not been elucidated. Here, we used phosphorylated ER peptide and semisynthetic protein constructs in a combined biochemical and structural study to, for the first time, provide a quantitative and structural characterization of the cSrc SH2-ER interaction. Fluorescence polarization experiments delineated the SH2 binding motif in the ER sequence. Chemical shift perturbation analysis by nuclear magnetic resonance (NMR) together with molecular dynamics (MD) simulations allowed us to put forward a 3D model of the ER-SH2 interaction. The structural basis of this protein-protein interaction has been compared with that of the high affinity SH2 binding sequence GpYEEI. The ER features a different binding mode from that of the "two-pronged plug two-hole socket" model in the so-called specificity determining region. This alternative binding mode is modulated via the folding of ER helix 12, a structural element directly C-terminal of the key phosphorylated tyrosine. The present findings provide novel molecular entries for understanding nongenomic ER signaling and targeting the corresponding disease states.

Chromosomal localization of the mouse Src-like adapter protein (Slap) gene and its putative human homolog SLA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Angrist, M.; Chakravarti, A.; Wells, D.E.

1995-12-10

Molecules containing Src-homology 2 (SH2) and Src-homology 3 (SH3) domains are critical components of signal transduction pathways that serve to relay signals originating from the cell surface to the interior of the cell. Src-like adapter protein (SLAP) is a recently described adapter protein that binds activated the Eck receptor protein-tyrosine kinase. Although SLAP bears a striking homology to the SH3 and SH2 domains of the Src family of nonreceptor tyrosine kinases, it does not contain a tyrosine kinase catalytic domain. In this report, the Slap gene was mapped by linkage analysis to mouse chromosome 15, while its putative human homologmore » (SLA) was identified and mapped to human 8q22.3-qter using a panel of somatic cell hybrids. 10 refs., 2 figs.« less

[NMR structure and dynamics of the chimeric protein SH3-F2].

PubMed

Kutyshenko, V P; Gushchina, L V; Khristoforov, V S; Prokhorov, D A; Timchenko, M A; Kudrevatykh, Iu A; Fediukina, D V; Filimonov, V V

2010-01-01

For the further elucidation of structural and dynamic principles of protein self-organization and protein-ligand interactions the design of new chimeric protein SH3-F2 was made and genetically engineered construct was created. The SH3-F2 amino acid sequence consists of polyproline ligand mgAPPLPPYSA, GG linker and the sequence of spectrin SH3 domain circular permutant S19-P20s. Structural and dynamics properties of the protein were studied by high-resolution NMR. According to NMR data the tertiary structure of the chimeric protein SH3-F2 has the topology which is typical of SH3 domains in the complex with the ligand, forming polyproline type II helix, located in the conservative region of binding in the orientation II. The polyproline ligand closely adjoins with the protein globule and is stabilized by hydrophobic interactions. However the interaction of ligand and the part of globule relative to SH3 domain is not too large because the analysis of protein dynamic characteristics points to the low amplitude, high-frequency ligand tumbling in relation to the slow intramolecular motions of the main globule. The constructed chimera permits to carry out further structural and thermodynamic investigations of polyproline helix properties and its interaction with regulatory domains.

Expression of the Grb2-related protein of the lymphoid system in B cell subsets enhances B cell antigen receptor signaling through mitogen-activated protein kinase pathways.

PubMed

Yankee, Thomas M; Solow, Sasha A; Draves, Kevin D; Clark, Edward A

2003-01-01

Adapter proteins play a critical role in regulating signals triggered by Ag receptor cross-linking. These small molecules link receptor proximal events with downstream signaling pathways. In this study, we explore the expression and function of the Grb2-related protein of the lymphoid system (GrpL)/Grb2-related adaptor downstream of Shc adapter protein in human B cells. GrpL is expressed in naive B cells and is down-regulated following B cell Ag receptor ligation. By contrast, germinal center and memory B cells express little or no GrpL. Using human B cell lines, we detected constitutive interactions between GrpL and B cell linker protein, Src homology (SH)2 domain-containing leukocyte protein of 76 kDa, hemopoietic progenitor kinase 1, and c-Cbl. The N-terminal SH3 domain of GrpL binds c-Cbl while the C-terminal SH3 domain binds B cell linker protein and SH2 domain-containing leukocyte protein of 76 kDa. Exogenous expression of GrpL in a GrpL-negative B cell line leads to enhanced Ag receptor-induced extracellular signal-related kinase and p38 mitogen-activated protein kinase phosphorylation. Thus, GrpL expression in human B cell subsets appears to regulate Ag receptor-mediated signaling events.

Site-specific protein backbone and side-chain NMR chemical shift and relaxation analysis of human vinexin SH3 domain using a genetically encoded {sup 15}N/{sup 19}F-labeled unnatural amino acid

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Pan; School of Life Science, University of Science and Technology of China, Hefei, Anhui 230026; Xi, Zhaoyong

Research highlights: {yields} Chemical synthesis of {sup 15}N/{sup 19}F-trifluomethyl phenylalanine. {yields} Site-specific incorporation of {sup 15}N/{sup 19}F-trifluomethyl phenylalanine to SH3. {yields} Site-specific backbone and side chain chemical shift and relaxation analysis. {yields} Different internal motions at different sites of SH3 domain upon ligand binding. -- Abstract: SH3 is a ubiquitous domain mediating protein-protein interactions. Recent solution NMR structural studies have shown that a proline-rich peptide is capable of binding to the human vinexin SH3 domain. Here, an orthogonal amber tRNA/tRNA synthetase pair for {sup 15}N/{sup 19}F-trifluoromethyl-phenylalanine ({sup 15}N/{sup 19}F-tfmF) has been applied to achieve site-specific labeling of SH3 at threemore » different sites. One-dimensional solution NMR spectra of backbone amide ({sup 15}N){sup 1}H and side-chain {sup 19}F were obtained for SH3 with three different site-specific labels. Site-specific backbone amide ({sup 15}N){sup 1}H and side-chain {sup 19}F chemical shift and relaxation analysis of SH3 in the absence or presence of a peptide ligand demonstrated different internal motions upon ligand binding at the three different sites. This site-specific NMR analysis might be very useful for studying large-sized proteins or protein complexes.« less

Mutation of CD2AP and SH3KBP1 Binding Motif in Alphavirus nsP3 Hypervariable Domain Results in Attenuated Virus.

PubMed

Mutso, Margit; Morro, Ainhoa Moliner; Smedberg, Cecilia; Kasvandik, Sergo; Aquilimeba, Muriel; Teppor, Mona; Tarve, Liisi; Lulla, Aleksei; Lulla, Valeria; Saul, Sirle; Thaa, Bastian; McInerney, Gerald M; Merits, Andres; Varjak, Margus

2018-04-27

Infection by Chikungunya virus (CHIKV) of the Old World alphaviruses (family Togaviridae) in humans can cause arthritis and arthralgia. The virus encodes four non-structural proteins (nsP) (nsP1, nsp2, nsP3 and nsP4) that act as subunits of the virus replicase. These proteins also interact with numerous host proteins and some crucial interactions are mediated by the unstructured C-terminal hypervariable domain (HVD) of nsP3. In this study, a human cell line expressing EGFP tagged with CHIKV nsP3 HVD was established. Using quantitative proteomics, it was found that CHIKV nsP3 HVD can bind cytoskeletal proteins, including CD2AP, SH3KBP1, CAPZA1, CAPZA2 and CAPZB. The interaction with CD2AP was found to be most evident; its binding site was mapped to the second SH3 ligand-like element in nsP3 HVD. Further assessment indicated that CD2AP can bind to nsP3 HVDs of many different New and Old World alphaviruses. Mutation of the short binding element hampered the ability of the virus to establish infection. The mutation also abolished ability of CD2AP to co-localise with nsP3 and replication complexes of CHIKV; the same was observed for Semliki Forest virus (SFV) harbouring a similar mutation. Similar to CD2AP, its homolog SH3KBP1 also bound the identified motif in CHIKV and SFV nsP3.

Interaction of the p85 subunit of PI 3-kinase and its N-terminal SH2 domain with a PDGF receptor phosphorylation site: structural features and analysis of conformational changes.

PubMed Central

Panayotou, G; Bax, B; Gout, I; Federwisch, M; Wroblowski, B; Dhand, R; Fry, M J; Blundell, T L; Wollmer, A; Waterfield, M D

1992-01-01

Circular dichroism and fluorescence spectroscopy were used to investigate the structure of the p85 alpha subunit of the PI 3-kinase, a closely related p85 beta protein, and a recombinant SH2 domain-containing fragment of p85 alpha. Significant spectral changes, indicative of a conformational change, were observed on formation of a complex with a 17 residue peptide containing a phosphorylated tyrosine residue. The sequence of this peptide is identical to the sequence surrounding Tyr751 in the kinase-insert region of the platelet-derived growth factor beta-receptor (beta PDGFR). The rotational correlation times measured by fluorescence anisotropy decay indicated that phosphopeptide binding changed the shape of the SH2 domain-containing fragment. The CD and fluorescence spectroscopy data support the secondary structure prediction based on sequence analysis and provide evidence for flexible linker regions between the various domains of the p85 proteins. The significance of these results for SH2 domain-containing proteins is discussed. Images PMID:1330535

An Investigation of Hierachical Protein Recruitment to the Inhibitory Platelet Receptor, G6B-b

PubMed Central

Coxon, Carmen H.; Sadler, Amanda J.; Huo, Jiandong; Campbell, R. Duncan

2012-01-01

Platelet activation is regulated by both positive and negative signals. G6B-b is an inhibitory platelet receptor with an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM). The molecular basis of inhibition by G6B-b is currently unknown but thought to involve the SH2 domain-containing tyrosine phosphatase SHP-1. Here we show that G6B-b also associates with SHP-2, as well as SHP-1, in human platelets. Using a number of biochemical approaches, we found these interactions to be direct and that the tandem SH2 domains of SHP-2 demonstrated a binding affinity for G6B-b 100-fold higher than that of SHP-1. It was also observed that while SHP-1 has an absolute requirement for phosphorylation at both motifs to bind, SHP-2 can associate with G6B-b when only one motif is phosphorylated, with the N-terminal SH2 domain and the ITIM being most important for the interaction. A number of other previously unreported SH2 domain-containing proteins, including Syk and PLCγ2, also demonstrated specificity for G6B-b phosphomotifs and may serve to explain the observation that G6B-b remains inhibitory in the absence of both SHP-1 and SHP-2. In addition, the presence of dual phosphorylated G6B-b in washed human platelets can reduce the EC50 for both CRP and collagen. PMID:23185356

Scaffold Protein SLP-76 Primes PLCγ1 for Activation by ITK-Mediated Phosphorylation.

PubMed

Devkota, Sujan; Joseph, Raji E; Min, Lie; Bruce Fulton, D; Andreotti, Amy H

2015-08-28

Activation of the phospholipase, PLCγ1, is critical for proper T cell signaling following antigen receptor engagement. In T cells, the Tec family kinase, interleukin-2-induced tyrosine kinase (ITK), phosphorylates PLCγ1 at tyrosine 783 (Y783) leading to activation of phospholipase function and subsequent production of the second messengers inositol 1,4,5-trisphosphate and diacylglycerol. In this work, we demonstrate that PLCγ1 can be primed for ITK-mediated phosphorylation on Y783 by a specific region of the adaptor protein, SLP-76. The SLP-76 phosphotyrosine-containing sequence, pY(173)IDR, does not conform to the canonical recognition motif for an SH2 domain yet binds with significant affinity to the C-terminal SH2 domain of PLCγ1 (SH2C). The SLP-76 pY(173) motif competes with the autoinhibited conformation surrounding the SH2C domain of PLCγ1 leading to exposure of the ITK recognition element on the PLCγ1 SH2 domain and release of the target tyrosine, Y783. These data contribute to the evolving model for the molecular events occurring early in the T cell activation process. Copyright © 2015 Elsevier Ltd. All rights reserved.

Specificity profiling of protein-binding domains using one-bead-one-compound Peptide libraries.

PubMed

Kunys, Andrew R; Lian, Wenlong; Pei, Dehua

2012-12-01

One-bead-one-compound (OBOC) libraries consist of structurally related compounds (e.g., peptides) covalently attached to a solid support, with each resin bead carrying a unique compound. OBOC libraries of high structural diversity can be rapidly synthesized and screened without the need for any special equipment, and therefore can be employed in any chemical or biochemical laboratory. OBOC peptide libraries have been widely used to map the ligand specificity of proteins, to determine the substrate specificity of enzymes, and to develop inhibitors against macromolecular targets. They have proven particularly useful in profiling the binding specificity of protein modular domains (e.g., SH2 domains, BIR domains, and PDZ domains); subsequently, the specificity information can be used to predict the protein targets of these domains. The protocols outlined in this article describe the methodologies for synthesizing and screening OBOC peptide libraries against SH2 and PDZ domains, and the related data analysis. Curr. Protoc. Chem. Biol. 4:331-355 © 2012 by John Wiley & Sons, Inc.

Crystal structure of the tyrosine kinase domain of the hepatocyte growth factor receptor c-Met and its complex with the microbial alkaloid K-252a.

PubMed

Schiering, Nikolaus; Knapp, Stefan; Marconi, Marina; Flocco, Maria M; Cui, Jean; Perego, Rita; Rusconi, Luisa; Cristiani, Cinzia

2003-10-28

The protooncogene c-met codes for the hepatocyte growth factor receptor tyrosine kinase. Binding of its ligand, hepatocyte growth factor/scatter factor, stimulates receptor autophosphorylation, which leads to pleiotropic downstream signaling events in epithelial cells, including cell growth, motility, and invasion. These events are mediated by interaction of cytoplasmic effectors, generally through Src homology 2 (SH2) domains, with two phosphotyrosine-containing sequence motifs in the unique C-terminal tail of c-Met (supersite). There is a strong link between aberrant c-Met activity and oncogenesis, which makes this kinase an important cancer drug target. The furanosylated indolocarbazole K-252a belongs to a family of microbial alkaloids that also includes staurosporine. It was recently shown to be a potent inhibitor of c-Met. Here we report the crystal structures of an unphosphorylated c-Met kinase domain harboring a human cancer mutation and its complex with K-252a at 1.8-A resolution. The structure follows the well established architecture of protein kinases. It adopts a unique, inhibitory conformation of the activation loop, a catalytically noncompetent orientation of helix alphaC, and reveals the complete C-terminal docking site. The first SH2-binding motif (1349YVHV) adopts an extended conformation, whereas the second motif (1356YVNV), a binding site for Grb2-SH2, folds as a type II Beta-turn. The intermediate portion of the supersite (1353NATY) assumes a type I Beta-turn conformation as in an Shc-phosphotyrosine binding domain peptide complex. K-252a is bound in the adenosine pocket with an analogous binding mode to those observed in previously reported structures of protein kinases in complex with staurosporine.

Microinjection of the SH2 domain of the 85-kilodalton subunit of phosphatidylinositol 3-kinase inhibits insulin-induced DNA synthesis and c-fos expression.

PubMed Central

Jhun, B H; Rose, D W; Seely, B L; Rameh, L; Cantley, L; Saltiel, A R; Olefsky, J M

1994-01-01

We have investigated the functional role of the SH2 domain of the 85-kDa subunit (p85) of the phosphatidylinositol 3-kinase in the insulin signal transduction pathway. Microinjection of a bacterial fusion protein containing the N-terminal SH2 domain of p85 inhibited insulin- and other growth factor-induced DNA synthesis by 90% and c-fos protein expression by 80% in insulin-responsive rat fibroblasts. The specificity of the fusion protein was examined by in vitro precipitation experiments, which showed that the SH2 domain of p85 can independently associate with both insulin receptor substrate 1 and the insulin receptor itself in the absence of detectable binding to other phosphoproteins. The microinjection results were confirmed through the use of an affinity-purified antibody directed against p85, which gave the same phenotype. Additional studies were carried out in another cell line expressing mutant insulin receptors which lack the cytoplasmic tyrosine residues with which p85 interacts. Microinjection of the SH2 domain fusion protein also inhibited insulin signaling in these cells, suggesting that association of p85 with insulin receptor substrate 1 is a key element in insulin-mediated cell cycle progression. In addition, coinjection of purified p21ras protein with the p85 fusion protein or the antibody restored DNA synthesis, suggesting that ras function is either downstream or independent of p85 SH2 domain interaction. Images PMID:7935461

A recombined fusion protein PTD-Grb2-SH2 inhibits the proliferation of breast cancer cells in vitro.

PubMed

Yin, Jikai; Cai, Zhongliang; Zhang, Li; Zhang, Jian; He, Xianli; Du, Xilin; Wang, Qing; Lu, Jianguo

2013-03-01

The growth factor receptor bound protein 2 (Grb2) is one of the affirmative targets for cancer therapy, especially for breast cancer. In this study, we hypothesized the Src-homology 2 (SH2) domain in Grb2 may serve as a competitive protein-binding agent to interfere with the proliferation of breast cancer cells in vitro. We designed, constructed, expressed and purified a novel fusion protein containing the protein transduction domain (PTD) and Grb2-SH2 domain (we named it after PTD-Grb2-SH2). An immunofluorescence assay was used to investigate the location of PTD-Grb2-SH2 in cells. MTT assay and EdU experiments were applied to detect the proliferation of breast cancer cells. The ultra-structure was observed using transmission electron microscopy. Flow cytometry was used to determine the cytotoxicity of PTD-Grb2-SH2 on cell proliferation. We successfully obtained the PTD-Grb2-SH2 fusion protein in soluble form using a prokaryotic expression system. The new fusion protein successfully passed through both the cellular and nuclear membranes of breast cancer cells. The MTT assay showed that PTD-Grb2-SH2 exhibited significant toxicity to breast cancer cells in a dose- and time-dependent manner in vitro. EdU identified the decreased proliferation rates in treated MDA-MB-231 and SK-BR-3 cells. Observation by transmission electron microscopy and flow cytometry further confirmed the cytotoxicity as apoptosis. Our results show that the HIV1-TAT domain is a useful tool for transporting a low molecular weight protein across the cell membrane in vitro. The PTD-Grb2-SH2 may be a novel agent for breast cancer therapy.

A Steric-inhibition model for regulation of nucleotide exchange via the Dock180 family of GEFs.

PubMed

Lu, Mingjian; Kinchen, Jason M; Rossman, Kent L; Grimsley, Cynthia; Hall, Matthew; Sondek, John; Hengartner, Michael O; Yajnik, Vijay; Ravichandran, Kodi S

2005-02-22

CDM (CED-5, Dock180, Myoblast city) family members have been recently identified as novel, evolutionarily conserved guanine nucleotide exchange factors (GEFs) for Rho-family GTPases . They regulate multiple processes, including embryonic development, cell migration, apoptotic-cell engulfment, tumor invasion, and HIV-1 infection, in diverse model systems . However, the mechanism(s) of regulation of CDM proteins has not been well understood. Here, our studies on the prototype member Dock180 reveal a steric-inhibition model for regulating the Dock180 family of GEFs. At basal state, the N-terminal SH3 domain of Dock180 binds to the distant catalytic Docker domain and negatively regulates the function of Dock180. Further studies revealed that the SH3:Docker interaction sterically blocks Rac access to the Docker domain. Interestingly, ELMO binding to the SH3 domain of Dock180 disrupted the SH3:Docker interaction, facilitated Rac access to the Docker domain, and contributed to the GEF activity of the Dock180/ELMO complex. Additional genetic rescue studies in C. elegans suggested that the regulation of the Docker-domain-mediated GEF activity by the SH3 domain and its adjoining region is evolutionarily conserved. This steric-inhibition model may be a general mechanism for regulating multiple SH3-domain-containing Dock180 family members and may have implications for a variety of biological processes.

Interaction between focal adhesion kinase and Crk-associated tyrosine kinase substrate p130Cas.

PubMed

Polte, T R; Hanks, S K

1995-11-07

The focal adhesion kinase (FAK) has been implicated in integrin-mediated signaling events and in the mechanism of cell transformation by the v-Src and v-Crk oncoproteins. To gain further insight into FAK signaling pathways, we used a two-hybrid screen to identify proteins that interact with mouse FAK. The screen identified two proteins that interact with FAK via their Src homology 3 (SH3) domains: a v-Crk-associated tyrosine kinase substrate (Cas), p130Cas, and a still uncharacterized protein, FIPSH3-2, which contains an SH3 domain closely related to that of p130Cas. These SH3 domains bind to the same proline-rich region of FAK (APPKPSR) encompassing residues 711-717. The mouse p130Cas amino acid sequence was deduced from cDNA clones, revealing an overall high degree of similarity to the recently reported rat sequence. Coimmunoprecipitation experiments confirmed that p130Cas and FAK are associated in mouse fibroblasts. The stable interaction between p130Cas and FAK emerges as a likely key element in integrin-mediated signal transduction and further represents a direct molecular link between the v-Src and v-Crk oncoproteins. The Src family kinase Fyn, whose Src homology 2 (SH2) domain binds to the major FAK autophosphorylation site (tyrosine 397), was also identified in the two-hybrid screen.

SH3-binding Protein 5 Mediates the Neuroprotective Effect of the Secreted Bioactive Peptide Humanin by Inhibiting c-Jun NH2-terminal Kinase*

PubMed Central

Takeshita, Yuji; Hashimoto, Yuichi; Nawa, Mikiro; Uchino, Hiroyuki; Matsuoka, Masaaki

2013-01-01

Humanin is a secreted bioactive peptide that suppresses cell toxicity caused by a variety of insults. The neuroprotective effect of Humanin against Alzheimer disease (AD)-related death is mediated by the binding of Humanin to its heterotrimeric Humanin receptor composed of ciliary neurotrophic receptor α, WSX-1, and gp130, as well as the activation of intracellular signaling pathways including a JAK2 and STAT3 signaling axis. Despite the elucidation of the signaling pathways by which Humanin mediates its neuroprotection, the transcriptional targets of Humanin that behaves as effectors of Humanin remains undefined. In the present study, Humanin increased the mRNA and protein expression of SH3 domain-binding protein 5 (SH3BP5), which has been known to be a JNK interactor, in neuronal cells. Similar to Humanin treatment, overexpression of SH3BP5 inhibited AD-related neuronal death, while siRNA-mediated knockdown of endogenous SH3BP5 expression attenuated the neuroprotective effect of Humanin. These results indicate that SH3BP5 is a downstream effector of Humanin. Furthermore, biochemical analysis has revealed that SH3BP5 binds to JNK and directly inhibits JNK through its two putative mitogen-activated protein kinase interaction motifs (KIMs). PMID:23861391

Staphylococcus haemolyticus prophage ΦSH2 endolysin relies on Cysteine, Histidine-dependent Amidohydrolases/Peptidases activity for lysis ‘from without’

PubMed Central

Schmelcher, Mathias; Korobova, Olga; Schischkova, Nina; Kiseleva, Natalia; Kopylov, Paul; Pryamchuk, Sergey; Donovan, David M.; Abaev, Igor

2014-01-01

Staphylococcus aureus is an important pathogen, with methicillin-resistant (MRSA) and multi-drug resistant strains becoming increasingly prevalent in both human and veterinary clinics. S. aureus causing bovine mastitis yields high annual losses to the dairy industry. Conventional treatment of mastitis by broad range antibiotics is often not successful and may contribute to development of antibiotic resistance. Bacteriophage endolysins present a promising new source of antimicrobials. The endolysin of prophage ΦSH2 of Staphylococcus haemolyticus strain JCSC1435 (ΦSH2 lysin) is a peptidoglycan hydrolase consisting of two catalytic domains (CHAP and amidase) and an SH3b cell wall binding domain. In this work, we demonstrated its lytic activity against live staphylococcal cells and investigated the contribution of each functional module to bacterial lysis by testing a series of deletion constructs in zymograms and turbidity reduction assays. The CHAP domain exhibited three-fold higher activity than the full length protein and optimum activity in physiological saline. This activity was further enhanced by the presence of bivalent calcium ions. The SH3b domain was shown to be required for full activity of the complete ΦSH2 lysin. The full length enzyme and the CHAP domain showed activity against multiple staphylococcal strains, including MRSA strains, mastitis isolates, and CoNS. PMID:23026556

Staphylococcus haemolyticus prophage ΦSH2 endolysin relies on cysteine, histidine-dependent amidohydrolases/peptidases activity for lysis 'from without'.

PubMed

Schmelcher, Mathias; Korobova, Olga; Schischkova, Nina; Kiseleva, Natalia; Kopylov, Paul; Pryamchuk, Sergey; Donovan, David M; Abaev, Igor

2012-12-31

Staphylococcus aureus is an important pathogen, with methicillin-resistant (MRSA) and multi-drug resistant strains becoming increasingly prevalent in both human and veterinary clinics. S. aureus causing bovine mastitis yields high annual losses to the dairy industry. Conventional treatment of mastitis by broad range antibiotics is often not successful and may contribute to development of antibiotic resistance. Bacteriophage endolysins present a promising new source of antimicrobials. The endolysin of prophage ΦSH2 of Staphylococcus haemolyticus strain JCSC1435 (ΦSH2 lysin) is a peptidoglycan hydrolase consisting of two catalytic domains (CHAP and amidase) and an SH3b cell wall binding domain. In this work, we demonstrated its lytic activity against live staphylococcal cells and investigated the contribution of each functional module to bacterial lysis by testing a series of deletion constructs in zymograms and turbidity reduction assays. The CHAP domain exhibited three-fold higher activity than the full length protein and optimum activity in physiological saline. This activity was further enhanced by the presence of bivalent calcium ions. The SH3b domain was shown to be required for full activity of the complete ΦSH2 lysin. The full length enzyme and the CHAP domain showed activity against multiple staphylococcal strains, including MRSA strains, mastitis isolates, and CoNS. Published by Elsevier B.V.

Growth of chronic myeloid leukemia cells is inhibited by infection with Ad-SH2-HA adenovirus that disrupts Grb2-Bcr-Abl complexes.

PubMed

Peng, Zhi; Luo, Hong-Wei; Yuan, Ying; Shi, Jing; Huang, Shi-Feng; Li, Chun-Li; Cao, Wei-Xi; Huang, Zong-Gan; Feng, Wen-Li

2011-05-01

The persistence of Bcr-Abl-positive cells in patients on imatinib therapy indicates that inhibition of the Bcr-Abl kinase activity alone might not be sufficient to eradicate the leukemia cells. Many downstream effectors of Bcr-Abl have been described, including activation of both the Grb2-SoS-Ras-MAPK and Grb2-Gab2-PI3K-Akt pathways. The Bcr-Abl-Grb2 interaction, which is mediated by the direct interaction of the Grb2 SH2 domain with the phospho-Bcr-Abl Y177, is required for activation of these signaling pathways. Therefore, disrupting their interaction represents a potential therapeutic strategy for inhibiting the oncogenic downstream signals of Bcr-Abl. Adenovirus Ad-SH2-HA expressing the Grb2 SH2 domain was constructed and applied in this study. As expected, Ad-SH2-HA efficiently infected CML cells and functioned by binding to the phospho-Bcr-Abl Y177 site, competitively disrupting the Grb2 SH2-phospho-Bcr-Abl Y177 complex. They induced potent anti-proliferation and apoptosis-inducing effects in CML cell lines. Moreover, the Ras, MAPK and Akt activities were significantly reduced in the Ad-SH2-HA treated cells. These were not observed with the point-mutated control adenovirus Ad-Sm-HA with abolished phospho-Bcr-Abl Y177 binding sites. These data indicate that, in addition to the direct targeting of Bcr-Abl, selective inhibition of its downstream signaling pathways may be a therapeutic option for CML, and the Ad-SH2-HA-mediated killing strategy could be explored as a promising anti-leukemia agent in CML.

The Dictyostelium Carmil Protein Links Capping Protein and the Arp2/3 Complex to Type I Myosins through Their Sh3 Domains

PubMed Central

Jung, Goeh; Remmert, Kirsten; Wu, Xufeng; Volosky, Joanne M.; III, John A. Hammer

2001-01-01

Fusion proteins containing the Src homology (SH)3 domains of Dictyostelium myosin IB (myoB) and IC (myoC) bind a 116-kD protein (p116), plus nine other proteins identified as the seven member Arp2/3 complex, and the α and β subunits of capping protein. Immunoprecipitation reactions indicate that myoB and myoC form a complex with p116, Arp2/3, and capping protein in vivo, that the myosins bind to p116 through their SH3 domains, and that capping protein and the Arp2/3 complex in turn bind to p116. Cloning of p116 reveals a protein dominated by leucine-rich repeats and proline-rich sequences, and indicates that it is a homologue of Acan 125. Studies using p116 fusion proteins confirm the location of the myosin I SH3 domain binding site, implicate NH2-terminal sequences in binding capping protein, and show that a region containing a short sequence found in several G-actin binding proteins, as well as an acidic stretch, can activate Arp2/3-dependent actin nucleation. p116 localizes along with the Arp2/3 complex, myoB, and myoC in dynamic actin-rich cellular extensions, including the leading edge of cells undergoing chemotactic migration, and dorsal, cup-like, macropinocytic extensions. Cells lacking p116 exhibit a striking defect in the formation of these macropinocytic structures, a concomitant reduction in the rate of fluid phase pinocytosis, a significant decrease in the efficiency of chemotactic aggregation, and a decrease in cellular F-actin content. These results identify a complex that links key players in the nucleation and termination of actin filament assembly with a ubiquitous barbed end–directed motor, indicate that the protein responsible for the formation of this complex is physiologically important, and suggest that previously reported myosin I mutant phenotypes in Dictyostelium may be due, at least in part, to defects in the assembly state of actin. We propose that p116 and Acan 125, along with homologues identified in Caenorhabditis elegans, Drosophila, mouse, and man, be named CARMIL proteins, for capping protein, Arp2/3, and myosin I linker. PMID:11425877

Truncated ALK derived from chromosomal translocation t(2;5)(p23;q35) binds to the SH3 domain of p85-PI3K.

PubMed

Polgar, Doris; Leisser, Christina; Maier, Susanne; Strasser, Stephan; Rüger, Beate; Dettke, Markus; Khorchide, Maya; Simonitsch, Ingrid; Cerni, Christa; Krupitza, Georg

2005-02-15

The chromosomal translocation t(2;5)(p23;q35) is associated with "Anaplastic large cell lymphomas" (ALCL), a Non Hodgkin Lymphoma occurring in childhood. The fusion of the tyrosine kinase gene-ALK (anaplastic lymphoma kinase) on chromosome 2p23 to the NPM (nucleophosmin/B23) gene on chromosome 5q35 results in a 80 kDa chimeric protein, which activates the "survival" kinase PI3K. However, the binding mechanism between truncated ALK and PI3K is poorly understood. Therefore, we attempted to elucidate the molecular interaction between ALK and the regulatory p85 subunit of PI3K. Here we provide evidence that the truncated ALK homodimer binds to the SH3 domain of p85. This finding may be useful for the development of a new target-specific intervention.

The SRC homology 2 domain of Rin1 mediates its binding to the epidermal growth factor receptor and regulates receptor endocytosis.

PubMed

Barbieri, M Alejandro; Kong, Chen; Chen, Pin-I; Horazdovsky, Bruce F; Stahl, Philip D

2003-08-22

Activated epidermal growth factor receptors (EGFRs) recruit intracellular proteins that mediate receptor signaling and endocytic trafficking. Rin1, a multifunctional protein, has been shown to regulate EGFR internalization (1). Here we show that EGF stimulation induces a specific, rapid, and transient membrane recruitment of Rin1 and that recruitment is dependent on the Src homology 2 (SH2) domain of Rin1. Immunoprecipitation of EGFR is accompanied by co-immunoprecipitation of Rin1 in a time- and ligand-dependent manner. Association of Rin1 and specifically the SH2 domain of Rin1 with the EGFR was dependent on tyrosine phosphorylation of the intracellular domain of the EGFR. The recruitment of Rin1, observed by light microscopy, indicated that although initially cytosolic, Rin1 was recruited to both plasma membrane and endosomes following EGF addition. Moreover, the expression of the SH2 domain of Rin1 substantially impaired the internalization of EGF without affecting internalization of transferrin. Finally, we found that Rin1 co-immunoprecipitated with a number of tyrosine kinase receptors but not with cargo endocytic receptors. These results indicate that Rin1 provides a link via its SH2 domain between activated tyrosine kinase receptors and the endocytic pathway through the recruitment and activation of Rab5a.

Identification and functional characterization of an Src homology domain 3 domain-binding site on Cbl.

PubMed

Sanjay, Archana; Miyazaki, Tsuyoshi; Itzstein, Cecile; Purev, Enkhtsetseg; Horne, William C; Baron, Roland

2006-12-01

Cbl is an adaptor protein and ubiquitin ligase that binds and is phosphorylated by the nonreceptor tyrosine kinase Src. We previously showed that the primary interaction between Src and Cbl is mediated by the Src homology domain 3 (SH3) of Src binding to proline-rich sequences of Cbl. The peptide Cbl RDLPPPPPPDRP(540-551), which corresponds to residues 540-551 of Cbl, inhibited the binding of a GST-Src SH3 fusion protein to Cbl, whereas RDLAPPAPPPDR(540-551) did not, suggesting that Src binds to this site on Cbl in a class I orientation. Mutating prolines 543-548 reduced Src binding to the Cbl 479-636 fragment significantly more than mutating the prolines in the PPVPPR(494-499) motif, which was previously reported to bind Src SH3. Mutating Cbl prolines 543-548 to alanines substantially reduced Src binding to Cbl, Src-induced phosphorylation of Cbl, and the inhibition of Src kinase activity by Cbl. Expressing the mutated Cbl in osteoclasts induced a moderate reduction in bone-resorbing activity and increased amounts of Src protein. In contrast, disabling the tyrosine kinase-binding domain of full-length Cbl by mutating glycine 306 to glutamic acid, and thereby preventing the previously described binding of the tyrosine kinase-binding domain to the Src phosphotyrosine 416, had no effect on Cbl phosphorylation, the inhibition of Src activity by full-length Cbl, or bone resorption. These data indicate that the Cbl RDLPPPP(540-546) sequence is a functionally important binding site for Src.

Proteome-wide inference of human endophilin 1-binding peptides.

PubMed

Wu, Gang; Zhang, Zeng-Li; Fu, Chun-Jiang; Lv, Feng-Lin; Tian, Fei-Fei

2012-10-01

Human endophilin 1 (hEndo1) is a multifunctional protein that was found to bind a wide spectrum of prolinerich endocytic proteins through its Src homology 3 (SH3) domain. In order to elucidate the unknown biological functions of hEndo1, it is essential to find out the cytoplasmic components that hEndo1 recognizes and binds. However, it is too time-consuming and expensive to synthesize all peptide candidates found in the human proteome and to perform hEndo1 SH3-peptide affinity assay to identify the hEndo1-binding partners. In the present work, we describe a structure/ sequence-hybrid approach to perform proteome-wide inference of human hEndo1-binding peptides using the information gained from both the primary sequence of affinity-known peptides and the interaction profile involved in hEndo1 SH3-peptide complex three-dimensional structures. Modeling results show that (i) different residue positions contribute distinctly to peptide affinity and specificity; P-1, P2 and P4 are most important, P1 and P3 are also effective, and P-3, P-2, P0, P5 and P6 are relatively insignificant, (ii) the consensus core PXXP motif is necessary but not sufficient for determining high affinity of peptides, and some other positions must be also essential in the hEndo1 SH3-peptide binding, and (iii) the alternating arrangement of polar and nonpolar amino acids along peptide sequence is critical for the high specificity of peptide recognition by hEndo1 SH3 domain. In addition, we also find that the residue type at a specific position of hEndo1-binding peptides is not stringently invariable; amino acids that possess similar polarity could replace each other without substantial influence on peptide affinity. In this way, hEndo1 presents a broad specificity in the peptide ligands that it binds.

The Cish SH2 domain is essential for PLC-γ1 regulation in TCR stimulated CD8+ T cells.

PubMed

Guittard, Geoffrey; Dios-Esponera, Ana; Palmer, Douglas C; Akpan, Itoro; Barr, Valarie A; Manna, Asit; Restifo, Nicholas P; Samelson, Lawrence E

2018-03-28

Cish, participates within a multi-molecular E3 ubiquitin ligase complex, which ubiquitinates target proteins. It has an inhibitory effect on T cell activation mediated by PLC-γ1 regulation, and it functions as a potent checkpoint in CD8 + T cell tumor immunotherapy. To study the structural and functional relationships between Cish and PLC-γ1 during CD8 + T cell activation, we tested mutants of the Cish-SH2 (R107K) and D/BC (L222Q, C226Q) domains. We confirmed that Cish-SH2-specific binding was essential for PLC-γ1 ubiquitination and degradation. This domain was essential for the Cish-mediated inhibition of Ca 2+ release upon TCR stimulation. No effect on inhibition of cytokine release was observed with SH2 or D/BC mutants, although the absence of Cish led to an increased release of IFN-γ and TNF-α. Using imaging we showed that Cish was expressed mostly in the cytoplasm and we did not see any Cish clustering at the plasma membrane upon stimulation. We conclude that the Cish-SH2 domain is essential for PLC-γ1 regulation in TCR-stimulated CD8 + T cells.

Differential Dynamic Engagement within 24 SH3 Domain: Peptide Complexes Revealed by Co-Linear Chemical Shift Perturbation Analysis

PubMed Central

Stollar, Elliott J.; Lin, Hong; Davidson, Alan R.; Forman-Kay, Julie D.

2012-01-01

There is increasing evidence for the functional importance of multiple dynamically populated states within single proteins. However, peptide binding by protein-protein interaction domains, such as the SH3 domain, has generally been considered to involve the full engagement of peptide to the binding surface with minimal dynamics and simple methods to determine dynamics at the binding surface for multiple related complexes have not been described. We have used NMR spectroscopy combined with isothermal titration calorimetry to comprehensively examine the extent of engagement to the yeast Abp1p SH3 domain for 24 different peptides. Over one quarter of the domain residues display co-linear chemical shift perturbation (CCSP) behavior, in which the position of a given chemical shift in a complex is co-linear with the same chemical shift in the other complexes, providing evidence that each complex exists as a unique dynamic rapidly inter-converting ensemble. The extent the specificity determining sub-surface of AbpSH3 is engaged as judged by CCSP analysis correlates with structural and thermodynamic measurements as well as with functional data, revealing the basis for significant structural and functional diversity amongst the related complexes. Thus, CCSP analysis can distinguish peptide complexes that may appear identical in terms of general structure and percent peptide occupancy but have significant local binding differences across the interface, affecting their ability to transmit conformational change across the domain and resulting in functional differences. PMID:23251481

Crystal structure of the C-terminal SH3 domain of the adaptor protein GADS in complex with SLP-76 motif peptide reveals a unique SH3-SH3 interaction.

PubMed

Dimasi, Nazzareno

2007-01-01

The Grb2-like adaptor protein GADS is essential for tyrosine kinase-dependent signaling in T lymphocytes. Following T cell receptor ligation, GADS interacts through its C-terminal SH3 domain with the adaptors SLP-76 and LAT, to form a multiprotein signaling complex that is crucial for T cell activation. To understand the structural basis for the selective recognition of GADS by SLP-76, herein is reported the crystal structure at 1.54 Angstrom of the C-terminal SH3 domain of GADS bound to the SLP-76 motif 233-PSIDRSTKP-241, which represents the minimal binding site. In addition to the unique structural features adopted by the bound SLP-76 peptide, the complex structure reveals a unique SH3-SH3 interaction. This homophilic interaction, which is observed in presence of the SLP-76 peptide and is present in solution, extends our understanding of the molecular mechanisms that could be employed by modular proteins to increase their signaling transduction specificity.

Evidence for a role for the phosphotyrosine-binding domain of Shc in interleukin 2 signaling.

PubMed Central

Ravichandran, K S; Igras, V; Shoelson, S E; Fesik, S W; Burakoff, S J

1996-01-01

Stimulation via the T-cell growth factor interleukin 2 (IL-2) leads to tyrosine phosphorylation of Shc, the interaction of Shc with Grb2, and the Ras GTP/GDP exchange factor, mSOS. Shc also coprecipitates with the IL-2 receptor (IL-2R), and therefore, may link IL-2R to Ras activation. We have further characterized the Shc-IL-2R interaction and have made the following observations. (i) Among the two phosphotyrosine-interaction domains present in Shc, the phosphotyrosine-binding (PTB) domain, rather than its SH2 domain, interacts with the tyrosine-phosphorylated IL-2R beta chain. Moreover, the Shc-PTB domain binds a phosphopeptide derived from the IL-2R beta chain (corresponding to residues surrounding Y338, SCFTNQGpYFF) with high affinity. (ii) In vivo, mutant IL-2R beta chains lacking the acidic region of IL-2Rbeta (which contains Y338) fail to phosphorylate Shc. Furthermore, when wild type or mutant Shc proteins that lack the PTB domain were expressed in the IL-2-dependent CTLL-20 cell line, an intact Shc-PTB domain was required for Shc phosphorylation by the IL-2R, which provides further support for a Shc-PTB-IL-2R interaction in vivo. (iii) PTB and SH2 domains of Shc associate with different proteins in IL-2- and T-cell-receptor-stimulated lysates, suggesting that Shc, through the concurrent use of its two different phosphotyrosine-binding domains, could assemble multiple protein complexes. Taken together, our in vivo and in vitro observations suggest that the PTB domain of Shc interacts with Y338 of the IL-2R and provide evidence for a functional role for the Shc-PTB domain in IL-2 signaling. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8643566

SHP-1 Binds and Negatively Modulates the c-Kit Receptor by Interaction with Tyrosine 569 in the c-Kit Juxtamembrane Domain

PubMed Central

Kozlowski, Maya; Larose, Louise; Lee, Fai; Le, Duc Mingh; Rottapel, Robert; Siminovitch, Katherine A.

1998-01-01

The SH2 domain-containing SHP-1 tyrosine phosphatase has been shown to negatively regulate a broad spectrum of growth factor- and cytokine-driven mitogenic signaling pathways. Included among these is the cascade of intracellular events evoked by stem cell factor binding to c-Kit, a tyrosine kinase receptor which associates with and is dephosphorylated by SHP-1. Using a series of glutathione S-transferase (GST) fusion proteins containing either tyrosine-phosphorylated segments of the c-Kit cytosolic region or the SH2 domains of SHP-1, we have shown that SHP-1 interacts with c-Kit by binding selectively to the phosphorylated c-Kit juxtamembrane region and that the association of c-Kit with the larger of the two SHP-1 isoforms may be mediated through either the N-terminal or C-terminal SHP-1 SH2 domain. The results of binding assays with mutagenized GST-Kit juxtamembrane fusion proteins and competitive inhibition assays with phosphopeptides encompassing each c-Kit juxtamembrane region identified the tyrosine residue at position 569 as the major site for binding of SHP-1 to c-Kit and suggested that tyrosine 567 contributes to, but is not required for, this interaction. By analysis of Ba/F3 cells retrovirally transduced to express c-Kit receptors, phenylalanine substitution of c-Kit tyrosine residue 569 was shown to be associated with disruption of c-Kit–SHP-1 binding and induction of hyperproliferative responses to stem cell factor. Although phenylalanine substitution of c-Kit tyrosine residue 567 in the Ba/F3–c-Kit cells did not alter SHP-1 binding to c-Kit, the capacity of a second c-Kit-binding tyrosine phosphatase, SHP-2, to associate with c-Kit was markedly reduced, and the cells again showed hyperproliferative responses to stem cell factor. These data therefore identify SHP-1 binding to tyrosine 569 on c-Kit as an interaction pivotal to SHP-1 inhibitory effects on c-Kit signaling, but they indicate as well that cytosolic protein tyrosine phosphatases other than SHP-1 may also negatively regulate the coupling of c-Kit engagement to proliferation. PMID:9528781

CD6 and Linker of Activated T Cells are Potential Interaction Partners for T Cell-Specific Adaptor Protein.

PubMed

Hem, C D; Ekornhol, M; Granum, S; Sundvold-Gjerstad, V; Spurkland, A

2017-02-01

The T cell-specific adaptor protein (TSAd) contains several protein interaction domains, and is merging as a modulator of T cell activation. Several interaction partners for the TSAd proline-rich region and phosphotyrosines have been identified, including the Src and Tec family kinases lymphocyte-specific protein tyrosine kinase and interleukin 2-inducible T cell kinase. Via its Src homology 2 (SH2) domain, TSAd may thus function as a link between these enzymes and other signalling molecules. However, few binding partners to the TSAd SH2 domain in T cells are hitherto known. Through the use of in silico ligand prediction, peptide spot arrays, pull-down and immunoprecipitation experiments, we here report novel interactions between the TSAd SH2 domain and CD6 phosphotyrosine (pTyr) 629 and linker of activated T cells (LAT) pTyr 171 , pTyr 191 and pTyr 226 . © 2016 The Foundation for the Scandinavian Journal of Immunology.

Proline Substitutions and Threonine Pseudophosphorylation of the SH3 Ligand of 18.5-kDa Myelin Basic Protein Decrease Its Affinity for the Fyn-SH3 Domain and Alter Process Development and Protein Localization in Oligodendrocytes

PubMed Central

Smith, Graham S.T.; De Avila, Miguel; Paez, Pablo M.; Spreuer, Vilma; Wills, Melanie K.B.; Jones, Nina; Boggs, Joan M.; Harauz, George

2012-01-01

The developmentally regulated myelin basic proteins (MBPs), which arise from the golli (gene of oligodendrocyte lineage) complex, are highly positively charged, intrinsically disordered, multifunctional proteins having several alternatively spliced isoforms and posttranslational modifications, and they play key roles in myelin compaction. The classic 18.5-kDa MBP isoform has a proline-rich region comprising amino acids 92–99 (murine sequence –T92PRTPPPS99–) that contains a minimal SH3 ligand domain. We have previously shown that 18.5-kDa MBP binds to several SH3 domains, including that of Fyn, a member of the Src family of tyrosine kinases involved in a number of signaling pathways during CNS development. To determine the physiological role of this binding as well as the role of phosphorylation of Thr92 and Thr95, in the current study we have produced several MBP variants specifically targeting phosphorylation sites and key structural regions of MBP’s SH3 ligand domain. Using isothermal titration calorimetry, we have demonstrated that, compared with the wild-type protein, these variants have lower affinity for the SH3 domain of Fyn. Moreover, overexpression of N-terminal-tagged GFP versions in immortalized oligodendroglial N19 and N20.1 cell cultures results in aberrant elongation of membrane processes and increased branching complexity and inhibits the ability of MBP to decrease Ca2+ influx. Phosphorylation of Thr92 can also cause MBP to traffic to the nucleus, where it may participate in additional protein–protein interactions. Coexpression of MBP with a constitutively active form of Fyn kinase resulted in membrane process elaboration, a phenomenon that was abolished by point amino acid substitutions in MBP’s SH3 ligand domain. These results suggest that MBP’s SH3 ligand domain plays a key role in intracellular protein interactions in vivo and may be required for proper membrane elaboration of developing oligodendrocytes and, further, that phosphorylation of Thr92 and Thr95 can regulate this function. PMID:21887699

Proline substitutions and threonine pseudophosphorylation of the SH3 ligand of 18.5-kDa myelin basic protein decrease its affinity for the Fyn-SH3 domain and alter process development and protein localization in oligodendrocytes.

PubMed

Smith, Graham S T; De Avila, Miguel; Paez, Pablo M; Spreuer, Vilma; Wills, Melanie K B; Jones, Nina; Boggs, Joan M; Harauz, George

2012-01-01

The developmentally regulated myelin basic proteins (MBPs), which arise from the golli (gene of oligodendrocyte lineage) complex, are highly positively charged, intrinsically disordered, multifunctional proteins having several alternatively spliced isoforms and posttranslational modifications, and they play key roles in myelin compaction. The classic 18.5-kDa MBP isoform has a proline-rich region comprising amino acids 92-99 (murine sequence -T(92)PRTPPPS(99)-) that contains a minimal SH3 ligand domain. We have previously shown that 18.5-kDa MBP binds to several SH3 domains, including that of Fyn, a member of the Src family of tyrosine kinases involved in a number of signaling pathways during CNS development. To determine the physiological role of this binding as well as the role of phosphorylation of Thr92 and Thr95, in the current study we have produced several MBP variants specifically targeting phosphorylation sites and key structural regions of MBP's SH3 ligand domain. Using isothermal titration calorimetry, we have demonstrated that, compared with the wild-type protein, these variants have lower affinity for the SH3 domain of Fyn. Moreover, overexpression of N-terminal-tagged GFP versions in immortalized oligodendroglial N19 and N20.1 cell cultures results in aberrant elongation of membrane processes and increased branching complexity and inhibits the ability of MBP to decrease Ca(2+) influx. Phosphorylation of Thr92 can also cause MBP to traffic to the nucleus, where it may participate in additional protein-protein interactions. Coexpression of MBP with a constitutively active form of Fyn kinase resulted in membrane process elaboration, a phenomenon that was abolished by point amino acid substitutions in MBP's SH3 ligand domain. These results suggest that MBP's SH3 ligand domain plays a key role in intracellular protein interactions in vivo and may be required for proper membrane elaboration of developing oligodendrocytes and, further, that phosphorylation of Thr92 and Thr95 can regulate this function. Copyright © 2011 Wiley Periodicals, Inc.

Identification of STAT1 and STAT3 Specific Inhibitors Using Comparative Virtual Screening and Docking Validation

PubMed Central

Szelag, Malgorzata; Czerwoniec, Anna; Wesoly, Joanna; Bluyssen, Hans A. R.

2015-01-01

Signal transducers and activators of transcription (STATs) facilitate action of cytokines, growth factors and pathogens. STAT activation is mediated by a highly conserved SH2 domain, which interacts with phosphotyrosine motifs for specific STAT-receptor contacts and STAT dimerization. The active dimers induce gene transcription in the nucleus by binding to a specific DNA-response element in the promoter of target genes. Abnormal activation of STAT signaling pathways is implicated in many human diseases, like cancer, inflammation and auto-immunity. Searches for STAT-targeting compounds, exploring the phosphotyrosine (pTyr)-SH2 interaction site, yielded many small molecules for STAT3 but sparsely for other STATs. However, many of these inhibitors seem not STAT3-specific, thereby questioning the present modeling and selection strategies of SH2 domain-based STAT inhibitors. We generated new 3D structure models for all human (h)STATs and developed a comparative in silico docking strategy to obtain further insight into STAT-SH2 cross-binding specificity of a selection of previously identified STAT3 inhibitors. Indeed, by primarily targeting the highly conserved pTyr-SH2 binding pocket the majority of these compounds exhibited similar binding affinity and tendency scores for all STATs. By comparative screening of a natural product library we provided initial proof for the possibility to identify STAT1 as well as STAT3-specific inhibitors, introducing the ‘STAT-comparative binding affinity value’ and ‘ligand binding pose variation’ as selection criteria. In silico screening of a multi-million clean leads (CL) compound library for binding of all STATs, likewise identified potential specific inhibitors for STAT1 and STAT3 after docking validation. Based on comparative virtual screening and docking validation, we developed a novel STAT inhibitor screening tool that allows identification of specific STAT1 and STAT3 inhibitory compounds. This could increase our understanding of the functional role of these STATs in different diseases and benefit the clinical need for more drugable STAT inhibitors with high specificity, potency and excellent bioavailability. PMID:25710482

The SH2-containing inositol polyphosphate 5-phosphatase, SHIP-2, binds filamin and regulates submembraneous actin

PubMed Central

Dyson, Jennifer M.; O'Malley, Cindy J.; Becanovic, Jelena; Munday, Adam D.; Berndt, Michael C.; Coghill, Imogen D.; Nandurkar, Harshal H.; Ooms, Lisa M.; Mitchell, Christina A.

2001-01-01

SHIP-2 is a phosphoinositidylinositol 3,4,5 trisphosphate (PtdIns[3,4,5]P3) 5-phosphatase that contains an NH2-terminal SH2 domain, a central 5-phosphatase domain, and a COOH-terminal proline-rich domain. SHIP-2 negatively regulates insulin signaling. In unstimulated cells, SHIP-2 localized in a perinuclear cytosolic distribution and at the leading edge of the cell. Endogenous and recombinant SHIP-2 localized to membrane ruffles, which were mediated by the COOH-terminal proline–rich domain. To identify proteins that bind to the SHIP-2 proline–rich domain, yeast two-hybrid screening was performed, which isolated actin-binding protein filamin C. In addition, both filamin A and B specifically interacted with SHIP-2 in this assay. SHIP-2 coimmunoprecipitated with filamin from COS-7 cells, and association between these species did not change after epidermal growth factor stimulation. SHIP-2 colocalized with filamin at Z-lines and the sarcolemma in striated muscle sections and at membrane ruffles in COS-7 cells, although the membrane ruffling response was reduced in cells overexpressing SHIP-2. SHIP-2 membrane ruffle localization was dependent on filamin binding, as SHIP-2 was expressed exclusively in the cytosol of filamin-deficient cells. Recombinant SHIP-2 regulated PtdIns(3,4,5)P3 levels and submembraneous actin at membrane ruffles after growth factor stimulation, dependent on SHIP-2 catalytic activity. Collectively these studies demonstrate that filamin-dependent SHIP-2 localization critically regulates phosphatidylinositol 3 kinase signaling to the actin cytoskeleton. PMID:11739414

MERTK interactions with SH2-domain proteins in the retinal pigment epithelium.

PubMed

Shelby, Shameka J; Colwill, Karen; Dhe-Paganon, Sirano; Pawson, Tony; Thompson, Debra A

2013-01-01

The receptor tyrosine kinase MERTK plays an essential role in the phagocytic uptake of shed photoreceptor membranes by the retinal pigment epithelium (RPE). A fundamental aspect of signal transduction by receptor tyrosine kinases involves autophosphorylation of tyrosine residues that recruit Src-homology 2 (SH2)-domain proteins to the receptor intracellular domain. The goal of the current study was to evaluate the interactions of human MERTK with SH2-domain proteins present in the RPE. The MERTK intracellular domain was expressed as a 6xHis-fusion protein (6xHis-rMERTK(571-999)), purified and phosphorylated. Ni(2+)-NTA pull downs were performed using 6xHis-rMERTK(571-999) in incubations with recombinant phosphotyrosine-recognition sequences expressed as GST-fusion proteins. In addition, pull downs of native SH2-domain proteins were performed using 6xHis-rMERTK(571-999) and protein homogenates from rat RPE/choroid. For both recombinant and native proteins, western analysis detected MERTK interactions with GRB2, PIK3R1 (P85α), VAV3, and SRC. Immunohistochemical analysis localized each protein to mouse RPE. In cultured RPE-J cells incubated with rod outer segments (OS), siRNA knockdown of Grb2 had no effect on OS binding, but significantly reduced OS uptake. Pik3r1 localized to early phagosomes along with Rab5 and Eea1. Phosphorylation and activation of Src was detected downstream of phagocytosis and Mertk activation. These findings suggest that MERTK signaling in the RPE involves a cohort of SH2-domain proteins with the potential to regulate both cytoskeletal rearrangement and membrane movement. Identification of the SH2-domain signaling partners of MERTK is an important step toward further defining the mechanism of RPE phagocytosis that is central to the function and survival of the retina.

Tyrosine phosphorylation of 3BP2 is indispensable for the interaction with VAV3 in chicken DT40 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chihara, Kazuyasu; Organization for Life Science Advancement Programs, University of Fukui, Fukui 910-1193; Kimura, Yukihiro

Adaptor protein c-Abl SH3 domain-binding protein-2 (3BP2) is known to play regulatory roles in immunoreceptor-mediated signal transduction. We have previously demonstrated that Tyr{sup 174}, Tyr{sup 183} and Tyr{sup 446} in mouse 3BP2 are predominantly phosphorylated by Syk, and the phosphorylation of Tyr{sup 183} and the Src homology 2 (SH2) domain of mouse 3BP2 are critical for B cell receptor (BCR)-induced activation of nuclear factor of activated T cells (NFAT) in human B cells. In this report, we have shown that Syk, but not Abl family protein-tyrosine kinases, is critical for BCR-mediated tyrosine phosphorylation of 3BP2 in chicken DT40 cells. Mutationalmore » analysis showed that Tyr{sup 174}, Tyr{sup 183} and Tyr{sup 426} of chicken 3BP2 are the major phosphorylation sites by Syk and the SH2 domain of 3BP2 is critical for tyrosine phosphorylation. In addition, phosphorylation of Tyr{sup 426} is required for the inducible interaction with the SH2 domain of Vav3. Moreover, the expression of the mutant form of 3BP2 in which Tyr{sup 426} was substituted to Phe resulted in the reduction in BCR-mediated Rac1 activation, when compared with the case of wild-type. Altogether, these data suggest that 3BP2 is involved in the activation of Rac1 through the regulation of Vav3 by Syk-dependent phosphorylation of Tyr{sup 426} following BCR stimulation. - Highlights: • 3BP2 is phosphorylated by Syk, but not Abl family kinases in BCR signaling. • Tyr183 and Tyr426 in chicken 3BP2 are the major phosphorylation sites by Syk. • The SH2 domain of 3BP2 is critical for tyrosine phosphorylation of 3BP2. • Phosphorylation of Tyr426 in 3BP2 is required for the inducible binding with Vav3. • 3BP2 is involved in the regulation of BCR-mediated Rac1 activation.« less

Real-Time Single Molecule Visualization of SH2 Domain Membrane Recruitment in Growth Factor Stimulated Cells.

PubMed

Oh, Dongmyung

2017-01-01

In the last decade, single molecule tracking (SMT) techniques have emerged as a versatile tool for molecular cell biology research. This approach allows researchers to monitor the real-time behavior of individual molecules in living cells with nanometer and millisecond resolution. As a result, it is possible to visualize biological processes as they occur at a molecular level in real time. Here we describe a method for the real-time visualization of SH2 domain membrane recruitment from the cytoplasm to epidermal growth factor (EGF) induced phosphotyrosine sites on the EGF receptor. Further, we describe methods that utilize SMT data to define SH2 domain membrane dynamics parameters such as binding (τ), dissociation (k d ), and diffusion (D) rates. Together these methods may allow us to gain greater understanding of signal transduction dynamics and the molecular basis of disease-related aberrant pathways.

Phosphotyrosine signaling proteins that drive oncogenesis tend to be highly interconnected.

PubMed

Koytiger, Grigoriy; Kaushansky, Alexis; Gordus, Andrew; Rush, John; Sorger, Peter K; MacBeath, Gavin

2013-05-01

Mutation and overexpression of receptor tyrosine kinases or the proteins they regulate serve as oncogenic drivers in diverse cancers. To better understand receptor tyrosine kinase signaling and its link to oncogenesis, we used protein microarrays to systematically and quantitatively measure interactions between virtually every SH2 or PTB domain encoded in the human genome and all known sites of tyrosine phosphorylation on 40 receptor tyrosine kinases and on most of the SH2 and PTB domain-containing adaptor proteins. We found that adaptor proteins, like RTKs, have many high affinity bindings sites for other adaptor proteins. In addition, proteins that drive cancer, including both receptors and adaptor proteins, tend to be much more highly interconnected via networks of SH2 and PTB domain-mediated interactions than nononcogenic proteins. Our results suggest that network topological properties such as connectivity can be used to prioritize new drug targets in this well-studied family of signaling proteins.

Interaction with the Src homology (SH3-SH2) region of the Src-family kinase Hck structures the HIV-1 Nef dimer for kinase activation and effector recruitment.

PubMed

Alvarado, John Jeff; Tarafdar, Sreya; Yeh, Joanne I; Smithgall, Thomas E

2014-10-10

HIV-1 Nef supports high titer viral replication in vivo and is essential for AIDS progression. Nef function depends on interactions with multiple host cell effectors, including Hck and other Src-family kinases. Here we describe the x-ray crystal structure of Nef in complex with the Hck SH3-SH2 regulatory region to a resolution of 1.86 Å. The complex crystallized as a dimer of complexes, with the conserved Nef PXXPXR motif engaging the Hck SH3 domain. A new intercomplex contact was found between SH3 Glu-93, and Nef Arg-105. Mutagenesis of Hck SH3 Glu-93 interfered with Nef·Hck complex formation and kinase activation in cells. The Hck SH2 domains impinge on the N-terminal region of Nef to stabilize a dimer conformation that exposes Asp-123, a residue critical for Nef function. Our results suggest that in addition to serving as a kinase effector for Nef, Hck binding may reorganize the Nef dimer for functional interaction with other signaling partners. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Interaction with the Src Homology (SH3-SH2) Region of the Src-family Kinase Hck Structures the HIV-1 Nef Dimer for Kinase Activation and Effector Recruitment*

PubMed Central

Alvarado, John Jeff; Tarafdar, Sreya; Yeh, Joanne I.; Smithgall, Thomas E.

2014-01-01

HIV-1 Nef supports high titer viral replication in vivo and is essential for AIDS progression. Nef function depends on interactions with multiple host cell effectors, including Hck and other Src-family kinases. Here we describe the x-ray crystal structure of Nef in complex with the Hck SH3-SH2 regulatory region to a resolution of 1.86 Å. The complex crystallized as a dimer of complexes, with the conserved Nef PXXPXR motif engaging the Hck SH3 domain. A new intercomplex contact was found between SH3 Glu-93, and Nef Arg-105. Mutagenesis of Hck SH3 Glu-93 interfered with Nef·Hck complex formation and kinase activation in cells. The Hck SH2 domains impinge on the N-terminal region of Nef to stabilize a dimer conformation that exposes Asp-123, a residue critical for Nef function. Our results suggest that in addition to serving as a kinase effector for Nef, Hck binding may reorganize the Nef dimer for functional interaction with other signaling partners. PMID:25122770

SH2/SH3 adaptor proteins can link tyrosine kinases to a Ste20-related protein kinase, HPK1.

PubMed

Anafi, M; Kiefer, F; Gish, G D; Mbamalu, G; Iscove, N N; Pawson, T

1997-10-31

Ste20-related protein kinases have been implicated as regulating a range of cellular responses, including stress-activated protein kinase pathways and the control of cytoskeletal architecture. An important issue involves the identities of the upstream signals and regulators that might control the biological functions of mammalian Ste20-related protein kinases. HPK1 is a protein-serine/threonine kinase that possesses a Ste20-like kinase domain, and in transfected cells activates a protein kinase pathway leading to the stress-activated protein kinase SAPK/JNK. Here we have investigated candidate upstream regulators that might interact with HPK1. HPK1 possesses an N-terminal catalytic domain and an extended C-terminal tail with four proline-rich motifs. The SH3 domains of Grb2 bound in vitro to specific proline-rich motifs in the HPK1 tail and functioned synergistically to direct the stable binding of Grb2 to HPK1 in transfected Cos1 cells. Epidermal growth factor (EGF) stimulation did not affect the binding of Grb2 to HPK1 but induced recruitment of the Grb2.HPK1 complex to the autophosphorylated EGF receptor and to the Shc docking protein. Several activated receptor and cytoplasmic tyrosine kinases, including the EGF receptor, stimulated the tyrosine phosphorylation of the HPK1 serine/threonine kinase. These results suggest that HPK1, a mammalian Ste20-related protein-serine/threonine kinase, can potentially associate with protein-tyrosine kinases through interactions mediated by SH2/SH3 adaptors such as Grb2. Such interaction may provide a possible mechanism for cross-talk between distinct biochemical pathways following the activation of tyrosine kinases.

The mechano-sensing role of the unique SH3 insertion in plakin domains revealed by Molecular Dynamics simulations.

PubMed

Daday, Csaba; Kolšek, Katra; Gräter, Frauke

2017-09-15

The plakin family of proteins, important actors in cross-linking force-bearing structures in the cell, contain a curious SH3 domain insertion in their chain of spectrin repeats (SRs). While SH3 domains are known to mediate protein-protein interactions, here, its canonical binding site is autoinhibited by the preceding SR. Under force, however, this SH3 domain could be released, and possibly launch a signaling cascade. We performed large-scale force-probe molecular dynamics simulations, across two orders of magnitude of loading rates, to test this hypothesis, on two prominent members of the plakin family: desmoplakin and plectin, obligate proteins at desmosomes and hemidesmosomes, respectively. Our simulations show that force unravels the SRs and abolishes the autoinhibition of the SH3 domain, an event well separated from the unfolding of this domain. The SH3 domain is free and fully functional for a significant portion of the unfolding trajectories. The rupture forces required for the two proteins significantly decrease when the SH3 domain is removed, which implies that the SH3 domain also stabilizes this junction. Our results persist across all simulations, and support a force-sensing as well as a stabilizing role of the unique SH3 insertion, putting forward this protein family as a new class of mechano-sensors.

Comprehensive Binary Interaction Mapping of SH2 Domains via Fluorescence Polarization Reveals Novel Functional Diversification of ErbB Receptors

PubMed Central

Ciaccio, Mark F.; Chuu, Chih-pin; Jones, Richard B.

2012-01-01

First-generation interaction maps of Src homology 2 (SH2) domains with receptor tyrosine kinase (RTK) phosphosites have previously been generated using protein microarray (PM) technologies. Here, we developed a large-scale fluorescence polarization (FP) methodology that was able to characterize interactions between SH2 domains and ErbB receptor phosphosites with higher fidelity and sensitivity than was previously achieved with PMs. We used the FP assay to query the interaction of synthetic phosphopeptides corresponding to 89 ErbB receptor intracellular tyrosine sites against 93 human SH2 domains and 2 phosphotyrosine binding (PTB) domains. From 358,944 polarization measurements, the affinities for 1,405 unique biological interactions were determined, 83% of which are novel. In contrast to data from previous reports, our analyses suggested that ErbB2 was not more promiscuous than the other ErbB receptors. Our results showed that each receptor displays unique preferences in the affinity and location of recruited SH2 domains that may contribute to differences in downstream signaling potential. ErbB1 was enriched versus the other receptors for recruitment of domains from RAS GEFs whereas ErbB2 was enriched for recruitment of domains from tyrosine and phosphatidyl inositol phosphatases. ErbB3, the kinase inactive ErbB receptor family member, was predictably enriched for recruitment of domains from phosphatidyl inositol kinases and surprisingly, was enriched for recruitment of domains from tyrosine kinases, cytoskeletal regulatory proteins, and RHO GEFs but depleted for recruitment of domains from phosphatidyl inositol phosphatases. Many novel interactions were also observed with phosphopeptides corresponding to ErbB receptor tyrosines not previously reported to be phosphorylated by mass spectrometry, suggesting the existence of many biologically relevant RTK sites that may be phosphorylated but below the detection threshold of standard mass spectrometry procedures. This dataset represents a rich source of testable hypotheses regarding the biological mechanisms of ErbB receptors. PMID:22973453

The Fyn tyrosine kinase binds Irs-1 and forms a distinct signaling complex during insulin stimulation.

PubMed

Sun, X J; Pons, S; Asano, T; Myers, M G; Glasheen, E; White, M F

1996-05-03

Irs-proteins link the receptors for insulin/IGF-1, growth hormones, and several interleukins and interferons to signaling proteins that contain Src homology-2 (SH2). To identify new Irs-1-binding proteins, we screened a mouse embryo expression library with recombinant [32P]Irs-1, which revealed a specific association between p59fyn and Irs-1. The SH2 domain in p59fyn bound to phosphorylated Tyr895 and Tyr1172, which are located in YXX(L/I) motifs. Mutation of p59fyn at the COOH-terminal tyrosine phosphorylation site (Tyr531) enhanced its binding to Irs-1 during insulin stimulation. Binding experiments with various SH2 protein revealed that Grb-2 was largely excluded from Irs-1 complexes containing p59fyn, whereas Grb-2 and p85 occurred in the same Irs-1 complex. By comparison with the insulin receptor, p59fyn kinase phosphorylated a unique cohort of tyrosine residues in Irs-1. These results outline a role for p59fyn or other related Src-kinases during insulin and cytokine signaling.

Purification, crystallization, small-angle X-ray scattering and preliminary X-ray diffraction analysis of the SH2 domain of the Csk-homologous kinase.

PubMed

Gunn, Natalie J; Gorman, Michael A; Dobson, Renwick C J; Parker, Michael W; Mulhern, Terrence D

2011-03-01

The C-terminal Src kinase (Csk) and Csk-homologous kinase (CHK) are endogenous inhibitors of the proto-oncogenic Src family of protein tyrosine kinases (SFKs). Phosphotyrosyl peptide binding to their Src-homology 2 (SH2) domains activates Csk and CHK, enhancing their ability to suppress SFK signalling; however, the detailed mechanistic basis of this activation event is unclear. The CHK SH2 was expressed in Escherichia coli and the purified protein was characterized as monomeric by synchrotron small-angle X-ray scattering in-line with size-exclusion chromatography. The CHK SH2 crystallized in 0.2 M sodium bromide, 0.1 M bis-Tris propane pH 6.5 and 20% polyethylene glycol 3350 and the best crystals diffracted to ∼1.6 Å resolution. The crystals belonged to space group P2, with unit-cell parameters a=25.8, b=34.6, c=63.2 Å, β=99.4°.

Using genome-wide measurements for computational prediction of SH2–peptide interactions

PubMed Central

Wunderlich, Zeba; Mirny, Leonid A.

2009-01-01

Peptide-recognition modules (PRMs) are used throughout biology to mediate protein–protein interactions, and many PRMs are members of large protein domain families. Recent genome-wide measurements describe networks of peptide–PRM interactions. In these networks, very similar PRMs recognize distinct sets of peptides, raising the question of how peptide-recognition specificity is achieved using similar protein domains. The analysis of individual protein complex structures often gives answers that are not easily applicable to other members of the same PRM family. Bioinformatics-based approaches, one the other hand, may be difficult to interpret physically. Here we integrate structural information with a large, quantitative data set of SH2 domain–peptide interactions to study the physical origin of domain–peptide specificity. We develop an energy model, inspired by protein folding, based on interactions between the amino-acid positions in the domain and peptide. We use this model to successfully predict which SH2 domains and peptides interact and uncover the positions in each that are important for specificity. The energy model is general enough that it can be applied to other members of the SH2 family or to new peptides, and the cross-validation results suggest that these energy calculations will be useful for predicting binding interactions. It can also be adapted to study other PRM families, predict optimal peptides for a given SH2 domain, or study other biological interactions, e.g. protein–DNA interactions. PMID:19502496

Comparison of SH3 and SH2 domain dynamics when expressed alone or in an SH(3+2) construct: the role of protein dynamics in functional regulation.

PubMed

Engen, J R; Smithgall, T E; Gmeiner, W H; Smith, D L

1999-04-02

Protein dynamics play an important role in protein function and regulation of enzymatic activity. To determine how additional interactions with surrounding structure affects local protein dynamics, we have used hydrogen exchange and mass spectrometry to investigate the SH2 and SH3 domains of the protein tyrosine kinase Hck. Exchange rates of isolated Hck SH3 and SH2 domains were compared with rates for the same domains when part of a larger SH(3+2) construct. Increased deuterium incorporation was observed for the SH3 domain in the joint construct, particularly near the SH2 interface and the short sequence that connects SH3 to SH2, implying greater flexibility of SH3 when it is part of SH(3+2). Slow cooperative unfolding of the SH3 domain occurred at the same rate in isolated SH3 as in the SH(3+2) construct, suggesting a functional significance for this unfolding. The SH2 domain displayed relatively smaller changes in flexibility when part of the SH(3+2) construct. These results suggest that the domains influence each other. Further, our results imply a link between functional regulation and structural dynamics of SH3 and SH2 domains. Copyright 1999 Academic Press.

BASH, a novel signaling molecule preferentially expressed in B cells of the bursa of Fabricius.

PubMed

Goitsuka, R; Fujimura, Y; Mamada, H; Umeda, A; Morimura, T; Uetsuka, K; Doi, K; Tsuji, S; Kitamura, D

1998-12-01

The bursa of Fabricius is a gut-associated lymphoid organ that is essential for the generation of a diversified B cell repertoire in the chicken. We describe here a novel gene preferentially expressed in bursal B cells. The gene encodes an 85-kDa protein, designated BASH (B cell adaptor containing SH2 domain), that contains N-terminal acidic domains with SH2 domain-binding phosphotyrosine-based motifs, a proline-rich domain, and a C-terminal SH2 domain. BASH shows a substantial sequence similarity to SLP-76, an adaptor protein functioning in TCR-signal transduction. BASH becomes tyrosine-phosphorylated with the B cell Ag receptor (BCR) cross-link or by coexpression with Syk and Lyn and associates with signaling molecules including Syk and a putative chicken Shc homologue. Overexpression of BASH results in suppression of the NF-AT activation induced by BCR-cross-linking. These findings suggest that BASH is involved in BCR-mediated signal transduction and could play a critical role in B cell development in the bursa.

A Cysteine-Rich CCG Domain Contains a Novel [4Fe-4S] Cluster Binding Motif As Deduced From Studies With Subunit B of Heterodisulfide Reductase From Methanothermobacter Marburgensis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hamann, N.; Mander, G.J.; Shokes, J.E.

Heterodisulfide reductase (HDR) of methanogenic archaea with its active-site [4Fe-4S] cluster catalyzes the reversible reduction of the heterodisulfide (CoM-S-S-CoB) of the methanogenic coenzyme M (CoM-SH) and coenzyme B (CoB-SH). CoM-HDR, a mechanistic-based paramagnetic intermediate generated upon half-reaction of the oxidized enzyme with CoM-SH, is a novel type of [4Fe-4S]{sup 3+} cluster with CoM-SH as a ligand. Subunit HdrB of the Methanothermobacter marburgensis HdrABC holoenzyme contains two cysteine-rich sequence motifs (CX{sub 31-39}CCX{sub 35-36}CXXC), designated as CCG domain in the Pfam database and conserved in many proteins. Here we present experimental evidence that the C-terminal CCG domain of HdrB binds this unusualmore » [4Fe-4S] cluster. HdrB was produced in Escherichia coli, and an iron-sulfur cluster was subsequently inserted by in vitro reconstitution. In the oxidized state the cluster without the substrate exhibited a rhombic EPR signal (g{sub zyx} = 2.015, 1.995, and 1.950) reminiscent of the CoM-HDR signal. {sup 57}Fe ENDOR spectroscopy revealed that this paramagnetic species is a [4Fe-4S] cluster with {sup 57}Fe hyperfine couplings very similar to that of CoM-HDR. CoM-{sup 33}SH resulted in a broadening of the EPR signal, and upon addition of CoM-SH the midpoint potential of the cluster was shifted to values observed for CoM-HDR, both indicating binding of CoM-SH to the cluster. Site-directed mutagenesis of all 12 cysteine residues in HdrB identified four cysteines of the C-terminal CCG domain as cluster ligands. Combined with the previous detection of CoM-HDR-like EPR signals in other CCG domain-containing proteins our data indicate a general role of the C-terminal CCG domain in coordination of this novel [4Fe-4S] cluster. In addition, Zn K-edge X-ray absorption spectroscopy identified an isolated Zn site with an S{sub 3}(O/N){sub 1} geometry in HdrB and the HDR holoenzyme. The N-terminal CCG domain is suggested to provide ligands to the Zn site.« less

A Drosophila protein-tyrosine phosphatase associates with an adapter protein required for axonal guidance.

PubMed

Clemens, J C; Ursuliak, Z; Clemens, K K; Price, J V; Dixon, J E

1996-07-19

We have used the yeast two-hybrid system to isolate a novel Drosophila adapter protein, which interacts with the Drosophila protein-tyrosine phosphatase (PTP) dPTP61F. Absence of this protein in Drosophila causes the mutant photoreceptor axon phenotype dreadlocks (dock) (Garrity, P. A., Rao, Y., Salecker, I., and Zipursky, S. L.(1996) Cell 85, 639-650). Dock is similar to the mammalian oncoprotein Nck and contains three Src homology 3 (SH3) domains and one Src homology 2 (SH2) domain. The interaction of dPTP61F with Dock was confirmed in vivo by immune precipitation experiments. A sequence containing five PXXP motifs from the non-catalytic domain of the PTP is sufficient for interaction with Dock. This suggests that binding to the PTP is mediated by one or more of the SH3 domains of Dock. Immune precipitations of Dock also co-precipitate two tyrosine-phosphorylated proteins having molecular masses of 190 and 145 kDa. Interactions between Dock and these tyrosine-phosphorylated proteins are likely mediated by the Dock SH2 domain. These findings identify potential signal-transducing partners of Dock and propose a role for dPTP61F and the unidentified phosphoproteins in axonal guidance.

Dynamics of the Tec‐family tyrosine kinase SH3 domains

PubMed Central

Roberts, Justin M.; Tarafdar, Sreya; Joseph, Raji E.; Andreotti, Amy H.; Smithgall, Thomas E.; Engen, John R.

2016-01-01

Abstract The Src Homology 3 (SH3) domain is an important regulatory domain found in many signaling proteins. X‐ray crystallography and NMR structures of SH3 domains are generally conserved but other studies indicate that protein flexibility and dynamics are not. We previously reported that based on hydrogen exchange mass spectrometry (HX MS) studies, there is variable flexibility and dynamics among the SH3 domains of the Src‐family tyrosine kinases and related proteins. Here we have extended our studies to the SH3 domains of the Tec family tyrosine kinases (Itk, Btk, Tec, Txk, Bmx). The SH3 domains of members of this family augment the variety in dynamics observed in previous SH3 domains. Txk and Bmx SH3 were found to be highly dynamic in solution by HX MS and Bmx was unstructured by NMR. Itk and Btk SH3 underwent a clear EX1 cooperative unfolding event, which was localized using pepsin digestion and mass spectrometry after hydrogen exchange labeling. The unfolding was localized to peptide regions that had been previously identified in the Src‐family and related protein SH3 domains, yet the kinetics of unfolding were not. Sequence alignment does not provide an easy explanation for the observed dynamics behavior, yet the similarity of location of EX1 unfolding suggests that higher‐order structural properties may play a role. While the exact reason for such dynamics is not clear, such motions can be exploited in intra‐ and intermolecular binding assays of proteins containing the domains. PMID:26808198

Preferred SH3 Domain Partners of ADAM Metalloproteases Include Shared and ADAM-Specific SH3 Interactions

PubMed Central

Kleino, Iivari; Järviluoma, Annika; Hepojoki, Jussi; Huovila, Ari Pekka; Saksela, Kalle

2015-01-01

A disintegrin and metalloproteinases (ADAMs) constitute a protein family essential for extracellular signaling and regulation of cell adhesion. Catalytic activity of ADAMs and their predicted potential for Src-homology 3 (SH3) domain binding show a strong correlation. Here we present a comprehensive characterization of SH3 binding capacity and preferences of the catalytically active ADAMs 8, 9, 10, 12, 15, 17, and 19. Our results revealed several novel interactions, and also confirmed many previously reported ones. Many of the identified SH3 interaction partners were shared by several ADAMs, whereas some were ADAM-specific. Most of the ADAM-interacting SH3 proteins were adapter proteins or kinases, typically associated with sorting and endocytosis. Novel SH3 interactions revealed in this study include TOCA1 and CIP4 as preferred partners of ADAM8, and RIMBP1 as a partner of ADAM19. Our results suggest that common as well as distinct mechanisms are involved in regulation and execution of ADAM signaling, and provide a useful framework for addressing the pathways that connect ADAMs to normal and aberrant cell behavior. PMID:25825872

Classification and Lineage Tracing of SH2 Domains Throughout Eukaryotes.

PubMed

Liu, Bernard A

2017-01-01

Today there exists a rapidly expanding number of sequenced genomes. Cataloging protein interaction domains such as the Src Homology 2 (SH2) domain across these various genomes can be accomplished with ease due to existing algorithms and predictions models. An evolutionary analysis of SH2 domains provides a step towards understanding how SH2 proteins integrated with existing signaling networks to position phosphotyrosine signaling as a crucial driver of robust cellular communication networks in metazoans. However organizing and tracing SH2 domain across organisms and understanding their evolutionary trajectory remains a challenge. This chapter describes several methodologies towards analyzing the evolutionary trajectory of SH2 domains including a global SH2 domain classification system, which facilitates annotation of new SH2 sequences essential for tracing the lineage of SH2 domains throughout eukaryote evolution. This classification utilizes a combination of sequence homology, protein domain architecture and the boundary positions between introns and exons within the SH2 domain or genes encoding these domains. Discrete SH2 families can then be traced across various genomes to provide insight into its origins. Furthermore, additional methods for examining potential mechanisms for divergence of SH2 domains from structural changes to alterations in the protein domain content and genome duplication will be discussed. Therefore a better understanding of SH2 domain evolution may enhance our insight into the emergence of phosphotyrosine signaling and the expansion of protein interaction domains.

Non-canonical dynamic mechanisms of interaction between the p66Shc protein and Met receptor

PubMed Central

Landry, Mélissa; Pomerleau, Véronique; Saucier, Caroline

2016-01-01

Met receptor tyrosine kinase (RTK) is known to bind to the three distinct protein isoforms encoded by the ShcA (Shc) gene. Structure–function studies have unveiled critical roles for p52Shc-dependent signalling pathways in Met-regulated biological functions. The molecular basis of the interaction between the Met and p52Shc proteins is well-defined, but not for the longest protein isoform, p66Shc. In the present study, co-immunoprecipitation assays were performed in human embryonic kidney 293 (HEK293) cells, transiently co-transfected with Met and p66Shc mutants, in order to define the molecular determinants involved in mediating Met–p66Shc interaction. Our results show that p66Shc interacts constitutively with the receptor Met, and the Grb2 (growth factor receptor-bound protein-2) and Gab1 (Grb2-associated binder-1) adaptor proteins. Although its phosphotyrosine-binding domain (PTB) and Src homology 2 (SH2) domains co-ordinate p66Shc binding to non-activated Met receptor, these phosphotyrosine-binding modules, and its collagen homology domain 2 (CH2) region, exert negative constraints. In contrast, p66Shc interaction with the activated Met depends mainly on the integrity of its PTB domain, and to a lesser extent of its SH2 domain. Even though not required for the recruitment of p66Shc, tyrosine phosphorylation of p66Shc by activated Met enhances these interactions by mechanisms not reliant on the integrity of the Met multisubstrate-binding site. In turn, this increases phosphotyrosine-dependent p66Shc–Grb2–Gab1 complex formation away from the receptor, while blocking Grb2 and Gab1 recruitment to activated Met. In conclusion, we identify, for the first time, a novel non-canonical dynamic mode of interaction between Met and the p66 protein isoform of Shc and its effects on rewiring binding effector complexes according to the activation state of the receptor. PMID:27048591

Can free energy calculations be fast and accurate at the same time? Binding of low-affinity, non-peptide inhibitors to the SH2 domain of the src protein

NASA Astrophysics Data System (ADS)

Chipot, Christophe; Rozanska, Xavier; Dixit, Surjit B.

2005-11-01

The usefulness of free-energy calculations in non-academic environments, in general, and in the pharmaceutical industry, in particular, is a long-time debated issue, often considered from the angle of cost/performance criteria. In the context of the rational drug design of low-affinity, non-peptide inhibitors to the SH2 domain of the pp60src tyrosine kinase, the continuing difficulties encountered in an attempt to obtain accurate free-energy estimates are addressed. free-energy calculations can provide a convincing answer, assuming that two key-requirements are fulfilled: (i) thorough sampling of the configurational space is necessary to minimize the statistical error, hence raising the question: to which extent can we sacrifice the computational effort, yet without jeopardizing the precision of the free-energy calculation? (ii) the sensitivity of binding free-energies to the parameters utilized imposes an appropriate parametrization of the potential energy function, especially for non-peptide molecules that are usually poorly described by multipurpose macromolecular force fields. Employing the free-energy perturbation method, accurate ranking, within ±0.7 kcal/mol, is obtained in the case of four non-peptide mimes of a sequence recognized by the pp60src SH2 domain.

Bio-mimicking of Proline-Rich Motif Applied to Carbon Nanotube Reveals Unexpected Subtleties Underlying Nanoparticle Functionalization

NASA Astrophysics Data System (ADS)

Zhang, Yuanzhao; Jimenez-Cruz, Camilo A.; Wang, Jian; Zhou, Bo; Yang, Zaixing; Zhou, Ruhong

2014-11-01

Here, we report computational studies of the SH3 protein domain interacting with various single-walled carbon nanotubes (SWCNT) either bare or functionalized by mimicking the proline-rich motif (PRM) ligand (PPPVPPRR) and compare it to the SH3-PRM complex binding. With prolines or a single arginine attached, the SWCNT gained slightly on specificity when compared with the bare control, whereas with multi-arginine systems the specificity dropped dramatically to our surprise. Although the electrostatic interaction provided by arginines is crucial in the recognition between PRM and SH3 domain, our results suggest that attaching multiple arginines to the SWCNT has a detrimental effect on the binding affinity. Detailed analysis of the MD trajectories found two main factors that modulate the specificity of the binding: the existence of competing acidic patches at the surface of SH3 that leads to ``trapping and clamping'' by the arginines, and the rigidity of the SWCNT introducing entropic penalties in the proper binding. Further investigation revealed that the same ``clamping'' phenomenon exits in the PRM-SH3 system, which has not been reported in previous literature. The competing effects between nanoparticle and its functionalization components revealed by our model system should be of value to current and future nanomedicine designs.

Bio-mimicking of Proline-Rich Motif Applied to Carbon Nanotube Reveals Unexpected Subtleties Underlying Nanoparticle Functionalization

PubMed Central

Zhang, Yuanzhao; Jimenez-Cruz, Camilo A.; Wang, Jian; Zhou, Bo; Yang, Zaixing; Zhou, Ruhong

2014-01-01

Here, we report computational studies of the SH3 protein domain interacting with various single-walled carbon nanotubes (SWCNT) either bare or functionalized by mimicking the proline-rich motif (PRM) ligand (PPPVPPRR) and compare it to the SH3-PRM complex binding. With prolines or a single arginine attached, the SWCNT gained slightly on specificity when compared with the bare control, whereas with multi-arginine systems the specificity dropped dramatically to our surprise. Although the electrostatic interaction provided by arginines is crucial in the recognition between PRM and SH3 domain, our results suggest that attaching multiple arginines to the SWCNT has a detrimental effect on the binding affinity. Detailed analysis of the MD trajectories found two main factors that modulate the specificity of the binding: the existence of competing acidic patches at the surface of SH3 that leads to “trapping and clamping” by the arginines, and the rigidity of the SWCNT introducing entropic penalties in the proper binding. Further investigation revealed that the same “clamping” phenomenon exits in the PRM-SH3 system, which has not been reported in previous literature. The competing effects between nanoparticle and its functionalization components revealed by our model system should be of value to current and future nanomedicine designs. PMID:25427563

Suppressor of cytokine signaling 1 interacts with oncogenic lymphocyte-specific protein tyrosine kinase.

PubMed

Venkitachalam, Srividya; Chueh, Fu-Yu; Leong, King-Fu; Pabich, Samantha; Yu, Chao-Lan

2011-03-01

Lymphocyte-specific protein tyrosine kinase (Lck) plays a key role in T cell signal transduction and is tightly regulated by phosphorylation and dephosphorylation. Lck can function as an oncoprotein when overexpressed or constantly activated by mutations. Our previous studies showed that Lck-induced cellular transformation could be suppressed by enforced expression of suppressor of cytokine signaling 1 (SOCS1), a SOCS family member involved in the negative feedback control of cytokine signaling. We observed attenuated Lck kinase activity in SOCS1-expressing cells, suggesting an important role of SOCS in regulating Lck functions. It remains largely unknown whether and how SOCS proteins interact with the oncogenic Lck kinase. Here, we report that among four SOCS family proteins, SOCS1, SOCS2, SOCS3 and CIS (cytokine-inducible SH2 domain containing protein), SOCS1 has the highest affinity in binding to the oncogenic Lck kinase. We identified the positive regulatory phosphotyrosine 394 residue in the kinase domain as the key interacting determinant in Lck. Additionally, the Lck kinase domain alone is sufficient to bind SOCS1. While the SH2 domain in SOCS1 is important in its association with the oncogenic Lck kinase, other functional domains may also contribute to overall binding affinity. These findings provide important mechanistic insights into the role of SOCS proteins as tumor suppressors in cells transformed by oncogenic protein tyrosine kinases.

Suppressor of cytokine signaling 1 interacts with oncogenic lymphocyte-specific protein tyrosine kinase

PubMed Central

VENKITACHALAM, SRIVIDYA; CHUEH, FU-YU; LEONG, KING-FU; PABICH, SAMANTHA; YU, CHAO-LAN

2011-01-01

Lymphocyte-specific protein tyrosine kinase (Lck) plays a key role in T cell signal transduction and is tightly regulated by phosphorylation and dephosphorylation. Lck can function as an oncoprotein when overexpressed or constantly activated by mutations. Our previous studies showed that Lck-induced cellular transformation could be suppressed by enforced expression of suppressor of cytokine signaling 1 (SOCS1), a SOCS family member involved in the negative feedback control of cytokine signaling. We observed attenuated Lck kinase activity in SOCS1-expressing cells, suggesting an important role of SOCS in regulating Lck functions. It remains largely unknown whether and how SOCS proteins interact with the oncogenic Lck kinase. Here we report that, among four SOCS family proteins, SOCS1, SOCS2, SOCS3 and CIS (cytokine–inducible SH2 domain containing protein), SOCS1 has the highest affinity in binding to the oncogenic Lck kinase. We identify the positive regulatory phospho-tyrosine 394 residue in the kinase domain as the key interacting determinant in Lck. Additionally, the Lck kinase domain alone is sufficient to bind SOCS1. While the SH2 domain in SOCS1 is important in its association with the oncogenic Lck kinase, other functional domains may also contribute to overall binding affinity. These findings provide important mechanistic insights into the role of SOCS proteins as tumor suppressors in cells transformed by oncogenic protein tyrosine kinases. PMID:21234523

Nckβ Adapter Regulates Actin Polymerization in NIH 3T3 Fibroblasts in Response to Platelet-Derived Growth Factor bb

PubMed Central

Chen, Min; She, Hongyun; Kim, Airie; Woodley, David T.; Li, Wei

2000-01-01

The SH3-SH3-SH3-SH2 adapter Nck represents a two-gene family that includes Nckα (Nck) and Nckβ (Grb4/Nck2), and it links receptor tyrosine kinases to intracellular signaling networks. The function of these mammalian Nck genes has not been established. We report here a specific role for Nckβ in platelet-derived growth factor (PDGF)-induced actin polymerization in NIH 3T3 cells. Overexpression of Nckβ but not Nckα blocks PDGF-stimulated membrane ruffling and formation of lamellipoda. Mutation in either the SH2 or the middle SH3 domain of Nckβ abolishes its interfering effect. Nckβ binds at Tyr-1009 in human PDGF receptor β (PDGFR-β) which is different from Nckα's binding site, Tyr-751, and does not compete with phosphatidylinositol-3 kinase for binding to PDGFR. Microinjection of an anti-Nckβ but not an anti-Nckα antibody inhibits PDGF-stimulated actin polymerization. Constitutively membrane-bound Nckβ but not Nckα blocks Rac1-L62-induced membrane ruffling and formation of lamellipodia, suggesting that Nckβ acts in parallel to or downstream of Rac1. This is the first report of Nckβ's role in receptor tyrosine kinase signaling to the actin cytoskeleton. PMID:11027258

SmShb, the SH2-Containing Adaptor Protein B of Schistosoma mansoni Regulates Venus Kinase Receptor Signaling Pathways

PubMed Central

Morel, Marion; Vanderstraete, Mathieu; Cailliau, Katia; Hahnel, Steffen; Grevelding, Christoph G.; Dissous, Colette

2016-01-01

Venus kinase receptors (VKRs) are invertebrate receptor tyrosine kinases (RTKs) formed by an extracellular Venus Fly Trap (VFT) ligand binding domain associated via a transmembrane domain with an intracellular tyrosine kinase (TK) domain. Schistosoma mansoni VKRs, SmVKR1 and SmVKR2, are both implicated in reproductive activities of the parasite. In this work, we show that the SH2 domain-containing protein SmShb is a partner of the phosphorylated form of SmVKR1. Expression of these proteins in Xenopus oocytes allowed us to demonstrate that the SH2 domain of SmShb interacts with the phosphotyrosine residue (pY979) located in the juxtamembrane region of SmVKR1. This interaction leads to phosphorylation of SmShb on tyrosines and promotes SmVKR1 signaling towards the JNK pathway. SmShb transcripts are expressed in all parasite stages and they were found in ovary and testes of adult worms, suggesting a possible colocalization of SmShb and SmVKR1 proteins. Silencing of SmShb in adult S. mansoni resulted in an accumulation of mature sperm in testes, indicating a possible role of SmShb in gametogenesis. PMID:27636711

SmShb, the SH2-Containing Adaptor Protein B of Schistosoma mansoni Regulates Venus Kinase Receptor Signaling Pathways.

PubMed

Morel, Marion; Vanderstraete, Mathieu; Cailliau, Katia; Hahnel, Steffen; Grevelding, Christoph G; Dissous, Colette

2016-01-01

Venus kinase receptors (VKRs) are invertebrate receptor tyrosine kinases (RTKs) formed by an extracellular Venus Fly Trap (VFT) ligand binding domain associated via a transmembrane domain with an intracellular tyrosine kinase (TK) domain. Schistosoma mansoni VKRs, SmVKR1 and SmVKR2, are both implicated in reproductive activities of the parasite. In this work, we show that the SH2 domain-containing protein SmShb is a partner of the phosphorylated form of SmVKR1. Expression of these proteins in Xenopus oocytes allowed us to demonstrate that the SH2 domain of SmShb interacts with the phosphotyrosine residue (pY979) located in the juxtamembrane region of SmVKR1. This interaction leads to phosphorylation of SmShb on tyrosines and promotes SmVKR1 signaling towards the JNK pathway. SmShb transcripts are expressed in all parasite stages and they were found in ovary and testes of adult worms, suggesting a possible colocalization of SmShb and SmVKR1 proteins. Silencing of SmShb in adult S. mansoni resulted in an accumulation of mature sperm in testes, indicating a possible role of SmShb in gametogenesis.

Functional Independence and Interdependence of the Src Homology Domains of Phospholipase C-γ1 in B-Cell Receptor Signal Transduction

PubMed Central

DeBell, Karen E.; Stoica, Bogdan A.; Verí, Maria-Concetta; Di Baldassarre, Angela; Miscia, Sebastiano; Graham, Laurie J.; Rellahan, Barbara L.; Ishiai, Masamichi; Kurosaki, Tomohiro; Bonvini, Ezio

1999-01-01

B-cell receptor (BCR)-induced activation of phospholipase C-γ1 (PLCγ1) and PLCγ2 is crucial for B-cell function. While several signaling molecules have been implicated in PLCγ activation, the mechanism coupling PLCγ to the BCR remains undefined. The role of PLCγ1 SH2 and SH3 domains at different steps of BCR-induced PLCγ1 activation was examined by reconstitution in a PLCγ-negative B-cell line. PLCγ1 membrane translocation required a functional SH2 N-terminal [SH2(N)] domain, was decreased by mutation of the SH3 domain, but was unaffected by mutation of the SH2(C) domain. Tyrosine phosphorylation did not require the SH2(C) or SH3 domains but depended exclusively on a functional SH2(N) domain, which mediated the association of PLCγ1 with the adapter protein, BLNK. Forcing PLCγ1 to the membrane via a myristoylation signal did not bypass the SH2(N) domain requirement for phosphorylation, indicating that the phosphorylation mediated by this domain is not due to membrane anchoring alone. Mutation of the SH2(N) or the SH2(C) domain abrogated BCR-stimulated phosphoinositide hydrolysis and signaling events, while mutation of the SH3 domain partially decreased signaling. PLCγ1 SH domains, therefore, have interrelated but distinct roles in BCR-induced PLCγ1 activation. PMID:10523627

HRD Motif as the Central Hub of the Signaling Network for Activation Loop Autophosphorylation in Abl Kinase.

PubMed

La Sala, Giuseppina; Riccardi, Laura; Gaspari, Roberto; Cavalli, Andrea; Hantschel, Oliver; De Vivo, Marco

2016-11-08

A number of structural factors modulate the activity of Abelson (Abl) tyrosine kinase, whose deregulation is often related to oncogenic processes. First, only the open conformation of the Abl kinase domain's activation loop (A-loop) favors ATP binding to the catalytic cleft. In this regard, the trans-autophosphorylation of the Y412 residue, which is located along the A-loop, favors the stability of the open conformation, in turn enhancing Abl activity. Another key factor for full Abl activity is the formation of active conformations of the catalytic DFG motif in the Abl kinase domain. Furthermore, binding of the SH2 domain to the N-lobe of the Abl kinase was recently demonstrated to have a long-range allosteric effect on the stabilization of the A-loop open state. Intriguingly, these distinct structural factors imply a complex signal transmission network for controlling the A-loop's flexibility and conformational preference for optimal Abl function. However, the exact dynamical features of this signal transmission network structure remain unclear. Here, we report on microsecond-long molecular dynamics coupled with enhanced sampling simulations of multiple Abl model systems, in the presence or absence of the SH2 domain and with the DFG motif flipped in two ways (in or out conformation). Through comparative analysis, our simulations augment the interpretation of the existing Abl experimental data, revealing a dynamical network of interactions that interconnect SH2 domain binding with A-loop plasticity and Y412 autophosphorylation in Abl. This signaling network engages the DFG motif and, importantly, other conserved structural elements of the kinase domain, namely, the EPK-ELK H-bond network and the HRD motif. Our results show that the signal propagation for modulating the A-loop spatial localization is highly dependent on the HRD motif conformation, which thus acts as the central hub of this (allosteric) signaling network controlling Abl activation and function.

Chemical Ligation of Folded Recombinant Proteins: Segmental Isotopic Labeling of Domains for NMR Studies

NASA Astrophysics Data System (ADS)

Xu, Rong; Ayers, Brenda; Cowburn, David; Muir, Tom W.

1999-01-01

A convenient in vitro chemical ligation strategy has been developed that allows folded recombinant proteins to be joined together. This strategy permits segmental, selective isotopic labeling of the product. The src homology type 3 and 2 domains (SH3 and SH2) of Abelson protein tyrosine kinase, which constitute the regulatory apparatus of the protein, were individually prepared in reactive forms that can be ligated together under normal protein-folding conditions to form a normal peptide bond at the ligation junction. This strategy was used to prepare NMR sample quantities of the Abelson protein tyrosine kinase-SH(32) domain pair, in which only one of the domains was labeled with 15N Mass spectrometry and NMR analyses were used to confirm the structure of the ligated protein, which was also shown to have appropriate ligand-binding properties. The ability to prepare recombinant proteins with selectively labeled segments having a single-site mutation, by using a combination of expression of fusion proteins and chemical ligation in vitro, will increase the size limits for protein structural determination in solution with NMR methods. In vitro chemical ligation of expressed protein domains will also provide a combinatorial approach to the synthesis of linked protein domains.

Crystal structure of the Rasputin NTF2-like domain from Drosophila melanogaster

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vognsen, Tina, E-mail: tv@farma.ku.dk; Kristensen, Ole, E-mail: ok@farma.ku.dk

2012-03-30

Highlights: Black-Right-Pointing-Pointer The crystal structure of the NTF2-like domain of Rasputin protein is presented. Black-Right-Pointing-Pointer Differences to known ligand binding sites of nuclear transport factor 2 are discussed. Black-Right-Pointing-Pointer A new ligand binding site for the Rasputin and G3BP proteins is proposed. -- Abstract: The crystal structure of the NTF2-like domain of the Drosophila homolog of Ras GTPase SH3 Binding Protein (G3BP), Rasputin, was determined at 2.7 A resolution. The overall structure is highly similar to nuclear transport factor 2: It is a homodimer comprised of a {beta}-sheet and three {alpha}-helices forming a cone-like shape. However, known binding sites formore » RanGDP and FxFG containing peptides show electrostatic and steric differences compared to nuclear transport factor 2. A HEPES molecule bound in the structure suggests a new, and possibly physiologically relevant, ligand binding site.« less

The proto-oncogene product p120CBL and the adaptor proteins CRKL and c-CRK link c-ABL, p190BCR/ABL and p210BCR/ABL to the phosphatidylinositol-3' kinase pathway.

PubMed

Sattler, M; Salgia, R; Okuda, K; Uemura, N; Durstin, M A; Pisick, E; Xu, G; Li, J L; Prasad, K V; Griffin, J D

1996-02-15

Chronic myelogenous leukemia (CML) and some acute lymphoblastic leukemias (ALL) are caused by the t(9;22) chromosome translocation, which produces the constitutively activated BCR/ABL tyrosine kinase. When introduced into factor dependent hematopoietic cell lines, BCR/ABL induces the tyrosine phosphorylation of many cellular proteins. One prominent BCR/ABL substrate is p120CBL, the cellular homolog of the v-Cbl oncoprotein. In an effort to understand the possible contribution of p120CBL to transformation by BCR/ABL, we looked for cellular proteins which associate with p120CBL in hematopoietic cell lines transformed by BCR/ABL. In addition to p210BCR/ABL and c-ABL, p120CBL coprecipitated with an 85 kDa phosphoprotein, which was identified as the p85 subunit of PI3K. Anti-p120CBL immunoprecipitates from BCR/ABL-transformed, but not from untransformed, cell lines contained PI3K lipid kinase activity. Interestingly, the adaptor proteins CRKL and c-CRK were also found in these complexes. In vitro binding studies indicated that the SH2 domains of CRKL and c-CRK bound directly to p120CBL, while the SH3 domains of c-CRK and CRKL bound to BCR/ABL and c-ABL. The N-terminal and the C-terminal SH2 and the SH3 domain of p85PI3K bound directly in vitro to p120CBL. The ABL-SH2, but not ABL-SH3, could also bind to p120CBL. These data suggest that BCR/ABL may induce the formation of multimeric complexes of signaling proteins which include p120CBL, PI3K, c-CRK or CRKL, c-ABL and BCR/ABL itself.

A non-chromatographic protein purification strategy using Src 3 homology domains as generalized capture domains.

PubMed

Kim, Heejae; Chen, Wilfred

2016-09-20

Protein purification using inverse phase transition of elastin-like polypeptide (ELP) domains is a useful alternative to chromatography. Genetic fusions of ELP domains to various proteins have the ability to reversibly transition between soluble monomers and micron-sized aggregates and this has been used to selectively purify many ELP fusions. Affinity domains can enhance this technology by using specific protein binding domains to enable ELP mediated affinity capture (EMAC) of proteins of interest (POI) that have been fused to corresponding affinity ligands. In this paper, we highlight the use of Src homology 3 (SH3) domains and corresponding peptide ligands in EMAC that have differential binding affinities towards SH3 for efficient capture and elution of proteins. Furthermore, differences between capture and elution of a monomeric and a multimeric protein were also studied. Copyright © 2016 Elsevier B.V. All rights reserved.

Nck adaptor proteins link Tks5 to invadopodia actin regulation and ECM degradation.

PubMed

Stylli, Stanley S; Stacey, T T I; Verhagen, Anne M; Xu, San San; Pass, Ian; Courtneidge, Sara A; Lock, Peter

2009-08-01

Invadopodia are actin-based projections enriched with proteases, which invasive cancer cells use to degrade the extracellular matrix (ECM). The Phox homology (PX)-Src homology (SH)3 domain adaptor protein Tks5 (also known as SH3PXD2A) cooperates with Src tyrosine kinase to promote invadopodia formation but the underlying pathway is not clear. Here we show that Src phosphorylates Tks5 at Y557, inducing it to associate directly with the SH3-SH2 domain adaptor proteins Nck1 and Nck2 in invadopodia. Tks5 mutants unable to bind Nck show reduced matrix degradation-promoting activity and recruit actin to invadopodia inefficiently. Conversely, Src- and Tks5-driven matrix proteolysis and actin assembly in invadopodia are enhanced by Nck1 or Nck2 overexpression and inhibited by Nck1 depletion. We show that clustering at the plasma membrane of the Tks5 inter-SH3 region containing Y557 triggers phosphorylation at this site, facilitating Nck recruitment and F-actin assembly. These results identify a Src-Tks5-Nck pathway in ECM-degrading invadopodia that shows parallels with pathways linking several mammalian and pathogen-derived proteins to local actin regulation.

p56Lck and p59Fyn Regulate CD28 Binding to Phosphatidylinositol 3-Kinase, Growth Factor Receptor-Bound Protein GRB-2, and T Cell-Specific Protein-Tyrosine Kinase ITK: Implications for T-Cell Costimulation

NASA Astrophysics Data System (ADS)

Raab, Monika; Cai, Yun-Cai; Bunnell, Stephen C.; Heyeck, Stephanie D.; Berg, Leslie J.; Rudd, Christopher E.

1995-09-01

T-cell activation requires cooperative signals generated by the T-cell antigen receptor ξ-chain complex (TCRξ-CD3) and the costimulatory antigen CD28. CD28 interacts with three intracellular proteins-phosphatidylinositol 3-kinase (PI 3-kinase), T cell-specific protein-tyrosine kinase ITK (formerly TSK or EMT), and the complex between growth factor receptor-bound protein 2 and son of sevenless guanine nucleotide exchange protein (GRB-2-SOS). PI 3-kinase and GRB-2 bind to the CD28 phosphotyrosine-based Tyr-Met-Asn-Met motif by means of intrinsic Src-homology 2 (SH2) domains. The requirement for tyrosine phosphorylation of the Tyr-Met-Asn-Met motif for SH2 domain binding implicates an intervening protein-tyrosine kinase in the recruitment of PI 3-kinase and GRB-2 by CD28. Candidate kinases include p56Lck, p59Fyn, ξ-chain-associated 70-kDa protein (ZAP-70), and ITK. In this study, we demonstrate in coexpression studies that p56Lck and p59Fyn phosphorylate CD28 primarily at Tyr-191 of the Tyr-Met-Asn-Met motif, inducing a 3- to 8-fold increase in p85 (subunit of PI 3-kinase) and GRB-2 SH2 binding to CD28. Phosphatase digestion of CD28 eliminated binding. In contrast to Src kinases, ZAP-70 and ITK failed to induce these events. Further, ITK binding to CD28 was dependent on the presence of p56Lck and is thus likely to act downstream of p56Lck/p59Fyn in a signaling cascade. p56Lck is therefore likely to be a central switch in T-cell activation, with the dual function of regulating CD28-mediated costimulation as well as TCR-CD3-CD4 signaling.

An SH2 domain model of STAT5 in complex with phospho-peptides define ``STAT5 Binding Signatures''

NASA Astrophysics Data System (ADS)

Gianti, Eleonora; Zauhar, Randy J.

2015-05-01

The signal transducer and activator of transcription 5 (STAT5) is a member of the STAT family of proteins, implicated in cell growth and differentiation. STAT activation is regulated by phosphorylation of protein monomers at conserved tyrosine residues, followed by binding to phospho-peptide pockets and subsequent dimerization. STAT5 is implicated in the development of severe pathological conditions, including many cancer forms. However, nowadays a few STAT5 inhibitors are known, and only one crystal structure of the inactive STAT5 dimer is publicly available. With a view to enabling structure-based drug design, we have: (1) analyzed phospho-peptide binding pockets on SH2 domains of STAT5, STAT1 and STAT3; (2) generated a model of STAT5 bound to phospho-peptides; (3) assessed our model by docking against a class of known STAT5 inhibitors (Müller et al. in ChemBioChem 9:723-727, 2008); (4) used molecular dynamics simulations to optimize the molecular determinants responsible for binding and (5) proposed unique "Binding Signatures" of STAT5. Our results put in place the foundations to address STAT5 as a target for rational drug design, from sequence, structural and functional perspectives.

Expression and Purification of Soluble STAT5b/STAT3 Proteins for SH2 Domain Binding Assay.

PubMed

Asai, Akira; Takakuma, Kazuyuki

2017-01-01

When a large hydrophobic full-length protein is expressed in bacteria, it is often challenging to obtain recombinant proteins in the soluble fraction. One way to overcome this challenge is expression of deletion mutants that have improved solubility while maintaining biological activity. In this chapter, we describe a protocol for expression of truncated forms of STAT5b and STAT3 proteins that are soluble and retain SH2-mediated activity for phospho-Tyr peptide recognition.

Methanethiosulfonate derivatives as ligands of the STAT3-SH2 domain.

PubMed

Gabriele, Elena; Ricci, Chiara; Meneghetti, Fiorella; Ferri, Nicola; Asai, Akira; Sparatore, Anna

2017-12-01

With the aim to discover new STAT3 direct inhibitors, potentially useful as anticancer agents, a set of methanethiosulfonate drug hybrids were synthesized. The in vitro tests showed that all the thiosulfonic compounds were able to strongly and selectively bind STAT3-SH2 domain, whereas the parent drugs were completely devoid of this ability. In addition, some of them showed a moderate antiproliferative activity on HCT-116 cancer cell line. These results suggest that methanethiosulfonate moiety can be considered a useful scaffold in the preparation of new direct STAT3 inhibitors. Interestingly, an unusual kind of organo-sulfur derivative, endowed with valuable antiproliferative activity, was occasionally isolated. [Formula: see text].

Interactions between relay helix and Src homology 1 domain helix (SH1) drive the converter domain rotation during the recovery stroke of myosin II

PubMed Central

Baumketner, Andrij

2012-01-01

Myosin motor protein exists in two alternative conformations, pre-recovery state M* and post-recovery state M**, upon ATP binding. The details of the M*-to-M** transition, known as the recovery stroke to reflect its role as the functional opposite of the force-generating power stroke, remain elusive. The defining feature of the post-recovery state is a kink in the relay helix, a key part of the protein involved in force generation. In this paper we determine the interactions that are responsible for the appearance of the kink. We design a series of computational models that contain three other segments, relay loop, converter domain and Src homology 1 domain helix (SH1), with which relay helix interacts, and determine their structure in accurate replica exchange molecular dynamics simulations in explicit solvent. By conducting an exhaustive combinatorial search among different models we find that: 1) the converter domain must be attached to the relay helix during the transition, so it does not interfere with other parts of the protein, 2) the structure of the relay helix is controlled by SH1 helix. The kink is strongly coupled to the position of SH1 helix. It arises as a result of direct interactions between SH1 and the relay helix and leads to a rotation of the C-terminal part of the relay helix which is subsequently transmitted to the converter domain. PMID:22411190

New approaches to high-throughput structure characterization of SH3 complexes: the example of Myosin-3 and Myosin-5 SH3 domains from S. cerevisiae.

PubMed

Musi, Valeria; Birdsall, Berry; Fernandez-Ballester, Gregorio; Guerrini, Remo; Salvatori, Severo; Serrano, Luis; Pastore, Annalisa

2006-04-01

SH3 domains are small protein modules that are involved in protein-protein interactions in several essential metabolic pathways. The availability of the complete genome and the limited number of clearly identifiable SH3 domains make the yeast Saccharomyces cerevisae an ideal proteomic-based model system to investigate the structural rules dictating the SH3-mediated protein interactions and to develop new tools to assist these studies. In the present work, we have determined the solution structure of the SH3 domain from Myo3 and modeled by homology that of the highly homologous Myo5, two myosins implicated in actin polymerization. We have then implemented an integrated approach that makes use of experimental and computational methods to characterize their binding properties. While accommodating their targets in the classical groove, the two domains have selectivity in both orientation and sequence specificity of the target peptides. From our study, we propose a consensus sequence that may provide a useful guideline to identify new natural partners and suggest a strategy of more general applicability that may be of use in other structural proteomic studies.

Mechanistic insights into the active site and allosteric communication pathways in human nonmuscle myosin-2C

PubMed Central

Preller, Matthias

2017-01-01

Despite a generic, highly conserved motor domain, ATP turnover kinetics and their activation by F-actin vary greatly between myosin-2 isoforms. Here, we present a 2.25 Å pre-powerstroke state (ADP⋅VO4) crystal structure of the human nonmuscle myosin-2C motor domain, one of the slowest myosins characterized. In combination with integrated mutagenesis, ensemble-solution kinetics, and molecular dynamics simulation approaches, the structure reveals an allosteric communication pathway that connects the distal end of the motor domain with the active site. Disruption of this pathway by mutation of hub residue R788, which forms the center of a cluster of interactions connecting the converter, the SH1-SH2 helix, the relay helix, and the lever, abolishes nonmuscle myosin-2 specific kinetic signatures. Our results provide insights into structural changes in the myosin motor domain that are triggered upon F-actin binding and contribute critically to the mechanochemical behavior of stress fibers, actin arcs, and cortical actin-based structures. PMID:29256864

Novel signal transducer and activator of transcription 3 (STAT3) mutations, reduced T(H)17 cell numbers, and variably defective STAT3 phosphorylation in hyper-IgE syndrome.

PubMed

Renner, Ellen D; Rylaarsdam, Stacey; Anover-Sombke, Stephanie; Rack, Anita L; Reichenbach, Janine; Carey, John C; Zhu, Qili; Jansson, Annette F; Barboza, Julia; Schimke, Lena F; Leppert, Mark F; Getz, Melissa M; Seger, Reinhard A; Hill, Harry R; Belohradsky, Bernd H; Torgerson, Troy R; Ochs, Hans D

2008-07-01

Hyper-IgE syndrome (HIES) is a rare, autosomal-dominant immunodeficiency characterized by eczema, Staphylococcus aureus skin abscesses, pneumonia with pneumatocele formation, Candida infections, and skeletal/connective tissue abnormalities. Recently it was shown that heterozygous signal transducer and activator of transcription 3 (STAT3) mutations cause autosomal-dominant HIES. To determine the spectrum and functional consequences of heterozygous STAT3 mutations in a cohort of patients with HIES. We sequenced the STAT3 gene in 38 patients with HIES (National Institutes of Health score >40 points) from 35 families, quantified T(H)17 cells in peripheral blood, and evaluated tyrosine phosphorylation of STAT3. Most STAT3 mutations in our cohort were in the DNA-binding domain (DBD; 22/35 families) or Src homology 2 (SH2) domain (10/35) and were missense mutations. We identified 2 intronic mutations resulting in exon skipping and in-frame deletions within the DBD. In addition, we identified 2 mutations located in the transactivation domain downstream of the SH2 domain: a 10-amino acid deletion and an amino acid substitution. In 1 patient, we were unable to identify a STAT3 mutation. T(H)17 cells were absent or low in the peripheral blood of all patients who were evaluated (n = 17). IL-6-induced STAT3-phosphorylation was consistently reduced in patients with SH2 domain mutations but comparable to normal controls in patients with mutations in the DBD. Heterozygous STAT3 mutations were identified in 34 of 35 unrelated HIES families. Patients had impaired T(H)17 cell development, and those with SH2 domain mutations had reduced STAT3 phosphorylation.

Two-state dynamics of the SH3-SH2 tandem of Abl kinase and the allosteric role of the N-cap.

PubMed

Corbi-Verge, Carles; Marinelli, Fabrizio; Zafra-Ruano, Ana; Ruiz-Sanz, Javier; Luque, Irene; Faraldo-Gómez, José D

2013-09-03

The regulation and localization of signaling enzymes is often mediated by accessory modular domains, which frequently function in tandems. The ability of these tandems to adopt multiple conformations is as important for proper regulation as the individual domain specificity. A paradigmatic example is Abl, a ubiquitous tyrosine kinase of significant pharmacological interest. SH3 and SH2 domains inhibit Abl by assembling onto the catalytic domain, allosterically clamping it in an inactive state. We investigate the dynamics of this SH3-SH2 tandem, using microsecond all-atom simulations and differential scanning calorimetry. Our results indicate that the Abl tandem is a two-state switch, alternating between the conformation observed in the structure of the autoinhibited enzyme and another configuration that is consistent with existing scattering data for an activated form. Intriguingly, we find that the latter is the most probable when the tandem is disengaged from the catalytic domain. Nevertheless, an amino acid stretch preceding the SH3 domain, the so-called N-cap, reshapes the free-energy landscape of the tandem and favors the interaction of this domain with the SH2-kinase linker, an intermediate step necessary for assembly of the autoinhibited complex. This allosteric effect arises from interactions between N-cap and the SH2 domain and SH3-SH2 connector, which involve a phosphorylation site. We also show that the SH3-SH2 connector plays a determinant role in the assembly equilibrium of Abl, because mutations thereof hinder the engagement of the SH2-kinase linker. These results provide a thermodynamic rationale for the involvement of N-cap and SH3-SH2 connector in Abl regulation and expand our understanding of the principles of modular domain organization.

Mechanism of IRSp53 inhibition and combinatorial activation by Cdc42 and downstream effectors.

PubMed

Kast, David J; Yang, Changsong; Disanza, Andrea; Boczkowska, Malgorzata; Madasu, Yadaiah; Scita, Giorgio; Svitkina, Tatyana; Dominguez, Roberto

2014-04-01

The Rho family GTPase effector IRSp53 has essential roles in filopodia formation and neuronal development, but its regulatory mechanism is poorly understood. IRSp53 contains a membrane-binding BAR domain followed by an unconventional CRIB motif that overlaps with a proline-rich region (CRIB-PR) and an SH3 domain that recruits actin cytoskeleton effectors. Using a fluorescence reporter assay, we show that human IRSp53 adopts a closed inactive conformation that opens synergistically with the binding of human Cdc42 to the CRIB-PR and effector proteins, such as the tumor-promoting factor Eps8, to the SH3 domain. The crystal structure of Cdc42 bound to the CRIB-PR reveals a new mode of effector binding to Rho family GTPases. Structure-inspired mutations disrupt autoinhibition and Cdc42 binding in vitro and decouple Cdc42- and IRSp53-dependent filopodia formation in cells. The data support a combinatorial mechanism of IRSp53 activation.

Focal adhesion kinase-dependent focal adhesion recruitment of SH2 domains directs SRC into focal adhesions to regulate cell adhesion and migration

PubMed Central

Wu, Jui-Chung; Chen, Yu-Chen; Kuo, Chih-Ting; Wenshin Yu, Helen; Chen, Yin-Quan; Chiou, Arthur; Kuo, Jean-Cheng

2015-01-01

Directed cell migration requires dynamical control of the protein complex within focal adhesions (FAs) and this control is regulated by signaling events involving tyrosine phosphorylation. We screened the SH2 domains present in tyrosine-specific kinases and phosphatases found within FAs, including SRC, SHP1 and SHP2, and examined whether these enzymes transiently target FAs via their SH2 domains. We found that the SRC_SH2 domain and the SHP2_N-SH2 domain are associated with FAs, but only the SRC_SH2 domain is able to be regulated by focal adhesion kinase (FAK). The FAK-dependent association of the SRC_SH2 domain is necessary and sufficient for SRC FA targeting. When the targeting of SRC into FAs is inhibited, there is significant suppression of SRC-mediated phosphorylation of paxillin and FAK; this results in an inhibition of FA formation and maturation and a reduction in cell migration. This study reveals an association between FAs and the SRC_SH2 domain as well as between FAs and the SHP2_N-SH2 domains. This supports the hypothesis that the FAK-regulated SRC_SH2 domain plays an important role in directing SRC into FAs and that this SRC-mediated FA signaling drives cell migration. PMID:26681405

Focal adhesion kinase-dependent focal adhesion recruitment of SH2 domains directs SRC into focal adhesions to regulate cell adhesion and migration.

PubMed

Wu, Jui-Chung; Chen, Yu-Chen; Kuo, Chih-Ting; Wenshin Yu, Helen; Chen, Yin-Quan; Chiou, Arthur; Kuo, Jean-Cheng

2015-12-18

Directed cell migration requires dynamical control of the protein complex within focal adhesions (FAs) and this control is regulated by signaling events involving tyrosine phosphorylation. We screened the SH2 domains present in tyrosine-specific kinases and phosphatases found within FAs, including SRC, SHP1 and SHP2, and examined whether these enzymes transiently target FAs via their SH2 domains. We found that the SRC_SH2 domain and the SHP2_N-SH2 domain are associated with FAs, but only the SRC_SH2 domain is able to be regulated by focal adhesion kinase (FAK). The FAK-dependent association of the SRC_SH2 domain is necessary and sufficient for SRC FA targeting. When the targeting of SRC into FAs is inhibited, there is significant suppression of SRC-mediated phosphorylation of paxillin and FAK; this results in an inhibition of FA formation and maturation and a reduction in cell migration. This study reveals an association between FAs and the SRC_SH2 domain as well as between FAs and the SHP2_N-SH2 domains. This supports the hypothesis that the FAK-regulated SRC_SH2 domain plays an important role in directing SRC into FAs and that this SRC-mediated FA signaling drives cell migration.

Tyrosine phosphorylation of 3BP2 is indispensable for the interaction with VAV3 in chicken DT40 cells.

PubMed

Chihara, Kazuyasu; Kimura, Yukihiro; Honjoh, Chisato; Yamauchi, Shota; Takeuchi, Kenji; Sada, Kiyonao

2014-03-10

Adaptor protein c-Abl SH3 domain-binding protein-2 (3BP2) is known to play regulatory roles in immunoreceptor-mediated signal transduction. We have previously demonstrated that Tyr(174), Tyr(183) and Tyr(446) in mouse 3BP2 are predominantly phosphorylated by Syk, and the phosphorylation of Tyr(183) and the Src homology 2 (SH2) domain of mouse 3BP2 are critical for B cell receptor (BCR)-induced activation of nuclear factor of activated T cells (NFAT) in human B cells. In this report, we have shown that Syk, but not Abl family protein-tyrosine kinases, is critical for BCR-mediated tyrosine phosphorylation of 3BP2 in chicken DT40 cells. Mutational analysis showed that Tyr(174), Tyr(183) and Tyr(426) of chicken 3BP2 are the major phosphorylation sites by Syk and the SH2 domain of 3BP2 is critical for tyrosine phosphorylation. In addition, phosphorylation of Tyr(426) is required for the inducible interaction with the SH2 domain of Vav3. Moreover, the expression of the mutant form of 3BP2 in which Tyr(426) was substituted to Phe resulted in the reduction in BCR-mediated Rac1 activation, when compared with the case of wild-type. Altogether, these data suggest that 3BP2 is involved in the activation of Rac1 through the regulation of Vav3 by Syk-dependent phosphorylation of Tyr(426) following BCR stimulation. Copyright © 2014 Elsevier Inc. All rights reserved.

Grb-IR: A SH2-Domain-Containing Protein that Binds to the Insulin Receptor and Inhibits Its Function

NASA Astrophysics Data System (ADS)

Liu, Feng; Roth, Richard A.

1995-10-01

To identify potential signaling molecules involved in mediating insulin-induced biological responses, a yeast two-hybrid screen was performed with the cytoplasmic domain of the human insulin receptor (IR) as bait to trap high-affinity interacting proteins encoded by human liver or HeLa cDNA libraries. A SH2-domain-containing protein was identified that binds with high affinity in vitro to the autophosphorylated IR. The mRNA for this protein was found by Northern blot analyses to be highest in skeletal muscle and was also detected in fat by PCR. To study the role of this protein in insulin signaling, a full-length cDNA encoding this protein (called Grb-IR) was isolated and stably expressed in Chinese hamster ovary cells overexpressing the human IR. Insulin treatment of these cells resulted in the in situ formation of a complex of the IR and the 60-kDa Grb-IR. Although almost 75% of the Grb-IR protein was bound to the IR, it was only weakly tyrosine-phosphorylated. The formation of this complex appeared to inhibit the insulin-induced increase in tyrosine phosphorylation of two endogenous substrates, a 60-kDa GTPase-activating-protein-associated protein and, to a lesser extent, IR substrate 1. The subsequent association of this latter protein with phosphatidylinositol 3-kinase also appeared to be inhibited. These findings raise the possibility that Grb-IR is a SH2-domain-containing protein that directly complexes with the IR and serves to inhibit signaling or redirect the IR signaling pathway.

Adaptor protein GRB2 promotes Src tyrosine kinase activation and podosomal organization by protein-tyrosine phosphatase ϵ in osteoclasts.

PubMed

Levy-Apter, Einat; Finkelshtein, Eynat; Vemulapalli, Vidyasiri; Li, Shawn S-C; Bedford, Mark T; Elson, Ari

2014-12-26

The non-receptor isoform of protein-tyrosine phosphatase ϵ (cyt-PTPe) supports adhesion of bone-resorbing osteoclasts by activating Src downstream of integrins. Loss of cyt-PTPe reduces Src activity in osteoclasts, reduces resorption of mineralized matrix both in vivo and in cell culture, and induces mild osteopetrosis in young female PTPe KO mice. Activation of Src by cyt-PTPe is dependent upon this phosphatase undergoing phosphorylation at its C-terminal Tyr-638 by partially active Src. To understand how cyt-PTPe activates Src, we screened 73 Src homology 2 (SH2) domains for binding to Tyr(P)-638 of cyt-PTPe. The SH2 domain of GRB2 bound Tyr(P)-638 of cyt-PTPe most prominently, whereas the Src SH2 domain did not bind at all, suggesting that GRB2 may link PTPe with downstream molecules. Further studies indicated that GRB2 is required for activation of Src by cyt-PTPe in osteoclast-like cells (OCLs) in culture. Overexpression of GRB2 in OCLs increased activating phosphorylation of Src at Tyr-416 and of cyt-PTPe at Tyr-638; opposite results were obtained when GRB2 expression was reduced by shRNA or by gene inactivation. Phosphorylation of cyt-PTPe at Tyr-683 and its association with GRB2 are integrin-driven processes in OCLs, and cyt-PTPe undergoes autodephosphorylation at Tyr-683, thus limiting Src activation by integrins. Reduced GRB2 expression also reduced the ability of bone marrow precursors to differentiate into OCLs and reduced the fraction of OCLs in which podosomal adhesion structures assume organization typical of active, resorbing cells. We conclude that GRB2 physically links cyt-PTPe with Src and enables cyt-PTPe to activate Src downstream of activated integrins in OCLs. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

The Human Metapneumovirus Small Hydrophobic Protein Has Properties Consistent with Those of a Viroporin and Can Modulate Viral Fusogenic Activity

PubMed Central

Masante, Cyril; El Najjar, Farah; Chang, Andres; Jones, Angela; Moncman, Carole L.

2014-01-01

ABSTRACT Human metapneumovirus (HMPV) encodes three glycoproteins: the glycoprotein, which plays a role in glycosaminoglycan binding, the fusion (F) protein, which is necessary and sufficient for both viral binding to the target cell and fusion between the cellular plasma membrane and the viral membrane, and the small hydrophobic (SH) protein, whose function is unclear. The SH protein of the closely related respiratory syncytial virus has been suggested to function as a viroporin, as it forms oligomeric structures consistent with a pore and alters membrane permeability. Our analysis indicates that both the full-length HMPV SH protein and the isolated SH protein transmembrane domain can associate into higher-order oligomers. In addition, HMPV SH expression resulted in increases in permeability to hygromycin B and alteration of subcellular localization of a fluorescent dye, indicating that SH affects membrane permeability. These results suggest that the HMPV SH protein has several characteristics consistent with a putative viroporin. Interestingly, we also report that expression of the HMPV SH protein can significantly decrease HMPV F protein-promoted membrane fusion activity, with the SH extracellular domain and transmembrane domain playing a key role in this inhibition. These results suggest that the HMPV SH protein could regulate both membrane permeability and fusion protein function during viral infection. IMPORTANCE Human metapneumovirus (HMPV), first identified in 2001, is a causative agent of severe respiratory tract disease worldwide. The small hydrophobic (SH) protein is one of three glycoproteins encoded by all strains of HMPV, but the function of the HMPV SH protein is unknown. We have determined that the HMPV SH protein can alter the permeability of cellular membranes, suggesting that HMPV SH is a member of a class of proteins termed viroporins, which modulate membrane permeability to facilitate critical steps in a viral life cycle. We also demonstrated that HMPV SH can inhibit the membrane fusion function of the HMPV fusion protein. This work suggests that the HMPV SH protein has several functions, though the steps in the HMPV life cycle impacted by these functions remain to be clarified. PMID:24672047

Neuronal SH2B1 is essential for controlling energy and glucose homeostasis.

PubMed

Ren, Decheng; Zhou, Yingjiang; Morris, David; Li, Minghua; Li, Zhiqin; Rui, Liangyou

2007-02-01

SH2B1 (previously named SH2-B), a cytoplasmic adaptor protein, binds via its Src homology 2 (SH2) domain to a variety of protein tyrosine kinases, including JAK2 and the insulin receptor. SH2B1-deficient mice are obese and diabetic. Here we demonstrated that multiple isoforms of SH2B1 (alpha, beta, gamma, and/or delta) were expressed in numerous tissues, including the brain, hypothalamus, liver, muscle, adipose tissue, heart, and pancreas. Rat SH2B1beta was specifically expressed in neural tissue in SH2B1-transgenic (SH2B1(Tg)) mice. SH2B1(Tg) mice were crossed with SH2B1-knockout (SH2B1(KO)) mice to generate SH2B1(TgKO) mice expressing SH2B1 only in neural tissue but not in other tissues. Systemic deletion of the SH2B1 gene resulted in metabolic disorders in SH2B1(KO) mice, including hyperlipidemia, leptin resistance, hyperphagia, obesity, hyperglycemia, insulin resistance, and glucose intolerance. Neuron-specific restoration of SH2B1beta not only corrected the metabolic disorders in SH2B1(TgKO) mice, but also improved JAK2-mediated leptin signaling and leptin regulation of orexigenic neuropeptide expression in the hypothalamus. Moreover, neuron-specific overexpression of SH2B1 dose-dependently protected against high-fat diet-induced leptin resistance and obesity. These observations suggest that neuronal SH2B1 regulates energy balance, body weight, peripheral insulin sensitivity, and glucose homeostasis at least in part by enhancing hypothalamic leptin sensitivity.

Structure-based design of an osteoclast-selective, nonpeptide Src homology 2 inhibitor with in vivo antiresorptive activity

PubMed Central

Shakespeare, William; Yang, Michael; Bohacek, Regine; Cerasoli, Franklin; Stebbins, Karin; Sundaramoorthi, Raji; Azimioara, Mihai; Vu, Chi; Pradeepan, Selvi; Metcalf, Chester; Haraldson, Chad; Merry, Taylor; Dalgarno, David; Narula, Surinder; Hatada, Marcos; Lu, Xiaode; van Schravendijk, Marie Rose; Adams, Susan; Violette, Shelia; Smith, Jeremy; Guan, Wei; Bartlett, Catherine; Herson, Jay; Iuliucci, John; Weigele, Manfred; Sawyer, Tomi

2000-01-01

Targeted disruption of the pp60src (Src) gene has implicated this tyrosine kinase in osteoclast-mediated bone resorption and as a therapeutic target for the treatment of osteoporosis and other bone-related diseases. Herein we describe the discovery of a nonpeptide inhibitor (AP22408) of Src that demonstrates in vivo antiresorptive activity. Based on a cocrystal structure of the noncatalytic Src homology 2 (SH2) domain of Src complexed with citrate [in the phosphotyrosine (pTyr) binding pocket], we designed 3′,4′-diphosphonophenylalanine (Dpp) as a pTyr mimic. In addition to its design to bind Src SH2, the Dpp moiety exhibits bone-targeting properties that confer osteoclast selectivity, hence minimizing possible undesired effects on other cells that have Src-dependent activities. The chemical structure AP22408 also illustrates a bicyclic template to replace the post-pTyr sequence of cognate Src SH2 phosphopeptides such as Ac-pTyr-Glu-Glu-Ile (1). An x-ray structure of AP22408 complexed with Lck (S164C) SH2 confirmed molecular interactions of both the Dpp and bicyclic template of AP22408 as predicted from molecular modeling. Relative to the cognate phosphopeptide, AP22408 exhibits significantly increased Src SH2 binding affinity (IC50 = 0.30 μM for AP22408 and 5.5 μM for 1). Furthermore, AP22408 inhibits rabbit osteoclast-mediated resorption of dentine in a cellular assay, exhibits bone-targeting properties based on a hydroxyapatite adsorption assay, and demonstrates in vivo antiresorptive activity in a parathyroid hormone-induced rat model. PMID:10944210

Structural basis of IFNα receptor recognition by TYK2

PubMed Central

Wallweber, Heidi J.A.; Tam, Christine; Franke, Yvonne; Starovasnik, Melissa A.; Lupardus, Patrick J.

2014-01-01

Tyrosine kinase 2 (TYK2) is a member of the Janus kinase (JAK) family of non-receptor tyrosine kinases, which are essential for proper signaling in immune responses and development. Here we present a 2.0 angstrom resolution crystal structure of a receptor-binding fragment of human TYK2 encompassing the FERM and SH2 domains in complex with a so-called “box2” containing intracellular peptide motif from the IFNα receptor (IFNAR1). The TYK2–IFNAR1 interface reveals an unexpected receptor-binding mode that mimics a SH2 domain–phosphopeptide interaction, with a glutamate replacing the canonical phosphotyrosine residue. This structure provides the first view to our knowledge of a JAK in complex with its cognate receptor and defines the molecular logic through which JAKs evolved to interact with divergent receptor sequences. PMID:24704786

The SH2 Domain–Containing Proteins in 21 Species Establish the Provenance and Scope of Phosphotyrosine Signaling in Eukaryotes

PubMed Central

Liu, Bernard A.; Shah, Eshana; Jablonowski, Karl; Stergachis, Andrew; Engelmann, Brett; Nash, Piers D.

2014-01-01

The Src homology 2 (SH2) domains are participants in metazoan signal transduction, acting as primary mediators for regulated protein-protein interactions with tyrosine-phosphorylated substrates. Here, we describe the origin and evolution of SH2 domain proteins by means of sequence analysis from 21 eukaryotic organisms from the basal unicellular eukaryotes, where SH2 domains first appeared, through the multicellular animals and increasingly complex metazoans. On the basis of our results, SH2 domains and phosphotyrosine signaling emerged in the early Unikonta, and the numbers of SH2 domains expanded in the choanoflagellate and metazoan lineages with the development of tyrosine kinases, leading to rapid elaboration of phosphotyrosine signaling in early multicellular animals. Our results also indicated that SH2 domains coevolved and the number of the domains expanded alongside protein tyrosine kinases and tyrosine phosphatases, thereby coupling phosphotyrosine signaling to downstream signaling networks. Gene duplication combined with domain gain or loss produced novel SH2-containing proteins that function within phosphotyrosine signaling, which likely have contributed to diversity and complexity in metazoans. We found that intra- and intermolecular interactions within and between SH2 domain proteins increased in prevalence along with organismal complexity and may function to generate more highly connected and robust phosphotyrosine signaling networks. PMID:22155787

Activation of PI3K/Akt signaling by n-terminal SH2 domain mutants of the p85α regulatory subunit of PI3K is enhanced by deletion of its c-terminal SH2 domain.

PubMed

Hofmann, Bianca T; Jücker, Manfred

2012-10-01

The phosphoinositide 3-kinase (PI3K) is frequently activated in human cancer cells due to gain of function mutations in the catalytic (p110) and the regulatory (p85) subunits. The regulatory subunit consists of an SH3 domain and two SH2 domains. An oncogenic form of p85α named p65 lacking the c-terminal SH2 domain (cSH2) has been cloned from an irradiation-induced murine thymic lymphoma and transgenic mice expressing p65 in T lymphocytes develop a lymphoproliferative disorder. We have recently detected a c-terminal truncated form of p85α named p76α in a human lymphoma cell line lacking most of the cSH2 domain due to a frame shift mutation. Here, we report that the deletion of the cSH2 domain enhances the activating effects of the n-terminal SH2 domain (nSH2) mutants K379E and R340E on the PI3K/Akt pathway and micro tumor formation in a focus assay. Further analysis revealed that this transforming effect is mediated by activation of the catalytic PI3K isoform p110α and downstream signaling through mTOR. Our data further support a mechanistic model in which mutations of the cSH2 domain of p85α can abrogate its negative regulatory function on PI3K activity via the nSH2 domain of p85α. Copyright © 2012 Elsevier Inc. All rights reserved.

Targeting the SH2-Kinase Interface in Bcr-Abl Inhibits Leukemogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Grebien, Florian; Hantschel, Oliver; Wojcik, John

2012-10-25

Chronic myelogenous leukemia (CML) is caused by the constitutively active tyrosine kinase Bcr-Abl and treated with the tyrosine kinase inhibitor (TKI) imatinib. However, emerging TKI resistance prevents complete cure. Therefore, alternative strategies targeting regulatory modules of Bcr-Abl in addition to the kinase active site are strongly desirable. Here, we show that an intramolecular interaction between the SH2 and kinase domains in Bcr-Abl is both necessary and sufficient for high catalytic activity of the enzyme. Disruption of this interface led to inhibition of downstream events critical for CML signaling and, importantly, completely abolished leukemia formation in mice. Furthermore, disruption of themore » SH2-kinase interface increased sensitivity of imatinib-resistant Bcr-Abl mutants to TKI inhibition. An engineered Abl SH2-binding fibronectin type III monobody inhibited Bcr-Abl kinase activity both in vitro and in primary CML cells, where it induced apoptosis. This work validates the SH2-kinase interface as an allosteric target for therapeutic intervention.« less

Targeting the SH2-kinase interface in Bcr-Abl inhibits leukemogenesis.

PubMed

Grebien, Florian; Hantschel, Oliver; Wojcik, John; Kaupe, Ines; Kovacic, Boris; Wyrzucki, Arkadiusz M; Gish, Gerald D; Cerny-Reiterer, Sabine; Koide, Akiko; Beug, Hartmut; Pawson, Tony; Valent, Peter; Koide, Shohei; Superti-Furga, Giulio

2011-10-14

Chronic myelogenous leukemia (CML) is caused by the constitutively active tyrosine kinase Bcr-Abl and treated with the tyrosine kinase inhibitor (TKI) imatinib. However, emerging TKI resistance prevents complete cure. Therefore, alternative strategies targeting regulatory modules of Bcr-Abl in addition to the kinase active site are strongly desirable. Here, we show that an intramolecular interaction between the SH2 and kinase domains in Bcr-Abl is both necessary and sufficient for high catalytic activity of the enzyme. Disruption of this interface led to inhibition of downstream events critical for CML signaling and, importantly, completely abolished leukemia formation in mice. Furthermore, disruption of the SH2-kinase interface increased sensitivity of imatinib-resistant Bcr-Abl mutants to TKI inhibition. An engineered Abl SH2-binding fibronectin type III monobody inhibited Bcr-Abl kinase activity both in vitro and in primary CML cells, where it induced apoptosis. This work validates the SH2-kinase interface as an allosteric target for therapeutic intervention. Copyright © 2011 Elsevier Inc. All rights reserved.

Targeting the SH2-Kinase Interface in Bcr-Abl Inhibits Leukemogenesis

PubMed Central

Grebien, Florian; Hantschel, Oliver; Wojcik, John; Kaupe, Ines; Kovacic, Boris; Wyrzucki, Arkadiusz M.; Gish, Gerald D.; Cerny-Reiterer, Sabine; Koide, Akiko; Beug, Hartmut; Pawson, Tony; Valent, Peter; Koide, Shohei; Superti-Furga, Giulio

2011-01-01

Summary Chronic myelogenous leukemia (CML) is caused by the constitutively active tyrosine kinase Bcr-Abl and treated with the tyrosine kinase inhibitor (TKI) imatinib. However, emerging TKI resistance prevents complete cure. Therefore, alternative strategies targeting regulatory modules of Bcr-Abl in addition to the kinase active site are strongly desirable. Here, we show that an intramolecular interaction between the SH2 and kinase domains in Bcr-Abl is both necessary and sufficient for high catalytic activity of the enzyme. Disruption of this interface led to inhibition of downstream events critical for CML signaling and, importantly, completely abolished leukemia formation in mice. Furthermore, disruption of the SH2-kinase interface increased sensitivity of imatinib-resistant Bcr-Abl mutants to TKI inhibition. An engineered Abl SH2-binding fibronectin type III monobody inhibited Bcr-Abl kinase activity both in vitro and in primary CML cells, where it induced apoptosis. This work validates the SH2-kinase interface as an allosteric target for therapeutic intervention. PaperFlick PMID:22000011

Crystal structure of an SH2-kinase construct of c-Abl and effect of the SH2 domain on kinase activity

PubMed Central

Lorenz, Sonja; Deng, Patricia; Hantschel, Oliver; Superti-Furga, Giulio; Kuriyan, John

2018-01-01

Constitutive activation of the non-receptor tyrosine kinase c-Abl (Abl1) in the Bcr-Abl1 fusion oncoprotein is the molecular cause of chronic myeloid leukemia. Recent studies have indicated that an interaction between the SH2 domain and the N-lobe of the c-Abl kinase domain has a critical role in leukemogenesis. To dissect the structural basis of this phenomenon we studied c-Abl constructs comprising the SH2 and kinase domains in vitro. We present a crystal structure of an SH2-kinase domain construct bound to dasatinib, which contains the relevant interface between the SH2 domain and the N-lobe of the kinase domain. We show that the presence of the SH2 domain enhances kinase activity moderately and that this effect depends on contacts in the SH2-N-lobe interface and is abrogated by specific mutations. Consistently, formation of the interface decreases slightly the association rate of imatinib with the kinase domain. That the effects are small compared to the dramatic in vivo consequences suggests an important function of the SH2-N-lobe interaction might be to help disassemble the autoinhibited conformation of c-Abl and promote processive phosphorylation, rather than substantially stimulate kinase activity. PMID:25779001

Domain requirements for the Dock adapter protein in growth- cone signaling.

PubMed

Rao, Y; Zipursky, S L

1998-03-03

Tyrosine phosphorylation has been implicated in growth-cone guidance through genetic, biochemical, and pharmacological studies. Adapter proteins containing src homology 2 (SH2) domains and src homology 3 (SH3) domains provide a means of linking guidance signaling through phosphotyrosine to downstream effectors regulating growth-cone motility. The Drosophila adapter, Dreadlocks (Dock), the homolog of mammalian Nck containing three N-terminal SH3 domains and a single SH2 domain, is highly specialized for growth-cone guidance. In this paper, we demonstrate that Dock can couple signals in either an SH2-dependent or an SH2-independent fashion in photoreceptor (R cell) growth cones, and that Dock displays different domain requirements in different neurons.

Two-state dynamics of the SH3–SH2 tandem of Abl kinase and the allosteric role of the N-cap

PubMed Central

Corbi-Verge, Carles; Marinelli, Fabrizio; Zafra-Ruano, Ana; Ruiz-Sanz, Javier; Luque, Irene; Faraldo-Gómez, José D.

2013-01-01

The regulation and localization of signaling enzymes is often mediated by accessory modular domains, which frequently function in tandems. The ability of these tandems to adopt multiple conformations is as important for proper regulation as the individual domain specificity. A paradigmatic example is Abl, a ubiquitous tyrosine kinase of significant pharmacological interest. SH3 and SH2 domains inhibit Abl by assembling onto the catalytic domain, allosterically clamping it in an inactive state. We investigate the dynamics of this SH3–SH2 tandem, using microsecond all-atom simulations and differential scanning calorimetry. Our results indicate that the Abl tandem is a two-state switch, alternating between the conformation observed in the structure of the autoinhibited enzyme and another configuration that is consistent with existing scattering data for an activated form. Intriguingly, we find that the latter is the most probable when the tandem is disengaged from the catalytic domain. Nevertheless, an amino acid stretch preceding the SH3 domain, the so-called N-cap, reshapes the free-energy landscape of the tandem and favors the interaction of this domain with the SH2-kinase linker, an intermediate step necessary for assembly of the autoinhibited complex. This allosteric effect arises from interactions between N-cap and the SH2 domain and SH3–SH2 connector, which involve a phosphorylation site. We also show that the SH3–SH2 connector plays a determinant role in the assembly equilibrium of Abl, because mutations thereof hinder the engagement of the SH2-kinase linker. These results provide a thermodynamic rationale for the involvement of N-cap and SH3–SH2 connector in Abl regulation and expand our understanding of the principles of modular domain organization. PMID:23959873

CARMA2sh and ULK2 control pathogen-associated molecular patterns recognition in human keratinocytes: psoriasis-linked CARMA2sh mutants escape ULK2 censorship.

PubMed

Scudiero, Ivan; Mazzone, Pellegrino; D'Andrea, Luca E; Ferravante, Angela; Zotti, Tiziana; Telesio, Gianluca; De Rubis, Gabriele; Reale, Carla; Pizzulo, Maddalena; Muralitharan, Shanmugakonar; Vito, Pasquale; Stilo, Romania

2017-02-23

The molecular complexes formed by specific members of the family of CARMA proteins, the CARD domain-containing adapter molecule BCL10 and MALT1 (CBM complex) represent a central hub in regulating activation of the pleiotropic transcription factor NF-κB. Recently, missense mutations in CARMA2sh have been shown to cause psoriasis in a dominant manner and with high penetrancy. Here, we demonstrate that in human keratinocytes CARMA2sh plays an essential role in the signal transduction pathway that connects pathogen-associated molecular patterns recognition to NF-κB activation. We also find that the serine/threonine kinase ULK2 binds to and phosphorylates CARMA2sh, thereby inhibiting its capacity to activate NF-κB by promoting lysosomal degradation of BCL10, which is essential for CARMA2sh-mediated NF-κB signaling. Remarkably, CARMA2sh mutants associated with psoriasis escape ULK2 inhibition. Finally, we show that a peptide blocking CARD-mediated BCL10 interactions reduces the capacity of psoriasis-linked CARMA2sh mutants to activate NF-κB. Our work elucidates a fundamental signaling mechanism operating in human keratinocytes and opens to novel potential tools for the therapeutical treatment of human skin disorders.

Electrostatic Interactions in the Binding Pathway of a Transient Protein Complex Studied by NMR and Isothermal Titration Calorimetry*

PubMed Central

Meneses, Erick; Mittermaier, Anthony

2014-01-01

Much of our knowledge of protein binding pathways is derived from extremely stable complexes that interact very tightly, with lifetimes of hours to days. Much less is known about weaker interactions and transient complexes because these are challenging to characterize experimentally. Nevertheless, these types of interactions are ubiquitous in living systems. The combination of NMR relaxation dispersion Carr–Purcell–Meiboom–Gill (CPMG) experiments and isothermal titration calorimetry allows the quantification of rapid binding kinetics for complexes with submillisecond lifetimes that are difficult to study using conventional techniques. We have used this approach to investigate the binding pathway of the Src homology 3 (SH3) domain from the Fyn tyrosine kinase, which forms complexes with peptide targets whose lifetimes are on the order of about a millisecond. Long range electrostatic interactions have been shown to play a critical role in the binding pathways of tightly binding complexes. The role of electrostatics in the binding pathways of transient complexes is less well understood. Similarly to previously studied tight complexes, we find that SH3 domain association rates are enhanced by long range electrostatics, whereas short range interactions are formed late in the docking process. However, the extent of electrostatic association rate enhancement is several orders of magnitudes less, whereas the electrostatic-free basal association rate is significantly greater. Thus, the SH3 domain is far less reliant on electrostatic enhancement to achieve rapid association kinetics than are previously studied systems. This suggests that there may be overall differences in the role played by electrostatics in the binding pathways of extremely stable versus transient complexes. PMID:25122758

Expression, refolding and crystallizations of the Grb2-like (GADS) C-terminal SH3 domain complexed with a SLP-76 motif peptide

DOE Office of Scientific and Technical Information (OSTI.GOV)

Faravelli, Alessandro; Dimasi, Nazzareno, E-mail: ndimasi@gmail.com

Several crystals of the Grb2-like C-terminal SH3 domain in complex with a motif peptide from the SLP-76 protein were obtained and characterized. The Grb2-like adaptor protein GADS is composed of an N-terminal SH3 domain, an SH2 domain, a proline-rich region and a C-terminal SH3 domain. GADS interacts through its C-terminal SH3 domain with the adaptor protein SLP-76, thus recruiting this protein and other associated molecules to the linker for activation of T-cell (LAT) protein. The DNA encoding the C-terminal SH3 domain of GADS (GADS-cSH3) was assembled synthetically using a recursive PCR technique and the protein was overexpressed in Escherichia coli,more » refolded and purified. Several crystals of this domain in complex with the SLP-76 peptide were obtained and characterized.« less

Characterization of a new disease-causing mutation of SH2D1A in a family with X-linked lymphoproliferative disease.

PubMed

Erdõs, Melinda; Uzvölgyi, Eva; Nemes, Zoltán; Török, Olga; Rákóczi, Eva; Went-Sümegi, Nils; Sümegi, János; Maródi, László

2005-05-01

Males with an expressed mutation in the SH2D1A gene that encodes an SH2 domain protein named SH2D1A or SAP (NP_002342; signaling lymphocyte activating molecule [SLAM]-associated protein), have an X-linked syndrome characterized by an increased vulnerability to infection with Epstein-Barr virus (EBV). We evaluated two related male patients with fatal infectious mononucleosis (FIM) and mutation in the SH2D1A gene. Sequence analysis revealed a hemizygous c.47G>A mutation in one of the patients, and heterozygosity for this mutation in the genomic DNA from his mother and maternal grandmother. This mutation resulted in p.G16D amino acid change in the sequence of the SAP protein. To analyze the effect of this missense mutation on protein function cDNA was generated by site-directed mutagenesis and expressed in COS cells. We found that half-life of the p.G16D protein was comparable to that of wild type SAP. However, the mutant protein was defective in binding to its physiological ligands SLAM and 2B4. These results suggest that a defect in ligand binding contributes to the loss of function of the SAP protein in patients carrying p.G16D mutation.

Expression, Refolding and Crystallizations of the Grb2-like (GADS) C-Terminal SH3 Domain Complexed with a SLP-76 Motif Peptide

DOE Office of Scientific and Technical Information (OSTI.GOV)

Faravelli,A.; Dimasi, N.

The Grb2-like adaptor protein GADS is composed of an N-terminal SH3 domain, an SH2 domain, a proline-rich region and a C-terminal SH3 domain. GADS interacts through its C-terminal SH3 domain with the adaptor protein SLP-76, thus recruiting this protein and other associated molecules to the linker for activation of T-cell (LAT) protein. The DNA encoding the C-terminal SH3 domain of GADS (GADS-cSH3) was assembled synthetically using a recursive PCR technique and the protein was overexpressed in Escherichia coli, refolded and purified. Several crystals of this domain in complex with the SLP-76 peptide were obtained and characterized.

Identification of polyproline II regions derived from the proline-rich nuclear receptor coactivators PNRC and PNRC2: new insights for ERα coactivator interactions.

PubMed

Byrne, C; Miclet, E; Broutin, I; Gallo, D; Pelekanou, V; Kampa, M; Castanas, E; Leclercq, G; Jacquot, Y

2013-10-01

Protein-protein interactions are crucial for signal transductions required for cell differentiation and proliferation. Their modulation is therefore key to the development of therapeutic alternatives, particularly in the context of cancer. According to literature data, the polyproline-rich nuclear receptor coactivators PNRC and PNRC2 interact with estrogen receptor (ERα) through their PxxP SH3-binding motifs. In a search to identify the molecular features governing this interaction, we explored using electronic circular dichroism (ECD) spectroscopy and molecular dynamics (MD) calculations, the capacity of a range of putative biologically active peptides derived from these proteins and containing this PxxP motif(s) to form polyproline II (PPII) domains. An additional more exhaustive structural study on a lead PPII peptide was also performed using 2D nuclear magnetic resonance (NMR) spectroscopy. With the exception of one of all the investigated peptides (PNRC-D), binding assays failed to detect any affinity for Grb2 SH3 domains, suggesting that PPII motifs issued from Grb2 antagonists have a binding mode distinct from those derived from Grb2 agonists. Instead, the peptides revealed a competitive binding ability against a synthetic peptide (ERα17p) with a putative PPII-cognate domain located within a coregulator recruitment region of ERα (AF-2 site). Our work, which constitutes the first structure-related interaction study concerning PNRC and PNRC2, supports not only the existence of PxxP-induced PPII sequences in these coregulators, but also confirms the presence of a PPII recognition site in the AF-2 of the steroid receptor ERα, a region important for transcription regulation. © 2013 Wiley Periodicals, Inc.

Domain requirements for the Dock adapter protein in growth- cone signaling

PubMed Central

Rao, Yong; Zipursky, S. Lawrence

1998-01-01

Tyrosine phosphorylation has been implicated in growth-cone guidance through genetic, biochemical, and pharmacological studies. Adapter proteins containing src homology 2 (SH2) domains and src homology 3 (SH3) domains provide a means of linking guidance signaling through phosphotyrosine to downstream effectors regulating growth-cone motility. The Drosophila adapter, Dreadlocks (Dock), the homolog of mammalian Nck containing three N-terminal SH3 domains and a single SH2 domain, is highly specialized for growth-cone guidance. In this paper, we demonstrate that Dock can couple signals in either an SH2-dependent or an SH2-independent fashion in photoreceptor (R cell) growth cones, and that Dock displays different domain requirements in different neurons. PMID:9482841

Flexible docking of a ligand peptide to a receptor protein by multicanonical molecular dynamics simulation

NASA Astrophysics Data System (ADS)

Nakajima, Nobuyuki; Higo, Junichi; Kidera, Akinori; Nakamura, Haruki

1997-10-01

A new method for flexible docking by multicanonical molecular dynamics simulation is presented. The method was applied to the binding of a short proline-rich peptide to a Src homology 3 (SH3) domain. The peptide and the side-chains at the ligand binding cleft of SH3 were completely flexible and the large number of possible conformations and dispositions of the peptide were sampled. The reweighted canonical resemble at 300 K resulted in only a few predominant binding modes, one of which was similar to the complex crystal structure. The inverted peptide orientation was also observed in the other binding modes.

Chemical shift assignments of the partially deuterated Fyn SH2-SH3 domain.

PubMed

Kieken, Fabien; Loth, Karine; van Nuland, Nico; Tompa, Peter; Lenaerts, Tom

2018-04-01

Src Homology 2 and 3 (SH2 and SH3) are two key protein interaction modules involved in regulating the activity of many proteins such as tyrosine kinases and phosphatases by respective recognition of phosphotyrosine and proline-rich regions. In the Src family kinases, the inactive state of the protein is the direct result of the interaction of the SH2 and the SH3 domain with intra-molecular regions, leading to a closed structure incompetent with substrate modification. Here, we report the 1 H, 15 N and 13 C backbone- and side-chain chemical shift assignments of the partially deuterated Fyn SH3-SH2 domain and structural differences between tandem and single domains. The BMRB accession number is 27165.

Molecular dissection of the interaction between the SH3 domain and the SH2-Kinase Linker region in PTK6.

PubMed

Kim, Han Ie; Jung, Jinwon; Lee, Eun-Saem; Kim, Yong-Chul; Lee, Weontae; Lee, Seung-Taek

2007-11-03

PTK6 (also known as Brk) is an intracellular tyrosine kinase that contains SH3, SH2, and tyrosine kinase catalytic (Kinase) domains. The SH3 domain of PTK6 interacts with the N-terminal half of the linker (Linker) region between the SH2 and Kinase domains. Site-directed mutagenesis and surface plasmon resonance studies showed that a tryptophan residue (Trp44) in the SH3 domain and proline residues in the Linker region, in the order of Pro177, Pro175, and Pro179, contribute to the interaction. The three-dimensional modeled structure of the SH3-Linker complex was in agreement with the biochemical data. Disruption of the intramolecular interaction between the SH3 domain and the Linker region by mutation of Trp44, Pro175, Pro177, and Pro179 markedly increased the catalytic activity of PTK6 in HEK 293 cells. These results demonstrate that Trp44 in the SH3 domain and Pro177, Pro175, and Pro179 in the N-terminal half of the Linker region play important roles in the SH3-Linker interaction to maintain the protein in an inactive conformation along with the phosphorylated Tyr447-SH2 interaction.

High-throughput analysis of peptide binding modules

PubMed Central

Liu, Bernard A.; Engelmann, Brett; Nash, Piers D.

2014-01-01

Modular protein interaction domains that recognize linear peptide motifs are found in hundreds of proteins within the human genome. Some protein interaction domains such as SH2, 14-3-3, Chromo and Bromo domains serve to recognize post-translational modification of amino acids (such as phosphorylation, acetylation, methylation etc.) and translate these into discrete cellular responses. Other modules such as SH3 and PDZ domains recognize linear peptide epitopes and serve to organize protein complexes based on localization and regions of elevated concentration. In both cases, the ability to nucleate specific signaling complexes is in large part dependent on the selectivity of a given protein module for its cognate peptide ligand. High throughput analysis of peptide-binding domains by peptide or protein arrays, phage display, mass spectrometry or other HTP techniques provides new insight into the potential protein-protein interactions prescribed by individual or even whole families of modules. Systems level analyses have also promoted a deeper understanding of the underlying principles that govern selective protein-protein interactions and how selectivity evolves. Lastly, there is a growing appreciation for the limitations and potential pitfalls of high-throughput analysis of protein-peptide interactomes. This review will examine some of the common approaches utilized for large-scale studies of protein interaction domains and suggest a set of standards for the analysis and validation of datasets from large-scale studies of peptide-binding modules. We will also highlight how data from large-scale studies of modular interaction domain families can provide insight into systems level properties such as the linguistics of selective interactions. PMID:22610655

Role of Interfacial Water Molecules in Proline-rich Ligand Recognition by the Src Homology 3 Domain of Abl*

PubMed Central

Palencia, Andres; Camara-Artigas, Ana; Pisabarro, M. Teresa; Martinez, Jose C.; Luque, Irene

2010-01-01

The interaction of Abl-Src homology 3 domain (SH3) with the high affinity peptide p41 is the most notable example of the inconsistency existing between the currently accepted description of SH3 complexes and their binding thermodynamic signature. We had previously hypothesized that the presence of interfacial water molecules is partially responsible for this thermodynamic behavior. We present here a thermodynamic, structural, and molecular dynamics simulation study of the interaction of p41 with Abl-SH3 and a set of mutants designed to alter the water-mediated interaction network. Our results provide a detailed description of the dynamic properties of the interfacial water molecules and a molecular interpretation of the thermodynamic effects elicited by the mutations in terms of the modulation of the water-mediated hydrogen bond network. In the light of these results, a new dual binding mechanism is proposed that provides a better description of proline-rich ligand recognition by Abl-SH3 and that has important implications for rational design. PMID:19906645

Role of interfacial water molecules in proline-rich ligand recognition by the Src homology 3 domain of Abl.

PubMed

Palencia, Andres; Camara-Artigas, Ana; Pisabarro, M Teresa; Martinez, Jose C; Luque, Irene

2010-01-22

The interaction of Abl-Src homology 3 domain (SH3) with the high affinity peptide p41 is the most notable example of the inconsistency existing between the currently accepted description of SH3 complexes and their binding thermodynamic signature. We had previously hypothesized that the presence of interfacial water molecules is partially responsible for this thermodynamic behavior. We present here a thermodynamic, structural, and molecular dynamics simulation study of the interaction of p41 with Abl-SH3 and a set of mutants designed to alter the water-mediated interaction network. Our results provide a detailed description of the dynamic properties of the interfacial water molecules and a molecular interpretation of the thermodynamic effects elicited by the mutations in terms of the modulation of the water-mediated hydrogen bond network. In the light of these results, a new dual binding mechanism is proposed that provides a better description of proline-rich ligand recognition by Abl-SH3 and that has important implications for rational design.

The chimeric ubiquitin ligase SH2-U-box inhibits the growth of imatinib-sensitive and resistant CML by targeting the native and T315I-mutant BCR-ABL

PubMed Central

Ru, Yi; Wang, Qinhao; Liu, Xiping; Zhang, Mei; Zhong, Daixing; Ye, Mingxiang; Li, Yuanchun; Han, Hua; Yao, Libo; Li, Xia

2016-01-01

Chronic myeloid leukemia (CML) is characterized by constitutively active fusion protein tyrosine kinase BCR-ABL. Although the tyrosine kinase inhibitor (TKI) against BCR-ABL, imatinib, is the first-line therapy for CML, acquired resistance almost inevitably emerges. The underlying mechanism are point mutations within the BCR-ABL gene, among which T315I is notorious because it resists to almost all currently available inhibitors. Here we took use of a previously generated chimeric ubiquitin ligase, SH2-U-box, in which SH2 from the adaptor protein Grb2 acts as a binding domain for activated BCR-ABL, while U-box from CHIP functions as an E3 ubiquitin ligase domain, so as to target the ubiquitination and degradation of both native and T315I-mutant BCR-ABL. As such, SH2-U-box significantly inhibited proliferation and induced apoptosis in CML cells harboring either the wild-type or T315I-mutant BCR-ABL (K562 or K562R), with BCR-ABL-dependent signaling pathways being repressed. Moreover, SH2-U-box worked in concert with imatinib in K562 cells. Importantly, SH2-U-box-carrying lentivirus could markedly suppress the growth of K562-xenografts in nude mice or K562R-xenografts in SCID mice, as well as that of primary CML cells. Collectively, by degrading the native and T315I-mutant BCR-ABL, the chimeric ubiquitin ligase SH2-U-box may serve as a potential therapy for both imatinib-sensitive and resistant CML. PMID:27329306

The chimeric ubiquitin ligase SH2-U-box inhibits the growth of imatinib-sensitive and resistant CML by targeting the native and T315I-mutant BCR-ABL.

PubMed

Ru, Yi; Wang, Qinhao; Liu, Xiping; Zhang, Mei; Zhong, Daixing; Ye, Mingxiang; Li, Yuanchun; Han, Hua; Yao, Libo; Li, Xia

2016-06-22

Chronic myeloid leukemia (CML) is characterized by constitutively active fusion protein tyrosine kinase BCR-ABL. Although the tyrosine kinase inhibitor (TKI) against BCR-ABL, imatinib, is the first-line therapy for CML, acquired resistance almost inevitably emerges. The underlying mechanism are point mutations within the BCR-ABL gene, among which T315I is notorious because it resists to almost all currently available inhibitors. Here we took use of a previously generated chimeric ubiquitin ligase, SH2-U-box, in which SH2 from the adaptor protein Grb2 acts as a binding domain for activated BCR-ABL, while U-box from CHIP functions as an E3 ubiquitin ligase domain, so as to target the ubiquitination and degradation of both native and T315I-mutant BCR-ABL. As such, SH2-U-box significantly inhibited proliferation and induced apoptosis in CML cells harboring either the wild-type or T315I-mutant BCR-ABL (K562 or K562R), with BCR-ABL-dependent signaling pathways being repressed. Moreover, SH2-U-box worked in concert with imatinib in K562 cells. Importantly, SH2-U-box-carrying lentivirus could markedly suppress the growth of K562-xenografts in nude mice or K562R-xenografts in SCID mice, as well as that of primary CML cells. Collectively, by degrading the native and T315I-mutant BCR-ABL, the chimeric ubiquitin ligase SH2-U-box may serve as a potential therapy for both imatinib-sensitive and resistant CML.

Feasibility of SH2 Binding as a Prognostic and Diagnostic Indicator: Probing the Tyrosine Phosphorylation State in Breast Cancer by Src Homology 2 Domain Binding

DTIC Science & Technology

2005-08-01

osteoclastic bone resorption, respectively. Despite formidable difficulties in drug design due to the lability and poor cell permeability of negatively...with LysO?,D6, generally been treated with two categories of drugs : bone displacing water molecules found in the previous X-ray resorption inhibitors...and Dpp [236-238]. cancer drugs , of which only 12 were shown to have survival Researchers from Aventis reported the superimposition benefit in

A novel disulfide bond in the SH2 Domain of the C-terminal Src kinase controls catalytic activity.

PubMed

Mills, Jamie E; Whitford, Paul C; Shaffer, Jennifer; Onuchic, Jose N; Adams, Joseph A; Jennings, Patricia A

2007-02-02

The SH2 domain of the C-terminal Src kinase [Csk] contains a unique disulfide bond that is not present in other known SH2 domains. To investigate whether this unusual disulfide bond serves a novel function, the effects of disulfide bond formation on catalytic activity of the full-length protein and on the structure of the SH2 domain were investigated. The kinase activity of full-length Csk decreases by an order of magnitude upon formation of the disulfide bond in the distal SH2 domain. NMR spectra of the fully oxidized and fully reduced SH2 domains exhibit similar chemical shift patterns and are indicative of similar, well-defined tertiary structures. The solvent-accessible disulfide bond in the isolated SH2 domain is highly stable and far from the small lobe of the kinase domain. However, reduction of this bond results in chemical shift changes of resonances that map to a cluster of residues that extend from the disulfide bond across the molecule to a surface that is in direct contact with the small lobe of the kinase domain in the intact molecule. Normal mode analyses and molecular dynamics calculations suggest that disulfide bond formation has large effects on residues within the kinase domain, most notably within the active-site cleft. Overall, the data indicate that reversible cross-linking of two cysteine residues in the SH2 domain greatly impacts catalytic function and interdomain communication in Csk.

Paralog-Specific Patterns of Structural Disorder and Phosphorylation in the Vertebrate SH3-SH2-Tyrosine Kinase Protein Family.

PubMed

Dos Santos, Helena G; Siltberg-Liberles, Jessica

2016-09-19

One of the largest multigene families in Metazoa are the tyrosine kinases (TKs). These are important multifunctional proteins that have evolved as dynamic switches that perform tyrosine phosphorylation and other noncatalytic activities regulated by various allosteric mechanisms. TKs interact with each other and with other molecules, ultimately activating and inhibiting different signaling pathways. TKs are implicated in cancer and almost 30 FDA-approved TK inhibitors are available. However, specific binding is a challenge when targeting an active site that has been conserved in multiple protein paralogs for millions of years. A cassette domain (CD) containing SH3-SH2-Tyrosine Kinase domains reoccurs in vertebrate nonreceptor TKs. Although part of the CD function is shared between TKs, it also presents TK specific features. Here, the evolutionary dynamics of sequence, structure, and phosphorylation across the CD in 17 TK paralogs have been investigated in a large-scale study. We establish that TKs often have ortholog-specific structural disorder and phosphorylation patterns, while secondary structure elements, as expected, are highly conserved. Further, domain-specific differences are at play. Notably, we found the catalytic domain to fluctuate more in certain secondary structure elements than the regulatory domains. By elucidating how different properties evolve after gene duplications and which properties are specifically conserved within orthologs, the mechanistic understanding of protein evolution is enriched and regions supposedly critical for functional divergence across paralogs are highlighted. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Allosteric Inhibition of the nonMyristoylated c-Abl Tyrosine Kinase by Phosphopeptides Derived from Abi1/Hssh3bp1

PubMed Central

Xiong, Xiaoling; Cui, Ping; Hossain, Sajjad; Xu, Rong; Warner, Brian; Guo, Xinhua; An, Xiuli; Debnath, Asim K.; Cowburn, David; Kotula, Leszek

2008-01-01

Here we report c-Abl kinase inhibition mediated by a phosphotyrosine located in trans in the c-Abl substrate, Abi1. The mechanism, which is pertinent to the nonmyristoylated c-Abl kinase, involves high affinity concurrent binding of the phosphotyrosine pY213 to the Abl SH2 domain and binding of a proximal PXXP motif to the Abl SH3 domain. Abi1 regulation of c-Abl in vivo appears to play a critical role, as demonstrated by inhibition of pY412 phosphorylation of the nonmyristoylated Abl by coexpression of Abi1. Pervanadate-induced c-Abl kinase activity was also reduced upon expression of the wild type Abi1 but not by expression of the Y213 to F213 mutant Abi1 in LNCaP cells, which are naturally deficient in the regulatory pY213. Our findings suggest a novel mechanism by which Abl kinase is regulated in cells. PMID:18328268

Comparison of binding energies of SrcSH2-phosphotyrosyl peptides with structure-based prediction using surface area based empirical parameterization.

PubMed Central

Henriques, D. A.; Ladbury, J. E.; Jackson, R. M.

2000-01-01

The prediction of binding energies from the three-dimensional (3D) structure of a protein-ligand complex is an important goal of biophysics and structural biology. Here, we critically assess the use of empirical, solvent-accessible surface area-based calculations for the prediction of the binding of Src-SH2 domain with a series of tyrosyl phosphopeptides based on the high-affinity ligand from the hamster middle T antigen (hmT), where the residue in the pY+ 3 position has been changed. Two other peptides based on the C-terminal regulatory site of the Src protein and the platelet-derived growth factor receptor (PDGFR) are also investigated. Here, we take into account the effects of proton linkage on binding, and test five different surface area-based models that include different treatments for the contributions to conformational change and protein solvation. These differences relate to the treatment of conformational flexibility in the peptide ligand and the inclusion of proximal ordered solvent molecules in the surface area calculations. This allowed the calculation of a range of thermodynamic state functions (deltaCp, deltaS, deltaH, and deltaG) directly from structure. Comparison with the experimentally derived data shows little agreement for the interaction of SrcSH2 domain and the range of tyrosyl phosphopeptides. Furthermore, the adoption of the different models to treat conformational change and solvation has a dramatic effect on the calculated thermodynamic functions, making the predicted binding energies highly model dependent. While empirical, solvent-accessible surface area based calculations are becoming widely adopted to interpret thermodynamic data, this study highlights potential problems with application and interpretation of this type of approach. There is undoubtedly some agreement between predicted and experimentally determined thermodynamic parameters: however, the tolerance of this approach is not sufficient to make it ubiquitously applicable. PMID:11106171

Dimerization of the docking/adaptor protein HEF1 via a carboxy-terminal helix-loop-helix domain.

PubMed

Law, S F; Zhang, Y Z; Fashena, S J; Toby, G; Estojak, J; Golemis, E A

1999-10-10

HEF1, p130(Cas), and Efs define a family of multidomain docking proteins which plays a central coordinating role for tyrosine-kinase-based signaling related to cell adhesion. HEF1 function has been specifically implicated in signaling pathways important for cell adhesion and differentiation in lymphoid and epithelial cells. While the SH3 domains and SH2-binding site domains (substrate domains) of HEF1 family proteins are well characterized and binding partners known, to date the highly conserved carboxy-terminal domains of the three proteins have lacked functional definition. In this study, we have determined that the carboxy-terminal domain of HEF1 contains a divergent helix-loop-helix (HLH) motif. This motif mediates HEF1 homodimerization and HEF1 heterodimerization with a recognition specificity similar to that of the transcriptional regulatory HLH proteins Id2, E12, and E47. We had previously demonstrated that the HEF1 carboxy-terminus expressed as a separate domain in yeast reprograms cell division patterns, inducing constitutive pseudohyphal growth. Here we show that pseudohyphal induction by HEF1 requires an intact HLH, further supporting the idea that this motif has an effector activity for HEF1, and implying that HEF1 pseudohyphal activity derives in part from interactions with yeast helix-loop-helix proteins. These combined results provide initial insight into the mode of function of the HEF1 carboxy-terminal domain and suggest that the HEF1 protein may interact with cellular proteins which control differentiation. Copyright 1999 Academic Press.

Distinctive functions of Syk N-terminal and C-terminal SH2 domains in the signaling cascade elicited by oxidative stress in B cells.

PubMed

Ding, J; Takano, T; Hermann, P; Gao, S; Han, W; Noda, C; Yanagi, S; Yamamura, H

2000-05-01

Syk plays a crucial role in the transduction of oxidative stress signaling. In this paper, we investigated the roles of Src homology 2 (SH2) domains of Syk in oxidative stress signaling, using Syk-negative DT40 cells expressing the N- or C-terminal SH2 domain mutant [mSH2(N) or mSH2(C)] of Syk. Tyrosine phosphorylation of Syk in cells expressing mSH2(N) Syk after H(2)O(2) treatment was higher than that in cells expressing wild-type Syk or mSH2(C) Syk. The tyrosine phosphorylation of wild-type Syk and mSH2(C) Syk, but not that of mSH2(N), was sensitive to PP2, a specific inhibitor of Src-family protein-tyrosine kinase. In oxidative stress, the C-terminal SH2 domain of Syk was demonstrated to be required for induction of tyrosine phosphorylation of cellular proteins, phospholipase C (PLC)-gamma2 phosphorylation, inositol 1,4, 5-triphosphate (IP(3)) generation, Ca(2)(+) release from intracellular stores, and c-Jun N-terminal kinase activation. In contrast, in mSH2(N) Syk-expressing cells, tyrosine phosphorylation of intracellular proteins including PLC-gamma2 was markedly induced in oxidative stress. The enhanced phosphorylation of mSH2(N) Syk and PLC-gamma2, however, did not link to Ca(2)(+) mobilization from intracellular pools and IP(3) generation. Thus, the N- and C-terminal SH2 domains of Syk possess distinctive functions in oxidative stress signaling.

Intramolecular Dynamics within the N-Cap-SH3-SH2 Regulatory Unit of the c-Abl Tyrosine Kinase Reveal Targeting to the Cellular Membrane*♦

PubMed Central

de Oliveira, Guilherme A. P.; Pereira, Elen G.; Ferretti, Giulia D. S.; Valente, Ana Paula; Cordeiro, Yraima; Silva, Jerson L.

2013-01-01

c-Abl is a key regulator of cell signaling and is under strict control via intramolecular interactions. In this study, we address changes in the intramolecular dynamics coupling within the c-Abl regulatory unit by presenting its N-terminal segment (N-Cap) with an alternative function in the cell as c-Abl becomes activated. Using small angle x-ray scattering, nuclear magnetic resonance, and confocal microscopy, we demonstrate that the N-Cap and the Src homology (SH) 3 domain acquire μs-ms motions upon N-Cap association with the SH2-L domain, revealing a stabilizing synergy between these segments. The N-Cap-myristoyl tether likely triggers the protein to anchor to the membrane because of these flip-flop dynamics, which occur in the μs-ms time range. This segment not only presents the myristate during c-Abl inhibition but may also trigger protein localization inside the cell in a functional and stability-dependent mechanism that is lost in Bcr-Abl+ cells, which underlie chronic myeloid leukemia. This loss of intramolecular dynamics and binding to the cellular membrane is a potential therapeutic target. PMID:23928308

Intramolecular dynamics within the N-Cap-SH3-SH2 regulatory unit of the c-Abl tyrosine kinase reveal targeting to the cellular membrane.

PubMed

de Oliveira, Guilherme A P; Pereira, Elen G; Ferretti, Giulia D S; Valente, Ana Paula; Cordeiro, Yraima; Silva, Jerson L

2013-09-27

c-Abl is a key regulator of cell signaling and is under strict control via intramolecular interactions. In this study, we address changes in the intramolecular dynamics coupling within the c-Abl regulatory unit by presenting its N-terminal segment (N-Cap) with an alternative function in the cell as c-Abl becomes activated. Using small angle x-ray scattering, nuclear magnetic resonance, and confocal microscopy, we demonstrate that the N-Cap and the Src homology (SH) 3 domain acquire μs-ms motions upon N-Cap association with the SH2-L domain, revealing a stabilizing synergy between these segments. The N-Cap-myristoyl tether likely triggers the protein to anchor to the membrane because of these flip-flop dynamics, which occur in the μs-ms time range. This segment not only presents the myristate during c-Abl inhibition but may also trigger protein localization inside the cell in a functional and stability-dependent mechanism that is lost in Bcr-Abl(+) cells, which underlie chronic myeloid leukemia. This loss of intramolecular dynamics and binding to the cellular membrane is a potential therapeutic target.

Oncogenic JAK2V617F requires an intact SH2-like domain for constitutive activation and induction of a myeloproliferative disease in mice.

PubMed

Gorantla, Sivahari P; Dechow, Tobias N; Grundler, Rebekka; Illert, Anna Lena; Zum Büschenfelde, Christian Meyer; Kremer, Marcus; Peschel, Christian; Duyster, Justus

2010-11-25

The oncogenic JAK2V617F mutation is found in myeloproliferative neoplasms (MPNs) and is believed to be critical for leukemogenesis. Here we show that JAK2V617F requires an intact SH2 domain for constitutive activation of downstream signaling pathways. In addition, there is a strict requirement of cytokine receptor expression for the activation of this oncogene. Further analysis showed that the SH2 domain mutation did not interfere with JAK2 membrane distribution. However, coimmunoprecipitated experiments revealed a role for the SH2 domain in the aggregation and cross-phosphorylation of JAK2V617F at the cell membrane. Forced overexpression of cytokine receptors could rescue the JAK2V617F SH2 mutant supporting a critical role of JAK2V617F abundance for constitutive activation. However, under physiologic cytokine receptor expression the SH2 domain is absolutely necessary for oncogenic JAK2V617F activation. This is demonstrated in a bone marrow transplantation model, in which an intact SH2 domain in JAK2V617F is required for the induction of an MPN-like disease. Thus, our results points to an indispensable role of the SH2 domain in JAK2V617F-induced MPNs.

Drosophila Ack targets its substrate, the sorting nexin DSH3PX1, to a protein complex involved in axonal guidance.

PubMed

Worby, Carolyn A; Simonson-Leff, Nancy; Clemens, James C; Huddler, Donald; Muda, Marco; Dixon, Jack E

2002-03-15

Dock, the Drosophila orthologue of Nck, is an adaptor protein that is known to function in axonal guidance paradigms in the fly including proper development of neuronal connections in photoreceptor cells and axonal tracking in Bolwig's organ. To develop a better understanding of axonal guidance at the molecular level, we purified proteins in a complex with the SH2 domain of Dock from fly Schneider 2 cells. A protein designated p145 was identified and shown to be a tyrosine kinase with sequence similarity to mammalian Cdc-42-associated tyrosine kinases. We demonstrate that Drosophila Ack (DAck) can be co-immunoprecipitated with Dock and DSH3PX1 from fly cell extracts. The domains responsible for the in vitro interaction between Drosophila Ack and Dock were identified, and direct protein-protein interactions between complex members were established. We conclude that DSH3PX1 is a substrate for DAck in vivo and in vitro and define one of the major in vitro sites of DSH3PX1 phosphorylation to be Tyr-56. Tyr-56 is located within the SH3 domain of DSH3PX1, placing it in an important position for regulating the binding of proline-rich targets. We demonstrate that Tyr-56 phosphorylation by DAck diminishes the DSH3PX1 SH3 domain interaction with the Wiskott-Aldrich Syndrome protein while enabling DSH3PX1 to associate with Dock. Furthermore, when Tyr-56 is mutated to aspartate or glutamate, the binding to Wiskott-Aldrich Syndrome protein is abrogated. These results suggest that the phosphorylation of DSH3PX1 by DAck targets this sorting nexin to a protein complex that includes Dock, an adaptor protein important for axonal guidance.

Purification and molecular cloning of SH2- and SH3-containing inositol polyphosphate-5-phosphatase, which is involved in the signaling pathway of granulocyte-macrophage colony-stimulating factor, erythropoietin, and Bcr-Abl.

PubMed

Odai, H; Sasaki, K; Iwamatsu, A; Nakamoto, T; Ueno, H; Yamagata, T; Mitani, K; Yazaki, Y; Hirai, H

1997-04-15

Grb2/Ash and Shc are the adapter proteins that link tyrosine-kinase receptors to Ras and make tyrosine-kinase functionally associated with receptors and Ras in fibroblasts and hematopoietic cells. Grb2/Ash and Shc have the SH3, SH2, or phosphotyrosine binding domains. These domains bind to proteins containing proline-rich regions or tyrosine-phosphorylated proteins and contribute to the association of Grb2/Ash and Shc with other signaling molecules. However, there could remain unidentified signaling molecules that physically and functionally interact with these adapter proteins and have biologically important roles in the signaling pathways. By using the GST fusion protein including the full length of Grb2/Ash, we have found that c-Cbl and an unidentified 135-kD protein (pp135) are associated with Grb2/Ash. We have also found that they become tyrosine-phosphorylated by treatment of a human leukemia cell line, UT-7, with granulocyte-macrophage colony-stimulating factor (GM-CSF). We have purified the pp135 by using GST-Grb2/Ash affinity column and have isolated the full-length complementary DNA (cDNA) encoding the pp135 using a cDNA probe, which was obtained by the degenerate polymerase chain reaction based on a peptide sequence of the purified pp135. The cloned cDNA has 3,958 nucleotides that contain a single long open reading frame of 3,567 nucleotides, encoding a 1,189 amino acid protein with a predicted molecular weight of approximately 133 kD. The deduced amino acid sequence reveals that pp135 is a protein that has one SH2, one SH3, and one proline-rich domain. The pp135, which contains two motifs conserved among the inositol polyphosphate-5-phosphatase proteins, was shown to have the inositol polyphosphate-5-phosphatase activity. The pp135 was revealed to associate constitutively with Grb2/Ash and inducibly with Shc using UT-7 cells stimulated with GM-CSF. In the cell lines derived from human chronic myelogenous leukemia, pp135 was constitutively tyrosine-phosphorylated and associated with Shc and Bcr-Abl. These facts suggest that pp135 is a signaling molecule that has a unique enzymatic activity and should play an important role in the signaling pathway triggered by GM-CSF and in the transformation of hematopoietic cells caused by Bcr-Abl.

A high affinity RIM-binding protein/Aplip1 interaction prevents the formation of ectopic axonal active zones.

PubMed

Siebert, Matthias; Böhme, Mathias A; Driller, Jan H; Babikir, Husam; Mampell, Malou M; Rey, Ulises; Ramesh, Niraja; Matkovic, Tanja; Holton, Nicole; Reddy-Alla, Suneel; Göttfert, Fabian; Kamin, Dirk; Quentin, Christine; Klinedinst, Susan; Andlauer, Till Fm; Hell, Stefan W; Collins, Catherine A; Wahl, Markus C; Loll, Bernhard; Sigrist, Stephan J

2015-08-14

Synaptic vesicles (SVs) fuse at active zones (AZs) covered by a protein scaffold, at Drosophila synapses comprised of ELKS family member Bruchpilot (BRP) and RIM-binding protein (RBP). We here demonstrate axonal co-transport of BRP and RBP using intravital live imaging, with both proteins co-accumulating in axonal aggregates of several transport mutants. RBP, via its C-terminal Src-homology 3 (SH3) domains, binds Aplip1/JIP1, a transport adaptor involved in kinesin-dependent SV transport. We show in atomic detail that RBP C-terminal SH3 domains bind a proline-rich (PxxP) motif of Aplip1/JIP1 with submicromolar affinity. Pointmutating this PxxP motif provoked formation of ectopic AZ-like structures at axonal membranes. Direct interactions between AZ proteins and transport adaptors seem to provide complex avidity and shield synaptic interaction surfaces of pre-assembled scaffold protein transport complexes, thus, favouring physiological synaptic AZ assembly over premature assembly at axonal membranes.

Structural Characterization of Monomeric/Dimeric State of p59fyn SH2 Domain.

PubMed

Huculeci, Radu; Kieken, Fabien; Garcia-Pino, Abel; Buts, Lieven; van Nuland, Nico; Lenaerts, Tom

2017-01-01

Src homology 2 (SH2) domains are key modulators in various signaling pathways allowing the recognition of phosphotyrosine sites of different proteins. Despite the fact that SH2 domains acquire their biological functions in a monomeric state, a multitude of reports have shown their tendency to dimerize. Here, we provide a technical description on how to isolate and characterize by gel filtration, circular dichroism (CD), and nuclear magnetic resonance (NMR) each conformational state of p59 fyn SH2 domain.

Chimeric Ply187 endolysin kills Staphylococcus aureus more effectively than the parental enzyme.

USDA-ARS?s Scientific Manuscript database

Peptidoglycan hydrolases are an effective new source of antimicrobials. A chimeric fusion protein of the Ply187 endopeptidase domain and LysK SH3b cell wall binding domain is a potent agent against Staphylococcus aureus in three functional assays....

The CDM Superfamily Protein MBC Directs Myoblast Fusion through a Mechanism That Requires Phosphatidylinositol 3,4,5-Triphosphate Binding but Is Independent of Direct Interaction with DCrk▿§

PubMed Central

Balagopalan, Lakshmi; Chen, Mei-Hui; Geisbrecht, Erika R.; Abmayr, Susan M.

2006-01-01

myoblast city (mbc), a member of the CDM superfamily, is essential in the Drosophila melanogaster embryo for fusion of myoblasts into multinucleate fibers. Using germ line clones in which both maternal and zygotic contributions were eliminated and rescue of the zygotic loss-of-function phenotype, we established that mbc is required in the fusion-competent subset of myoblasts. Along with its close orthologs Dock180 and CED-5, MBC has an SH3 domain at its N terminus, conserved internal domains termed DHR1 and DHR2 (or “Docker”), and C-terminal proline-rich domains that associate with the adapter protein DCrk. The importance of these domains has been evaluated by the ability of MBC mutations and deletions to rescue the mbc loss-of-function muscle phenotype. We demonstrate that the SH3 and Docker domains are essential. Moreover, ethyl methanesulfonate-induced mutations that change amino acids within the MBC Docker domain to residues that are conserved in other CDM family members nevertheless eliminate MBC function in the embryo, which suggests that these sites may mediate interactions specific to Drosophila MBC. A functional requirement for the conserved DHR1 domain, which binds to phosphatidylinositol 3,4,5-triphosphate, implicates phosphoinositide signaling in myoblast fusion. Finally, the proline-rich C-terminal sites mediate strong interactions with DCrk, as expected. These sites are not required for MBC to rescue the muscle loss-of-function phenotype, however, which suggests that MBC's role in myoblast fusion can be carried out independently of direct DCrk binding. PMID:17030600

Phospholipase C and D regulation of Src, calcium release and membrane fusion during Xenopus laevis development

PubMed Central

Stith, Bradley J.

2015-01-01

This review emphasizes how lipids regulate membrane fusion and the proteins involved in three developmental stages: oocyte maturation to the fertilizable egg, fertilization and during first cleavage. Decades of work show that phosphatidic acid (PA) releases intracellular calcium, and recent work shows that the lipid can activate Src tyrosine kinase or phospholipase C during Xenopus fertilization. Numerous reports are summarized to show three levels of increase in lipid second messengers inositol 1,4,5-trisphosphate and sn 1,2-diacylglycerol (DAG) during the three different developmental stages. In addition, possible roles for PA, ceramide, lysophosphatidylcholine, plasmalogens, phosphatidylinositol 4-phosphate, phosphatidylinositol 5-phosphate, phosphatidylinositol 4,5-bisphosphate, membrane microdomains (rafts) and phosphatidylinositol 3,4,5-trisphosphate in regulation of membrane fusion (acrosome reaction, sperm-egg fusion, cortical granule exocytosis), inositol 1,4,5-trisphosphate receptors, and calcium release are discussed. The role of six lipases involved in generating putative lipid second messengers during fertilization is also discussed: phospholipase D, autotaxin, lipin1, sphingomyelinase, phospholipase C, and phospholipase A2. More specifically, proteins involved in developmental events and their regulation through lipid binding to SH3, SH4, PH, PX, or C2 protein domains is emphasized. New models are presented for PA activation of Src (through SH3, SH4 and a unique domain), that this may be why the SH2 domain of PLCγ is not required for Xenopus fertilization, PA activation of phospholipase C, a role for PA during the calcium wave after fertilization, and that calcium/calmodulin may be responsible for the loss of Src from rafts after fertilization. Also discussed is that the large DAG increase during fertilization derives from phospholipase D production of PA and lipin dephosphorylation to DAG. PMID:25748412

Effect of the SH3-SH2 domain linker sequence on the structure of Hck kinase.

PubMed

Meiselbach, Heike; Sticht, Heinrich

2011-08-01

The coordination of activity in biological systems requires the existence of different signal transduction pathways that interact with one another and must be precisely regulated. The Src-family tyrosine kinases, which are found in many signaling pathways, differ in their physiological function despite their high overall structural similarity. In this context, the differences in the SH3-SH2 domain linkers might play a role for differential regulation, but the structural consequences of linker sequence remain poorly understood. We have therefore performed comparative molecular dynamics simulations of wildtype Hck and of a mutant Hck in which the SH3-SH2 domain linker is replaced by the corresponding sequence from the homologous kinase Lck. These simulations reveal that linker replacement not only affects the orientation of the SH3 domain itself, but also leads to an alternative conformation of the activation segment in the Hck kinase domain. The sequence of the SH3-SH2 domain linker thus exerts a remote effect on the active site geometry and might therefore play a role in modulating the structure of the inactive kinase or in fine-tuning the activation process itself.

Characterization of the Lytic Capability of a LysK-Like Endolysin, Lys-phiSA012, Derived from a Polyvalent Staphylococcus aureus Bacteriophage

PubMed Central

Nakamura, Tomohiro; Furusawa, Takaaki; Ohno, Hazuki; Takahashi, Hiromichi; Kitana, Junya; Usui, Masaru; Higuchi, Hidetoshi; Tamura, Yutaka

2018-01-01

Antibiotic-resistant bacteria (ARB) have spread widely and rapidly, with their increased occurrence corresponding with the increased use of antibiotics. Infections caused by Staphylococcus aureus have a considerable negative impact on human and livestock health. Bacteriophages and their peptidoglycan hydrolytic enzymes (endolysins) have received significant attention as novel approaches against ARB, including S. aureus. In the present study, we purified an endolysin, Lys-phiSA012, which harbors a cysteine/histidine-dependent amidohydrolase/peptidase (CHAP) domain, an amidase domain, and a SH3b cell wall binding domain, derived from a polyvalent S. aureus bacteriophage which we reported previously. We demonstrate that Lys-phiSA012 exhibits high lytic activity towards staphylococcal strains, including methicillin-resistant S. aureus (MRSA). Analysis of deletion mutants showed that only mutants possessing the CHAP and SH3b domains could lyse S. aureus, indicating that lytic activity of the CHAP domain depended on the SH3b domain. The presence of at least 1 mM Ca2+ and 100 µM Zn2+ enhanced the lytic activity of Lys-phiSA012 in a turbidity reduction assay. Furthermore, a minimum inhibitory concentration (MIC) assay showed that the addition of Lys-phiSA012 decreased the MIC of oxacillin. Our results suggest that endolysins are a promising approach for replacing current antimicrobial agents and may contribute to the proper use of antibiotics, leading to the reduction of ARB. PMID:29495305

Structural basis of unique ligand specificity of KAI2-like protein from parasitic weed Striga hermonthica.

PubMed

Xu, Yuqun; Miyakawa, Takuya; Nakamura, Hidemitsu; Nakamura, Akira; Imamura, Yusaku; Asami, Tadao; Tanokura, Masaru

2016-08-10

The perception of two plant germination inducers, karrikins and strigolactones, are mediated by the proteins KAI2 and D14. Recently, KAI2-type proteins from parasitic weeds, which are possibly related to seed germination induced by strigolactone, have been classified into three clades characterized by different responses to karrikin/strigolactone. Here we characterized a karrikin-binding protein in Striga (ShKAI2iB) that belongs to intermediate-evolving KAI2 and provided the structural bases for its karrikin-binding specificity. Binding assays showed that ShKAI2iB bound karrikins but not strigolactone, differing from other KAI2 and D14. The crystal structures of ShKAI2iB and ShKAI2iB-karrikin complex revealed obvious structural differences in a helix located at the entry of its ligand-binding cavity. This results in a smaller closed pocket, which is also the major cause of ShKAI2iB's specificity of binding karrikin. Our structural study also revealed that a few non-conserved amino acids led to the distinct ligand-binding profile of ShKAI2iB, suggesting that the evolution of KAI2 resulted in its diverse functions.

The Activation of c-Src Tyrosine Kinase: Conformational Transition Pathway and Free Energy Landscape.

PubMed

Fajer, Mikolai; Meng, Yilin; Roux, Benoît

2017-04-20

Tyrosine kinases are important cellular signaling allosteric enzymes that regulate cell growth, proliferation, metabolism, differentiation, and migration. Their activity must be tightly controlled, and malfunction can lead to a variety of diseases, particularly cancer. The nonreceptor tyrosine kinase c-Src, a prototypical model system and a representative member of the Src-family, functions as complex multidomain allosteric molecular switches comprising SH2 and SH3 domains modulating the activity of the catalytic domain. The broad picture of self-inhibition of c-Src via the SH2 and SH3 regulatory domains is well characterized from a structural point of view, but a detailed molecular mechanism understanding is nonetheless still lacking. Here, we use advanced computational methods based on all-atom molecular dynamics simulations with explicit solvent to advance our understanding of kinase activation. To elucidate the mechanism of regulation and self-inhibition, we have computed the pathway and the free energy landscapes for the "inactive-to-active" conformational transition of c-Src for different configurations of the SH2 and SH3 domains. Using the isolated c-Src catalytic domain as a baseline for comparison, it is observed that the SH2 and SH3 domains, depending upon their bound orientation, promote either the inactive or active state of the catalytic domain. The regulatory structural information from the SH2-SH3 tandem is allosterically transmitted via the N-terminal linker of the catalytic domain. Analysis of the conformational transition pathways also illustrates the importance of the conserved tryptophan 260 in activating c-Src, and reveals a series of concerted events during the activation process.

Molecular Basis of Gain-of-Function LEOPARD Syndrome-Associated SHP2 Mutations

PubMed Central

2015-01-01

The Src homology 2 (SH2) domain-containing protein tyrosine phosphatase 2 (SHP2) is a critical signal transducer downstream of growth factors that promotes the activation of the RAS-ERK1/2 cascade. In its basal state, SHP2 exists in an autoinhibited closed conformation because of an intramolecular interaction between its N-SH2 and protein tyrosine phosphatase (PTP) domains. Binding to pTyr ligands present on growth factor receptors and adaptor proteins with its N-SH2 domain localizes SHP2 to its substrates and frees the active site from allosteric inhibition. Germline mutations in SHP2 are known to cause both Noonan syndrome (NS) and LEOPARD syndrome (LS), two clinically similar autosomal dominant developmental disorders. NS-associated SHP2 mutants display elevated phosphatase activity, while LS-associated SHP2 mutants exhibit reduced catalytic activity. A conundrum in how clinically similar diseases result from mutations to SHP2 that have opposite effects on this enzyme’s catalytic functionality exists. Here we report a comprehensive investigation of the kinetic, structural, dynamic, and biochemical signaling properties of the wild type as well as all reported LS-associated SHP2 mutants. The results reveal that LS-causing mutations not only affect SHP2 phosphatase activity but also induce a weakening of the intramolecular interaction between the N-SH2 and PTP domains, leading to mutants that are more readily activated by competing pTyr ligands. Our data also indicate that the residual phosphatase activity associated with the LS SHP2 mutant is required for enhanced ERK1/2 activation. Consequently, catalytically impaired SHP2 mutants could display gain-of-function properties because of their ability to localize to the vicinity of substrates for longer periods of time, thereby affording the opportunity for prolonged substrate turnover and sustained RAS-ERK1/2 activation. PMID:24935154

Preparation of crystals for characterizing the Grb7 SH2 domain before and after complex formation with a bicyclic peptide antagonist.

PubMed

Ambaye, Nigus D; Gunzburg, Menachem J; Traore, Daouda A K; Del Borgo, Mark P; Perlmutter, Patrick; Wilce, Matthew C J; Wilce, Jacqueline A

2014-02-01

Human growth factor receptor-bound protein 7 (Grb7) is an adapter protein involved in cell growth, migration and proliferation. It is now recognized that Grb7 is an emerging therapeutic target in specific cancer subtypes. Recently, the discovery of a bicyclic peptide inhibitor that targets the Grb7 SH2 domain, named G7-B1, was reported. In an attempt to probe the foundation of its interaction with Grb7, the crystallization and preliminary data collection of both the apo and G7-B1-bound forms of the Grb7 SH2 domain are reported here. Diffraction-quality crystals were obtained using the hanging-drop vapour-diffusion method. After several rounds of microseeding, crystals of the apo Grb7 SH2 domain were obtained that diffracted to 1.8 Å resolution, while those of the G7-B1-Grb7 SH2 domain complex diffracted to 2.2 Å resolution. The apo Grb7 SH2 domain crystallized in the trigonal space group P63, whereas the G7-B1-Grb7 SH2 domain complex crystallized in the monoclinic space group P21. The experimental aspects of crystallization, crystal optimization and data collection and the preliminary data are reported.

Expression, purification and crystallization of a human protein SH3BGRL at atomic resolution

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yin, Lei; Zhu, De-Yu; Yang, Na

2005-04-01

The protein SH3BGRL, containing both SH3-binding and Homer EVH1-binding motifs, has been crystallized using the hanging-drop vapour-diffusion method. The protein SH3BGRL, containing both SH3-binding and Homer EVH1-binding motifs, has been crystallized using the hanging-drop vapour-diffusion method. The crystals diffract to 0.88 Å resolution and belong to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 28.8886, b = 34.9676, c = 98.0016 Å. Preliminary analysis indicates that the asymmetric unit contains one molecule and has a solvent content of about 34%.

Direct association of Csk homologous kinase (CHK) with the diphosphorylated site Tyr568/570 of the activated c-KIT in megakaryocytes.

PubMed

Price, D J; Rivnay, B; Fu, Y; Jiang, S; Avraham, S; Avraham, H

1997-02-28

The Csk homologous kinase (CHK), formerly MATK, has previously been shown to bind to activated c-KIT. In this report, we characterize the binding of SH2(CHK) to specific phosphotyrosine sites on the c-KIT protein sequence. Phosphopeptide inhibition of the in vitro interaction of SH2(CHK)-glutathione S-transferase fusion protein/c-KIT from SCF/KL-treated Mo7e megakaryocytic cells indicated that two sites on c-KIT were able to bind SH2(CHK). These sites were the Tyr568/570 diphosphorylated sequence and the monophosphorylated Tyr721 sequence. To confirm this, we precipitated native CHK from cellular extracts using phosphorylated peptides linked to Affi-Gel 15. In addition, purified SH2(CHK)-glutathione S-transferase fusion protein was precipitated with the same peptide beads. All of the peptide bead-binding studies were consistent with the direct binding of SH2(CHK) to phosphorylated Tyr568/570 and Tyr721 sites. Binding of FYN and SHC to the diphosphorylated Tyr568/570 site was observed, while binding of Csk to this site was not observed. The SH2(CHK) binding to the two sites is direct and not through phosphorylated intermediates such as FYN or SHC. Site-directed mutagenesis of the full-length c-KIT cDNA followed by transient transfection indicated that only the Tyr568/570, and not the Tyr721, is able to bind SH2(CHK). This indicates that CHK binds to the same site on c-KIT to which FYN binds, possibly bringing the two into proximity on associated c-KIT subunits and leading to the down-regulation of FYN by CHK.

Gab-family adapter molecules in signal transduction of cytokine and growth factor receptors, and T and B cell antigen receptors.

PubMed

Hibi, M; Hirano, T

2000-04-01

Gab1 and Gab2 (Grb2 associated binder 1 and 2) are scaffolding adapter molecules that display sequence similarity with Drosophila DOS (daughter of sevenless), which is a potential substrate for the protein tyrosine phosphatase, Corkscrew, Both Gab1 and Gab2, like DOS, have a pleckstrin homology domain and potential binding sites for SH2 and SH3 domains. Gab1 and Gab2 are phosphorylated on tyrosine upon the stimulation of various cytokines, growth factors, and antigen receptors, and interact with signaling molecules, such as Grb2, SHP-2, and PI-3 kinase. Overexpression of Gab1 or Gab2 mimics or enhances growth factor or cytokine-mediated biological processes and activates ERK MAP kinase. These data imply that Gab1 and Gab2 act downstream of a broad range of cytokine and growth factor receptors, as well as T and B antigen receptors, and link these receptors to ERK MAP kinase and biological actions.

Progesterone receptor (PR) polyproline domain (PPD) mediates inhibition of epidermal growth factor receptor (EGFR) signaling in non-small cell lung cancer cells.

PubMed

Kawprasertsri, Sornsawan; Pietras, Richard J; Marquez-Garban, Diana C; Boonyaratanakornkit, Viroj

2016-05-01

Recent evidence has suggested a possible role for progesterone receptor (PR) in the progression of non-small cell lung cancer (NSCLC). However, little is known concerning roles of PR in NSCLC. PR contains a polyproline domain (PPD), which directly binds to the SH3 domain of signaling molecules. Because PPD-SH3 interactions are essential for EGFR signaling, we hypothesized that the presence of PR-PPD interfered with EGFR-mediated signaling and cell proliferation. We examined the role of PR-PPD in cell proliferation and signaling by stably expressing PR-B, or PR-B with disrupting mutations in the PPD (PR-BΔSH3), from a tetracycline-regulated promoter in A549 NSCLC cells. PR-B dose-dependently inhibited cell growth in the absence of ligand, and progestin (R5020) treatment further suppressed the growth. Treatment with RU486 abolished PR-B- and R5020-mediated inhibition of cell proliferation. Expression of PR-BΔSH3 and treatment with R5020 or RU486 had no effect on cell proliferation. Furthermore, PR-B expression but not PR-BΔSH3 expression reduced EGF-induced A549 proliferation and activation of ERK1/2, in the absence of ligand. Taken together, our data demonstrated the significance of PR extranuclear signaling through PPD interactions in EGFR-mediated proliferation and signaling in NSCLC. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Protein-phosphotyrosine proteome profiling by superbinder-SH2 domain affinity purification mass spectrometry, sSH2-AP-MS.

PubMed

Tong, Jiefei; Cao, Biyin; Martyn, Gregory D; Krieger, Jonathan R; Taylor, Paul; Yates, Bradley; Sidhu, Sachdev S; Li, Shawn S C; Mao, Xinliang; Moran, Michael F

2017-03-01

Recently, "superbinder" SH2 domain variants with three amino acid substitutions (sSH2) were reported to have 100-fold or greater affinity for protein-phosphotyrosine (pY) than natural SH2 domains. Here we report a protocol in which His-tagged Src sSH2 efficiently captures pY-peptides from protease-digested HeLa cell total protein extracts. Affinity purification of pY-peptides by this method shows little bias for pY-proximal amino acid sequences, comparable to that achieved by using antibodies to pY, but with equal or higher yield. Superbinder-SH2 affinity purification mass spectrometry (sSH2-AP-MS) therefore provides an efficient and economical approach for unbiased pY-directed phospho-proteome profiling without the use of antibodies. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Structural basis of unique ligand specificity of KAI2-like protein from parasitic weed Striga hermonthica

PubMed Central

Xu, Yuqun; Miyakawa, Takuya; Nakamura, Hidemitsu; Nakamura, Akira; Imamura, Yusaku; Asami, Tadao; Tanokura, Masaru

2016-01-01

The perception of two plant germination inducers, karrikins and strigolactones, are mediated by the proteins KAI2 and D14. Recently, KAI2-type proteins from parasitic weeds, which are possibly related to seed germination induced by strigolactone, have been classified into three clades characterized by different responses to karrikin/strigolactone. Here we characterized a karrikin-binding protein in Striga (ShKAI2iB) that belongs to intermediate-evolving KAI2 and provided the structural bases for its karrikin-binding specificity. Binding assays showed that ShKAI2iB bound karrikins but not strigolactone, differing from other KAI2 and D14. The crystal structures of ShKAI2iB and ShKAI2iB-karrikin complex revealed obvious structural differences in a helix located at the entry of its ligand-binding cavity. This results in a smaller closed pocket, which is also the major cause of ShKAI2iB’s specificity of binding karrikin. Our structural study also revealed that a few non-conserved amino acids led to the distinct ligand-binding profile of ShKAI2iB, suggesting that the evolution of KAI2 resulted in its diverse functions. PMID:27507097

SH3BP1-induced Rac-Wave2 pathway activation regulates cervical cancer cell migration, invasion, and chemoresistance to cisplatin.

PubMed

Wang, Jingjing; Feng, Yeqian; Chen, Xishan; Du, Zheng; Jiang, Shaijun; Ma, Shuyun; Zou, Wen

2018-02-01

Cervical cancer still remains the fourth most common cancer, affecting women worldwide with large geographic variations in cervical cancer incidence and mortality rates. SH3-domain binding protein-1 (SH3BP1) specifically inactivating Rac1 and its target Wave2 is required for cell motility, thus regarded as an essential regulator of cancer cell metastasis. However, the exact effects and molecular mechanisms of SH3BP1 in cervical cancer progression are still unknown. The present study is aimed to investigate the mechanism of SH3BP1 in regulation of cervical cancer cell metastasis and chemoresistance. In the present study, we demonstrated a high SH3BP1 expression in cervical cancer tissues; a higher SH3BP1 expression is also correlated with a shorter overall survival of patients with cervical cancer. Further, we revealed that SH3BP1 overexpression promoted the invasion, migration, and chemoresistance of cervical cancer cell through increasing Rac1 activity and Wave2 protein level. The promotive effect of SH3BP1 could be partially reversed by a Rac1 inhibitor, NSC 23766. In cisplatin-resistant cervical cancer tissues, SH3BP1, Rac1, and Wave2 mRNA expression was significantly up-regulated compared to that of the cisplatin-sensitive cervical cancer tissues. Taken together, SH3BP1/Rac1/Wave2 pathway may potentially act as an effective therapeutic target combined with traditional cisplatin-based chemotherapy for cervical cancer. © 2017 Wiley Periodicals, Inc.

NS1-binding protein abrogates the elevation of cell viability by the influenza A virus NS1 protein in association with CRKL

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miyazaki, Masaya; Nishihara, Hiroshi, E-mail: hnishihara@med.hokudai.ac.jp; Hasegawa, Hideki

Highlights: •NS1 induced excessive phosphorylation of ERK and elevated cell viability. •NS1-BP expression and CRKL knockdown abolished survival effect of NS1. •NS1-BP and NS1 formed the complex through the interaction with CRKL-SH3(N). -- Abstract: The influenza A virus non-structural protein 1 (NS1) is a multifunctional virulence factor consisting of an RNA binding domain and several Src-homology (SH) 2 and SH3 binding motifs, which promotes virus replication in the host cell and helps to evade antiviral immunity. NS1 modulates general host cell physiology in association with various cellular molecules including NS1-binding protein (NS1-BP) and signaling adapter protein CRK-like (CRKL), while themore » physiological role of NS1-BP during influenza A virus infection especially in association with NS1 remains unclear. In this study, we analyzed the intracellular association of NS1-BP, NS1 and CRKL to elucidate the physiological roles of these molecules in the host cell. In HEK293T cells, enforced expression of NS1 of A/Beijing (H1N1) and A/Indonesia (H5N1) significantly induced excessive phosphorylation of ERK and elevated cell viability, while the over-expression of NS1-BP and the abrogation of CRKL using siRNA abolished such survival effect of NS1. The pull-down assay using GST-fusion CRKL revealed the formation of intracellular complexes of NS1-BP, NS1 and CRKL. In addition, we identified that the N-terminus SH3 domain of CRKL was essential for binding to NS1-BP using GST-fusion CRKL-truncate mutants. This is the first report to elucidate the novel function of NS1-BP collaborating with viral protein NS1 in modulation of host cell physiology. In addition, an alternative role of adaptor protein CRKL in association with NS1 and NS1-BP during influenza A virus infection is demonstrated.« less

Backbone dynamics in an intramolecular prolylpeptide-SH3 complex from the diphtheria toxin repressor, DtxR

PubMed Central

Bhattacharya, Nilakshee; Yi, Myunggi; Zhou, Huan-Xiang; Logan, Timothy M.

2008-01-01

Summary The diphtheria toxin repressor contains an SH3-like domain that forms an intramolecular complex with a proline-rich (Pr) peptide segment and stabilizes the inactive state of the repressor. Upon activation of DtxR by transition metals, this intramolecular complex must dissociate as the SH3 domain and Pr segment form different interactions in the active repressor. In this study we investigate the dynamics of this intramolecular complex using backbone amide nuclear spin relaxation rates determined using NMR spectroscopy and molecular dynamics trajectories. The SH3 domain in the unbound and bound states showed typical dynamics in that the secondary structures were fairly ordered with high generalized order parameters and low effective correlation times while residues in the loops connecting β-strands exhibited reduced generalized order parameters and required additional motional terms to adequately model the relaxation rates. Residues forming the Pr segment exhibited low order parameters with internal rotational correlation times on the order of 0.6 – 1 ns. Further analysis showed that the SH3 domain was rich in millisecond timescale motions while the Pr segment was rich in motions on the 100s μs timescale. Molecular dynamics simultations indicated structural rearrangements that may contribute to the observed relaxation rates and, together with the observed relaxation rate data, suggested that the Pr segment exhibits a binding ↔ unbinding equilibrium. The results of this study provide new insights into the nature of the intramolecular complex and provide a better understanding of the biological role of the SH3 domain in regulating DtxR activity. PMID:17976643

Evidence for the requirement of ITAM domains but not SLP-76/Gads interaction for integrin signaling in hematopoietic cells.

PubMed

Abtahian, Farhad; Bezman, Natalie; Clemens, Regina; Sebzda, Eric; Cheng, Lan; Shattil, Sanford J; Kahn, Mark L; Koretzky, Gary A

2006-09-01

Syk tyrosine kinase and Src homology 2 (SH2) domain-containing leukocyte-specific phosphoprotein of 76 kDa (SLP-76) are signaling mediators activated downstream of immunoreceptor tyrosine-based activation motif (ITAM)-containing immunoreceptors and integrins. While the signaling cascades descending from integrins are similar to immunoreceptors, the mechanism of Syk activation and SLP-76 recruitment remains unclear. We used an in vivo structure-function approach to study the requirements for the domains of Syk and SLP-76 in immunoreceptor and integrin signaling. We found that both SH2 domains and the kinase domain of Syk are required for immunoreceptor-dependent signaling and cellular response via integrins. While the Gads-binding domain of SLP-76 is needed for immunoreceptor signaling, it appears dispensable for integrin signaling. Syk and SLP-76 also are required for initiating and/or maintaining separation between the blood and lymphatic vasculature. Therefore, we correlated the signaling requirement of the various domains of Syk and SLP-76 to their requirement in regulating vascular separation. Our data suggest ITAMs are required in Syk-dependent integrin signaling, demonstrate the separation of the structural features of SLP-76 to selectively support immunoreceptor versus integrin signaling, and provide evidence that the essential domains of SLP-76 for ITAM signals are those which most efficiently support separation between lymphatic and blood vessels.

Theoretical Insights Reveal Novel Motions in Csk’s SH3 Domain That Control Kinase Activation

PubMed Central

Barkho, Sulyman; Pierce, Levi C. T.; Li, Sheng; Adams, Joseph A.; Jennings, Patricia A.

2015-01-01

The Src family of tyrosine kinases (SFKs) regulate numerous aspects of cell growth and differentiation and are under the principal control of the C-terminal Src Kinase (Csk). Although Csk and SFKs share conserved kinase, SH2 and SH3 domains, they differ considerably in three-dimensional structure, regulatory mechanism, and the intrinsic kinase activities. Although the SH2 and SH3 domains are known to up- or down-regulate tyrosine kinase function, little is known about the global motions in the full-length kinase that govern these catalytic variations. We use a combination of accelerated Molecular Dynamics (aMD) simulations and experimental methods to provide a new view of functional motions in the Csk scaffold. These computational studies suggest that high frequency vibrations in the SH2 domain are coupled through the N-terminal lobe of the kinase domain to motions in the SH3 domain. The effects of these reflexive movements on the kinase domain can be viewed using both Deuterium Exchange Mass Spectrometry (DXMS) and steady-state kinetic methods. Removal of several contacts, including a crystallographically unobserved N-terminal segment, between the SH3 and kinase domains short-circuit these coupled motions leading to reduced catalytic efficiency and stability of N-lobe motifs within the kinase domain. The data expands the model of Csk’s activation whereby separate domains productively interact with two diametrically opposed surfaces of the kinase domain. Such reversible transitions may organize the active structure of the tyrosine kinase domain of Csk. PMID:26030592

Auto-inhibition and phosphorylation-induced activation of PLC-γ isozymes

PubMed Central

Hajicek, Nicole; Charpentier, Thomas H.; Rush, Jeremy R.; Harden, T. Kendall; Sondek, John

2013-01-01

Multiple extracellular stimuli, such as growth factors and antigens, initiate signaling cascades through tyrosine phosphorylation and activation of phospholipase C (PLC)-γ isozymes. Like most other PLCs, PLC-γ1 is basally auto-inhibited by its X-Y linker, which separates the X-and Y-boxes of the catalytic core. The C-terminal SH2 (cSH2) domain within the X-Y linker is the critical determinant for auto-inhibition of phospholipase activity. Release of auto-inhibition requires an intramolecular interaction between the cSH2 domain and a phosphorylated tyrosine, Tyr783, also located within the X-Y linker. The molecular mechanisms that mediate auto-inhibition and phosphorylation-induced activation have not been defined. Here, we describe structures of the cSH2 domain both alone and bound to a PLC-γ1 peptide encompassing phosphorylated Tyr783. The cSH2 domain remains largely unaltered by peptide engagement. Point mutations in the cSH2 domain located at the interface with the peptide were sufficient to constitutively activate PLC-γ1 suggesting that peptide engagement directly interferes with the capacity of the cSH2 domain to block the lipase active site. This idea is supported by mutations in a complimentary surface of the catalytic core that also enhanced phospholipase activity. PMID:23777354

Regulation of synaptic vesicle recycling by complex formation between intersectin 1 and the clathrin adaptor complex AP2

PubMed Central

Pechstein, Arndt; Bacetic, Jelena; Vahedi-Faridi, Ardeschir; Gromova, Kira; Sundborger, Anna; Tomlin, Nikolay; Krainer, Georg; Vorontsova, Olga; Schäfer, Johannes G.; Owe, Simen G.; Cousin, Michael A.; Saenger, Wolfram; Shupliakov, Oleg; Haucke, Volker

2010-01-01

Clathrin-mediated synaptic vesicle (SV) recycling involves the spatiotemporally controlled assembly of clathrin coat components at phosphatidylinositiol (4, 5)-bisphosphate [PI(4,5)P2]-enriched membrane sites within the periactive zone. Such spatiotemporal control is needed to coordinate SV cargo sorting with clathrin/AP2 recruitment and to restrain membrane fission and synaptojanin-mediated uncoating until membrane deformation and clathrin coat assembly are completed. The molecular events underlying these control mechanisms are unknown. Here we show that the endocytic SH3 domain-containing accessory protein intersectin 1 scaffolds the endocytic process by directly associating with the clathrin adaptor AP2. Acute perturbation of the intersectin 1-AP2 interaction in lamprey synapses in situ inhibits the onset of SV recycling. Structurally, complex formation can be attributed to the direct association of hydrophobic peptides within the intersectin 1 SH3A-B linker region with the “side sites” of the AP2 α- and β-appendage domains. AP2 appendage association of the SH3A-B linker region inhibits binding of the inositol phosphatase synaptojanin 1 to intersectin 1. These data identify the intersectin-AP2 complex as an important regulator of clathrin-mediated SV recycling in synapses. PMID:20160082

Lytic activity of the staphylolytic Twort phage endolysin CHAP domain is enhanced by the SH3b cell wall binding domain

USDA-ARS?s Scientific Manuscript database

Increases in the prevalence of antibiotic resistant strains of Staphylococcus (S.) aureus have elicited efforts to develop novel antimicrobials to treat these drug-resistant pathogens. One potential treatment repurposes the lytic enzymes produced by bacteriophages as antimicrobials. The phage Twor...

The cyanobacterial cytochrome b6f subunit PetP adopts an SH3 fold in solution.

PubMed

Veit, Sebastian; Nagadoi, Aritaka; Rögner, Matthias; Rexroth, Sascha; Stoll, Raphael; Ikegami, Takahisa

2016-06-01

PetP is a peripheral subunit of the cytochrome b(6)f complex (b(6)f) present in both, cyanobacteria and red algae. It is bound to the cytoplasmic surface of this membrane protein complex where it greatly affects the efficiency of the linear photosynthetic electron flow although it is not directly involved in the electron transfer reactions. Despite the crystal structures of the b(6)f core complex, structural information for the transient regulatory b(6)f subunits is still missing. Here we present the first structure of PetP at atomic resolution as determined by solution NMR. The protein adopts an SH3 fold, which is a common protein motif in eukaryotes but comparatively rare in prokaryotes. The structure of PetP enabled the identification of the potential interaction site for b(6)f binding by conservation mapping. The interaction surface is mainly formed by two large loop regions and one short 310 helix which also exhibit an increased flexibility as indicated by heteronuclear steady-state {(1)H}-(15)N NOE and random coil index parameters. The properties of this potential b(6)f binding site greatly differ from the canonical peptide binding site which is highly conserved in eukaryotic SH3 domains. Interestingly, three other proteins of the photosynthetic electron transport chain share this SH3 fold with PetP: NdhS of the photosynthetic NADH dehydrogenase-like complex (NDH-1), PsaE of the photosystem 1 and subunit α of the ferredoxin-thioredoxin reductase have, similar to PetP, a great impact on the photosynthetic electron transport. Finally, a model is presented to illustrate how SH3 domains modulate the photosynthetic electron transport processes in cyanobacteria. Copyright © 2016 Elsevier B.V. All rights reserved.

Crystal structure of an SH2-kinase construct of c-Abl and effect of the SH2 domain on kinase activity.

PubMed

Lorenz, Sonja; Deng, Patricia; Hantschel, Oliver; Superti-Furga, Giulio; Kuriyan, John

2015-06-01

Constitutive activation of the non-receptor tyrosine kinase c-Abl (cellular Abelson tyrosine protein kinase 1, Abl1) in the Bcr (breakpoint cluster region)-Abl1 fusion oncoprotein is the molecular cause of chronic myeloid leukaemia (CML). Recent studies have indicated that an interaction between the SH2 (Src-homology 2) domain and the N-lobe (N-terminal lobe) of the c-Abl kinase domain (KD) has a critical role in leukaemogenesis [Grebien et al. (2011) Cell 147, 306-319; Sherbenou et al. (2010) Blood 116, 3278-3285]. To dissect the structural basis of this phenomenon, we studied c-Abl constructs comprising the SH2 and KDs in vitro. We present a crystal structure of an SH2-KD construct bound to dasatinib, which contains the relevant interface between the SH2 domain and the N-lobe of the KD. We show that the presence of the SH2 domain enhances kinase activity moderately and that this effect depends on contacts in the SH2/N-lobe interface and is abrogated by specific mutations. Consistently, formation of the interface decreases slightly the association rate of imatinib with the KD. That the effects are small compared with the dramatic in vivo consequences suggests an important function of the SH2-N-lobe interaction might be to help disassemble the auto-inhibited conformation of c-Abl and promote processive phosphorylation, rather than substantially stimulate kinase activity.

A graph kernel approach for alignment-free domain-peptide interaction prediction with an application to human SH3 domains.

PubMed

Kundu, Kousik; Costa, Fabrizio; Backofen, Rolf

2013-07-01

State-of-the-art experimental data for determining binding specificities of peptide recognition modules (PRMs) is obtained by high-throughput approaches like peptide arrays. Most prediction tools applicable to this kind of data are based on an initial multiple alignment of the peptide ligands. Building an initial alignment can be error-prone, especially in the case of the proline-rich peptides bound by the SH3 domains. Here, we present a machine-learning approach based on an efficient graph-kernel technique to predict the specificity of a large set of 70 human SH3 domains, which are an important class of PRMs. The graph-kernel strategy allows us to (i) integrate several types of physico-chemical information for each amino acid, (ii) consider high-order correlations between these features and (iii) eliminate the need for an initial peptide alignment. We build specialized models for each human SH3 domain and achieve competitive predictive performance of 0.73 area under precision-recall curve, compared with 0.27 area under precision-recall curve for state-of-the-art methods based on position weight matrices. We show that better models can be obtained when we use information on the noninteracting peptides (negative examples), which is currently not used by the state-of-the art approaches based on position weight matrices. To this end, we analyze two strategies to identify subsets of high confidence negative data. The techniques introduced here are more general and hence can also be used for any other protein domains, which interact with short peptides (i.e. other PRMs). The program with the predictive models can be found at http://www.bioinf.uni-freiburg.de/Software/SH3PepInt/SH3PepInt.tar.gz. We also provide a genome-wide prediction for all 70 human SH3 domains, which can be found under http://www.bioinf.uni-freiburg.de/Software/SH3PepInt/Genome-Wide-Predictions.tar.gz. Supplementary data are available at Bioinformatics online.

The Adaptor Molecule Nck Localizes the WAVE Complex to Promote Actin Polymerization during CEACAM3-Mediated Phagocytosis of Bacteria

PubMed Central

Delgado Tascón, Julia; Nyffenegger-Jann, Naja J.; Hauck, Christof R.

2012-01-01

Background CEACAM3 is a granulocyte receptor mediating the opsonin-independent recognition and phagocytosis of human-restricted CEACAM-binding bacteria. CEACAM3 function depends on an intracellular immunoreceptor tyrosine-based activation motif (ITAM)-like sequence that is tyrosine phosphorylated by Src family kinases upon receptor engagement. The phosphorylated ITAM-like sequence triggers GTP-loading of Rac by directly associating with the guanine nucleotide exchange factor (GEF) Vav. Rac stimulation in turn is critical for actin cytoskeleton rearrangements that generate lamellipodial protrusions and lead to bacterial uptake. Principal Findings In our present study we provide biochemical and microscopic evidence that the adaptor proteins Nck1 and Nck2, but not CrkL, Grb2 or SLP-76, bind to tyrosine phosphorylated CEACAM3. The association is phosphorylation-dependent and requires the Nck SH2 domain. Overexpression of the isolated Nck1 SH2 domain, RNAi-mediated knock-down of Nck1, or genetic deletion of Nck1 and Nck2 interfere with CEACAM3-mediated bacterial internalization and with the formation of lamellipodial protrusions. Nck is constitutively associated with WAVE2 and directs the actin nucleation promoting WAVE complex to tyrosine phosphorylated CEACAM3. In turn, dominant-negative WAVE2 as well as shRNA-mediated knock-down of WAVE2 or the WAVE-complex component Nap1 reduce internalization of bacteria. Conclusions Our results provide novel mechanistic insight into CEACAM3-initiated phagocytosis. We suggest that the CEACAM3 ITAM-like sequence is optimized to co-ordinate a minimal set of cellular factors needed to efficiently trigger actin-based lamellipodial protrusions and rapid pathogen engulfment. PMID:22448228

The adaptor molecule Nck localizes the WAVE complex to promote actin polymerization during CEACAM3-mediated phagocytosis of bacteria.

PubMed

Pils, Stefan; Kopp, Kathrin; Peterson, Lisa; Delgado Tascón, Julia; Nyffenegger-Jann, Naja J; Hauck, Christof R

2012-01-01

CEACAM3 is a granulocyte receptor mediating the opsonin-independent recognition and phagocytosis of human-restricted CEACAM-binding bacteria. CEACAM3 function depends on an intracellular immunoreceptor tyrosine-based activation motif (ITAM)-like sequence that is tyrosine phosphorylated by Src family kinases upon receptor engagement. The phosphorylated ITAM-like sequence triggers GTP-loading of Rac by directly associating with the guanine nucleotide exchange factor (GEF) Vav. Rac stimulation in turn is critical for actin cytoskeleton rearrangements that generate lamellipodial protrusions and lead to bacterial uptake. In our present study we provide biochemical and microscopic evidence that the adaptor proteins Nck1 and Nck2, but not CrkL, Grb2 or SLP-76, bind to tyrosine phosphorylated CEACAM3. The association is phosphorylation-dependent and requires the Nck SH2 domain. Overexpression of the isolated Nck1 SH2 domain, RNAi-mediated knock-down of Nck1, or genetic deletion of Nck1 and Nck2 interfere with CEACAM3-mediated bacterial internalization and with the formation of lamellipodial protrusions. Nck is constitutively associated with WAVE2 and directs the actin nucleation promoting WAVE complex to tyrosine phosphorylated CEACAM3. In turn, dominant-negative WAVE2 as well as shRNA-mediated knock-down of WAVE2 or the WAVE-complex component Nap1 reduce internalization of bacteria. Our results provide novel mechanistic insight into CEACAM3-initiated phagocytosis. We suggest that the CEACAM3 ITAM-like sequence is optimized to co-ordinate a minimal set of cellular factors needed to efficiently trigger actin-based lamellipodial protrusions and rapid pathogen engulfment.

Insulin stimulates movement of sorting nexin 9 between cellular compartments: a putative role mediating cell surface receptor expression and insulin action.

PubMed Central

MaCaulay, S Lance; Stoichevska, Violet; Grusovin, Julian; Gough, Keith H; Castelli, Laura A; Ward, Colin W

2003-01-01

SNX9 (sorting nexin 9) is one member of a family of proteins implicated in protein trafficking. This family is characterized by a unique PX (Phox homology) domain that includes a proline-rich sequence and an upstream phospholipid binding domain. Many sorting nexins, including SNX9, also have a C-terminal coiled region. SNX9 additionally has an N-terminal SH3 (Src homology 3) domain. Here we have investigated the cellular localization of SNX9 and the potential role it plays in insulin action. SNX9 had a cytosolic and punctate distribution, consistent with endosomal and cytosolic localization, in 3T3L1 adipocytes. It was excluded from the nucleus. The SH3 domain was responsible, at least in part, for the membrane localization of SNX9, since expression of an SH3-domain-deleted GFP (green fluorescent protein)-SNX9 fusion protein in HEK293T cells rendered the protein cytosolic. Membrane localization may also be attributed in part to the PX domain, since in vitro phospholipid binding studies demonstrated SNX9 binding to polyphosphoinositides. Insulin induced movement of SNX9 to membrane fractions from the cytosol. A GST (glutathione S-transferase)-SNX9 fusion protein was associated with IGF1 (insulin-like growth factor 1) and insulin receptors in vitro. A GFP-SNX9 fusion protein, overexpressed in 3T3L1 adipocytes, co-immunoprecipitated with insulin receptors. Furthermore, overexpression of this GFP-SNX9 fusion protein in CHOT cells decreased insulin binding, consistent with a role for SNX9 in the trafficking of insulin receptors. Microinjection of 3T3L1 cells with an antibody against SNX9 inhibited stimulation by insulin of GLUT4 translocation. These results support the involvement of SNX9 in insulin action, via an influence on the processing/trafficking of insulin receptors. A secondary role in regulation of the cellular processing, transport and/or subcellular localization of GLUT4 is also suggested. PMID:12917015

Alternate binding modes for a ubiquitin-SH3 domain interaction studied by NMR spectroscopy.

PubMed

Korzhnev, Dmitry M; Bezsonova, Irina; Lee, Soyoung; Chalikian, Tigran V; Kay, Lewis E

2009-02-20

Surfaces of many binding domains are plastic, enabling them to interact with multiple targets. An understanding of how they bind and recognize their partners is therefore predicated on characterizing such dynamic interfaces. Yet, these interfaces are difficult to study by standard biophysical techniques that often 'freeze' out conformations or that produce data averaged over an ensemble of conformers. In this study, we used NMR spectroscopy to study the interaction between the C-terminal SH3 domain of CIN85 and ubiquitin that involves the 'classical' binding sites of these proteins. Notably, chemical shift titration data of one target with another and relaxation dispersion data that report on millisecond time scale exchange processes are both well fit to a simple binding model in which free protein is in equilibrium with a single bound conformation. However, dissociation constants and chemical shift differences between free and bound states measured from both classes of experiment are in disagreement. It is shown that the data can be reconciled by considering three-state binding models involving two distinct bound conformations. By combining titration and dispersion data, kinetic and thermodynamic parameters of the three-state binding reaction are obtained along with chemical shifts for each state. A picture emerges in which one bound conformer has increased entropy and enthalpy relative to the second and chemical shifts similar to that of the free state, suggesting a less packed interface. This study provides an example of the interplay between entropy and enthalpy to fine-tune molecular interactions involving the same binding surfaces.

Domain cooperativity in the β1a subunit is essential for dihydropyridine receptor voltage sensing in skeletal muscle

PubMed Central

Dayal, Anamika; Bhat, Vinayakumar; Franzini-Armstrong, Clara; Grabner, Manfred

2013-01-01

The dihydropyridine receptor (DHPR) β1a subunit is crucial for enhancement of DHPR triad expression, assembly of DHPRs in tetrads, and elicitation of DHPRα1S charge movement—the three prerequisites of skeletal muscle excitation–contraction coupling. Despite the ability to fully target α1S into triadic junctions and tetradic arrays, the neuronal isoform β3 was unable to restore considerable charge movement (measure of α1S voltage sensing) upon expression in β1-null zebrafish relaxed myotubes, unlike the other three vertebrate β-isoforms (β1a, β2a, and β4). Thus, we used β3 for chimerization with β1a to investigate whether any of the five distinct molecular regions of β1a is dominantly involved in inducing the voltage-sensing function of α1S. Surprisingly, systematic domain swapping between β1a and β3 revealed a pivotal role of the src homology 3 (SH3) domain and C terminus of β1a in charge movement restoration. More interestingly, β1a SH3 domain and C terminus, when simultaneously engineered into β3 sequence background, were able to fully restore charge movement together with proper intracellular Ca2+ release, suggesting cooperativity of these two domains in induction of the α1S voltage-sensing function in skeletal muscle excitation–contraction coupling. Furthermore, substitution of a proline by alanine in the putative SH3-binding polyproline motif in the proximal C terminus of β1a (also of β2a and β4) fully obstructed α1S charge movement. Consequently, we postulate a model according to which β subunits, probably via the SH3–C-terminal polyproline interaction, adapt a discrete conformation required to modify the α1S conformation apt for voltage sensing in skeletal muscle. PMID:23589859

Domain cooperativity in the β1a subunit is essential for dihydropyridine receptor voltage sensing in skeletal muscle.

PubMed

Dayal, Anamika; Bhat, Vinayakumar; Franzini-Armstrong, Clara; Grabner, Manfred

2013-04-30

The dihydropyridine receptor (DHPR) β1a subunit is crucial for enhancement of DHPR triad expression, assembly of DHPRs in tetrads, and elicitation of DHPRα1S charge movement--the three prerequisites of skeletal muscle excitation-contraction coupling. Despite the ability to fully target α1S into triadic junctions and tetradic arrays, the neuronal isoform β3 was unable to restore considerable charge movement (measure of α1S voltage sensing) upon expression in β1-null zebrafish relaxed myotubes, unlike the other three vertebrate β-isoforms (β1a, β2a, and β4). Thus, we used β3 for chimerization with β1a to investigate whether any of the five distinct molecular regions of β1a is dominantly involved in inducing the voltage-sensing function of α1S. Surprisingly, systematic domain swapping between β1a and β3 revealed a pivotal role of the src homology 3 (SH3) domain and C terminus of β1a in charge movement restoration. More interestingly, β1a SH3 domain and C terminus, when simultaneously engineered into β3 sequence background, were able to fully restore charge movement together with proper intracellular Ca(2+) release, suggesting cooperativity of these two domains in induction of the α1S voltage-sensing function in skeletal muscle excitation-contraction coupling. Furthermore, substitution of a proline by alanine in the putative SH3-binding polyproline motif in the proximal C terminus of β1a (also of β2a and β4) fully obstructed α1S charge movement. Consequently, we postulate a model according to which β subunits, probably via the SH3-C-terminal polyproline interaction, adapt a discrete conformation required to modify the α1S conformation apt for voltage sensing in skeletal muscle.

The proline-rich region of 18.5 kDa myelin basic protein binds to the SH3-domain of Fyn tyrosine kinase with the aid of an upstream segment to form a dynamic complex in vitro

PubMed Central

De Avila, Miguel; Vassall, Kenrick A.; Smith, Graham S. T.; Bamm, Vladimir V.; Harauz, George

2014-01-01

The intrinsically disordered 18.5 kDa classic isoform of MBP (myelin basic protein) interacts with Fyn kinase during oligodendrocyte development and myelination. It does so primarily via a central proline-rich SH3 (Src homology 3) ligand (T92–R104, murine 18.5 kDa MBP sequence numbering) that is part of a molecular switch due to its high degree of conservation and modification by MAP (mitogen-activated protein) and other kinases, especially at residues T92 and T95. Here, we show using co-transfection experiments of an early developmental oligodendroglial cell line (N19) that an MBP segment upstream of the primary ligand is involved in MBP–Fyn–SH3 association in cellula. Using solution NMR spectroscopy in vitro, we define this segment to comprise MBP residues (T62–L68), and demonstrate further that residues (V83–P93) are the predominant SH3-target, assessed by the degree of chemical shift change upon titration. We show by chemical shift index analysis that there is no formation of local poly-proline type II structure in the proline-rich segment upon binding, and by NOE (nuclear Overhauser effect) and relaxation measurements that MBP remains dynamic even while complexed with Fyn–SH3. The association is a new example first of a non-canonical SH3-domain interaction and second of a fuzzy MBP complex. PMID:25343306

The proline-rich region of 18.5 kDa myelin basic protein binds to the SH3-domain of Fyn tyrosine kinase with the aid of an upstream segment to form a dynamic complex in vitro.

PubMed

De Avila, Miguel; Vassall, Kenrick A; Smith, Graham S T; Bamm, Vladimir V; Harauz, George

2014-12-08

The intrinsically disordered 18.5 kDa classic isoform of MBP (myelin basic protein) interacts with Fyn kinase during oligodendrocyte development and myelination. It does so primarily via a central proline-rich SH3 (Src homology 3) ligand (T92-R104, murine 18.5 kDa MBP sequence numbering) that is part of a molecular switch due to its high degree of conservation and modification by MAP (mitogen-activated protein) and other kinases, especially at residues T92 and T95. Here, we show using co-transfection experiments of an early developmental oligodendroglial cell line (N19) that an MBP segment upstream of the primary ligand is involved in MBP-Fyn-SH3 association in cellula. Using solution NMR spectroscopy in vitro, we define this segment to comprise MBP residues (T62-L68), and demonstrate further that residues (V83-P93) are the predominant SH3-target, assessed by the degree of chemical shift change upon titration. We show by chemical shift index analysis that there is no formation of local poly-proline type II structure in the proline-rich segment upon binding, and by NOE (nuclear Overhauser effect) and relaxation measurements that MBP remains dynamic even while complexed with Fyn-SH3. The association is a new example first of a non-canonical SH3-domain interaction and second of a fuzzy MBP complex.

The conservation pattern of short linear motifs is highly correlated with the function of interacting protein domains.

PubMed

Ren, Siyuan; Yang, Guang; He, Youyu; Wang, Yiguo; Li, Yixue; Chen, Zhengjun

2008-10-01

Many well-represented domains recognize primary sequences usually less than 10 amino acids in length, called Short Linear Motifs (SLiMs). Accurate prediction of SLiMs has been difficult because they are short (often < 10 amino acids) and highly degenerate. In this study, we combined scoring matrixes derived from peptide library and conservation analysis to identify protein classes enriched of functional SLiMs recognized by SH2, SH3, PDZ and S/T kinase domains. Our combined approach revealed that SLiMs are highly conserved in proteins from functional classes that are known to interact with a specific domain, but that they are not conserved in most other protein groups. We found that SLiMs recognized by SH2 domains were highly conserved in receptor kinases/phosphatases, adaptor molecules, and tyrosine kinases/phosphatases, that SLiMs recognized by SH3 domains were highly conserved in cytoskeletal and cytoskeletal-associated proteins, that SLiMs recognized by PDZ domains were highly conserved in membrane proteins such as channels and receptors, and that SLiMs recognized by S/T kinase domains were highly conserved in adaptor molecules, S/T kinases/phosphatases, and proteins involved in transcription or cell cycle control. We studied Tyr-SLiMs recognized by SH2 domains in more detail, and found that SH2-recognized Tyr-SLiMs on the cytoplasmic side of membrane proteins are more highly conserved than those on the extra-cellular side. Also, we found that SH2-recognized Tyr-SLiMs that are associated with SH3 motifs and a tyrosine kinase phosphorylation motif are more highly conserved. The interactome of protein domains is reflected by the evolutionary conservation of SLiMs recognized by these domains. Combining scoring matrixes derived from peptide libraries and conservation analysis, we would be able to find those protein groups that are more likely to interact with specific domains.

The conservation pattern of short linear motifs is highly correlated with the function of interacting protein domains

PubMed Central

Ren, Siyuan; Yang, Guang; He, Youyu; Wang, Yiguo; Li, Yixue; Chen, Zhengjun

2008-01-01

Background Many well-represented domains recognize primary sequences usually less than 10 amino acids in length, called Short Linear Motifs (SLiMs). Accurate prediction of SLiMs has been difficult because they are short (often < 10 amino acids) and highly degenerate. In this study, we combined scoring matrixes derived from peptide library and conservation analysis to identify protein classes enriched of functional SLiMs recognized by SH2, SH3, PDZ and S/T kinase domains. Results Our combined approach revealed that SLiMs are highly conserved in proteins from functional classes that are known to interact with a specific domain, but that they are not conserved in most other protein groups. We found that SLiMs recognized by SH2 domains were highly conserved in receptor kinases/phosphatases, adaptor molecules, and tyrosine kinases/phosphatases, that SLiMs recognized by SH3 domains were highly conserved in cytoskeletal and cytoskeletal-associated proteins, that SLiMs recognized by PDZ domains were highly conserved in membrane proteins such as channels and receptors, and that SLiMs recognized by S/T kinase domains were highly conserved in adaptor molecules, S/T kinases/phosphatases, and proteins involved in transcription or cell cycle control. We studied Tyr-SLiMs recognized by SH2 domains in more detail, and found that SH2-recognized Tyr-SLiMs on the cytoplasmic side of membrane proteins are more highly conserved than those on the extra-cellular side. Also, we found that SH2-recognized Tyr-SLiMs that are associated with SH3 motifs and a tyrosine kinase phosphorylation motif are more highly conserved. Conclusion The interactome of protein domains is reflected by the evolutionary conservation of SLiMs recognized by these domains. Combining scoring matrixes derived from peptide libraries and conservation analysis, we would be able to find those protein groups that are more likely to interact with specific domains. PMID:18828911

SH2-B promotes insulin receptor substrate 1 (IRS1)- and IRS2-mediated activation of the phosphatidylinositol 3-kinase pathway in response to leptin.

PubMed

Duan, Chaojun; Li, Minghua; Rui, Liangyou

2004-10-15

Leptin regulates energy homeostasis primarily by binding and activating its long form receptor (LRb). Deficiency of either leptin or LRb causes morbid obesity. Leptin stimulates LRb-associated JAK2, thus initiating multiple pathways including the Stat3 and phosphatidylinositol (PI) 3-kinase pathways that mediate leptin biological actions. Here we report that SH2-B, a JAK2-interacting protein, promotes activation of the PI 3-kinase pathway by recruiting insulin receptor substrate 1 (IRS1) and IRS2 in response to leptin. SH2-B directly bound, via its PH and SH2 domain, to both IRS1 and IRS2 both in vitro and in intact cells and mediated formation of a JAK2/SH2-B/IRS1 or IRS2 tertiary complex. Consequently, SH2-B dramatically enhanced leptin-stimulated tyrosine phosphorylation of IRS1 and IRS2 in HEK293 cells stably expressing LRb, thus promoting association of IRS1 and IRS2 with the p85 regulatory subunit of PI 3-kinase and phosphorylation and activation of Akt. SH2-B mutants with lower affinity for IRS1 and IRS2 exhibited reduced ability to promote association of JAK2 with IRS1, tyrosine phosphorylation of IRS1, and association of IRS1 with p85 in response to leptin. Moreover, deletion of the SH2-B gene impaired leptin-stimulated tyrosine phosphorylation of endogenous IRS1 in mouse embryonic fibroblasts (MEF), which was reversed by reintroduction of SH2-B. Similarly, SH2-B promoted growth hormone-stimulated tyrosine phosphorylation of IRS1 in both HEK293 and MEF cells. Our data suggest that SH2-B is a novel mediator of the PI 3-kinase pathway in response to leptin or other hormones and cytokines that activate JAK2.

SH2 Domain Histochemistry.

PubMed

Buhs, Sophia; Nollau, Peter

2017-01-01

Among posttranslational modifications, the phosphorylation of tyrosine residues is a key modification in cell signaling. Because of its biological importance, characterization of the cellular state of tyrosine phosphorylation is of great interest. Based on the unique properties of endogenously expressed SH2 domains recognizing tyrosine phosphorylated signaling proteins with high specificity we have developed an alternative approach, coined SH2 profiling, enabling us to decipher complex patterns of tyrosine phosphorylation in various normal and cancerous tissues. So far, SH2 profiling has largely been applied for the analysis of protein extracts with the limitation that information on spatial distribution and intensity of tyrosine phosphorylation within a tissue is lost. Here, we describe a novel SH2 domain based strategy for differential characterization of the state of tyrosine phosphorylation in formaldehyde-fixed and paraffin-embedded tissues. This approach demonstrates that SH2 domains may serve as very valuable tools for the analysis of the differential state of tyrosine phosphorylation in primary tissues fixed and processed under conditions frequently applied by routine pathology laboratories.

High-Throughput Quantification of SH2 Domain-Phosphopeptide Interactions with Cellulose-Peptide Conjugate Microarrays.

PubMed

Engelmann, Brett W

2017-01-01

The Src Homology 2 (SH2) domain family primarily recognizes phosphorylated tyrosine (pY) containing peptide motifs. The relative affinity preferences among competing SH2 domains for phosphopeptide ligands define "specificity space," and underpins many functional pY mediated interactions within signaling networks. The degree of promiscuity exhibited and the dynamic range of affinities supported by individual domains or phosphopeptides is best resolved by a carefully executed and controlled quantitative high-throughput experiment. Here, I describe the fabrication and application of a cellulose-peptide conjugate microarray (CPCMA) platform to the quantitative analysis of SH2 domain specificity space. Included herein are instructions for optimal experimental design with special attention paid to common sources of systematic error, phosphopeptide SPOT synthesis, microarray fabrication, analyte titrations, data capture, and analysis.

Resolution of Site-Specific Conformational Heterogeneity in Proline-Rich Molecular Recognition by Src Homology 3 Domains.

PubMed

Horness, Rachel E; Basom, Edward J; Mayer, John P; Thielges, Megan C

2016-02-03

Conformational heterogeneity and dynamics are increasingly evoked in models of protein molecular recognition but are challenging to experimentally characterize. Here we combine the inherent temporal resolution of infrared (IR) spectroscopy with the spatial resolution afforded by selective incorporation of carbon-deuterium (C-D) bonds, which provide frequency-resolved absorptions within a protein IR spectrum, to characterize the molecular recognition of the Src homology 3 (SH3) domain of the yeast protein Sho1 with its cognate proline-rich (PR) sequence of Pbs2. The IR absorptions of C-D bonds introduced at residues along a peptide of the Pbs2 PR sequence report on the changes in the local environments upon binding to the SH3 domain. Interestingly, upon forming the complex the IR spectra of the peptides labeled with C-D bonds at either of the two conserved prolines of the PXXP consensus recognition sequence show more absorptions than there are C-D bonds, providing evidence for the population of multiple states. In contrast, the NMR spectra of the peptides labeled with (13)C at the same residues show only single resonances, indicating rapid interconversion on the NMR time scale. Thus, the data suggest that the SH3 domain recognizes its cognate peptide with a component of induced fit molecular recognition involving the adoption of multiples states, which have previously gone undetected due to interconversion between the populated states that is too fast to resolve using conventional methods.

Requirement of Nck adaptors for actin dynamics and cell migration stimulated by platelet-derived growth factor B.

PubMed

Rivera, G M; Antoku, S; Gelkop, S; Shin, N Y; Hanks, S K; Pawson, T; Mayer, B J

2006-06-20

The Nck family of Src homology (SH) 2/SH3 domain adaptors functions to link tyrosine phosphorylation induced by extracellular signals with downstream regulators of actin dynamics. We investigated the role of mammalian Nck adaptors in signaling from the activated platelet-derived growth factor (PDGF) receptor (PDGFbetaR) to the actin cytoskeleton. We report here that Nck adaptors are required for cytoskeletal reorganization and chemotaxis stimulated by PDGF-B. Analysis of tyrosine-phosphorylated proteins demonstrated that Crk-associated substrate (p130(Cas)), not the activated PDGFbetaR itself, is the major Nck SH2 domain-binding protein in PDGF-B-stimulated cells. Both Nck- and p130(Cas)-deficient cells fail to display cytoskeletal rearrangements, including the formation of membrane ruffles and the disassembly of actin bundles, typically shown by their WT counterparts in response to PDGF-B. Furthermore, Nck and p130(Cas) colocalize in phosphotyrosine-enriched membrane ruffles induced by PDGF-B in NIH 3T3 cells. These results suggest that Nck adaptors play an essential role in linking the activated PDGFbetaR with actin dynamics through a pathway that involves p130(Cas).

A graph kernel approach for alignment-free domain–peptide interaction prediction with an application to human SH3 domains

PubMed Central

Kundu, Kousik; Costa, Fabrizio; Backofen, Rolf

2013-01-01

Motivation: State-of-the-art experimental data for determining binding specificities of peptide recognition modules (PRMs) is obtained by high-throughput approaches like peptide arrays. Most prediction tools applicable to this kind of data are based on an initial multiple alignment of the peptide ligands. Building an initial alignment can be error-prone, especially in the case of the proline-rich peptides bound by the SH3 domains. Results: Here, we present a machine-learning approach based on an efficient graph-kernel technique to predict the specificity of a large set of 70 human SH3 domains, which are an important class of PRMs. The graph-kernel strategy allows us to (i) integrate several types of physico-chemical information for each amino acid, (ii) consider high-order correlations between these features and (iii) eliminate the need for an initial peptide alignment. We build specialized models for each human SH3 domain and achieve competitive predictive performance of 0.73 area under precision-recall curve, compared with 0.27 area under precision-recall curve for state-of-the-art methods based on position weight matrices. We show that better models can be obtained when we use information on the noninteracting peptides (negative examples), which is currently not used by the state-of-the art approaches based on position weight matrices. To this end, we analyze two strategies to identify subsets of high confidence negative data. The techniques introduced here are more general and hence can also be used for any other protein domains, which interact with short peptides (i.e. other PRMs). Availability: The program with the predictive models can be found at http://www.bioinf.uni-freiburg.de/Software/SH3PepInt/SH3PepInt.tar.gz. We also provide a genome-wide prediction for all 70 human SH3 domains, which can be found under http://www.bioinf.uni-freiburg.de/Software/SH3PepInt/Genome-Wide-Predictions.tar.gz. Contact: backofen@informatik.uni-freiburg.de Supplementary information: Supplementary data are available at Bioinformatics online. PMID:23813002

Csk Homologous Kinase, a Potential Regulator of CXCR4-Medicated Breast Cancer Cell Metastasis

DTIC Science & Technology

2011-08-01

is a non-receptor tyrosine kinase and a second member of the Csk family. Like Csk, CHK has Src homology 2 ( SH2 ) and SH3 domains and lacks the...MSCV-retroviral vectors encoding either wild-type CHK or kinase -dead CHK or wild type SH2 domain or SH2 -R147A or SH2 -G129A. All these constructs were... Kinase , a Potential Regulator of CXCR4-Medicated Breast Cancer Cell Metastasis Byeong-Chel Lee The University of Pittsburgh Pittsburgh, PA 15213

A novel inhibitor of STAT3 homodimerization selectively suppresses STAT3 activity and malignant transformation.

PubMed

Zhang, Xiaolei; Sun, Ying; Pireddu, Roberta; Yang, Hua; Urlam, Murali K; Lawrence, Harshani R; Guida, Wayne C; Lawrence, Nicholas J; Sebti, Saïd M

2013-03-15

STAT3-STAT3 dimerization, which involves reciprocal binding of the STAT3-SH2 domain to phosphorylated tyrosine-705 (Y-705), is required for STAT3 nuclear translocation, DNA binding, and transcriptional regulation of downstream target genes. Here, we describe a small molecule S3I-1757 capable of disrupting STAT3-STAT3 dimerization, activation, and malignant transforming activity. Fluorescence polarization assay and molecular modeling suggest that S3I-1757 interacts with the phospho-Y-705-binding site in the SH2 domain and displaces fluorescein-labeled GpYLPQTV phosphotyrosine peptide from binding to STAT3. We generated hemagglutinin (HA)-tagged STAT3 and FLAG-tagged STAT3 and showed using coimmunoprecipitation and colocalization studies that S3I-1757 inhibits STAT3 dimerization and STAT3-EGF receptor (EGFR) binding in intact cells. Treatment of human cancer cells with S3I-1757 (but not a closely related analog, S3I-1756, which does not inhibit STAT3 dimerization), inhibits selectively the phosphorylation of STAT3 over AKT1 and ERK1/2 (MAPK3/1), nuclear accumulation of P-Y705-STAT3, STAT3-DNA binding, and transcriptional activation and suppresses the expression levels of STAT3 target genes, such as Bcl-xL (BCL2L1), survivin (BIRC5), cyclin D1 (CCND1), and matrix metalloproteinase (MMP)-9. Furthermore, S3I-1757, but not S3I-1756, inhibits anchorage-dependent and -independent growth, migration, and invasion of human cancer cells, which depend on STAT3. Finally, STAT3-C, a genetically engineered mutant of STAT3 that forms a constitutively dimerized STAT3, rescues cells from the effects of S3I-1757 inhibition. Thus, we have developed S3I-1757 as a STAT3-STAT3 dimerization inhibitor capable of blocking hyperactivated STAT3 and suppressing malignant transformation in human cancer cells that depend on STAT3.

A novel inhibitor of STAT3 homodimerization selectively suppresses STAT3 activity and malignant transformation

PubMed Central

Zhang, Xiaolei; Sun, Ying; Pireddu, Roberta; Yang, Hua; Urlam, Murali K.; Lawrence, Harshani R.; Guida, Wayne C.; Lawrence, Nicholas J.; Sebti, Saïd M.

2014-01-01

STAT3-STAT3 dimerization, which involves reciprocal binding of the STAT3-SH2 domain to phosphorylated tyrosine-705 (Y-705), is required for STAT3 nuclear translocation, DNA binding and transcriptional regulation of downstream target genes. Here we describe a small molecule S3I-1757 capable of disrupting STAT3-STAT3 dimerization, activation and malignant transforming activity. Fluorescence polarization assays and molecular modeling suggest that S3I-1757 interacts with the Y-705 binding site in the SH2 domain and displaces fluorescein-labelled GpYLPQTV phosphotyrosine peptide from binding to STAT3. We generated HA-tagged STAT3 and FLAG-tagged STAT3 and showed using co-immunoprecipitation and co-localization studies that S3I-1757 inhibits STAT3 dimerization and STAT3-EGF receptor binding in intact cells. Treatment of human cancer cells with S3I-1757 (but not a closely related analogue, S3I-1756, that does not inhibit STAT3 dimerization), inhibits selectively the phosphorylation of STAT3 over AKT1 and ERK1/2 (MAPK3/1), nuclear accumulation of P-Y705-STAT3, STAT3-DNA binding and transcriptional activation and suppresses the expression levels of STAT3 target genes such as Bcl-xL (BCL2L1), survivin (BIRC5), cyclin D1 (CCND1) and MMP9. Furthermore, S3I-1757 but not S3I-1756 inhibits anchorage-dependent and -independent growth, migration and invasion of human cancer cells which depend on STAT3. Finally, STAT3-C, a genetically engineered mutant of STAT3 that forms a constitutively dimerized STAT3, rescues cells from the effects of S3I-1757 inhibition. Thus, we have developed S3I-1757 as a STAT3-STAT3 dimerization inhibitor capable of blocking hyper activated STAT3 and suppressing malignant transformation in human cancer cells that depend on STAT3. PMID:23322008

The human phosphotyrosine signaling network: Evolution and hotspots of hijacking in cancer

PubMed Central

Li, Lei; Tibiche, Chabane; Fu, Cong; Kaneko, Tomonori; Moran, Michael F.; Schiller, Martin R.; Li, Shawn Shun-Cheng; Wang, Edwin

2012-01-01

Phosphotyrosine (pTyr) signaling, which plays a central role in cell–cell and cell–environment interactions, has been considered to be an evolutionary innovation in multicellular metazoans. However, neither the emergence nor the evolution of the human pTyr signaling system is currently understood. Tyrosine kinase (TK) circuits, each of which consists of a TK writer, a kinase substrate, and a related reader, such as Src homology (SH) 2 domains and pTyr-binding (PTB) domains, comprise the core machinery of the pTyr signaling network. In this study, we analyzed the evolutionary trajectories of 583 literature-derived and 50,000 computationally predicted human TK circuits in 19 representative eukaryotic species and assigned their evolutionary origins. We found that human TK circuits for intracellular pTyr signaling originated largely from primitive organisms, whereas the inter- or extracellular signaling circuits experienced significant expansion in the bilaterian lineage through the “back-wiring” of newly evolved kinases to primitive substrates and SH2/PTB domains. Conversely, the TK circuits that are involved in tissue-specific signaling evolved mainly in vertebrates by the back-wiring of vertebrate substrates to primitive kinases and SH2/PTB domains. Importantly, we found that cancer signaling preferentially employs the pTyr sites, which are linked to more TK circuits. Our work provides insights into the evolutionary paths of the human pTyr signaling circuits and suggests the use of a network approach for cancer intervention through the targeting of key pTyr sites and their associated signaling hubs in the network. PMID:22194470

MMPP Attenuates Non-Small Cell Lung Cancer Growth by Inhibiting the STAT3 DNA-Binding Activity via Direct Binding to the STAT3 DNA-Binding Domain.

PubMed

Son, Dong Ju; Zheng, Jie; Jung, Yu Yeon; Hwang, Chul Ju; Lee, Hee Pom; Woo, Ju Rang; Baek, Song Yi; Ham, Young Wan; Kang, Min Woong; Shong, Minho; Kweon, Gi Ryang; Song, Min Jong; Jung, Jae Kyung; Han, Sang-Bae; Kim, Bo Yeon; Yoon, Do Young; Choi, Bu Young; Hong, Jin Tae

2017-01-01

Rationale: Signal transducer and activator of transcription-3 (STAT3) plays a pivotal role in cancer biology. Many small-molecule inhibitors that target STAT3 have been developed as potential anticancer drugs. While designing small-molecule inhibitors that target the SH2 domain of STAT3 remains the leading focus for drug discovery, there has been a growing interest in targeting the DNA-binding domain (DBD) of the protein. Methods: We demonstrated the potential antitumor activity of a novel, small-molecule (E)-2-methoxy-4-(3-(4-methoxyphenyl)prop-1-en-1-yl)phenol (MMPP) that directly binds to the DBD of STAT3, in patient-derived non-small cell lung cancer (NSCLC) xenograft model as well as in NCI-H460 cell xenograft model in nude mice. Results: MMPP effectively inhibited the phosphorylation of STAT3 and its DNA binding activity in vitro and in vivo . It induced G1-phase cell cycle arrest and apoptosis through the regulation of cell cycle- and apoptosis-regulating genes by directly binding to the hydroxyl residue of threonine 456 in the DBD of STAT3. Furthermore, MMPP showed a similar or better antitumor activity than that of docetaxel or cisplatin. Conclusion: MMPP is suggested to be a potential candidate for further development as an anticancer drug that targets the DBD of STAT3.

Purification, crystallization and preliminary crystallographic analysis of the SH2 domain of IL-2-inducible T-cell kinase.

PubMed

Joseph, Raji E; Ginder, Nathaniel D; Hoy, Julie A; Nix, Jay C; Honzatko, Richard B; Andreotti, Amy H

2011-02-01

Proline is a unique amino acid owing to the relatively small energy difference between the cis and trans conformations of its peptide bond. The X-Pro imide bond readily undergoes cis-trans isomerization in the context of short peptides as well as some proteins. However, the direct detection of cis-trans proline isomerization in folded proteins is technically challenging. NMR spectroscopy is well suited to the direct detection of proline isomerization in folded proteins. It is less clear how well X-ray crystallography can reveal this conformational exchange event in folded proteins. Conformational heterogeneity owing to cis-trans proline isomerization in the Src homology 2 (SH2) domain of the IL-2-inducible T-cell kinase (ITK) has been extensively characterized by NMR. Using the ITK SH2 domain as a test system, an attempt was made to determine whether proline isomerization could be detected in a crystal structure of the ITK SH2 domain. As a first step towards this goal, the purification, crystallization and preliminary characterization of the ITK SH2 domain are described.

An update on the LIM and SH3 domain protein 1 (LASP1): a versatile structural, signaling, and biomarker protein

PubMed Central

Orth, Martin F.; Cazes, Alex; Butt, Elke; Grunewald, Thomas G. P.

2015-01-01

The gene encoding the LIM and SH3 domain protein (LASP1) was cloned two decades ago from a cDNA library of breast cancer metastases. As the first protein of a class comprising one N-terminal LIM and one C-terminal SH3 domain, LASP1 founded a new LIM-protein subfamily of the nebulin group. Since its discovery LASP1 proved to be an extremely versatile protein because of its exceptional structure allowing interaction with various binding partners, its ubiquitous expression in normal tissues, albeit with distinct expression patterns, and its ability to transmit signals from the cytoplasm into the nucleus. As a result, LASP1 plays key roles in cell structure, physiological processes, and cell signaling. Furthermore, LASP1 overexpression contributes to cancer aggressiveness hinting to a potential value of LASP1 as a cancer biomarker. In this review we summarize published data on structure, regulation, function, and expression pattern of LASP1, with a focus on its role in human cancer and as a biomarker protein. In addition, we provide a comprehensive transcriptome analysis of published microarrays (n=2,780) that illustrates the expression profile of LASP1 in normal tissues and its overexpression in a broad range of human cancer entities. PMID:25622104

Signaling by Kit protein-tyrosine kinase--the stem cell factor receptor.

PubMed

Roskoski, Robert

2005-11-11

Signaling by stem cell factor and Kit, its receptor, plays important roles in gametogenesis, hematopoiesis, mast cell development and function, and melanogenesis. Moreover, human and mouse embryonic stem cells express Kit transcripts. Stem cell factor exists as both a soluble and a membrane-bound glycoprotein while Kit is a receptor protein-tyrosine kinase. The complete absence of stem cell factor or Kit is lethal. Deficiencies of either produce defects in red and white blood cell production, hypopigmentation, and sterility. Gain-of-function mutations of Kit are associated with several human neoplasms including acute myelogenous leukemia, gastrointestinal stromal tumors, and mastocytomas. Kit consists of an extracellular domain, a transmembrane segment, a juxtamembrane segment, and a protein kinase domain that contains an insert of about 80 amino acid residues. Binding of stem cell factor to Kit results in receptor dimerization and activation of protein kinase activity. The activated receptor becomes autophosphorylated at tyrosine residues that serve as docking sites for signal transduction molecules containing SH2 domains. The adaptor protein APS, Src family kinases, and Shp2 tyrosyl phosphatase bind to phosphotyrosine 568. Shp1 tyrosyl phosphatase and the adaptor protein Shc bind to phosphotyrosine 570. C-terminal Src kinase homologous kinase and the adaptor Shc bind to both phosphotyrosines 568 and 570. These residues occur in the juxtamembrane segment of Kit. Three residues in the kinase insert domain are phosphorylated and attract the adaptor protein Grb2 (Tyr703), phosphatidylinositol 3-kinase (Tyr721), and phospholipase Cgamma (Tyr730). Phosphotyrosine 900 in the distal kinase domain binds phosphatidylinositol 3-kinase which in turn binds the adaptor protein Crk. Phosphotyrosine 936, also in the distal kinase domain, binds the adaptor proteins APS, Grb2, and Grb7. Kit has the potential to participate in multiple signal transduction pathways as a result of interaction with several enzymes and adaptor proteins.

WW domain-containing oxidoreductase promotes neuronal differentiation via negative regulation of glycogen synthase kinase 3β.

PubMed

Wang, H-Y; Juo, L-I; Lin, Y-T; Hsiao, M; Lin, J-T; Tsai, C-H; Tzeng, Y-H; Chuang, Y-C; Chang, N-S; Yang, C-N; Lu, P-J

2012-06-01

WW domain-containing oxidoreductase (WWOX), a putative tumour suppressor, is suggested to be involved in the hyperphosphorylation of Alzheimer's Tau. Tau is a microtubule-associated protein that has an important role in microtubule assembly and stability. Glycogen synthase kinase 3β (GSK3β) has a vital role in Tau hyperphosphorylation at its microtubule-binding domains. Hyperphosphorylated Tau has a low affinity for microtubules, thus disrupting microtubule stability. Bioinformatics analysis indicated that WWOX contains two potential GSK3β-binding FXXXLI/VXRLE motifs. Immunofluorescence, immunoprecipitation and molecular modelling showed that WWOX interacts physically with GSK3β. We demonstrated biochemically that WWOX can bind directly to GSK3β through its short-chain alcohol dehydrogenase/reductase domain. Moreover, the overexpression of WWOX inhibited GSK3β-stimulated S396 and S404 phosphorylation within the microtubule domains of Tau, indicating that WWOX is involved in regulating GSK3β activity in cells. WWOX repressed GSK3β activity, restored the microtubule assembly activity of Tau and promoted neurite outgrowth in SH-SY5Y cells. Conversely, RNAi-mediated knockdown of WWOX in retinoic acid (RA)-differentiated SH-SY5Y cells inhibited neurite outgrowth. These results suggest that WWOX is likely to be involved in regulating GSK3β activity, reducing the level of phosphorylated Tau, and subsequently promoting neurite outgrowth during neuron differentiation. In summary, our data reveal a novel mechanism by which WWOX promotes neuronal differentiation in response to RA.

Modular evolution of phosphorylation-based signalling systems

PubMed Central

Jin, Jing; Pawson, Tony

2012-01-01

Phosphorylation sites are formed by protein kinases (‘writers’), frequently exert their effects following recognition by phospho-binding proteins (‘readers’) and are removed by protein phosphatases (‘erasers’). This writer–reader–eraser toolkit allows phosphorylation events to control a broad range of regulatory processes, and has been pivotal in the evolution of new functions required for the development of multi-cellular animals. The proteins that comprise this system of protein kinases, phospho-binding targets and phosphatases are typically modular in organization, in the sense that they are composed of multiple globular domains and smaller peptide motifs with binding or catalytic properties. The linkage of these binding and catalytic modules in new ways through genetic recombination, and the selection of particular domain combinations, has promoted the evolution of novel, biologically useful processes. Conversely, the joining of domains in aberrant combinations can subvert cell signalling and be causative in diseases such as cancer. Major inventions such as phosphotyrosine (pTyr)-mediated signalling that flourished in the first multi-cellular animals and their immediate predecessors resulted from stepwise evolutionary progression. This involved changes in the binding properties of interaction domains such as SH2 and their linkage to new domain types, and alterations in the catalytic specificities of kinases and phosphatases. This review will focus on the modular aspects of signalling networks and the mechanism by which they may have evolved. PMID:22889906

Modulating drug resistance by targeting BCRP/ABCG2 using retrovirus-mediated RNA interference.

PubMed

Xie, Ni; Mou, Lisha; Yuan, Jianhui; Liu, Wenlan; Deng, Tingting; Li, Zigang; Jing, Yi; Jin, Yi; Hu, Zhangli

2014-01-01

The BCRP/ABCG2 transporter, which mediates drug resistance in many types of cells, depends on energy provided by ATP hydrolysis. Here, a retrovirus encoding a shRNA targeting the ATP-binding domain of this protein was used to screen for highly efficient agents that could reverse drug resistance and improve cell sensitivity to drugs, thus laying the foundation for further studies and applications. To target the ATP-binding domain of BCRP/ABCG2, pLenti6/BCRPsi shRNA recombinant retroviruses, with 20 bp target sequences starting from the 270th, 745th and 939th bps of the 6th exon, were constructed and packaged. The pLenti6/BCRPsi retroviruses (V-BCRPi) that conferred significant knockdown effects were screened using a drug-sensitivity experiment and flow cytometry. The human choriocarcinoma cell line JAR, which highly expresses endogenous BCRP/ABCG2, was injected under the dorsal skin of a hairless mouse to initiate a JAR cytoma. After injecting V-BCRPi-infected JAR tumor cells into the dorsal skin of hairless mice, BCRP/ABCG2 expression in the tumor tissue was determined using immunohistochemistry, fluorescent quantitative RT-PCR and Western blot analyses. After intraperitoneal injection of BCRP/ABCG2-tolerant 5-FU, the tumor volume, weight change, and apoptosis rate of the tumor tissue were determined using in situ hybridization. V-BCRPi increased the sensitivity of the tumor histiocytes to 5-FU and improved the cell apoptosis-promoting effects of 5-FU in the tumor. The goal of the in vivo and in vitro studies was to screen for an RNA interference recombinant retrovirus capable of stably targeting the ATP-binding domain of BCRP/ABCG2 (V-BCRPi) to inhibit its function. A new method to improve the chemo-sensitivity of breast cancer and other tumor cells was discovered, and this method could be used for gene therapy and functional studies of malignant tumors.

SH2 domain containing leukocyte phosphoprotein of 76-kDa (SLP-76) feedback regulation of ZAP-70 microclustering.

PubMed

Liu, Hebin; Purbhoo, Marco A; Davis, Daniel M; Rudd, Christopher E

2010-06-01

T cell receptor (TCR) signaling involves CD4/CD8-p56lck recruitment of ZAP-70 to the TCR receptor, ZAP-70 phosphorylation of LAT that is followed by LAT recruitment of the GADS-SLP-76 complex. Back regulation of ZAP-70 by SLP-76 has not been documented. In this paper, we show that anti-CD3 induced ZAP-70 cluster formation is significantly reduced in the absence of SLP-76 (i.e., J14 cells) and in the presence of a mutant of SLP-76 (4KE) in Jurkat and primary T cells. Both the number of cells with clusters and the number of clusters per cell were reduced. This effect was not mediated by SLP-76 SH2 domain binding to ZAP-70 because SLP-76 failed to precipitate ZAP-70 and an inactivating SH2 domain mutation (i.e., R448L) on SLP-76 4KE did not reverse the inhibition of ZAP-70 clustering. Mutation of R448 on WT SLP-76 still supported ZAP-70 clustering. Intriguingly, by contrast, LAT clustering occurred normally in the absence of SLP-76, or the presence of 4KE SLP-76 indicating that this transmembrane adaptor can operate independently of ZAP-70-GADS-SLP-76. Our findings reconfigure the TCR signaling pathway by showing SLP-76 back-regulation of ZAP-70, an event that could ensure that signaling components are in balance for optimal T cell activation.

Allosteric Fine-Tuning of the Binding Pocket Dynamics in the ITK SH2 Domain by a Distal Molecular Switch: An Atomistic Perspective.

PubMed

Momin, Mohamed; Xin, Yao; Hamelberg, Donald

2017-06-29

Although the regulation of function of proteins by allosteric interactions has been identified in many subcellular processes, molecular switches are also known to induce long-range conformational changes in proteins. A less well understood molecular switch involving cis-trans isomerization of a peptidyl-prolyl bond could induce a conformational change directly to the backbone that is propagated to other parts of the protein. However, these switches are elusive and hard to identify because they are intrinsic to biomolecules that are inherently dynamic. Here, we explore the conformational dynamics and free energy landscape of the SH2 domain of interleukin-2-inducible T-cell or tyrosine kinase (ITK) to fully understand the conformational coupling between the distal cis-trans molecular switch and its binding pocket of the phosphotyrosine motif. We use multiple microsecond-long all-atom molecular dynamics simulations in explicit water for over a total of 60 μs. We show that cis-trans isomerization of the Asn286-Pro287 peptidyl-prolyl bond is directly coupled to the dynamics of the binding pocket of the phosphotyrosine motif, in agreement with previous NMR experiments. Unlike the cis state that is localized and less dynamic in a single free energy basin, the trans state samples two distinct conformations of the binding pocket-one that recognizes the phosphotyrosine motif and the other that is somewhat similar to that of the cis state. The results provide an atomic-level description of a less well understood allosteric regulation by a peptidyl-prolyl cis-trans molecular switch that could aid in the understanding of normal and aberrant subcellular processes and the identification of these elusive molecular switches in other proteins.

SH3 Domain-Containing Protein 2 Plays a Crucial Role at the Step of Membrane Tubulation during Cell Plate Formation

PubMed Central

Ahn, Gyeongik; Kim, Hyeran; Kim, Dae Heon; Hanh, Hong; Yoon, Youngdae; Singaram, Indira; Wijesinghe, Kaveesha J.; Johnson, Kristen A.; Liang, Zizhen; Stahelin, Robert V.; Jiang, Liwen; Cho, Wonhwa; Kang, Byung-Ho

2017-01-01

During cytokinesis in plants, trans-Golgi network-derived vesicles accumulate at the center of dividing cells and undergo various structural changes to give rise to the planar cell plate. However, how this conversion occurs at the molecular level remains elusive. In this study, we report that SH3 Domain-Containing Protein 2 (SH3P2) in Arabidopsis thaliana plays a crucial role in converting vesicles to the planar cell plate. SH3P2 RNAi plants showed cytokinesis-defective phenotypes and produced aggregations of vesicles at the leading edge of the cell plate. SH3P2 localized to the leading edge of the cell plate, particularly the constricted or curved regions of the cell plate. The BAR domain of SH3P2 induced tubulation of vesicles. SH3P2 formed a complex with dynamin-related protein 1A (DRP1A) and affected DRP1A accumulation to the cell plate. Based on these results, we propose that SH3P2 functions together with DRP1A to convert the fused vesicles to tubular structures during cytokinesis. PMID:28584166

Affinity modulation of small-molecule ligands by borrowing endogenous protein surfaces

PubMed Central

Briesewitz, Roger; Ray, Gregory T.; Wandless, Thomas J.; Crabtree, Gerald R.

1999-01-01

A general strategy is described for improving the binding properties of small-molecule ligands to protein targets. A bifunctional molecule is created by chemically linking a ligand of interest to another small molecule that binds tightly to a second protein. When the ligand of interest is presented to the target protein by the second protein, additional protein–protein interactions outside of the ligand-binding sites serve either to increase or decrease the affinity of the binding event. We have applied this approach to an intractable target, the SH2 domain, and demonstrate a 3-fold enhancement over the natural peptide. This approach provides a way to modulate the potency and specificity of biologically active compounds. PMID:10051576

Role of electrostatic interactions in binding of peptides and intrinsically disordered proteins to their folded targets. 1. NMR and MD characterization of the complex between the c-Crk N-SH3 domain and the peptide Sos.

PubMed

Xue, Yi; Yuwen, Tairan; Zhu, Fangqiang; Skrynnikov, Nikolai R

2014-10-21

Intrinsically disordered proteins (IDPs) often rely on electrostatic interactions to bind their structured targets. To obtain insight into the mechanism of formation of the electrostatic encounter complex, we investigated the binding of the peptide Sos (PPPVPPRRRR), which serves as a minimal model for an IDP, to the c-Crk N-terminal SH3 domain. Initially, we measured ¹⁵N relaxation rates at two magnetic field strengths and determined the binding shifts for the complex of Sos with wild-type SH3. We have also recorded a 3 μs molecular dynamics (MD) trajectory of this complex using the Amber ff99SB*-ILDN force field. The comparison of the experimental and simulated data shows that MD simulation consistently overestimates the strength of salt bridge interactions at the binding interface. The series of simulations using other advanced force fields also failed to produce any satisfactory results. To address this issue, we have devised an empirical correction to the Amber ff99SB*-ILDN force field whereby the Lennard-Jones equilibrium distance for the nitrogen-oxygen pair across the Arg-to-Asp and Arg-to-Glu salt bridges has been increased by 3%. Implementing this correction resulted in a good agreement between the simulations and the experiment. Adjusting the strength of salt bridge interactions removed a certain amount of strain contained in the original MD model, thus improving the binding of the hydrophobic N-terminal portion of the peptide. The arginine-rich C-terminal portion of the peptide, freed from the effect of the overstabilized salt bridges, was found to interconvert more rapidly between its multiple conformational states. The modified MD protocol has also been successfully used to simulate the entire binding process. In doing so, the peptide was initially placed high above the protein surface. It then arrived at the correct bound pose within ∼2 Å of the crystallographic coordinates. This simulation allowed us to analyze the details of the dynamic binding intermediate, i.e., the electrostatic encounter complex. However, an experimental characterization of this transient, weakly populated state remains out of reach. To overcome this problem, we designed the double mutant of c-Crk N-SH3 in which mutations Y186L and W169F abrogate tight Sos binding and shift the equilibrium toward the intermediate state resembling the electrostatic encounter complex. The results of the combined NMR and MD study of this engineered system will be reported in the next part of this paper.

p120Ras-GAP binds the DLC1 Rho-GAP tumor suppressor protein and inhibits its RhoA GTPase and growth-suppressing activities.

PubMed

Yang, X-Y; Guan, M; Vigil, D; Der, C J; Lowy, D R; Popescu, N C

2009-03-19

DLC1 (deleted in liver cancer 1), which encodes a Rho GTPase-activating protein (Rho-GAP), is a potent tumor suppressor gene that is frequently inactivated in several human cancers. DLC1 is a multidomain protein that has been shown previously to bind members of the tensin gene family. Here we show that p120Ras-GAP (Ras-GAP; also known as RASA1) interacts and extensively colocalizes with DLC1 in focal adhesions. The binding was mapped to the SH3 domain located in the N terminus of Ras-GAP and to the Rho-GAP catalytic domain located in the C terminus of the DLC1. In vitro analyses with purified proteins determined that the isolated Ras-GAP SH3 domain inhibits DLC1 Rho-GAP activity, suggesting that Ras-GAP is a negative regulator of DLC1 Rho-GAP activity. Consistent with this possibility, we found that ectopic overexpression of Ras-GAP in a Ras-GAP-insensitive tumor line impaired the growth-suppressing activity of DLC1 and increased RhoA activity in vivo. Our observations expand the complexity of proteins that regulate DLC1 function and define a novel mechanism of the cross talk between Ras and Rho GTPases.1R01CA129610

Fast and reliable prediction of domain-peptide binding affinity using coarse-grained structure models.

PubMed

Tian, Feifei; Tan, Rui; Guo, Tailin; Zhou, Peng; Yang, Li

2013-07-01

Domain-peptide recognition and interaction are fundamentally important for eukaryotic signaling and regulatory networks. It is thus essential to quantitatively infer the binding stability and specificity of such interaction based upon large-scale but low-accurate complex structure models which could be readily obtained from sophisticated molecular modeling procedure. In the present study, a new method is described for the fast and reliable prediction of domain-peptide binding affinity with coarse-grained structure models. This method is designed to tolerate strong random noises involved in domain-peptide complex structures and uses statistical modeling approach to eliminate systematic bias associated with a group of investigated samples. As a paradigm, this method was employed to model and predict the binding behavior of various peptides to four evolutionarily unrelated peptide-recognition domains (PRDs), i.e. human amph SH3, human nherf PDZ, yeast syh GYF and yeast bmh 14-3-3, and moreover, we explored the molecular mechanism and biological implication underlying the binding of cognate and noncognate peptide ligands to their domain receptors. It is expected that the newly proposed method could be further used to perform genome-wide inference of domain-peptide binding at three-dimensional structure level. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

MDC9, a widely expressed cellular disintegrin containing cytoplasmic SH3 ligand domains

PubMed Central

1996-01-01

Cellular disintegrins are a family of proteins that are related to snake venom integrin ligands and metalloproteases. We have cloned and sequenced the mouse and human homologue of a widely expressed cellular disintegrin, which we have termed MDC9 (for metalloprotease/disintegrin/cysteine-rich protein 9). The deduced mouse and human protein sequences are 82% identical. MDC9 contains several distinct protein domains: a signal sequence is followed by a prodomain and a domain with sequence similarity to snake venom metalloproteases, a disintegrin domain, a cysteine-rich region, an EGF repeat, a membrane anchor, and a cytoplasmic tail. The cytoplasmic tail of MDC9 has two proline-rich sequences which can bind the SH3 domain of Src, and may therefore function as SH3 ligand domains. Western blot analysis shows that MDC9 is an approximately 84-kD glycoprotein in all mouse tissues examined, and in NIH 3T3 fibroblast and C2C12 myoblast mouse cell lines. MDC9 can be both cell surface biotinylated and 125I-labeled in NIH 3T3 mouse fibroblasts, indicating that the protein is present on the plasma membrane. Expression of MDC9 in COS-7 cells yields an 84-kD protein, and immunofluorescence analysis of COS-7 cells expressing MDC9 shows a staining pattern that is consistent with a plasma membrane localization. The apparent molecular mass of 84 kD suggests that MDC9 contains a membrane-anchored metalloprotease and disintegrin domain. We propose that MDC9 might function as a membrane-anchored integrin ligand or metalloprotease, or that MDC9 may combine both activities in one protein. PMID:8647900

Kinome signaling through regulated protein-protein interactions in normal and cancer cells.

PubMed

Pawson, Tony; Kofler, Michael

2009-04-01

The flow of molecular information through normal and oncogenic signaling pathways frequently depends on protein phosphorylation, mediated by specific kinases, and the selective binding of the resulting phosphorylation sites to interaction domains present on downstream targets. This physical and functional interplay of catalytic and interaction domains can be clearly seen in cytoplasmic tyrosine kinases such as Src, Abl, Fes, and ZAP-70. Although the kinase and SH2 domains of these proteins possess similar intrinsic properties of phosphorylating tyrosine residues or binding phosphotyrosine sites, they also undergo intramolecular interactions when linked together, in a fashion that varies from protein to protein. These cooperative interactions can have diverse effects on substrate recognition and kinase activity, and provide a variety of mechanisms to link the stimulation of catalytic activity to substrate recognition. Taken together, these data have suggested how protein kinases, and the signaling pathways in which they are embedded, can evolve complex properties through the stepwise linkage of domains within single polypeptides or multi-protein assemblies.

Development and Application of a High Throughput Protein Unfolding Kinetic Assay

PubMed Central

Wang, Qiang; Waterhouse, Nicklas; Feyijinmi, Olusegun; Dominguez, Matthew J.; Martinez, Lisa M.; Sharp, Zoey; Service, Rachel; Bothe, Jameson R.; Stollar, Elliott J.

2016-01-01

The kinetics of folding and unfolding underlie protein stability and quantification of these rates provides important insights into the folding process. Here, we present a simple high throughput protein unfolding kinetic assay using a plate reader that is applicable to the studies of the majority of 2-state folding proteins. We validate the assay by measuring kinetic unfolding data for the SH3 (Src Homology 3) domain from Actin Binding Protein 1 (AbpSH3) and its stabilized mutants. The results of our approach are in excellent agreement with published values. We further combine our kinetic assay with a plate reader equilibrium assay, to obtain indirect estimates of folding rates and use these approaches to characterize an AbpSH3-peptide hybrid. Our high throughput protein unfolding kinetic assays allow accurate screening of libraries of mutants by providing both kinetic and equilibrium measurements and provide a means for in-depth ϕ-value analyses. PMID:26745729

Modulation of fatty acid synthase degradation by concerted action of p38 MAP kinase, E3 ligase COP1, and SH2-tyrosine phosphatase Shp2.

PubMed

Yu, Jianxiu; Deng, Rong; Zhu, Helen H; Zhang, Sharon S; Zhu, Changhong; Montminy, Marc; Davis, Roger; Feng, Gen-Sheng

2013-02-08

The Src-homology 2 (SH2) domain-containing tyrosine phosphatase Shp2 has been known to regulate various signaling pathways triggered by receptor and cytoplasmic tyrosine kinases. Here we describe a novel function of Shp2 in control of lipid metabolism by mediating degradation of fatty acid synthase (FASN). p38-phosphorylated COP1 accumulates in the cytoplasm and subsequently binds FASN through Shp2 here as an adapter, leading to FASN-Shp2-COP1 complex formation and FASN degradation mediated by ubiquitination pathway. By fasting p38 is activated and stimulates FASN protein degradation in mice. Consistently, the FASN protein levels are dramatically elevated in mouse liver and pancreas in which Shp2/Ptpn11 is selectively deleted. Thus, this study identifies a new activity for Shp2 in lipid metabolism.

HRV signaling in airway epithelial cells is regulated by ITAM-mediated recruitment and activation of Syk.

PubMed

Lau, Christine; Castellanos, Patricia; Ranev, Dimitre; Wang, Xiaomin; Chow, Chung-Wai

2011-05-01

Human rhinovirus (HRV), cause of the common cold, is a leading cause of exacerbations of asthma and chronic obstruction pulmonary disease (COPD). Binding of HRV to ICAM (intercellular adhesion molecule)-1, its major receptor, induces a profound inflammatory response from airway epithelial cells. My laboratory has identified Syk tyrosine kinase to be an early regulator of HRV-ICAM-1 signalling: Syk mediates replication-independent p38 mitogen-activated protein (MAP) kinase and phosphatidyl-inositol 3 (PI3)-kinase activation, interleukin (IL)-8 expression, as well as HRV internalization via clathrin-mediated endocytosis. Syk activation is accompanied by formation of a protein complex consisting of ICAM-1, ezrin and Syk at the plasma membrane. However, the molecular mechanisms that regulate this process are not understood. In this report, we investigated the role of the Syk-SH2 domains and the ezrin ITAM (immuno-tyrosine activation motif)-like motif in HRV-induced cell activation using the human BEAS-2B airway epithelial cells. Our observations suggest that the ezrin-ITAM plays a role in Syk recruitment and activation by binding to the Syk tandem SH2 domains, as originally described in the canonical ITAM-mediating signal transduction pathway in hematopoietic cells. This report is the first to demonstrate ITAM-mediated signaling in non-hematopoietic cells, suggesting that this signaling paradigm may be more ubiquitous than previously recognized.

Identification of the kinase that activates a nonmetazoan STAT gives insights into the evolution of phosphotyrosine-SH2 domain signaling.

PubMed

Araki, Tsuyoshi; Kawata, Takefumi; Williams, Jeffrey G

2012-07-10

SH2 domains are integral to many animal signaling pathways. By interacting with specific phosphotyrosine residues, they provide regulatable protein-protein interaction domains. Dictyostelium is the only nonmetazoan with functionally characterized SH2 domains, but the cognate tyrosine kinases are unknown. There are no orthologs of the animal tyrosine kinases, but there are very many tyrosine kinase-like kinases (TKLs), a group of kinases which, despite their family name, are classified mainly as serine-threonine kinases. STATs are transcription factors that dimerize via phosphotyrosine-SH2 domain interactions. STATc is activated by phosphorylation on Tyr922 when cells are exposed to the prestalk inducer differentiation inducing factor (DIF-1), a chlorinated hexaphenone. We show that in a null mutant for Pyk2, a tyrosine-specific TKL, exposure to DIF-1 does not activate STATc. Conversely, overexpression of Pyk2 causes constitutive STATc activation. Pyk2 phosphorylates STATc on Tyr922 in vitro and complexes with STATc both in vitro and in vivo. This demonstration that a TKL directly activates a STAT has significant implications for understanding the evolutionary origins of SH2 domain-phosphotyrosine signaling. It also has mechanistic implications. Our previous work suggested that a predicted constitutive STATc tyrosine kinase activity is counterbalanced in vivo by the DIF-1-regulated activity of PTP3, a Tyr922 phosphatase. Here we show that the STATc-Pyk2 complex is formed constitutively by an interaction between the STATc SH2 domain and phosphotyrosine residues on Pyk2 that are generated by autophosphorylation. Also, as predicted, Pyk2 is constitutively active as a STATc kinase. This observation provides further evidence for this highly atypical, possibly ancestral, STAT regulation mechanism.

SH003 represses tumor angiogenesis by blocking VEGF binding to VEGFR2

PubMed Central

Choi, Hyeong Sim; Kim, Min Kyoung; Lee, Kangwook; Lee, Kang Min; Choi, Youn Kyung; Shin, Yong Cheol; Cho, Sung-Gook; Ko, Seong-Gyu

2016-01-01

Tumor angiogenesis is a key feature of cancer progression, because a tumor requires abundant oxygen and nutrition to grow. Here, we demonstrate that SH003, a mixed herbal extract containing Astragalus membranaceus (Am), Angelica gigas (Ag) and Trichosanthes Kirilowii Maximowicz (Tk), represses VEGF-induced tumor angiogenesis both in vitro and in vivo. SH003 inhibited VEGF-induced migration, invasion and tube formation in human umbilical vein endothelial cells (HUVEC) with no effect on the proliferation. SH003 reduced CD31-positive vessel numbers in tumor tissues and retarded tumor growth in our xenograft mouse tumor model, while SH003 did not affect pancreatic tumor cell viability. Consistently, SH003 inhibited VEGF-stimulated vascular permeability in ears and back skins. Moreover, SH003 inhibited VEGF-induced VEGFR2-dependent signaling by blocking VEGF binding to VEGFR2. Therefore, our data conclude that SH003 represses tumor angiogenesis by inhibiting VEGF-induced VEGFR2 activation, and suggest that SH003 may be useful for treating cancer. PMID:27105528

Discovery of novel STAT3 small molecule inhibitors via in silico site-directed fragment-based drug design.

PubMed

Yu, Wenying; Xiao, Hui; Lin, Jiayuh; Li, Chenglong

2013-06-13

Constitutive activation of signal transducer and activator of transcription 3 (STAT3) has been validated as an attractive therapeutic target for cancer therapy. To stop both STAT3 activation and dimerization, a viable strategy is to design inhibitors blocking its SH2 domain phosphotyrosine binding site that is responsible for both actions. A new fragment-based drug design (FBDD) strategy, in silico site-directed FBDD, was applied in this study. A designed novel compound, 5,8-dioxo-6-(pyridin-3-ylamino)-5,8-dihydronaphthalene-1-sulfonamide (LY5), was confirmed to bind to STAT3 SH2 by fluorescence polarization assay. In addition, four out of the five chosen compounds have IC50 values lower than 5 μM for the U2OS cancer cells. 8 (LY5) has an IC50 range in 0.5-1.4 μM in various cancer cell lines. 8 also suppresses tumor growth in an in vivo mouse model. This study has demonstrated the utility of this approach and could be used to other drug targets in general.

Conformational Entropy from NMR Relaxation in Proteins: The SRLS Perspective.

PubMed

Tchaicheeyan, Oren; Meirovitch, Eva

2017-02-02

Conformational entropy changes associated with bond-vector motions in proteins contribute to the free energy of ligand-binding. To derive such contributions, we apply the slowly relaxing local structure (SRLS) approach to NMR relaxation from 15 N-H bonds or C-CDH 2 moieties of several proteins in free and ligand-bound form. The spatial restraints on probe motion, which determine the extent of local order, are expressed in SRLS by a well-defined potential, u(θ). The latter yields the orientational probability density, P eq  = exp(-u(θ)), and hence the related conformational entropy, Ŝ = -∫P eq (θ) ln[P eq (θ)] sin θ dθ (Ŝ is "entropy" in units of k B T, and θ represents the bond-vector orientation in the protein). SRLS is applied to 4-oxalocrotonate tautomerase (4-OT), the acyl-coenzyme A binding protein (ACBP), the C-terminal SH2 domain of phospholipase C γ 1 (PLC γ 1C SH2), the construct dihydrofolate reductase-E:folate (DHFR-E:folate), and their complexes with appropriate ligands, to determine ΔŜ. Eglin C and its V18A and V34A mutants are also studied. Finally, SRLS is applied to the structurally homologous proteins TNfn3 and FNfn10 to characterize within its scope the unusual "dynamics" of the TNfn3 core. Upon ligand-binding, the backbones of 4-OT, ACBP, and PLC γ 1C SH2 show limited, increased, and decreased order, respectively; the cores of DHFR-E:folate and PLC γ 1C SH2 become more ordered. The V18A (V34A) mutation increases (decreases) the order within the eglin C core. The core of TNfn3 is less ordered structurally and more mobile kinetically. Secondary structure versus loops, surface-binding versus core insertion, and ligand size emerged as being important in rationalizing ΔŜ. The consistent and general tool developed herein is expected to provide further insights in future work.

Structure, organization and expression of common carp (Cyprinus carpio L.) SLP-76 gene.

PubMed

Huang, Rong; Sun, Xiao-Feng; Hu, Wei; Wang, Ya-Ping; Guo, Qiong-Lin

2008-05-01

SLP-76 is an important member of the SLP-76 family of adapters, and it plays a key role in TCR signaling and T cell function. Partial cDNA sequence of SLP-76 of common carp (Cyprinus carpio L.) was isolated from thymus cDNA library by the method of suppression subtractive hybridization (SSH). Subsequently, the full length cDNA of carp SLP-76 was obtained by means of 3' RACE and 5' RACE, respectively. The full length cDNA of carp SLP-76 was 2007 bp, consisting of a 5'-terminal untranslated region (UTR) of 285 bp, a 3'-terminal UTR of 240 bp, and an open reading frame of 1482 bp. Sequence comparison showed that the deduced amino acid sequence of carp SLP-76 had an overall similarity of 34-73% to that of other species homologues, and it was composed of an NH2-terminal domain, a central proline-rich domain, and a C-terminal SH2 domain. Amino acid sequence analysis indicated the existence of a Gads binding site R-X-X-K, a 10-aa-long sequence which binds to the SH3 domain of LCK in vitro, and three conserved tyrosine-containing sequence in the NH2-terminal domain. Then we used PCR to obtain a genomic DNA which covers the entire coding region of carp SLP-76. In the 9.2k-long genomic sequence, twenty one exons and twenty introns were identified. RT-PCR results showed that carp SLP-76 was expressed predominantly in hematopoietic tissues, and was upregulated in thymus tissue of four-month carp compared to one-year old carp. RT-PCR and virtual northern hybridization results showed that carp SLP-76 was also upregulated in thymus tissue of GH transgenic carp at the age of four-months. These results suggest that the expression level of SLP-76 gene may be related to thymocyte development in teleosts.

Cherubism Mice Also Deficient in c-Fos Exhibit Inflammatory Bone Destruction Executed by Macrophages That Express MMP14 Despite the Absence of TRAP+ Osteoclasts.

PubMed

Kittaka, Mizuho; Mayahara, Kotoe; Mukai, Tomoyuki; Yoshimoto, Tetsuya; Yoshitaka, Teruhito; Gorski, Jeffrey P; Ueki, Yasuyoshi

2018-01-01

Currently, it is believed that osteoclasts positive for tartrate-resistant acid phosphatase (TRAP+) are the exclusive bone-resorbing cells responsible for focal bone destruction in inflammatory arthritis. Recently, a mouse model of cherubism (Sh3bp2 KI/KI ) with a homozygous gain-of-function mutation in the SH3-domain binding protein 2 (SH3BP2) was shown to develop auto-inflammatory joint destruction. Here, we demonstrate that Sh3bp2 KI/KI mice also deficient in the FBJ osteosarcoma oncogene (c-Fos) still exhibit noticeable bone erosion at the distal tibia even in the absence of osteoclasts at 12 weeks old. Levels of serum collagen I C-terminal telopeptide (ICTP), a marker of bone resorption generated by matrix metalloproteinases (MMPs), were elevated, whereas levels of serum cross-linked C-telopeptide (CTX), another resorption marker produced by cathepsin K, were not increased. Collagenolytic MMP levels were increased in the inflamed joints of the Sh3bp2 KI/KI mice deficient in c-Fos. Resorption pits contained a large number of F4/80+ macrophages and genetic depletion of macrophages rescued these erosive changes. Importantly, administration of NSC405020, an MMP14 inhibitor targeted to the hemopexin (PEX) domain, suppressed bone erosion in c-Fos-deficient Sh3bp2 KI/KI mice. After activation of the NF-κB pathway, macrophage colony-stimulating factor (M-CSF)-dependent macrophages from c-Fos-deficient Sh3bp2 KI/KI mice expressed increased amounts of MMP14 compared with wild-type macrophages. Interestingly, receptor activator of NF-κB ligand (RANKL)-deficient Sh3bp2 KI/KI mice failed to show notable bone erosion, whereas c-Fos deletion did restore bone erosion to the RANKL-deficient Sh3bp2 KI/KI mice, suggesting that osteolytic transformation of macrophages requires both loss-of-function of c-Fos and gain-of-function of SH3BP2 in this model. These data provide the first genetic evidence that cells other than osteoclasts can cause focal bone destruction in inflammatory bone disease and suggest that MMP14 is a key mediator conferring pathological bone-resorbing capacity on c-Fos-deficient Sh3bp2 KI/KI macrophages. In summary, the paradigm that osteoclasts are the exclusive cells executing inflammatory bone destruction may need to be reevaluated based on our findings with c-Fos-deficient cherubism mice lacking osteoclasts. © 2017 American Society for Bone and Mineral Research. © 2017 American Society for Bone and Mineral Research.

Roles for SH2 and SH3 domains in Lyn kinase association with activated FcepsilonRI in RBL mast cells revealed by patterned surface analysis.

PubMed

Hammond, Stephanie; Wagenknecht-Wiesner, Alice; Veatch, Sarah L; Holowka, David; Baird, Barbara

2009-10-01

In mast cells, antigen-mediated cross-linking of IgE bound to its high-affinity surface receptor, FcepsilonRI, initiates a signaling cascade that culminates in degranulation and release of allergic mediators. Antigen-patterned surfaces, in which the antigen is deposited in micron-sized features on a silicon substrate, were used to examine the spatial relationship between clustered IgE-FcepsilonRI complexes and Lyn, the signal-initiating tyrosine kinase. RBL mast cells expressing wild-type Lyn-EGFP showed co-redistribution of this protein with clustered IgE receptors on antigen-patterned surfaces, whereas Lyn-EGFP containing an inhibitory point mutation in its SH2 domain did not significantly accumulate with the patterned antigen, and Lyn-EGFP with an inhibitory point mutation in its SH3 domain exhibited reduced interactions. Our results using antigen-patterned surfaces and quantitative cross-correlation image analysis reveal that both the SH2 and SH3 domains contribute to interactions between Lyn kinase and cross-linked IgE receptors in stimulated mast cells.

Phosphopeptide occupancy and photoaffinity cross-linking of the v-Src SH2 domain attenuates tyrosine kinase activity.

PubMed

Garcia, P; Shoelson, S E; Drew, J S; Miller, W T

1994-12-02

Phosphorylation of c-Src at carboxyl-terminal Tyr-527 suppresses tyrosine kinase activity and transforming potential, presumably by facilitating the intramolecular interaction of the C terminus of Src with its SH2 domain. In addition, it has been shown previously that occupancy of the c-Src SH2 domain with a phosphopeptide stimulates c-Src kinase catalytic activity. We have performed analogous studies with v-Src, the transforming protein from Rous sarcoma virus, which has extensive homology with c-Src. v-Src lacks an autoregulatory phosphorylation site, and its kinase domain is constitutively active. Phosphopeptides corresponding to the sequences surrounding c-Src Tyr-527 and a Tyr-Glu-Glu-Ile motif from the hamster polyoma virus middle T antigen inhibit tyrosine kinase activity of baculovirus-expressed v-Src 2- and 4-fold, respectively. To determine the mechanism of this regulation, the Tyr-527 phosphopeptide was substituted with the photoactive amino acid p-benzoylphenylalanine at the adjacent positions (N- and C-terminal) to phosphotyrosine. These peptides photoinactivate the v-Src tyrosine kinase 5-fold in a time- and concentration-dependent manner. Furthermore, the peptides cross-link an isolated Src SH2 domain with similar rates and specificity. These data indicate that occupancy of the v-Src SH2 domain induces a conformational change that is transmitted to the kinase domain and attenuates tyrosine kinase activity.

p68 Sam is a substrate of the insulin receptor and associates with the SH2 domains of p85 PI3K.

PubMed

Sánchez-Margalet, V; Najib, S

1999-07-23

The 68 kDa Src substrate associated during mitosis is an RNA binding protein with Src homology 2 and 3 domain binding sites. A role for Src associated in mitosis 68 as an adaptor protein in signaling transduction has been proposed in different systems such as T-cell receptors. In the present work, we have sought to assess the possible role of Src associated in mitosis 68 in insulin receptor signaling. We performed in vivo studies in HTC-IR cells and in vitro studies using recombinant Src associated in mitosis 68, purified insulin receptor and fusion proteins containing either the N-terminal or the C-terminal Src homology 2 domain of p85 phosphatidylinositol-3-kinase. We have found that Src associated in mitosis 68 is a substrate of the insulin receptor both in vivo and in vitro. Moreover, tyrosine-phosphorylated Src associated in mitosis 68 was found to associate with p85 phosphatidylinositol-3-kinase in response to insulin, as assessed by co-immunoprecipitation studies. Therefore, Src associated in mitosis 68 may be part of the signaling complexes of insulin receptor along with p85. In vitro studies demonstrate that Src associated in mitosis 68 associates with the Src homology 2 domains of p85 after tyrosine phosphorylation by the activated insulin receptor. Moreover, tyr-phosphorylated Src associated in mitosis 68 binds with a higher affinity to the N-terminal Src homology 2 domain of p85 compared to the C-terminal Src homology 2 domain of p85, suggesting a preferential association of Src associated in mitosis 68 with the N-terminal Src homology 2 domain of p85. This association may be important for the link of the signaling with RNA metabolism.

Crystal structure of the motor domain of a class-I myosin

PubMed Central

Kollmar, Martin; Dürrwang, Ulrike; Kliche, Werner; Manstein, Dietmar J.; Kull, F.Jon

2002-01-01

The crystal structure of the motor domain of Dictyostelium discoideum myosin-IE, a monomeric unconventional myosin, was determined. The crystallographic asymmetric unit contains four independently resolved molecules, highlighting regions that undergo large conformational changes. Differences are particularly pronounced in the actin binding region and the converter domain. The changes in position of the converter domain reflect movements both parallel to and perpendicular to the actin axis. The orientation of the converter domain is ∼30° further up than in other myosin structures, indicating that MyoE can produce a larger power stroke by rotating its lever arm through a larger angle. The role of extended loops near the actin-binding site is discussed in the context of cellular localization. The core regions of the motor domain are similar, and the structure reveals how that core is stabilized in the absence of an N-terminal SH3-like domain. PMID:12032065

Binding of polarity-sensitive hydrophobic ligands to erythroid and nonerythroid spectrin: fluorescence and molecular modeling studies.

PubMed

Patra, Malay; Mitra, Madhurima; Chakrabarti, Abhijit; Mukhopadhyay, Chaitali

2014-01-01

We have used three polarity-sensitive fluorescence probes, 6-propionyl 2-(N,N-dimethyl-amino) naphthalene (Prodan), pyrene and 8-anilino 1-naphthalene sulphonic acid, to study their binding with erythroid and nonerythroid spectrin, using fluorescence spectroscopy. We have found that both bind to prodan and pyrene with high affinities with apparent dissociation constants (Kd) of .50 and .17 μM, for prodan, and .04 and .02 μM, for pyrene, respectively. The most striking aspect of these bindings have been that the binding stoichiometry have been equal to 1 in erythroid spectrin, both in dimeric and tetrameric form, and in tetrameric nonerythroid spectrin. From an estimate of apparent dielectric constants, the polarity of the binding site in both erythroid and nonerythroid forms have been found to be extremely hydrophobic. Thermodynamic parameters associated with such binding revealed that the binding is favored by positive change in entropy. Molecular docking studies alone indicate that both prodan and pyrene bind to the four major structural domains, following the order in the strength of binding to the Ankyrin binding domain > SH3 domain > Self-association domain > N-terminal domain of α-spectrin of both forms of spectrin. The binding experiments, particularly with the tetrameric nonerythroid spectrin, however, indicate more toward the self association domain in offering the unique binding site, since the binding stoichiometry have been 1 in all forms of dimeric and tetrameric spectrin, so far studied by us. Further studies are needed to characterize the hydrophobic binding sites in both forms of spectrin.

Ibrutinib inhibition of Bruton protein-tyrosine kinase (BTK) in the treatment of B cell neoplasms.

PubMed

Roskoski, Robert

2016-11-01

The Bruton non-receptor protein-tyrosine kinase (BTK), a deficiency of which leads to X-linked agammaglobulinemia, plays a central role in B cell antigen receptor signaling. Owing to the exclusivity of this enzyme in B cells, the acronym could represent B cell tyrosine kinase. BTK is activated by the Lyn and SYK protein kinases following activation of the B cell receptor. BTK in turn catalyzes the phosphorylation and activation of phospholipase Cγ2 leading to the downstream activation of the Ras/RAF/MEK/ERK pathway and the NF-κB pathways. Both pathways participate in the maturation of antibody-producing B cells. The BTK domains include a PH (pleckstrin homology) domain that interacts with membrane-associated phosphatidyl inositol trisphosphate, a TH (TEC homology) domain, which is followed by an SH3, SH2, and finally a protein kinase domain. Dysregulation of B cell receptor signaling occurs in several B cell neoplasms including mantle cell lymphoma, chronic lymphocytic leukemia, and Waldenström macroglobulinemia. Ibrutinib is FDA-approved as first-line or second line treatment for these diseases. The drug binds tightly in the ATP-binding pocket of BTK making salt bridges with residues within the hinge that connects the two lobes of the enzyme; then its unsaturated acrylamide group forms a covalent bond with BTK cysteine 481 to form an inactive adduct. In addition to the treatment of various B cell lymphomas, ibrutinib is under clinical trials for the treatment of numerous solid tumors owing to the role of tumor-promoting inflammation in the pathogenesis of neoplastic diseases. Copyright © 2016 Elsevier Ltd. All rights reserved.

Involvement of stress-activated protein kinase in the cellular response to 1-beta-D-arabinofuranosylcytosine and other DNA-damaging agents.

PubMed

Saleem, A; Datta, R; Yuan, Z M; Kharbanda, S; Kufe, D

1995-12-01

The cellular response to 1-beta-D-arabinofuranosylcytosine (ara-C) includes activation of Jun/AP-1, induction of c-jun transcription, and programmed cell death. The stress-activated protein (SAP) kinases stimulate the transactivation function of c-jun by amino terminal phosphorylation. The present work demonstrates that ara-C activates p54 SAP kinase. The finding that SAP kinase is also activated by alkylating agents (mitomycin C and cisplatinum) and the topoisomerase I inhibitor 9-amino-camptothecin supports DNA damage as an initial signal in this cascade. The results demonstrate that ara-C also induces binding of SAP kinase to the SH2/SH3-containing adapter protein Grb2. SAP kinase binds to the SH3 domains of Grb2, while interaction of the p85 alpha-subunit of phosphatidylinositol 3-kinase complex. The results also demonstrate that ara-C treatment is associated with inhibition of lipid and serine kinase activities of PI 3-kinase. The potential significance of the ara-C-induced interaction between SAP kinase and PI 3-kinase is further supported by the demonstration that Wortmannin, an inhibitor of PI 3-kinase, stimulates SAP kinase activity. The finding that Wortmannin treatment is also associated with internucleosomal DNA fragmentation may support a potential link between PI 3-kinase and regulation of both SAP kinase and programmed cell death.

ADAM binding protein Eve-1 is required for ectodomain shedding of epidermal growth factor receptor ligands.

PubMed

Tanaka, Motonari; Nanba, Daisuke; Mori, Seiji; Shiba, Fumio; Ishiguro, Hiroshi; Yoshino, Koichiro; Matsuura, Nariaki; Higashiyama, Shigeki

2004-10-01

A disintegrin and metalloproteases (ADAMs) are implicated in the ectodomain shedding of epidermal growth factor receptor (EGFR) ligands in EGFR transactivation. However, the activation mechanisms of ADAMs remain elusive. To analyze the regulatory mechanisms of ADAM activation, we performed yeast two-hybrid screening using the cytoplasmic domain of ADAM12 as bait, and identified a protein that we designated Eve-1. Two cDNAs were cloned and characterized. They encode alternatively spliced isoforms of Eve-1, called Eve-1a and Eve-1b, that have four and five tandem Src homology 3 (SH3) domains in the carboxyl-terminal region, respectively, and seven proline-rich SH3 domain binding motifs in the amino-terminal region. The short forms of Eve-1, Eve-1c and Eve-1d, translated at Met-371 are human counterparts of mouse Sh3d19. Northern blot analysis demonstrated that Eve-1 is abundantly expressed in skeletal muscle and heart. Western blot analysis revealed the dominant production of Eve-1c in human cancer cell lines. Knockdown of Eve-1 by small interfering RNA in HT1080 cells reduced the shedding of proHB-EGF induced by angiotensin II and 12-O-tetradecanoylphorbol-13-acetate, as well as the shedding of pro-transforming growth factor-alpha, promphiregulin, and proepiregulin by 12-O-tetradecanoylphorbol-13-acetate, suggesting that Eve-1 plays a role in positively regulating the activity of ADAMs in the signaling of EGFR-ligand shedding.

Cbl Associates with Pyk2 and Src to Regulate Src Kinase Activity, αvβ3 Integrin-Mediated Signaling, Cell Adhesion, and Osteoclast Motility

PubMed Central

Sanjay, Archana; Houghton, Adam; Neff, Lynn; DiDomenico, Emilia; Bardelay, Chantal; Antoine, Evelyne; Levy, Joan; Gailit, James; Bowtell, David; Horne, William C.; Baron, Roland

2001-01-01

The signaling events downstream of integrins that regulate cell attachment and motility are only partially understood. Using osteoclasts and transfected 293 cells, we find that a molecular complex comprising Src, Pyk2, and Cbl functions to regulate cell adhesion and motility. The activation of integrin αvβ3 induces the [Ca2+]i-dependent phosphorylation of Pyk2 Y402, its association with Src SH2, Src activation, and the Src SH3-dependent recruitment and phosphorylation of c-Cbl. Furthermore, the PTB domain of Cbl is shown to bind to phosphorylated Tyr-416 in the activation loop of Src, the autophosphorylation site of Src, inhibiting Src kinase activity and integrin-mediated adhesion. Finally, we show that deletion of c Src or c-Cbl leads to a decrease in osteoclast migration. Thus, binding of αvβ3 integrin induces the formation of a Pyk2/Src/Cbl complex in which Cbl is a key regulator of Src kinase activity and of cell adhesion and migration. These findings may explain the osteopetrotic phenotype in the Src−/− mice. PMID:11149930

Identification and molecular profiling of DC-SIGN-like from big belly seahorse (Hippocampus abdominalis) inferring its potential relevancy in host immunity.

PubMed

Jo, Eunyoung; Elvitigala, Don Anushka Sandaruwan; Wan, Qiang; Oh, Minyoung; Oh, Chulhong; Lee, Jehee

2017-12-01

Dendritic-cell-specific ICAM-3-grabbing non-integrin (DC-SIGN) is a C-type lectin that functions as a pattern recognition receptor by recognizing pathogen-associated molecular patterns (PAMPs). It is also involved in various events of the dendritic cell (DC) life cycle, such as DC migration, antigen capture and presentation, and T cell priming. In this study, a DC-SIGN-like gene from the big belly seahorse Hippocampus abdominalis (designated as ShDCS-like) was identified and molecularly characterized. The putative, complete ORF was found to be 1368 bp in length, encoding a protein of 462 amino acids with a molecular mass of 52.6 kDa and a theoretical isoelectric point of 8.26. The deduced amino acid sequence contains a single carbohydrate recognition domain (CRD), in which six conserved cysteine residues and two Ca 2+ -binding site motifs (QPN, WND) were identified. Based on pairwise sequence analysis, ShDCS-like exhibits the highest amino acid identity (94.6%) and similarity (97.4%) with DC-SIGN-like counterpart from tiger tail seahorse Hippocampus comes. Quantitative real-time PCR revealed that ShDCS-like mRNA is transcribed universally in all tissues examined, but with abundance in kidney and gill tissues. The basal mRNA expression of ShDCS-like was modulated in blood cell, kidney, gill and liver tissues in response to the stimulation of healthy fish with lipopolysaccharides (LPS), Edwardsiella tarda, or Streptococcus iniae. Moreover, recombinant ShDCS-like-CRD domain exhibited detectable agglutination activity against different bacteria. Collectively, these results suggest that ShDCS-like may potentially involve in immune function in big belly seahorses. Copyright © 2017 Elsevier Ltd. All rights reserved.

Role of Crk Adaptor Proteins in Cellular Migration and Invasion in Human Breast Cancer

DTIC Science & Technology

2007-03-01

nucleus. To confirm the staining is indeed specific, another antibody specific for CrkII is being tested. Furthermore, cytoplasmic and nuclear...the endogenous CrkL binding partner, Gab1 , which is enhanced upon HGF stimulation (Appendix 16). One final experiment, showing that the CrkLV5 tag...receptor tyrosine kinases, and a docking protein Gab1 , involved in epithelial dispersal and morphogenesis (5, 11, 12). The NH2-terminal SH3 domain of

Autoinhibition of Bruton's tyrosine kinase (Btk) and activation by soluble inositol hexakisphosphate

PubMed Central

Wang, Qi; Vogan, Erik M; Nocka, Laura M; Rosen, Connor E; Zorn, Julie A; Harrison, Stephen C; Kuriyan, John

2015-01-01

Bruton's tyrosine kinase (Btk), a Tec-family tyrosine kinase, is essential for B-cell function. We present crystallographic and biochemical analyses of Btk, which together reveal molecular details of its autoinhibition and activation. Autoinhibited Btk adopts a compact conformation like that of inactive c-Src and c-Abl. A lipid-binding PH-TH module, unique to Tec kinases, acts in conjunction with the SH2 and SH3 domains to stabilize the inactive conformation. In addition to the expected activation of Btk by membranes containing phosphatidylinositol triphosphate (PIP3), we found that inositol hexakisphosphate (IP6), a soluble signaling molecule found in both animal and plant cells, also activates Btk. This activation is a consequence of a transient PH-TH dimerization induced by IP6, which promotes transphosphorylation of the kinase domains. Sequence comparisons with other Tec-family kinases suggest that activation by IP6 is unique to Btk. DOI: http://dx.doi.org/10.7554/eLife.06074.001 PMID:25699547

SHIP2 associates with intersectin and recruits it to the plasma membrane in response to EGF.

PubMed

Xie, Jingwei; Vandenbroere, Isabelle; Pirson, Isabelle

2008-09-03

We identified intersectin1 (ITSN1) as a new binding partner of the SH2 domain containing inositol 5-phosphatase 2 (SHIP2). The interaction between SHIP2 and ITSN1 was confirmed in vivo. Src homology 3D, A, C, and E domains of ITSN1 were shown to be implicated in the interaction. In response to epidermal growth factor, SHIP2 expression could recruit the ITSN1 short form (ITSN1-S) to the cell membrane, while SHIP2 overexpression did not modulate the ITSN-mediated extracellular signal-regulated kinase1/2 and c-Jun NH2-terminal kinase activation. Our data provide a molecular link between SHIP2 and ITSN1 which are involved in receptor endocytosis regulation.

Structural studies and SH3 domain binding properties of a human antiviral salivary proline-rich peptide.

PubMed

Righino, Benedetta; Pirolli, Davide; Radicioni, Giorgia; Marzano, Valeria; Longhi, Renato; Arcovito, Alessandro; Sanna, Maria Teresa; De Rosa, Maria Cristina; Paoluzi, Serena; Cesareni, Gianni; Messana, Irene; Castagnola, Massimo; Vitali, Alberto

2016-09-01

Human saliva contains hundreds of small proline-rich peptides originated by the proteolytic cleavage of the salivary basic Proline-Rich Proteins. Nevertheless only for few of them a specific biological activity has been assigned to date. Among them, the 1932 Da peptide (p1932) has been patented as an anti-HIV agent. In order to shed light on the possible mechanism of action of this peptide, we assessed in this study, by means of molecular dynamics calculations, circular dichroism and FTIR spectroscopic techniques, that p1932 has an intrinsic propensity to adopt a polyproline-II helix arrangement. This structural feature combined with the presence of PxxP motifs in its primary structure, represents an essential property for the exploitation of several biological activities. Next to these findings, we recently demonstrated the ability of this peptide to be internalized within cells of the oral mucosa, thus we focused onto a possible intracellular target, represented by the SH3 domains family. Its ability to interact with selected SH3 domains was finally assayed by Surface Plasmon Resonance spectroscopy. As a result, only Fyn, Hck, and c-Src SH3 domains gave positive results in terms of interaction, showing dissociation constants ranging from nanomolar to micromolar values having the best performer a KD of 148 nM. It is noteworthy that all the interacting domains belong to the Src kinases family, suggesting a role for p1932 as a modulator of the signal transduction pathways mediated by these kinases. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 714-725, 2016. © 2016 Wiley Periodicals, Inc.

Regulation of Nucleocytoplasmic Shuttling of Bruton's Tyrosine Kinase (Btk) through a Novel SH3-Dependent Interaction with Ankyrin Repeat Domain 54 (ANKRD54)

PubMed Central

Hussain, Alamdar; Mohammad, Dara K.; Mohamed, Abdalla J.; Nguyen, Vivian; Metalnikov, Pavel; Colwill, Karen; Pawson, Tony; Nore, Beston F.

2012-01-01

Bruton's tyrosine kinase (Btk), belonging to the Tec family of tyrosine kinases (TFKs), is essential for B-lymphocyte development. Abrogation of Btk signaling causes human X-linked agammaglobulinemia (XLA) and murine X-linked immunodeficiency (Xid). We employed affinity purification of Flag-tagged Btk, combined with tandem mass spectrometry, to capture and identify novel interacting proteins. We here characterize the interaction with ankryin repeat domain 54 protein (ANKRD54), also known as Lyn-interacting ankyrin repeat protein (Liar). While Btk is a nucleocytoplasmic protein, the Liar pool was found to shuttle at a higher rate than Btk. Importantly, our results suggest that Liar mediates nuclear export of both Btk and another TFK, Txk/Rlk. Liar-mediated Btk shuttling was enriched for activation loop, nonphosphorylated Btk and entirely dependent on Btk's SH3 domain. Liar also showed reduced binding to an aspartic acid phosphomimetic SH3 mutant. Three other investigated nucleus-located proteins, Abl, estrogen receptor β (ERβ), and transcription factor T-bet, were all unaffected by Liar. We mapped the interaction site to the C terminus of the Btk SH3 domain. A biotinylated, synthetic Btk peptide, ARDKNGQEGYIPSNYVTEAEDS, was sufficient for this interaction. Liar is the first protein identified that specifically influences the nucleocytoplasmic shuttling of Btk and Txk and belongs to a rare group of known proteins carrying out this activity in a Crm1-dependent manner. PMID:22527282

Mechanical coupling in myosin V: a simulation study

PubMed Central

Ovchinnikov, Victor; Trout, Bernhardt L.

2009-01-01

Myosin motor function depends on the interaction between different domains that transmit information from one part of the molecule to another. The inter-domain coupling in myosin V is studied with Restrained Targeted Molecular Dynamics (RTMD) using an all-atom representation in explicit solvent. To elucidate the origin of the conformational change due to the binding of ATP, targeting forces are applied to small sets of atoms (the forcing sets, FS) in the direction of their displacement from the rigor conformation, which has a closed actin-binding cleft, to the post-rigor conformation, in which the cleft is open. The ‘minimal’ FS that results in extensive structural changes in the overall myosin conformation is comprised of the ATP, Switch 1, and the nearby HF, HG and HH helices. Addition of switch 2 to the forcing set is required to achieve a complete opening of the actin-binding cleft. The RTMD simulations reveal the mechanical coupling pathways between (i) the nucleotide-binding pocket (NBP) and the actin-binding cleft, (ii) the NBP and the converter, and (iii) the actin-binding cleft and the converter. Closing of the NBP due to ATP binding is tightly coupled to the opening of the cleft, and leads to the rupture of a key hydrogen bond (F441N/A684O) between switch 2 and the SH1 helix. The actin-binding cleft may mediate the rupture of this bond via a connection between the HW helix, the Relay helix, and Switch 2. The findings are consistent with experimental studies and a recent normal mode analysis. The present method is expected to be useful more generally in studies of inter-domain coupling in proteins. PMID:19853615

The SH2 domain of Abl kinases regulates kinase autophosphorylation by controlling activation loop accessibility.

PubMed

Lamontanara, Allan Joaquim; Georgeon, Sandrine; Tria, Giancarlo; Svergun, Dmitri I; Hantschel, Oliver

2014-11-17

The activity of protein kinases is regulated by multiple molecular mechanisms, and their disruption is a common driver of oncogenesis. A central and almost universal control element of protein kinase activity is the activation loop that utilizes both conformation and phosphorylation status to determine substrate access. In this study, we use recombinant Abl tyrosine kinases and conformation-specific kinase inhibitors to quantitatively analyse structural changes that occur after Abl activation. Allosteric SH2-kinase domain interactions were previously shown to be essential for the leukemogenesis caused by the Bcr-Abl oncoprotein. We find that these allosteric interactions switch the Abl activation loop from a closed to a fully open conformation. This enables the trans-autophosphorylation of the activation loop and requires prior phosphorylation of the SH2-kinase linker. Disruption of the SH2-kinase interaction abolishes activation loop phosphorylation. Our analysis provides a molecular mechanism for the SH2 domain-dependent activation of Abl that may also regulate other tyrosine kinases.

The SH2 domain of Abl kinases regulates kinase autophosphorylation by controlling activation loop accessibility

NASA Astrophysics Data System (ADS)

Lamontanara, Allan Joaquim; Georgeon, Sandrine; Tria, Giancarlo; Svergun, Dmitri I.; Hantschel, Oliver

2014-11-01

The activity of protein kinases is regulated by multiple molecular mechanisms, and their disruption is a common driver of oncogenesis. A central and almost universal control element of protein kinase activity is the activation loop that utilizes both conformation and phosphorylation status to determine substrate access. In this study, we use recombinant Abl tyrosine kinases and conformation-specific kinase inhibitors to quantitatively analyse structural changes that occur after Abl activation. Allosteric SH2-kinase domain interactions were previously shown to be essential for the leukemogenesis caused by the Bcr-Abl oncoprotein. We find that these allosteric interactions switch the Abl activation loop from a closed to a fully open conformation. This enables the trans-autophosphorylation of the activation loop and requires prior phosphorylation of the SH2-kinase linker. Disruption of the SH2-kinase interaction abolishes activation loop phosphorylation. Our analysis provides a molecular mechanism for the SH2 domain-dependent activation of Abl that may also regulate other tyrosine kinases.

Insights into the Folding and Unfolding Processes of Wild-Type and Mutated SH3 Domain by Molecular Dynamics and Replica Exchange Molecular Dynamics Simulations

PubMed Central

Chu, Wen-Ting; Zhang, Ji-Long; Zheng, Qing-Chuan; Chen, Lin; Zhang, Hong-Xing

2013-01-01

Src-homology regions 3 (SH3) domain is essential for the down-regulation of tyrosine kinase activity. Mutation A39V/N53P/V55L of SH3 is found to be relative to the urgent misfolding diseases. To gain insight, the human and gallus SH3 domains (PDB ID: 1NYG and 2LP5), including 58 amino acids in each protein, were selected for MD simulations (Amber11, ff99SB force field) and cluster analysis to investigate the influence of mutations on the spatial structure of the SH3 domain. It is found that the large conformational change of mutations mainly exists in three areas in the vicinity of protein core: RT loop, N-src loop, distal β-hairpin to 310 helix. The C-terminus of the mutated gallus SH3 is disordered after simulation, which represents the intermediate state of aggregation. The disappeared strong Hbond net in the mutated human and gallus systems will make these mutated proteins looser than the wild-type proteins. Additionally, by performing the REMD simulations on the gallus SH3 domain, the mutated domain is found to have an obvious effect on the unfolding process. These studies will be helpful for further aggregation mechanisms investigations on SH3 family. PMID:23734224

Insights into the folding and unfolding processes of wild-type and mutated SH3 domain by molecular dynamics and replica exchange molecular dynamics simulations.

PubMed

Chu, Wen-Ting; Zhang, Ji-Long; Zheng, Qing-Chuan; Chen, Lin; Zhang, Hong-Xing

2013-01-01

Src-homology regions 3 (SH3) domain is essential for the down-regulation of tyrosine kinase activity. Mutation A39V/N53P/V55L of SH3 is found to be relative to the urgent misfolding diseases. To gain insight, the human and gallus SH3 domains (PDB ID: 1NYG and 2LP5), including 58 amino acids in each protein, were selected for MD simulations (Amber11, ff99SB force field) and cluster analysis to investigate the influence of mutations on the spatial structure of the SH3 domain. It is found that the large conformational change of mutations mainly exists in three areas in the vicinity of protein core: RT loop, N-src loop, distal β-hairpin to 310 helix. The C-terminus of the mutated gallus SH3 is disordered after simulation, which represents the intermediate state of aggregation. The disappeared strong Hbond net in the mutated human and gallus systems will make these mutated proteins looser than the wild-type proteins. Additionally, by performing the REMD simulations on the gallus SH3 domain, the mutated domain is found to have an obvious effect on the unfolding process. These studies will be helpful for further aggregation mechanisms investigations on SH3 family.

Immunoreceptor Tyrosine-based Inhibitory Motif (ITIM)-mediated Inhibitory Signaling is Regulated by Sequential Phosphorylation Mediated by Distinct Nonreceptor Tyrosine Kinases: A Case Study Involving PECAM-1

PubMed Central

Tourdot, Benjamin E.; Brenner, Michelle K.; Keough, Kathleen C.; Holyst, Trudy; Newman, Peter J.; Newman, Debra K.

2013-01-01

The activation state of many blood and vascular cells is tightly controlled by a delicate balance between receptors that contain immunoreceptor tyrosine-based activation motifs (ITAMs) and those that contain immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Precisely how the timing of cellular activation by ITAM-coupled receptors is regulated by ITIM-containing receptors is, however, poorly understood. Using platelet endothelial cell adhesion molecule 1 (PECAM-1) as a prototypical ITIM-bearing receptor, we demonstrate that initiation of inhibitory signaling occurs via a novel, sequential process in which Src family kinases phosphorylate the C-terminal ITIM, thereby enabling phosphorylation of the N-terminal ITIM of PECAM-1 by other Src homology 2 domain-containing non-receptor tyrosine kinases (NRTKs). NRTKs capable of mediating the second phosphorylation event include C-terminal Src kinase (Csk) and Bruton’s tyrosine kinase (Btk). Btk and Csk function downstream of phosphatidylinositol 3-kinase (PI3K) activation during ITAM-dependent platelet activation. In ITAM-activated platelets that were treated with a PI3K inhibitor, PECAM-1 was phosphorylated but did not bind the tandem SH2 domain-containing tyrosine phosphatase SHP-2, indicating that it was not phosphorylated on its N-terminal ITIM. Csk bound to and phosphorylated PECAM-1 more efficiently than did Btk, and required its SH2 domain to perform these functions. Additionally, the phosphorylation of the N-terminal ITIM of Siglec-9 by Csk is enhanced by the prior phosphorylation of its C-terminal ITIM, providing evidence that the ITIMs of other dual ITIM-containing receptors are also sequentially phosphorylated. On the basis of these findings, we propose that sequential ITIM phosphorylation provides a general mechanism for precise temporal control over the recruitment and activation of tandem SH2 domain-containing tyrosine phosphatases that dampen ITAM-dependent signals. PMID:23418871

SAP97 Controls the Trafficking and Resensitization of the Beta-1-Adrenergic Receptor through Its PDZ2 and I3 Domains

PubMed Central

Nooh, Mohammed M.; Naren, Anjaparavanda P.; Kim, Sung-Jin; Xiang, Yang K.; Bahouth, Suleiman W.

2013-01-01

Previous studies have determined that the type-1 PDZ sequence at the extreme carboxy-terminus of the ß1-adrenergic receptor (ß1-AR) binds SAP97 and AKAP79 to organize a scaffold involved in trafficking of the ß1-AR. In this study we focused on characterizing the domains in SAP97 that were involved in recycling and resensitization of the ß1-AR in HEK-293 cells. Using a SAP97 knockdown and rescue strategy, we determined that PDZ-deletion mutants of SAP97 containing PDZ2 rescued the recycling and resensitization of the ß1-AR. Among the three PDZs of SAP97, PDZ2 displayed the highest affinity in binding to the ß1-AR. Expression of isolated PDZ2, but not the other PDZs, inhibited the recycling of the ß1-AR by destabilizing the macromolecular complex involved in trafficking and functional resensitization of the ß1-AR. In addition to its PDZs, SAP97 contains other protein interacting domains, such as the I3 sequence in the SRC homology-3 (SH3) domain, which binds to AKAP79. Deletion of I3 from SAP97 (ΔI3-SAP97) did not affect the binding of SAP97 to the ß1-AR. However, ΔI3-SAP97 could not rescue the recycling of the ß1-AR because it failed to incorporate AKAP79/PKA into the SAP97-ß1-AR complex. Therefore, bipartite binding of SAP97 to the ß1-AR and to AKAP79 is necessary for SAP97-mediated effects on recycling, externalization and functional resensitization of the ß1-AR. These data establish a prominent role for PDZ2 and I3 domains of SAP97 in organizing the ß1-adrenergic receptosome involved in connecting the ß1-AR to trafficking and signaling networks. PMID:23696820

Structural Basis for Activation of ZAP-70 by Phosphorylation of the SH2-Kinase Linker

PubMed Central

Yan, Qingrong; Barros, Tiago; Visperas, Patrick R.; Deindl, Sebastian; Kadlecek, Theresa A.; Weiss, Arthur

2013-01-01

Serial activation of the tyrosine kinases Lck and ZAP-70 initiates signaling downstream of the T cell receptor. We previously reported the structure of an autoinhibited ZAP-70 variant in which two regulatory tyrosine residues (315 and 319) in the SH2-kinase linker were replaced by phenylalanine. We now present a crystal structure of ZAP-70 in which Tyr 315 and Tyr 319 are not mutated, leading to the recognition of a five-residue sequence register error in the SH2-kinase linker of the original crystallographic model. The revised model identifies distinct roles for these two tyrosines. As seen in a recently reported structure of the related tyrosine kinase Syk, Tyr 315 of ZAP-70 is part of a hydrophobic interface between the regulatory apparatus and the kinase domain, and the integrity of this interface would be lost upon engagement of doubly phosphorylated peptides by the SH2 domains. Tyr 319 is not necessarily dislodged by SH2 engagement, which activates ZAP-70 only ∼5-fold in vitro. In contrast, phosphorylation by Lck activates ZAP-70 ∼100-fold. This difference is due to the ability of Tyr 319 to suppress ZAP-70 activity even when the SH2 domains are dislodged from the kinase domain, providing stringent control of ZAP-70 activity downstream of Lck. PMID:23530057

Structural basis for activation of ZAP-70 by phosphorylation of the SH2-kinase linker.

PubMed

Yan, Qingrong; Barros, Tiago; Visperas, Patrick R; Deindl, Sebastian; Kadlecek, Theresa A; Weiss, Arthur; Kuriyan, John

2013-06-01

Serial activation of the tyrosine kinases Lck and ZAP-70 initiates signaling downstream of the T cell receptor. We previously reported the structure of an autoinhibited ZAP-70 variant in which two regulatory tyrosine residues (315 and 319) in the SH2-kinase linker were replaced by phenylalanine. We now present a crystal structure of ZAP-70 in which Tyr 315 and Tyr 319 are not mutated, leading to the recognition of a five-residue sequence register error in the SH2-kinase linker of the original crystallographic model. The revised model identifies distinct roles for these two tyrosines. As seen in a recently reported structure of the related tyrosine kinase Syk, Tyr 315 of ZAP-70 is part of a hydrophobic interface between the regulatory apparatus and the kinase domain, and the integrity of this interface would be lost upon engagement of doubly phosphorylated peptides by the SH2 domains. Tyr 319 is not necessarily dislodged by SH2 engagement, which activates ZAP-70 only ∼5-fold in vitro. In contrast, phosphorylation by Lck activates ZAP-70 ∼100-fold. This difference is due to the ability of Tyr 319 to suppress ZAP-70 activity even when the SH2 domains are dislodged from the kinase domain, providing stringent control of ZAP-70 activity downstream of Lck.

NSP-CAS Protein Complexes: Emerging Signaling Modules in Cancer.

PubMed

Wallez, Yann; Mace, Peter D; Pasquale, Elena B; Riedl, Stefan J

2012-05-01

The CAS (CRK-associated substrate) family of adaptor proteins comprises 4 members, which share a conserved modular domain structure that enables multiple protein-protein interactions, leading to the assembly of intracellular signaling platforms. Besides their physiological role in signal transduction downstream of a variety of cell surface receptors, CAS proteins are also critical for oncogenic transformation and cancer cell malignancy through associations with a variety of regulatory proteins and downstream effectors. Among the regulatory partners, the 3 recently identified adaptor proteins constituting the NSP (novel SH2-containing protein) family avidly bind to the conserved carboxy-terminal focal adhesion-targeting (FAT) domain of CAS proteins. NSP proteins use an anomalous nucleotide exchange factor domain that lacks catalytic activity to form NSP-CAS signaling modules. Additionally, the NSP SH2 domain can link NSP-CAS signaling assemblies to tyrosine-phosphorylated cell surface receptors. NSP proteins can potentiate CAS function by affecting key CAS attributes such as expression levels, phosphorylation state, and subcellular localization, leading to effects on cell adhesion, migration, and invasion as well as cell growth. The consequences of these activities are well exemplified by the role that members of both families play in promoting breast cancer cell invasiveness and resistance to antiestrogens. In this review, we discuss the intriguing interplay between the NSP and CAS families, with a particular focus on cancer signaling networks.

Brain Region and Isoform-Specific Phosphorylation Alters Kalirin SH2 Domain Interaction Sites and Calpain Sensitivity

PubMed Central

Miller, Megan B.; Yan, Yan; Machida, Kazuya; Kiraly, Drew D.; Levy, Aaron D.; Wu, Yi I.; Lam, TuKiet T.; Abbott, Thomas; Koleske, Anthony J.; Eipper, Betty A.; Mains, Richard E.

2017-01-01

Kalirin7 (Kal7), a postsynaptic Rho GDP/GTP exchange factor (RhoGEF), plays a crucial role in long term potentiation and in the effects of cocaine on behavior and spine morphology. The KALRN gene has been linked to schizophrenia and other disorders of synaptic function. Mass spectrometry was used to quantify phosphorylation at 26 sites in Kal7 from individual adult rat nucleus accumbens and prefrontal cortex before and after exposure to acute or chronic cocaine. Region- and isoform-specific phosphorylation was observed along with region-specific effects of cocaine on Kal7 phosphorylation. Evaluation of the functional significance of multi-site phosphorylation in a complex protein like Kalirin is difficult. With the identification of five tyrosine phosphorylation (pY) sites, a panel of 71 SH2 domains was screened, identifying subsets that interacted with multiple pY sites in Kal7. In addition to this type of reversible interaction, endoproteolytic cleavage by calpain plays an essential role in long-term potentiation. Calpain cleaved Kal7 at two sites, separating the N-terminal domain, which affects spine length, and the PDZ binding motif from the GEF domain. Mutations preventing phosphorylation did not affect calpain sensitivity or GEF activity; phosphomimetic mutations at specific sites altered protein stability, increased calpain sensitivity and reduced GEF activity. PMID:28418645

Phosphorylated c-Mpl tyrosine 591 regulates thrombopoietin-induced signaling.

PubMed

Sangkhae, Veena; Saur, Sebastian Jonas; Kaushansky, Alexis; Kaushansky, Kenneth; Hitchcock, Ian Stuart

2014-06-01

Thrombopoietin (TPO) is the primary regulator of platelet production, affecting cell survival, proliferation, and differentiation through binding to and stimulation of the cell surface receptor the cellular myeloproliferative leukemia virus oncogene (c-Mpl). Activating mutations in c-Mpl constitutively stimulate downstream signaling pathways, leading to aberrant hematopoiesis, and contribute to development of myeloproliferative neoplasms. Several studies have mapped the tyrosine residues within the cytoplasmic domain of c-Mpl that mediate these cellular signals; however, secondary signaling pathways are incompletely understood. In this study, we focused on c-Mpl tyrosine 591 (Y591). We found Y591 of wild-type c-Mpl to be phosphorylated in the presence of TPO. Additionally, eliminating Y591 phosphorylation by mutation to Phe resulted in decreased total receptor phosphorylation. Using a Src homology 2/phosphotyrosine-binding (SH2/PTB) domain binding microarray, we identified novel c-Mpl binding partners for phosphorylated Y591, including Src homology region 2 domain-containing phosphatase-1 (SHP-1), spleen tyrosine kinase (SYK) and Bruton's tyrosine kinase (BTK). The functional significance of binding partners was determined through small interfering RNA treatment of Ba/F3-Mpl cells, confirming that the increase in pERK1/2 resulting from removal of Y591 may be mediated by spleen tyrosine kinase. These findings identify a novel negative regulatory pathway that controls TPO-mediated signaling, advancing our understanding of the mechanisms required for successful maintenance of hematopoietic stem cells and megakaryocyte development. Copyright © 2014 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.

The measles virus phosphoprotein interacts with the linker domain of STAT1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Devaux, Patricia, E-mail: devaux.patricia@mayo.edu; Priniski, Lauren; Cattaneo, Roberto

2013-09-15

The measles virus (MV) phosphoprotein (P) and V proteins block the interferon (IFN) response by impeding phosphorylation of the signal transducer and activator of transcription 1 (STAT1) by the Janus kinase 1 (JAK1). We characterized how STAT1 mutants interact with P and JAK1 phosphorylation. Certain mutants of the linker, the Src-homology 2 domain (SH2), or the transactivation domain had reduced or abolished phosphorylation through JAK1 after IFN treatment. Other mutants, mainly localized in the linker, failed to interact with P as documented by the lack of interference with nuclear translocation. Thus the functional footprint of P on STAT1 localizes mainlymore » to the linker domain; there is also some overlap with the STAT1 phosphorylation functional footprint on the SH2 domain. Based on these observations, we discuss how the MV-P might operate to inhibit the JAK/STAT pathway. - Highlights: • Residue in the linker and SH2 domains of STAT1 are important for MV-P interaction. • Residue in the linker and SH2 domains of STAT1 are important for STAT1 phosphorylation. • Residues interferring with both functions have similar location on STAT1. • The viral P and V proteins may operate in concert to inhibit the JAK/STAT pathway.« less

The tight junction protein ZO-1 and an interacting transcription factor regulate ErbB-2 expression

PubMed Central

Balda, Maria S.; Matter, Karl

2000-01-01

Epithelial tight junctions regulate paracellular diffusion and restrict the intermixing of apical and basolateral plasma membrane components. We now identify a Y-box transcription factor, ZONAB (ZO-1-associated nucleic acid-binding protein), that binds to the SH3 domain of ZO-1, a submembrane protein of tight junctions. ZONAB localizes to the nucleus and at tight junctions, and binds to sequences of specific promoters containing an inverted CCAAT box. In reporter assays, ZONAB and ZO-1 functionally interact in the regulation of the ErbB-2 promoter in a cell density-dependent manner. In stably transfected overexpressing cells, ZO-1 and ZONAB control expression of endogenous ErbB-2 and function in the regulation of paracellular permeability. These data indicate that tight junctions directly participate in the control of gene expression and suggest that they function in the regulation of epithelial cell differentiation. PMID:10790369

The Ste20 kinase misshapen regulates both photoreceptor axon targeting and dorsal closure, acting downstream of distinct signals.

PubMed

Su, Y C; Maurel-Zaffran, C; Treisman, J E; Skolnik, E Y

2000-07-01

We have previously shown that the Ste20 kinase encoded by misshapen (msn) functions upstream of the c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase module in Drosophila. msn is required to activate the Drosophila JNK, Basket (Bsk), to promote dorsal closure of the embryo. A mammalian homolog of Msn, Nck interacting kinase, interacts with the SH3 domains of the SH2-SH3 adapter protein Nck. We now show that Msn likewise interacts with Dreadlocks (Dock), the Drosophila homolog of Nck. dock is required for the correct targeting of photoreceptor axons. We have performed a structure-function analysis of Msn in vivo in Drosophila in order to elucidate the mechanism whereby Msn regulates JNK and to determine whether msn, like dock, is required for the correct targeting of photoreceptor axons. We show that Msn requires both a functional kinase and a C-terminal regulatory domain to activate JNK in vivo in Drosophila. A mutation in a PXXP motif on Msn that prevents it from binding to the SH3 domains of Dock does not affect its ability to rescue the dorsal closure defect in msn embryos, suggesting that Dock is not an upstream regulator of msn in dorsal closure. Larvae with only this mutated form of Msn show a marked disruption in photoreceptor axon targeting, implicating an SH3 domain protein in this process; however, an activated form of Msn is not sufficient to rescue the dock mutant phenotype. Mosaic analysis reveals that msn expression is required in photoreceptors in order for their axons to project correctly. The data presented here genetically link msn to two distinct biological events, dorsal closure and photoreceptor axon pathfinding, and thus provide the first evidence that Ste20 kinases of the germinal center kinase family play a role in axonal pathfinding. The ability of Msn to interact with distinct classes of adapter molecules in dorsal closure and photoreceptor axon pathfinding may provide the flexibility that allows it to link to distinct upstream signaling systems.

The Ste20 Kinase Misshapen Regulates Both Photoreceptor Axon Targeting and Dorsal Closure, Acting Downstream of Distinct Signals

PubMed Central

Su, Yi-Chi; Maurel-Zaffran, Corinne; Treisman, Jessica E.; Skolnik, Edward Y.

2000-01-01

We have previously shown that the Ste20 kinase encoded by misshapen (msn) functions upstream of the c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase module in Drosophila. msn is required to activate the Drosophila JNK, Basket (Bsk), to promote dorsal closure of the embryo. A mammalian homolog of Msn, Nck interacting kinase, interacts with the SH3 domains of the SH2-SH3 adapter protein Nck. We now show that Msn likewise interacts with Dreadlocks (Dock), the Drosophila homolog of Nck. dock is required for the correct targeting of photoreceptor axons. We have performed a structure-function analysis of Msn in vivo in Drosophila in order to elucidate the mechanism whereby Msn regulates JNK and to determine whether msn, like dock, is required for the correct targeting of photoreceptor axons. We show that Msn requires both a functional kinase and a C-terminal regulatory domain to activate JNK in vivo in Drosophila. A mutation in a PXXP motif on Msn that prevents it from binding to the SH3 domains of Dock does not affect its ability to rescue the dorsal closure defect in msn embryos, suggesting that Dock is not an upstream regulator of msn in dorsal closure. Larvae with only this mutated form of Msn show a marked disruption in photoreceptor axon targeting, implicating an SH3 domain protein in this process; however, an activated form of Msn is not sufficient to rescue the dock mutant phenotype. Mosaic analysis reveals that msn expression is required in photoreceptors in order for their axons to project correctly. The data presented here genetically link msn to two distinct biological events, dorsal closure and photoreceptor axon pathfinding, and thus provide the first evidence that Ste20 kinases of the germinal center kinase family play a role in axonal pathfinding. The ability of Msn to interact with distinct classes of adapter molecules in dorsal closure and photoreceptor axon pathfinding may provide the flexibility that allows it to link to distinct upstream signaling systems. PMID:10848599

DNA binding of the p21 repressor ZBTB2 is inhibited by cytosine hydroxymethylation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lafaye, Céline; Barbier, Ewa; Miscioscia, Audrey

2014-03-28

Highlights: • 5-hmC epigenetic modification is measurable in HeLa, SH-SY5Y and UT7-MPL cell lines. • ZBTB2 binds to DNA probes containing 5-mC but not to sequences containing 5-hmC. • This differential binding is verified with DNA sequences involved in p21 regulation. - Abstract: Recent studies have demonstrated that the modified base 5-hydroxymethylcytosine (5-hmC) is detectable at various rates in DNA extracted from human tissues. This oxidative product of 5-methylcytosine (5-mC) constitutes a new and important actor of epigenetic mechanisms. We designed a DNA pull down assay to trap and identify nuclear proteins bound to 5-hmC and/or 5-mC. We applied thismore » strategy to three cancerous cell lines (HeLa, SH-SY5Y and UT7-MPL) in which we also measured 5-mC and 5-hmC levels by HPLC-MS/MS. We found that the putative oncoprotein Zinc finger and BTB domain-containing protein 2 (ZBTB2) is associated with methylated DNA sequences and that this interaction is inhibited by the presence of 5-hmC replacing 5-mC. As published data mention ZBTB2 recognition of p21 regulating sequences, we verified that this sequence specific binding was also alleviated by 5-hmC. ZBTB2 being considered as a multifunctional cell proliferation activator, notably through p21 repression, this work points out new epigenetic processes potentially involved in carcinogenesis.« less

The Association of Myosin IB with Actin Waves in Dictyostelium Requires Both the Plasma Membrane-Binding Site and Actin-Binding Region in the Myosin Tail

PubMed Central

Brzeska, Hanna; Pridham, Kevin; Chery, Godefroy; Titus, Margaret A.; Korn, Edward D.

2014-01-01

F-actin structures and their distribution are important determinants of the dynamic shapes and functions of eukaryotic cells. Actin waves are F-actin formations that move along the ventral cell membrane driven by actin polymerization. Dictyostelium myosin IB is associated with actin waves but its role in the wave is unknown. Myosin IB is a monomeric, non-filamentous myosin with a globular head that binds to F-actin and has motor activity, and a non-helical tail comprising a basic region, a glycine-proline-glutamine-rich region and an SH3-domain. The basic region binds to acidic phospholipids in the plasma membrane through a short basic-hydrophobic site and the Gly-Pro-Gln region binds F-actin. In the current work we found that both the basic-hydrophobic site in the basic region and the Gly-Pro-Gln region of the tail are required for the association of myosin IB with actin waves. This is the first evidence that the Gly-Pro-Gln region is required for localization of myosin IB to a specific actin structure in situ. The head is not required for myosin IB association with actin waves but binding of the head to F-actin strengthens the association of myosin IB with waves and stabilizes waves. Neither the SH3-domain nor motor activity is required for association of myosin IB with actin waves. We conclude that myosin IB contributes to anchoring actin waves to the plasma membranes by binding of the basic-hydrophobic site to acidic phospholipids in the plasma membrane and binding of the Gly-Pro-Gln region to F-actin in the wave. PMID:24747353

Human SLP-65 isoforms contribute differently to activation and apoptosis of B lymphocytes.

PubMed

Grabbe, Annika; Wienands, Jürgen

2006-12-01

The SH2 domain-containing leukocyte adaptor protein of 65 kDa (SLP-65) is the key effector for signaling downstream of the B-cell antigen receptor (BCR). SLP-65 controls not only B lymphopoiesis and humoral immunity but also possesses a yet poorly defined tumor suppressor activity that is lost in many cases of acute lymphoblastic leukemia. We found that the 2 isoforms of human SLP-65 are differentially involved in positive and negative B-cell signaling. Reconstitution experiments revealed that an atypical SH3 domain-binding motif, which is present in the long but not in the short SLP-65 isoform, mediates association to Grb2 and suppresses activation of mitogen-activated protein kinases p38 and JNK as well as up-regulation of c-Fos expression. In turn, the short isoform activates not only AP1-driven but also NF-kappaB-driven gene transcription more potently than the long isoform. Conversely, the long rather than the short SLP-65 isoform promotes BCR-induced B-cell apoptosis. Our data further delineate the structural requirements of positive and negative SLP-65 signal transduction in normal and neoplastic cells.

Capns1, a new binding partner of RasGAP-SH3 domain in K-Ras(V12) oncogenic cells: modulation of cell survival and migration.

PubMed

Pamonsinlapatham, Perayot; Gril, Brunilde; Dufour, Sylvie; Hadj-Slimane, Réda; Gigoux, Véronique; Pethe, Stéphanie; L'hoste, Sébastien; Camonis, Jacques; Garbay, Christiane; Raynaud, Françoise; Vidal, Michel

2008-11-01

Ras GTPase-activating protein (RasGAP) is hypothesized to be an effector of oncogenic Ras stimulating numerous downstream cellular signaling cascades involved in survival, proliferation and motility. In this study, we identified calpain small subunit-1 (Capns1) as a new RasGAP-SH3 domain binding partner, using yeast two-hybrid screening. The interaction was confirmed by co-immunoprecipitation assay and was found specific to cells expressing oncogenic K-Ras. We used confocal microscopy to analyze our stably transfected cell model producing mutant Ras (PC3Ras(V12)). Staining for RasGAP-SH3/Capns1 co-localization was two-fold stronger in the protrusions of Ras(V12) cells than in PC3 cells. RasGAP or Capns1 knockdown in PC3Ras(V12) cells induced a two- to three-fold increase in apoptosis. Capns1 gene silencing reduced the speed and increased the persistence of movement in PC3Ras(V12) cells. In contrast, RasGAP knockdown in PC3Ras(V12) cells increased cell migration. Knockdown of both proteins altered the speed and directionality of cell motility. Our findings suggest that RasGAP and Capns1 interaction in oncogenic Ras cells is involved in regulating migration and cell survival.

Mechanistic insights into phosphoprotein-binding FHA domains.

PubMed

Liang, Xiangyang; Van Doren, Steven R

2008-08-01

[Structure: see text]. FHA domains are protein modules that switch signals in diverse biological pathways by monitoring the phosphorylation of threonine residues of target proteins. As part of the effort to gain insight into cellular avoidance of cancer, FHA domains involved in the cellular response to DNA damage have been especially well-characterized. The complete protein where the FHA domain resides and the interaction partners determine the nature of the signaling. Thus, a key biochemical question is how do FHA domains pick out their partners from among thousands of alternatives in the cell? This Account discusses the structure, affinity, and specificity of FHA domains and the formation of their functional structure. Although FHA domains share sequence identity at only five loop residues, they all fold into a beta-sandwich of two beta-sheets. The conserved arginine and serine of the recognition loops recognize the phosphorylation of the threonine targeted. Side chains emanating from loops that join beta-strand 4 with 5, 6 with 7, or 10 with 11 make specific contacts with amino acids of the ligand that tailor sequence preferences. Many FHA domains choose a partner in extended conformation, somewhat according to the residue three after the phosphothreonine in sequence (pT + 3 position). One group of FHA domains chooses a short carboxylate-containing side chain at pT + 3. Another group chooses a long, branched aliphatic side chain. A third group prefers other hydrophobic or uncharged polar side chains at pT + 3. However, another FHA domain instead chooses on the basis of pT - 2, pT - 3, and pT + 1 positions. An FHA domain from a marker of human cancer instead chooses a much longer protein fragment that adds a beta-strand to its beta-sheet and that presents hydrophobic residues from a novel helix to the usual recognition surface. This novel recognition site and more remote sites for the binding of other types of protein partners were predicted for the entire family of FHA domains by a bioinformatics approach. The phosphopeptide-dependent dynamics of an FHA domain, SH2 domain, and PTB domain suggest a common theme: rigid, preformed binding surfaces support van der Waals contacts that provide favorable binding enthalpy. Despite the lack of pronounced conformational changes in FHA domains linked to binding events, more subtle adjustments may be possible. In the one FHA domain tested, phosphothreonine peptide binding is accompanied by increased flexibility just outside the binding site and increased rigidity across the beta-sandwich. The folding of the same FHA domain progresses through near-native intermediates that stabilize the recognition loops in the center of the phosphoprotein-binding surface; this may promote rigidity in the interface and affinity for targets phosphorylated on threonine.

The SH3BGR/STAT3 Pathway Regulates Cell Migration and Angiogenesis Induced by a Gammaherpesvirus MicroRNA

PubMed Central

Ding, Xiangya; Shen, Chenyou; Hu, Minmin; Zhu, Ying; Qin, Di; Lu, Hongmei; Krueger, Brian J.; Renne, Rolf; Gao, Shou-Jiang; Lu, Chun

2016-01-01

Kaposi’s sarcoma (KS)-associated herpesvirus (KSHV) is a gammaherpesvirus etiologically associated with KS, a highly disseminated angiogenic tumor of hyperproliferative spindle endothelial cells. KSHV encodes 25 mature microRNAs but their roles in KSHV-induced tumor dissemination and angiogenesis remain unknown. Here, we investigated KSHV-encoded miR-K12-6-3p (miR-K6-3p) promotion of endothelial cell migration and angiogenesis, which are the underlying mechanisms of tumor dissemination and angiogenesis. We found that ectopic expression of miR-K6-3p promoted endothelial cell migration and angiogenesis. Mass spectrometry, bioinformatics and luciferase reporter analyses revealed that miR-K6-3p directly targeted sequence in the 3’ untranslated region (UTR) of SH3 domain binding glutamate-rich protein (SH3BGR). Overexpression of SH3BGR reversed miR-K6-3p induction of cell migration and angiogenesis. Mechanistically, miR-K6-3p downregulated SH3BGR, hence relieved STAT3 from SH3BGR direct binding and inhibition, which was required for miR-K6-3p maximum activation of STAT3 and induction of cell migration and angiogenesis. Finally, deletion of miR-K6 from the KSHV genome abrogated its effect on the SH3BGR/STAT3 pathway, and KSHV-induced migration and angiogenesis. Our results illustrated that, by inhibiting SH3BGR, miR-K6-3p enhances cell migration and angiogenesis by activating the STAT3 pathway, and thus contributes to the dissemination and angiogenesis of KSHV-induced malignancies. PMID:27128969

Identification of N-Terminal Lobe Motifs that Determine the Kinase Activity of the Catalytic Domains and Regulatory Strategies of Src and Csk Protein Tyrosine Kinases†

PubMed Central

Huang, Kezhen; Wang, Yue-Hao; Brown, Alex; Sun, Gongqin

2009-01-01

Csk and Src protein tyrosine kinases are structurally homologous, but use opposite regulatory strategies. The isolated catalytic domain of Csk is intrinsically inactive and is activated by interactions with the regulatory SH3 and SH2 domains, while the isolated catalytic domain of Src is intrinsically active and is suppressed by interactions with the regulatory SH3 and SH2 domains. The structural basis for why one isolated catalytic domain is intrinsically active while the other is inactive is not clear. In this current study, we identify the structural elements in the N-terminal lobe of the catalytic domain that render the Src catalytic domain active. These structural elements include the α-helix C region, a β-turn between the β-4 and β-5 strands, and an Arg residue at the beginning of the catalytic domain. These three motifs interact with each other to activate the Src catalytic domain, but the equivalent motifs in Csk directly interact with the regulatory domains that are important for Csk activation. The Src motifs can be grafted to the Csk catalytic domain to obtain an active Csk catalytic domain. These results, together with available Src and Csk tertiary structures, reveal an important structural switch that determines the kinase activity of a catalytic domain and dictates the regulatory strategy of a kinase. PMID:19244618

Cell Signaling by a Novel SH2 Domain Protein that is Overexpressed with Her2 in Breast Cancer

DTIC Science & Technology

1998-01-01

1990) Methods Enzymol. 185, 51. Wary, K. K, Mainiero, F ., Isakoff, S. J., Marcantonio , E. E., and Giancotti 527-537 F . G. (1996) Cell 87, 733-743...family of SH2 domain proteins and acts cell nonautonomously in excretory canal development. Dev. Biol. 184:150-164. 3. Pelicci, G., L. Lanfrancone, F ...Grignani, J. McGlade, F . Cavallo, G. Forni, I. Nicoletti, T. Pawson, and P. G. Pelicci. 1992. A novel transforming protein (SHC) with an SH2

Identification of amino acids in the N-terminal SH2 domain of phospholipase C gamma 1 important in the interaction with epidermal growth factor receptor.

PubMed

Gergel, J R; McNamara, D J; Dobrusin, E M; Zhu, G; Saltiel, A R; Miller, W T

1994-12-13

Photoaffinity labeling and site-directed mutagenesis have been used to identify amino acid residues of the phospholipase C gamma 1 (PLC gamma 1) N-terminal SH2 domain involved in recognition of the activated epidermal growth factor receptor (EGFR). The photoactive amino acid p-benzoylphenylalanine (Bpa) was incorporated into phosphotyrosine-containing peptides derived from EGFR autophosphorylation sites Tyr992 and Tyr1068. Irradiation of these labels in the presence of SH2 domains showed cross-linking which was time-dependent and specific; labeling was inhibited with non-Bpa-containing peptides from EGFR in molar excess. The phosphotyrosine residue on the peptides was important for SH2 recognition, as dephosphorylated peptides did not cross-link. Radiolabeled peptides were used to identify sites of cross-linking to the N-terminal SH2 of PLC gamma 1. Bpa peptide-SH2 complexes were digested with trypsin, and radioactive fragments were purified by HPLC and analyzed by Edman sequencing. These experiments showed Arg562 and an additional site in the alpha A-beta B region of the SH2 domain, most likely Glu587, to be labeled by the Tyr992-derived peptide. Similar analysis of the reaction with the Tyr1068-derived photoaffinity label identified Leu653 as the cross-linked site. Mutation of the neighboring residues of Glu587 decreased photo-cross-linking, emphasizing the importance of this region of the molecule for recognition. These results are consistent with evidence from the v-Src crystal structure and implicate the loop spanning residues Gln640-Ser654 of PLC gamma 1 in specific recognition of phosphopeptides.

The novel SH3 domain protein Dlish/CG10933 mediates fat signaling in Drosophila by binding and regulating Dachs

PubMed Central

Zhang, Yifei; Wang, Xing; Matakatsu, Hitoshi; Fehon, Richard; Blair, Seth S

2016-01-01

Much of the Hippo and planar cell polarity (PCP) signaling mediated by the Drosophila protocadherin Fat depends on its ability to change the subcellular localization, levels and activity of the unconventional myosin Dachs. To better understand this process, we have performed a structure-function analysis of Dachs, and used this to identify a novel and important mediator of Fat and Dachs activities, a Dachs-binding SH3 protein we have named Dlish. We found that Dlish is regulated by Fat and Dachs, that Dlish also binds Fat and the Dachs regulator Approximated, and that Dlish is required for Dachs localization, levels and activity in both wild type and fat mutant tissue. Our evidence supports dual roles for Dlish. Dlish tethers Dachs to the subapical cell cortex, an effect partly mediated by the palmitoyltransferase Approximated under the control of Fat. Conversely, Dlish promotes the Fat-mediated degradation of Dachs. DOI: http://dx.doi.org/10.7554/eLife.16624.001 PMID:27692068

Cooperative interactions at the SLP-76 complex are critical for actin polymerization.

PubMed

Barda-Saad, Mira; Shirasu, Naoto; Pauker, Maor H; Hassan, Nirit; Perl, Orly; Balbo, Andrea; Yamaguchi, Hiroshi; Houtman, Jon C D; Appella, Ettore; Schuck, Peter; Samelson, Lawrence E

2010-07-21

T-cell antigen receptor (TCR) engagement induces formation of multi-protein signalling complexes essential for regulating T-cell functions. Generation of a complex of SLP-76, Nck and VAV1 is crucial for regulation of the actin machinery. We define the composition, stoichiometry and specificity of interactions in the SLP-76, Nck and VAV1 complex. Our data reveal that this complex can contain one SLP-76 molecule, two Nck and two VAV1 molecules. A direct interaction between Nck and VAV1 is mediated by binding between the C-terminal SH3 domain of Nck and the VAV1 N-terminal SH3 domain. Disruption of the VAV1:Nck interaction deleteriously affected actin polymerization. These novel findings shed new light on the mechanism of actin polymerization after T-cell activation.

SH3-like motif-containing C-terminal domain of staphylococcal teichoic acid transporter suggests possible function.

PubMed

Ko, Tzu-Ping; Tseng, Shih-Ting; Lai, Shu-Jung; Chen, Sheng-Chia; Guan, Hong-Hsiang; Shin Yang, Chia; Jung Chen, Chun; Chen, Yeh

2016-09-01

The negatively charged bacterial polysaccharides-wall teichoic acids (WTAs) are synthesized intracellularly and exported by a two-component transporter, TagGH, comprising a transmembrane subunit TagG and an ATPase subunit TagH. We determined the crystal structure of the C-terminal domain of TagH (TagH-C) to investigate its function. The structure shows an N-terminal SH3-like subdomain wrapped by a C-terminal subdomain with an anti-parallel β-sheet and an outer shell of α-helices. A stretch of positively charged surface across the subdomain interface is flanked by two negatively charged regions, suggesting a potential binding site for negatively charged polymers, such as WTAs or acidic peptide chains. Proteins 2016; 84:1328-1332. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

BIN1/M-Amphiphysin2 induces clustering of phosphoinositides to recruit its downstream partner dynamin

NASA Astrophysics Data System (ADS)

Picas, Laura; Viaud, Julien; Schauer, Kristine; Vanni, Stefano; Hnia, Karim; Fraisier, Vincent; Roux, Aurélien; Bassereau, Patricia; Gaits-Iacovoni, Frédérique; Payrastre, Bernard; Laporte, Jocelyn; Manneville, Jean-Baptiste; Goud, Bruno

2014-12-01

Phosphoinositides play a central role in many physiological processes by assisting the recruitment of proteins to membranes through specific phosphoinositide-binding motifs. How this recruitment is coordinated in space and time is not well understood. Here we show that BIN1/M-Amphiphysin2, a protein involved in T-tubule biogenesis in muscle cells and frequently mutated in centronuclear myopathies, clusters PtdIns(4,5)P2 to recruit its downstream partner dynamin. By using several mutants associated with centronuclear myopathies, we find that the N-BAR and the SH3 domains of BIN1 control the kinetics and the accumulation of dynamin on membranes, respectively. We show that phosphoinositide clustering is a mechanism shared by other proteins that interact with PtdIns(4,5)P2, but do not contain a BAR domain. Our numerical simulations point out that clustering is a diffusion-driven process in which phosphoinositide molecules are not sequestered. We propose that this mechanism plays a key role in the recruitment of downstream phosphoinositide-binding proteins.

Structural basis for interdomain communication in SHIP2 providing high phosphatase activity.

PubMed

Le Coq, Johanne; Camacho-Artacho, Marta; Velázquez, José Vicente; Santiveri, Clara M; Gallego, Luis Heredia; Campos-Olivas, Ramón; Dölker, Nicole; Lietha, Daniel

2017-08-09

SH2-containing-inositol-5-phosphatases (SHIPs) dephosphorylate the 5-phosphate of phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P 3 ) and play important roles in regulating the PI3K/Akt pathway in physiology and disease. Aiming to uncover interdomain regulatory mechanisms in SHIP2, we determined crystal structures containing the 5-phosphatase and a proximal region adopting a C2 fold. This reveals an extensive interface between the two domains, which results in significant structural changes in the phosphatase domain. Both the phosphatase and C2 domains bind phosphatidylserine lipids, which likely helps to position the active site towards its substrate. Although located distant to the active site, the C2 domain greatly enhances catalytic turnover. Employing molecular dynamics, mutagenesis and cell biology, we identify two distinct allosteric signaling pathways, emanating from hydrophobic or polar interdomain interactions, differentially affecting lipid chain or headgroup moieties of PI(3,4,5)P 3 . Together, this study reveals details of multilayered C2-mediated effects important for SHIP2 activity and points towards interesting new possibilities for therapeutic interventions.

Artificial proteins as allosteric modulators of PDZ3 and SH3 in two-domain constructs: A computational characterization of novel chimeric proteins.

PubMed

Kirubakaran, Palani; Pfeiferová, Lucie; Boušová, Kristýna; Bednarova, Lucie; Obšilová, Veronika; Vondrášek, Jiří

2016-10-01

Artificial multidomain proteins with enhanced structural and functional properties can be utilized in a broad spectrum of applications. The design of chimeric fusion proteins utilizing protein domains or one-domain miniproteins as building blocks is an important advancement for the creation of new biomolecules for biotechnology and medical applications. However, computational studies to describe in detail the dynamics and geometry properties of two-domain constructs made from structurally and functionally different proteins are lacking. Here, we tested an in silico design strategy using all-atom explicit solvent molecular dynamics simulations. The well-characterized PDZ3 and SH3 domains of human zonula occludens (ZO-1) (3TSZ), along with 5 artificial domains and 2 types of molecular linkers, were selected to construct chimeric two-domain molecules. The influence of the artificial domains on the structure and dynamics of the PDZ3 and SH3 domains was determined using a range of analyses. We conclude that the artificial domains can function as allosteric modulators of the PDZ3 and SH3 domains. Proteins 2016; 84:1358-1374. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

SH2 Domain-Based FRET Biosensor for Measuring BCR-ABL Activity in Living CML Cells.

PubMed

Fujioka, Mari; Asano, Yumi; Nakada, Shigeyuki; Ohba, Yusuke

2017-01-01

Fluorescent proteins (FPs) displaying distinct spectra have shed their light on a wide range of biological functions. Moreover, sophisticated biosensors engineered to contain single or multiple FPs, including Förster resonance energy transfer (FRET)-based biosensors, spatiotemporally reveal the molecular mechanisms underlying a variety of pathophysiological processes. However, their usefulness for applied life sciences has yet to be fully explored. Recently, our research group has begun to expand the potential of FPs from basic biological research to the clinic. Here, we describe a method to evaluate the responsiveness of leukemia cells from patients to tyrosine kinase inhibitors using a biosensor based on FP technology and the principle of FRET. Upon phosphorylation of the tyrosine residue of the biosensor, binding of the SH2 domain to phosphotyrosine induces conformational change of the biosensor and brings the donor and acceptor FPs into close proximity. Therefore, kinase activity and response to kinase inhibitors can be monitored by an increase and a decrease in FRET efficiency, respectively. As in basic research, this biosensor resolves hitherto arduous tasks and may provide innovative technological advances in clinical laboratory examinations. State-of-the-art detection devices that enable such innovation are also introduced.

Role of IRS-2 in insulin and cytokine signalling.

PubMed

Sun, X J; Wang, L M; Zhang, Y; Yenush, L; Myers, M G; Glasheen, E; Lane, W S; Pierce, J H; White, M F

1995-09-14

The protein IRS-1 acts as an interface between signalling proteins with Src-homology-2 domains (SH2 proteins) and the receptors for insulin, IGF-1, growth hormone, several interleukins (IL-4, IL-9, IL-13) and other cytokines. It regulates gene expression and stimulates mitogenesis, and appears to mediate insulin/IGF-1-stimulated glucose transport. Thus, survival of the IRS-1-/- mouse with only mild resistance to insulin was surprising. This dilemma is provisionally resolved with our discovery of a second IRS-signalling protein. We purified and cloned a likely candidate called 4PS from myeloid progenitor cells and, because of its resemblance to IRS-1, we designate it IRS-2. Alignment of the sequences of IRS-2 and IRS-1 revealed a highly conserved amino terminus containing a pleckstrin-homology domain and a phosphotyrosine-binding domain, and a poorly conserved carboxy terminus containing several tyrosine phosphorylation motifs. IRS-2 is expressed in many cells, including tissues from IRS-1-/- mice, and may be essential for signalling by several receptor systems.

A protein-tyrosine phosphatase with sequence similarity to the SH2 domain of the protein-tyrosine kinases.

PubMed

Shen, S H; Bastien, L; Posner, B I; Chrétien, P

1991-08-22

The phosphorylation of proteins at tyrosine residues is critical in cellular signal transduction, neoplastic transformation and control of the mitotic cycle. These mechanisms are regulated by the activities of both protein-tyrosine kinases (PTKs) and protein-tyrosine phosphatases (PTPases). As in the PTKs, there are two classes of PTPases: membrane associated, receptor-like enzymes and soluble proteins. Here we report the isolation of a complementary DNA clone encoding a new form of soluble PTPase, PTP1C. The enzyme possesses a large noncatalytic region at the N terminus which unexpectedly contains two adjacent copies of the Src homology region 2 (the SH2 domain) found in various nonreceptor PTKs and other cytoplasmic signalling proteins. As with other SH2 sequences, the SH2 domains of PTP1C formed high-affinity complexes with the activated epidermal growth factor receptor and other phosphotyrosine-containing proteins. These results suggest that the SH2 regions in PTP1C may interact with other cellular components to modulate its own phosphatase activity against interacting substrates. PTPase activity may thus directly link growth factor receptors and other signalling proteins through protein-tyrosine phosphorylation.

The C-terminus SH3-binding domain of Kv1.3 is required for the actin-mediated immobilization of the channel via cortactin

PubMed Central

Hajdu, Peter; Martin, Geoffrey V.; Chimote, Ameet A.; Szilagyi, Orsolya; Takimoto, Koichi; Conforti, Laura

2015-01-01

Kv1.3 channels play a pivotal role in the activation and migration of T-lymphocytes. These functions are accompanied by the channels' polarization, which is essential for associated downstream events. However, the mechanisms that govern the membrane movement of Kv1.3 channels remain unclear. F-actin polymerization occurs concomitantly to channel polarization, implicating the actin cytoskeleton in this process. Here we show that cortactin, a factor initiating the actin network, controls the membrane mobilization of Kv1.3 channels. FRAP with EGFP-tagged Kv1.3 channels demonstrates that knocking down cortactin decreases the actin-based immobilization of the channels. Using various deletion and mutation constructs, we show that the SH3 motif of Kv1.3 mediates the channel immobilization. Proximity ligation assays indicate that deletion or mutation of the SH3 motif also disrupts interaction of the channel with cortactin. In T-lymphocytes, the interaction between HS1 (the cortactin homologue) and Kv1.3 occurs at the immune synapse and requires the channel's C-terminal domain. These results show that actin dynamics regulates the membrane motility of Kv1.3 channels. They also provide evidence that the SH3 motif of the channel and cortactin plays key roles in this process. PMID:25739456

Potent and Selective Phosphopeptide Mimetic Prodrugs Targeted to the Src Homology 2 (SH2) Domain of Signal Transducer and Activator of Transcription 3

PubMed Central

Mandal, Pijus K.; Gao, Fengqin; Lu, Zhen; Ren, Zhiyong; Ramesh, Rajagopal; Birtwistle, J. Sanderson; Kaluarachchi, Kumaralal K.; Chen, Xiaomin; Bast, Robert C.; Liao, Warren S.; McMurray, John S.

2011-01-01

Signal transducer and activator of transcription 3 (Stat3), a target for anticancer drug design, is activated by recruitment to phosphotyrosine residues on growth factor and cytokine receptors via its SH2 domain. We report here structure-activity relationship studies on phosphopeptide mimics targeted to the SH2 domain of Stat3. Inclusion of a methyl group on the β-position of the pTyr mimic, 4-phosphocinfnamide, enhanced affinity 2–3 fold. Bis-pivaloyloxymethyl prodrugs containing β-methyl cinnamide, dipeptide scaffolds Haic and Nle-cis-3,4-methanoproline, and glutamine surrogates were highly potent, completely inhibiting phosphorylation of Stat3 Tyr705 at 0.5–1 µM in a variety of cancer cell lines. The inhibitors were selective for Stat3 over Stat1, Stat5, Src, and p85 of PI3K, indicating ability to discriminate individual SH2 domains in intact cells. At concentrations that completely inhibited Stat3 phosphorylation, the prodrugs were not cytotoxic to a panel of tumor cells, thereby showing clear distinction between cytotoxicity and effects downstream of activated Stat3. PMID:21486047

Mechanisms of inhibition of zinc-finger transcription factors by selenium compounds ebselen and selenite.

PubMed

Larabee, Jason L; Hocker, James R; Hanas, Jay S

2009-03-01

The anti-inflammatory selenium compounds, ebselen (2-phenyl-1,2-benzisoselenazol-3[2H]-one) and selenite, were found to alter the DNA binding mechanisms and structures of cysteine-rich zinc-finger transcription factors. As assayed by DNase I protection, DNA binding by TFIIIA (transcription factor IIIA, prototypical Cys(2)His(2) zinc finger protein), was inhibited by micromolar amounts of ebselen. In a gel shift assay, ebselen inhibited the Cys(2)His(2) zinc finger-containing DNA binding domain (DBD) of the NF-kappaB mediated transcription factor Sp1. Ebselen also inhibited DNA binding by the p50 subunit of the pro-inflammatory Cys-containing NF-kappaB transcription factor. Electrospray ionization mass spectrometry (ESI-MS) was utilized to elucidate mechanisms of chemical interaction between ebselen and a zinc-bound Cys(2)His(2) zinc finger polypeptide modeled after the third finger of Sp1 (Sp1-3). Exposing Sp1-3 to micromolar amounts of ebselen resulted in Zn(2+) release from this peptide and the formation of a disulfide bond by oxidation of zinc finger SH groups, the likely mechanism for DNA binding inhibition. Selenite was shown by ESI-MS to also eject zinc from Sp1-3 as well as induce disulfide bond formation through SH oxidation. The selenite-dependent inhibition/oxidation mechanism differed from that of ebselen by inducing the formation of a stable selenotrisulfide bond. Selenite-induced selenotrisulfide formation was dependent upon the structure of the Cys(2)His(2) zinc finger as alteration in the finger structure enhanced this reaction as well as selenite-dependent zinc release. Ebselen and selenite-dependent inhibition/oxidation of Cys-rich zinc finger proteins, with concomitant release of zinc and finger structural changes, points to mechanisms at the atomic and protein level for selenium-induced alterations in Cys-rich proteins, and possible amelioration of certain inflammatory, neurodegenerative, and oncogenic responses.

Role of Electrostatic Interactions in Binding of Peptides and Intrinsically Disordered Proteins to Their Folded Targets: 2. The Model of Encounter Complex Involving the Double Mutant of the c-Crk N-SH3 Domain and Peptide Sos.

PubMed

Yuwen, Tairan; Xue, Yi; Skrynnikov, Nikolai R

2016-03-29

In the first part of this work (paper 1, Xue, Y. et al. Biochemistry 2014 , 53 , 6473 ), we have studied the complex between the 10-residue peptide Sos and N-terminal SH3 domain from adaptor protein c-Crk. In the second part (this paper), we designed the double mutant of the c-Crk N-SH3 domain, W169F/Y186L, with the intention to eliminate the interactions responsible for tight peptide-protein binding, while retaining the interactions that create the initial electrostatic encounter complex. The resulting system was characterized experimentally by measuring the backbone and side-chain (15)N relaxation rates, as well as binding shifts and (1)H(N) temperature coefficients. In addition, it was also modeled via a series of ∼5 μs molecular dynamics (MD) simulations recorded in a large water box under an Amber ff99SB*-ILDN force field. Similar to paper 1, we have found that the strength of arginine-aspartate and arginine-glutamate salt bridges is overestimated in the original force field. To address this problem we have applied the empirical force-field correction described in paper 1. Specifically, the Lennard-Jones equilibrium distance for the nitrogen-oxygen pair across Arg-to-Asp/Glu salt bridges has been increased by 3%. This modification led to MD models in good agreement with the experimental data. The emerging picture is that of a fuzzy complex, where the peptide "dances" over the surface of the protein, making transient contacts via salt-bridge interactions. Every once in a while the peptide assumes a certain more stable binding pose, assisted by a number of adventitious polar and nonpolar contacts. On the other hand, occasionally Sos flies off the protein surface; it is then guided by electrostatic steering to quickly reconnect with the protein. The dynamic interaction between Sos and the double mutant of c-Crk N-SH3 gives rise to only small binding shifts. The peptide retains a high degree of conformational mobility, although it is appreciably slowed down due to its (loose) association with the protein. Note that spin relaxation data are indispensable in determining the dynamic status of the peptide. Such data can be properly modeled only on a basis of bona fide MD simulations, as shown in our study. We anticipate that in future the field will move away from the ensemble view of protein disorder and toward more sophisticated MD models. This will require further optimization of force fields, aimed specifically at disordered systems. Efforts in this direction have been recently initiated by several research groups; the empirical salt-bridge correction proposed in our work falls in the same category. MD models obtained with the help of suitably refined force fields and guided by experimental NMR data will provide a powerful insight into an intricate world of disordered biomolecules.

MUSI: an integrated system for identifying multiple specificity from very large peptide or nucleic acid data sets.

PubMed

Kim, Taehyung; Tyndel, Marc S; Huang, Haiming; Sidhu, Sachdev S; Bader, Gary D; Gfeller, David; Kim, Philip M

2012-03-01

Peptide recognition domains and transcription factors play crucial roles in cellular signaling. They bind linear stretches of amino acids or nucleotides, respectively, with high specificity. Experimental techniques that assess the binding specificity of these domains, such as microarrays or phage display, can retrieve thousands of distinct ligands, providing detailed insight into binding specificity. In particular, the advent of next-generation sequencing has recently increased the throughput of such methods by several orders of magnitude. These advances have helped reveal the presence of distinct binding specificity classes that co-exist within a set of ligands interacting with the same target. Here, we introduce a software system called MUSI that can rapidly analyze very large data sets of binding sequences to determine the relevant binding specificity patterns. Our pipeline provides two major advances. First, it can detect previously unrecognized multiple specificity patterns in any data set. Second, it offers integrated processing of very large data sets from next-generation sequencing machines. The results are visualized as multiple sequence logos describing the different binding preferences of the protein under investigation. We demonstrate the performance of MUSI by analyzing recent phage display data for human SH3 domains as well as microarray data for mouse transcription factors.

Regulation of the Src Kinase-associated Phosphoprotein 55 Homologue by the Protein Tyrosine Phosphatase PTP-PEST in the Control of Cell Motility*

PubMed Central

Ayoub, Emily; Hall, Anita; Scott, Adam M.; Chagnon, Mélanie J.; Miquel, Géraldine; Hallé, Maxime; Noda, Masaharu; Bikfalvi, Andreas; Tremblay, Michel L.

2013-01-01

PTP-PEST is a cytosolic ubiquitous protein tyrosine phosphatase (PTP) that contains, in addition to its catalytic domain, several protein-protein interaction domains that allow it to interface with several signaling pathways. Among others, PTP-PEST is a key regulator of cellular motility and cytoskeleton dynamics. The complexity of the PTP-PEST interactome underscores the necessity to identify its interacting partners and physiological substrates in order to further understand its role in focal adhesion complex turnover and actin organization. Using a modified yeast substrate trapping two-hybrid system, we identified a cytosolic adaptor protein named Src kinase-associated phosphoprotein 55 homologue (SKAP-Hom) as a novel substrate of PTP-PEST. To confirm PTP-PEST interaction with SKAP-Hom, in vitro pull down assays were performed demonstrating that the PTP catalytic domain and Proline-rich 1 (P1) domain are respectively binding to the SKAP-Hom Y260 and Y297 residues and its SH3 domain. Subsequently, we generated and rescued SKAP-Hom-deficient mouse embryonic fibroblasts (MEFs) with WT SKAP-Hom, SKAP-Hom tyrosine mutants (Y260F, Y260F/Y297F), or SKAP-Hom SH3 domain mutant (W335K). Given the role of PTP-PEST, wound-healing and trans-well migration assays were performed using the generated lines. Indeed, SKAP-Hom-deficient MEFs showed a defect in migration compared with WT-rescued MEFs. Interestingly, the SH3 domain mutant-rescued MEFs showed an enhanced cell migration corresponding potentially with higher tyrosine phosphorylation levels of SKAP-Hom. These findings suggest a novel role of SKAP-Hom and its phosphorylation in the regulation of cellular motility. Moreover, these results open new avenues by which PTP-PEST regulates cellular migration, a hallmark of metastasis. PMID:23897807

Docking and molecular dynamics simulations of the Fyn-SH3 domain with free and phospholipid bilayer-associated 18.5-kDa myelin basic protein (MBP)-Insights into a noncanonical and fuzzy interaction.

PubMed

Bessonov, Kyrylo; Vassall, Kenrick A; Harauz, George

2017-07-01

The molecular details of the association between the human Fyn-SH3 domain, and the fragment of 18.5-kDa myelin basic protein (MBP) spanning residues S38-S107 (denoted as xα2-peptide, murine sequence numbering), were studied in silico via docking and molecular dynamics over 50-ns trajectories. The results show that interaction between the two proteins is energetically favorable and heavily dependent on the MBP proline-rich region (P93-P98) in both aqueous and membrane environments. In aqueous conditions, the xα2-peptide/Fyn-SH3 complex adopts a "sandwich"-like structure. In the membrane context, the xα2-peptide interacts with the Fyn-SH3 domain via the proline-rich region and the β-sheets of Fyn-SH3, with the latter wrapping around the proline-rich region in a form of a clip. Moreover, the simulations corroborate prior experimental evidence of the importance of upstream segments beyond the canonical SH3-ligand. This study thus provides a more-detailed glimpse into the context-dependent interaction dynamics and importance of the β-sheets in Fyn-SH3 and proline-rich region of MBP. Proteins 2017; 85:1336-1350. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

The Vps27/Hrs/STAM (VHS) Domain of the Signal-transducing Adaptor Molecule (STAM) Directs Associated Molecule with the SH3 Domain of STAM (AMSH) Specificity to Longer Ubiquitin Chains and Dictates the Position of Cleavage*

PubMed Central

Baiady, Nardeen; Padala, Prasanth; Mashahreh, Bayan; Cohen-Kfir, Einav; Todd, Emily A.; Du Pont, Kelly E.; Berndsen, Christopher E.; Wiener, Reuven

2016-01-01

The deubiquitinating enzyme associated molecule with the SH3 domain of STAM (AMSH) is crucial for the removal of ubiquitin molecules during receptor-mediated endocytosis and lysosomal receptor sorting. AMSH interacts with signal transducing adapter molecule (STAM) 1 or 2, which enhances the activity of AMSH through an unknown mechanism. This stimulation is dependent on the ubiquitin-interacting motif of STAM. Here we investigate the specific mechanism of AMSH stimulation by STAM proteins and the role of the STAM Vps27/Hrs/STAM domain. We show that, in the presence of STAM, the length of the ubiquitin chains affects the apparent cleavage rate. Through measurement of the chain cleavage kinetics, we found that, although the kcat of Lys63-linked ubiquitin chain cleavage was comparable for di- and tri-ubiquitin, the Km value was lower for tri-ubiquitin. This increased affinity for longer chains was dependent on the Vps27/Hrs/STAM domain of STAM and required that the substrate ubiquitin chain contain homogenous Lys63-linkages. In addition, STAM directed AMSH cleavage toward the distal isopeptide bond in tri-ubiquitin chains. Finally, we generated a structural model of AMSH-STAM to show how the complex binds Lys63-linked ubiquitin chains and cleaves at the distal end. These data show how a deubiquitinating enzyme-interacting protein dictates the efficiency and specificity of substrate cleavage. PMID:26601948

Structural basis for activation of trimeric Gi proteins by multiple growth factor receptors via GIV/Girdin

PubMed Central

Lin, Changsheng; Ear, Jason; Midde, Krishna; Lopez-Sanchez, Inmaculada; Aznar, Nicolas; Garcia-Marcos, Mikel; Kufareva, Irina; Abagyan, Ruben; Ghosh, Pradipta

2014-01-01

A long-standing issue in the field of signal transduction is to understand the cross-talk between receptor tyrosine kinases (RTKs) and heterotrimeric G proteins, two major and distinct signaling hubs that control eukaryotic cell behavior. Although stimulation of many RTKs leads to activation of trimeric G proteins, the molecular mechanisms behind this phenomenon remain elusive. We discovered a unifying mechanism that allows GIV/Girdin, a bona fide metastasis-related protein and a guanine-nucleotide exchange factor (GEF) for Gαi, to serve as a direct platform for multiple RTKs to activate Gαi proteins. Using a combination of homology modeling, protein–protein interaction, and kinase assays, we demonstrate that a stretch of ∼110 amino acids within GIV C-terminus displays structural plasticity that allows folding into a SH2-like domain in the presence of phosphotyrosine ligands. Using protein–protein interaction assays, we demonstrated that both SH2 and GEF domains of GIV are required for the formation of a ligand-activated ternary complex between GIV, Gαi, and growth factor receptors and for activation of Gαi after growth factor stimulation. Expression of a SH2-deficient GIV mutant (Arg 1745→Leu) that cannot bind RTKs impaired all previously demonstrated functions of GIV—Akt enhancement, actin remodeling, and cell migration. The mechanistic and structural insights gained here shed light on the long-standing questions surrounding RTK/G protein cross-talk, set a novel paradigm, and characterize a unique pharmacological target for uncoupling GIV-dependent signaling downstream of multiple oncogenic RTKs. PMID:25187647

High-resolution frequency-domain second-harmonic optical coherence tomography

NASA Astrophysics Data System (ADS)

Su, Jianping; Tomov, Ivan V.; Jiang, Yi; Chen, Zhongping

2007-04-01

We used continuum generated in an 8.5 cm long fiber by a femtosecond Yb fiber laser to improve threefold the axial resolution of frequency domain second-harmonic optical coherence tomography (SH-OCT) to 12 μm. The acquisition time was shortened by more than 2 orders of magnitude compared to the time-domain SH-OCT. The system was applied to image biological tissue of fish scales, pig leg tendon, and rabbit eye sclera. Highly organized collagen fibrils can be visualized in the recorded images. Polarization dependence on the SH has been used to obtain polarization resolved images.

The SH2B1 obesity locus is associated with myocardial infarction in diabetic patients and with NO synthase activity in endothelial cells.

PubMed

Prudente, Sabrina; Morini, Eleonora; Larmon, Jay; Andreozzi, Francesco; Di Pietro, Natalia; Nigro, Angela; Gervino, Ernest V; Mannino, Gaia Chiara; Bacci, Simonetta; Hauser, Thomas H; Bellacchio, Emanuele; Formoso, Gloria; Pellegrini, Fabio; Proto, Vittoria; Menzaghi, Claudia; Frittitta, Lucia; Pandolfi, Assunta; Sesti, Giorgio; Doria, Alessandro; Trischitta, Vincenzo

2011-12-01

Obesity and cardiovascular disease recognize a common metabolic soil and may therefore share part of their genetic background. Genome-wide association studies have identified variability at the SH2B1 locus as a predictor of obesity. We investigated whether SNP rs4788102, which captures the entire SH2B1 variability, is associated with coronary artery disease (CAD) and/or myocardial infarction (MI) in patients with type 2 diabetes mellitus (T2DM). SNP rs4788102 was typed in 2015 White subjects with T2DM from three CAD case-control studies [n=740 from the Gargano Hearth Study (GHS, Italy); n=818 from the Joslin Hearth Study (JHS, Boston); n=457 from the University of Catanzaro (CZ, Italy)]. SNP rs4788102 (G/A) was not associated with CAD (overall allelic OR=1.06, 95% CI=0.93-1.21; p=0.37). On the contrary, it was associated with MI in GHS (1.42, 1.12-1.81; p=0.004) and in the three samples analyzed together (1.21, 1.04-1.41; p=0.016). Insulin stimulated nitric oxide synthase (NOS) activity in human vein endothelial cells from G/G (n=4, p=0.03) but not the G/A (n=5, p=0.83) genotype. Of the SNPs in perfect LD with rs4788102, one (rs7498665) affects amino acid polarity (Ala484Thr) and falls into a highly conserved protein segment of SH2B1 containing a class II SH3 domain binding site. Variability at the SH2B1 obesity locus is associated with MI in diabetic patients and with reduced insulin-stimulated NOS activity in human endothelial cells. Further studies are needed to replicate this association and dissect the biology underlying this finding. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

The murine Nck SH2/SH3 adaptors are important for the development of mesoderm-derived embryonic structures and for regulating the cellular actin network.

PubMed

Bladt, Friedhelm; Aippersbach, Elke; Gelkop, Sigal; Strasser, Geraldine A; Nash, Piers; Tafuri, Anna; Gertler, Frank B; Pawson, Tony

2003-07-01

Mammalian Nck1 and Nck2 are closely related adaptor proteins that possess three SH3 domains, followed by an SH2 domain, and are implicated in coupling phosphotyrosine signals to polypeptides that regulate the actin cytoskeleton. However, the in vivo functions of Nck1 and Nck2 have not been defined. We have mutated the murine Nck1 and Nck2 genes and incorporated beta-galactosidase reporters into the mutant loci. In mouse embryos, the two Nck genes have broad and overlapping expression patterns. They are functionally redundant in the sense that mice deficient for either Nck1 or Nck2 are viable, whereas inactivation of both Nck1 and Nck2 results in profound defects in mesoderm-derived notochord and embryonic lethality at embryonic day 9.5. Fibroblast cell lines derived from Nck1(-/-) Nck2(-/-) embryos have defects in cell motility and in the organization of the lamellipodial actin network. These data suggest that the Nck SH2/SH3 adaptors have important functions in the development of mesodermal structures during embryogenesis, potentially linked to a role in cell movement and cytoskeletal organization.

The Carboxyl Terminus of v-Abl Protein Can Augment SH2 Domain Function

PubMed Central

Warren, David; Heilpern, Andrew J.; Berg, Kent; Rosenberg, Naomi

2000-01-01

Abelson murine leukemia virus (Ab-MLV) transforms NIH 3T3 and pre-B cells via expression of the v-Abl tyrosine kinase. Although the enzymatic activity of this molecule is absolutely required for transformation, other regions of the protein are also important for this response. Among these are the SH2 domain, involved in phosphotyrosine-dependent protein-protein interactions, and the long carboxyl terminus, which plays an important role in transformation of hematopoietic cells. Important signals are sent from each of these regions, and transformation is most likely orchestrated by the concerted action of these different parts of the protein. To explore this idea, we compared the ability of the v-Src SH2 domain to substitute for that of v-Abl in the full-length P120 v-Abl protein and in P70 v-Abl, a protein that lacks the carboxyl terminus characteristic of Abl family members. Ab-MLV strains expressing P70/S2 failed to transform NIH 3T3 cells and demonstrated a greatly reduced capacity to mediate signaling events associated with the Ras-dependent mitogen-activated protein (MAP) kinase pathway. In contrast, Ab-MLV strains expressing P120/S2 were indistinguishable from P120 with respect to these features. Analyses of additional mutants demonstrated that the last 162 amino acids of the carboxyl terminus were sufficient to restore transformation. These data demonstrate that an SH2 domain with v-Abl substrate specificity is required for NIH 3T3 transformation in the absence of the carboxyl terminus and suggest that cooperativity between the extreme carboxyl terminus and the SH2 domain facilitates the transmission of transforming signals via the MAP kinase pathway. PMID:10775585

Structural and sequencing analysis of local target DNA recognition by MLV integrase.

PubMed

Aiyer, Sriram; Rossi, Paolo; Malani, Nirav; Schneider, William M; Chandar, Ashwin; Bushman, Frederic D; Montelione, Gaetano T; Roth, Monica J

2015-06-23

Target-site selection by retroviral integrase (IN) proteins profoundly affects viral pathogenesis. We describe the solution nuclear magnetic resonance structure of the Moloney murine leukemia virus IN (M-MLV) C-terminal domain (CTD) and a structural homology model of the catalytic core domain (CCD). In solution, the isolated MLV IN CTD adopts an SH3 domain fold flanked by a C-terminal unstructured tail. We generated a concordant MLV IN CCD structural model using SWISS-MODEL, MMM-tree and I-TASSER. Using the X-ray crystal structure of the prototype foamy virus IN target capture complex together with our MLV domain structures, residues within the CCD α2 helical region and the CTD β1-β2 loop were predicted to bind target DNA. The role of these residues was analyzed in vivo through point mutants and motif interchanges. Viable viruses with substitutions at the IN CCD α2 helical region and the CTD β1-β2 loop were tested for effects on integration target site selection. Next-generation sequencing and analysis of integration target sequences indicate that the CCD α2 helical region, in particular P187, interacts with the sequences distal to the scissile bonds whereas the CTD β1-β2 loop binds to residues proximal to it. These findings validate our structural model and disclose IN-DNA interactions relevant to target site selection. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

The p85α regulatory subunit of PI3K mediates cAMP-PKA and retinoic acid biological effects on MCF7 cell growth and migration.

PubMed

Donini, Caterina F; Di Zazzo, Erika; Zuchegna, Candida; Di Domenico, Marina; D'Inzeo, Sonia; Nicolussi, Arianna; Avvedimento, Enrico V; Coppa, Anna; Porcellini, Antonio

2012-05-01

Phosphoinositide-3-OH kinase (PI3K) signalling regulates various cellular processes, including cell survival, growth, proliferation and motility, and is among the most frequently mutated pathways in cancer. Although the involvement of p85αPI3K SH2 domain in signal transduction has been extensively studied, the function of the SH3 domain at the N-terminus remains elusive. A serine (at codon 83) adjacent to the N-terminal SH3 domain in the PI3K regulatory subunit p85αPI3K that is phosphorylated by protein kinase A (PKA) in vivo and in vitro has been identified. Virtually all receptors binding p85αPI3K can cooperate with cAMP-PKA signals via phosphorylation of p85αPI3KSer83. To analyse the role of p85αPI3KSer83 in retinoic acid (RA) and cAMP signalling, in MCF7 cells, we used p85αPI3K mutated forms, in which Ser83 has been substituted with alanine (p85A) to prevent phosphorylation or with aspartic acid (p85D) to mimic the phosphorylated residue. We demonstrated that p85αPI3KSer83 is crucial for the synergistic enhancement of RARα/p85αPI3K binding induced by cAMP/RA co-treatment in MCF7 cells. Growth curves, colorimetric MTT assay and cell cycle analysis demonstrated that phosphorylation of p85αPI3KSer83 plays an important role in the control of MCF7 cell proliferation and in RA-induced inhibition of proliferation. Wound healing and transwell experiments demonstrated that p85αPI3KSer83 was also essential both for the control of migratory behaviour and for the reduction of motility induced by RA. This study points to p85αPI3KSer83 as the physical link between different pathways (cAMP-PKA, RA and FAK), and as an important regulator of MCF7 cell proliferation and migration.

Protein Kinase A (PKA) Phosphorylation of Shp2 Protein Inhibits Its Phosphatase Activity and Modulates Ligand Specificity

PubMed Central

Burmeister, Brian T.; Wang, Li; Gold, Matthew G.; Skidgel, Randal A.; O'Bryan, John P.; Carnegie, Graeme K.

2015-01-01

Pathological cardiac hypertrophy (an increase in cardiac mass resulting from stress-induced cardiac myocyte growth) is a major factor underlying heart failure. Src homology 2 domain-containing phosphatase (Shp2) is critical for cardiac function because mutations resulting in loss of Shp2 catalytic activity are associated with congenital cardiac defects and hypertrophy. We identified a novel mechanism of Shp2 inhibition that may promote cardiac hypertrophy. We demonstrate that Shp2 is a component of the protein kinase A anchoring protein (AKAP)-Lbc complex. AKAP-Lbc facilitates PKA phosphorylation of Shp2, which inhibits Shp2 phosphatase activity. We identified two key amino acids in Shp2 that are phosphorylated by PKA. Thr-73 contributes a helix cap to helix αB within the N-terminal SH2 domain of Shp2, whereas Ser-189 occupies an equivalent position within the C-terminal SH2 domain. Utilizing double mutant PKA phosphodeficient (T73A/S189A) and phosphomimetic (T73D/S189D) constructs, in vitro binding assays, and phosphatase activity assays, we demonstrate that phosphorylation of these residues disrupts Shp2 interaction with tyrosine-phosphorylated ligands and inhibits its protein-tyrosine phosphatase activity. Overall, our data indicate that AKAP-Lbc integrates PKA and Shp2 signaling in the heart and that AKAP-Lbc-associated Shp2 activity is reduced in hypertrophic hearts in response to chronic β-adrenergic stimulation and PKA activation. Therefore, although induction of cardiac hypertrophy is a multifaceted process, inhibition of Shp2 activity through AKAP-Lbc-anchored PKA is a previously unrecognized mechanism that may promote this compensatory response. PMID:25802336

Protein Kinase A (PKA) Phosphorylation of Shp2 Protein Inhibits Its Phosphatase Activity and Modulates Ligand Specificity.

PubMed

Burmeister, Brian T; Wang, Li; Gold, Matthew G; Skidgel, Randal A; O'Bryan, John P; Carnegie, Graeme K

2015-05-08

Pathological cardiac hypertrophy (an increase in cardiac mass resulting from stress-induced cardiac myocyte growth) is a major factor underlying heart failure. Src homology 2 domain-containing phosphatase (Shp2) is critical for cardiac function because mutations resulting in loss of Shp2 catalytic activity are associated with congenital cardiac defects and hypertrophy. We identified a novel mechanism of Shp2 inhibition that may promote cardiac hypertrophy. We demonstrate that Shp2 is a component of the protein kinase A anchoring protein (AKAP)-Lbc complex. AKAP-Lbc facilitates PKA phosphorylation of Shp2, which inhibits Shp2 phosphatase activity. We identified two key amino acids in Shp2 that are phosphorylated by PKA. Thr-73 contributes a helix cap to helix αB within the N-terminal SH2 domain of Shp2, whereas Ser-189 occupies an equivalent position within the C-terminal SH2 domain. Utilizing double mutant PKA phosphodeficient (T73A/S189A) and phosphomimetic (T73D/S189D) constructs, in vitro binding assays, and phosphatase activity assays, we demonstrate that phosphorylation of these residues disrupts Shp2 interaction with tyrosine-phosphorylated ligands and inhibits its protein-tyrosine phosphatase activity. Overall, our data indicate that AKAP-Lbc integrates PKA and Shp2 signaling in the heart and that AKAP-Lbc-associated Shp2 activity is reduced in hypertrophic hearts in response to chronic β-adrenergic stimulation and PKA activation. Therefore, although induction of cardiac hypertrophy is a multifaceted process, inhibition of Shp2 activity through AKAP-Lbc-anchored PKA is a previously unrecognized mechanism that may promote this compensatory response. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Slit stimulation recruits Dock and Pak to the roundabout receptor and increases Rac activity to regulate axon repulsion at the CNS midline.

PubMed

Fan, Xueping; Labrador, Juan Pablo; Hing, Huey; Bashaw, Greg J

2003-09-25

Drosophila Roundabout (Robo) is the founding member of a conserved family of repulsive axon guidance receptors that respond to secreted Slit proteins. Here we present evidence that the SH3-SH2 adaptor protein Dreadlocks (Dock), the p21-activated serine-threonine kinase (Pak), and the Rac1/Rac2/Mtl small GTPases can function during Robo repulsion. Loss-of-function and genetic interaction experiments suggest that limiting the function of Dock, Pak, or Rac partially disrupts Robo repulsion. In addition, Dock can directly bind to Robo's cytoplasmic domain, and the association of Dock and Robo is enhanced by stimulation with Slit. Furthermore, Slit stimulation can recruit a complex of Dock and Pak to the Robo receptor and trigger an increase in Rac1 activity. These results provide a direct physical link between the Robo receptor and an important cytoskeletal regulatory protein complex and suggest that Rac can function in both attractive and repulsive axon guidance.

Cloning and characterization of a G protein-activated human phosphoinositide-3 kinase.

PubMed

Stoyanov, B; Volinia, S; Hanck, T; Rubio, I; Loubtchenkov, M; Malek, D; Stoyanova, S; Vanhaesebroeck, B; Dhand, R; Nürnberg, B

1995-08-04

Phosphoinositide-3 kinase activity is implicated in diverse cellular responses triggered by mammalian cell surface receptors and in the regulation of protein sorting in yeast. Receptors with intrinsic and associated tyrosine kinase activity recruit heterodimeric phosphoinositide-3 kinases that consist of p110 catalytic subunits and p85 adaptor molecules containing Src homology 2 (SH2) domains. A phosphoinositide-3 kinase isotype, p110 gamma, was cloned and characterized. The p110 gamma enzyme was activated in vitro by both the alpha and beta gamma subunits of heterotrimeric guanosine triphosphate (GTP)-binding proteins (G proteins) and did not interact with p85. A potential pleckstrin homology domain is located near its amino terminus. The p110 gamma isotype may link signaling through G protein-coupled receptors to the generation of phosphoinositide second messengers phosphorylated in the D-3 position.

Structural basis for interdomain communication in SHIP2 providing high phosphatase activity

PubMed Central

Le Coq, Johanne; Camacho-Artacho, Marta; Velázquez, José Vicente; Santiveri, Clara M; Gallego, Luis Heredia; Campos-Olivas, Ramón; Dölker, Nicole; Lietha, Daniel

2017-01-01

SH2-containing-inositol-5-phosphatases (SHIPs) dephosphorylate the 5-phosphate of phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3) and play important roles in regulating the PI3K/Akt pathway in physiology and disease. Aiming to uncover interdomain regulatory mechanisms in SHIP2, we determined crystal structures containing the 5-phosphatase and a proximal region adopting a C2 fold. This reveals an extensive interface between the two domains, which results in significant structural changes in the phosphatase domain. Both the phosphatase and C2 domains bind phosphatidylserine lipids, which likely helps to position the active site towards its substrate. Although located distant to the active site, the C2 domain greatly enhances catalytic turnover. Employing molecular dynamics, mutagenesis and cell biology, we identify two distinct allosteric signaling pathways, emanating from hydrophobic or polar interdomain interactions, differentially affecting lipid chain or headgroup moieties of PI(3,4,5)P3. Together, this study reveals details of multilayered C2-mediated effects important for SHIP2 activity and points towards interesting new possibilities for therapeutic interventions. DOI: http://dx.doi.org/10.7554/eLife.26640.001 PMID:28792888

Enigma interacts with adaptor protein with PH and SH2 domains to control insulin-induced actin cytoskeleton remodeling and glucose transporter 4 translocation.

PubMed

Barrès, Romain; Grémeaux, Thierry; Gual, Philippe; Gonzalez, Teresa; Gugenheim, Jean; Tran, Albert; Le Marchand-Brustel, Yannick; Tanti, Jean-François

2006-11-01

APS (adaptor protein with PH and SH2 domains) initiates a phosphatidylinositol 3-kinase-independent pathway involved in insulin-stimulated glucose transport. We recently identified Enigma, a PDZ and LIM domain-containing protein, as a partner of APS and showed that APS-Enigma complex plays a critical role in actin cytoskeleton organization in fibroblastic cells. Because actin rearrangement is important for insulin-induced glucose transporter 4 (Glut 4) translocation, we studied the potential involvement of Enigma in insulin-induced glucose transport in 3T3-L1 adipocytes. Enigma mRNA was expressed in differentiated adipocytes and APS and Enigma were colocalized with cortical actin. Expression of an APS mutant unable to bind Enigma increased the insulin-induced Glut 4 translocation to the plasma membrane. By contrast, overexpression of Enigma inhibited insulin-stimulated glucose transport and Glut 4 translocation without alterations in proximal insulin signaling. This inhibitory effect was prevented with the deletion of the LIM domains of Enigma. Using time-lapse fluorescent microscopy of green fluorescent protein-actin, we demonstrated that the overexpression of Enigma altered insulin-induced actin rearrangements, whereas the expression of Enigma without its LIM domains was without effect. A physiological link between increased expression of Enigma and an alteration in insulin-induced glucose uptake was suggested by the increase in Enigma mRNA expression in adipose tissue of diabetic obese patients. Taken together, these data strongly suggest that the interaction between APS and Enigma is involved in insulin-induced Glut 4 translocation by regulating cortical actin remodeling and raise the possibility that modification of APS/Enigma ratio could participate in the alteration of insulin-induced glucose uptake in adipose tissue.

Phosphorylated c-MPL tyrosine 591 regulates thrombopoietin-induced signaling

PubMed Central

Sangkhae, Veena; Saur, Sebastian Jonas; Kaushansky, Alexis; Kaushansky, Kenneth; Hitchcock, Ian Stuart

2018-01-01

Thrombopoietin (TPO) is the primary regulator of platelet production, affecting cell survival, proliferation and differentiation through binding to and stimulation of the cell surface receptor c-MPL. Activating mutations in c-MPL constitutively stimulate downstream signaling pathways, leading to aberrant hematopoiesis and contribute to development of myeloproliferative neoplasms. Several studies have mapped the tyrosine residues within the cytoplasmic domain of c-MPL that mediate these cellular signals; however, secondary signaling pathways are incompletely understood. In this study we focused on c-MPL tyrosine 591 (Y591). We found Y591 of wild-type c-MPL to be phosphorylated in the presence of TPO. Additionally, eliminating Y591 phosphorylation by mutation to Phe resulted in decreased total receptor phosphorylation. Using an SH2/PTB domain binding microarray, we identified novel c-MPL binding partners for phosphorylated Y591, including Src homology phosphatase-1 (SHP-1), spleen tyrosine kinase (SYK) and Bruton's tyrosine kinase (BTK). The functional significance of binding partners was determined through siRNA treatment of Ba/F3-MPL cells, confirming that the increase in pERK1/2 resulting from removal of Y591 may be mediated by SYK. These findings identify a novel negative regulatory pathway that controls TPO-mediated signaling, advancing our understanding of the mechanisms required for successful maintenance of hematopoietic stem cells and megakaryocyte development. PMID:24607955

Structure of the dimeric exonuclease TREX1 in complex with DNA displays a proline-rich binding site for WW Domains.

PubMed

Brucet, Marina; Querol-Audí, Jordi; Serra, Maria; Ramirez-Espain, Ximena; Bertlik, Kamila; Ruiz, Lidia; Lloberas, Jorge; Macias, Maria J; Fita, Ignacio; Celada, Antonio

2007-05-11

TREX1 is the most abundant mammalian 3' --> 5' DNA exonuclease. It has been described to form part of the SET complex and is responsible for the Aicardi-Goutières syndrome in humans. Here we show that the exonuclease activity is correlated to the binding preferences toward certain DNA sequences. In particular, we have found three motifs that are selected, GAG, ACA, and CTGC. To elucidate how the discrimination occurs, we determined the crystal structures of two murine TREX1 complexes, with a nucleotide product of the exonuclease reaction, and with a single-stranded DNA substrate. Using confocal microscopy, we observed TREX1 both in nuclear and cytoplasmic subcellular compartments. Remarkably, the presence of TREX1 in the nucleus requires the loss of a C-terminal segment, which we named leucine-rich repeat 3. Furthermore, we detected the presence of a conserved proline-rich region on the surface of TREX1. This observation points to interactions with proline-binding domains. The potential interacting motif "PPPVPRPP" does not contain aromatic residues and thus resembles other sequences that select SH3 and/or Group 2 WW domains. By means of nuclear magnetic resonance titration experiments, we show that, indeed, a polyproline peptide derived from the murine TREX1 sequence interacted with the WW2 domain of the elongation transcription factor CA150. Co-immunoprecipitation studies confirmed this interaction with the full-length TREX1 protein, thereby suggesting that TREX1 participates in more functional complexes than previously thought.

In vivo significance of ITK-SLP-76 interaction in cytokine production.

PubMed

Grasis, Juris A; Guimond, David M; Cam, Nicholas R; Herman, Krystal; Magotti, Paola; Lambris, John D; Tsoukas, Constantine D

2010-07-01

In vitro data have suggested that activation of the inducible T-cell kinase (ITK) requires an interaction with the adaptor protein SLP-76. One means for this interaction involves binding of the ITK SH3 domain to the polyproline-rich (PR) region of SLP-76. However, the biological significance of this association in live cells and the consequences of its disruption have not been demonstrated. Here, we utilized a polyarginine-rich, cell-permeable peptide that represents the portion of the SLP-76 PR region that interacts with the ITK SH3 domain as a competitive inhibitor to disrupt the association between ITK and SLP-76 in live cells. We demonstrate that treatment of cells with this peptide, by either in vitro incubation or intraperitoneal injection of the peptide in mice, inhibits the T-cell receptor (TCR)-induced association between ITK and SLP-76, recruitment and transphosphorylation of ITK, actin polarization at the T-cell contact site, and expression of Th2 cytokines. The inhibition is specific, as indicated by lack of effects by the polyarginine vehicle alone or a scrambled sequence of the cargo peptide. In view of the role of ITK as a regulator of Th2 cytokine expression, the data underscore the significance of ITK as a target for pharmacological intervention.

Role of Gab1 in Heart, Placenta, and Skin Development and Growth Factor- and Cytokine-Induced Extracellular Signal-Regulated Kinase Mitogen-Activated Protein Kinase Activation

PubMed Central

Itoh, Motoyuki; Yoshida, Yuichi; Nishida, Keigo; Narimatsu, Masahiro; Hibi, Masahiko; Hirano, Toshio

2000-01-01

Gab1 is a member of the Gab/DOS (Daughter of Sevenless) family of adapter molecules, which contain a pleckstrin homology (PH) domain and potential binding sites for SH2 and SH3 domains. Gab1 is tyrosine phosphorylated upon stimulation of various cytokines, growth factors, and antigen receptors in cell lines and interacts with signaling molecules, such as SHP-2 and phosphatidylinositol 3-kinase, although its biological roles have not yet been established. To reveal the functions of Gab1 in vivo, we generated mice lacking Gab1 by gene targeting. Gab1-deficient embryos died in utero and displayed developmental defects in the heart, placenta, and skin, which were similar to phenotypes observed in mice lacking signals of the hepatocyte growth factor/scatter factor, platelet-derived growth factor, and epidermal growth factor pathways. Consistent with these observations, extracellular signal-regulated kinase mitogen-activated protein (ERK MAP) kinases were activated at much lower levels in cells from Gab1-deficient embryos in response to these growth factors or to stimulation of the cytokine receptor gp130. These results indicate that Gab1 is a common player in a broad range of growth factor and cytokine signaling pathways linking ERK MAP kinase activation. PMID:10779359

Germline cytotoxic lymphocytes defective mutations in Chinese patients with lymphoma.

PubMed

Chen, Xue; Zhang, Yang; Wang, Fang; Wang, Mangju; Teng, Wen; Lin, Yuehui; Han, Xiangping; Jin, Fangyuan; Xu, Yuanli; Cao, Panxiang; Fang, Jiancheng; Zhu, Ping; Tong, Chunrong; Liu, Hongxing

2017-11-01

Certain patients with lymphoma may harbor mutations in perforin 1 (PRF1), unc-13 homolog D (UNC13D), syntaxin 11 (STX11), STXBP2 (syntaxin binding protein 2) or SH2 domain containing 1A (SH2D1A), which causes functional defects of cytotoxic lymphocytes. Data regarding the association between genetic defects and the development of lymphoma in Chinese patients are limited to date. In the present study, 90 patients with lymphoma were analyzed for UNC13D, PRF1, STXBP2, STX11, SH2D1A and X-linked inhibitor of apoptosis. Mutations were observed in 24 (26.67%) patients; 16 patients exhibited mutations in UNC13D, 7 exhibited PRF1 mutations, and 1 exhibited monoallelic mutation in STX11. UNC13D c.2588G>A/p.G863D mutation was detected in 9 patients (10.00%) and in 4/210 controls (1.90%). This mutation was predicted to be pathogenic and it predominantly existed in the Chinese population. These findings suggest that impaired cytotoxic machinery may represent a predisposing factor for the development of lymphoma. Furthermore, these data describe a distinct mutation spectrum in Chinese patients with lymphoma, whereby UNC13D is the most frequently mutated gene. In addition, these findings suggest UNC13D c.2588G>A mutation is a founder mutation in Chinese patients.

Germline cytotoxic lymphocytes defective mutations in Chinese patients with lymphoma

PubMed Central

Chen, Xue; Zhang, Yang; Wang, Fang; Wang, Mangju; Teng, Wen; Lin, Yuehui; Han, Xiangping; Jin, Fangyuan; Xu, Yuanli; Cao, Panxiang; Fang, Jiancheng; Zhu, Ping; Tong, Chunrong; Liu, Hongxing

2017-01-01

Certain patients with lymphoma may harbor mutations in perforin 1 (PRF1), unc-13 homolog D (UNC13D), syntaxin 11 (STX11), STXBP2 (syntaxin binding protein 2) or SH2 domain containing 1A (SH2D1A), which causes functional defects of cytotoxic lymphocytes. Data regarding the association between genetic defects and the development of lymphoma in Chinese patients are limited to date. In the present study, 90 patients with lymphoma were analyzed for UNC13D, PRF1, STXBP2, STX11, SH2D1A and X-linked inhibitor of apoptosis. Mutations were observed in 24 (26.67%) patients; 16 patients exhibited mutations in UNC13D, 7 exhibited PRF1 mutations, and 1 exhibited monoallelic mutation in STX11. UNC13D c.2588G>A/p.G863D mutation was detected in 9 patients (10.00%) and in 4/210 controls (1.90%). This mutation was predicted to be pathogenic and it predominantly existed in the Chinese population. These findings suggest that impaired cytotoxic machinery may represent a predisposing factor for the development of lymphoma. Furthermore, these data describe a distinct mutation spectrum in Chinese patients with lymphoma, whereby UNC13D is the most frequently mutated gene. In addition, these findings suggest UNC13D c.2588G>A mutation is a founder mutation in Chinese patients. PMID:29113160

The Murine Nck SH2/SH3 Adaptors Are Important for the Development of Mesoderm-Derived Embryonic Structures and for Regulating the Cellular Actin Network

PubMed Central

Bladt, Friedhelm; Aippersbach, Elke; Gelkop, Sigal; Strasser, Geraldine A.; Nash, Piers; Tafuri, Anna; Gertler, Frank B.; Pawson, Tony

2003-01-01

Mammalian Nck1 and Nck2 are closely related adaptor proteins that possess three SH3 domains, followed by an SH2 domain, and are implicated in coupling phosphotyrosine signals to polypeptides that regulate the actin cytoskeleton. However, the in vivo functions of Nck1 and Nck2 have not been defined. We have mutated the murine Nck1 and Nck2 genes and incorporated β-galactosidase reporters into the mutant loci. In mouse embryos, the two Nck genes have broad and overlapping expression patterns. They are functionally redundant in the sense that mice deficient for either Nck1 or Nck2 are viable, whereas inactivation of both Nck1 and Nck2 results in profound defects in mesoderm-derived notochord and embryonic lethality at embryonic day 9.5. Fibroblast cell lines derived from Nck1−/− Nck2−/− embryos have defects in cell motility and in the organization of the lamellipodial actin network. These data suggest that the Nck SH2/SH3 adaptors have important functions in the development of mesodermal structures during embryogenesis, potentially linked to a role in cell movement and cytoskeletal organization. PMID:12808099

Polymer brushes on nanoparticles: their positioning in and influence on block copolymer morphology.

NASA Astrophysics Data System (ADS)

Kim, Bumjoon

2007-03-01

Polymers brushes grafted to the nanoparticle surface enable the precise positioning of particles within a block copolymer matrix by determining the compatibility of nanoparticles within a polymeric matrix and modifying the interfacial properties between polymers and inorganic nanoparticle. Short thiol terminated polystyrene (PS-SH), poly(2-vinylpyridine) (P2VP-SH) and PS-r-P2VP with the molecular weight (Mn) of 3 kg/mol were used to control the location of Au nanoparticles over PS-b-P2VP diblock copolymer template. We will discuss further the approach of varying the areal chain density (σ) of PS-SH brushes on the PS coated particles, which utilizes the preferential wetting of one block of a copolymer (P2VP) on the Au substrate. Such favorable interaction provides the strong binding of Au particles to the PS/P2VP interface as σ of PS chains on the Au particle decreases. We find that at σ above a certain value, the nanoparticles are segregated to the center of the PS domains while below this value they are segregated to the interface. The transition σ for PS-SH chains (Mn = 3.4 kg/mol) is 1.3 chains/nm^2 but unexpectedly scales as Mn-0.55 as Mn is varied from 1.5 to 13 kg/mol. In addition, we will discuss changes in block copolymer morphology that occur as the nanoparticle volume fraction (φ) is increased for nanoparticles that segregate to the domain center as well as those that segregate to the interface, the latter behaving as nanoparticle surfactants. Small φ of such surfactants added to lamellar diblock copolymers lead initially to a decrease in lamellar thickness, a consequence of decreasing interfacial tension, up to a critical value of φ beyond which the block copolymer adopts a bicontinuous morphology. I thank my collaborators G. H. Fredrickson, J. Bang, C. J. Hawker, and E. J. Kramer as well as funding by the MRL as UCSB from the NSF-MRSEC-Program Award DMR05-20418.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pennington, M.W.

We have determined that ShN I, a 48-residue type 2 sea anemone toxin, delays the inactivation of the Na channel in lobster olfactory somas. The receptor for ShN I was identified in vesicle preparations of neuronal tissues from both crustaceans and mammals; however, the K{sub D} values for the former is more than 1,000 fold lower for the later. The binding of ({sup 125}I)-ShN I to this receptor was determined to be unaffected by Anemonia sulcata II, depolarization of the membrane, or veratridine. ShN I was unable to displace ({sup 125}I)-Androctonus austrialis Hector II, whereas unlabeled AaH II and Asmore » II displaced the labeled scorpion toxin from rat brain synaptosomes. This is the first characterization of a new Na channel receptor site which specifically binds type 2 anemone toxins. To study the interactions that specific amino acid residues of ShN I have with this receptor, we developed a strategy using solid phase peptide synthesis. Prior to the synthesis of analogs to ShN I, we assembled the native ShN I sequence and reoxidized the three intramolecular disulfide bonds. Chemical, physical, and pharmacological characterization of the purified synthetic ShN I showed it to be indistinguishable from the natural toxin.« less

Structural basis for the recognition of Asef by adenomatous polyposis coli

PubMed Central

Zhang, Zhenyi; Chen, Leyi; Gao, Lei; Lin, Kui; Zhu, Liang; Lu, Yang; Shi, Xiaoshan; Gao, Yuan; Zhou, Jing; Xu, Ping; Zhang, Jian; Wu, Geng

2012-01-01

Adenomatous polyposis coli (APC) regulates cell-cell adhesion and cell migration through activating the APC-stimulated guanine nucleotide-exchange factor (GEF; Asef), which is usually autoinhibited through the binding between its Src homology 3 (SH3) and Dbl homology (DH) domains. The APC-activated Asef stimulates the small GTPase Cdc42, which leads to decreased cell-cell adherence and enhanced cell migration. In colorectal cancers, truncated APC constitutively activates Asef and promotes cancer cell migration and angiogenesis. Here, we report crystal structures of the human APC/Asef complex. We find that the armadillo repeat domain of APC uses a highly conserved surface groove to recognize the APC-binding region (ABR) of Asef, conformation of which changes dramatically upon binding to APC. Key residues on APC and Asef for the complex formation were mutated and their importance was demonstrated by binding and activity assays. Structural superimposition of the APC/Asef complex with autoinhibited Asef suggests that the binding between APC and Asef might create a steric clash between Asef-DH domain and APC, which possibly leads to a conformational change in Asef that stimulates its GEF activity. Our structures thus elucidate the molecular mechanism of Asef recognition by APC, as well as provide a potential target for pharmaceutical intervention against cancers. PMID:21788986

Caenorhabditis elegans fibroblast growth factor receptor signaling can occur independently of the multi-substrate adaptor FRS2.

PubMed

Lo, Te-Wen; Bennett, Daniel C; Goodman, S Jay; Stern, Michael J

2010-06-01

The components of receptor tyrosine kinase signaling complexes help to define the specificity of the effects of their activation. The Caenorhabditis elegans fibroblast growth factor receptor (FGFR), EGL-15, regulates a number of processes, including sex myoblast (SM) migration guidance and fluid homeostasis, both of which require a Grb2/Sos/Ras cassette of signaling components. Here we show that SEM-5/Grb2 can bind directly to EGL-15 to mediate SM chemoattraction. A yeast two-hybrid screen identified SEM-5 as able to interact with the carboxy-terminal domain (CTD) of EGL-15, a domain that is specifically required for SM chemoattraction. This interaction requires the SEM-5 SH2-binding motifs present in the CTD (Y(1009) and Y(1087)), and these sites are required for the CTD role of EGL-15 in SM chemoattraction. SEM-5, but not the SEM-5 binding sites located in the CTD, is required for the fluid homeostasis function of EGL-15, indicating that SEM-5 can link to EGL-15 through an alternative mechanism. The multi-substrate adaptor protein FRS2 serves to link vertebrate FGFRs to Grb2. In C. elegans, an FRS2-like gene, rog-1, functions upstream of a Ras/MAPK pathway for oocyte maturation but is not required for EGL-15 function. Thus, unlike the vertebrate FGFRs, which require the multi-substrate adaptor FRS2 to recruit Grb2, EGL-15 can recruit SEM-5/Grb2 directly.

The JH2 domain and SH2-JH2 linker regulate JAK2 activity: A detailed kinetic analysis of wild type and V617F mutant kinase domains.

PubMed

Sanz Sanz, Arturo; Niranjan, Yashavanthi; Hammarén, Henrik; Ungureanu, Daniela; Ruijtenbeek, Rob; Touw, Ivo P; Silvennoinen, Olli; Hilhorst, Riet

2014-10-01

JAK2 tyrosine kinase regulates many cellular functions. Its activity is controlled by the pseudokinase (JH2) domain by still poorly understood mechanisms. The V617F mutation in the pseudokinase domain activates JAK2 and causes myeloproliferative neoplasms. We conducted a detailed kinetic analysis of recombinant JAK2 tyrosine kinase domain (JH1) and wild-type and V617F tandem kinase (JH1JH2) domains using peptide microarrays to define the functions of the kinase domains. The results show that i) JAK2 follows a random Bi-Bi reaction mechanism ii) JH2 domain restrains the activity of the JH1 domain by reducing the affinity for ATP and ATP competitive inhibitors iii) V617F decreases affinity for ATP but increases catalytic activity compared to wild-type and iv) the SH2-JH2 linker region participates in controlling activity by reducing the affinity for ATP. Copyright © 2014 Elsevier B.V. All rights reserved.

SIALIC ACIDS AND AUTOIMMUNE DISEASE

PubMed Central

Mahajan, Vinay S.; Pillai, Shiv

2016-01-01

summary An important underlying mechanism that contributes to autoimmunity is the loss of inhibitory signaling in the immune system. Sialic acid-recognizing Ig superfamily lectins or Siglecs are a family of cell surface proteins largely expressed in hematopoietic cells. The majority of Siglecs are inhibitory receptors expressed in immune cells that bind to sialic acid containing ligands and recruit SH2-domain containing tyrosine phosphatases to their cytoplasmic tails. They deliver inhibitory signals that can contribute to the constraining of immune cells and thus protect the host from autoimmunity. The inhibitory functions of CD22/Siglec-2 and Siglec-G and their contributions to tolerance and autoimmunity, primarily in the B lymphocyte context, are considered in some detail in this review. The relevance to autoimmunity and unregulated inflammation of modified sialic acids, enzymes that modify sialic acid, and other sialic acid binding proteins are also reviewed. PMID:26683151

SH3BP4, a novel pigmentation gene, is inversely regulated by miR-125b and MITF

PubMed Central

Kim, Kyu-Han; Lee, Tae Ryong; Cho, Eun-Gyung

2017-01-01

Our previous work has identified miR-125b as a negative regulator of melanogenesis. However, the specific melanogenesis-related genes targeted by this miRNA had not been identified. In this study, we established a screening strategy involving three consecutive analytical approaches—analysis of target genes of miR-125b, expression correlation analysis between each target gene and representative pigmentary genes, and functional analysis of candidate genes related to melanogenesis—to discover melanogenesis-related genes targeted by miR-125b. Through these analyses, we identified SRC homology 3 domain-binding protein 4 (SH3BP4) as a novel pigmentation gene. In addition, by combining bioinformatics analysis and experimental validation, we demonstrated that SH3BP4 is a direct target of miR-125b. Finally, we found that SH3BP4 is transcriptionally regulated by microphthalmia-associated transcription factor as its direct target. These findings provide important insights into the roles of miRNAs and their targets in melanogenesis. PMID:28819321

Phosphorylation of the Grb2- and phosphatidylinositol 3-kinase p85-binding p36/38 by Syk in Lck-negative T cells.

PubMed

von Willebrand, M; Williams, S; Tailor, P; Mustelin, T

1998-06-01

Activation of the mitogen-activated protein kinase (MAPK) pathway by the T-cell antigen receptor (TCR) in T cells involves a positive role for phosphatidylinositol 3-kinase (PI3K) activity. We recently reported that over-expression of the Syk protein tyrosine kinase in the Lck-negative JCaM1 cells enabled the TCR to induce a normal activation of the Erk2 MAPK and enhanced transcription of a reporter gene driven by the nuclear factor of activated T cells and AP-1. Because this system allows us to analyse the targets for Syk in receptor-mediated signalling, we examined the role of PI3K in signalling events between the TCR-regulated Syk and the downstream activation of Erk2. We report that inhibition of PI3K by wortmannin or an inhibitory p85 construct, p85deltaiSH2, reduced the TCR-induced Syk-dependent activation of Erk2, as well as the appearance of phospho-Erk and phospho-Mek. At the same time, expression of Syk resulted in the activation-dependent phosphorylation of three proteins that bound to the src homology 2 (SH2) domains of PI3K p85. The strongest of these bands had an apparent molecular mass of 36-38 kDa on SDS gels, and it was quantitatively removed from the lysates by adsorption to a fusion protein containing the SH2 domain of Grb2. The appearance of this band was Syk dependent, and it was seen only upon triggering of the TCR complex. Thus, p36/38 was phosphorylated by Syk or a Syk-regulated kinase, and this protein may provide a link to the recruitment and activation of PI3K, as well as to the Ras-MAPK pathway, in TCR-triggered T cells.

On the importance of a funneled energy landscape for the assembly and regulation of multidomain Src tyrosine kinases.

PubMed

Faraldo-Gómez, José D; Roux, Benoît

2007-08-21

Regulation of signaling pathways in the cell often involves multidomain allosteric enzymes that are able to adopt alternate active or inactive conformations in response to specific stimuli. It is therefore of great interest to elucidate the energetic and structural determinants that govern the conformational plasticity of these proteins. In this study, free-energy computations have been used to address this fundamental question, focusing on one important family of signaling enzymes, the Src tyrosine kinases. Inactivation of these enzymes depends on the formation of an assembly comprising a tandem of SH3 and SH2 modules alongside a catalytic domain. Activation results from the release of the SH3 and SH2 domains, which are then believed to be structurally uncoupled by virtue of a flexible peptide link. In contrast to this view, this analysis shows that inactivation depends critically on the intrinsic propensity of the SH3-SH2 tandem to adopt conformations that are conducive to the assembled inactive state, even when no interactions with the rest of the kinase are possible. This funneling of the available conformational space is encoded within the SH3-SH2 connector, which appears to have evolved to modulate the flexibility of the tandem in solution. To further substantiate this notion, we show how constitutively activating mutations in the SH3-SH2 connector shift the assembly equilibrium toward the disassembled, active state. Based on a similar analysis of several constructs of the kinase complex, we propose that assembly is characterized by the progressive optimization of the protein's conformational energy, with little or no energetic frustration.

The sorting nexin, DSH3PX1, connects the axonal guidance receptor, Dscam, to the actin cytoskeleton.

PubMed

Worby, C A; Simonson-Leff, N; Clemens, J C; Kruger, R P; Muda, M; Dixon, J E

2001-11-09

Dock, an adaptor protein that functions in Drosophila axonal guidance, consists of three tandem Src homology 3 (SH3) domains preceding an SH2 domain. To develop a better understanding of axonal guidance at the molecular level, we used the SH2 domain of Dock to purify a protein complex from fly S2 cells. Five proteins were obtained in pure form from this protein complex. The largest protein in the complex was identified as Dscam (Down syndrome cell adhesion molecule), which was subsequently shown to play a key role in directing neurons of the fly embryo to correct positions within the nervous system (Schmucker, D., Clemens, J. C., Shu, H., Worby, C. A., Xiao, J., Muda, M., Dixon, J. E., and Zipursky, S. L. (2000) Cell 101, 671-684). The smallest protein in this complex (p63) has now been identified. We have named p63 DSH3PX1 because it appears to be the Drosophila orthologue of the human protein known as SH3PX1. DSH3PX1 is comprised of an NH(2)-terminal SH3 domain, an internal PHOX homology (PX) domain, and a carboxyl-terminal coiled-coil region. Because of its PX domain, DSH3PX1 is considered to be a member of a growing family of proteins known collectively as sorting nexins, some of which have been shown to be involved in vesicular trafficking. We demonstrate that DSH3PX1 immunoprecipitates with Dock and Dscam from S2 cell extracts. The domains responsible for the in vitro interaction between DSH3PX1 and Dock were also identified. We further show that DSH3PX1 interacts with the Drosophila orthologue of Wasp, a protein component of actin polymerization machinery, and that DSH3PX1 co-immunoprecipitates with AP-50, the clathrin-coat adapter protein. This evidence places DSH3PX1 in a complex linking cell surface receptors like Dscam to proteins involved in cytoskeletal rearrangements and/or receptor trafficking.

Essential roles of Gab1 tyrosine phosphorylation in growth factor-mediated signaling and angiogenesis.

PubMed

Wang, Weiye; Xu, Suowen; Yin, Meimei; Jin, Zheng Gen

2015-02-15

Growth factors and their downstream receptor tyrosine kinases (RTKs) mediate a number of biological processes controlling cell function. Adaptor (docking) proteins, which consist exclusively of domains and motifs that mediate molecular interactions, link receptor activation to downstream effectors. Recent studies have revealed that Grb2-associated-binders (Gab) family members (including Gab1, Gab2, and Gab3), when phosphorylated on tyrosine residues, provide binding sites for multiple effector proteins, such as Src homology-2 (SH2)-containing protein tyrosine phosphatase 2 (SHP2) and phosphatidylinositol 3-kinase (PI3K) regulatory subunit p85, thereby playing important roles in transducing RTKs-mediated signals into pathways with diversified biological functions. Here, we provide an up-to-date overview on the domain structure and biological functions of Gab1, the most intensively studied Gab family protein, in growth factor signaling and biological functions, with a special focus on angiogenesis. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

The B-Cell Specific Transcription Factor, Oct-2, Promotes Epstein-Barr Virus Latency by Inhibiting the Viral Immediate-Early Protein, BZLF1

PubMed Central

Robinson, Amanda R.; Kwek, Swee Sen; Kenney, Shannon C.

2012-01-01

The Epstein-Barr virus (EBV) latent-lytic switch is mediated by the BZLF1 immediate-early protein. EBV is normally latent in memory B cells, but cellular factors which promote viral latency specifically in B cells have not been identified. In this report, we demonstrate that the B-cell specific transcription factor, Oct-2, inhibits the function of the viral immediate-early protein, BZLF1, and prevents lytic viral reactivation. Co-transfected Oct-2 reduces the ability of BZLF1 to activate lytic gene expression in two different latently infected nasopharyngeal carcinoma cell lines. Furthermore, Oct-2 inhibits BZLF1 activation of lytic EBV promoters in reporter gene assays, and attenuates BZLF1 binding to lytic viral promoters in vivo. Oct-2 interacts directly with BZLF1, and this interaction requires the DNA-binding/dimerization domain of BZLF1 and the POU domain of Oct-2. An Oct-2 mutant (Δ262–302) deficient for interaction with BZLF1 is unable to inhibit BZLF1-mediated lytic reactivation. However, an Oct-2 mutant defective for DNA-binding (Q221A) retains the ability to inhibit BZLF1 transcriptional effects and DNA-binding. Importantly, shRNA-mediated knockdown of endogenous Oct-2 expression in several EBV-positive Burkitt lymphoma and lymphoblastoid cell lines increases the level of lytic EBV gene expression, while decreasing EBNA1 expression. Moreover, treatments which induce EBV lytic reactivation, such as anti-IgG cross-linking and chemical inducers, also decrease the level of Oct-2 protein expression at the transcriptional level. We conclude that Oct-2 potentiates establishment of EBV latency in B cells. PMID:22346751

Dock mediates Scar- and WASp-dependent actin polymerization through interaction with cell adhesion molecules in founder cells and fusion-competent myoblasts.

PubMed

Kaipa, Balasankara Reddy; Shao, Huanjie; Schäfer, Gritt; Trinkewitz, Tatjana; Groth, Verena; Liu, Jianqi; Beck, Lothar; Bogdan, Sven; Abmayr, Susan M; Önel, Susanne-Filiz

2013-01-01

The formation of the larval body wall musculature of Drosophila depends on the asymmetric fusion of two myoblast types, founder cells (FCs) and fusion-competent myoblasts (FCMs). Recent studies have established an essential function of Arp2/3-based actin polymerization during myoblast fusion, formation of a dense actin focus at the site of fusion in FCMs, and a thin sheath of actin in FCs and/or growing muscles. The formation of these actin structures depends on recognition and adhesion of myoblasts that is mediated by cell surface receptors of the immunoglobulin superfamily. However, the connection of the cell surface receptors with Arp2/3-based actin polymerization is poorly understood. To date only the SH2-SH3 adaptor protein Crk has been suggested to link cell adhesion with Arp2/3-based actin polymerization in FCMs. Here, we propose that the SH2-SH3 adaptor protein Dock, like Crk, links cell adhesion with actin polymerization. We show that Dock is expressed in FCs and FCMs and colocalizes with the cell adhesion proteins Sns and Duf at cell-cell contact points. Biochemical data in this study indicate that different domains of Dock are involved in binding the cell adhesion molecules Duf, Rst, Sns and Hbs. We emphasize the importance of these interactions by quantifying the enhanced myoblast fusion defects in duf dock, sns dock and hbs dock double mutants. Additionally, we show that Dock interacts biochemically and genetically with Drosophila Scar, Vrp1 and WASp. Based on these data, we propose that Dock links cell adhesion in FCs and FCMs with either Scar- or Vrp1-WASp-dependent Arp2/3 activation.

The structural coupling between ATPase activation and recovery stroke in the myosin II motor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Koppole, Sampath; Smith, Jeremy C; Fischer, S.

2007-07-01

Before the myosin motor head can perform the next power stroke, it undergoes a large conformational transition in which the converter domain, bearing the lever arm, rotates {approx} 65{sup o}. Simultaneous with this 'recovery stroke', myosin activates its ATPase function by closing the Switch-2 loop over the bound ATP. This coupling between the motions of the converter domain and of the 40 {angstrom}-distant Switch-2 loop is essential to avoid unproductive ATP hydrolysis. The coupling mechanism is determined here by finding a series of optimized intermediates between crystallographic end structures of the recovery stroke (Dictyostelium discoideum), yielding movies of the transitionmore » at atomic detail. The successive formation of two hydrogen bonds by the Switch-2 loop is correlated with the successive see-saw motions of the relay and SH1 helices that hold the converter domain. SH1 helix and Switch-2 loop communicate via a highly conserved loop that wedges against the SH1-helix upon Switch-2 closing.« less

The structural coupling between ATPase activation and recovery stroke in the myosin II motor.

PubMed

Koppole, Sampath; Smith, Jeremy C; Fischer, Stefan

2007-07-01

Before the myosin motor head can perform the next power stroke, it undergoes a large conformational transition in which the converter domain, bearing the lever arm, rotates approximately 65 degrees . Simultaneous with this "recovery stroke," myosin activates its ATPase function by closing the Switch-2 loop over the bound ATP. This coupling between the motions of the converter domain and of the 40 A-distant Switch-2 loop is essential to avoid unproductive ATP hydrolysis. The coupling mechanism is determined here by finding a series of optimized intermediates between crystallographic end structures of the recovery stroke (Dictyostelium discoideum), yielding movies of the transition at atomic detail. The successive formation of two hydrogen bonds by the Switch-2 loop is correlated with the successive see-saw motions of the relay and SH1 helices that hold the converter domain. SH1 helix and Switch-2 loop communicate via a highly conserved loop that wedges against the SH1-helix upon Switch-2 closing.

Antitumour effects of PLC-gamma1-(SH2)2-TAT fusion proteins on EGFR/c-erbB-2-positive breast cancer cells.

PubMed

Katterle, Y; Brandt, B H; Dowdy, S F; Niggemann, B; Zänker, K S; Dittmar, T

2004-01-12

Due to its pivotal role in the growth factor-mediated tumour cell migration, the adaptor protein phospholipase C-gamma1 (PLC-gamma1) is an appropriate target to block ultimately the spreading of EGFR/c-erbB-2-positive tumour cells, thereby minimising metastasis formation. Here, we present an approach to block PLC-gamma1 activity by using protein-based PLC-gamma1 inhibitors consisting of PLC-gamma1 SH2 domains, which were fused to the TAT-transduction domain to ensure a high protein transduction efficiency. Two proteins were generated containing one PLC-gamma1-SH2-domain (PS1-TAT) or two PLC-gamma1-SH2 domains (PS2-TAT). PS2-TAT treatment of the EGFR/c-erbB-2-positive cell line MDA-HER2 resulted in a reduction of the EGF-mediated PLC-gamma1 tyrosine phosphorylation of about 30%, concomitant with a complete abrogation of the EGF-driven calcium influx. In addition to this, long-term PS2-TAT treatment both reduces the EGF-mediated migration of about 75% combined with a markedly decreased time locomotion of single MDA-HER2 cells as well as decreases the proliferation of MDA-HER2 cells by about 50%. Due to its antitumoral capacity on EGFR/c-erbB-2-positive breast cancer cells, we conclude from our results that the protein-based PLC-gamma1 inhibitor PS2-TAT may be a means for novel adjuvant antitumour strategies to minimise metastasis formation because of the blockade of cell migration and proliferation.

[Syk inhibitors].

PubMed

Kimura, Yukihiro; Chihara, Kazuyasu; Takeuchi, Kenji; Sada, Kiyonao

2013-07-01

Non-receptor type of protein-tyrosine kinase Syk (spleen tyrosine kinase) was isolated in the University of Fukui in 1991. Syk is known to be essential for the various physiological functions, especially in hematopoietic lineage cells. Moreover, ectopic expression of Syk by epigenetic changes is reported to cause retinoblastoma. Recently, novel Syk inhibitors were developed and its usefulness has been evaluated in the treatment of allergic rhinitis, rheumatoid arthritis, and idiopathic thrombocytopenic purpura. In this review, we will summarize the history, structure, and function of Syk, and then describe the novel Syk inhibitors and their current status. Furthermore, we will introduce our findings of the adaptor protein 3BP2 (c-Abl SH3 domain-binding protein-2), as a novel target of Syk.

Two variants of the major serine protease inhibitor from the sea anemone Stichodactyla helianthus, expressed in Pichia pastoris.

PubMed

García-Fernández, Rossana; Ziegelmüller, Patrick; González, Lidice; Mansur, Manuel; Machado, Yoan; Redecke, Lars; Hahn, Ulrich; Betzel, Christian; Chávez, María de Los Ángeles

2016-07-01

The major protease inhibitor from the sea anemone Stichodactyla helianthus (ShPI-1) is a non-specific inhibitor that binds trypsin and other trypsin-like enzymes, as well as chymotrypsin, and human neutrophil elastase. We performed site-directed mutagenesis of ShPI-1 to produce two variants (rShPI-1/K13L and rShPI/Y15S) that were expressed in Pichia pastoris, purified, and characterized. After a single purification step, 65 mg and 15 mg of protein per liter of culture supernatant were obtained for rShPI-1/K13L and rShPI/Y15S, respectively. Functional studies demonstrated a 100-fold decreased trypsin inhibitory activity as result of the K13L substitution at the reactive (P1) site. This protein variant has a novel tight-binding inhibitor activity of pancreatic elastase and increased activity toward neutrophil elastase in comparison to rShPI-1A. In contrast, the substitution Y15S at P2' site did not affect the Ki value against trypsin, but did reduce activity 10-fold against chymotrypsin and neutrophil elastase. Our results provide two new ShPI-1 variants with modified inhibitory activities, one of them with increased biomedical potential. This study also offers new insight into the functional impact of the P1 and P2' sites on ShPI-1 specificity. Copyright © 2016 Elsevier Inc. All rights reserved.

Syk-dependent tyrosine phosphorylation of 3BP2 is required for optimal FcRγ-mediated phagocytosis and chemokine expression in U937 cells.

PubMed

Chihara, Kazuyasu; Kato, Yuji; Yoshiki, Hatsumi; Takeuchi, Kenji; Fujieda, Shigeharu; Sada, Kiyonao

2017-09-13

The adaptor protein c-Abl SH3 domain binding protein-2 (3BP2) is tyrosine phosphorylated by Syk in response to cross-linking of antigen receptors, which in turn activates various immune responses. Recently, a study using the mouse model of cherubism, a dominant inherited disorder caused by mutations in the gene encoding 3BP2, showed that 3BP2 is involved in the regulation of phagocytosis mediated by Fc receptor for IgG (FcγR) in macrophages. However, the molecular mechanisms underlying 3BP2-mediated regulation of phagocytosis and the physiological relevance of 3BP2 tyrosine phosphorylation remains elusive. In this study, we established various gene knockout U937 cell lines using the CRISPR/Cas9 system and found that 3BP2 is rapidly tyrosine phosphorylated by Syk in response to cross-linking of FcγRI. Depletion of 3BP2 caused significant reduction in the Fc receptor γ chain (FcRγ)-mediated phagocytosis in addition to the FcγRI-mediated induction of chemokine mRNA for IL-8, CCL3L3 and CCL4L2. Syk-dependent tyrosine phosphorylation of 3BP2 was required for overcoming these defects. Finally, we found that the PH and SH2 domains play important roles on FcγRI-mediated tyrosine phosphorylation of 3BP2 in HL-60 cells. Taken together, these results indicate that Syk-dependent tyrosine phosphorylation of 3BP2 is required for optimal FcRγ-mediated phagocytosis and chemokine expression.

Water-refined solution structure of the human Grb7-SH2 domain in complex with the erbB2 receptor peptide pY1139.

PubMed

Pias, Sally C; Johnson, Dennis L; Smith, David E; Lyons, Barbara A

2012-08-01

We report a refinement in implicit water of the previously published solution structure of the Grb7-SH2 domain bound to the erbB2 receptor peptide pY1139. Structure quality measures indicate substantial improvement, with residues in the most favored regions of the Ramachandran plot increasing by 14 % and with WHAT IF statistics (Vriend, G. J. Mol. Graph., 1990, 8(1), 52-56) falling closer to expected values for well-refined structures.

Interactions between the bud emergence proteins Bem1p and Bem2p and Rho-type GTPases in yeast.

PubMed

Peterson, J; Zheng, Y; Bender, L; Myers, A; Cerione, R; Bender, A

1994-12-01

The SH3 domain-containing protein Bem1p is needed for normal bud emergence and mating projection formation, two processes that require asymmetric reorganizations of the cortical cytoskeleton in Saccharomyces cerevisiae. To identify proteins that functionally and/or physically interact with Bem1p, we screened for mutations that display synthetic lethality with a mutant allele of the BEM1 gene and for genes whose products display two-hybrid interactions with the Bem1 protein. CDC24, which is required for bud emergence and encodes a GEF (guanine-nucleotide exchange factor) for the essential Rho-type GTPase Cdc42p, was identified during both screens. The COOH-terminal 75 amino acids of Cdc24p, outside of the GEF domain, can interact with a portion of Bem1p that lacks both SH3 domains. Bacterially expressed Cdc24p and Bem1p bind to each other in vitro, indicating that no other yeast proteins are required for this interaction. The most frequently identified gene that arose from the bem1 synthetic-lethal screen was the bud-emergence gene BEM2 (Bender and Pringle. 1991. Mol. Cell Biol. 11:1295-1395), which is allelic with IPL2 (increase in ploidy; Chan and Botstein, 1993. Genetics. 135:677-691). Here we show that Bem2p contains a GAP (GTPase-activating protein) domain for Rho-type GTPases, and that this portion of Bem2p can stimulate in vitro the GTPase activity of Rho1p, a second essential yeast Rho-type GTPase. Cells deleted for BEM2 become large and multinucleate. These and other genetic, two-hybrid, biochemical, and phenotypic data suggest that multiple Rho-type GTPases control the reorganization of the cortical cytoskeleton in yeast and that the functions of these GTPases are tightly coupled. Also, these findings raise the possibility that Bem1p may regulate or be a target of action of one or more of these GTPases.

Peptide Array X-Linking (PAX): A New Peptide-Protein Identification Approach

PubMed Central

Okada, Hirokazu; Uezu, Akiyoshi; Soderblom, Erik J.; Moseley, M. Arthur; Gertler, Frank B.; Soderling, Scott H.

2012-01-01

Many protein interaction domains bind short peptides based on canonical sequence consensus motifs. Here we report the development of a peptide array-based proteomics tool to identify proteins directly interacting with ligand peptides from cell lysates. Array-formatted bait peptides containing an amino acid-derived cross-linker are photo-induced to crosslink with interacting proteins from lysates of interest. Indirect associations are removed by high stringency washes under denaturing conditions. Covalently trapped proteins are subsequently identified by LC-MS/MS and screened by cluster analysis and domain scanning. We apply this methodology to peptides with different proline-containing consensus sequences and show successful identifications from brain lysates of known and novel proteins containing polyproline motif-binding domains such as EH, EVH1, SH3, WW domains. These results suggest the capacity of arrayed peptide ligands to capture and subsequently identify proteins by mass spectrometry is relatively broad and robust. Additionally, the approach is rapid and applicable to cell or tissue fractions from any source, making the approach a flexible tool for initial protein-protein interaction discovery. PMID:22606326

In brain, Axl recruits Grb2 and the p85 regulatory subunit of Pl3 kinase; in vitro mutagenesis defines th requisite binding sites for downstream Akt activation

PubMed Central

Weinger, Jason G.; Gohari, Pouyan; Yan, Ying; Backer, Jonathan M.; Varnum, Brian; Shafit-Zagardo, Bridget

2010-01-01

Axl is a receptor tyrosine kinase implicated in cell survival following growth factor withdrawal and other stressors. The binding of Axl's ligand, growth arrest-specific protein 6 (Gas6), results in Axl autophosphorylation, recruitment of signaling molecules, and activation of downstream survival pathways. Pull-down assays and immunoprecipitations using wildtype and mutant Axl transfected cells determined that Axl directly binds growth factor receptor-bound protein 2 (Grb2) at pYVN and the p85 subunit of phosphatidylinositol-3 kinase (PI3 kinase) at two pYXXM sites (pY779 and pY821). Also, p85 can indirectly bind to Axl via an interaction between p85's second proline-rich region and the N-terminal SH3 domain of Grb2. Further, Grb2 and p85 can compete for binding at the pY821VNM site. Gas6-stimulation of Axl-transfected COS7 cells recruited activated PI3 kinase and phosphorylated Akt. An interaction between Axl, p85 and Grb2 was confirmed in brain homogenates, enriched populations of O4+ oligodendrocytes, and O4– flow-through prepared from day 10 mouse brain, indicating that cells with active Gas6/Axl signal through Grb2 and the PI3 kinase/Akt pathways. PMID:18346204

Targets of B-cell antigen receptor signaling: the phosphatidylinositol 3-kinase/Akt/glycogen synthase kinase-3 signaling pathway and the Rap1 GTPase.

PubMed

Gold, M R; Ingham, R J; McLeod, S J; Christian, S L; Scheid, M P; Duronio, V; Santos, L; Matsuuchi, L

2000-08-01

In this review, we discuss the role of phosphatidylinositol 3-kinase (PI3K) and Rap 1 in B-cell receptor (BCR) signaling. PI3K produces lipids that recruit pleckstrin homology domain-containing proteins to the plasma membrane. Akt is a kinase that the BCR activates in this manner. Akt phosphorylates several transcription factors as well as proteins that regulate apoptosis and protein synthesis. Akt also regulates glycogen synthase kinase-3, a kinase whose substrates include the nuclear factor of activated T cells (NF-AT)cl and beta-catenin transcriptional activators. In addition to Akt, PI3K-derived lipids also regulate the activity and localization of other targets of BCR signaling. Thus, a key event in BCR signaling is the recruitment of PI3K to the plasma membrane where its substrates are located. This is mediated by binding of the Src homology (SH) 2 domains in PI3K to phosphotyrosine-containing sequences on membrane-associated docking proteins. The docking proteins that the BCR uses to recruit PI3K include CD19, Cbl, Gab1, and perhaps Gab2. We have shown that Gab1 colocalizes PI3K with SH2 domain-containing inositol phosphatase (SHIP) and SHP2, two enzymes that regulate PI3K-dependent signaling. In contrast to PI3K, little is known about the Rap1 GTPase. We showed that the BCR activates Rap1 via phospholipase C-dependent production of diacylglycerol. Since Rap1 is thought to regulate cell adhesion and cell polarity, it may be involved in B-cell migration.

Characterizing SHP2 as a Novel Therapeutic Target in Breast Cancer

DTIC Science & Technology

2013-02-01

attempted to elucidate interactions with molecular docking (5). The peptide was docked into the SH2 active site of 2SHP.pdb (with SH2 domains...activated protein kinase (MAPK) pathway, which is read as a drop in phosphorylated ERK protein(3). 5 First, the problem of cell permeability

Structural insights into the intertwined dimer of fyn SH2.

PubMed

Huculeci, Radu; Garcia-Pino, Abel; Buts, Lieven; Lenaerts, Tom; van Nuland, Nico

2015-12-01

Src homology 2 domains are interaction modules dedicated to the recognition of phosphotyrosine sites incorporated in numerous proteins found in intracellular signaling pathways. Here we provide for the first time structural insight into the dimerization of Fyn SH2 both in solution and in crystalline conditions, providing novel crystal structures of both the dimer and peptide-bound structures of Fyn SH2. Using nuclear magnetic resonance chemical shift analysis, we show how the peptide is able to eradicate the dimerization, leading to monomeric SH2 in its bound state. Furthermore, we show that Fyn SH2's dimer form differs from other SH2 dimers reported earlier. Interestingly, the Fyn dimer can be used to construct a completed dimer model of Fyn without any steric clashes. Together these results extend our understanding of SH2 dimerization, giving structural details, on one hand, and suggesting a possible physiological relevance of such behavior, on the other hand. © 2015 The Protein Society.