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Sample records for adaptor protein regulating

  1. Anti-adaptors provide multiple modes for regulation of the RssB adaptor protein

    PubMed Central

    Battesti, Aurelia; Hoskins, Joel R.; Tong, Song; Milanesio, Paola; Mann, Jessica M.; Kravats, Andrea; Tsegaye, Yodit M.; Bougdour, Alexandre; Wickner, Sue; Gottesman, Susan

    2013-01-01

    RpoS, an RNA polymerase σ factor, controls the response of Escherichia coli and related bacteria to multiple stress responses. During nonstress conditions, RpoS is rapidly degraded by ClpXP, mediated by the adaptor protein RssB, a member of the response regulator family. In response to stress, RpoS degradation ceases. Small anti-adaptor proteins—IraP, IraM, and IraD, each made under a different stress condition—block RpoS degradation. RssB mutants resistant to either IraP or IraM were isolated and analyzed in vivo and in vitro. Each of the anti-adaptors is unique in its interaction with RssB and sensitivity to RssB mutants. One class of mutants defined an RssB N-terminal region close to the phosphorylation site and critical for interaction with IraP but unnecessary for IraM and IraD function. A second class, in the RssB C-terminal PP2C-like domain, led to activation of RssB function. These mutants allowed the response regulator to act in the absence of phosphorylation but did not abolish interaction with anti-adaptors. This class of mutants is broadly resistant to the anti-adaptors and bears similarity to constitutively activated mutants found in a very different PP2C protein. The mutants provide insight into how the anti-adaptors perturb RssB response regulator function and activation. PMID:24352426

  2. Differential Regulation of Clathrin and Its Adaptor Proteins during Membrane Recruitment for Endocytosis.

    PubMed

    Wang, Chao; Hu, Tianwei; Yan, Xu; Meng, Tingting; Wang, Yutong; Wang, Qingmei; Zhang, Xiaoyue; Gu, Ying; Sánchez-Rodríguez, Clara; Gadeyne, Astrid; Lin, Jinxing; Persson, Staffan; Van Damme, Daniël; Li, Chuanyou; Bednarek, Sebastian Y; Pan, Jianwei

    2016-05-01

    In plants, clathrin-mediated endocytosis (CME) is dependent on the function of clathrin and its accessory heterooligomeric adaptor protein complexes, ADAPTOR PROTEIN2 (AP-2) and the TPLATE complex (TPC), and is negatively regulated by the hormones auxin and salicylic acid (SA). The details for how clathrin and its adaptor complexes are recruited to the plasma membrane (PM) to regulate CME, however, are poorly understood. We found that SA and the pharmacological CME inhibitor tyrphostin A23 reduce the membrane association of clathrin and AP-2, but not that of the TPC, whereas auxin solely affected clathrin membrane association, in Arabidopsis (Arabidopsis thaliana). Genetic and pharmacological experiments revealed that loss of AP2μ or AP2σ partially affected the membrane association of other AP-2 subunits and that the AP-2 subunit AP2σ, but not AP2μ, was required for SA- and tyrphostin A23-dependent inhibition of CME Furthermore, we show that although AP-2 and the TPC are both required for the PM recruitment of clathrin in wild-type cells, the TPC is necessary for clathrin PM association in AP-2-deficient cells. These results indicate that developmental signals may differentially modulate the membrane recruitment of clathrin and its core accessory complexes to regulate the process of CME in plant cells. PMID:26945051

  3. Differential Regulation of Clathrin and Its Adaptor Proteins during Membrane Recruitment for Endocytosis1[OPEN

    PubMed Central

    Wang, Chao; Hu, Tianwei; Yan, Xu; Meng, Tingting; Wang, Yutong; Wang, Qingmei; Zhang, Xiaoyue; Gu, Ying; Sánchez-Rodríguez, Clara; Gadeyne, Astrid; Lin, Jinxing

    2016-01-01

    In plants, clathrin-mediated endocytosis (CME) is dependent on the function of clathrin and its accessory heterooligomeric adaptor protein complexes, ADAPTOR PROTEIN2 (AP-2) and the TPLATE complex (TPC), and is negatively regulated by the hormones auxin and salicylic acid (SA). The details for how clathrin and its adaptor complexes are recruited to the plasma membrane (PM) to regulate CME, however, are poorly understood. We found that SA and the pharmacological CME inhibitor tyrphostin A23 reduce the membrane association of clathrin and AP-2, but not that of the TPC, whereas auxin solely affected clathrin membrane association, in Arabidopsis (Arabidopsis thaliana). Genetic and pharmacological experiments revealed that loss of AP2μ or AP2σ partially affected the membrane association of other AP-2 subunits and that the AP-2 subunit AP2σ, but not AP2μ, was required for SA- and tyrphostin A23-dependent inhibition of CME. Furthermore, we show that although AP-2 and the TPC are both required for the PM recruitment of clathrin in wild-type cells, the TPC is necessary for clathrin PM association in AP-2-deficient cells. These results indicate that developmental signals may differentially modulate the membrane recruitment of clathrin and its core accessory complexes to regulate the process of CME in plant cells. PMID:26945051

  4. Alternative Splicing in CaV2.2 Regulates Neuronal Trafficking via Adaptor Protein Complex-1 Adaptor Protein Motifs

    PubMed Central

    Macabuag, Natsuko

    2015-01-01

    N-type voltage-gated calcium (CaV2.2) channels are expressed in neurons and targeted to the plasma membrane of presynaptic terminals, facilitating neurotransmitter release. Here, we find that the adaptor protein complex-1 (AP-1) mediates trafficking of CaV2.2 from the trans-Golgi network to the cell surface. Examination of splice variants of CaV2.2, containing either exon 37a (selectively expressed in nociceptors) or 37b in the proximal C terminus, reveal that canonical AP-1 binding motifs, YxxΦ and [DE]xxxL[LI], present only in exon 37a, enhance intracellular trafficking of exon 37a-containing CaV2.2 to the axons and plasma membrane of rat DRG neurons. Finally, we identify differential effects of dopamine-2 receptor (D2R) and its agonist-induced activation on trafficking of CaV2.2 isoforms. D2R slowed the endocytosis of CaV2.2 containing exon 37b, but not exon 37a, and activation by the agonist quinpirole reversed the effect of the D2R. Our work thus reveals key mechanisms involved in the trafficking of N-type calcium channels. SIGNIFICANCE STATEMENT CaV2.2 channels are important for neurotransmitter release, but how they are trafficked is still poorly understood. Here, we describe a novel mechanism for trafficking of CaV2.2 from the trans-Golgi network to the cell surface which is mediated by the adaptor protein AP-1. Alternative splicing of exon 37 produces CaV2.2-exon 37a, selectively expressed in nociceptors, or CaV2.2-exon 37b, which is the major splice isoform. Our study reveals that canonical AP-1 binding motifs (YxxΦ and [DE]xxxL[LI]), present in exon 37a, but not 37b, enhance intracellular trafficking of exon 37a-containing CaV2.2 to axons and plasma membrane of DRG neurons. Interaction of APs with CaV2.2 channels may also be key underlying mechanisms for differential effects of the dopamine D2 receptor on trafficking of CaV2.2 splice variants. PMID:26511252

  5. The polarity protein Par3 regulates APP trafficking and processing through the endocytic adaptor protein Numb.

    PubMed

    Sun, Miao; Asghar, Suwaiba Z; Zhang, Huaye

    2016-09-01

    The processing of amyloid precursor protein (APP) into β-amyloid peptide (Aβ) is a key step in the pathogenesis of Alzheimer's disease (AD), and trafficking dysregulations of APP and its secretases contribute significantly to altered APP processing. Here we show that the cell polarity protein Par3 plays an important role in APP processing and trafficking. We found that the expression of full length Par3 is significantly decreased in AD patients. Overexpression of Par3 promotes non-amyloidogenic APP processing, while depletion of Par3 induces intracellular accumulation of Aβ. We further show that Par3 functions by regulating APP trafficking. Loss of Par3 decreases surface expression of APP by targeting APP to the late endosome/lysosome pathway. Finally, we show that the effects of Par3 are mediated through the endocytic adaptor protein Numb, and Par3 functions by interfering with the interaction between Numb and APP. Together, our studies show a novel role for Par3 in regulating APP processing and trafficking. PMID:27072891

  6. Phosphorylation-Dependent Regulation of the DNA Damage Response of Adaptor Protein KIBRA in Cancer Cells.

    PubMed

    Mavuluri, Jayadev; Beesetti, Swarnalatha; Surabhi, Rohan; Kremerskothen, Joachim; Venkatraman, Ganesh; Rayala, Suresh K

    2016-05-01

    Multifunctional adaptor proteins encompassing various protein-protein interaction domains play a central role in the DNA damage response pathway. In this report, we show that KIBRA is a physiologically interacting reversible substrate of ataxia telangiectasia mutated (ATM) kinase. We identified the site of phosphorylation in KIBRA as threonine 1006, which is embedded within the serine/threonine (S/T) Q consensus motif, by site-directed mutagenesis, and we further confirmed the same with a phospho-(S/T) Q motif-specific antibody. Results from DNA repair functional assays such as the γ-H2AX assay, pulsed-field gel electrophoresis (PFGE), Comet assay, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, and clonogenic cell survival assay using stable overexpression clones of wild-type (wt.) KIBRA and active (T1006E) and inactive (T1006A) KIBRA phosphorylation mutants showed that T1006 phosphorylation on KIBRA is essential for optimal DNA double-strand break repair in cancer cells. Further, results from stable retroviral short hairpin RNA-mediated knockdown (KD) clones of KIBRA and KIBRA knockout (KO) model cells generated by a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system showed that depleting KIBRA levels compromised the DNA repair functions in cancer cells upon inducing DNA damage. All these phenotypic events were reversed upon reconstitution of KIBRA into cells lacking KIBRA knock-in (KI) model cells. All these results point to the fact that phosphorylated KIBRA might be functioning as a scaffolding protein/adaptor protein facilitating the platform for further recruitment of other DNA damage response factors. In summary, these data demonstrate the imperative functional role of KIBRAper se(KIBRA phosphorylation at T1006 site as a molecular switch that regulates the DNA damage response, possibly via the nonhomologous end joining [NHEJ] pathway), suggesting that KIBRA could be a potential

  7. Adaptor Protein 2 Regulates Receptor-Mediated Endocytosis and Cyst Formation in Giardia lamblia

    PubMed Central

    Rivero, Maria R.; Vranych, Cecilia V.; Bisbal, Mariano; Maletto, Belkys A.; Ropolo, Andrea S.; Touz, Maria C.

    2010-01-01

    Synopsis The parasite Giardia lamblia possesses peripheral vacuoles (PVs) that function as both endosomes and lysosomes and are implicated in the adaptation, differentiation, and survival of the parasite in different environments. The mechanisms by which Giardia traffics essential proteins to these organelles and regulates their secretion have important implications in the control of parasite dissemination. In this study, we describe the participation of the heterotetrameric clathrin-adaptor protein gAP2 complex in lysosomal protein trafficking. A specific monoclonal antibody against the medium subunit (gμ2) of gAP2 showed localization of this complex to the PVs, cytoplasm, and plasma membrane in the growing trophozoites. gAP2 also colocalized with clathrin in the PVs, suggesting its involvement in endocytosis. Uptake experiments using standard molecules for the study of endocytosis revealed that gAP2 specifically participated in the endocytosis of LDL. Targeted downregulation of the gene encoding gμ2 in growing and encysting trophozoites resulted in a large decrease in the amount of cell growth and cyst wall formation, suggesting a distinct mechanism in which gAP2 is directly involved in both endocytosis and vesicular trafficking. PMID:20199400

  8. Stargazin regulates AMPA receptor trafficking through adaptor protein complexes during long-term depression

    NASA Astrophysics Data System (ADS)

    Matsuda, Shinji; Kakegawa, Wataru; Budisantoso, Timotheus; Nomura, Toshihiro; Kohda, Kazuhisa; Yuzaki, Michisuke

    2013-11-01

    Long-term depression (LTD) underlies learning and memory in various brain regions. Although postsynaptic AMPA receptor trafficking mediates LTD, its underlying molecular mechanisms remain largely unclear. Here we show that stargazin, a transmembrane AMPA receptor regulatory protein, forms a ternary complex with adaptor proteins AP-2 and AP-3A in hippocampal neurons, depending on its phosphorylation state. Inhibiting the stargazin-AP-2 interaction disrupts NMDA-induced AMPA receptor endocytosis, and inhibiting that of stargazin-AP-3A abrogates the late endosomal/lysosomal trafficking of AMPA receptors, thereby upregulating receptor recycling to the cell surface. Similarly, stargazin’s interaction with AP-2 or AP-3A is necessary for low-frequency stimulus-evoked LTD in CA1 hippocampal neurons. Thus, stargazin has a crucial role in NMDA-dependent LTD by regulating two trafficking pathways of AMPA receptors—transport from the cell surface to early endosomes and from early endosomes to late endosomes/lysosomes—through its sequential binding to AP-2 and AP-3A.

  9. Adaptor protein LNK is a negative regulator of brain neural stem cell proliferation after stroke.

    PubMed

    Ahlenius, Henrik; Devaraju, Karthikeyan; Monni, Emanuela; Oki, Koichi; Wattananit, Somsak; Darsalia, Vladimer; Iosif, Robert E; Torper, Olof; Wood, James C; Braun, Sebastian; Jagemann, Lucas; Nuber, Ulrike A; Englund, Elisabet; Jacobsen, Sten-Eirik W; Lindvall, Olle; Kokaia, Zaal

    2012-04-11

    Ischemic stroke causes transient increase of neural stem and progenitor cell (NSPC) proliferation in the subventricular zone (SVZ), and migration of newly formed neuroblasts toward the damaged area where they mature to striatal neurons. The molecular mechanisms regulating this plastic response, probably involved in structural reorganization and functional recovery, are poorly understood. The adaptor protein LNK suppresses hematopoietic stem cell self-renewal, but its presence and role in the brain are poorly understood. Here we demonstrate that LNK is expressed in NSPCs in the adult mouse and human SVZ. Lnk(-/-) mice exhibited increased NSPC proliferation after stroke, but not in intact brain or following status epilepticus. Deletion of Lnk caused increased NSPC proliferation while overexpression decreased mitotic activity of these cells in vitro. We found that Lnk expression after stroke increased in SVZ through the transcription factors STAT1/3. LNK attenuated insulin-like growth factor 1 signaling by inhibition of AKT phosphorylation, resulting in reduced NSPC proliferation. Our findings identify LNK as a stroke-specific, endogenous negative regulator of NSPC proliferation, and suggest that LNK signaling is a novel mechanism influencing plastic responses in postischemic brain. PMID:22496561

  10. Activity-Regulated Cytoskeleton-Associated Protein Controls AMPAR Endocytosis through a Direct Interaction with Clathrin-Adaptor Protein 2123

    PubMed Central

    Wall, Mark J.; P. de Almeida, Luciana; Wauters, Sandrine C.; Januário, Yunan C.; Müller, Jürgen

    2016-01-01

    Abstract The activity-regulated cytoskeleton-associated (Arc) protein controls synaptic strength by facilitating AMPA receptor (AMPAR) endocytosis. Here we demonstrate that Arc targets AMPAR to be internalized through a direct interaction with the clathrin-adaptor protein 2 (AP-2). We show that Arc overexpression in dissociated hippocampal neurons obtained from C57BL/6 mouse reduces the density of AMPAR GluA1 subunits at the cell surface and reduces the amplitude and rectification of AMPAR-mediated miniature-EPSCs (mEPSCs). Mutations of Arc, that prevent the AP-2 interaction reduce Arc-mediated endocytosis of GluA1 and abolish the reduction in AMPAR-mediated mEPSC amplitude and rectification. Depletion of the AP-2 subunit µ2 blocks the Arc-mediated reduction in mEPSC amplitude, an effect that is restored by reintroducing µ2. The Arc–AP-2 interaction plays an important role in homeostatic synaptic scaling as the Arc-dependent decrease in mEPSC amplitude, induced by a chronic increase in neuronal activity, is inhibited by AP-2 depletion. These data provide a mechanism to explain how activity-dependent expression of Arc decisively controls the fate of AMPAR at the cell surface and modulates synaptic strength, via the direct interaction with the endocytic clathrin adaptor AP-2. PMID:27257628

  11. Btn3 regulates the endosomal sorting function of the yeast Ent3 epsin, an adaptor for SNARE proteins.

    PubMed

    Morvan, Joëlle; de Craene, Johan-Owen; Rinaldi, Bruno; Addis, Vanessa; Misslin, Cédric; Friant, Sylvie

    2015-02-15

    Ent3 and Ent5 are yeast epsin N-terminal homology (ENTH) domain-containing proteins involved in protein trafficking between the Golgi and late endosomes. They interact with clathrin, clathrin adaptors at the Golgi (AP-1 and GGA) and different SNAREs (Vti1, Snc1, Pep12 and Syn8) required for vesicular transport at the Golgi and endosomes. To better understand the role of these epsins in membrane trafficking, we performed a protein-protein interaction screen. We identified Btn3 (also known as Tda3), a putative oxidoreductase, as a new partner of both Ent3 and Ent5. Btn3 is a negative regulator of the Batten-disease-linked protein Btn2 involved in the retrieval of specific SNAREs (Vti1, Snc1, Tlg1 and Tlg2) from the late endosome to the Golgi. We show that Btn3 endosomal localization depends on the epsins Ent3 and Ent5. We demonstrated that in btn3Δ mutant cells, endosomal sorting of ubiquitylated cargos and endosomal recycling of the Snc1 SNARE are delayed. We thus propose that Btn3 regulates the sorting function of two adaptors for SNARE proteins, the epsin Ent3 and the Batten-disease-linked protein Btn2. PMID:25512335

  12. Endostatin-induced tyrosine kinase signaling through the Shb adaptor protein regulates endothelial cell apoptosis.

    PubMed

    Dixelius, J; Larsson, H; Sasaki, T; Holmqvist, K; Lu, L; Engström, A; Timpl, R; Welsh, M; Claesson-Welsh, L

    2000-06-01

    Endostatin, which corresponds to the C-terminal fragment of collagen XVIII, is a potent inhibitor of angiogenesis. Fibroblast growth factor-2 (FGF-2)-induced angiogenesis in the chicken chorioallantoic membrane was inhibited by endostatin, but not by an endostatin mutant R158/270A, lacking heparin-binding ability. Endostatin was internalized by endothelial cells, but not by mouse fibroblasts. Treatment of murine brain endothelial (IBE) cells with endostatin reduced the proportion of cells in S phase, whereas growth-arrested IBE cells in collagen gels treated with endostatin displayed enhanced tubular morphogenesis. IBE cells overexpressing Shb, an adaptor protein implicated in angiostatin-induced apoptosis, displayed elevated apoptosis and decreased tubular morphogenesis in collagen gels in response to endostatin when added together with FGF-2. Induction of apoptosis was dependent on the heparin-binding ability of endostatin and the expression of Shb with a functional Src homology 2 (SH2)-domain. Endostatin treatment for 10 minutes or 24 hours induced tyrosine phosphorylation of Shb and formation of multiprotein complexes. An Shb SH2 domain fusion protein precipitated a 125-kd phosphotyrosyl protein in endostatin-treated cells. The 125-kd component either contained intrinsic tyrosine kinase activity or occurred in complex with a tyrosine kinase. In conclusion, our data show that endostatin induces tyrosine kinase activity and enhanced apoptosis in FGF-treated endothelial cells. PMID:10828022

  13. Cysteine-based regulation of the CUL3 adaptor protein Keap1

    SciTech Connect

    Sekhar, Konjeti R.; Rachakonda, Girish; Freeman, Michael L.

    2010-04-01

    Nrf2 (NF-E2-related factor 2) is a master transcription factor containing a powerful acidic transcriptional activation domain. Nrf2-dependent gene expression impacts cancer chemoprevention strategies, inflammatory responses, and progression of neurodegenerative diseases. Under basal conditions, association of Nrf2 with the CUL3 adaptor protein Keap1 results in the rapid Nrf2 ubiquitylation and proteasome-dependent degradation. Inhibition of Keap1 function blocks ubiquitylation of Nrf2, allowing newly synthesized Nrf2 to translocate into the nucleus, bind to ARE sites and direct target gene expression. Site-directed mutagenesis experiments coupled with proteomic analysis support a model in which Keap1 contains at least 2 distinct cysteine motifs. The first is located at Cys 151 in the BTB domain. The second is located in the intervening domain and centers around Cys 273 and 288. Adduction or oxidation at Cys151 has been shown to produce a conformational change in Keap1 that results in dissociation of Keap1 from CUL3, thereby inhibiting Nrf2 ubiquitylation. Thus, adduction captures specific chemical information and translates it into biochemical information via changes in structural conformation.

  14. Toll-Interleukin 1 Receptor domain-containing adaptor protein positively regulates BV2 cell M1 polarization.

    PubMed

    Gong, Leilei; Wang, Hanxiang; Sun, Xiaolei; Liu, Chun; Duan, Chengwei; Cai, Rixin; Gu, Xingxing; Zhu, Shunxing

    2016-06-01

    Microglial activation, including classical (M1) and alternative (M2) activation, plays important roles in the development of several central nervous system disorders and promotes tissue reconstruction. Toll-like receptor (TLR)4 is important for microglial polarization. TIR domain-containing adaptor protein (TIRAP) is an intracellular adaptor protein, which is responsible for the early phase of TLR4 activation. The role of TIRAP in BV2 cell M1 polarization is still unknown. In this study, we showed that TIRAP expression is greatly elevated in lipopolysaccharide (LPS)/interferon (IFN)-γ-treated microglia. TIRAP overexpression promoted BV2 microglial M1 polarization by increasing M1-related marker production (inducible nitric oxide synthase, CD86, interleukin-6, interleukin-1β and tumour necrosis factor-α). In contrast, TIRAP knockdown prevented M1-related marker production. Mechanistically, TIRAP could interact with TNF Receptor-Associated Factor 6 (TRAF6) to increase M1-related marker production in TIRAP overexpressed and LPS/IFN-γ-treated BV2 cells. In addition, silencing of TIRAP effectively inhibited the activation of the Transforming Growth Factor-Beta-Activated Kinase 1/I-Kappa-B Kinase /Nuclear Factor of Kappa Light Polypeptide Gene Enhancer in B-Cells (TAK1/IKK/NF-κB) signalling pathway and the phosphorylation of Akt and mitogen-activated protein kinases, which were activated by LPS/IFN-γ stimulation. Thus, our results suggest that TIRAP positively regulated BV2 microglial M1 polarization through TLR4-mediated TAK1/IKK/NF-κB, mitogen-activated protein kinases and Akt signalling pathways. PMID:27061018

  15. Transmembrane Adaptor Protein PAG/CBP Is Involved in both Positive and Negative Regulation of Mast Cell Signaling

    PubMed Central

    Draberova, Lubica; Bugajev, Viktor; Potuckova, Lucie; Halova, Ivana; Bambouskova, Monika; Polakovicova, Iva; Xavier, Ramnik J.; Seed, Brian

    2014-01-01

    The transmembrane adaptor protein PAG/CBP (here, PAG) is expressed in multiple cell types. Tyrosine-phosphorylated PAG serves as an anchor for C-terminal SRC kinase, an inhibitor of SRC-family kinases. The role of PAG as a negative regulator of immunoreceptor signaling has been examined in several model systems, but no functions in vivo have been determined. Here, we examined the activation of bone marrow-derived mast cells (BMMCs) with PAG knockout and PAG knockdown and the corresponding controls. Our data show that PAG-deficient BMMCs exhibit impaired antigen-induced degranulation, extracellular calcium uptake, tyrosine phosphorylation of several key signaling proteins (including the high-affinity IgE receptor subunits, spleen tyrosine kinase, and phospholipase C), production of several cytokines and chemokines, and chemotaxis. The enzymatic activities of the LYN and FYN kinases were increased in nonactivated cells, suggesting the involvement of a LYN- and/or a FYN-dependent negative regulatory loop. When BMMCs from PAG-knockout mice were activated via the KIT receptor, enhanced degranulation and tyrosine phosphorylation of the receptor were observed. In vivo experiments showed that PAG is a positive regulator of passive systemic anaphylaxis. The combined data indicate that PAG can function as both a positive and a negative regulator of mast cell signaling, depending upon the signaling pathway involved. PMID:25246632

  16. A Cyclic di-GMP-binding Adaptor Protein Interacts with Histidine Kinase to Regulate Two-component Signaling.

    PubMed

    Xu, Linghui; Venkataramani, Prabhadevi; Ding, Yichen; Liu, Yang; Deng, Yinyue; Yong, Grace Lisi; Xin, Lingyi; Ye, Ruijuan; Zhang, Lianhui; Yang, Liang; Liang, Zhao-Xun

    2016-07-29

    The bacterial messenger cyclic di-GMP (c-di-GMP) binds to a diverse range of effectors to exert its biological effect. Despite the fact that free-standing PilZ proteins are by far the most prevalent c-di-GMP effectors known to date, their physiological function and mechanism of action remain largely unknown. Here we report that the free-standing PilZ protein PA2799 from the opportunistic pathogen Pseudomonas aeruginosa interacts directly with the hybrid histidine kinase SagS. We show that PA2799 (named as HapZ: histidine kinase associated PilZ) binds directly to the phosphoreceiver (REC) domain of SagS, and that the SagS-HapZ interaction is further enhanced at elevated c-di-GMP concentration. We demonstrate that binding of HapZ to SagS inhibits the phosphotransfer between SagS and the downstream protein HptB in a c-di-GMP-dependent manner. In accordance with the role of SagS as a motile-sessile switch and biofilm growth factor, we show that HapZ impacts surface attachment and biofilm formation most likely by regulating the expression of a large number of genes. The observations suggest a previously unknown mechanism whereby c-di-GMP mediates two-component signaling through a PilZ adaptor protein. PMID:27231351

  17. SorLA/LR11 regulates processing of amyloid precursor protein via interaction with adaptors GGA and PACS-1.

    PubMed

    Schmidt, Vanessa; Sporbert, Anje; Rohe, Michael; Reimer, Tatjana; Rehm, Armin; Andersen, Olav M; Willnow, Thomas E

    2007-11-01

    SorLA has been recognized as a novel sorting receptor that regulates trafficking and processing of the amyloid precursor protein (APP) and that represents a significant risk factor for sporadic Alzheimer disease. Here, we investigated the cellular mechanisms that control intracellular trafficking of sorLA and their relevance for APP processing. We demonstrate that sorLA acts as a retention factor for APP in trans-Golgi compartments/trans-Golgi network, preventing release of the precursor into regular processing pathways. Proper localization and activity of sorLA are dependent on functional interaction with GGA and PACS-1, adaptor proteins involved in protein transport to and from the trans-Golgi network. Aberrant targeting of sorLA to the recycling compartment or the plasma membrane causes faulty APP trafficking and imbalance in non-amyloidogenic and amyloidogenic processing fates. Thus, our findings identified altered routing of sorLA as a major cellular mechanism contributing to abnormal APP processing and enhanced amyloid beta-peptide formation. PMID:17855360

  18. The Wnt Adaptor Protein ATP6AP2 Regulates Multiple Stages of Adult Hippocampal Neurogenesis

    PubMed Central

    Han, Jinju; Pena, Monique; von Bohlen und Halbach, Oliver; Peters, Jörg; Gage, Fred H.

    2015-01-01

    In the mammalian hippocampus, canonical Wnt signals provided by the microenvironment regulate the differentiation of adult neural stem cells (NSCs) toward the neuronal lineage. Wnts are part of a complex and diverse set of signaling pathways and the role of Wnt/Planar cell polarity (PCP) signaling in adult neurogenesis remains unknown. Using in vitro assays on differentiating adult NSCs, we identified a transition of Wnt signaling responsiveness from Wnt/β-catenin to Wnt/PCP signaling. In mice, retroviral knockdown strategies against ATP6AP2, a recently discovered core protein involved in both signaling pathways, revealed that its dual role is critical for granule cell fate and morphogenesis. We were able to confirm its dual role in neurogenic Wnt signaling in vitro for both canonical Wnt signaling in proliferating adult NSCs and non-canonical Wnt signaling in differentiating neuroblasts. Although LRP6 appeared to be critical for granule cell fate determination, in vivo knockdown of PCP core proteins FZD3 and CELSR1-3 revealed severe maturational defects without changing the identity of newborn granule cells. Furthermore, we found that CELSR1-3 control distinctive aspects of PCP-mediated granule cell morphogenesis with CELSR1 regulating the direction of dendrite initiation sites and CELSR2/3 controlling radial migration and dendritic patterning. The data presented here characterize distinctive roles for Wnt/β-catenin signaling in granule cell fate determination and for Wnt/PCP signaling in controlling the morphological maturation of differentiating neuroblasts. PMID:25810528

  19. The Wnt adaptor protein ATP6AP2 regulates multiple stages of adult hippocampal neurogenesis.

    PubMed

    Schafer, Simon T; Han, Jinju; Pena, Monique; von Bohlen Und Halbach, Oliver; Peters, Jörg; Gage, Fred H

    2015-03-25

    In the mammalian hippocampus, canonical Wnt signals provided by the microenvironment regulate the differentiation of adult neural stem cells (NSCs) toward the neuronal lineage. Wnts are part of a complex and diverse set of signaling pathways and the role of Wnt/Planar cell polarity (PCP) signaling in adult neurogenesis remains unknown. Using in vitro assays on differentiating adult NSCs, we identified a transition of Wnt signaling responsiveness from Wnt/β-catenin to Wnt/PCP signaling. In mice, retroviral knockdown strategies against ATP6AP2, a recently discovered core protein involved in both signaling pathways, revealed that its dual role is critical for granule cell fate and morphogenesis. We were able to confirm its dual role in neurogenic Wnt signaling in vitro for both canonical Wnt signaling in proliferating adult NSCs and non-canonical Wnt signaling in differentiating neuroblasts. Although LRP6 appeared to be critical for granule cell fate determination, in vivo knockdown of PCP core proteins FZD3 and CELSR1-3 revealed severe maturational defects without changing the identity of newborn granule cells. Furthermore, we found that CELSR1-3 control distinctive aspects of PCP-mediated granule cell morphogenesis with CELSR1 regulating the direction of dendrite initiation sites and CELSR2/3 controlling radial migration and dendritic patterning. The data presented here characterize distinctive roles for Wnt/β-catenin signaling in granule cell fate determination and for Wnt/PCP signaling in controlling the morphological maturation of differentiating neuroblasts. PMID:25810528

  20. The adaptor protein alpha-syntrophin regulates adipocyte lipid droplet growth.

    PubMed

    Eisinger, Kristina; Rein-Fischboeck, Lisa; Pohl, Rebekka; Meier, Elisabeth M; Krautbauer, Sabrina; Buechler, Christa

    2016-07-01

    The scaffold protein alpha-syntrophin (SNTA) regulates lipolysis indicating a role in lipid homeostasis. Adipocytes are the main lipid storage cells in the body, and here, the function of SNTA has been analyzed in 3T3-L1 cells. SNTA is expressed in preadipocytes and is induced early during adipogenesis. Knock-down of SNTA in preadipocytes increases their proliferation. Proteins which are induced during adipogenesis like adiponectin and caveolin-1, and the inflammatory cytokine IL-6 are at normal levels in the mature cells differentiated from preadipocytes with low SNTA. This suggests that SNTA does neither affect differentiation nor inflammation. Expression of proteins with a role in cholesterol and triglyceride homeostasis is unchanged. Consequently, basal and epinephrine induced lipolysis as well as insulin stimulated phosphorylation of Akt and ERK1/2 are normal. Importantly, adipocytes with low SNTA form smaller lipid droplets and store less triglycerides. Stearoyl-CoA reductase and MnSOD are reduced upon SNTA knock-down but do not contribute to lower lipid levels. Oleate uptake is even increased in cells with SNTA knock-down. In summary, current data show that SNTA is involved in the expansion of lipid droplets independent of adipogenesis. Enhanced preadipocyte proliferation and capacity to store surplus fatty acids may protect adipocytes with low SNTA from lipotoxicity in obesity. PMID:27242274

  1. The adaptor protein Cindr regulates JNK activity to maintain epithelial sheet integrity.

    PubMed

    Yasin, Hannah W R; van Rensburg, Samuel H; Feiler, Christina E; Johnson, Ruth I

    2016-02-15

    Epithelia are essential barrier tissues that must be appropriately maintained for their correct function. To achieve this a plethora of protein interactions regulate epithelial cell number, structure and adhesion, and differentiation. Here we show that Cindr (the Drosophila Cin85 and Cd2ap ortholog) is required to maintain epithelial integrity. Reducing Cindr triggered cell delamination and movement. Most delaminating cells died. These behaviors were consistent with JNK activation previously associated with loss of epithelial integrity in response to ectopic oncogene activity. We confirmed a novel interaction between Cindr and Drosophila JNK (dJNK), which when perturbed caused inappropriate JNK signaling. Genetically reducing JNK signaling activity suppressed the effects of reducing Cindr. Furthermore, ectopic JNK signaling phenocopied loss of Cindr and was partially rescued by concomitant cindr over-expression. Thus, correct Cindr-dJNK stoichiometry is essential to maintain epithelial integrity and disturbing this balance may contribute to the pathogenesis of disease states, including cancer. PMID:26772997

  2. A combinatorial F box protein directed pathway controls TRAF adaptor stability to regulate inflammation.

    PubMed

    Chen, Bill B; Coon, Tiffany A; Glasser, Jennifer R; McVerry, Bryan J; Zhao, Jing; Zhao, Yutong; Zou, Chunbin; Ellis, Bryon; Sciurba, Frank C; Zhang, Yingze; Mallampalli, Rama K

    2013-05-01

    Uncontrolled activation of tumor necrosis factor receptor-associated factor (TRAF) proteins may result in profound tissue injury by linking surface signals to cytokine release. Here we show that a ubiquitin E3 ligase component, Fbxo3, potently stimulates cytokine secretion from human inflammatory cells by destabilizing a sentinel TRAF inhibitor, Fbxl2. Fbxo3 and TRAF protein in circulation positively correlated with cytokine responses in subjects with sepsis, and we identified a polymorphism in human Fbxo3, with one variant being hypofunctional. A small-molecule inhibitor targeting Fbxo3 was sufficient to lessen severity of cytokine-driven inflammation in several mouse disease models. These studies identified a pathway of innate immunity that may be useful to detect subjects with altered immune responses during critical illness or provide a basis for therapeutic intervention targeting TRAF protein abundance. PMID:23542741

  3. Adaptor Protein MecA Is a Negative Regulator of the Expression of Late Competence Genes in Streptococcus thermophilus

    PubMed Central

    Boutry, Céline; Wahl, Astrid; Delplace, Brigitte; Clippe, André; Fontaine, Laetitia

    2012-01-01

    In Streptococcus thermophilus, the ComRS regulatory system governs the transcriptional level of comX expression and, hence, controls the early stage of competence development. The present work focuses on the posttranslational control of the activity of the sigma factor ComX and, therefore, on the late stage of competence regulation. In silico analysis performed on the S. thermophilus genome revealed the presence of a homolog of mecA (mecASt), which codes for the adaptor protein that is involved in ComK degradation by ClpCP in Bacillus subtilis. Using reporter strains and microarray experiments, we showed that MecASt represses late competence genes without affecting the early competence stage under conditions that are not permissive for competence development. In addition, this repression mechanism was found not only to act downstream of comX expression but also to be fully dependent on the presence of a functional comX gene. This negative control was similarly released in strains deleted for clpC, mecA, and clpC-mecA. Under artificial conditions of comX expression, we next showed that the abundance of ComX is higher in the absence of MecA or ClpC. Finally, results of bacterial two-hybrid assays strongly suggested that MecA interacts with both ComX and ClpC. Based on these results, we proposed that ClpC and MecA act together in the same regulatory circuit to control the abundance of ComX in S. thermophilus. PMID:22287513

  4. The AP2 clathrin adaptor protein complex regulates the abundance of GLR-1 glutamate receptors in the ventral nerve cord of Caenorhabditis elegans

    PubMed Central

    Garafalo, Steven D.; Luth, Eric S.; Moss, Benjamin J.; Monteiro, Michael I.; Malkin, Emily; Juo, Peter

    2015-01-01

    Regulation of glutamate receptor (GluR) abundance at synapses by clathrin-mediated endocytosis can control synaptic strength and plasticity. We take advantage of viable, null mutations in subunits of the clathrin adaptor protein 2 (AP2) complex in Caenorhabditis elegans to characterize the in vivo role of AP2 in GluR trafficking. In contrast to our predictions for an endocytic adaptor, we found that levels of the GluR GLR-1 are decreased at synapses in the ventral nerve cord (VNC) of animals with mutations in the AP2 subunits APM-2/μ2, APA-2/α, or APS-2/σ2. Rescue experiments indicate that APM-2/μ2 functions in glr-1–expressing interneurons and the mature nervous system to promote GLR-1 levels in the VNC. Genetic analyses suggest that APM-2/μ2 acts upstream of GLR-1 endocytosis in the VNC. Consistent with this, GLR-1 accumulates in cell bodies of apm-2 mutants. However, GLR-1 does not appear to accumulate at the plasma membrane of the cell body as expected, but instead accumulates in intracellular compartments including Syntaxin-13– and RAB-14–labeled endosomes. This study reveals a novel role for the AP2 clathrin adaptor in promoting the abundance of GluRs at synapses in vivo, and implicates AP2 in the regulation of GluR trafficking at an early step in the secretory pathway. PMID:25788288

  5. MEK Kinase 2 and the Adaptor Protein Lad Regulate Extracellular Signal-Regulated Kinase 5 Activation by Epidermal Growth Factor via Src

    PubMed Central

    Sun, Weiyong; Wei, Xudong; Kesavan, Kamala; Garrington, Timothy P.; Fan, Ruihua; Mei, Junjie; Anderson, Steven M.; Gelfand, Erwin W.; Johnson, Gary L.

    2003-01-01

    Lad is an SH2 domain-containing adaptor protein that binds MEK kinase 2 (MEKK2), a mitogen-activated protein kinase (MAPK) kinase kinase for the extracellular signal-regulated kinase 5 (ERK5) and JNK pathways. Lad and MEKK2 are in a complex in resting cells. Antisense knockdown of Lad expression and targeted gene disruption of MEKK2 expression results in loss of epidermal growth factor (EGF) and stress stimuli-induced activation of ERK5. Activation of MEKK2 and the ERK5 pathway by EGF and stress stimuli is dependent on Src kinase activity. The Lad-binding motif is encoded within amino acids 228 to 282 in the N terminus of MEKK2, and expression of this motif blocks Lad-MEKK2 interaction, resulting in inhibition of Src-dependent activation of MEKK2 and ERK5. JNK activation by EGF is similarly inhibited by loss of Lad or MEKK2 expression and by blocking the interaction of MEKK2 and Lad. Our studies demonstrate that Src kinase activity is required for ERK5 activation in response to EGF, MEKK2 expression is required for ERK5 activation by Src, Lad and MEKK2 association is required for Src activation of ERK5, and EGF and Src stimulation of ERK5-regulated MEF2-dependent promoter activity requires a functional Lad-MEKK2 signaling complex. PMID:12640115

  6. Role of Adaptor Protein Toll-Like Interleukin Domain Containing Adaptor Inducing Interferon β in Toll-Like Receptor 3- and 4-Mediated Regulation of Hepatic Drug Metabolizing Enzyme and Transporter Genes.

    PubMed

    Shah, Pranav; Omoluabi, Ozozoma; Moorthy, Bhagavatula; Ghose, Romi

    2016-01-01

    The expressions and activities of hepatic drug-metabolizing enzymes and transporters (DMETs) are altered during infection and inflammation. Inflammatory responses in the liver are mediated primarily by Toll-like receptor (TLR)-signaling, which involves recruitment of Toll/interleukin (IL)-1 receptor (TIR) domain containing adaptor protein (TIRAP) and TIR domain containing adaptor inducing interferon (IFN)-β (TRIF) that eventually leads to induction of proinflammatory cytokines and mitogen-activated protein kinases (MAPKs). Lipopolysaccharide (LPS) activates the Gram-negative bacterial receptor TLR4 and polyinosinic:polycytidylic acid (polyI:C) activates the viral receptor TLR3. TLR4 signaling involves TIRAP and TRIF, whereas TRIF is the only adaptor protein involved in the TLR3 pathway. We have shown previously that LPS-mediated downregulation of DMETs is independent of TIRAP. To determine the role of TRIF, we treated TRIF(+/+) and TRIF(-/-) mice with LPS or polyI:C. LPS downregulated (∼40%-60%) Cyp3a11, Cyp2a4, Ugt1a1, Mrp2 mRNA levels, whereas polyI:C downregulated (∼30%-60%) Cyp3a11, Cyp2a4, Cyp1a2, Cyp2b10, Ugt1a1, Mrp2, and Mrp3 mRNA levels in TRIF(+/+) mice. This downregulation was not attenuated in TRIF(-/-) mice. Induction of cytokines by LPS was observed in both TRIF(+/+) and TRIF(-/-) mice. Cytokine induction was delayed in polyI:C-treated TRIF(-/-) mice, indicating that multiple mechanisms mediating polyI:C signaling exist. To assess the role of MAPKs, primary hepatocytes were pretreated with specific inhibitors before treatment with LPS/polyI:C. We found that only the c-jun-N-terminal kinase (JNK) inhibitor attenuated the down-regulation of DMETs. These results show that TRIF-independent pathways can be involved in the downregulation of DMETs through TLR4 and 3. JNK-dependent mechanisms likely mediate this downregulation. PMID:26470915

  7. Adaptor protein containing PH domain, PTB domain and leucine zipper (APPL1) regulates the protein level of EGFR by modulating its trafficking

    SciTech Connect

    Lee, Jae-Rin; Hahn, Hwa-Sun; Kim, Young-Hoon; Nguyen, Hong-Hoa; Yang, Jun-Mo; Kang, Jong-Sun; Hahn, Myong-Joon

    2011-11-11

    Highlights: Black-Right-Pointing-Pointer APPL1 regulates the protein level of EGFR in response to EGF stimulation. Black-Right-Pointing-Pointer Depletion of APPL1 accelerates the movement of EGF/EGFR from the cell surface to the perinuclear region in response to EGF. Black-Right-Pointing-Pointer Knockdown of APPL1 enhances the activity of Rab5. -- Abstract: The EGFR-mediated signaling pathway regulates multiple biological processes such as cell proliferation, survival and differentiation. Previously APPL1 (adaptor protein containing PH domain, PTB domain and leucine zipper 1) has been reported to function as a downstream effector of EGF-initiated signaling. Here we demonstrate that APPL1 regulates EGFR protein levels in response to EGF stimulation. Overexpression of APPL1 enhances EGFR stabilization while APPL1 depletion by siRNA reduces EGFR protein levels. APPL1 depletion accelerates EGFR internalization and movement of EGF/EGFR from cell surface to the perinuclear region in response to EGF treatment. Conversely, overexpression of APPL1 decelerates EGFR internalization and translocation of EGF/EGFR to the perinuclear region. Furthermore, APPL1 depletion enhances the activity of Rab5 which is involved in internalization and trafficking of EGFR and inhibition of Rab5 in APPL1-depleted cells restored EGFR levels. Consistently, APPL1 depletion reduced activation of Akt, the downstream signaling effector of EGFR and this is restored by inhibition of Rab5. These findings suggest that APPL1 is required for EGFR signaling by regulation of EGFR stabilities through inhibition of Rab5.

  8. Protein Modifications Regulate the Role of 14-3-3γ Adaptor Protein in cAMP-induced Steroidogenesis in MA-10 Leydig Cells*

    PubMed Central

    Aghazadeh, Yasaman; Ye, Xiaoying; Blonder, Josip; Papadopoulos, Vassilios

    2014-01-01

    The 14-3-3 protein family comprises adaptors and scaffolds that regulate intracellular signaling pathways. The 14-3-3γ isoform is a negative regulator of steroidogenesis that is hormonally induced and transiently functions at the initiation of steroidogenesis by delaying maximal steroidogenesis in MA-10 mouse tumor Leydig cells. Treatment of MA-10 cells with the cAMP analog 8-bromo-cAMP (8-Br-cAMP), which stimulates steroidogenesis, triggers the interaction of 14-3-3γ with the steroidogenic acute regulatory protein (STAR) in the cytosol, limiting STAR activity to basal levels. Over time, this interaction ceases, allowing for a 2-fold induction in STAR activity and maximal increase in the rate of steroid formation. The 14-3-3γ/STAR pattern of interaction was found to be opposite that of the 14-3-3γ homodimerization pattern. Phosphorylation and acetylation of 14-3-3γ showed similar patterns to homodimerization and STAR binding, respectively. 14-3-3γ Ser58 phosphorylation and 14-3-3γ Lys49 acetylation were blocked using trans-activator of HIV transcription factor 1 peptides coupled to 14-3-3γ sequences containing Ser58 or Lys49. Blocking either one of these modifications further induced 8-Br-cAMP-induced steroidogenesis while reducing lipid storage, suggesting that the stored cholesterol is used for steroid formation. Taken together, these results indicate that Ser58 phosphorylation and Lys49 acetylation of 14-3-3γ occur in a coordinated time-dependent manner to regulate 14-3-3γ homodimerization. 14-3-3γ Ser58 phosphorylation is required for STAR interactions under control conditions, and 14-3-3γ Lys49 acetylation is important for the cAMP-dependent induction of these interactions. PMID:25086053

  9. Skb5, an SH3 adaptor protein, regulates Pmk1 MAPK signaling by controlling the intracellular localization of the MAPKKK Mkh1.

    PubMed

    Kanda, Yuki; Satoh, Ryosuke; Matsumoto, Saki; Ikeda, Chisato; Inutsuka, Natsumi; Hagihara, Kanako; Matzno, Sumio; Tsujimoto, Sho; Kita, Ayako; Sugiura, Reiko

    2016-08-15

    The mitogen-activated protein kinase (MAPK) cascade is a highly conserved signaling module composed of MAPK kinase kinases (MAPKKKs), MAPK kinases (MAPKK) and MAPKs. The MAPKKK Mkh1 is an initiating kinase in Pmk1 MAPK signaling, which regulates cell integrity in fission yeast (Schizosaccharomyces pombe). Our genetic screen for regulators of Pmk1 signaling identified Shk1 kinase binding protein 5 (Skb5), an SH3-domain-containing adaptor protein. Here, we show that Skb5 serves as an inhibitor of Pmk1 MAPK signaling activation by downregulating Mkh1 localization to cell tips through its interaction with the SH3 domain. Consistent with this, the Mkh1(3PA) mutant protein, with impaired Skb5 binding, remained in the cell tips, even when Skb5 was overproduced. Intriguingly, Skb5 needs Mkh1 to localize to the growing ends as Mkh1 deletion and disruption of Mkh1 binding impairs Skb5 localization. Deletion of Pck2, an upstream activator of Mkh1, impaired the cell tip localization of Mkh1 and Skb5 as well as the Mkh1-Skb5 interaction. Interestingly, both Pck2 and Mkh1 localized to the cell tips at the G1/S phase, which coincided with Pmk1 MAPK activation. Taken together, Mkh1 localization to cell tips is important for transmitting upstream signaling to Pmk1, and Skb5 spatially regulates this process. PMID:27451356

  10. AP-1/σ1A and AP-1/σ1B adaptor-proteins differentially regulate neuronal early endosome maturation via the Rab5/Vps34-pathway

    PubMed Central

    Candiello, Ermes; Kratzke, Manuel; Wenzel, Dirk; Cassel, Dan; Schu, Peter

    2016-01-01

    The σ1 subunit of the AP-1 clathrin-coated-vesicle adaptor-protein complex is expressed as three isoforms. Tissues express σ1A and one of the σ1B and σ1C isoforms. Brain is the tissue with the highest σ1A and σ1B expression. σ1B-deficiency leads to severe mental retardation, accumulation of early endosomes in synapses and fewer synaptic vesicles, whose recycling is slowed down. AP-1/σ1A and AP-1/σ1B regulate maturation of these early endosomes into multivesicular body late endosomes, thereby controlling synaptic vesicle protein transport into a degradative pathway. σ1A binds ArfGAP1, and with higher affinity brain-specific ArfGAP1, which bind Rabex-5. AP-1/σ1A-ArfGAP1-Rabex-5 complex formation leads to more endosomal Rabex-5 and enhanced, Rab5GTP-stimulated Vps34 PI3-kinase activity, which is essential for multivesicular body endosome formation. Formation of AP-1/σ1A-ArfGAP1-Rabex-5 complexes is prevented by σ1B binding of Rabex-5 and the amount of endosomal Rabex-5 is reduced. AP-1 complexes differentially regulate endosome maturation and coordinate protein recycling and degradation, revealing a novel molecular mechanism by which they regulate protein transport besides their established function in clathrin-coated-vesicle formation. PMID:27411398

  11. A novel class of antihyperlipidemic agents with low density lipoprotein receptor up-regulation via the adaptor protein autosomal recessive hypercholesterolemia.

    PubMed

    Asano, Shigehiro; Ban, Hitoshi; Tsuboya, Norie; Uno, Shinsaku; Kino, Kouichi; Ioriya, Katsuhisa; Kitano, Masafumi; Ueno, Yoshihide

    2010-04-22

    We have previously reported compound 2 as a inhibitor of acyl-coenzyme A:cholesterol O-acyltransferase (ACAT) and up-regulator of the low density lipoprotein receptor (LDL-R) expression. In this study we focused on compound 2, a unique LDL-R up-regulator, and describe the discovery of a novel class of up-regulators of LDL-R. Replacement the methylene urea linker in compound 2 with an acylsulfonamide linker kept a potent LDL-R up-regulatory activity, and subsequent optimization work gave compound 39 as a highly potent LDL-R up-regulator (39; EC(25) = 0.047 microM). Compound 39 showed no ACAT inhibitory activity even at 1 microM. The sodium salts of compound 39 reduced plasma total and LDL cholesterol levels in a dose-dependent manner in an experimental animal model of hyperlipidemia. Moreover, we revealed in this study using RNA interference that autosomal recessive hypercholesterolemia (ARH), an adaptor protein of LDL-R, is essential for compound 39 up-regulation of LDL-R expression. PMID:20356098

  12. The Adaptor Complex AP-4 Regulates Vacuolar Protein Sorting at the trans-Golgi Network by Interacting with VACUOLAR SORTING RECEPTOR1.

    PubMed

    Fuji, Kentaro; Shirakawa, Makoto; Shimono, Yuki; Kunieda, Tadashi; Fukao, Yoichiro; Koumoto, Yasuko; Takahashi, Hideyuki; Hara-Nishimura, Ikuko; Shimada, Tomoo

    2016-01-01

    Adaptor protein (AP) complexes play critical roles in protein sorting among different post-Golgi pathways by recognizing specific cargo protein motifs. Among the five AP complexes (AP-1-AP-5) in plants, AP-4 is one of the most poorly understood; the AP-4 components, AP-4 cargo motifs, and AP-4 functional mechanism are not known. Here, we identify the AP-4 components and show that the AP-4 complex regulates receptor-mediated vacuolar protein sorting by recognizing VACUOLAR SORTING RECEPTOR1 (VSR1), which was originally identified as a sorting receptor for seed storage proteins to target protein storage vacuoles in Arabidopsis (Arabidopsis thaliana). From the vacuolar sorting mutant library GREEN FLUORESCENT SEED (GFS), we isolated three gfs mutants that accumulate abnormally high levels of VSR1 in seeds and designated them as gfs4, gfs5, and gfs6. Their responsible genes encode three (AP4B, AP4M, and AP4S) of the four subunits of the AP-4 complex, respectively, and an Arabidopsis mutant (ap4e) lacking the fourth subunit, AP4E, also had the same phenotype. Mass spectrometry demonstrated that these four proteins form a complex in vivo. The four mutants showed defects in the vacuolar sorting of the major storage protein 12S globulins, indicating a role for the AP-4 complex in vacuolar protein transport. AP4M bound to the tyrosine-based motif of VSR1. AP4M localized at the trans-Golgi network (TGN) subdomain that is distinct from the AP-1-localized TGN subdomain. This study provides a novel function for the AP-4 complex in VSR1-mediated vacuolar protein sorting at the specialized domain of the TGN. PMID:26546666

  13. The interaction of Kinesin-1 with its adaptor protein JIP1 can be regulated via proteins binding to the JIP1-PTB domain

    PubMed Central

    2013-01-01

    Background The regulatory mechanisms of motor protein-dependent intracellular transport are still not fully understood. The kinesin-1-binding protein, JIP1, can function as an adaptor protein that links kinesin-1 and other JIP1-binding “cargo” proteins. However, it is unknown whether these “cargo” proteins influence the JIP1–kinesin-1 binding. Results We show here that JIP1–kinesin-1 binding in Neuro2a cells was dependent on conserved amino acid residues in the JIP1-phosphotyrosine binding (PTB) domain, including F687. In addition, mutation of F687 severely affected the neurite tip localization of JIP1. Proteomic analysis revealed another kinesin-1 binding protein, JIP3, as a major JIP1 binding protein. The association between JIP1 and JIP3 was dependent on the F687 residue in JIP1, and this association induced the formation of a stable ternary complex with kinesin-1. On the other hand, the binding of JIP1 and JIP3 was independent of kinesin-1 binding. We also show that other PTB binding proteins can interrupt the formation of the ternary complex. Conclusions The formation of the JIP1–kinesin-1 complex depends on the protein binding-status of the JIP1 PTB domain. This may imply a regulatory mechanism of kinesin-1-dependent intracellular transport. PMID:23496950

  14. REGULATION OF PROCESS RETRACTION AND CELL MIGRATION BY EPHA3 IS MEDIATED BY THE ADAPTOR PROTEIN NCK1†

    PubMed Central

    Hu, Tianjing; Shi, Guanfang; Larose, Louise; Rivera, Gonzalo M.; Mayer, Bruce J.; Zhou, Renping

    2009-01-01

    The Eph family of tyrosine kinase receptors and their ligands, the ephrins, participates in the regulation of a wide variety of biological functions under normal and pathological conditions. During embryonic development, interactions between the ligands and receptors define tissue boundaries, guide migrating axons, and regulate angiogenesis, as well as bone morphogenesis. These molecules have also been shown to modify neural activity in the adult nervous system, and influence tumor progression. However, the molecular mechanisms underlying these diverse functions are not completely understood. In the present study, a yeast two-hybrid screen has been conducted to identify molecules that physically interact with Eph receptors using the cytoplasmic domain of EphA3 as “bait”. This study identified Nck1 as a strong binding partner of EphA3 as assayed using both GST-fusion protein pull down and co-immunoprecipitation techniques. The interaction is mediated through binding of the Nck1 SH2 domain to the phosphotyrosine residue at position 602 (Y602) of EphA3 receptor. The removal of the SH2 domain or the mutation of the Y602 residue abolishes the interaction. It is further demonstrated that EphA3 activation inhibits cell migration and process outgrowth, and these inhibiting effects are partially alleviated by dominant-negative Nck1 mutants that lack functional SH2 or SH3 domains, but not by the wild type Nck1 gene. These results suggest that Nck1 interacts with EphA3 to regulate cell migration and process retraction. PMID:19505147

  15. The adaptor protein SAP directly associates with PECAM-1 and regulates PECAM-1-mediated-cell adhesion in T-like cell lines.

    PubMed

    Proust, Richard; Crouin, Catherine; Gandji, Leslie Yewakon; Bertoglio, Jacques; Gesbert, Franck

    2014-04-01

    SAP is a small cytosolic adaptor protein expressed in hematopoietic lineages whose main function is to regulate intracellular signaling pathways induced by the triggering of members of the SLAM receptor family. In this paper, we have identified the adhesion molecule PECAM-1 as a new partner for SAP in a conditional yeast two-hybrid screen. PECAM-1 is an immunoglobulin-like molecule expressed by endothelial cells and leukocytes, which possesses both pro- and anti-inflammatory properties. However, little is known about PECAM-1 functions in T cells. We show that SAP directly and specifically interacts with the cytosolic tyrosine 686 of PECAM-1. We generated different T-like cell lines in which SAP or PECAM-1 are expressed or down modulated and we demonstrate that a diminished SAP expression correlates with a diminished PECAM-1-mediated adhesion. Although SAP has mainly been shown to associate with SLAM receptors, we evidence here that SAP is a new actor downstream of PECAM-1. PMID:24388971

  16. Protease-activated Receptor-4 Signaling and Trafficking Is Regulated by the Clathrin Adaptor Protein Complex-2 Independent of β-Arrestins.

    PubMed

    Smith, Thomas H; Coronel, Luisa J; Li, Julia G; Dores, Michael R; Nieman, Marvin T; Trejo, JoAnn

    2016-08-26

    Protease-activated receptor-4 (PAR4) is a G protein-coupled receptor (GPCR) for thrombin and is proteolytically activated, similar to the prototypical PAR1. Due to the irreversible activation of PAR1, receptor trafficking is intimately linked to signal regulation. However, unlike PAR1, the mechanisms that control PAR4 trafficking are not known. Here, we sought to define the mechanisms that control PAR4 trafficking and signaling. In HeLa cells depleted of clathrin by siRNA, activated PAR4 failed to internalize. Consistent with clathrin-mediated endocytosis, expression of a dynamin dominant-negative K44A mutant also blocked activated PAR4 internalization. However, unlike most GPCRs, PAR4 internalization occurred independently of β-arrestins and the receptor's C-tail domain. Rather, we discovered a highly conserved tyrosine-based motif in the third intracellular loop of PAR4 and found that the clathrin adaptor protein complex-2 (AP-2) is important for internalization. Depletion of AP-2 inhibited PAR4 internalization induced by agonist. In addition, mutation of the critical residues of the tyrosine-based motif disrupted agonist-induced PAR4 internalization. Using Dami megakaryocytic cells, we confirmed that AP-2 is required for agonist-induced internalization of endogenous PAR4. Moreover, inhibition of activated PAR4 internalization enhanced ERK1/2 signaling, whereas Akt signaling was markedly diminished. These findings indicate that activated PAR4 internalization requires AP-2 and a tyrosine-based motif and occurs independent of β-arrestins, unlike most classical GPCRs. Moreover, these findings are the first to show that internalization of activated PAR4 is linked to proper ERK1/2 and Akt activation. PMID:27402844

  17. Small-molecule control of protein degradation using split adaptors

    PubMed Central

    Davis, Joseph H.; Baker, Tania A.; Sauer, Robert T.

    2011-01-01

    Targeted intracellular degradation provides a method to study the biological function of proteins and has numerous applications in biotechnology. One promising approach uses adaptor proteins to target substrates with genetically encoded degradation tags for proteolysis. Here, we describe an engineered split-adaptor system, in which adaptor assembly and delivery of substrates to the ClpXP protease depends on a small molecule (rapamycin). This degradation system does not require modification of endogenous proteases, functions robustly over a wide range of adaptor concentrations, and does not require new synthesis of adaptors or proteases to initiate degradation. We demonstrate the efficacy of this system in E. coli by degrading tagged variants of LacI repressor and FtsA, an essential cell-division protein. In the latter case, addition of rapamycin causes pronounced filamentation because daughter cells cannot divide. Strikingly, washing rapamycin away reverses this phenotype. Our system is highly modular, with clearly-defined interfaces for substrate binding, protease binding, and adaptor assembly, providing a clear path to extend this system to other degradation tags, proteases, or induction systems. Together, these new reagents should be useful in controlling protein degradation in bacteria. PMID:21866931

  18. Small-molecule control of protein degradation using split adaptors.

    PubMed

    Davis, Joseph H; Baker, Tania A; Sauer, Robert T

    2011-11-18

    Targeted intracellular degradation provides a method to study the biological function of proteins and has numerous applications in biotechnology. One promising approach uses adaptor proteins to target substrates with genetically encoded degradation tags for proteolysis. Here, we describe an engineered split-adaptor system, in which adaptor assembly and delivery of substrates to the ClpXP protease depends on a small molecule (rapamycin). This degradation system does not require modification of endogenous proteases, functions robustly over a wide range of adaptor concentrations, and does not require new synthesis of adaptors or proteases to initiate degradation. We demonstrate the efficacy of this system in E. coli by degrading tagged variants of LacI repressor and FtsA, an essential cell-division protein. In the latter case, addition of rapamycin causes pronounced filamentation because daughter cells cannot divide. Strikingly, washing rapamycin away reverses this phenotype. Our system is highly modular, with clearly defined interfaces for substrate binding, protease binding, and adaptor assembly, providing a clear path to extend this system to other degradation tags, proteases, or induction systems. Together, these new reagents should be useful in controlling protein degradation in bacteria. PMID:21866931

  19. The Emerging and Diverse Roles of Src-Like Adaptor Proteins in Health and Disease

    PubMed Central

    Marton, Nikolett; Baricza, Eszter; Érsek, Barbara; Buzás, Edit I.; Nagy, György

    2015-01-01

    Although Src-like adaptor proteins (SLAP-1 and SLAP-2) were mainly studied in lymphocytes, where they act as negative regulators and provide fine control of receptor signaling, recently, several other functions of these proteins were discovered. In addition to the well-characterized immunoregulatory functions, SLAP proteins appear to have an essential role in the pathogenesis of type I hypersensitivity, osteoporosis, and numerous malignant diseases. Both adaptor proteins are expressed in a wide variety of tissues, where they have mostly inhibitory effects on multiple intracellular signaling pathways. In this review, we summarize the diverse effects of SLAP proteins. PMID:26339145

  20. Energy metabolism regulates clathrin adaptors at the trans-Golgi network and endosomes

    PubMed Central

    Aoh, Quyen L.; Hung, Chao-wei; Duncan, Mara C.

    2013-01-01

    Glucose is a master regulator of cell behavior in the yeast Saccharomyces cerevisiae. It acts as both a metabolic substrate and a potent regulator of intracellular signaling cascades. Glucose starvation induces the transient delocalization and then partial relocalization of clathrin adaptors at the trans-Golgi network and endosomes. Although these localization responses are known to depend on the protein kinase A (PKA) signaling pathway, the molecular mechanism of this regulation is unknown. Here we demonstrate that PKA and the AMP-regulated kinase regulate adaptor localization through changes in energy metabolism. We show that genetic and chemical manipulation of intracellular ATP levels cause corresponding changes in adaptor localization. In permeabilized cells, exogenous ATP is sufficient to induce adaptor localization. Furthermore, we reveal distinct energy-dependent steps in adaptor localization: a step that requires the ADP-ribosylation factor ARF, an ATP-dependent step that requires the phosphatidyl-inositol-4 kinase Pik1, and third ATP-dependent step for which we provide evidence but for which the mechanism is unknown. We propose that these energy-dependent mechanisms precisely synchronize membrane traffic with overall proliferation rates and contribute a crucial aspect of energy conservation during acute glucose starvation. PMID:23345590

  1. Interchangeable adaptors regulate mitochondrial dynamin assembly for membrane scission

    PubMed Central

    Koirala, Sajjan; Guo, Qian; Kalia, Raghav; Bui, Huyen T.; Eckert, Debra M.; Frost, Adam; Shaw, Janet M.

    2013-01-01

    Mitochondrial fission is mediated by the dynamin-related GTPases Dnm1/Drp1 (yeast/mammals), which form spirals around constricted sites on mitochondria. Additional membrane-associated adaptor proteins (Fis1, Mdv1, Mff, and MiDs) are required to recruit these GTPases from the cytoplasm to the mitochondrial surface. Whether these adaptors participate in both GTPase recruitment and membrane scission is not known. Here we use a yeast strain lacking all fission proteins to identify the minimal combinations of GTPases and adaptors sufficient for mitochondrial fission. Although Fis1 is dispensable for fission, membrane-anchored Mdv1, Mff, or MiDs paired individually with their respective GTPases are sufficient to divide mitochondria. In addition to their role in Drp1 membrane recruitment, MiDs coassemble with Drp1 in vitro. The resulting heteropolymer adopts a dramatically different structure with a narrower diameter than Drp1 homopolymers assembled in isolation. This result demonstrates that an adaptor protein alters the architecture of a mitochondrial dynamin GTPase polymer in a manner that could facilitate membrane constriction and severing activity. PMID:23530241

  2. SR proteins are NXF1 adaptors that link alternative RNA processing to mRNA export.

    PubMed

    Müller-McNicoll, Michaela; Botti, Valentina; de Jesus Domingues, Antonio M; Brandl, Holger; Schwich, Oliver D; Steiner, Michaela C; Curk, Tomaz; Poser, Ina; Zarnack, Kathi; Neugebauer, Karla M

    2016-03-01

    Nuclear export factor 1 (NXF1) exports mRNA to the cytoplasm after recruitment to mRNA by specific adaptor proteins. How and why cells use numerous different export adaptors is poorly understood. Here we critically evaluate members of the SR protein family (SRSF1-7) for their potential to act as NXF1 adaptors that couple pre-mRNA processing to mRNA export. Consistent with this proposal, >1000 endogenous mRNAs required individual SR proteins for nuclear export in vivo. To address the mechanism, transcriptome-wide RNA-binding profiles of NXF1 and SRSF1-7 were determined in parallel by individual-nucleotide-resolution UV cross-linking and immunoprecipitation (iCLIP). Quantitative comparisons of RNA-binding sites showed that NXF1 and SR proteins bind mRNA targets at adjacent sites, indicative of cobinding. SRSF3 emerged as the most potent NXF1 adaptor, conferring sequence specificity to RNA binding by NXF1 in last exons. Interestingly, SRSF3 and SRSF7 were shown to bind different sites in last exons and regulate 3' untranslated region length in an opposing manner. Both SRSF3 and SRSF7 promoted NXF1 recruitment to mRNA. Thus, SRSF3 and SRSF7 couple alternative splicing and polyadenylation to NXF1-mediated mRNA export, thereby controlling the cytoplasmic abundance of transcripts with alternative 3' ends. PMID:26944680

  3. SR proteins are NXF1 adaptors that link alternative RNA processing to mRNA export

    PubMed Central

    Müller-McNicoll, Michaela; Botti, Valentina; de Jesus Domingues, Antonio M.; Brandl, Holger; Schwich, Oliver D.; Steiner, Michaela C.; Curk, Tomaz; Poser, Ina; Zarnack, Kathi; Neugebauer, Karla M.

    2016-01-01

    Nuclear export factor 1 (NXF1) exports mRNA to the cytoplasm after recruitment to mRNA by specific adaptor proteins. How and why cells use numerous different export adaptors is poorly understood. Here we critically evaluate members of the SR protein family (SRSF1–7) for their potential to act as NXF1 adaptors that couple pre-mRNA processing to mRNA export. Consistent with this proposal, >1000 endogenous mRNAs required individual SR proteins for nuclear export in vivo. To address the mechanism, transcriptome-wide RNA-binding profiles of NXF1 and SRSF1–7 were determined in parallel by individual-nucleotide-resolution UV cross-linking and immunoprecipitation (iCLIP). Quantitative comparisons of RNA-binding sites showed that NXF1 and SR proteins bind mRNA targets at adjacent sites, indicative of cobinding. SRSF3 emerged as the most potent NXF1 adaptor, conferring sequence specificity to RNA binding by NXF1 in last exons. Interestingly, SRSF3 and SRSF7 were shown to bind different sites in last exons and regulate 3′ untranslated region length in an opposing manner. Both SRSF3 and SRSF7 promoted NXF1 recruitment to mRNA. Thus, SRSF3 and SRSF7 couple alternative splicing and polyadenylation to NXF1-mediated mRNA export, thereby controlling the cytoplasmic abundance of transcripts with alternative 3′ ends. PMID:26944680

  4. Adaptor Protein-3-Mediated Trafficking of TLR2 Ligands Controls Specificity of Inflammatory Responses but Not Adaptor Complex Assembly.

    PubMed

    Petnicki-Ocwieja, Tanja; Kern, Aurelie; Killpack, Tess L; Bunnell, Stephen C; Hu, Linden T

    2015-11-01

    Innate immune engagement results in the activation of host defenses that produce microbe-specific inflammatory responses. A long-standing interest in the field of innate immunity is to understand how varied host responses are generated through the signaling of just a limited number of receptors. Recently, intracellular trafficking and compartmental partitioning have been identified as mechanisms that provide signaling specificity for receptors by regulating signaling platform assembly. We show that cytokine activation as a result of TLR2 stimulation occurs at different intracellular locations and is mediated by the phagosomal trafficking molecule adaptor protein-3 (AP-3). AP-3 is required for trafficking TLR2 purified ligands or the Lyme disease causing bacterium, Borrelia burgdorferi, to LAMP-1 lysosomal compartments. The presence of AP-3 is necessary for the activation of cytokines such as IL-6 but not TNF-α or type I IFNs, suggesting induction of these cytokines occurs from a different compartment. Lack of AP-3 does not interfere with the recruitment of TLR signaling adaptors TRAM and MyD88 to the phagosome, indicating that the TLR-MyD88 signaling complex is assembled at a prelysosomal stage and that IL-6 activation depends on proper localization of signaling molecules downstream of MyD88. Finally, infection of AP-3-deficient mice with B. burgdorferi resulted in altered joint inflammation during murine Lyme arthritis. Our studies further elucidate the effects of phagosomal trafficking on tailoring immune responses in vitro and in vivo. PMID:26423153

  5. Glucose regulates clathrin adaptors at the trans-Golgi network and endosomes

    PubMed Central

    Aoh, Quyen L.; Graves, Lee M.; Duncan, Mara C.

    2011-01-01

    Glucose is a rich source of energy and the raw material for biomass increase. Many eukaryotic cells remodel their physiology in the presence and absence of glucose. The yeast Saccharomyces cerevisiae undergoes changes in transcription, translation, metabolism, and cell polarity in response to glucose availability. Upon glucose starvation, translation initiation and cell polarity are immediately inhibited, and then gradually recover. In this paper, we provide evidence that, as in cell polarity and translation, traffic at the trans-Golgi network (TGN) and endosomes is regulated by glucose via an unknown mechanism that depends on protein kinase A (PKA). Upon glucose withdrawal, clathrin adaptors exhibit a biphasic change in localization: they initially delocalize from the membrane within minutes and later partially recover onto membranes. Additionally, the removal of glucose induces changes in posttranslational modifications of adaptors. Ras and Gpr1 signaling pathways, which converge on PKA, are required for changes in adaptor localization and changes in posttranslational modifications. Acute inhibition of PKA demonstrates that inhibition of PKA prior to glucose withdrawal prevents several adaptor responses to starvation. This study demonstrates that PKA activity prior to glucose starvation primes membrane traffic at the TGN and endosomes in response to glucose starvation. PMID:21832155

  6. Exploring structure and interactions of the bacterial adaptor protein YjbH by crosslinking mass spectrometry.

    PubMed

    Al-Eryani, Yusra; Ib Rasmussen, Morten; Kjellström, Sven; Højrup, Peter; Emanuelsson, Cecilia; von Wachenfeldt, Claes

    2016-09-01

    Adaptor proteins assist proteases in degrading specific proteins under appropriate conditions. The adaptor protein YjbH promotes the degradation of an important global transcriptional regulator Spx, which controls the expression of hundreds of genes and operons in response to thiol-specific oxidative stress in Bacillus subtilis. Under normal growth conditions, the transcription factor is bound to the adaptor protein and therefore degraded by the AAA+ protease ClpXP. If this binding is alleviated during stress, the transcription factor accumulates and turns on genes encoding stress-alleviating proteins. The adaptor protein YjbH is thus a key player involved in these interactions but its structure is unknown. To gain insight into its structure and interactions we have used chemical crosslinking mass spectrometry. Distance constraints obtained from the crosslinked monomer were used to select and validate a structure model of YjbH and then to probe its interactions with other proteins. The core structure of YjbH is reminiscent of DsbA family proteins. One lysine residue in YjbH (K177), located in one of the α-helices outside the thioredoxin fold, crosslinked to both Spx K99 and Spx K117, thereby suggesting one side of the YjbH for the interaction with Spx. Another lysine residue that crosslinked to Spx was YjbH K5, located in the long and presumably very flexible N-terminal arm of YjbH. Our crosslinking data lend support to a model proposed based on site-directed mutagenesis where the YjbH interaction with Spx can stabilize and present the C-terminal region of Spx for protease recognition and proteolysis. Proteins 2016; 84:1234-1245. © 2016 Wiley Periodicals, Inc. PMID:27191337

  7. Science Signaling Podcast for 12 July 2016: Adaptor proteins limit signaling.

    PubMed

    Wiley, H Steven; VanHook, Annalisa M

    2016-01-01

    This Podcast features an interview with Steven Wiley, senior author of a Research Article that appears in the 12 July 2016 issue of Science Signaling, about how the abundance of adaptor proteins and feedback regulators affect the flow of information downstream of the epidermal growth factor receptor (EGFR). Information flows through a signaling pathway by sequential interactions between core components of the pathway, many of which have enzymatic activity. Adaptor proteins do not directly participate in relaying the signal and do not have enzymatic activity, but are important for signaling because they facilitate interactions between the core components. Using quantitative methods, Shi et al demonstrated that core components of the EGFR pathway were highly abundant in both normal cells and cancer cells. However, adaptor proteins were present in much lower abundance in both cell types, indicating that it is the abundance of these proteins that limit signaling downstream of EGFR. The authors also found that differences in EGFR signaling between different cell types likely resulted from the variable abundance of feedback regulators.Listen to Podcast. PMID:27405978

  8. Two Clathrin Adaptor Protein Complexes Instruct Axon-Dendrite Polarity.

    PubMed

    Li, Pengpeng; Merrill, Sean A; Jorgensen, Erik M; Shen, Kang

    2016-05-01

    The cardinal feature of neuronal polarization is the establishment and maintenance of axons and dendrites. How axonal and dendritic proteins are sorted and targeted to different compartments is poorly understood. Here, we identified distinct dileucine motifs that are necessary and sufficient to target transmembrane proteins to either the axon or the dendrite through direct interactions with the clathrin-associated adaptor protein complexes (APs) in C. elegans. Axonal targeting requires AP-3, while dendritic targeting is mediated by AP-1. The axonal dileucine motif binds to AP-3 with higher efficiency than to AP-1. Both AP-3 and AP-1 are localized to the Golgi but occupy adjacent domains. We propose that AP-3 and AP-1 directly select transmembrane proteins and target them to axon and dendrite, respectively, by sorting them into distinct vesicle pools. PMID:27151641

  9. Regulation of ubiquitin-dependent cargo sorting by multiple endocytic adaptors at the plasma membrane

    PubMed Central

    Mayers, Jonathan R.; Wang, Lei; Pramanik, Jhuma; Johnson, Adam; Sarkeshik, Ali; Wang, Yueju; Saengsawang, Witchuda; Yates, John R.; Audhya, Anjon

    2013-01-01

    Endocytic protein trafficking is directed by sorting signals on cargo molecules that are recognized by cytosolic adaptor proteins. However, the steps necessary to segregate the variety of cargoes during endocytosis remain poorly defined. Using Caenorhabditis elegans, we demonstrate that multiple plasma membrane endocytic adaptors function redundantly to regulate clathrin-mediated endocytosis and to recruit components of the endosomal sorting complex required for transport (ESCRT) machinery to the cell surface to direct the sorting of ubiquitin-modified substrates. Moreover, our data suggest that preassembly of cargoes with the ESCRT-0 complex at the plasma membrane enhances the efficiency of downstream sorting events in the endolysosomal system. In the absence of a heterooligomeric adaptor complex composed of FCHO, Eps15, and intersectin, ESCRT-0 accumulation at the cell surface is diminished, and the degradation of a ubiquitin-modified cargo slows significantly without affecting the rate of its clathrin-mediated internalization. Consistent with a role for the ESCRT machinery during cargo endocytosis, we further show that the ESCRT-0 complex accumulates at a subset of clathrin-coated pits on the surface of human cells. Our findings suggest a unique mechanism by which ubiquitin-modified cargoes are sequestered into the endolysosomal pathway. PMID:23818590

  10. A novel interaction between the SH2 domain of signaling adaptor protein Nck-1 and the upstream regulator of the Rho family GTPase Rac1 engulfment and cell motility 1 (ELMO1) promotes Rac1 activation and cell motility.

    PubMed

    Zhang, Guo; Chen, Xia; Qiu, Fanghua; Zhu, Fengxin; Lei, Wenjing; Nie, Jing

    2014-08-15

    Nck family proteins function as adaptors to couple tyrosine phosphorylation signals to actin cytoskeleton reorganization. Several lines of evidence indicate that Nck family proteins involve in regulating the activity of Rho family GTPases. In the present study, we characterized a novel interaction between Nck-1 with engulfment and cell motility 1 (ELMO1). GST pull-down and co-immunoprecipitation assay demonstrated that the Nck-1-ELMO1 interaction is mediated by the SH2 domain of Nck-1 and the phosphotyrosine residues at position 18, 216, 395, and 511 of ELMO1. A R308K mutant of Nck-1 (in which the SH2 domain was inactive), or a 4YF mutant of ELMO1 lacking these four phosphotyrosine residues, diminished Nck-1-ELMO1 interaction. Conversely, tyrosine phosphatase inhibitor treatment and overexpression of Src family kinase Hck significantly enhanced Nck-1-ELMO1 interaction. Moreover, wild type Nck-1, but not R308K mutant, significantly augmented the interaction between ELMO1 and constitutively active RhoG (RhoG(V12A)), thus promoted Rac1 activation and cell motility. Taken together, the present study characterized a novel Nck-1-ELMO1 interaction and defined a new role for Nck-1 in regulating Rac1 activity. PMID:24928514

  11. Targeted decay of a regulatory small RNA by an adaptor protein for RNase E and counteraction by an anti-adaptor RNA

    PubMed Central

    Göpel, Yvonne; Papenfort, Kai; Reichenbach, Birte; Vogel, Jörg; Görke, Boris

    2013-01-01

    Bacterial small RNAs (sRNAs) are well established to regulate diverse cellular processes, but how they themselves are regulated is less understood. Recently, we identified a regulatory circuit wherein the GlmY and GlmZ sRNAs of Escherichia coli act hierarchically to activate mRNA glmS, which encodes glucosamine-6-phosphate (GlcN6P) synthase. Although the two sRNAs are highly similar, only GlmZ is a direct activator that base-pairs with the glmS mRNA, aided by protein Hfq. GlmY, however, does not bind Hfq and activates glmS indirectly by protecting GlmZ from RNA cleavage. This complex regulation feedback controls the levels of GlmS protein in response to its product, GlcN6P, a key metabolite in cell wall biosynthesis. Here, we reveal the molecular basis for the regulated turnover of GlmZ, identifying RapZ (RNase adaptor protein for sRNA GlmZ; formerly YhbJ) as a novel type of RNA-binding protein that recruits the major endoribonuclease RNase E to GlmZ. This involves direct interaction of RapZ with the catalytic domain of RNase E. GlmY binds RapZ through a secondary structure shared by both sRNAs and therefore acts by molecular mimicry as a specific decoy for RapZ. Thus, in analogy to regulated proteolysis, RapZ is an adaptor, and GlmY is an anti-adaptor in regulated turnover of a regulatory small RNA. PMID:23475961

  12. Nuclear Translocation of Crk Adaptor Proteins by the Influenza A Virus NS1 Protein

    PubMed Central

    Ylösmäki, Leena; Fagerlund, Riku; Kuisma, Inka; Julkunen, Ilkka; Saksela, Kalle

    2016-01-01

    The non-structural protein-1 (NS1) of many influenza A strains, especially those of avian origin, contains an SH3 ligand motif, which binds tightly to the cellular adaptor proteins Crk (Chicken tumor virus number 10 (CT10) regulator of kinase) and Crk-like adapter protein (CrkL). This interaction has been shown to potentiate NS1-induced activation of the phosphatidylinositol 3-kinase (PI3K), but additional effects on the host cell physiology may exist. Here we show that NS1 can induce an efficient translocation of Crk proteins from the cytoplasm into the nucleus, which results in an altered pattern of nuclear protein tyrosine phosphorylation. This was not observed using NS1 proteins deficient in SH3 binding or engineered to be exclusively cytoplasmic, indicating a physical role for NS1 as a carrier in the nuclear translocation of Crk. These data further emphasize the role of Crk proteins as host cell interaction partners of NS1, and highlight the potential for host cell manipulation gained by a viral protein simply via acquiring a short SH3 binding motif. PMID:27092521

  13. Nuclear Translocation of Crk Adaptor Proteins by the Influenza A Virus NS1 Protein.

    PubMed

    Ylösmäki, Leena; Fagerlund, Riku; Kuisma, Inka; Julkunen, Ilkka; Saksela, Kalle

    2016-01-01

    The non-structural protein-1 (NS1) of many influenza A strains, especially those of avian origin, contains an SH3 ligand motif, which binds tightly to the cellular adaptor proteins Crk (Chicken tumor virus number 10 (CT10) regulator of kinase) and Crk-like adapter protein (CrkL). This interaction has been shown to potentiate NS1-induced activation of the phosphatidylinositol 3-kinase (PI3K), but additional effects on the host cell physiology may exist. Here we show that NS1 can induce an efficient translocation of Crk proteins from the cytoplasm into the nucleus, which results in an altered pattern of nuclear protein tyrosine phosphorylation. This was not observed using NS1 proteins deficient in SH3 binding or engineered to be exclusively cytoplasmic, indicating a physical role for NS1 as a carrier in the nuclear translocation of Crk. These data further emphasize the role of Crk proteins as host cell interaction partners of NS1, and highlight the potential for host cell manipulation gained by a viral protein simply via acquiring a short SH3 binding motif. PMID:27092521

  14. Systematic VCP-UBXD Adaptor Network Proteomics Identifies a Role for UBXN10 in Regulating Ciliogenesis

    PubMed Central

    Raman, Malavika; Sergeev, Mikhail; Garnaas, Maija; Lydeard, John R.; Huttlin, Edward L.; Goessling, Wolfram; Shah, Jagesh V.; Harper, J. Wade

    2015-01-01

    The AAA-ATPase VCP (also known as p97 or CDC48) uses ATP hydrolysis to “segregate” ubiquitinated proteins from their binding partners. VCP acts via UBX-domain containing adaptors that provide target specificity, but targets and functions of UBXD proteins remain poorly understood. Through systematic proteomic analysis of UBXD proteins in human cells, we reveal a network of over 195 interacting proteins, implicating VCP in diverse cellular pathways. We have explored one such complex between an unstudied adaptor UBXN10 and the intraflagellar transport B (IFT-B) complex, which regulates anterograde transport into cilia. UBXN10 localizes to cilia in a VCP-dependent manner and both VCP and UBXN10 are required for ciliogenesis. Pharmacological inhibition of VCP destabilized the IFT-B complex and increased trafficking rates. Depletion of UBXN10 in zebrafish embryos causes defects in left-right asymmetry, which depends on functional cilia. This study provides a resource for exploring the landscape of UBXD proteins in biology and identifies an unexpected requirement for VCP-UBXN10 in ciliogenesis. PMID:26389662

  15. A Role for the Adaptor Proteins TRAM and TRIF in Toll-like Receptor 2 Signaling*

    PubMed Central

    Nilsen, Nadra J.; Vladimer, Gregory I.; Stenvik, Jørgen; Orning, M. Pontus A.; Zeid-Kilani, Maria V.; Bugge, Marit; Bergstroem, Bjarte; Conlon, Joseph; Husebye, Harald; Hise, Amy G.; Fitzgerald, Katherine A.; Espevik, Terje; Lien, Egil

    2015-01-01

    Toll-like receptors (TLRs) are involved in sensing invading microbes by host innate immunity. TLR2 recognizes bacterial lipoproteins/lipopeptides, and lipopolysaccharide activates TLR4. TLR2 and TLR4 signal via the Toll/interleukin-1 receptor adaptors MyD88 and MAL, leading to NF-κB activation. TLR4 also utilizes the adaptors TRAM and TRIF, resulting in activation of interferon regulatory factor (IRF) 3. Here, we report a new role for TRAM and TRIF in TLR2 regulation and signaling. Interestingly, we observed that TLR2-mediated induction of the chemokine Ccl5 was impaired in TRAM or TRIF deficient macrophages. Inhibition of endocytosis reduced Ccl5 release, and the data also suggested that TRAM and TLR2 co-localize in early endosomes, supporting the hypothesis that signaling may occur from an intracellular compartment. Ccl5 release following lipoprotein challenge additionally involved the kinase Tbk-1 and Irf3, as well as MyD88 and Irf1. Induction of Interferon-β and Ccl4 by lipoproteins was also partially impaired in cells lacking TRIF cells. Our results show a novel function of TRAM and TRIF in TLR2-mediated signal transduction, and the findings broaden our understanding of how Toll/interleukin-1 receptor adaptor proteins may participate in signaling downstream from TLR2. PMID:25505250

  16. Valosin-containing protein (VCP)-Adaptor Interactions are Exceptionally Dynamic and Subject to Differential Modulation by a VCP Inhibitor.

    PubMed

    Xue, Liang; Blythe, Emily E; Freiberger, Elyse C; Mamrosh, Jennifer L; Hebert, Alexander S; Reitsma, Justin M; Hess, Sonja; Coon, Joshua J; Deshaies, Raymond J

    2016-09-01

    Protein quality control (PQC) plays an important role in stemming neurodegenerative diseases and is essential for the growth of some cancers. Valosin-containing protein (VCP)/p97 plays a pivotal role in multiple PQC pathways by interacting with numerous adaptors that link VCP to specific PQC pathways and substrates and influence the post-translational modification state of substrates. However, our poor understanding of the specificity and architecture of the adaptors, and the dynamic properties of their interactions with VCP hinders our understanding of fundamental features of PQC and how modulation of VCP activity can best be exploited therapeutically. In this study we use multiple mass spectrometry-based proteomic approaches combined with biophysical studies to characterize the interaction of adaptors with VCP. Our results reveal that most VCP-adaptor interactions are characterized by rapid dynamics that in some cases are modulated by the VCP inhibitor NMS873. These findings have significant implications for both the regulation of VCP function and the impact of VCP inhibition on different VCP-adaptor complexes. PMID:27406709

  17. Loss of PDZ-adaptor protein NHERF2 affects membrane localization and cGMP- and [Ca2+]- but not cAMP-dependent regulation of Na+/H+ exchanger 3 in murine intestine

    PubMed Central

    Chen, Mingmin; Sultan, Ayesha; Cinar, Ayhan; Yeruva, Sunil; Riederer, Brigitte; Singh, Anurag Kumar; Li, Junhua; Bonhagen, Janina; Chen, Gang; Yun, Chris; Donowitz, Mark; Hogema, Boris; deJonge, Hugo; Seidler, Ursula

    2010-01-01

    Trafficking and regulation of the epithelial brush border membrane (BBM) Na+/H+ exchanger 3 (NHE3) in the intestine involves interaction with four different members of the NHERF family in a signal-dependent and possibly segment-specific fashion. The aim of this research was to study the role of NHERF2 (E3KARP) in intestinal NHE3 BBM localization and second messenger-mediated and receptor-mediated inhibition of NHE3. Immunolocalization of NHE3 in WT mice revealed predominant microvillar localization in jejunum and colon, a mixed distribution in the proximal ileum but localization near the terminal web in the distal ileum. The terminal web localization of NHE3 in the distal ileum correlated with reduced acid-activated NHE3 activity (fluorometrically assessed). NHERF2 ablation resulted in a shift of NHE3 to the microvilli and higher basal fluid absorption rates in the ileum, but no change in overall NHE3 protein or mRNA expression. Forskolin-induced NHE3 inhibition was preserved in the absence of NHERF2, whereas Ca2+ ionophore- or carbachol-mediated inhibition was abolished. Likewise, Escherichia coli heat stable enterotoxin peptide (STp) lost its inhibitory effect on intestinal NHE3. It is concluded that in native murine intestine, the NHE3 adaptor protein NHERF2 plays important roles in tethering NHE3 to a position near the terminal web and in second messenger inhibition of NHE3 in a signal- and segment-specific fashion, and is therefore an important regulator of intestinal fluid transport. PMID:20962002

  18. Nephrin Suppresses Hippo Signaling through the Adaptor Proteins Nck and WTIP.

    PubMed

    Keyvani Chahi, Ava; Martin, Claire E; Jones, Nina

    2016-06-10

    Podocytes are key components of the kidney blood filtration barrier, and their ability to withstand hemodynamic strain is proposed to be closely tied to their unique and flexible cytoarchitecture. However, the mechanisms that control such mechanotransduction are poorly understood. We have previously established that tyrosine phosphorylation of the transmembrane protein nephrin promotes recruitment of the Nck1/2 cytoskeletal adaptor proteins and downstream actin remodeling. We now reveal that Nck integrates nephrin with the Hippo kinase cascade through association with the adaptor protein WTIP. Using mutational analysis, we show that Nck sequesters WTIP and its binding partner Lats1 to phosphorylated nephrin, resulting in decreased phospho-activation of Lats1. We further demonstrate that, coincident with nephrin dephosphorylation in a transient model of podocyte injury in mice, Lats1 is rapidly activated, and this precedes significant down-regulation of the transcription regulator Yap. Moreover, we show reduced levels of Yap protein in mice with chronic disruption of nephrin phospho-signaling. Together, these findings support the existence of a dynamic molecular link between nephrin signaling and the canonical Hippo pathway in podocytes, which may facilitate the conversion of mechanical cues to biochemical signals promoting podocyte viability. PMID:27033705

  19. The E3 Ubiquitin Ligase Adaptor Protein Skp1 Is Glycosylated by an Evolutionarily Conserved Pathway That Regulates Protist Growth and Development.

    PubMed

    Rahman, Kazi; Zhao, Peng; Mandalasi, Msano; van der Wel, Hanke; Wells, Lance; Blader, Ira J; West, Christopher M

    2016-02-26

    Toxoplasma gondii is a protist parasite of warm-blooded animals that causes disease by proliferating intracellularly in muscle and the central nervous system. Previous studies showed that a prolyl 4-hydroxylase related to animal HIFα prolyl hydroxylases is required for optimal parasite proliferation, especially at low O2. We also observed that Pro-154 of Skp1, a subunit of the Skp1/Cullin-1/F-box protein (SCF)-class of E3-ubiquitin ligases, is a natural substrate of this enzyme. In an unrelated protist, Dictyostelium discoideum, Skp1 hydroxyproline is modified by five sugars via the action of three glycosyltransferases, Gnt1, PgtA, and AgtA, which are required for optimal O2-dependent development. We show here that TgSkp1 hydroxyproline is modified by a similar pentasaccharide, based on mass spectrometry, and that assembly of the first three sugars is dependent on Toxoplasma homologs of Gnt1 and PgtA. Reconstitution of the glycosyltransferase reactions in extracts with radioactive sugar nucleotide substrates and appropriate Skp1 glycoforms, followed by chromatographic analysis of acid hydrolysates of the reaction products, confirmed the predicted sugar identities as GlcNAc, Gal, and Fuc. Disruptions of gnt1 or pgtA resulted in decreased parasite growth. Off target effects were excluded based on restoration of the normal glycan chain and growth upon genetic complementation. By analogy to Dictyostelium Skp1, the mechanism may involve regulation of assembly of the SCF complex. Understanding the mechanism of Toxoplasma Skp1 glycosylation is expected to help develop it as a drug target for control of the pathogen, as the glycosyltransferases are absent from mammalian hosts. PMID:26719340

  20. Surfactant Protein A Enhances Constitutive Immune Functions of Clathrin Heavy Chain and Clathrin Adaptor Protein 2.

    PubMed

    Moulakakis, Christina; Steinhäuser, Christine; Biedziak, Dominika; Freundt, Katja; Reiling, Norbert; Stamme, Cordula

    2016-07-01

    NF-κB transcription factors are key regulators of pulmonary inflammatory disorders and repair. Constitutive lung cell type- and microenvironment-specific NF-κB/inhibitor κBα (IκB-α) regulation, however, is poorly understood. Surfactant protein (SP)-A provides both a critical homeostatic and lung defense control, in part by immune instruction of alveolar macrophages (AMs) via clathrin-mediated endocytosis. The central endocytic proteins, clathrin heavy chain (CHC) and the clathrin adaptor protein (AP) complex AP2, have pivotal alternative roles in cellular homeostasis that are endocytosis independent. Here, we dissect endocytic from alternative functions of CHC, the α-subunit of AP2, and dynamin in basal and SP-A-modified LPS signaling of macrophages. As revealed by pharmacological inhibition and RNA interference in primary AMs and RAW264.7 macrophages, respectively, CHC and α-adaptin, but not dynamin, prevent IκB-α degradation and TNF-α release, independent of their canonical role in membrane trafficking. Kinetics studies employing confocal microscopy, Western analysis, and immunomagnetic sorting revealed that SP-A transiently enhances the basal protein expression of CHC and α-adaptin, depending on early activation of protein kinase CK2 (former casein kinase II) and Akt1 in primary AMs from rats, SP-A(+/+), and SP-A(-/-) mice, as well as in vivo when intratracheally administered to SP-A(+/+) mice. Constitutive immunomodulation by SP-A, but not SP-A-mediated inhibition of LPS-induced NF-κB activity and TNF-α release, requires CHC, α-adaptin, and dynamin. Our data demonstrate that endocytic proteins constitutively restrict NF-κB activity in macrophages and provide evidence that SP-A enhances the immune regulatory capacity of these proteins, revealing a previously unknown pathway of microenvironment-specific NF-κB regulation in the lung. PMID:26771574

  1. Adaptor Protein 1A Facilitates Dengue Virus Replication

    PubMed Central

    Yasamut, Umpa; Tongmuang, Nopprarat; Yenchitsomanus, Pa-thai; Junking, Mutita; Noisakran, Sansanee; Puttikhunt, Chunya; Chu, Justin Jang Hann; Limjindaporn, Thawornchai

    2015-01-01

    Rearrangement of membrane structure induced by dengue virus (DENV) is essential for replication, and requires host cellular machinery. Adaptor protein complex (AP)-1 is a host component, which can be recruited to components required for membrane rearrangement. Therefore, dysfunction of AP-1 may affect membrane organization, thereby decreasing replication of virus in infected cells. In the present study, AP-1-dependent traffic inhibitor inhibited DENV protein expression and virion production. We further clarified the role of AP-1A in the life cycle of DENV by RNA interference. AP-1A was not involved in DENV entry into cells. However, it facilitated DENV RNA replication. Viral RNA level was reduced significantly in Huh7 cells transfected with AP-1A small interfering RNA (siRNA) compared with control siRNA. Transfection of naked DENV viral RNA into Huh7 cells transfected with AP-1A siRNA resulted in less viral RNA and virion production than transfection into Huh7 cells transfected with control siRNA. Huh7 cells transfected with AP-1A siRNA showed greater modification of membrane structures and fewer vesicular packets compared with cells transfected with control siRNA. Therefore, AP-1A may partly control DENV-induced rearrangement of membrane structures required for viral replication. PMID:26090672

  2. Structure of the periplasmic adaptor protein from a major facilitator superfamily (MFS) multidrug efflux pump.

    PubMed

    Hinchliffe, Philip; Greene, Nicholas P; Paterson, Neil G; Crow, Allister; Hughes, Colin; Koronakis, Vassilis

    2014-08-25

    Periplasmic adaptor proteins are key components of bacterial tripartite efflux pumps. The 2.85 Å resolution structure of an MFS (major facilitator superfamily) pump adaptor, Aquifex aeolicus EmrA, shows linearly arranged α-helical coiled-coil, lipoyl, and β-barrel domains, but lacks the fourth membrane-proximal domain shown in other pumps to interact with the inner membrane transporter. The adaptor α-hairpin, which binds outer membrane TolC, is exceptionally long at 127 Å, and the β-barrel contains a conserved disordered loop. The structure extends the view of adaptors as flexible, modular components that mediate diverse pump assembly, and suggests that in MFS tripartite pumps a hexamer of adaptors could provide a periplasmic seal. PMID:24996185

  3. Structure of the periplasmic adaptor protein from a major facilitator superfamily (MFS) multidrug efflux pump

    PubMed Central

    Hinchliffe, Philip; Greene, Nicholas P.; Paterson, Neil G.; Crow, Allister; Hughes, Colin; Koronakis, Vassilis

    2014-01-01

    Periplasmic adaptor proteins are key components of bacterial tripartite efflux pumps. The 2.85 Å resolution structure of an MFS (major facilitator superfamily) pump adaptor, Aquifex aeolicus EmrA, shows linearly arranged α-helical coiled-coil, lipoyl, and β-barrel domains, but lacks the fourth membrane-proximal domain shown in other pumps to interact with the inner membrane transporter. The adaptor α-hairpin, which binds outer membrane TolC, is exceptionally long at 127 Å, and the β-barrel contains a conserved disordered loop. The structure extends the view of adaptors as flexible, modular components that mediate diverse pump assembly, and suggests that in MFS tripartite pumps a hexamer of adaptors could provide a periplasmic seal. PMID:24996185

  4. The adaptor protein CIN85 assembles intracellular signaling clusters for B cell activation.

    PubMed

    Kühn, Julius; Wong, Leo E; Pirkuliyeva, Sona; Schulz, Kathrin; Schwiegk, Claudia; Fünfgeld, Kevser Gencalp; Keppler, Selina; Batista, Facundo D; Urlaub, Henning; Habeck, Michael; Becker, Stefan; Griesinger, Christian; Wienands, Jürgen

    2016-01-01

    The adaptor molecule Cbl-interacting protein of 85 kD (CIN85) regulates signaling from a number of cell surface receptors, such as growth factor receptors and antigen receptors on lymphocytes. Because of its multidomain structure, CIN85 is thought to act as a classical adaptor protein that connects functionally distinct components of a given signaling pathway through diverse protein domains. However, we found that in B lymphocytes, CIN85 functions to oligomerize SLP-65, which is the central effector protein of the B cell receptor (BCR). Therefore, CIN85 trimerizes through a carboxyl-terminal, coiled-coil domain. The multiple Src homology 3 (SH3) domains of trimeric CIN85 molecules associated with multiple SLP-65 molecules, which recruited further CIN85 trimers, thereby perpetuating the oligomerization process. Formation of this oligomeric signaling complex in resting B cells rendered the cells poised for the efficient initiation of intracellular signaling upon BCR stimulation. Our data suggest that the functionality of signaling cascades does not rely solely on the qualitative linkage of their various components but requires a critical number of effectors to become concentrated in signaling complexes. PMID:27353366

  5. Oncogenic transformation by the signaling adaptor proteins insulin receptor substrate (IRS)-1 and IRS-2

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Insulin receptor substrates (IRSs) are adaptor proteins that link signaling from upstream activators to multiple downstream effectors to modulate normal growth, metabolism, survival, and differentiation. Recent cell culture studies have shown that IRSs can interact with, and are functionally require...

  6. The Hypoxic Regulator of Sterol Synthesis Nro1 Is a Nuclear Import Adaptor

    SciTech Connect

    T Yeh; C Lee; L Amzel; P Espenshade; M Bianchet

    2011-12-31

    Fission yeast protein Sre1, the homolog of the mammalian sterol regulatory element-binding protein (SREBP), is a hypoxic transcription factor required for sterol homeostasis and low-oxygen growth. Nro1 regulates the stability of the N-terminal transcription factor domain of Sre1 (Sre1N) by inhibiting the action of the prolyl 4-hydroxylase-like Ofd1 in an oxygen-dependent manner. The crystal structure of Nro1 determined at 2.2 {angstrom} resolution shows an all-{alpha}-helical fold that can be divided into two domains: a small N-terminal domain, and a larger C-terminal HEAT-repeat domain. Follow-up studies showed that Nro1 defines a new class of nuclear import adaptor that functions both in Ofd1 nuclear localization and in the oxygen-dependent inhibition of Ofd1 to control the hypoxic response.

  7. Recruitment of the Adaptor Protein Grb2 to EGFR Tetramers

    PubMed Central

    2015-01-01

    Adaptor protein Grb2 binds phosphotyrosines in the epidermal growth factor (EGF) receptor (EGFR) and thereby links receptor activation to intracellular signaling cascades. Here, we investigated how recruitment of Grb2 to EGFR is affected by the spatial organization and quaternary state of activated EGFR. We used the techniques of image correlation spectroscopy (ICS) and lifetime-detected Förster resonance energy transfer (also known as FLIM-based FRET or FLIM–FRET) to measure ligand-induced receptor clustering and Grb2 binding to activated EGFR in BaF/3 cells. BaF/3 cells were stably transfected with fluorescently labeled forms of Grb2 (Grb2–mRFP) and EGFR (EGFR–eGFP). Following stimulation of the cells with EGF, we detected nanometer-scale association of Grb2–mRFP with EGFR–eGFP clusters, which contained, on average, 4 ± 1 copies of EGFR–eGFP per cluster. In contrast, the pool of EGFR–eGFP without Grb2–mRFP had an average cluster size of 1 ± 0.3 EGFR molecules per punctum. In the absence of EGF, there was no association between EGFR–eGFP and Grb2–mRFP. To interpret these data, we extended our recently developed model for EGFR activation, which considers EGFR oligomerization up to tetramers, to include recruitment of Grb2 to phosphorylated EGFR. The extended model, with adjustment of one new parameter (the ratio of the Grb2 and EGFR copy numbers), is consistent with a cluster size distribution where 2% of EGFR monomers, 5% of EGFR dimers, <1% of EGFR trimers, and 94% of EGFR tetramers are associated with Grb2. Together, our experimental and modeling results further implicate tetrameric EGFR as the key signaling unit and call into question the widely held view that dimeric EGFR is the predominant signaling unit. PMID:24697349

  8. Vaccinia virus protein A46R targets multiple Toll-like-interleukin-1 receptor adaptors and contributes to virulence.

    PubMed

    Stack, Julianne; Haga, Ismar R; Schröder, Martina; Bartlett, Nathan W; Maloney, Geraldine; Reading, Patrick C; Fitzgerald, Katherine A; Smith, Geoffrey L; Bowie, Andrew G

    2005-03-21

    Viral immune evasion strategies target key aspects of the host antiviral response. Recently, it has been recognized that Toll-like receptors (TLRs) have a role in innate defense against viruses. Here, we define the function of the vaccinia virus (VV) protein A46R and show it inhibits intracellular signalling by a range of TLRs. TLR signalling is triggered by homotypic interactions between the Toll-like-interleukin-1 resistance (TIR) domains of the receptors and adaptor molecules. A46R contains a TIR domain and is the only viral TIR domain-containing protein identified to date. We demonstrate that A46R targets the host TIR adaptors myeloid differentiation factor 88 (MyD88), MyD88 adaptor-like, TIR domain-containing adaptor inducing IFN-beta (TRIF), and the TRIF-related adaptor molecule and thereby interferes with downstream activation of mitogen-activated protein kinases and nuclear factor kappaB. TRIF mediates activation of interferon (IFN) regulatory factor 3 (IRF3) and induction of IFN-beta by TLR3 and TLR4 and suppresses VV replication in macrophages. Here, A46R disrupted TRIF-induced IRF3 activation and induction of the TRIF-dependent gene regulated on activation, normal T cell expressed and secreted. Furthermore, we show that A46R is functionally distinct from another described VV TLR inhibitor, A52R. Importantly, VV lacking the A46R gene was attenuated in a murine intranasal model, demonstrating the importance of A46R for VV virulence. PMID:15767367

  9. The Adaptor Protein Rai/ShcC Promotes Astrocyte-Dependent Inflammation during Experimental Autoimmune Encephalomyelitis.

    PubMed

    Ulivieri, Cristina; Savino, Maria Teresa; Luccarini, Ilaria; Fanigliulo, Emanuela; Aldinucci, Alessandra; Bonechi, Elena; Benagiano, Marisa; Ortensi, Barbara; Pelicci, Giuliana; D'Elios, Mario Milco; Ballerini, Clara; Baldari, Cosima Tatiana

    2016-07-15

    Th17 cells have been casually associated to the pathogenesis of autoimmune disease. We have previously demonstrated that Rai/ShcC, a member of the Shc family of adaptor proteins, negatively regulates Th17 cell differentiation and lupus autoimmunity. In this study, we have investigated the pathogenic outcome of the Th17 bias associated with Rai deficiency on multiple sclerosis development, using the experimental autoimmune encephalomyelitis (EAE) mouse model. We found that, unexpectedly, EAE was less severe in Rai(-/-) mice compared with their wild-type counterparts despite an enhanced generation of myelin-specific Th17 cells that infiltrated into the CNS. Nevertheless, when adoptively transferred into immunodeficient Rai(+/+) mice, these cells promoted a more severe disease compared with wild-type encephalitogenic Th17 cells. This paradoxical phenotype was caused by a dampened inflammatory response of astrocytes, which were found to express Rai, to IL-17. The results provide evidence that Rai plays opposite roles in Th17 cell differentiation and astrocyte activation, with the latter dominant over the former in EAE, highlighting this adaptor as a potential novel target for the therapy of multiple sclerosis. PMID:27288534

  10. The adaptor proteins p140CAP and p130CAS as molecular hubs in cell migration and invasion of cancer cells

    PubMed Central

    Di Stefano, Paola; Leal, Maria Pilar Camacho; Tornillo, Giusy; Bisaro, Brigitte; Repetto, Daniele; Pincini, Alessandra; Santopietro, Emanuela; Sharma, Nanaocha; Turco, Emilia; Cabodi, Sara; Defilippi, Paola

    2011-01-01

    The assembly of molecular hubs upon integrin and growth factor stimulation represents a preferential way to transduce signals throughout the cell. Among the intracellular kinases that are responsive to integrin and growth factor activation, Src Family Kinases (SFKs) are crucial regulators of cell migration and invasion. Increasing evidence highlight the importance of adaptor proteins in these processes, based on their ability to create signalling platforms that control downstream signals. Among these adaptors we will discuss the molecular features of p130Cas and p140Cap proteins in terms of regulation of cell migration and invasion in normal and transformed cells. PMID:21994904

  11. Distinct adaptor proteins assist exit of Kre2-family proteins from the yeast ER

    PubMed Central

    Noda, Yoichi; Hara, Takehiro; Ishii, Minako; Yoda, Koji

    2014-01-01

    ABSTRACT The Svp26 protein of S. cerevisiae is an ER- and Golgi-localized integral membrane protein with 4 potential membrane-spanning domains. It functions as an adaptor protein that facilitates the ER exit of Ktr3, a mannosyltransferase required for biosynthesis of O-linked oligosaccharides, and the ER exit of Mnn2 and Mnn5, mannosyltransferases, which participate in the biosynthesis of N-linked oligosaccharides. Ktr3 belongs to the Kre2 family, which consists of 9 members of type-II membrane proteins sharing sequence similarities. In this report, we examined all Kre2 family members and found that the Golgi localizations of two others, Kre2 and Ktr1, were dependent on Svp26 by immunofluorescence microscopy and cell fractionations in sucrose density gradients. We show that Svp26 functions in facilitating the ER exit of Kre2 and Ktr1 by an in vitro COPII budding assay. Golgi localization of Ktr4 was not dependent on Svp26. Screening null mutants of the genes encoding abundant COPII membrane proteins for those showing mislocalization of Ktr4 in the ER revealed that Erv41 and Erv46 are required for the correct Golgi localization of Ktr4. We provide biochemical evidence that the Erv41-Erv46 complex functions as an adaptor protein for ER exit of Ktr4. This is the first demonstration of the molecular function of this evolutionally conserved protein complex. The domain switching experiments show that the lumenal domain of Ktr4 is responsible for recognition by the Erv41-Erv46 complex. Thus, ER exit of Kre2-family proteins is dependent on distinct adaptor proteins and our results provide new insights into the traffic of Kre2-family mannosyltransferases. PMID:24585773

  12. PHF6 Degrees of Separation: The Multifaceted Roles of a Chromatin Adaptor Protein

    PubMed Central

    Todd, Matthew A.M.; Ivanochko, Danton; Picketts, David J.

    2015-01-01

    The importance of chromatin regulation to human disease is highlighted by the growing number of mutations identified in genes encoding chromatin remodeling proteins. While such mutations were first identified in severe developmental disorders, or in specific cancers, several genes have been implicated in both, including the plant homeodomain finger protein 6 (PHF6) gene. Indeed, germline mutations in PHF6 are the cause of the Börjeson–Forssman–Lehmann X-linked intellectual disability syndrome (BFLS), while somatic PHF6 mutations have been identified in T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML). Studies from different groups over the last few years have made a significant impact towards a functional understanding of PHF6 protein function. In this review, we summarize the current knowledge of PHF6 with particular emphasis on how it interfaces with a distinct set of interacting partners and its functional roles in the nucleoplasm and nucleolus. Overall, PHF6 is emerging as a key chromatin adaptor protein critical to the regulation of neurogenesis and hematopoiesis. PMID:26103525

  13. Identification of a new transmembrane adaptor protein that constitutively binds Grb2 in B cells

    PubMed Central

    Liu, Yan; Zhang, Weiguo

    2008-01-01

    Transmembrane adaptor proteins couple antigen receptor engagement to downstream signaling cascades in lymphocytes. One example of these proteins is the linker for activation of T cells (LAT), which plays an indispensable role in T cell activation and development. Here, we report identification of a new transmembrane adaptor molecule, namely growth factor receptor-bound protein 2 (Grb2)-binding adaptor protein, transmembrane (GAPT), which is expressed in B cells and myeloid cells. Similar to LAT, GAPT has an extracellular domain, a transmembrane domain, and a cytoplasmic tail with multiple Grb2-binding motifs. In contrast to other transmembrane adaptor proteins, GAPT is not phosphorylated upon BCR ligation but associates with Grb2 constitutively through its proline-rich region. Targeted disruption of the gapt gene in mice affects neither B cell development nor a nitrophenylacetyl-specific antibody response. However, in the absence of GAPT, B cell proliferation after BCR cross-linking is enhanced. In aged GAPT−/− mice, the number of marginal zone (MZ) B cells is increased, and other B cell subsets are normal. The serum concentrations of IgM, IgG2b, and IgG3 are also elevated in these mice. These data indicate that GAPT might play an important role in control of B cell activation and proper maintenance of MZ B cells. PMID:18559951

  14. The clathrin adaptor AP-1 complex and Arf1 regulate planar cell polarity in vivo

    PubMed Central

    Mendoza, Meg; Dussert, Aurore; Collu, Giovanna; Roman, Angel-Carlos; Weber, Ursula; Ciruna, Brian; Mlodzik, Marek

    2015-01-01

    A key step in generating planar cell polarity (PCP) is the formation of restricted junctional domains containing Frizzled/Dishevelled/Diego (Fz/Dsh/Dgo) or Van Gogh/Prickle (Vang/Pk) complexes within the same cell, stabilized via Flamingo (Fmi) across cell membranes. Although models have been proposed for how these complexes acquire and maintain their polarized localization, the machinery involved in moving core PCP proteins around cells remains unknown. We describe the AP-1 adaptor complex and Arf1 as major regulators of PCP protein trafficking in vivo. AP-1 and Arf1 disruption affects the accumulation of Fz/Fmi and Vang/Fmi complexes in the proximo–distal axis, producing severe PCP phenotypes. Using novel tools, we demonstrate a direct and specific Arf1 involvement in Fz trafficking in vivo. Moreover, we uncover a conserved Arf1 PCP function in vertebrates. Our data support a model whereby the trafficking machinery plays an important part during PCP establishment, promoting formation of polarized PCP-core complexes in vivo. PMID:25849195

  15. Identification of human proteins functionally conserved with the yeast putative adaptors ADA2 and GCN5.

    PubMed Central

    Candau, R; Moore, P A; Wang, L; Barlev, N; Ying, C Y; Rosen, C A; Berger, S L

    1996-01-01

    Transcriptional adaptor proteins are required for full function of higher eukaryotic acidic activators in the yeast Saccharomyces cerevisiae, suggesting that this pathway of activation is evolutionarily conserved. Consistent with this view, we have identified possible human homologs of yeast ADA2 (yADA2) and yeast GCN5 (yGCN5), components of a putative adaptor complex. While there is overall sequence similarity between the yeast and human proteins, perhaps more significant is conservation of key sequence features with other known adaptors. We show several functional similarities between the human and yeast adaptors. First, as shown for yADA2 and yGCN5, human ADA2 (hADA2) and human GCN5 (hGCN5) interacted in vivo in a yeast two-hybrid assay. Moreover, hGCN5 interacted with yADA2 in this assay, suggesting that the human proteins form similar complexes. Second, both yADA2 and hADA2 contain cryptic activation domains. Third, hGCN5 and yGCN5 had similar stabilizing effects on yADA2 in vivo. Furthermore, the region of yADA2 that interacted with yGCN5 mapped to the amino terminus of yADA2, which is highly conserved in hADA2. Most striking, is the behavior of the human proteins in human cells. First, GAL4-hADA2 activated transcription in HeLa cells, and second, either hADA2 or hGCN5 augmented GAL4-VP16 activation. These data indicated that the human proteins correspond to functional homologs of the yeast adaptors, suggesting that these cofactors play a key role in transcriptional activation. PMID:8552087

  16. Merkel cell polyomavirus small T antigen targets the NEMO adaptor protein to disrupt inflammatory signaling.

    PubMed

    Griffiths, David A; Abdul-Sada, Hussein; Knight, Laura M; Jackson, Brian R; Richards, Kathryn; Prescott, Emma L; Peach, A Howard S; Blair, G Eric; Macdonald, Andrew; Whitehouse, Adrian

    2013-12-01

    Merkel cell carcinoma (MCC) is a highly aggressive nonmelanoma skin cancer arising from epidermal mechanoreceptor Merkel cells. In 2008, a novel human polyomavirus, Merkel cell polyomavirus (MCPyV), was identified and is strongly implicated in MCC pathogenesis. Currently, little is known regarding the virus-host cell interactions which support virus replication and virus-induced mechanisms in cellular transformation and metastasis. Here we identify a new function of MCPyV small T antigen (ST) as an inhibitor of NF-κB-mediated transcription. This effect is due to an interaction between MCPyV ST and the NF-κB essential modulator (NEMO) adaptor protein. MCPyV ST expression inhibits IκB kinase α (IKKα)/IKKβ-mediated IκB phosphorylation, which limits translocation of the NF-κB heterodimer to the nucleus. Regulation of this process involves a previously undescribed interaction between MCPyV ST and the cellular phosphatase subunits, protein phosphatase 4C (PP4C) and/or protein phosphatase 2A (PP2A) Aβ, but not PP2A Aα. Together, these results highlight a novel function of MCPyV ST to subvert the innate immune response, allowing establishment of early or persistent infection within the host cell. PMID:24109239

  17. Merkel Cell Polyomavirus Small T Antigen Targets the NEMO Adaptor Protein To Disrupt Inflammatory Signaling

    PubMed Central

    Griffiths, David A.; Abdul-Sada, Hussein; Knight, Laura M.; Jackson, Brian R.; Richards, Kathryn; Prescott, Emma L.; Peach, A. Howard S.; Blair, G. Eric

    2013-01-01

    Merkel cell carcinoma (MCC) is a highly aggressive nonmelanoma skin cancer arising from epidermal mechanoreceptor Merkel cells. In 2008, a novel human polyomavirus, Merkel cell polyomavirus (MCPyV), was identified and is strongly implicated in MCC pathogenesis. Currently, little is known regarding the virus-host cell interactions which support virus replication and virus-induced mechanisms in cellular transformation and metastasis. Here we identify a new function of MCPyV small T antigen (ST) as an inhibitor of NF-κB-mediated transcription. This effect is due to an interaction between MCPyV ST and the NF-κB essential modulator (NEMO) adaptor protein. MCPyV ST expression inhibits IκB kinase α (IKKα)/IKKβ-mediated IκB phosphorylation, which limits translocation of the NF-κB heterodimer to the nucleus. Regulation of this process involves a previously undescribed interaction between MCPyV ST and the cellular phosphatase subunits, protein phosphatase 4C (PP4C) and/or protein phosphatase 2A (PP2A) Aβ, but not PP2A Aα. Together, these results highlight a novel function of MCPyV ST to subvert the innate immune response, allowing establishment of early or persistent infection within the host cell. PMID:24109239

  18. Insulin Receptor Substrate Adaptor Proteins Mediate Prognostic Gene Expression Profiles in Breast Cancer

    PubMed Central

    Becker, Marc A.; Ibrahim, Yasir H.; Oh, Annabell S.; Fagan, Dedra H.; Byron, Sara A.; Sarver, Aaron L.; Lee, Adrian V.; Shaw, Leslie M.; Fan, Cheng; Perou, Charles M.; Yee, Douglas

    2016-01-01

    Therapies targeting the type I insulin-like growth factor receptor (IGF-1R) have not been developed with predictive biomarkers to identify tumors with receptor activation. We have previously shown that the insulin receptor substrate (IRS) adaptor proteins are necessary for linking IGF1R to downstream signaling pathways and the malignant phenotype in breast cancer cells. The purpose of this study was to identify gene expression profiles downstream of IGF1R and its two adaptor proteins. IRS-null breast cancer cells (T47D-YA) were engineered to express IRS-1 or IRS-2 alone and their ability to mediate IGF ligand-induced proliferation, motility, and gene expression determined. Global gene expression signatures reflecting IRS adaptor specific and primary vs. secondary ligand response were derived (Early IRS-1, Late IRS-1, Early IRS-2 and Late IRS-2) and functional pathway analysis examined. IRS isoforms mediated distinct gene expression profiles, functional pathways, and breast cancer subtype association. For example, IRS-1/2-induced TGFb2 expression and blockade of TGFb2 abrogated IGF-induced cell migration. In addition, the prognostic value of IRS proteins was significant in the luminal B breast tumor subtype. Univariate and multivariate analyses confirmed that IRS adaptor signatures correlated with poor outcome as measured by recurrence-free and overall survival. Thus, IRS adaptor protein expression is required for IGF ligand responses in breast cancer cells. IRS-specific gene signatures represent accurate surrogates of IGF activity and could predict response to anti-IGF therapy in breast cancer. PMID:26991655

  19. The Late Endosomal Adaptor Molecule p14 (LAMTOR2) Regulates TGFβ1-Mediated Homeostasis of Langerhans Cells

    PubMed Central

    Sparber, Florian; Tripp, Christoph H.; Komenda, Kerstin; Scheffler, Julia M.; Clausen, Björn E.; Huber, Lukas A.; Romani, Nikolaus; Stoitzner, Patrizia

    2014-01-01

    Langerhans cells (LCs), a sub-population of dendritic cells (DCs) in the skin, participate in the regulation of immunity and peripheral tolerance. The adaptor molecule p14 is part of the late endosomal/lysosomal adaptor and mitogen-activated protein kinase and mammalian target of rapamycin (mTOR) activator/regulator (LAMTOR) complex, which mediates the activation of lysosome-associated extracellular signaling-regulated kinase (ERK) and the mTOR cascade. In previous work, we demonstrated that CD11c-specific deficiency of p14 disrupts LC homeostasis by affecting the LAMTOR-mediated ERK and mTOR signaling. In this study, we extended our analysis on p14 deficiency specifically in LCs. Langerin-specific ablation of p14 caused a complete loss of LCs, accompanied by an increased maturational phenotype of LCs. The absence of LCs in p14-deficient mice reduced contact hypersensitivity (CHS) responses to the contact sensitizer trinitrochlorobenzene. Analysis using bone marrow-derived DCs (BMDCs) revealed that p14 deficiency in DCs/LCs interfered with the LC-relevant transforming growth factor β1 (TGFβ1) pathway, by lowering TGFβ receptor II expression on BMDCs and LCs, as well as surface binding of TGFβ1 on BMDCs. We conclude that p14 deficiency affects TGFβ1 sensitivity of LCs, which is mandatory for their homeostasis and subsequently for their immunological function during CHS. PMID:25078666

  20. Nck adaptors are positive regulators of the size and sensitivity of the T-cell repertoire

    PubMed Central

    Roy, Edwige; Togbe, Dieudonnée; Holdorf, Amy D.; Trubetskoy, Dmitry; Nabti, Sabrina; Küblbeck, Günter; Klevenz, Alexandra; Kopp-Schneider, Annette; Leithäuser, Frank; Möller, Peter; Bladt, Friedhelm; Hämmerling, Günter; Arnold, Bernd; Pawson, Tony; Tafuri, Anna

    2010-01-01

    The size and sensitivity of the T-cell repertoire governs the effectiveness of immune responses against invading pathogens. Both are modulated by T-cell receptor (TCR) activity through molecular mechanisms, which remain unclear. Here, we provide genetic evidence that the SH2/SH3 domain containing proteins Nck lower the threshold of T-cell responsiveness. The hallmarks of Nck deletion were T-cell lymphopenia and hyporeactivity to TCR-mediated stimulation. In the absence of the Nck adaptors, peripheral T cells expressing a TCR with low avidity for self-antigens were strongly reduced, whereas an overall impairment of T-cell activation by weak antigenic stimulation was observed. Mechanistically, Nck deletion resulted in a significant decrease in calcium mobilization and ERK phosphorylation upon TCR engagement. Taken together, our findings unveil a crucial role for the Nck adaptors in shaping the T-cell repertoire to ensure maximal antigenic coverage and optimal T cell excitability. PMID:20709959

  1. Nck adaptors are positive regulators of the size and sensitivity of the T-cell repertoire.

    PubMed

    Roy, Edwige; Togbe, Dieudonnée; Holdorf, Amy D; Trubetskoy, Dmitry; Nabti, Sabrina; Küblbeck, Günter; Klevenz, Alexandra; Kopp-Schneider, Annette; Leithäuser, Frank; Möller, Peter; Bladt, Friedhelm; Hämmerling, Günter; Arnold, Bernd; Pawson, Tony; Tafuri, Anna

    2010-08-31

    The size and sensitivity of the T-cell repertoire governs the effectiveness of immune responses against invading pathogens. Both are modulated by T-cell receptor (TCR) activity through molecular mechanisms, which remain unclear. Here, we provide genetic evidence that the SH2/SH3 domain containing proteins Nck lower the threshold of T-cell responsiveness. The hallmarks of Nck deletion were T-cell lymphopenia and hyporeactivity to TCR-mediated stimulation. In the absence of the Nck adaptors, peripheral T cells expressing a TCR with low avidity for self-antigens were strongly reduced, whereas an overall impairment of T-cell activation by weak antigenic stimulation was observed. Mechanistically, Nck deletion resulted in a significant decrease in calcium mobilization and ERK phosphorylation upon TCR engagement. Taken together, our findings unveil a crucial role for the Nck adaptors in shaping the T-cell repertoire to ensure maximal antigenic coverage and optimal T cell excitability. PMID:20709959

  2. New Insights to Clathrin and Adaptor Protein 2 for the Design and Development of Therapeutic Strategies.

    PubMed

    Poulsen, Ebbe Toftgaard; Larsen, Agnete; Zollo, Alen; Jørgensen, Arne L; Sanggaard, Kristian W; Enghild, Jan J; Matrone, Carmela

    2015-01-01

    The Amyloid Precursor Protein (APP) has been extensively studied for its role as the precursor of the β-amyloid protein (Aβ) in Alzheimer's disease (AD). However, our understanding of the normal function of APP is still patchy. Emerging evidence indicates that a dysfunction in APP trafficking and degradation can be responsible for neuronal deficits and progressive degeneration in humans. We recently reported that the Y682 mutation in the 682YENPTY687 domain of APP affects its binding to specific adaptor proteins and leads to its anomalous trafficking, to defects in the autophagy machinery and to neuronal degeneration. In order to identify adaptors that influence APP function, we performed pull-down experiments followed by quantitative mass spectrometry (MS) on hippocampal tissue extracts of three month-old mice incubated with either the 682YENPTY687 peptide, its mutated form, 682GENPTY687 or its phosphorylated form, 682pYENPTY687. Our experiments resulted in the identification of two proteins involved in APP internalization and trafficking: Clathrin heavy chain (hc) and its Adaptor Protein 2 (AP-2). Overall our results consolidate and refine the importance of Y682 in APP normal functions from an animal model of premature aging and dementia. Additionally, they open the perspective to consider Clathrin hc and AP-2 as potential targets for the design and development of new therapeutic strategies. PMID:26690411

  3. New Insights to Clathrin and Adaptor Protein 2 for the Design and Development of Therapeutic Strategies

    PubMed Central

    Poulsen, Ebbe Toftgaard; Larsen, Agnete; Zollo, Alen; Jørgensen, Arne L.; Sanggaard, Kristian W.; Enghild, Jan J.; Matrone, Carmela

    2015-01-01

    The Amyloid Precursor Protein (APP) has been extensively studied for its role as the precursor of the β-amyloid protein (Aβ) in Alzheimer’s disease (AD). However, our understanding of the normal function of APP is still patchy. Emerging evidence indicates that a dysfunction in APP trafficking and degradation can be responsible for neuronal deficits and progressive degeneration in humans. We recently reported that the Y682 mutation in the 682YENPTY687 domain of APP affects its binding to specific adaptor proteins and leads to its anomalous trafficking, to defects in the autophagy machinery and to neuronal degeneration. In order to identify adaptors that influence APP function, we performed pull-down experiments followed by quantitative mass spectrometry (MS) on hippocampal tissue extracts of three month-old mice incubated with either the 682YENPTY687 peptide, its mutated form, 682GENPTY687 or its phosphorylated form, 682pYENPTY687. Our experiments resulted in the identification of two proteins involved in APP internalization and trafficking: Clathrin heavy chain (hc) and its Adaptor Protein 2 (AP-2). Overall our results consolidate and refine the importance of Y682 in APP normal functions from an animal model of premature aging and dementia. Additionally, they open the perspective to consider Clathrin hc and AP-2 as potential targets for the design and development of new therapeutic strategies. PMID:26690411

  4. Lymphocyte adaptor protein LNK deficiency exacerbates hypertension and end-organ inflammation.

    PubMed

    Saleh, Mohamed A; McMaster, William G; Wu, Jing; Norlander, Allison E; Funt, Samuel A; Thabet, Salim R; Kirabo, Annet; Xiao, Liang; Chen, Wei; Itani, Hana A; Michell, Danielle; Huan, Tianxiao; Zhang, Yahua; Takaki, Satoshi; Titze, Jens; Levy, Daniel; Harrison, David G; Madhur, Meena S

    2015-03-01

    The lymphocyte adaptor protein LNK (also known as SH2B3) is primarily expressed in hematopoietic and endothelial cells, where it functions as a negative regulator of cytokine signaling and cell proliferation. Single-nucleotide polymorphisms in the gene encoding LNK are associated with autoimmune and cardiovascular disorders; however, it is not known how LNK contributes to hypertension. Here, we determined that loss of LNK exacerbates angiotensin II-induced (Ang II-induced) hypertension and the associated renal and vascular dysfunction. At baseline, kidneys from Lnk-/- mice exhibited greater levels of inflammation, oxidative stress, and glomerular injury compared with WT animals, and these parameters were further exacerbated by Ang II infusion. Aortas from Lnk-/- mice exhibited enhanced inflammation, reduced nitric oxide levels, and impaired endothelial-dependent relaxation. Bone marrow transplantation studies demonstrated that loss of LNK in hematopoietic cells is primarily responsible for the observed renal and vascular inflammation and predisposition to hypertension. Ang II infusion increased IFN-γ-producing CD8+ T cells in the spleen and kidneys of Lnk-/- mice compared with WT mice. Moreover, IFN-γ deficiency resulted in blunted hypertension in response to Ang II infusion. Together, these results suggest that LNK is a potential therapeutic target for hypertension and its associated renal and vascular sequela. PMID:25664851

  5. Lymphocyte adaptor protein LNK deficiency exacerbates hypertension and end-organ inflammation

    PubMed Central

    Saleh, Mohamed A.; McMaster, William G.; Wu, Jing; Norlander, Allison E.; Funt, Samuel A.; Thabet, Salim R.; Kirabo, Annet; Xiao, Liang; Chen, Wei; Itani, Hana A.; Michell, Danielle; Huan, Tianxiao; Zhang, Yahua; Takaki, Satoshi; Titze, Jens; Levy, Daniel; Harrison, David G.; Madhur, Meena S.

    2015-01-01

    The lymphocyte adaptor protein LNK (also known as SH2B3) is primarily expressed in hematopoietic and endothelial cells, where it functions as a negative regulator of cytokine signaling and cell proliferation. Single-nucleotide polymorphisms in the gene encoding LNK are associated with autoimmune and cardiovascular disorders; however, it is not known how LNK contributes to hypertension. Here, we determined that loss of LNK exacerbates angiotensin II–induced (Ang II–induced) hypertension and the associated renal and vascular dysfunction. At baseline, kidneys from Lnk–/– mice exhibited greater levels of inflammation, oxidative stress, and glomerular injury compared with WT animals, and these parameters were further exacerbated by Ang II infusion. Aortas from Lnk–/– mice exhibited enhanced inflammation, reduced nitric oxide levels, and impaired endothelial-dependent relaxation. Bone marrow transplantation studies demonstrated that loss of LNK in hematopoietic cells is primarily responsible for the observed renal and vascular inflammation and predisposition to hypertension. Ang II infusion increased IFN-γ–producing CD8+ T cells in the spleen and kidneys of Lnk–/– mice compared with WT mice. Moreover, IFN-γ deficiency resulted in blunted hypertension in response to Ang II infusion. Together, these results suggest that LNK is a potential therapeutic target for hypertension and its associated renal and vascular sequela. PMID:25664851

  6. Large hepatitis delta antigen is a novel clathrin adaptor-like protein.

    PubMed

    Huang, Cheng; Chang, Shin C; Yu, I-Chen; Tsay, Yeou-Guang; Chang, Ming-Fu

    2007-06-01

    Clathrin-mediated endocytosis is a common pathway for viral entry, but little is known about the direct association of viral protein with clathrin in the cytoplasm. In this study, a putative clathrin box known to be conserved in clathrin adaptors was identified at the C terminus of the large hepatitis delta antigen (HDAg-L). Similar to clathrin adaptors, HDAg-L directly interacted with the N terminus of the clathrin heavy chain through the clathrin box. HDAg-L is a nucleocytoplasmic shuttle protein important for the assembly of hepatitis delta virus (HDV). Here, we demonstrated that brefeldin A and wortmannin, inhibitors of clathrin-mediated exocytosis and endosomal trafficking, respectively, specifically blocked HDV assembly but had no effect on the assembly of the small surface antigen of hepatitis B virus. In addition, cytoplasm-localized HDAg-L inhibited the clathrin-mediated endocytosis of transferrin and the degradation of epidermal growth factor receptor. These results indicate that HDAg-L is a new clathrin adaptor-like protein, and it may be involved in the maturation and pathogenesis of HDV coinfection or superinfection with hepatitis B virus through interaction with clathrin. PMID:17376909

  7. Molecular physiology of the tensin brotherhood of integrin adaptor proteins.

    PubMed

    Haynie, Donald T

    2014-07-01

    Numerous proteins have been identified as constituents of the adhesome, the totality of molecular components in the supramolecular assemblies known as focal adhesions, fibrillar adhesions and other kinds of adhesive contact. The transmembrane receptor proteins called integrins are pivotal adhesome members, providing a physical link between the extracellular matrix (ECM) and the actin cytoskeleton. Tensins are ever more widely investigated intracellular adhesome constituents. Involved in cell attachment and migration, cytoskeleton reorganization, signal transduction and other processes relevant to cancer research, tensins have recently been linked to functional properties of deleted in liver cancer 1 (DLC1) and a mitogen-activated protein kinases (MAPK), to cell migration in breast cancer, and to metastasis suppression in the kidney. Tensins are close relatives of phosphatase homolog/tensin homolog (PTEN), an extensively studied tumor suppressor. Such findings are recasting the earlier vision of tensin (TNS) as an actin-filament (F-actin) capping protein in a different light. This critical review aims to summarize current knowledge on tensins and thus to highlight key points concerning the expression, structure, function, and evolution of the various members of the TNS brotherhood. Insight is sought by comparisons with homologous proteins. Some historical points are added for perspective. PMID:24634006

  8. RNF11 is a GGA protein cargo and acts as a molecular adaptor for GGA3 ubiquitination mediated by Itch.

    PubMed

    Santonico, E; Mattioni, A; Panni, S; Belleudi, F; Mattei, M; Torrisi, M R; Cesareni, G; Castagnoli, L

    2015-06-01

    Ring finger protein 11 (RNF11) is a RING (really interesting new gene)-H2 E3 ligase that is overexpressed in several human tumor tissues. The mature protein, which is anchored to membranes via a double acylation, localizes to early endosome and recycling compartments. Apart from its subcellular localization, additional lines of evidence implicate RNF11 in the mechanisms underlying vesicle traffic. Here we identify two acidic-cluster dileucine (Ac-LL) motifs, which are recognized by the VHS domains of Golgi-localized, gamma adaptin era-containing, ADP-ribosylation factor-binding protein (GGA) adaptors, as the molecular determinants governing RNF11 sorting at the trans-Golgi network and its internalization from the plasma membrane. We also show that RNF11 recruits itch to drive the ubiquitination of GGA3. This function is experimentally detectable only in cells overexpressing an RNF11 variant that is inactivated in the RING domain, indicating that RNF11 recruits GGA3 and controls its ubiquitination by regulating itch activity. Accordingly, our data demonstrate the involvement of itch in regulating GGA3 stability. Indeed, we observe that the endogenous levels of GGA3 are increased in cells knocked down for itch and endogenous GGA3 is hyperubiquitinated in an itch-dependent manner in a cell line expressing catalytically inactive RNF11. Our data are consistent with a model whereby the RING E3 ligase RNF11 is a novel GGA cargo actively participating in regulating the ubiquitination of the GGA protein family. The results that we are presenting put RNF11 at the center of a finally regulated system where it acts both as an adaptor and a modulator of itch-mediated control of ubiquitination events underlying membrane traffic. PMID:25195858

  9. Your personalized protein structure: Andrei N. Lupas fused to GCN4 adaptors.

    PubMed

    Deiss, Silvia; Hernandez Alvarez, Birte; Bär, Kerstin; Ewers, Carolin P; Coles, Murray; Albrecht, Reinhard; Hartmann, Marcus D

    2014-06-01

    This work presents a protein structure that has been designed purely for aesthetic reasons, symbolizing decades of coiled-coil research and praising its most fundamental model system, the GCN4 leucine zipper. The GCN4 leucine zipper is a highly stable coiled coil which can be tuned to adopt different oligomeric states via mutation of its core residues. For these reasons it is used in structural studies as a stabilizing fusion adaptor. On the occasion of the 50th birthday of Andrei N. Lupas, we used it to create the first personalized protein structure: we fused the sequence ANDREI-N-LVPAS in heptad register to trimeric GCN4 adaptors and determined its structure by X-ray crystallography. The structure demonstrates the robustness and versatility of GCN4 as a fusion adaptor. We learn how proline can be accommodated in trimeric coiled coils, and put the structure into the context of the other GCN4-fusion structures known to date. PMID:24486584

  10. Single Amino Acid Substitutions Confer the Antiviral Activity of the TRAF3 Adaptor Protein onto TRAF5

    PubMed Central

    Zhang, Peng; Reichardt, Anna; Liang, Huanhuan; Aliyari, Roghiyh; Cheng, David; Wang, Yaya; Xu, Feng

    2014-01-01

    The TRAF [tumor necrosis factor receptor–associated factor] family of cytoplasmic adaptor proteins link cell-surface receptors to intracellular signaling pathways that regulate innate and adaptive immune responses. In response to activation of RIG-I (retinoic acid–inducible gene I), a component of a pattern recognition receptor that detects viruses, TRAF3 binds to the adaptor protein Cardif [caspase activation and recruitment domain (CARD) adaptor–inducing interferon-b (IFN-b)], leading to induction of type I IFNs. We report the crystal structures of the TRAF domain of TRAF5 and that of TRAF3 bound to a peptide from the TRAF-interacting motif of Cardif. By comparing these structures, we identified two residues located near the Cardif binding pocket in TRAF3 (Tyr440 and Phe473) that potentially contributed to Cardif recognition. In vitro and cellular experiments showed that forms of TRAF5 with mutation of the corresponding residues to those of TRAF3 had TRAF3-like antiviral activity. Our results provide a structural basis for the critical role of TRAF3 in activating RIG-I–mediated IFN production. PMID:23150880

  11. Dissecting nuclear Wingless signalling: recruitment of the transcriptional co-activator Pygopus by a chain of adaptor proteins.

    PubMed

    Städeli, Reto; Basler, Konrad

    2005-11-01

    Members of the Wingless (Wg)/Wnt family of secreted glycoproteins control cell fate during embryonic development and adult homeostasis. Wnt signals regulate the expression of target genes by activating a conserved signal transduction pathway. Upon receptor activation, the signal is transmitted intracellularly by stabilization of Armadillo (Arm)/beta-catenin. Arm/beta-catenin translocates to the nucleus, interacts with DNA-binding factors of the Pangolin (Pan)/TCF/LEF class and activates transcription of target genes in cooperation with the recently identified proteins Legless/BCL9 (Lgs) and Pygopus (Pygo). Here, we analyse the mode of action of Pan, Arm, Lgs, and Pygo in Drosophila cultured cells. We provide evidence that together these four proteins form a 'chain of adaptors' linking the NH2-terminal homology domain (NHD) of Pygo to the DNA-binding domain of Pan. We show that the NHD has potent transcriptional activation capacity, which differs from that of acidic activator domains and depends on a conserved NPF tripeptide. A single point mutation within this NPF motif abolishes the transcriptional activity of the Pygo NHD in vitro and strongly reduces Wg signalling in vivo. Together, our results suggest that the transcriptional output of Wg pathway activity largely relies on a 'chain of adaptors' design to direct the Pygo NHD to Wg target promoters in an Arm-dependent manner. PMID:16169192

  12. Structural basis for concerted recruitment and activation of IRF-3 by innate immune adaptor proteins.

    PubMed

    Zhao, Baoyu; Shu, Chang; Gao, Xinsheng; Sankaran, Banumathi; Du, Fenglei; Shelton, Catherine L; Herr, Andrew B; Ji, Jun-Yuan; Li, Pingwei

    2016-06-14

    Type I IFNs are key cytokines mediating innate antiviral immunity. cGMP-AMP synthase, ritinoic acid-inducible protein 1 (RIG-I)-like receptors, and Toll-like receptors recognize microbial double-stranded (ds)DNA, dsRNA, and LPS to induce the expression of type I IFNs. These signaling pathways converge at the recruitment and activation of the transcription factor IRF-3 (IFN regulatory factor 3). The adaptor proteins STING (stimulator of IFN genes), MAVS (mitochondrial antiviral signaling), and TRIF (TIR domain-containing adaptor inducing IFN-β) mediate the recruitment of IRF-3 through a conserved pLxIS motif. Here we show that the pLxIS motif of phosphorylated STING, MAVS, and TRIF binds to IRF-3 in a similar manner, whereas residues upstream of the motif confer specificity. The structure of the IRF-3 phosphomimetic mutant S386/396E bound to the cAMP response element binding protein (CREB)-binding protein reveals that the pLxIS motif also mediates IRF-3 dimerization and activation. Moreover, rotavirus NSP1 (nonstructural protein 1) employs a pLxIS motif to target IRF-3 for degradation, but phosphorylation of NSP1 is not required for its activity. These results suggest a concerted mechanism for the recruitment and activation of IRF-3 that can be subverted by viral proteins to evade innate immune responses. PMID:27302953

  13. The adaptor protein Dab2 sorts LDL receptors into coated pits independently of AP-2 and ARH.

    PubMed

    Maurer, Meghan E; Cooper, Jonathan A

    2006-10-15

    Clathrin-mediated endocytosis requires cargo-specific adaptor proteins that recognize specific receptors and recruit them into coated pits. ARH [also called low-density lipoprotein receptor (LDLR) adaptor protein] serves as an adaptor for LDLR endocytosis in liver. However, ARH is dispensable for LDL uptake by some other cell types. Here, we show that the adaptor Dab2 plays a major role in LDLR internalization in HeLa cells and fibroblasts. Dab2 mediates internalization of LDLRs but not transferrin receptors independently of ARH and the classic clathrin adaptor AP-2. If Dab2 is absent, ARH can mediate LDLR endocytosis, but its action requires AP-2. Furthermore, the rate of LDLR endocytosis is decreased when Dab2 is absent and Dab2, but not ARH, catalyzes the efficient clustering of LDLR into coated pits. Dab2 activity requires its binding to clathrin, LDLR and phospholipids. Dab2 is also involved in moving LDLRs off filopodia. We suggest that Dab2 is a cargo-specific endocytic adaptor protein, stably associating with phospholipids and clathrin to sort LDLR to nascent-coated pits, whereas ARH might accelerate later steps in LDLR endocytosis in cooperation with AP-2. PMID:16984970

  14. The SH2B1 Adaptor Protein Associates with a Proximal Region of the Erythropoietin Receptor*

    PubMed Central

    Javadi, Mojib; Hofstätter, Edda; Stickle, Natalie; Beattie, Bryan K.; Jaster, Robert; Carter-Su, Christin; Barber, Dwayne L.

    2012-01-01

    Gene targeting experiments have shown that the cytokine erythropoietin (EPO), its cognate erythropoietin receptor (EPO-R), and associated Janus tyrosine kinase, JAK2, are all essential for erythropoiesis. Structural-functional and murine knock-in experiments have suggested that EPO-R Tyr-343 is important in EPO-mediated mitogenesis. Although Stat5 binds to EPO-R phosphotyrosine 343, the initial Stat5-deficient mice did not have profound erythroid abnormalities suggesting that additional Src homology 2 (SH2) domain-containing effectors may bind to EPO-R Tyr-343 and couple to downstream signaling pathways. We have utilized cloning of ligand target (COLT) screening to demonstrate that EPO-R Tyr(P)-343 and Tyr(P)-401 bind to the SH2 domain-containing adaptor protein SH2B1β. Immunoprecipitation and in vitro mixing experiments reveal that EPO-R binds to SH2B1 in an SH2 domain-dependent manner and that the sequence that confers SH2B1 binding to the EPO-R is pYXXL. Previous studies have shown that SH2B1 binds directly to JAK2, but we show that in hematopoietic cells, SH2B1β preferentially associates with the EPO-R. SH2B1 is capable of constitutive association with EPO-R, which is necessary for its optimal SH2-dependent recruitment to EPO-R-Tyr(P)-343/Tyr(P)-401. We also demonstrate that SH2B1 is responsive to EPO stimulation and becomes phosphorylated, most likely on serines/threonines, in an EPO dose- and time-dependent manner. In the absence of SH2B1, we observe enhanced activation of signaling pathways downstream of the EPO-R, indicating that SH2B1 is a negative regulator of EPO signaling. PMID:22669948

  15. The SH2B1 adaptor protein associates with a proximal region of the erythropoietin receptor.

    PubMed

    Javadi, Mojib; Hofstätter, Edda; Stickle, Natalie; Beattie, Bryan K; Jaster, Robert; Carter-Su, Christin; Barber, Dwayne L

    2012-07-27

    Gene targeting experiments have shown that the cytokine erythropoietin (EPO), its cognate erythropoietin receptor (EPO-R), and associated Janus tyrosine kinase, JAK2, are all essential for erythropoiesis. Structural-functional and murine knock-in experiments have suggested that EPO-R Tyr-343 is important in EPO-mediated mitogenesis. Although Stat5 binds to EPO-R phosphotyrosine 343, the initial Stat5-deficient mice did not have profound erythroid abnormalities suggesting that additional Src homology 2 (SH2) domain-containing effectors may bind to EPO-R Tyr-343 and couple to downstream signaling pathways. We have utilized cloning of ligand target (COLT) screening to demonstrate that EPO-R Tyr(P)-343 and Tyr(P)-401 bind to the SH2 domain-containing adaptor protein SH2B1β. Immunoprecipitation and in vitro mixing experiments reveal that EPO-R binds to SH2B1 in an SH2 domain-dependent manner and that the sequence that confers SH2B1 binding to the EPO-R is pYXXL. Previous studies have shown that SH2B1 binds directly to JAK2, but we show that in hematopoietic cells, SH2B1β preferentially associates with the EPO-R. SH2B1 is capable of constitutive association with EPO-R, which is necessary for its optimal SH2-dependent recruitment to EPO-R-Tyr(P)-343/Tyr(P)-401. We also demonstrate that SH2B1 is responsive to EPO stimulation and becomes phosphorylated, most likely on serines/threonines, in an EPO dose- and time-dependent manner. In the absence of SH2B1, we observe enhanced activation of signaling pathways downstream of the EPO-R, indicating that SH2B1 is a negative regulator of EPO signaling. PMID:22669948

  16. The role of small adaptor proteins in the control of oncogenic signaling driven by tyrosine kinases in human cancer

    PubMed Central

    Naudin, Cécile; Chevalier, Clément; Roche, Serge

    2016-01-01

    Protein phosphorylation on tyrosine (Tyr) residues has evolved as an important mechanism to coordinate cell communication in multicellular organisms. The importance of this process has been revealed by the discovery of the prominent oncogenic properties of tyrosine kinases (TK) upon deregulation of their physiological activities, often due to protein overexpression and/or somatic mutation. Recent reports suggest that TK oncogenic signaling is also under the control of small adaptor proteins. These cytosolic proteins lack intrinsic catalytic activity and signal by linking two functional members of a catalytic pathway. While most adaptors display positive regulatory functions, a small group of this family exerts negative regulatory functions by targeting several components of the TK signaling cascade. Here, we review how these less studied adaptor proteins negatively control TK activities and how their loss of function induces abnormal TK signaling, promoting tumor formation. We also discuss the therapeutic consequences of this novel regulatory mechanism in human oncology. PMID:26788993

  17. Nrf2 reduces levels of phosphorylated tau protein by inducing autophagy adaptor protein NDP52

    NASA Astrophysics Data System (ADS)

    Jo, Chulman; Gundemir, Soner; Pritchard, Susanne; Jin, Youngnam N.; Rahman, Irfan; Johnson, Gail V. W.

    2014-03-01

    Nuclear factor erythroid 2-related factor 2 (Nrf2) is a pivotal transcription factor in the defence against oxidative stress. Here we provide evidence that activation of the Nrf2 pathway reduces the levels of phosphorylated tau by induction of an autophagy adaptor protein NDP52 (also known as CALCOCO2) in neurons. The expression of NDP52, which we show has three antioxidant response elements (AREs) in its promoter region, is strongly induced by Nrf2, and its overexpression facilitates clearance of phosphorylated tau in the presence of an autophagy stimulator. In Nrf2-knockout mice, phosphorylated and sarkosyl-insoluble tau accumulates in the brains concurrent with decreased levels of NDP52. Moreover, NDP52 associates with phosphorylated tau from brain cortical samples of Alzheimer disease cases, and the amount of phosphorylated tau in sarkosyl-insoluble fractions is inversely proportional to that of NDP52. These results suggest that NDP52 plays a key role in autophagy-mediated degradation of phosphorylated tau in vivo.

  18. Proteins recruited by SH3 domains of Ruk/CIN85 adaptor identified by LC-MS/MS

    PubMed Central

    Havrylov, Serhiy; Rzhepetskyy, Yuriy; Malinowska, Agata; Drobot, Lyudmyla; Redowicz, Maria Jolanta

    2009-01-01

    Background Ruk/CIN85 is a mammalian adaptor molecule with three SH3 domains. Using its SH3 domains Ruk/CIN85 can cluster multiple proteins and protein complexes, and, consequently, facilitates organisation of elaborate protein interaction networks with diverse regulatory roles. Previous research linked Ruk/CIN85 with the regulation of vesicle-mediated transport and cancer cell invasiveness. Despite the recent findings, precise molecular functions of Ruk/CIN85 in these processes remain largely elusive and further research is hampered by a lack of complete lists of its partner proteins. Results In the present study we employed a LC-MS/MS-based experimental pipeline to identify a considerable number (over 100) of proteins recruited by the SH3 domains of Ruk/CIN85 in vitro. Most of these identifications are novel Ruk/CIN85 interaction candidates. The identified proteins have diverse molecular architectures and can interact with other proteins, as well as with lipids and nucleic acids. Some of the identified proteins possess enzymatic activities. Functional profiling analyses and literature mining demonstrate that many of the proteins recruited by the SH3 domains of Ruk/CIN85 identified in this work were involved in the regulation of membranes and cytoskeletal structures necessary for vesicle-mediated transport and cancer cell invasiveness. Several groups of the proteins were also associated with few other cellular processes not previously related to Ruk/CIN85, most prominently with cell division. Conclusion Obtained data support the notion that Ruk/CIN85 regulates vesicle-mediated transport and cancer cell invasiveness through the assembly of multimeric protein complexes governing coordinated remodelling of membranes and underlying cytoskeletal structures, and imply its important roles in formation of coated vesicles and biogenesis of invadopodia. In addition, this study points to potential involvement of Ruk/CIN85 in other cellular processes, chiefly in cell division

  19. Interaction of atypical cadherin Fat1 with SoHo adaptor proteins CAP/ponsin and ArgBP2.

    PubMed

    Braun, Gerald S; Kuszka, Andrzej; Dau, Cécile; Kriz, Wilhelm; Moeller, Marcus J

    2016-03-25

    Mammalian Fat1 is a giant atypical cadherin/tumor suppressor involved in the regulation of cellular orientation, migration, and growth. Fat1 is implicated in the development of the brain, eye, and kidney. Altered expression or mutations of FAT1 are also associated with cancer and facioscapulohumeral muscular dystrophy (FSHD). Yet, the mechanistic functions of this pathway remain incompletely understood. Here, we report the identification of Sorbin-homology (SoHo) proteins as novel interaction partners of Fat1 by virtue of a yeast-two-hybrid screen. SoHo proteins play diverse roles as adaptor proteins in cell signaling, cell adhesion and sarcomere architecture, including altered expression in cancer and FSHD. Specifically, we found SoHo proteins CAP/ponsin-1 and -2 (Sorbs1) and ArgBP2 (Sorbs2) to interact with the cytoplasmic domain of Fat1. We mapped the interaction to a prolin-rich classic type II PXXP motif within Fat1 and to the three Src-homology (SH3) domains within SoHo proteins using mutant expression in yeast, pulldown assays, and cell culture. Functionally, endogenous ponsin-2 expression of NRK-52E cells at cellular leading edges was lost upon knockdown of Fat1. In summary, our data point to an interaction of Fat1 with SoHo proteins that is able to recruit SoHo proteins to sites of Fat1 expression. PMID:26903299

  20. Synthetic Protein Scaffolds Based on Peptide Motifs and Cognate Adaptor Domains for Improving Metabolic Productivity.

    PubMed

    Horn, Anselm H C; Sticht, Heinrich

    2015-01-01

    The efficiency of many cellular processes relies on the defined interaction among different proteins within the same metabolic or signaling pathway. Consequently, a spatial colocalization of functionally interacting proteins has frequently emerged during evolution. This concept has been adapted within the synthetic biology community for the purpose of creating artificial scaffolds. A recent advancement of this concept is the use of peptide motifs and their cognate adaptor domains. SH2, SH3, GBD, and PDZ domains have been used most often in research studies to date. The approach has been successfully applied to the synthesis of a variety of target molecules including catechin, D-glucaric acid, H2, hydrochinone, resveratrol, butyrate, gamma-aminobutyric acid, and mevalonate. Increased production levels of up to 77-fold have been observed compared to non-scaffolded systems. A recent extension of this concept is the creation of a covalent linkage between peptide motifs and adaptor domains, which leads to a more stable association of the scaffolded systems and thus bears the potential to further enhance metabolic productivity. PMID:26636078

  1. Synthetic Protein Scaffolds Based on Peptide Motifs and Cognate Adaptor Domains for Improving Metabolic Productivity

    PubMed Central

    Horn, Anselm H. C.; Sticht, Heinrich

    2015-01-01

    The efficiency of many cellular processes relies on the defined interaction among different proteins within the same metabolic or signaling pathway. Consequently, a spatial colocalization of functionally interacting proteins has frequently emerged during evolution. This concept has been adapted within the synthetic biology community for the purpose of creating artificial scaffolds. A recent advancement of this concept is the use of peptide motifs and their cognate adaptor domains. SH2, SH3, GBD, and PDZ domains have been used most often in research studies to date. The approach has been successfully applied to the synthesis of a variety of target molecules including catechin, D-glucaric acid, H2, hydrochinone, resveratrol, butyrate, gamma-aminobutyric acid, and mevalonate. Increased production levels of up to 77-fold have been observed compared to non-scaffolded systems. A recent extension of this concept is the creation of a covalent linkage between peptide motifs and adaptor domains, which leads to a more stable association of the scaffolded systems and thus bears the potential to further enhance metabolic productivity. PMID:26636078

  2. Asc1p, a WD40-domain containing adaptor protein, is required for the interaction of the RNA-binding protein Scp160p with polysomes.

    PubMed Central

    Baum, Sonja; Bittins, Margarethe; Frey, Steffen; Seedorf, Matthias

    2004-01-01

    Scp160p interacts in an mRNA-dependent manner with translating ribosomes via multiple RNA-binding heterogeneous nuclear ribonucleoprotein K-homology (KH) domains. In the present study, we show by protein-protein cross-linking that Scp160p is in close proximity to translation elongation factor 1A and the WD40 (Trp-Asp 40)-repeat containing protein Asc1p at ribosomes. Analysis of a truncation mutant revealed that the C-terminus of Scp160p is essential for ribosome binding and that Cys(1067) at the C-terminus of Scp160p is required to obtain these cross-links. The interaction of Scp160p with ribosomes depends on Asc1p. In fast-growing yeast cells, nearly all Asc1p is tightly bound to ribosomes, but it can also be present in a ribosome-free form depending on growth conditions. The functional homologue of Asc1p, mammalian RACK1 (receptor of activated C kinase), was previously characterized as an adaptor protein bridging activated signalling molecules with their substrates. Our results suggest that Scp160p connects specific mRNAs, ribosomes and a translation factor with an adaptor for signalling molecules. These interactions might regulate the translation activity of ribosomes programmed with specific mRNAs. PMID:15012629

  3. Novel Toll/IL-1 Receptor Homologous Region Adaptors Act as Negative Regulators in Amphioxus TLR Signaling.

    PubMed

    Peng, Jian; Tao, Xin; Li, Rui; Hu, Jingru; Ruan, Jie; Wang, Ruihua; Yang, Manyi; Yang, Rirong; Dong, Xiangru; Chen, Shangwu; Xu, Anlong; Yuan, Shaochun

    2015-10-01

    Studies have shown that the basal chordate amphioxus possesses an extraordinarily complex TLR system, including 39 TLRs and at least 40 Toll/IL-1R homologous region (TIR) adaptors. Besides homologs to MyD88 and TIR domain-containing adaptor molecule (TICAM), most amphioxus TIR adaptors exhibit domain architectures that are not observed in other species. To reveal how these novel TIR adaptors function in amphioxus Branchiostoma belcheri tsingtauense (bbt), four representatives, bbtTIRA, bbtTIRB, bbtTIRC, and bbtTIRD, were selected for functional analyses. We found bbtTIRA to show a unique inhibitory role in amphioxus TICAM-mediated pathway by interacting with bbtTICAM and bbt receptor interacting protein 1b, whereas bbtTIRC specifically inhibits the amphioxus MyD88-dependent pathway by interacting with bbtMyD88 and depressing the polyubiquitination of bbt TNFR-associated factor 6. Although both bbtTIRB and bbtTIRD are located on endosomes, the TIR domain of bbtTIRB can interact with bbtMyD88 in the cytosol, whereas the TIR domain of bbtTIRD is enclosed in endosome, suggesting that bbtTIRD may be a redundant gene in amphioxus. This study indicated that most expanded TIR adaptors play nonredundant regulatory roles in amphioxus TLR signaling, adding a new layer to understanding the diversity and complexity of innate immunity at basal chordate. PMID:26324776

  4. The SH3-SAM adaptor HACS1 is up-regulated in B cell activation signaling cascades.

    PubMed

    Zhu, Yuan Xiao; Benn, Sally; Li, Zhi Hua; Wei, Ellen; Masih-Khan, Esther; Trieu, Young; Bali, Meenakshi; McGlade, C Jane; Claudio, Jaime O; Stewart, A Keith

    2004-09-20

    HACS1 is a Src homology 3 and sterile alpha motif domain-containing adaptor that is preferentially expressed in normal hematopoietic tissues and malignancies including myeloid leukemia, lymphoma, and myeloma. Microarray data showed HACS1 expression is up-regulated in activated human B cells treated with interleukin (IL)-4, CD40L, and anti-immunoglobulin (Ig)M and clustered with genes involved in signaling, including TNF receptor-associated protein 1, signaling lymphocytic activation molecule, IL-6, and DEC205. Immunoblot analysis demonstrated that HACS1 is up-regulated by IL-4, IL-13, anti-IgM, and anti-CD40 in human peripheral blood B cells. In murine spleen B cells, Hacs1 can also be up-regulated by lipopolysaccharide but not IL-13. Induction of Hacs1 by IL-4 is dependent on Stat6 signaling and can also be impaired by inhibitors of phosphatidylinositol 3-kinase, protein kinase C, and nuclear factor kappaB. HACS1 associates with tyrosine-phosphorylated proteins after B cell activation and binds in vitro to the inhibitory molecule paired Ig-like receptor B. Overexpression of HACS1 in murine spleen B cells resulted in a down-regulation of the activation marker CD23 and enhancement of CD138 expression, IgM secretion, and Xbp-1 expression. Knock down of HACS1 in a human B lymphoma cell line by small interfering ribonucleic acid did not significantly change IL-4-stimulated B cell proliferation. Our study demonstrates that HACS1 is up-regulated by B cell activation signals and is a participant in B cell activation and differentiation. PMID:15381729

  5. The role of palmitoylation and transmembrane domain in sorting of transmembrane adaptor proteins.

    PubMed

    Chum, Tomáš; Glatzová, Daniela; Kvíčalová, Zuzana; Malínský, Jan; Brdička, Tomáš; Cebecauer, Marek

    2016-01-01

    Plasma membrane proteins synthesised at the endoplasmic reticulum are delivered to the cell surface via sorting pathways. Hydrophobic mismatch theory based on the length of the transmembrane domain (TMD) dominates discussion about determinants required for protein sorting to the plasma membrane. Transmembrane adaptor proteins (TRAP) are involved in signalling events which take place at the plasma membrane. Members of this protein family have TMDs of varying length. We were interested in whether palmitoylation or other motifs contribute to the effective sorting of TRAP proteins. We found that palmitoylation is essential for some, but not all, TRAP proteins independent of their TMD length. We also provide evidence that palmitoylation and proximal sequences can modulate sorting of artificial proteins with TMDs of suboptimal length. Our observations point to a unique character of each TMD defined by its primary amino acid sequence and its impact on membrane protein localisation. We conclude that, in addition to the TMD length, secondary sorting determinants such as palmitoylation or flanking sequences have evolved for the localisation of membrane proteins. PMID:26585312

  6. Immune adaptor ADAP in T cells regulates HIV-1 transcription and cell-cell viral spread via different co-receptors

    PubMed Central

    2013-01-01

    Background Immune cell adaptor protein ADAP (adhesion and degranulation-promoting adaptor protein) mediates aspects of T-cell adhesion and proliferation. Despite this, a connection between ADAP and infection by the HIV-1 (human immunodeficiency virus-1) has not been explored. Results In this paper, we show for the first time that ADAP and its binding to SLP-76 (SH2 domain-containing leukocyte protein of 76 kDa) regulate HIV-1 infection via two distinct mechanisms and co-receptors. siRNA down-regulation of ADAP, or expression of a mutant that is defective in associating to its binding partner SLP-76 (termed M12), inhibited the propagation of HIV-1 in T-cell lines and primary human T-cells. In one step, ADAP and its binding to SLP-76 were needed for the activation of NF-κB and its transcription of the HIV-1 long terminal repeat (LTR) in cooperation with ligation of co-receptor CD28, but not LFA-1. In a second step, the ADAP-SLP-76 module cooperated with LFA-1 to regulate conjugate formation between T-cells and dendritic cells or other T-cells as well as the development of the virological synapse (VS) and viral spread between immune cells. Conclusions These findings indicate that ADAP regulates two steps of HIV-1 infection cooperatively with two distinct receptors, and as such, serves as a new potential target in the blockade of HIV-1 infection. PMID:24047317

  7. Molecular basis of substrate selection by the N-end rule adaptor protein ClpS

    SciTech Connect

    Román-Hernández, Giselle; Grant, Robert A.; Sauer, Robert T.; Baker, Tania A.

    2009-06-19

    The N-end rule is a conserved degradation pathway that relates the stability of a protein to its N-terminal amino acid. Here, we present crystal structures of ClpS, the bacterial N-end rule adaptor, alone and engaged with peptides containing N-terminal phenylalanine, leucine, and tryptophan. These structures, together with a previous structure of ClpS bound to an N-terminal tyrosine, illustrate the molecular basis of recognition of the complete set of primary N-end rule amino acids. In each case, the alpha-amino group and side chain of the N-terminal residue are the major determinants of recognition. The binding pocket for the N-end residue is preformed in the free adaptor, and only small adjustments are needed to accommodate N-end rule residues having substantially different sizes and shapes. M53A ClpS is known to mediate degradation of an expanded repertoire of substrates, including those with N-terminal valine or isoleucine. A structure of Met53A ClpS engaged with an N-end rule tryptophan reveals an essentially wild-type mechanism of recognition, indicating that the Met(53) side chain directly enforces specificity by clashing with and excluding beta-branched side chains. Finally, experimental and structural data suggest mechanisms that make proteins with N-terminal methionine bind very poorly to ClpS, explaining why these high-abundance proteins are not degraded via the N-end rule pathway in the cell.

  8. Adaptors in toll-like receptor signaling and their potential as therapeutic targets.

    PubMed

    Ve, Thomas; Gay, Nicholas J; Mansell, Ashley; Kobe, Bostjan; Kellie, Stuart

    2012-10-01

    To initiate the innate immune response, Toll-like receptors (TLRs) associate with cytoplasmic adaptor proteins through TIR (Toll/interleukin-1 receptor) domain interactions. The four principal signaling adaptor proteins include MyD88, MAL, TRIF and TRAM, and the fifth protein SARM, involved in negative regulation of TLR pathways, is usually considered a part of the TIR domain-containing adaptor protein group. Other TIR domain-containing proteins have also been shown to regulate these signaling pathways, including ST2 and SIGIRR, as well as several bacterial and viral TIR domain-containing proteins that modulate these pathways as virulence factors. TLR pathways and the adaptor proteins are associated with a number of diseases, including infection, sepsis, inflammatory, allergic and autoimmune diseases and cancer. We review our current understanding of the structure and function of adaptor proteins and their regulatory proteins, their association with disease and their potential as therapeutic targets in human disease. PMID:22664090

  9. 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

  10. The adaptor protein TRAF3 inhibits interleukin-6 receptor signaling in B cells to limit plasma cell development

    PubMed Central

    Lin, Wai W.; Yi, Zuoan; Stunz, Laura L.; Maine, Christian J.; Sherman, Linda A.; Bishop, Gail A.

    2016-01-01

    Tumor necrosis factor receptor–associated factor 3 (TRAF3) is an adaptor protein that inhibits signaling by CD40 and by the receptor for B cell–activating factor (BAFF) and negatively regulates homeostatic B cell survival. Loss-of-function mutations in TRAF3 are associated with human B cell malignancies, in particular multiple myeloma. The cytokine interleukin-6 (IL-6) supports the differentiation and survival of normal and neoplastic plasma cells. We found that mice with a deficiency in TRAF3 specifically in B cells (B-Traf3−/− mice) had about twice as many plasma cells as did their littermate controls. TRAF3-deficient B cells had enhanced responsiveness to IL-6, and genetic loss of IL-6 in B-Traf3−/− mice restored their plasma cell numbers to normal. TRAF3 inhibited IL-6 receptor (IL-6R)–mediated signaling by facilitating the association of PTPN22 (a nonreceptor protein tyrosine phosphatase) with the kinase Janus-activated kinase 1 (Jak1), which in turn blocked phosphorylation of the transcription factor STAT3 (signal transducer and activator of transcription 3). Consistent with these results, the number of plasma cells in the PTPN22-deficient mice was increased compared to that in the wild-type mice. Our findings identify TRAF3 and PTPN22 as inhibitors of IL-6R signaling in B cells and reveal a previously uncharacterized role for TRAF3 in the regulation of plasma cell differentiation. PMID:26329582

  11. The clathrin adaptor Numb regulates intestinal cholesterol absorption through dynamic interaction with NPC1L1.

    PubMed

    Li, Pei-Shan; Fu, Zhen-Yan; Zhang, Ying-Yu; Zhang, Jin-Hui; Xu, Chen-Qi; Ma, Yi-Tong; Li, Bo-Liang; Song, Bao-Liang

    2014-01-01

    Hypercholesterolemia, typically due to excessive cholesterol uptake, is a major risk factor for cardiovascular disease, which is responsible for ∼50% of all deaths in developed societies. Although it has been shown that intestinal cholesterol absorption is mediated by vesicular endocytosis of the Niemann-Pick C1-like 1 (NPC1L1) protein, the mechanism of sterol-stimulated NPC1L1 internalization is still mysterious. Here, we identified an endocytic peptide signal, YVNXXF (where X stands for any amino acid), in the cytoplasmic C-terminal tail of NPC1L1. Cholesterol binding on the N-terminal domain of NPC1L1 released the YVNXXF-containing region of NPC1L1 from association with the plasma membrane and enabled Numb binding. We also found that Numb, a clathrin adaptor, specifically recognized this motif and recruited clathrin for internalization. Disrupting the NPC1L1-Numb interaction decreased cholesterol uptake. Ablation of Numb in mouse intestine significantly reduced dietary cholesterol absorption and plasma cholesterol level. Together, these data show that Numb is a pivotal protein for intestinal cholesterol absorption and may provide a therapeutic target for hypercholesterolemia. PMID:24336247

  12. Regulation of natural cytotoxicity by the adaptor SAP and the Src-related kinase Fyn

    PubMed Central

    Bloch-Queyrat, Coralie; Fondanèche, Marie-Claude; Chen, Riyan; Yin, Luo; Relouzat, Francis; Veillette, André; Fischer, Alain; Latour, Sylvain

    2005-01-01

    SAP is an adaptor protein that is expressed in NK and T cells. It is mutated in humans who have X-linked lymphoproliferative (XLP) disease. By interacting with SLAM family receptors, SAP enables tyrosine phosphorylation signaling of these receptors by its ability to recruit the Src-related kinase, Fyn. Here, we analyzed the role of SAP in NK cell functions using the SAP-deficient mouse model. Our results showed that SAP was required for the ability of NK cells to eliminate tumor cells in vitro and in vivo. This effect strongly correlated with expression of CD48 on tumor cells, the ligand of 2B4, a SLAM-related receptor expressed in NK cells. In keeping with earlier reports that studied human NK cells, we showed that SAP was necessary for the ability of 2B4 to trigger cytotoxicity and IFN-γ secretion. In the absence of SAP, 2B4 function was shifted toward inhibition of NK cell–mediated cytotoxicity. By analyzing mice lacking Fyn, we showed that similarly to SAP, Fyn was strictly required for 2B4 function. Taken together, these results provide evidence that the 2B4-SAP-Fyn cascade defines a potent activating pathway of natural cytotoxicity. They also could help to explain the high propensity of patients who have XLP disease to develop lymphoproliferative disorders. PMID:15998796

  13. A Common Variant in the Adaptor Mal Regulates Interferon Gamma Signaling.

    PubMed

    Ní Cheallaigh, Clíona; Sheedy, Frederick J; Harris, James; Muñoz-Wolf, Natalia; Lee, Jinhee; West, Kim; McDermott, Eva Palsson; Smyth, Alicia; Gleeson, Laura E; Coleman, Michelle; Martinez, Nuria; Hearnden, Claire H A; Tynan, Graham A; Carroll, Elizabeth C; Jones, Sarah A; Corr, Sinéad C; Bernard, Nicholas J; Hughes, Mark M; Corcoran, Sarah E; O'Sullivan, Mary; Fallon, Ciara M; Kornfeld, Hardy; Golenbock, Douglas; Gordon, Stephen V; O'Neill, Luke A J; Lavelle, Ed C; Keane, Joseph

    2016-02-16

    Humans that are heterozygous for the common S180L polymorphism in the Toll-like receptor (TLR) adaptor Mal (encoded by TIRAP) are protected from a number of infectious diseases, including tuberculosis (TB), whereas those homozygous for the allele are at increased risk. The reason for this difference in susceptibility is not clear. We report that Mal has a TLR-independent role in interferon-gamma (IFN-γ) receptor signaling. Mal-dependent IFN-γ receptor (IFNGR) signaling led to mitogen-activated protein kinase (MAPK) p38 phosphorylation and autophagy. IFN-γ signaling via Mal was required for phagosome maturation and killing of intracellular Mycobacterium tuberculosis (Mtb). The S180L polymorphism, and its murine equivalent S200L, reduced the affinity of Mal for the IFNGR, thereby compromising IFNGR signaling in macrophages and impairing responses to TB. Our findings highlight a role for Mal outside the TLR system and imply that genetic variation in TIRAP may be linked to other IFN-γ-related diseases including autoimmunity and cancer. PMID:26885859

  14. A Common Variant in the Adaptor Mal Regulates Interferon Gamma Signaling

    PubMed Central

    Ní Cheallaigh, Clíona; Sheedy, Frederick J.; Harris, James; Muñoz-Wolf, Natalia; Lee, Jinhee; West, Kim; McDermott, Eva Palsson; Smyth, Alicia; Gleeson, Laura E.; Coleman, Michelle; Martinez, Nuria; Hearnden, Claire H.A.; Tynan, Graham A.; Carroll, Elizabeth C.; Jones, Sarah A.; Corr, Sinéad C.; Bernard, Nicholas J.; Hughes, Mark M.; Corcoran, Sarah E.; O’Sullivan, Mary; Fallon, Ciara M.; Kornfeld, Hardy; Golenbock, Douglas; Gordon, Stephen V.; O’Neill, Luke A.J.; Lavelle, Ed C.; Keane, Joseph

    2016-01-01

    Summary Humans that are heterozygous for the common S180L polymorphism in the Toll-like receptor (TLR) adaptor Mal (encoded by TIRAP) are protected from a number of infectious diseases, including tuberculosis (TB), whereas those homozygous for the allele are at increased risk. The reason for this difference in susceptibility is not clear. We report that Mal has a TLR-independent role in interferon-gamma (IFN-γ) receptor signaling. Mal-dependent IFN-γ receptor (IFNGR) signaling led to mitogen-activated protein kinase (MAPK) p38 phosphorylation and autophagy. IFN-γ signaling via Mal was required for phagosome maturation and killing of intracellular Mycobacterium tuberculosis (Mtb). The S180L polymorphism, and its murine equivalent S200L, reduced the affinity of Mal for the IFNGR, thereby compromising IFNGR signaling in macrophages and impairing responses to TB. Our findings highlight a role for Mal outside the TLR system and imply that genetic variation in TIRAP may be linked to other IFN-γ-related diseases including autoimmunity and cancer. PMID:26885859

  15. Losses, Expansions, and Novel Subunit Discovery of Adaptor Protein Complexes in Haptophyte Algae.

    PubMed

    Lee, Laura J Y; Klute, Mary J; Herman, Emily K; Read, Betsy; Dacks, Joel B

    2015-11-01

    The phylum Haptophyta (Diaphoratickes) contains marine algae that perform biomineralization, extruding large, distinctive calcium carbonate scales (coccoliths) that completely cover the cell. Coccolith production is an important part of global carbon cycling; however, the membrane trafficking pathway by which they are secreted has not yet been elucidated. In most eukaryotes, post-Golgi membrane trafficking involves five heterotetrameric adaptor protein (AP) complexes, which impart cargo selection specificity. To better understand coccolith secretion, we performed comparative genomic, phylogenetic, and transcriptomic analyses of the AP complexes in Emiliania huxleyi strains 92A, Van556, EH2, and CCMP1516, and related haptophytes Gephyrocapsa oceanica and Isochrysis galbana; the latter has lost the ability to biomineralize. We show that haptophytes have a modified membrane trafficking system (MTS), as we found both AP subunit losses and duplications. Additionally, we identified a single conserved subunit of the AP-related TSET complex, whose expression suggests a functional role in membrane trafficking. Finally, we detected novel alpha adaptin ear and gamma adaptin ear proteins, the first of their kind to be described outside of opisthokonts. These novel ear proteins and the sculpting of the MTS may support the capacity for biomineralization in haptophytes, enhancing their ability to perform this highly specialized form of secretion. PMID:26519625

  16. Adaptor Protein Complex 2 (AP-2) Mediated, Clathrin Dependent Endocytosis, And Related Gene Activities, Are A Prominent Feature During Maturation Stage Amelogenesis

    PubMed Central

    LACRUZ, Rodrigo S.; BROOKES, Steven J.; WEN, Xin; JIMENEZ, Jaime M.; VIKMAN, Susanna; HU, Ping; WHITE, Shane N.; LYNGSTADAAS, S. Petter; OKAMOTO, Curtis T.; SMITH, Charles E.; PAINE, Michael L.

    2012-01-01

    Molecular events defining enamel matrix removal during amelogenesis are poorly understood. Early reports have suggested that adaptor proteins (AP) participate in ameloblast-mediated endocytosis. Enamel formation involves the secretory and maturation stages, with an increase in resorptive function during the latter. Here, using real time PCR, we show that the expression of clathrin and adaptor protein subunits are up-regulated in maturation stage rodent enamel organ cells. AP-2 is the most up-regulated of the four distinct adaptor protein complexes. Immunolocalization confirms the presence of AP-2 and clathrin in ameloblasts with strongest reactivity at the apical pole. These data suggest that the resorptive functions of enamel cells involve AP-2 mediated, clathrin dependent endocytosis, thus implying the likelihood of a specific membrane-bound receptor(s) of enamel matrix protein debris. The mRNA expression of other endocytosis-related gene products is also up-regulated during maturation including: lysosomal-associated membrane protein 1 (Lamp1), cluster of differentiation 63 and 68 (Cd63 and Cd68), ATPase, H+ transporting, lysosomal V0 subunit D2 (Atp6v0d2), ATPase, H+ transporting, lysosomal V1 subunit B2 (Atp6v1b2), chloride channel, voltage-sensitive 7 (Clcn7) and cathepsin K (Ctsk). Immunohistological data confirms the expression of a number of these proteins in maturation stage ameloblasts. The enamel of Cd63-null mice was also examined. Despite increased mRNA and protein expression in the enamel organ during maturation, the enamel of Cd63-null mice appeared normal. This may suggest inherent functional redundancies between Cd63 and related gene products, such as Lamp1 and Cd68. Ameloblast-like LS8 cells treated with the enamel matrix protein complex Emdogain® showed up-regulation of AP-2 and clathrin subunits, further supporting the existence of a membrane-bound receptor regulated pathway for the endocytosis of enamel matrix proteins. These data together

  17. Adaptor Protein Complex 2–Mediated Endocytosis Is Crucial for Male Reproductive Organ Development in Arabidopsis[W

    PubMed Central

    Kim, Soo Youn; Xu, Zheng-Yi; Song, Kyungyoung; Kim, Dae Heon; Kang, Hyangju; Reichardt, Ilka; Sohn, Eun Ju; Friml, Jiří; Juergens, Gerd; Hwang, Inhwan

    2013-01-01

    Fertilization in flowering plants requires the temporal and spatial coordination of many developmental processes, including pollen production, anther dehiscence, ovule production, and pollen tube elongation. However, it remains elusive as to how this coordination occurs during reproduction. Here, we present evidence that endocytosis, involving heterotetrameric adaptor protein complex 2 (AP-2), plays a crucial role in fertilization. An Arabidopsis thaliana mutant ap2m displays multiple defects in pollen production and viability, as well as elongation of staminal filaments and pollen tubes, all of which are pivotal processes needed for fertilization. Of these abnormalities, the defects in elongation of staminal filaments and pollen tubes were partially rescued by exogenous auxin. Moreover, DR5rev:GFP (for green fluorescent protein) expression was greatly reduced in filaments and anthers in ap2m mutant plants. At the cellular level, ap2m mutants displayed defects in both endocytosis of N-(3-triethylammonium-propyl)-4-(4-diethylaminophenylhexatrienyl) pyridinium dibromide, a lypophilic dye used as an endocytosis marker, and polar localization of auxin-efflux carrier PIN FORMED2 (PIN2) in the stamen filaments. Moreover, these defects were phenocopied by treatment with Tyrphostin A23, an inhibitor of endocytosis. Based on these results, we propose that AP-2–dependent endocytosis plays a crucial role in coordinating the multiple developmental aspects of male reproductive organs by modulating cellular auxin level through the regulation of the amount and polarity of PINs. PMID:23975898

  18. Phosphorylation of APP-CTF-AICD domains and interaction with adaptor proteins: signal transduction and/or transcriptional role--relevance for Alzheimer pathology.

    PubMed

    Schettini, Gennaro; Govoni, Stefano; Racchi, Marco; Rodriguez, Guido

    2010-12-01

    In recent decades, the study of the amyloid precursor protein (APP) and of its proteolytic products carboxy terminal fragment (CTF), APP intracellular C-terminal domain (AICD) and amyloid beta has been mostly focussed on the role of APP as a producer of the toxic amyloid beta peptide. Here, we reconsider the role of APP suggesting, in a provocative way, the protein as a central player in a putative signalling pathway. We highlight the presence in the cytosolic tail of APP of the YENPTY motif which is typical of tyrosine kinase receptors, the phosphorylation of the tyrosine, serine and threonine residues, the kinases involved and the interaction with intracellular adaptor proteins. In particular, we examine the interaction with Shc and Grb2 regulators, which through the activation of Ras proteins elicit downstream signalling events such as the MAPK pathway. The review also addresses the interaction of APP, CTFs and AICD with other adaptor proteins and in particular with Fe65 for nuclear transcriptional activity and the importance of phosphorylation for sorting the secretases involved in the amyloidogenic or non-amyloidogenic pathways. We provide a novel perspective on Alzheimer's disease pathogenesis, focussing on the perturbation of the physiological activities of APP-CTFs and AICD as an alternative perspective from that which normally focuses on the accumulation of neurotoxic proteolytic fragments. PMID:21039524

  19. The COPII adaptor protein TMED7 is required to initiate and mediate the anterograde trafficking of Toll-like receptor 4 to the plasma membrane

    PubMed Central

    Liaunardy-Jopeace, Ardiyanto; Bryant, Clare E.; Gay, Nicholas J.

    2015-01-01

    Toll-like receptor 4 (TLR4), the receptor for the bacterial product endotoxin, is subject to multiple points of regulation at the levels of signaling, biogenesis, and trafficking. Dysregulation of TLR4 signaling can cause serious inflammatory diseases, such as sepsis. We found that the p24 family protein TMED7 (transmembrane emp24 protein transport domain containing 7) is required for the trafficking of TLR4 from the endoplasmic reticulum to the cell surface through the Golgi. TMED7 formed a stable complex with the ectodomain of TLR4, an interaction that required the coiled-coil and GOLD domains, but not the cytosolic, COP II sorting motif, of TMED7. Depletion of TMED7 reduced TLR4 signaling mediated by the adaptor protein MyD88, but not that mediated by the adaptor proteins TRAM and TRIF. Truncated forms of TMED7 lacking the COP II sorting motif or the transmembrane domain were mislocalized and resulted in constitutive activation of TLR4 signaling. Together, these results support the hypothesis that p24 proteins perform a quality control step by recognizing correctly folded anterograde cargo, such as TLR4, in early secretory compartments and facilitating the translocation of this cargo to the cell surface. PMID:25074978

  20. Regulation of ITAM adaptor molecules and their receptors by inhibition of calcineurin-NFAT signalling during late stage osteoclast differentiation

    SciTech Connect

    Zawawi, M.S.F.; Dharmapatni, A.A.S.S.K.; Cantley, M.D.; McHugh, K.P.; Haynes, D.R.; Crotti, T.N.

    2012-10-19

    Highlights: Black-Right-Pointing-Pointer Calcineurin/NFAT inhibitors FK506 and VIVIT treated human PBMC derived osteoclasts in vitro. Black-Right-Pointing-Pointer Differential regulation of ITAM receptors and adaptor molecules by calcineurin/NFAT inhibitors. Black-Right-Pointing-Pointer FK506 and VIVIT suppress ITAM factors during late phase osteoclast differentiation. -- Abstract: Osteoclasts are specialised bone resorptive cells responsible for both physiological and pathological bone loss. Osteoclast differentiation and activity is dependent upon receptor activator NF-kappa-B ligand (RANKL) interacting with its receptor RANK to induce the transcription factor, nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1). The immunoreceptor tyrosine-based activation motif (ITAM)-dependent pathway has been identified as a co-stimulatory pathway in osteoclasts. Osteoclast-associated receptor (OSCAR) and triggering receptor expressed in myeloid cells (TREM2) are essential receptors that pair with adaptor molecules Fc receptor common gamma chain (FcR{gamma}) and DNAX-activating protein 12 kDa (DAP12) respectively to induce calcium signalling. Treatment with calcineurin-NFAT inhibitors, Tacrolimus (FK506) and the 11R-VIVIT (VIVIT) peptide, reduces NFATc1 expression consistent with a reduction in osteoclast differentiation and activity. This study aimed to investigate the effects of inhibiting calcineurin-NFAT signalling on the expression of ITAM factors and late stage osteoclast genes including cathepsin K (CathK), Beta 3 integrin ({beta}3) and Annexin VIII (AnnVIII). Human peripheral blood mononuclear cells (PBMCs) were differentiated with RANKL and macrophage-colony stimulating factor (M-CSF) over 10 days in the presence or absence of FK506 or VIVIT. Osteoclast formation (as assessed by tartrate resistant acid phosphatase (TRAP)) and activity (assessed by dentine pit resorption) were significantly reduced with treatment. Quantitative real

  1. Architecture and roles of periplasmic adaptor proteins in tripartite efflux assemblies

    PubMed Central

    Symmons, Martyn F.; Marshall, Robert L.

    2015-01-01

    Recent years have seen major advances in the structural understanding of the different components of tripartite efflux assemblies, which encompass the multidrug efflux (MDR) pumps and type I secretion systems. The majority of these investigations have focused on the role played by the inner membrane transporters and the outer membrane factor (OMF), leaving the third component of the system – the Periplasmic Adaptor Proteins (PAPs) – relatively understudied. Here we review the current state of knowledge of these versatile proteins which, far from being passive linkers between the OMF and the transporter, emerge as active architects of tripartite assemblies, and play diverse roles in the transport process. Recognition between the PAPs and OMFs is essential for pump assembly and function, and targeting this interaction may provide a novel avenue for combating multidrug resistance. With the recent advances elucidating the drug efflux and energetics of the tripartite assemblies, the understanding of the interaction between the OMFs and PAPs is the last piece remaining in the complete structure of the tripartite pump assembly puzzle. PMID:26074901

  2. Architecture and roles of periplasmic adaptor proteins in tripartite efflux assemblies.

    PubMed

    Symmons, Martyn F; Marshall, Robert L; Bavro, Vassiliy N

    2015-01-01

    Recent years have seen major advances in the structural understanding of the different components of tripartite efflux assemblies, which encompass the multidrug efflux (MDR) pumps and type I secretion systems. The majority of these investigations have focused on the role played by the inner membrane transporters and the outer membrane factor (OMF), leaving the third component of the system - the Periplasmic Adaptor Proteins (PAPs) - relatively understudied. Here we review the current state of knowledge of these versatile proteins which, far from being passive linkers between the OMF and the transporter, emerge as active architects of tripartite assemblies, and play diverse roles in the transport process. Recognition between the PAPs and OMFs is essential for pump assembly and function, and targeting this interaction may provide a novel avenue for combating multidrug resistance. With the recent advances elucidating the drug efflux and energetics of the tripartite assemblies, the understanding of the interaction between the OMFs and PAPs is the last piece remaining in the complete structure of the tripartite pump assembly puzzle. PMID:26074901

  3. The adaptor protein insulin receptor substrate 2 inhibits alternative macrophage activation and allergic lung inflammation.

    PubMed

    Dasgupta, Preeta; Dorsey, Nicolas J; Li, Jiaqi; Qi, Xiulan; Smith, Elizabeth P; Yamaji-Kegan, Kazuyo; Keegan, Achsah D

    2016-01-01

    Insulin receptor substrate 2 (IRS2) is an adaptor protein that becomes tyrosine-phosphorylated in response to the cytokines interleukin-4 (IL-4) and IL-13, which results in activation of the phosphoinositide 3-kinase (PI3K)-Akt pathway. IL-4 and IL-13 contribute to allergic lung inflammation. To examine the role of IRS2 in allergic disease, we evaluated the responses of IRS2-deficient (IRS2(-/-)) mice. Unexpectedly, loss of IRS2 resulted in a substantial increase in the expression of a subset of genes associated with the generation of alternatively activated macrophages (AAMs) in response to IL-4 or IL-13 in vitro. AAMs secrete factors that enhance allergic responses and promote airway remodeling. Moreover, compared to IRS2(+/+) mice, IRS2(+/-) and IRS2(-/-) mice developed enhanced pulmonary inflammation, accumulated eosinophils and AAMs, and exhibited airway and vascular remodeling upon allergen stimulation, responses that partially depended on macrophage-intrinsic IRS2 signaling. Both in unstimulated and IL-4-stimulated macrophages, lack of IRS2 enhanced phosphorylation of Akt and ribosomal S6 protein. Thus, we identified a critical inhibitory loop downstream of IRS2, demonstrating an unanticipated and previously unrecognized role for IRS2 in suppressing allergic lung inflammation and remodeling. PMID:27330190

  4. Impairment of dendritic cell functions in patients with adaptor protein-3 complex deficiency.

    PubMed

    Prandini, Alberto; Salvi, Valentina; Colombo, Francesca; Moratto, Daniele; Lorenzi, Luisa; Vermi, William; De Francesco, Maria Antonia; Notarangelo, Lucia Dora; Porta, Fulvio; Plebani, Alessandro; Facchetti, Fabio; Sozzani, Silvano; Badolato, Raffaele

    2016-06-30

    Hermansky-Pudlak syndrome type 2 (HPS2) is a primary immunodeficiency due to adaptor protein-3 (AP-3) complex deficiency. HPS2 patients present neutropenia, partial albinism, and impaired lysosomal vesicles formation in hematopoietic cells. Given the role of dendritic cells (DCs) in the immune response, we studied monocyte-derived DCs (moDCs) and plasmacytoid DCs (pDCs) in two HPS2 siblings. Mature HPS2 moDCs showed impaired expression of CD83 and DC-lysosome-associated membrane protein (LAMP), low levels of MIP1-β/CCL4, MIG/CXCL9, and severe defect of interleukin-12 (IL-12) secretion. DCs in lymph-node biopsies from the same patients showed a diffuse cytoplasm reactivity in a large fraction of DC-LAMP(+) cells, instead of the classical dot-like stain. In addition, analysis of pDC-related functions of blood-circulating mononuclear cells revealed reduced interferon-α secretion in response to herpes simplex virus-1 (HSV-1), whereas granzyme-B induction upon IL-3/IL-10 stimulation was normal. Finally, T-cell costimulatory activity, as measured by mixed lymphocyte reaction assay, was lower in patients, suggesting that function and maturation of DCs is abnormal in patients with HPS2. PMID:27207797

  5. Molecular protein adaptor with genetically encoded interaction sites guiding the hierarchical assembly of plasmonically active nanoparticle architectures

    NASA Astrophysics Data System (ADS)

    Schreiber, Andreas; Huber, Matthias C.; Cölfen, Helmut; Schiller, Stefan M.

    2015-03-01

    The control over the defined assembly of nano-objects with nm-precision is important to create systems and materials with enhanced properties, for example, metamaterials. In nature, the precise assembly of inorganic nano-objects with unique features, for example, magnetosomes, is accomplished by efficient and reliable recognition schemes involving protein effectors. Here we present a molecular approach using protein-based ‘adaptors/connectors’ with genetically encoded interaction sites to guide the assembly and functionality of different plasmonically active gold nanoparticle architectures (AuNP). The interaction of the defined geometricaly shaped protein adaptors with the AuNP induces the self-assembly of nanoarchitectures ranging from AuNP encapsulation to one-dimensional chain-like structures, complex networks and stars. Synthetic biology and bionanotechnology are applied to co-translationally encode unnatural amino acids as additional site-specific modification sites to generate functionalized biohybrid nanoarchitectures. This protein adaptor-based nano-object assembly approach might be expanded to other inorganic nano-objects creating biohybrid materials with unique electronic, photonic, plasmonic and magnetic properties.

  6. Molecular protein adaptor with genetically encoded interaction sites guiding the hierarchical assembly of plasmonically active nanoparticle architectures.

    PubMed

    Schreiber, Andreas; Huber, Matthias C; Cölfen, Helmut; Schiller, Stefan M

    2015-01-01

    The control over the defined assembly of nano-objects with nm-precision is important to create systems and materials with enhanced properties, for example, metamaterials. In nature, the precise assembly of inorganic nano-objects with unique features, for example, magnetosomes, is accomplished by efficient and reliable recognition schemes involving protein effectors. Here we present a molecular approach using protein-based 'adaptors/connectors' with genetically encoded interaction sites to guide the assembly and functionality of different plasmonically active gold nanoparticle architectures (AuNP). The interaction of the defined geometricaly shaped protein adaptors with the AuNP induces the self-assembly of nanoarchitectures ranging from AuNP encapsulation to one-dimensional chain-like structures, complex networks and stars. Synthetic biology and bionanotechnology are applied to co-translationally encode unnatural amino acids as additional site-specific modification sites to generate functionalized biohybrid nanoarchitectures. This protein adaptor-based nano-object assembly approach might be expanded to other inorganic nano-objects creating biohybrid materials with unique electronic, photonic, plasmonic and magnetic properties. PMID:25813537

  7. PLEKHA7: Cytoskeletal adaptor protein at center stage in junctional organization and signaling.

    PubMed

    Shah, Jimit; Guerrera, Diego; Vasileva, Ekaterina; Sluysmans, Sophie; Bertels, Eva; Citi, Sandra

    2016-06-01

    PLEKHA7 is a recently characterized component of the cytoplasmic region of epithelial adherens junctions (AJ). It comprises two WW domains, a pleckstrin-homology domain, and proline-rich and coiled-coil domains. PLEKHA7 interacts with cytoplasmic components of the AJ (p120-catenin, paracingulin, afadin), stabilizes the E-cadherin complex by linking it to the minus ends of noncentrosomal microtubules, and stabilizes junctional nectins through the newly identified interactor PDZD11. Similarly to afadin, and unlike E-cadherin and p120-catenin, the localization of PLEKHA7 at AJ is strictly zonular (in the zonula adhaerens subdomain of AJ), and does not extend along the basolateral contacts. Genome-wide association studies and experiments on animal and cellular models show that although PLEKHA7 is not required for organism viability, it is implicated in cardiovascular physiology, hypertension, primary angle closure glaucoma, susceptibility to staphylococcal α-toxin, and epithelial morphogenesis and growth. Thus, PLEKHA7 is a cytoskeletal adaptor protein important for AJ organization, and at the center of junction-associated signaling pathways which fine-tune important pathophysiological processes. PMID:27072621

  8. Tetraspanins and Transmembrane Adaptor Proteins As Plasma Membrane Organizers—Mast Cell Case

    PubMed Central

    Halova, Ivana; Draber, Petr

    2016-01-01

    The plasma membrane contains diverse and specialized membrane domains, which include tetraspanin-enriched domains (TEMs) and transmembrane adaptor protein (TRAP)-enriched domains. Recent biophysical, microscopic, and functional studies indicated that TEMs and TRAP-enriched domains are involved in compartmentalization of physicochemical events of such important processes as immunoreceptor signal transduction and chemotaxis. Moreover, there is evidence of a cross-talk between TEMs and TRAP-enriched domains. In this review we discuss the presence and function of such domains and their crosstalk using mast cells as a model. The combined data based on analysis of selected mast cell-expressed tetraspanins [cluster of differentiation (CD)9, CD53, CD63, CD81, CD151)] or TRAPs [linker for activation of T cells (LAT), non-T cell activation linker (NTAL), and phosphoprotein associated with glycosphingolipid-enriched membrane microdomains (PAG)] using knockout mice or specific antibodies point to a diversity within these two families and bring evidence of the important roles of these molecules in signaling events. An example of this diversity is physical separation of two TRAPs, LAT and NTAL, which are in many aspects similar but show plasma membrane location in different microdomains in both non-activated and activated cells. Although our understanding of TEMs and TRAP-enriched domains is far from complete, pharmaceutical applications of the knowledge about these domains are under way. PMID:27243007

  9. The methyltransferase adaptor protein Trm112 is involved in biogenesis of both ribosomal subunits

    PubMed Central

    Sardana, Richa; Johnson, Arlen W.

    2012-01-01

    We previously identified Bud23 as the methyltransferase that methylates G1575 of rRNA in the P-site of the small (40S) ribosomal subunit. In this paper, we show that Bud23 requires the methyltransferase adaptor protein Trm112 for stability in vivo. Deletion of Trm112 results in a bud23Δ-like mutant phenotype. Thus Trm112 is required for efficient small-subunit biogenesis. Genetic analysis suggests the slow growth of a trm112Δ mutant is due primarily to the loss of Bud23. Surprisingly, suppression of the bud23Δ-dependent 40S defect revealed a large (60S) biogenesis defect in a trm112Δ mutant. Using sucrose gradient sedimentation analysis and coimmunoprecipitation, we show that Trm112 is also involved in 60S subunit biogenesis. The 60S defect may be dependent on Nop2 and Rcm1, two additional Trm112 interactors that we identify. Our work extends the known range of Trm112 function from modification of tRNAs and translation factors to both ribosomal subunits, showing that its effects span all aspects of the translation machinery. Although Trm112 is required for Bud23 stability, our results suggest that Trm112 is not maintained in a stable complex with Bud23. We suggest that Trm112 stabilizes its free methyltransferase partners not engaged with substrate and/or helps to deliver its methyltransferase partners to their substrates. PMID:22956767

  10. The adaptor protein DCAF7 mediates the interaction of the adenovirus E1A oncoprotein with the protein kinases DYRK1A and HIPK2

    PubMed Central

    Glenewinkel, Florian; Cohen, Michael J.; King, Cason R.; Kaspar, Sophie; Bamberg-Lemper, Simone; Mymryk, Joe S.; Becker, Walter

    2016-01-01

    DYRK1A is a constitutively active protein kinase that has a critical role in growth and development which functions by regulating cell proliferation, differentiation and survival. DCAF7 (also termed WDR68 or HAN11) is a cellular binding partner of DYRK1A and also regulates signalling by the protein kinase HIPK2. DCAF7 is an evolutionarily conserved protein with a single WD40 repeat domain and has no catalytic activity. We have defined a DCAF7 binding motif of 12 amino acids in the N-terminal domain of class 1 DYRKs that is functionally conserved in DYRK1 orthologs from Xenopus, Danio rerio and the slime mold Dictyostelium discoideum. A similar sequence was essential for DCAF7 binding to HIPK2, whereas the closely related HIPK1 family member did not bind DCAF7. Immunoprecipitation and pulldown experiments identified DCAF7 as an adaptor for the association of the adenovirus E1A protein with DYRK1A and HIPK2. Furthermore, DCAF7 was required for the hyperphosphorylation of E1A in DYRK1A or HIPK2 overexpressing cells. Our results characterize DCAF7 as a substrate recruiting subunit of DYRK1A and HIPK2 and suggest that it is required for the negative effect of DYRK1A on E1A-induced oncogenic transformation. PMID:27307198

  11. The adaptor protein DCAF7 mediates the interaction of the adenovirus E1A oncoprotein with the protein kinases DYRK1A and HIPK2.

    PubMed

    Glenewinkel, Florian; Cohen, Michael J; King, Cason R; Kaspar, Sophie; Bamberg-Lemper, Simone; Mymryk, Joe S; Becker, Walter

    2016-01-01

    DYRK1A is a constitutively active protein kinase that has a critical role in growth and development which functions by regulating cell proliferation, differentiation and survival. DCAF7 (also termed WDR68 or HAN11) is a cellular binding partner of DYRK1A and also regulates signalling by the protein kinase HIPK2. DCAF7 is an evolutionarily conserved protein with a single WD40 repeat domain and has no catalytic activity. We have defined a DCAF7 binding motif of 12 amino acids in the N-terminal domain of class 1 DYRKs that is functionally conserved in DYRK1 orthologs from Xenopus, Danio rerio and the slime mold Dictyostelium discoideum. A similar sequence was essential for DCAF7 binding to HIPK2, whereas the closely related HIPK1 family member did not bind DCAF7. Immunoprecipitation and pulldown experiments identified DCAF7 as an adaptor for the association of the adenovirus E1A protein with DYRK1A and HIPK2. Furthermore, DCAF7 was required for the hyperphosphorylation of E1A in DYRK1A or HIPK2 overexpressing cells. Our results characterize DCAF7 as a substrate recruiting subunit of DYRK1A and HIPK2 and suggest that it is required for the negative effect of DYRK1A on E1A-induced oncogenic transformation. PMID:27307198

  12. Modulation of Hepatitis C Virus Genome Replication by Glycosphingolipids and Four-Phosphate Adaptor Protein 2

    PubMed Central

    Khan, Irfan; Katikaneni, Divya S.; Han, Qingxia; Sanchez-Felipe, Lorena; Hanada, Kentaro; Ambrose, Rebecca L.; Mackenzie, Jason M.

    2014-01-01

    ABSTRACT Hepatitis C virus (HCV) assembles its replication complex on cytosolic membrane vesicles often clustered in a membranous web (MW). During infection, HCV NS5A protein activates PI4KIIIα enzyme, causing massive production and redistribution of phosphatidylinositol 4-phosphate (PI4P) lipid to the replication complex. However, the role of PI4P in the HCV life cycle is not well understood. We postulated that PI4P recruits host effectors to modulate HCV genome replication or virus particle production. To test this hypothesis, we generated cell lines for doxycycline-inducible expression of short hairpin RNAs (shRNAs) targeting the PI4P effector, four-phosphate adaptor protein 2 (FAPP2). FAPP2 depletion attenuated HCV infectivity and impeded HCV RNA synthesis. Indeed, FAPP2 has two functional lipid-binding domains specific for PI4P and glycosphingolipids. While expression of the PI4P-binding mutant protein was expected to inhibit HCV replication, a marked drop in replication efficiency was observed unexpectedly with the glycosphingolipid-binding mutant protein. These data suggest that both domains are crucial for the role of FAPP2 in HCV genome replication. We also found that HCV significantly increases the level of some glycosphingolipids, whereas adding these lipids to FAPP2-depleted cells partially rescued replication, further arguing for the importance of glycosphingolipids in HCV RNA synthesis. Interestingly, FAPP2 is redistributed to the replication complex (RC) characterized by HCV NS5A, NS4B, or double-stranded RNA (dsRNA) foci. Additionally, FAPP2 depletion disrupts the RC and alters the colocalization of HCV replicase proteins. Altogether, our study implies that HCV coopts FAPP2 for virus genome replication via PI4P binding and glycosphingolipid transport to the HCV RC. IMPORTANCE Like most viruses with a positive-sense RNA genome, HCV replicates its RNA on remodeled host membranes composed of lipids hijacked from various internal membrane compartments

  13. Detachment-Based Equilibrium of Anoikic Cell Death and Autophagic Cell Survival Through Adaptor Protein p66(Shc).

    PubMed

    Cai, Zeyuan; Zhao, Dan; Sun, Yanan; Gao, Dan; Li, Xia; Yang, Jie; Ma, Zhenyi

    2016-03-01

    Anoikis (detachment-induced cell death) confers a tumor-suppressive function in metastatic cancer cells. Autophagy, a conserved self-degradative process, enhances the anoikis resistance of detached cancer cells by maintaining cellular homeostasis. However, the mechanism of regulating cell fate-decision by balancing anoikis and autophagy has been poorly understood. Our previous studies have shown that the adaptor protein p66(Shc) mediates anoikis through RhoA activation and inhibits tumor metastasis in vivo. We also found that p66(Shc) depletion mitigates nutrient-deprivation-induced autophagy. These findings suggest p66(Shc) may coordinately regulate these two processes. To verify this hypothesis, we investigated the effect of p66(Shc) on the cell death of detached lung cancer cells, and measured autophagy markers and autophagic flux. Results showed that p66(Shc) depletion significantly inhibited anoikis, and reduced the formation of LC3B-II and the degradation of Sequestosome 1 (SQSTM1, p62) in detachment-induced cells. Using monodansylcadaverine (MDC)-LysoTracker double staining and monomeric Cherry (mCherry)-GFP-LC3 assay, we found that the autophagic flux was also mitigated by p66(Shc) depletion. In addition, p66(Shc) knockdown increased the formation of full-length X-linked inhibitor of apoptosis (XIAP)-associated factor 1 (XAF1), which enhances anoikis sensitivity. In conclusion, p66(Shc) plays an essential role in detachment-based equilibrium of anoikic cell death and autophagic cell survival. Anat Rec, 299:325-333, 2016. © 2015 Wiley Periodicals, Inc. PMID:26643258

  14. The Adaptor Protein p62 Is Involved in RANKL-induced Autophagy and Osteoclastogenesis

    PubMed Central

    Li, Rui-Fang; Chen, Gang; Ren, Jian-Gang; Zhang, Wei; Wu, Zhong-Xing; Liu, Bing; Zhao, Yi-Fang

    2014-01-01

    Previous studies have implicated autophagy in osteoclast differentiation. The aim of this study was to investigate the potential role of p62, a characterized adaptor protein for autophagy, in RANKL-induced osteoclastogenesis. Real-time quantitative PCR and western blot analyses were used to evaluate the expression levels of autophagy-related markers during RANKL-induced osteoclastogenesis in mouse macrophage-like RAW264.7 cells. Meanwhile, the potential relationship between p62/LC3 localization and F-actin ring formation was tested using double-labeling immunofluorescence. Then, the expression of p62 in RAW264.7 cells was knocked down using small-interfering RNA (siRNA), followed by detecting its influence on RANKL-induced autophagy activation, osteoclast differentiation, and F-actin ring formation. The data showed that several key autophagy-related markers including p62 were significantly altered during RANKL-induced osteoclast differentiation. In addition, the expression and localization of p62 showed negative correlation with LC3 accumulation and F-actin ring formation, as demonstrated by western blot and immunofluorescence analyses, respectively. Importantly, the knockdown of p62 obviously attenuated RANKL-induced expression of autophagy- and osteoclastogenesis-related genes, formation of TRAP-positive multinuclear cells, accumulation of LC3, as well as formation of F-actin ring. Our study indicates that p62 may play essential roles in RANKL-induced autophagy and osteoclastogenesis, which may help to develop a novel therapeutic strategy against osteoclastogenesis-related diseases. PMID:25163928

  15. Shc adaptor proteins are key transducers of mitogenic signaling mediated by the G protein-coupled thrombin receptor.

    PubMed Central

    Chen, Y; Grall, D; Salcini, A E; Pelicci, P G; Pouysségur, J; Van Obberghen-Schilling, E

    1996-01-01

    The serine protease thrombin activates G protein signaling systems that lead to Ras activation and, in certain cells, proliferation. Whereas the steps leading to Ras activation by G protein-coupled receptors are not well defined, the mechanisms of Ras activation by receptor tyrosine kinases have recently been elucidated biochemically and genetically. The present study was undertaken to determine whether common signaling components are used by these two distinct classes of receptors. Here we report that the adaptor protein Shc, is phosphorylated on tyrosine residues following stimulation of the thrombin receptor in growth-responsive CCL39 fibroblasts. Shc phosphorylation by thrombin or the thrombin receptor agonist peptide is maximal by 15 min and persists for > or = 2 h. Following thrombin stimulation, phosphorylated Shc is recruited to Grb2 complexes. One or more pertussis toxin-insensitive proteins appear to mediate this effect, since (i) pertussis toxin pre-treatment of cells does not blunt the action of thrombin and (ii) Shc phosphorylation on tyrosine can be stimulated by the muscarinic m1 receptor. Shc phosphorylation does not appear to involve protein kinase C, since the addition of 4-beta-phorbol-12,13-dibutyrate has no effect. Rather, thrombin-induced Shc phosphorylation is enhanced in cells depleted of phorbol ester-sensitive protein kinase C isoforms. Expression of mutant Shc proteins defective in Grb2 binding displays a dominant-negative effect on thrombin-stimulated p44 MAP kinase activation, gene induction and cell growth. From these data, we conclude that Shc represents a crucial point of convergence between signaling pathways activated by receptor tyrosine kinases and G protein-coupled receptors. Images PMID:8605873

  16. High Fat Diet Enhances β-Site Cleavage of Amyloid Precursor Protein (APP) via Promoting β-Site APP Cleaving Enzyme 1/Adaptor Protein 2/Clathrin Complex Formation.

    PubMed

    Maesako, Masato; Uemura, Maiko; Tashiro, Yoshitaka; Sasaki, Kazuki; Watanabe, Kiwamu; Noda, Yasuha; Ueda, Karin; Asada-Utsugi, Megumi; Kubota, Masakazu; Okawa, Katsuya; Ihara, Masafumi; Shimohama, Shun; Uemura, Kengo; Kinoshita, Ayae

    2015-01-01

    Obesity and type 2 diabetes are risk factors of Alzheimer's disease (AD). We reported that a high fat diet (HFD) promotes amyloid precursor protein (APP) cleavage by β-site APP cleaving enzyme 1 (BACE1) without increasing BACE1 levels in APP transgenic mice. However, the detailed mechanism had remained unclear. Here we demonstrate that HFD promotes BACE1/Adaptor protein-2 (AP-2)/clathrin complex formation by increasing AP-2 levels in APP transgenic mice. In Swedish APP overexpressing Chinese hamster ovary (CHO) cells as well as in SH-SY5Y cells, overexpression of AP-2 promoted the formation of BACE1/AP-2/clathrin complex, increasing the level of the soluble form of APP β (sAPPβ). On the other hand, mutant D495R BACE1, which inhibits formation of this trimeric complex, was shown to decrease the level of sAPPβ. Overexpression of AP-2 promoted the internalization of BACE1 from the cell surface, thus reducing the cell surface BACE1 level. As such, we concluded that HFD may induce the formation of the BACE1/AP-2/clathrin complex, which is followed by its transport of BACE1 from the cell surface to the intracellular compartments. These events might be associated with the enhancement of β-site cleavage of APP in APP transgenic mice. Here we present evidence that HFD, by regulation of subcellular trafficking of BACE1, promotes APP cleavage. PMID:26414661

  17. The alternate AP-1 adaptor subunit Apm2 interacts with the Mil1 regulatory protein and confers differential cargo sorting

    PubMed Central

    Whitfield, Shawn T.; Burston, Helen E.; Bean, Björn D. M.; Raghuram, Nandini; Maldonado-Báez, Lymarie; Davey, Michael; Wendland, Beverly; Conibear, Elizabeth

    2016-01-01

    Heterotetrameric adaptor protein complexes are important mediators of cargo protein sorting in clathrin-coated vesicles. The cell type–specific expression of alternate μ chains creates distinct forms of AP-1 with altered cargo sorting, but how these subunits confer differential function is unclear. Whereas some studies suggest the μ subunits specify localization to different cellular compartments, others find that the two forms of AP-1 are present in the same vesicle but recognize different cargo. Yeast have two forms of AP-1, which differ only in the μ chain. Here we show that the variant μ chain Apm2 confers distinct cargo-sorting functions. Loss of Apm2, but not of Apm1, increases cell surface levels of the v-SNARE Snc1. However, Apm2 is unable to replace Apm1 in sorting Chs3, which requires a dileucine motif recognized by the γ/σ subunits common to both complexes. Apm2 and Apm1 colocalize at Golgi/early endosomes, suggesting that they do not associate with distinct compartments. We identified a novel, conserved regulatory protein that is required for Apm2-dependent sorting events. Mil1 is a predicted lipase that binds Apm2 but not Apm1 and contributes to its membrane recruitment. Interactions with specific regulatory factors may provide a general mechanism to diversify the functional repertoire of clathrin adaptor complexes. PMID:26658609

  18. Structure, Function and On-Off Switching of a Core Unit Contact between CheA Kinase and CheW Adaptor Protein in the Bacterial Chemosensory Array: A Disulfide Mapping and TAM-IDS Study

    PubMed Central

    Natale, Andrew M.; Duplantis, Jane L.; Piasta, Kene N.; Falke, Joseph J.

    2014-01-01

    The ultrasensitive, ultrastable bacterial chemosensory array of Escherichia coli and Salmonella typhimurium is representative of the large, conserved family of sensory arrays that control the cellular chemotaxis of motile bacteria and Archaea. The core framework of the membrane-bound array is a lattice assembled from three components: a transmembrane receptor, a cytoplasmic His kinase (CheA), and a cytoplasmic adaptor protein (CheW). Structural studies in the field have revealed the global architecture of the array and complexes between specific components, but much remains to be learned about the essential protein-protein interfaces that define array structure and transmit signals between components. This study has focused on the structure, function and on-off switching of a key contact between the kinase and adaptor proteins in the working, membrane-bound array. Specifically, the study addressed interface 1 in the putative kinase-adaptor ring where subdomain 1 of the kinase regulatory domain contacts subdomain 2 of the adaptor protein. Two independent approaches – disulfide mapping and site-directed Trp and Ala mutagenesis – were employed to (i) test the structural model of interface 1 and (ii) investigate its functional roles in both stable kinase incorporation and receptor-regulated kinase on-off switching. Studies were carried out in functional, membrane-bound arrays or in live cells. The findings reveal that crystal structures of binary and ternary complexes accurately depict the native interface in its kinase-activating on state. Furthermore, the findings indicate that at least part of the interface becomes less closely packed in its kinase-inhibiting off state. Together, the evidence shows the interface has a dual structural and signaling function that is crucial for stable kinase incorporation into the array, for kinase activation in the array on state, and likely for attractant-triggered kinase on-off switching. A model is presented that describes the

  19. Adaptor bypass mutations of Bacillus subtilis spx suggest a mechanism for YjbH-enhanced proteolysis of the regulator Spx by ClpXP

    PubMed Central

    Chan, Chio Mui; Hahn, Erik; Zuber, Peter

    2014-01-01

    Summary The global regulator, Spx, is under proteolytic control exerted by the adaptor YjbH and ATP-dependent protease ClpXP in Bacillus subtilis. While YjbH is observed to bind the Spx C-terminus, YjbH shows little affinity for ClpXP, indicating adaptor activity that does not operate by tethering. Chimeric proteins derived from B. subtilis AbrB and the Spx C-terminus showed that a 28 residue C-terminal section of Spx (AbrB28), but not the last 12 or 16 residues (AbrB12, AbrB16), was required for YjbH interaction and for ClpXP proteolysis, although the rate of AbrB28 proteolysis was not affected by YjbH addition. The result suggested that the YjbH-targeted 28 residue segment of the Spx C-terminus bears a ClpXP-recognition element(s) that is hidden in the intact Spx protein. Residue substitutions in the conserved helix α6 of the C-terminal region generated Spx substrates that were degraded by ClpXP at accelerated rates compared to wild type Spx, and showed reduced dependency on the YjbH activity. The residue substitutions also weakened the interaction between Spx and YjbH. The results suggest a model in which YjbH, through interaction with residues of α6 helix, exposes the C-terminus of Spx for recognition and proteolysis by ClpXP. PMID:24942655

  20. The 3A Protein from Multiple Picornaviruses Utilizes the Golgi Adaptor Protein ACBD3 To Recruit PI4KIIIβ

    PubMed Central

    Greninger, Alexander L.; Knudsen, Giselle M.; Betegon, Miguel; Burlingame, Alma L.

    2012-01-01

    The activity of phosphatidylinositol 4-kinase class III beta (PI4KIIIβ) has been shown to be required for the replication of multiple picornaviruses; however, it is unclear whether a physical association between PI4KIIIβ and the viral replication machinery exists and, if it does, whether association is necessary. We examined the ability of the 3A protein from 18 different picornaviruses to form a complex with PI4KIIIβ by affinity purification of Strep-Tagged transiently transfected constructs followed by mass spectrometry and Western blotting for putative interacting targets. We found that the 3A proteins of Aichi virus, bovine kobuvirus, poliovirus, coxsackievirus B3, and human rhinovirus 14 all copurify with PI4KIIIβ. Furthermore, we found that multiple picornavirus 3A proteins copurify with the Golgi adaptor protein acyl coenzyme A (acyl-CoA) binding domain protein 3 (ACBD3/GPC60), including those from Aichi virus, bovine kobuvirus, human rhinovirus 14, poliovirus, and coxsackievirus B2, B3, and B5. Affinity purification of ACBD3 confirmed interaction with multiple picornaviral 3A proteins and revealed the ability to bind PI4KIIIβ in the absence of 3A. Mass-spectrometric analysis of transiently expressed Aichi virus, bovine kobuvirus, and human klassevirus 3A proteins demonstrated that the N-terminal glycines of these 3A proteins are myristoylated. Alanine-scanning mutagenesis along the entire length of Aichi virus 3A followed by transient expression and affinity purification revealed that copurification of PI4KIIIβ could be eliminated by mutation of specific residues, with little or no effect on recruitment of ACBD3. One mutation at the N terminus, I5A, significantly reduced copurification of both ACBD3 and PI4KIIIβ. The dependence of Aichi virus replication on the activity of PI4KIIIβ was confirmed by both chemical and genetic inhibition. Knockdown of ACBD3 by small interfering RNA (siRNA) also prevented replication of both Aichi virus and poliovirus

  1. Regulation of Cell Migration and β1 Integrin Trafficking by the Endosomal Adaptor GGA3.

    PubMed

    Ratcliffe, Colin D H; Sahgal, Pranshu; Parachoniak, Christine A; Ivaska, Johanna; Park, Morag

    2016-06-01

    The integrin family of cell adhesion receptors link extracellular matrices to intracellular signaling pathways and the actin cytoskeleton; and regulate cell migration, proliferation and survival in normal and diseased tissues. The subcellular location of integrin receptors is critical for their function and deregulated trafficking is implicated in various human diseases. Here we identify a role for Golgi-localized gamma-ear containing Arf-binding protein 3 (GGA3), in regulating trafficking of β1 integrin. GGA3 knockdown reduces cell surface and total levels of α2, α5 and β1 integrin subunits, inhibits cell spreading, reduces focal adhesion number, as well as cell migration. In the absence of GGA3, integrins are increasingly retained inside the cell, traffic toward the perinuclear lysosomal compartment and their degradation is enhanced. Integrin traffic and maintenance of integrin levels are dependent on the integrity of the Arf binding site of GGA3. Furthermore, sorting nexin 17 (SNX17), a critical regulator of integrin recycling, becomes mislocalized to enlarged late endosomes upon GGA3 depletion. These data support a model whereby GGA3, through its ability to regulate SNX17 endosomal localization and through interaction with Arf6 diverts integrins from the degradative pathway supporting cell migration. PMID:26935970

  2. T-cell immune adaptor SKAP1 regulates the induction of collagen-induced arthritis in mice.

    PubMed

    Smith, Xin; Taylor, Alison; Rudd, Christopher E

    2016-08-01

    SKAP1 is an immune cell adaptor that couples the T-cell receptor with the 'inside-out' signalling pathway for LFA-1 mediated adhesion in T-cells. A connection of SKAP1 to the regulation of an autoimmune disorder has not previously been reported. In this study, we show that Skap1-deficient (skap1-/-) mice are highly resistant to the induction of collagen-induced arthritis (CIA), both in terms of incidence or severity. Skap1-/- T-cells were characterised by a selective reduction in the presence IL-17+ (Th17) in response to CII peptide and a marked reduction of joint infiltrating T-cells in Skap1-/- mice. SKAP1 therefore represents a novel connection to Th17 producing T-cells and is new potential target in the therapeutic intervention in autoimmune and inflammatory diseases. PMID:27181093

  3. A conserved serine residue regulates the stability of Drosophila Salvador and human WW domain-containing adaptor 45 through proteasomal degradation

    SciTech Connect

    Wu, Di Wu, Shian

    2013-04-19

    Highlights: •Ser-17 is key for the stability of Drosophila Sav. •Ala mutation of Ser-17 promotes the proteasomal degradation of Sav. •Ser-17 residue is not the main target of Hpo-induced Sav stabilization. •Hpo-dependent and -independent mechanisms regulate Sav stability. •This mechanism is conserved in the homologue of Sav, human WW45. -- Abstract: The Hippo (Hpo) pathway is a conserved tumor suppressor pathway that controls organ size through the coordinated regulation of apoptosis and proliferation. Drosophila Salvador (Sav), which limits organ size, is a core component of the Hpo pathway. In this study, Ser-17 was shown to be important for the stability of Sav. Alanine mutation of Ser-17 promoted the proteasomal degradation of Sav. Destabilization and stabilization of the Sav protein mediated by alanine mutation of Ser-17 and by Hpo, respectively, were independent of each other. This implies that the stability of Sav is controlled by two mechanisms, one that is Ser-17-dependent and Hpo-independent, and another that is Ser-17-independent and Hpo-dependent. These dual mechanisms also regulated the human counterpart of Drosophila Sav, WW domain-containing adaptor 45 (WW45). The conservation of this regulation adds to its significance in normal physiology and tumorigenesis.

  4. Multiple interactions drive adaptor-mediated recruitment of the ubiquitin ligase rsp5 to membrane proteins in vivo and in vitro.

    PubMed

    Sullivan, James A; Lewis, Michael J; Nikko, Elina; Pelham, Hugh R B

    2007-07-01

    Recognition of membrane proteins by the Nedd4/Rsp5 ubiquitin ligase family is a critical step in their targeting to the multivesicular body pathway. Some substrates contain "PY" motifs (PPxY), which bind to WW domains in the ligase. Others lack PY motifs and instead rely on adaptors that recruit the ligase to them. To investigate the mechanism of adaptor-mediated ubiquitination, we have characterized the interactions between the adaptor Bsd2, the ubiquitin ligase Rsp5, and the membrane proteins Cps1, Tre1, and Smf1 from Saccharomyces cerevisiae. We have reconstituted adaptor-mediated modification of Cps1 and Tre1 in vitro, and we show that two PY motifs in Bsd2 and two WW domains (WW2 and WW3) in Rsp5 are crucial for this. The binding of a weak noncanonical DMAPSY motif in Bsd2 to WW3 is an absolute requirement for Bsd2 adaptor function. We show that sorting of the manganese transporter Smf1, which requires both Bsd2 and Tre1, depends upon two PY motifs in Bsd2 and one motif in Tre1 but only two WW domains in Rsp5. We suggest that sequential assembly of first a Bsd2/Rsp5 complex, then a Tre1/Bsd2/Rsp5 complex followed by a rearrangement of PY-WW interactions is required for the ubiquitination of Smf1. PMID:17429078

  5. Nck adaptor proteins control the organization of neuronal circuits important for walking.

    PubMed

    Fawcett, James P; Georgiou, John; Ruston, Julie; Bladt, Friedhelm; Sherman, Andrew; Warner, Neil; Saab, Bechara J; Scott, Rizaldy; Roder, John C; Pawson, Tony

    2007-12-26

    The intracellular signaling targets used by mammalian axon guidance receptors to organize the nervous system in vivo are unclear. The Nck1 and Nck2 SH2/SH3 adaptors (collectively Nck) can couple phosphotyrosine (pTyr) signals to reorganization of the actin cytoskeleton and are therefore candidates for linking guidance cues to the regulatory machinery of the cytoskeleton. We find that selective inactivation of Nck in the murine nervous system causes a hopping gait and a defect in the spinal central pattern generator, which is characterized by synchronous firing of bilateral ventral motor neurons. Nck-deficient mice also show abnormal projections of corticospinal tract axons and defective development of the posterior tract of the anterior commissure. These phenotypes are consistent with a role for Nck in signaling initiated by different classes of guidance receptors, including the EphA4 receptor tyrosine kinase. Our data indicate that Nck adaptors couple pTyr guidance signals to cytoskeletal events required for the ipsilateral projections of spinal cord neurons and thus for normal limb movement. PMID:18093944

  6. Molecular basis for the specific recognition of the metazoan cyclic GMP-AMP by the innate immune adaptor protein STING

    PubMed Central

    Shi, Heping; Wu, Jiaxi; Chen, Zhijian J.; Chen, Chuo

    2015-01-01

    Cyclic GMP-AMP containing a unique combination of mixed phosphodiester linkages (2′3′-cGAMP) is an endogenous second messenger molecule that activates the type-I IFN pathway upon binding to the homodimer of the adaptor protein STING on the surface of endoplasmic reticulum membrane. However, the preferential binding of the asymmetric ligand 2′3′-cGAMP to the symmetric dimer of STING represents a physicochemical enigma. Here we show that 2′3′-cGAMP, but not its linkage isomers, adopts an organized free-ligand conformation that resembles the STING-bound conformation and pays low entropy and enthalpy costs in converting into the active conformation. Our results demonstrate that analyses of free-ligand conformations can be as important as analyses of protein conformations in understanding protein–ligand interactions. PMID:26150511

  7. Stability of HIB-Cul3 E3 ligase adaptor HIB Is Regulated by Self-degradation and Availability of Its Substrates

    PubMed Central

    Zhou, Zizhang; Xu, Congyu; Chen, Ping; Liu, Chen; Pang, Shu; Yao, Xia; Zhang, Qing

    2015-01-01

    The HIB-Cul3 complex E3 ligase regulates physiological homeostasis through regulating its substrate stability and its activity can be modulated by changing HIB abundance. However, regulation of HIB remains elusive. Here we provide evidence that HIB is degraded through the proteasome by Cul3-mediated polyubiquitination in K48 manner in Drosophila. Strikingly, HIB is targeted for degradation by itself. We further identify that three degrons (52LKSS56T, 76LDEE80S and 117MESQ121R) and K185 and K198 of HIB are essential for its auto-degradation. Finally, we demonstrate that HIB-Cul3 substrates, Ci and Puc, can effectively protect HIB from HIB-Cul3-mediated degradation. Taken together, our study indicates that there is an exquisite equilibrium between the adaptor and targets to achieve the tight control of the HIB, which is essential for maintaining suitable Hh and JNK signaling. And the mechanism of adaptor self-degradation and reciprocal control of the abundance between adaptor and its substrates is also applied to BTB-Cul3 E3 ligase adaptor dKeap1, dDiablo and dKLHL18. PMID:26263855

  8. A dual role for the adaptor protein DRK in Drosophila olfactory learning and memory

    PubMed Central

    Moressis, Anastasios; Friedrich, Anke R.; Pavlopoulos, Elias; Davis, Ronald L.; Skoulakis, Efthimios M. C.

    2009-01-01

    Participation of RAS, RAF and MAPK in learning and memory has been demonstrated in a number of studies, but the molecular events requisite for cascade activation and regulation have not been explored. We demonstrate that the adapter protein DRK which is essential for signaling to RAS in developmental contexts, is preferentially distributed in the adult mushroom bodies, centers for olfactory learning and memory. We demonstrate that drk mutant heterozygotes exhibit deficits in olfactory learning and memory, apparent under limited training conditions, but are not impaired in sensory responses requisite for the association of the stimuli, or brain neuroanatomy. Furthermore we demonstrate that the protein is required acutely within mushroom body neurons to mediate efficient learning, a process that requires RAF activation. Importantly, 90-minute memory remained impaired, even after differential training yielding equivalent learning in animals with compromised DRK levels and controls, and did not require RAF. Sustained MAPK activation is compromised in drk mutants and surprisingly is negatively regulated by constitutive RAF activity. The data establish a role for DRK in Drosophila behavioral neuroplasticity and suggest a dual role for the protein, first in RAF activation-dependent learning and additionally in RAF-inhibition dependent sustained MAPK activation essential for memory formation or stability. PMID:19244537

  9. Role of TAPP1 and TAPP2 adaptor binding to PtdIns(3,4)P2 in regulating insulin sensitivity defined by knock-in analysis

    PubMed Central

    Wullschleger, Stephan; Wasserman, David H.; Gray, Alex; Sakamoto, Kei; Alessi, Dario R.

    2015-01-01

    Insulin sensitivity is critically dependent on the activity of PI3K (phosphoinositide 3-kinase) and generation of the PtdIns(3,4,5)P3 second messenger. PtdIns(3,4,5)P3 can be broken down to PtdIns(3,4)P2 through the action of the SHIPs (Src-homology-2-domain-containing inositol phosphatases). As PtdIns(3,4)P2 levels peak after those of PtdIns(3,4,5)P3, it has been proposed that PtdIns(3,4)P2 controls a negative-feedback loop that down-regulates the insulin and PI3K network. Previously, we identified two related adaptor proteins termed TAPP [tandem PH (pleckstrin homology)-domain-containing protein] 1 and TAPP2 that specifically bind to PtdIns(3,4)P2 through their C-terminal PH domain. To determine whether TAPP1 and TAPP2 play a role in regulating insulin sensitivity, we generated knock-in mice that express normal endogenous levels of mutant TAPP1 and TAPP2 that are incapable of binding PtdIns(3,4)P2. These homozygous TAPP1R211L/R211LTAPP2R218L/R218L double knock-in mice are viable and exhibit significantly enhanced activation of Akt, a key downstream mediator of insulin signalling. Consistent with increased PI3K and Akt activity, the double knock-in mice display enhanced whole body insulin sensitivity and disposal of glucose uptake into muscle tissues. We also generated wild-type and double TAPP1R211L/R211LTAPP2R218L/R218L knock-in embryonic fibroblasts and found that insulin triggered enhanced production of PtdIns(3,4,5)P3 and Akt activity in the double knock-in fibroblasts. These observations provide the first genetic evidence to support the notion that binding of TAPP1 and TAPP2 adaptors to PtdIns(3,4)P2 function as negative regulators of the insulin and PI3K signalling pathways. PMID:21204784

  10. Brucella TIR-like protein TcpB/Btp1 specifically targets the host adaptor protein MAL/TIRAP to promote infection.

    PubMed

    Li, Wenna; Ke, Yuehua; Wang, Yufei; Yang, Mingjuan; Gao, Junguang; Zhan, Shaoxia; Xinying, Du; Huang, Liuyu; Li, Wenfeng; Chen, Zeliang; Li, Juan

    2016-08-26

    Brucella spp. are known to avoid host immune recognition and weaken the immune response to infection. Brucella like accomplish this by employing two clever strategies, called the stealth strategy and hijacking strategy. The TIR domain-containing protein (TcpB/Btp1) of Brucella melitensis is thought to be involved in inhibiting host NF-κB activation by binding to adaptors downstream of Toll-like receptors. However, of the five TIR domain-containing adaptors conserved in mammals, whether MyD88 or MAL, even other three adaptors, are specifically targeted by TcpB has not been identified. Here, we confirmed the effect of TcpB on B.melitensis virulence in mice and found that TcpB selectively targets MAL. By using siRNA against MAL, we found that TcpB from B.melitensis is involved in intracellular survival and that MAL affects intracellular replication of B.melitensis. Our results confirm that TcpB specifically targets MAL/TIRAP to disrupt downstream signaling pathways and promote intra-host survival of Brucella spp. PMID:27311859

  11. Adaptor protein complex 4 deficiency causes severe autosomal-recessive intellectual disability, progressive spastic paraplegia, shy character, and short stature.

    PubMed

    Abou Jamra, Rami; Philippe, Orianne; Raas-Rothschild, Annick; Eck, Sebastian H; Graf, Elisabeth; Buchert, Rebecca; Borck, Guntram; Ekici, Arif; Brockschmidt, Felix F; Nöthen, Markus M; Munnich, Arnold; Strom, Tim M; Reis, Andre; Colleaux, Laurence

    2011-06-10

    Intellectual disability inherited in an autosomal-recessive fashion represents an important fraction of severe cognitive-dysfunction disorders. Yet, the extreme heterogeneity of these conditions markedly hampers gene identification. Here, we report on eight affected individuals who were from three consanguineous families and presented with severe intellectual disability, absent speech, shy character, stereotypic laughter, muscular hypotonia that progressed to spastic paraplegia, microcephaly, foot deformity, decreased muscle mass of the lower limbs, inability to walk, and growth retardation. Using a combination of autozygosity mapping and either Sanger sequencing of candidate genes or next-generation exome sequencing, we identified one mutation in each of three genes encoding adaptor protein complex 4 (AP4) subunits: a nonsense mutation in AP4S1 (NM_007077.3: c.124C>T, p.Arg42(∗)), a frameshift mutation in AP4B1 (NM_006594.2: c.487_488insTAT, p.Glu163_Ser739delinsVal), and a splice mutation in AP4E1 (NM_007347.3: c.542+1_542+4delGTAA, r.421_542del, p.Glu181Glyfs(∗)20). Adaptor protein complexes (AP1-4) are ubiquitously expressed, evolutionarily conserved heterotetrameric complexes that mediate different types of vesicle formation and the selection of cargo molecules for inclusion into these vesicles. Interestingly, two mutations affecting AP4M1 and AP4E1 have recently been found to cause cerebral palsy associated with severe intellectual disability. Combined with previous observations, these results support the hypothesis that AP4-complex-mediated trafficking plays a crucial role in brain development and functioning and demonstrate the existence of a clinically recognizable syndrome due to deficiency of the AP4 complex. PMID:21620353

  12. Interaction of the protein tyrosine phosphatase PTPL1 with the PtdIns(3,4)P2-binding adaptor protein TAPP1.

    PubMed Central

    Kimber, Wendy A; Deak, Maria; Prescott, Alan R; Alessi, Dario R

    2003-01-01

    It has been postulated that PtdIns(3,4) P (2), one of the immediate breakdown products of PtdIns(3,4,5) P (3), functions as a signalling molecule in insulin- and growth-factor-stimulated pathways. To date, the t andem- P H-domain-containing p rotein- 1 (TAPP1) and related TAPP2 are still the only known PH-domain-containing proteins that interact strongly and specifically with PtdIns(3,4) P (2). In this study we demonstrate that endogenously expressed TAPP1, is constitutively associated with the protein-tyrosine-phosphatase-like protein-1 (PTPL1 also known as FAP-1). We show that PTPL1 binds to TAPP1 and TAPP2, principally though its first PDZ domain [where PDZ is postsynaptic density protein ( P SD-95)/ Drosophila disc large tumour suppressor ( d lg)/tight junction protein ( Z O1)] and show that this renders PTPL1 capable of associating with PtdIns(3,4) P (2) in vitro. Our data suggest that the binding of TAPP1 to PTPL1 does not influence PTPL1 phosphatase activity, but instead functions to maintain PTPL1 in the cytoplasm. Following stimulation of cells with hydrogen peroxide to induce PtdIns(3,4) P (2) production, PTPL1, complexed to TAPP1, translocates to the plasma membrane. This study provides the first evidence that TAPP1 and PtdIns(3,4) P (2) could function to regulate the membrane localization of PTPL1. We speculate that if PTPL1 was recruited to the plasma membrane by increasing levels of PtdIns(3,4) P (2), it could trigger a negative feedback loop in which phosphoinositide-3-kinase-dependent or other signalling pathways could be switched off by the phosphatase-catalysed dephosphorylation of receptor tyrosine kinases or tyrosine phosphorylated adaptor proteins such as IRS1 or IRS2. Consistent with this notion we observed RNA-interference-mediated knock-down of TAPP1 in HEK-293 cells, enhanced activation and phosphorylation of PKB following IGF1 stimulation. PMID:14516276

  13. Endoproteolytic cleavage of FE65 converts the adaptor protein to a potent suppressor of the sAPPalpha pathway in primates.

    PubMed

    Hu, Qubai; Wang, Lin; Yang, Zheng; Cool, Bethany H; Zitnik, Galynn; Martin, George M

    2005-04-01

    Adaptor protein FE65 (APBB1) specifically binds to the intracellular tail of the type I transmembrane protein, beta-amyloid precursor protein (APP). The formation of this complex may be important for modulation of the processing and function of APP. APP is proteolytically cleaved at multiple sites. The cleavages and their regulation are of central importance in the pathogenesis of dementias of the Alzheimer type. In cell cultures and perhaps in vivo, secretion of the alpha-cleaved APP ectodomain (sAPPalpha) is the major pathway in the most cells. Regulation of the process may require extracellular/intracellular cues. Neither extracellular ligands nor intracellular mediators have been identified, however. Here, we show novel evidence that the major isoform of FE65 (97-kDa FE65, p97FE65) can be converted to a 65-kDa N-terminally truncated C-terminal fragment (p65FE65) via endoproteolysis. The cleavage region locates immediately after an acidic residue cluster but before the three major protein-protein binding domains. The cleavage activity is particularly high in human and non-human primate cells and low in rodent cells; the activity appears to be triggered/enhanced by high cell density, presumably via cell-cell/cell-substrate contact cues. As a result, p65FE65 exhibits extraordinarily high affinity for APP (up to 40-fold higher than p97FE65) and potent suppression (up to 90%) of secretion of sAPPalpha. Strong p65FE65-APP binding is required for the suppression. The results suggest that p65FE65 may be an intracellular mediator in a signaling cascade regulating alpha-secretion of APP, particularly in primates. PMID:15647266

  14. Adaptor protein complexes AP-1 and AP-3 are required by the HHV-7 Immunoevasin U21 for rerouting of class I MHC molecules to the lysosomal compartment.

    PubMed

    Kimpler, Lisa A; Glosson, Nicole L; Downs, Deanna; Gonyo, Patrick; May, Nathan A; Hudson, Amy W

    2014-01-01

    The human herpesvirus-7 (HHV-7) U21 gene product binds to class I major histocompatibility complex (MHC) molecules and reroutes them to a lysosomal compartment. Trafficking of integral membrane proteins to lysosomes is mediated through cytoplasmic sorting signals that recruit heterotetrameric clathrin adaptor protein (AP) complexes, which in turn mediate protein sorting in post-Golgi vesicular transport. Since U21 can mediate rerouting of class I molecules to lysosomes even when lacking its cytoplasmic tail, we hypothesize the existence of a cellular protein that contains the lysosomal sorting information required to escort class I molecules to the lysosomal compartment. If such a protein exists, we expect that it might recruit clathrin adaptor protein complexes as a means of lysosomal sorting. Here we describe experiments demonstrating that the μ adaptins from AP-1 and AP-3 are involved in U21-mediated trafficking of class I molecules to lysosomes. These experiments support the idea that a cellular protein(s) is necessary for U21-mediated lysosomal sorting of class I molecules. We also examine the impact of transient versus chronic knockdown of these adaptor protein complexes, and show that the few remaining μ subunits in the cells are eventually able to reroute class I molecules to lysosomes. PMID:24901711

  15. The membrane-associated proteins FCHo and SGIP are allosteric activators of the AP2 clathrin adaptor complex

    PubMed Central

    Hollopeter, Gunther; Lange, Jeffrey J; Zhang, Ying; Vu, Thien N; Gu, Mingyu; Ailion, Michael; Lambie, Eric J; Slaughter, Brian D; Unruh, Jay R; Florens, Laurence; Jorgensen, Erik M

    2014-01-01

    The AP2 clathrin adaptor complex links protein cargo to the endocytic machinery but it is unclear how AP2 is activated on the plasma membrane. Here we demonstrate that the membrane-associated proteins FCHo and SGIP1 convert AP2 into an open, active conformation. We screened for Caenorhabditis elegans mutants that phenocopy the loss of AP2 subunits and found that AP2 remains inactive in fcho-1 mutants. A subsequent screen for bypass suppressors of fcho-1 nulls identified 71 compensatory mutations in all four AP2 subunits. Using a protease-sensitivity assay we show that these mutations restore the open conformation in vivo. The domain of FCHo that induces this rearrangement is not the F-BAR domain or the µ-homology domain, but rather is an uncharacterized 90 amino acid motif, found in both FCHo and SGIP proteins, that directly binds AP2. Thus, these proteins stabilize nascent endocytic pits by exposing membrane and cargo binding sites on AP2. DOI: http://dx.doi.org/10.7554/eLife.03648.001 PMID:25303366

  16. Structural basis of HIV-1 Vpu-mediated BST2 antagonism via hijacking of the clathrin adaptor protein complex 1

    PubMed Central

    Jia, Xiaofei; Weber, Erin; Tokarev, Andrey; Lewinski, Mary; Rizk, Maryan; Suarez, Marissa; Guatelli, John; Xiong, Yong

    2014-01-01

    BST2/tetherin, an antiviral restriction factor, inhibits the release of enveloped viruses from the cell surface. Human immunodeficiency virus-1 (HIV-1) antagonizes BST2 through viral protein u (Vpu), which downregulates BST2 from the cell surface. We report the crystal structure of a protein complex containing Vpu and BST2 cytoplasmic domains and the core of the clathrin adaptor protein complex 1 (AP1). This, together with our biochemical and functional validations, reveals how Vpu hijacks the AP1-dependent membrane trafficking pathways to mistraffick BST2. Vpu mimics a canonical acidic dileucine-sorting motif to bind AP1 in the cytosol, while simultaneously interacting with BST2 in the membrane. These interactions enable Vpu to build on an intrinsic interaction between BST2 and AP1, presumably causing the observed retention of BST2 in juxtanuclear endosomes and stimulating its degradation in lysosomes. The ability of Vpu to hijack AP-dependent trafficking pathways suggests a potential common theme for Vpu-mediated downregulation of host proteins. DOI: http://dx.doi.org/10.7554/eLife.02362.001 PMID:24843023

  17. Matrilin-2, an extracellular adaptor protein, is needed for the regeneration of muscle, nerve and other tissues

    PubMed Central

    Korpos, Éva; Deák, Ferenc; Kiss, Ibolya

    2015-01-01

    The extracellular matrix (ECM) performs essential functions in the differentiation, maintenance and remodeling of tissues during development and regeneration, and it undergoes dynamic changes during remodeling concomitant to alterations in the cell-ECM interactions. Here we discuss recent data addressing the critical role of the widely expressed ECM protein, matrilin-2 (Matn2) in the timely onset of differentiation and regeneration processes in myogenic, neural and other tissues and in tumorigenesis. As a multiadhesion adaptor protein, it interacts with other ECM proteins and integrins. Matn2 promotes neurite outgrowth, Schwann cell migration, neuromuscular junction formation, skeletal muscle and liver regeneration and skin wound healing. Matn2 deposition by myoblasts is crucial for the timely induction of the global switch toward terminal myogenic differentiation during muscle regeneration by affecting transforming growth factor beta/bone morphogenetic protein 7/Smad and other signal transduction pathways. Depending on the type of tissue and the pathomechanism, Matn2 can also promote or suppress tumor growth. PMID:26199591

  18. Molecular cloning of the mouse grb2 gene: differential interaction of the Grb2 adaptor protein with epidermal growth factor and nerve growth factor receptors.

    PubMed Central

    Suen, K L; Bustelo, X R; Pawson, T; Barbacid, M

    1993-01-01

    We report the isolation and molecular characterization of the mouse grb2 gene. The product of this gene, the Grb2 protein, is highly related to the Caenorhabditis elegans sem-5 gene product and the human GRB2 protein and displays the same SH3-SH2-SH3 structural motifs. In situ hybridization studies revealed that the mouse grb2 gene is widely expressed throughout embryonic development (E9.5 to P0). However, grb2 transcripts are not uniformly distributed, and in certain tissues (e.g., thymus) they appear to be regulated during development. Recent genetic and biochemical evidence has implicated the Grb2 protein in the signaling pathways that link cell surface tyrosine kinase receptors with Ras. We have investigated the association of the Grb2 protein with epidermal growth factor (EGF) and nerve growth factor (NGF) receptors in PC12 pheochromocytoma cells. EGF treatment of PC12 cells results in the rapid association of Grb2 with the activated EGF receptors, an interaction mediated by the Grb2 SH2 domain. However, Grb2 does not bind to NGF-activated Trk receptors. Mitogenic signaling of NGF in NIH 3T3 cells ectopically expressing Trk receptors also takes place without detectable association between Grb2 and Trk. These results suggest that whereas EGF and NGF can activate the Ras signaling pathway in PC12 cells, only the EGF receptor is likely to do so through a direct interaction with Grb2. Finally, binding studies with glutathione S-transferase fusion proteins indicate that Grb2 binds two distinct subsets of proteins which are individually recognized by its SH2 and SH3 domains. These observations add further support to the concept that Grb2 is a modular adaptor protein. Images PMID:7689150

  19. The cytoskeleton adaptor protein ankyrin-1 is upregulated by p53 following DNA damage and alters cell migration.

    PubMed

    Hall, A E; Lu, W-T; Godfrey, J D; Antonov, A V; Paicu, C; Moxon, S; Dalmay, T; Wilczynska, A; Muller, P A J; Bushell, M

    2016-01-01

    The integrity of the genome is maintained by a host of surveillance and repair mechanisms that are pivotal for cellular function. The tumour suppressor protein p53 is a major component of the DNA damage response pathway and plays a vital role in the maintenance of cell-cycle checkpoints. Here we show that a microRNA, miR-486, and its host gene ankyrin-1 (ANK1) are induced by p53 following DNA damage. Strikingly, the cytoskeleton adaptor protein ankyrin-1 was induced over 80-fold following DNA damage. ANK1 is upregulated in response to a variety of DNA damage agents in a range of cell types. We demonstrate that miR-486-5p is involved in controlling G1/S transition following DNA damage, whereas the induction of the ankyrin-1 protein alters the structure of the actin cytoskeleton and sustains limited cell migration during DNA damage. Importantly, we found that higher ANK1 expression correlates with decreased survival in cancer patients. Thus, these observations highlight ANK1 as an important effector downstream of the p53 pathway. PMID:27054339

  20. The cytoskeleton adaptor protein ankyrin-1 is upregulated by p53 following DNA damage and alters cell migration

    PubMed Central

    Hall, A E; Lu, W-T; Godfrey, J D; Antonov, A V; Paicu, C; Moxon, S; Dalmay, T; Wilczynska, A; Muller, P A J; Bushell, M

    2016-01-01

    The integrity of the genome is maintained by a host of surveillance and repair mechanisms that are pivotal for cellular function. The tumour suppressor protein p53 is a major component of the DNA damage response pathway and plays a vital role in the maintenance of cell-cycle checkpoints. Here we show that a microRNA, miR-486, and its host gene ankyrin-1 (ANK1) are induced by p53 following DNA damage. Strikingly, the cytoskeleton adaptor protein ankyrin-1 was induced over 80-fold following DNA damage. ANK1 is upregulated in response to a variety of DNA damage agents in a range of cell types. We demonstrate that miR-486-5p is involved in controlling G1/S transition following DNA damage, whereas the induction of the ankyrin-1 protein alters the structure of the actin cytoskeleton and sustains limited cell migration during DNA damage. Importantly, we found that higher ANK1 expression correlates with decreased survival in cancer patients. Thus, these observations highlight ANK1 as an important effector downstream of the p53 pathway. PMID:27054339

  1. Bicaudal D Family of Motor Adaptors: Linking Dynein Motility to Cargo Binding.

    PubMed

    Hoogenraad, Casper C; Akhmanova, Anna

    2016-05-01

    Transport of different intracellular cargoes along cytoskeleton filaments is essential for the morphogenesis and function of a broad variety of eukaryotic cells. Intracellular transport is mediated by cytoskeletal motors including myosin, kinesin, and dynein, which are typically linked to various cargoes by adaptor proteins. Recent studies suggest that adaptor proteins can also act as essential transport cofactors, which control motor activity and coordination. Characterization of the evolutionary conserved Bicaudal D (BICD) family of dynein adaptor proteins has provided important insights into the fundamental mechanisms governing cargo trafficking. This review highlights the advances in the current understanding of how BICD adaptors regulate microtubule-based transport and how they contribute to developmental processes and human disease. PMID:26822037

  2. An Inducible System for Rapid Degradation of Specific Cellular Proteins Using Proteasome Adaptors

    PubMed Central

    Wilmington, Shameika R.; Matouschek, Andreas

    2016-01-01

    A common way to study protein function is to deplete the protein of interest from cells and observe the response. Traditional methods involve disrupting gene expression but these techniques are only effective against newly synthesized proteins and leave previously existing and stable proteins untouched. Here, we introduce a technique that induces the rapid degradation of specific proteins in mammalian cells by shuttling the proteins to the proteasome for degradation in a ubiquitin-independent manner. We present two implementations of the system in human culture cells that can be used individually to control protein concentration. Our study presents a simple, robust, and flexible technology platform for manipulating intracellular protein levels. PMID:27043013

  3. The Ras suppressor Rsu-1 binds to the LIM 5 domain of the adaptor protein PINCH1 and participates in adhesion-related functions

    SciTech Connect

    Dougherty, Gerard W.; Chopp, Treasa; Qi Shengmei; Cutler, Mary Lou . E-mail: mcutler@usuhs.mil

    2005-05-15

    Rsu-1 is a highly conserved leucine rich repeat (LRR) protein that is expressed ubiquitously in mammalian cells. Rsu-1 was identified based on its ability to inhibit transformation by Ras, and previous studies demonstrated that ectopic expression of Rsu-1 inhibited anchorage-independent growth of Ras-transformed cells and human tumor cell lines. Using GAL4-based yeast two-hybrid screening, the LIM domain protein, PINCH1, was identified as the binding partner of Rsu-1. PINCH1 is an adaptor protein that localizes to focal adhesions and it has been implicated in the regulation of adhesion functions. Subdomain mapping in yeast revealed that Rsu-1 binds to the LIM 5 domain of PINCH1, a region not previously identified as a specific binding domain for any other protein. Additional testing demonstrated that PINCH2, which is highly homologous to PINCH1, except in the LIM 5 domain, does not interact with Rsu-1. Glutathione transferase fusion protein binding studies determined that the LRR region of Rsu-1 interacts with PINCH1. Transient expression studies using epitope-tagged Rsu-1 and PINCH1 revealed that Rsu-1 co-immunoprecipitated with PINCH1 and colocalized with vinculin at sites of focal adhesions in mammalian cells. In addition, endogenous P33 Rsu-1 from 293T cells co-immunoprecipitated with transiently expressed myc-tagged PINCH1. Furthermore, RNAi-induced reduction in Rsu-1 RNA and protein inhibited cell attachment, and while previous studies demonstrated that ectopic expression of Rsu-1 inhibited Jun kinase activation, the depletion of Rsu-1 resulted in activation of Jun and p38 stress kinases. These studies demonstrate that Rsu-1 interacts with PINCH1 in mammalian cells and functions, in part, by altering cell adhesion.

  4. Protein Interaction Profiling of the p97 Adaptor UBXD1 Points to a Role for the Complex in Modulating ERGIC-53 Trafficking*

    PubMed Central

    Haines, Dale S.; Lee, J. Eugene; Beauparlant, Stephen L.; Kyle, Dane B.; den Besten, Willem; Sweredoski, Michael J.; Graham, Robert L. J.; Hess, Sonja; Deshaies, Raymond J.

    2012-01-01

    UBXD1 is a member of the poorly understood subfamily of p97 adaptors that do not harbor a ubiquitin association domain or bind ubiquitin-modified proteins. Of clinical importance, p97 mutants found in familial neurodegenerative conditions Inclusion Body Myopathy Paget's disease of the bone and/or Frontotemporal Dementia and Amyotrophic Lateral Sclerosis are defective at interacting with UBXD1, indicating that functions regulated by a p97-UBXD1 complex are altered in these diseases. We have performed liquid chromatography-mass spectrometric analysis of UBXD1-interacting proteins to identify pathways in which UBXD1 functions. UBXD1 displays prominent association with ERGIC-53, a hexameric type I integral membrane protein that functions in protein trafficking. The UBXD1-ERGIC-53 interaction requires the N-terminal 10 residues of UBXD1 and the C-terminal cytoplasmic 12 amino acid tail of ERGIC-53. Use of p97 and E1 enzyme inhibitors indicate that complex formation between UBXD1 and ERGIC-53 requires the ATPase activity of p97, but not ubiquitin modification. We also performed SILAC-based quantitative proteomic profiling to identify ERGIC-53 interacting proteins. This analysis identified known (e.g. COPI subunits) and novel (Rab3GAP1/2 complex involved in the fusion of vesicles at the cell membrane) interactions that are also mediated through the C terminus of the protein. Immunoprecipitation and Western blotting analysis confirmed the proteomic interaction data and it also revealed that an UBXD1-Rab3GAP association requires the ERGIC-53 binding domain of UBXD1. Localization studies indicate that UBXD1 modules the sub-cellular trafficking of ERGIC-53, including promoting movement to the cell membrane. We propose that p97-UBXD1 modulates the trafficking of ERGIC-53-containing vesicles by controlling the interaction of transport factors with the cytoplasmic tail of ERGIC-53. PMID:22337587

  5. Distinct Involvement of the Gab1 and Grb2 Adaptor Proteins in Signal Transduction by the Related Receptor Tyrosine Kinases RON and MET

    PubMed Central

    Chaudhuri, Amitabha; Xie, Ming-Hong; Yang, Becky; Mahapatra, Kaushiki; Liu, Jinfeng; Marsters, Scot; Bodepudi, Sweta; Ashkenazi, Avi

    2011-01-01

    Although the signal transduction mechanisms of the receptor tyrosine kinase MET are well defined, less is known about its close relative RON. MET initiates intracellular signaling by autophosphorylation on specific cytoplasmic tyrosines that form docking sites for the adaptor proteins Grb2 and Gab1. Grb2 binds directly and is essential for all of the biological activities of MET. Gab1 docks either directly or indirectly via Grb2 and controls only a subset of MET functions. Because MET and RON possess similar adaptor binding sites, it was anticipated that their adaptor interactions would be conserved. Here we show that in contrast to MET, RON relies primarily on Gab1 for signal transmission. Surprisingly, disruption of the Grb2 docking site of RON or Grb2 depletion augments activity, whereas enhancement of Grb2 binding attenuates Gab1 recruitment and signaling. Hence, RON and MET differ in their adaptor interactions; furthermore, Grb2 performs a novel antagonistic role in the context of RON signaling. PMID:21784853

  6. Bivalent Motif-Ear Interactions Mediate the Association of the Accessory Protein Tepsin with the AP-4 Adaptor Complex.

    PubMed

    Mattera, Rafael; Guardia, Carlos M; Sidhu, Sachdev S; Bonifacino, Juan S

    2015-12-25

    The heterotetrameric (ϵ-β4-μ4-σ4) complex adaptor protein 4 (AP-4) is a component of a non-clathrin coat involved in protein sorting at the trans-Golgi network (TGN). Considerable interest in this complex has arisen from the recent discovery that mutations in each of its four subunits are the cause of a congenital intellectual disability and movement disorder in humans. Despite its physiological importance, the structure and function of this coat remain poorly understood. To investigate the assembly of the AP-4 coat, we dissected the determinants of interaction of AP-4 with its only known accessory protein, the ENTH/VHS-domain-containing protein tepsin. Using a variety of protein interaction assays, we found that tepsin comprises two phylogenetically conserved peptide motifs, [GS]LFXG[ML]X[LV] and S[AV]F[SA]FLN, within its C-terminal unstructured region, which interact with the C-terminal ear (or appendage) domains of the β4 and ϵ subunits of AP-4, respectively. Structure-based mutational analyses mapped the binding site for the [GS]LFXG[ML]X[LV] motif to a conserved, hydrophobic surface on the β4-ear platform fold. Both peptide-ear interactions are required for efficient association of tepsin with AP-4, and for recruitment of tepsin to the TGN. The bivalency of the interactions increases the avidity of tepsin for AP-4 and may enable cross-linking of multiple AP-4 heterotetramers, thus contributing to the assembly of the AP-4 coat. In addition to revealing critical aspects of this coat, our findings extend the paradigm of peptide-ear interactions, previously established for clathrin-AP-1/AP-2 coats, to a non-clathrin coat. PMID:26542808

  7. Cell biological characterization of a multidomain adaptor protein, ArgBP2, in epithelial NMuMG cells, and identification of a novel short isoform.

    PubMed

    Murase, Kana; Ito, Hidenori; Kanoh, Hiroyuki; Sudo, Kaori; Iwamoto, Ikuko; Morishita, Rika; Soubeyran, Philippe; Seishima, Mariko; Nagata, Koh-Ichi

    2012-12-01

    ArgBP2 is a member of the SoHo (sorbin-homology) family of adaptor proteins believed to play roles in cell adhesion, cytoskeletal organization, and signaling. We show here a novel splicing isoform of ArgBP2, i.e., ArgBP2™, composed of only three SH3 (src-homology 3) domains and structurally similar to vinexinß. We then characterized the biochemical and cell biological properties of ArgBP2 to compare these with vinexin. Similar to vinexin, ArgBP2 was enriched at focal adhesions in REF52 fibroblast cells and induced anchorage-dependent extracellular signal-regulated kinase activation in NIH3T3 fibroblast cells. In epithelial NMuMG cells, immunofluorescence analyses revealed localization of ArgBP2 at tight junctions (TJs), whereas vinexin was distributed in cytoplasm as well as cell-cell boundaries. During TJ formation, recruitment of ZO-1 to TJs was followed by ArgBP2. Based on mutation analyses, a second SH3 domain was found to be important for ArgBP2 localization to the cell-cell contact sites. These data suggest some role of ArgBP2 in NMuMG cells at TJs that may be distinct from the function of vinexin. PMID:22431180

  8. Early Loss of Telomerase Action in Yeast Creates a Dependence on the DNA Damage Response Adaptor Proteins

    PubMed Central

    Jay, Kyle A.; Smith, Dana L.

    2016-01-01

    Telomeres cap the ends of chromosomes, protecting them from degradation and inappropriate DNA repair processes that can lead to genomic instability. A short telomere elicits increased telomerase action on itself that replenishes telomere length, thereby stabilizing the telomere. In the prolonged absence of telomerase activity in dividing cells, telomeres eventually become critically short, inducing a permanent cell cycle arrest (senescence). We recently showed that even early after telomerase inactivation (ETI), yeast cells have accelerated mother cell aging and mildly perturbed cell cycles. Here, we show that the complete disruption of DNA damage response (DDR) adaptor proteins in ETI cells causes severe growth defects. This synthetic-lethality phenotype was as pronounced as that caused by extensive DNA damage in wild-type cells but showed genetic dependencies distinct from such damage and was completely alleviated by SML1 deletion, which increases deoxynucleoside triphosphate (dNTP) pools. Our results indicated that these deleterious effects in ETI cells cannot be accounted for solely by the slow erosion of telomeres due to incomplete replication that leads to senescence. We propose that normally occurring telomeric DNA replication stress is resolved by telomerase activity and the DDR in two parallel pathways and that deletion of Sml1 prevents this stress. PMID:27161319

  9. Early Loss of Telomerase Action in Yeast Creates a Dependence on the DNA Damage Response Adaptor Proteins.

    PubMed

    Jay, Kyle A; Smith, Dana L; Blackburn, Elizabeth H

    2016-07-15

    Telomeres cap the ends of chromosomes, protecting them from degradation and inappropriate DNA repair processes that can lead to genomic instability. A short telomere elicits increased telomerase action on itself that replenishes telomere length, thereby stabilizing the telomere. In the prolonged absence of telomerase activity in dividing cells, telomeres eventually become critically short, inducing a permanent cell cycle arrest (senescence). We recently showed that even early after telomerase inactivation (ETI), yeast cells have accelerated mother cell aging and mildly perturbed cell cycles. Here, we show that the complete disruption of DNA damage response (DDR) adaptor proteins in ETI cells causes severe growth defects. This synthetic-lethality phenotype was as pronounced as that caused by extensive DNA damage in wild-type cells but showed genetic dependencies distinct from such damage and was completely alleviated by SML1 deletion, which increases deoxynucleoside triphosphate (dNTP) pools. Our results indicated that these deleterious effects in ETI cells cannot be accounted for solely by the slow erosion of telomeres due to incomplete replication that leads to senescence. We propose that normally occurring telomeric DNA replication stress is resolved by telomerase activity and the DDR in two parallel pathways and that deletion of Sml1 prevents this stress. PMID:27161319

  10. The Adaptor Protein-1 μ1B Subunit Expands the Repertoire of Basolateral Sorting Signal Recognition in Epithelial Cells

    PubMed Central

    Guo, Xiaoli; Mattera, Rafael; Ren, Xuefeng; Chen, Yu; Retamal, Claudio; González, Alfonso; Bonifacino, Juan S.

    2014-01-01

    SUMMARY An outstanding question in protein sorting is why polarized epithelial cells express two isoforms of the μ1 subunit of the AP-1 clathrin adaptor complex: the ubiquitous μ1A and the epithelial-specific μ1B. Previous studies led to the notion that μ1A and μ1B mediate basolateral sorting predominantly from the trans-Golgi network (TGN) and recycling endosomes, respectively. Using improved analytical tools, however, we find that μ1A and μ1B largely colocalize with each other. They also colocalize to similar extents with TGN and recycling endosome markers, as well as with basolateral cargoes transiting biosynthetic and endocytic-recycling routes. Instead, the two isoforms differ in their signal-recognition specificity. In particular, μ1B preferentially binds a subset of signals from cargoes that are sorted basolaterally in a μ1B-dependent manner. We conclude that expression of distinct μ1 isoforms in epithelial cells expands the repertoire of signals recognized by AP-1 for sorting of a broader range of cargoes to the basolateral surface. PMID:24229647

  11. The clathrin adaptor proteins ARH, Dab2, and numb play distinct roles in Niemann-Pick C1-Like 1 versus low density lipoprotein receptor-mediated cholesterol uptake.

    PubMed

    Wei, Jian; Fu, Zhen-Yan; Li, Pei-Shan; Miao, Hong-Hua; Li, Bo-Liang; Ma, Yi-Tong; Song, Bao-Liang

    2014-11-28

    The uptake of circulating low density lipoproteins (LDL) is mediated by LDL receptor (LDLR) through clathrin-dependent endocytosis. At the early stage of this process, adaptor proteins ARH and Dab2 specifically bind the endocytic signal motif in LDLR and recruit clathrin/AP2 to initiate internalization. On the other hand, intestinal cholesterol is absorbed by Niemann-Pick C1-Like 1 (NPC1L1) through clathrin-dependent endocytosis. Another adaptor protein, Numb recognizes the endocytic motif in NPC1L1 C terminus and couples NPC1L1 to endocytic machinery. The ARH, Dab2, and Numb proteins contain a homogeneous phosphotyrosine binding (PTB) domain that directly binds endocytic motifs. Because ARH, Dab2, and Numb are all PTB domain family members, the emerging mystery is whether these adaptors act complementally in LDLR and NPC1L1 endocytosis. Here, we found that ARH and Dab2 did not bind NPC1L1 and were not required for NPC1L1 internalization. Similarly, Numb lacked the ability to interact with the LDLR C terminus and was dispensable for LDL uptake. Only the Numb isoforms with shorter PTB domain could facilitate NPC1L1 endocytosis. Besides the reported function in intestinal cholesterol absorption, Numb also mediated cholesterol reabsorption from bile in liver. We further identified a Numb variant with G595D substitution in humans of low blood LDL-cholesterol. The G595D substitution impaired NPC1L1 internalization and cholesterol reabsorption, due to attenuating affinity of Numb to clathrin/AP2. These results demonstrate that Numb specifically regulates NPC1L1-mediated cholesterol absorption both in human intestine and liver, distinct from ARH and Dab2, which selectively participate in LDLR-mediated LDL uptake. PMID:25331956

  12. Structure of a putative ClpS N-end rule adaptor protein from the malaria pathogen Plasmodium falciparum.

    PubMed

    AhYoung, Andrew P; Koehl, Antoine; Vizcarra, Christina L; Cascio, Duilio; Egea, Pascal F

    2016-03-01

    The N-end rule pathway uses an evolutionarily conserved mechanism in bacteria and eukaryotes that marks proteins for degradation by ATP-dependent chaperones and proteases such as the Clp chaperones and proteases. Specific N-terminal amino acids (N-degrons) are sufficient to target substrates for degradation. In bacteria, the ClpS adaptor binds and delivers N-end rule substrates for their degradation upon association with the ClpA/P chaperone/protease. Here, we report the first crystal structure, solved at 2.7 Å resolution, of a eukaryotic homolog of bacterial ClpS from the malaria apicomplexan parasite Plasmodium falciparum (Pfal). Despite limited sequence identity, Plasmodium ClpS is very similar to bacterial ClpS. Akin to its bacterial orthologs, plasmodial ClpS harbors a preformed hydrophobic pocket whose geometry and chemical properties are compatible with the binding of N-degrons. However, while the N-degron binding pocket in bacterial ClpS structures is open and accessible, the corresponding pocket in Plasmodium ClpS is occluded by a conserved surface loop that acts as a latch. Despite the closed conformation observed in the crystal, we show that, in solution, Pfal-ClpS binds and discriminates peptides mimicking bona fide N-end rule substrates. The presence of an apicoplast targeting peptide suggests that Pfal-ClpS localizes to this plastid-like organelle characteristic of all Apicomplexa and hosting most of its Clp machinery. By analogy with the related ClpS1 from plant chloroplasts and cyanobacteria, Plasmodium ClpS likely functions in association with ClpC in the apicoplast. Our findings open new venues for the design of novel anti-malarial drugs aimed at disrupting parasite-specific protein quality control pathways. PMID:26701219

  13. Structural Basis for Membrane Binding and Remodeling by the Exomer Secretory Vesicle Cargo Adaptor

    PubMed Central

    Paczkowski, Jon E.; Fromme, J. Christopher

    2014-01-01

    Summary Cargo adaptor subunits of vesicle coat protein complexes sort transmembrane proteins to distinct membrane compartments in eukaryotic cells. The exomer complex is the only cargo adaptor known to sort proteins at the trans-Golgi network into secretory vesicles. Exomer function is regulated by the Arf1 GTPase, a master regulator of trafficking at the Golgi. We report the structure of exomer bound to two copies of Arf1. Exomer interacts with each Arf1 molecule via two surfaces; one is a non-canonical interface that regulates GTP hydrolysis. The structure uncovers an unexpected membrane-proximal hydrophobic element that exomer uses in cooperation with Arf1 to remodel membranes. Given the constrained motion of the exomer hinge region, we envision that exomer dynamically positions multiple membrane insertion elements to drive membrane fission. In contrast to other known cargo adaptors, exomer therefore couples two functions, cargo sorting and membrane fission, into a single complex. PMID:25203211

  14. CD2v Interacts with Adaptor Protein AP-1 during African Swine Fever Infection

    PubMed Central

    Pérez-Núñez, Daniel; García-Urdiales, Eduardo; Martínez-Bonet, Marta; Nogal, María L.; Barroso, Susana; Revilla, Yolanda; Madrid, Ricardo

    2015-01-01

    African swine fever virus (ASFV) CD2v protein is believed to be involved in virulence enhancement, viral hemadsorption, and pathogenesis, although the molecular mechanisms of the function of this viral protein are still not fully understood. Here we describe that CD2v localized around viral factories during ASFV infection, suggesting a role in the generation and/or dynamics of these viral structures and hence in disturbing cellular traffic. We show that CD2v targeted the regulatory trans-Golgi network (TGN) protein complex AP-1, a key element in cellular traffic. This interaction was disrupted by brefeldin A even though the location of CD2v around the viral factory remained unchanged. CD2v-AP-1 binding was independent of CD2v glycosylation and occurred on the carboxy-terminal part of CD2v, where a canonical di-Leu motif previously reported to mediate AP-1 binding in eukaryotic cells, was identified. This motif was shown to be functionally interchangeable with the di-Leu motif present in HIV-Nef protein in an AP-1 binding assay. However, we demonstrated that it was not involved either in CD2v cellular distribution or in CD2v-AP-1 binding. Taken together, these findings shed light on CD2v function during ASFV infection by identifying AP-1 as a cellular factor targeted by CD2v and hence elucidate the cellular pathways used by the virus to enhance infectivity. PMID:25915900

  15. The deca-GX3 proteins Yae1-Lto1 function as adaptors recruiting the ABC protein Rli1 for iron-sulfur cluster insertion

    PubMed Central

    Paul, Viktoria Désirée; Mühlenhoff, Ulrich; Stümpfig, Martin; Seebacher, Jan; Kugler, Karl G; Renicke, Christian; Taxis, Christof; Gavin, Anne-Claude; Pierik, Antonio J; Lill, Roland

    2015-01-01

    Cytosolic and nuclear iron-sulfur (Fe-S) proteins are involved in many essential pathways including translation and DNA maintenance. Their maturation requires the cytosolic Fe-S protein assembly (CIA) machinery. To identify new CIA proteins we employed systematic protein interaction approaches and discovered the essential proteins Yae1 and Lto1 as binding partners of the CIA targeting complex. Depletion of Yae1 or Lto1 results in defective Fe-S maturation of the ribosome-associated ABC protein Rli1, but surprisingly no other tested targets. Yae1 and Lto1 facilitate Fe-S cluster assembly on Rli1 in a chain of binding events. Lto1 uses its conserved C-terminal tryptophan for binding the CIA targeting complex, the deca-GX3 motifs in both Yae1 and Lto1 facilitate their complex formation, and Yae1 recruits Rli1. Human YAE1D1 and the cancer-related ORAOV1 can replace their yeast counterparts demonstrating evolutionary conservation. Collectively, the Yae1-Lto1 complex functions as a target-specific adaptor that recruits apo-Rli1 to the generic CIA machinery. DOI: http://dx.doi.org/10.7554/eLife.08231.001 PMID:26182403

  16. Structural and Functional Characterization of Cargo-Binding Sites on the μ4-Subunit of Adaptor Protein Complex 4

    PubMed Central

    Ross, Breyan H.; Lin, Yimo; Corales, Esteban A.; Burgos, Patricia V.; Mardones, Gonzalo A.

    2014-01-01

    Adaptor protein (AP) complexes facilitate protein trafficking by playing key roles in the selection of cargo molecules to be sorted in post-Golgi compartments. Four AP complexes (AP-1 to AP-4) contain a medium-sized subunit (μ1-μ4) that recognizes YXXØ-sequences (Ø is a bulky hydrophobic residue), which are sorting signals in transmembrane proteins. A conserved, canonical region in μ subunits mediates recognition of YXXØ-signals by means of a critical aspartic acid. Recently we found that a non-canonical YXXØ-signal on the cytosolic tail of the Alzheimer's disease amyloid precursor protein (APP) binds to a distinct region of the μ4 subunit of the AP-4 complex. In this study we aimed to determine the functionality of both binding sites of μ4 on the recognition of the non-canonical YXXØ-signal of APP. We found that substitutions in either binding site abrogated the interaction with the APP-tail in yeast-two hybrid experiments. Further characterization by isothermal titration calorimetry showed instead loss of binding to the APP signal with only the substitution R283D at the non-canonical site, in contrast to a decrease in binding affinity with the substitution D190A at the canonical site. We solved the crystal structure of the C-terminal domain of the D190A mutant bound to this non-canonical YXXØ-signal. This structure showed no significant difference compared to that of wild-type μ4. Both differential scanning fluorimetry and limited proteolysis analyses demonstrated that the D190A substitution rendered μ4 less stable, suggesting an explanation for its lower binding affinity to the APP signal. Finally, in contrast to overexpression of the D190A mutant, and acting in a dominant-negative manner, overexpression of μ4 with either a F255A or a R283D substitution at the non-canonical site halted APP transport at the Golgi apparatus. Together, our analyses support that the functional recognition of the non-canonical YXXØ-signal of APP is limited to the non

  17. Structural and functional characterization of cargo-binding sites on the μ4-subunit of adaptor protein complex 4.

    PubMed

    Ross, Breyan H; Lin, Yimo; Corales, Esteban A; Burgos, Patricia V; Mardones, Gonzalo A

    2014-01-01

    Adaptor protein (AP) complexes facilitate protein trafficking by playing key roles in the selection of cargo molecules to be sorted in post-Golgi compartments. Four AP complexes (AP-1 to AP-4) contain a medium-sized subunit (μ1-μ4) that recognizes YXXØ-sequences (Ø is a bulky hydrophobic residue), which are sorting signals in transmembrane proteins. A conserved, canonical region in μ subunits mediates recognition of YXXØ-signals by means of a critical aspartic acid. Recently we found that a non-canonical YXXØ-signal on the cytosolic tail of the Alzheimer's disease amyloid precursor protein (APP) binds to a distinct region of the μ4 subunit of the AP-4 complex. In this study we aimed to determine the functionality of both binding sites of μ4 on the recognition of the non-canonical YXXØ-signal of APP. We found that substitutions in either binding site abrogated the interaction with the APP-tail in yeast-two hybrid experiments. Further characterization by isothermal titration calorimetry showed instead loss of binding to the APP signal with only the substitution R283D at the non-canonical site, in contrast to a decrease in binding affinity with the substitution D190A at the canonical site. We solved the crystal structure of the C-terminal domain of the D190A mutant bound to this non-canonical YXXØ-signal. This structure showed no significant difference compared to that of wild-type μ4. Both differential scanning fluorimetry and limited proteolysis analyses demonstrated that the D190A substitution rendered μ4 less stable, suggesting an explanation for its lower binding affinity to the APP signal. Finally, in contrast to overexpression of the D190A mutant, and acting in a dominant-negative manner, overexpression of μ4 with either a F255A or a R283D substitution at the non-canonical site halted APP transport at the Golgi apparatus. Together, our analyses support that the functional recognition of the non-canonical YXXØ-signal of APP is limited to the non

  18. Invertebrate and Vertebrate Class III Myosins Interact with MORN Repeat-Containing Adaptor Proteins

    PubMed Central

    Mecklenburg, Kirk L.; Freed, Stephanie A.; Raval, Manmeet; Quintero, Omar A.; Yengo, Christopher M.; O'Tousa, Joseph. E.

    2015-01-01

    In Drosophila photoreceptors, the NINAC-encoded myosin III is found in a complex with a small, MORN-repeat containing, protein Retinophilin (RTP). Expression of these two proteins in other cell types showed NINAC myosin III behavior is altered by RTP. NINAC deletion constructs were used to map the RTP binding site within the proximal tail domain of NINAC. In vertebrates, the RTP ortholog is MORN4. Co-precipitation experiments demonstrated that human MORN4 binds to human myosin IIIA (MYO3A). In COS7 cells, MORN4 and MYO3A, but not MORN4 and MYO3B, co-localize to actin rich filopodia extensions. Deletion analysis mapped the MORN4 binding to the proximal region of the MYO3A tail domain. MYO3A dependent MORN4 tip localization suggests that MYO3A functions as a motor that transports MORN4 to the filopodia tips and MORN4 may enhance MYO3A tip localization by tethering it to the plasma membrane at the protrusion tips. These results establish conserved features of the RTP/MORN4 family: they bind within the tail domain of myosin IIIs to control their behavior. PMID:25822849

  19. NRAGE, a p75NTR adaptor protein, is required for developmental apoptosis in vivo.

    PubMed

    Bertrand, M J M; Kenchappa, R S; Andrieu, D; Leclercq-Smekens, M; Nguyen, H N T; Carter, B D; Muscatelli, F; Barker, P A; De Backer, O

    2008-12-01

    NRAGE (also known as Maged1, Dlxin) is a member of the MAGE gene family that may play a role in the neuronal apoptosis that is regulated by the p75 neurotrophin receptor (p75NTR). To test this hypothesis in vivo, we generated NRAGE knockout mice and found that NRAGE deletion caused a defect in developmental apoptosis of sympathetic neurons of the superior cervical ganglia, similar to that observed in p75NTR knockout mice. Primary sympathetic neurons derived from NRAGE knockout mice were resistant to apoptosis induced by brain-derived neurotrophic factor (BDNF), a pro-apoptotic p75NTR ligand, and NRAGE-deficient sympathetic neurons show attenuated BDNF-dependent JNK activation. Hair follicle catagen is an apoptosis-like process that is dependent on p75NTR signaling; we show that NRAGE and p75NTR show regulated co-expression in the hair follicle and that identical defects in hair follicle catagen are present in NRAGE and p75NTR knockout mice. Interestingly, NRAGE knockout mice have severe defects in motoneuron apoptosis that are not observed in p75NTR knockout animals, raising the possibility that NRAGE may facilitate apoptosis induced by receptors other than p75NTR. Together, these studies demonstrate that NRAGE plays an important role in apoptotic-signaling in vivo. PMID:18772898

  20. Mechanisms Regulating Protein Localization.

    PubMed

    Bauer, Nicholas C; Doetsch, Paul W; Corbett, Anita H

    2015-10-01

    Cellular functions are dictated by protein content and activity. There are numerous strategies to regulate proteins varying from modulating gene expression to post-translational modifications. One commonly used mode of regulation in eukaryotes is targeted localization. By specifically redirecting the localization of a pool of existing protein, cells can achieve rapid changes in local protein function. Eukaryotic cells have evolved elegant targeting pathways to direct proteins to the appropriate cellular location or locations. Here, we provide a general overview of these localization pathways, with a focus on nuclear and mitochondrial transport, and present a survey of the evolutionarily conserved regulatory strategies identified thus far. We end with a description of several specific examples of proteins that exploit localization as an important mode of regulation. PMID:26172624

  1. Structural Biology and Regulation of Protein Import into the Nucleus.

    PubMed

    Christie, Mary; Chang, Chiung-Wen; Róna, Gergely; Smith, Kate M; Stewart, Alastair G; Takeda, Agnes A S; Fontes, Marcos R M; Stewart, Murray; Vértessy, Beáta G; Forwood, Jade K; Kobe, Bostjan

    2016-05-22

    Proteins are translated in the cytoplasm, but many need to access the nucleus to perform their functions. Understanding how these nuclear proteins are transported through the nuclear envelope and how the import processes are regulated is therefore an important aspect of understanding cell function. Structural biology has played a key role in understanding the molecular events during the transport processes and their regulation, including the recognition of nuclear targeting signals by the corresponding receptors. Here, we review the structural basis of the principal nuclear import pathways and the molecular basis of their regulation. The pathways involve transport factors that are members of the β-karyopherin family, which can bind cargo directly (e.g., importin-β, transportin-1, transportin-3, importin-13) or through adaptor proteins (e.g., importin-α, snurportin-1, symportin-1), as well as unrelated transport factors such as Hikeshi, involved in the transport of heat-shock proteins, and NTF2, involved in the transport of RanGDP. Solenoid proteins feature prominently in these pathways. Nuclear transport factors recognize nuclear targeting signals on the cargo proteins, including the classical nuclear localization signals, recognized by the adaptor importin-α, and the PY nuclear localization signals, recognized by transportin-1. Post-translational modifications, particularly phosphorylation, constitute key regulatory mechanisms operating in these pathways. PMID:26523678

  2. Impaired Fracture Healing Caused by Deficiency of the Immunoreceptor Adaptor Protein DAP12.

    PubMed

    Kamimura, Masayuki; Mori, Yu; Sugahara-Tobinai, Akiko; Takai, Toshiyuki; Itoi, Eiji

    2015-01-01

    Osteoclasts play an important role in bone metabolism, but their exact role in fracture healing remains unclear. DAP12 is an immunoadaptor protein with associated immunoreceptors on myeloid lineage cells, including osteoclasts. Its deficiency causes osteopetrosis due to suppression of osteoclast development and activation. In this report, we assessed the impact of DAP12 on the fracture healing process using C57BL/6 (B6) and DAP12-/- mice. Healing was evaluated using radiography, micro-CT, histology, immunohistochemistry and real-time RT-PCR. Radiography showed lower callus volume and lower callus radiolucency in DAP12-/- mice during later stages. Micro-CT images and quantitative structural analysis indicated that DAP12-/- mice developed calluses of dense trabecular structures and experienced deteriorated cortical shell formation on the surface. Histologically, DAP12-/- mice showed less cartilaginous resorption and woven bone formation. In addition, prominent cortical shell formation was much less in DAP12-/- mice. Immunohistochemistry revealed lower invasion of F4/80 positive monocytes and macrophages into the fracture hematoma in DAP12-/- mice. The expression levels of Col1a1, Col2a1 and Col10a1 in DAP12-/- mice increased and subsequently became higher than those in B6 mice. There was a decrease in the gene expression of Tnf during the early stages in DAP12-/- mice. Our results indicate that DAP12 deficiency impairs fracture healing, suggesting a significant role of DAP12 in the initial inflammatory response, bone remodeling and regeneration. PMID:26030755

  3. Siglec-15 regulates osteoclast differentiation by modulating RANKL-induced phosphatidylinositol 3-kinase/Akt and Erk pathways in association with signaling Adaptor DAP12.

    PubMed

    Kameda, Yusuke; Takahata, Masahiko; Komatsu, Miki; Mikuni, Shintaro; Hatakeyama, Shigetsugu; Shimizu, Tomohiro; Angata, Takashi; Kinjo, Masataka; Minami, Akio; Iwasaki, Norimasa

    2013-12-01

    Siglecs are a family of sialic acid-binding immunoglobulin-like lectins that regulate the functions of cells in the innate and adaptive immune systems through glycan recognition. Here we show that Siglec-15 regulates osteoclast development and bone resorption by modulating receptor activator of nuclear factor κB ligand (RANKL) signaling in association with DNAX-activating protein 12 kDa (DAP12), an adaptor protein bearing an immunoreceptor tyrosine-based activation motif (ITAM). Among the known Siglecs expressed in myeloid lineage cells, only Siglec-15 was upregulated by RANKL in mouse primary bone marrow macrophages. Siglec-15-deficient mice exhibit mild osteopetrosis resulting from impaired osteoclast development. Consistently, cells lacking Siglec-15 exhibit defective osteoclast development and resorptive activity in vitro. RANKL-induced activation of phosphatidylinositol 3-kinase (PI3K)/Akt and Erk pathways were impaired in Siglec-15-deficient cells. Retroviral transduction of Siglec-15-null osteoclast precursors with wild-type Siglec-15 or mutant Siglec-15 revealed that the association of Siglec-15 with DAP12 is involved in the downstream signal transduction of RANK. Furthermore, we found that the ability of osteoclast formation is preserved in the region adjacent to the growth plate in Siglec-15-deficient mice, indicating that there is a compensatory mechanism for Siglec-15-mediated osteoclastogenesis in the primary spongiosa. To clarify the mechanism of this compensation, we examined whether osteoclast-associated receptor (OSCAR)/Fc receptor common γ (FcRγ) signaling, an alternative ITAM-mediated signaling pathway to DAP12, rescues impaired osteoclastogenesis in Siglec-15-deficient cells. The ligands in type II collagen activate OSCAR and rescue impaired osteoclastogenesis in Siglec-15-deficient cells when cultured on bone slices, indicating that Siglec-15-mediated signaling can be compensated for by signaling activated by type II collagen and other bone

  4. Impaired Fracture Healing Caused by Deficiency of the Immunoreceptor Adaptor Protein DAP12

    PubMed Central

    Kamimura, Masayuki; Mori, Yu; Sugahara-Tobinai, Akiko; Takai, Toshiyuki; Itoi, Eiji

    2015-01-01

    Osteoclasts play an important role in bone metabolism, but their exact role in fracture healing remains unclear. DAP12 is an immunoadaptor protein with associated immunoreceptors on myeloid lineage cells, including osteoclasts. Its deficiency causes osteopetrosis due to suppression of osteoclast development and activation. In this report, we assessed the impact of DAP12 on the fracture healing process using C57BL/6 (B6) and DAP12–/– mice. Healing was evaluated using radiography, micro-CT, histology, immunohistochemistry and real-time RT-PCR. Radiography showed lower callus volume and lower callus radiolucency in DAP12–/– mice during later stages. Micro-CT images and quantitative structural analysis indicated that DAP12–/– mice developed calluses of dense trabecular structures and experienced deteriorated cortical shell formation on the surface. Histologically, DAP12–/– mice showed less cartilaginous resorption and woven bone formation. In addition, prominent cortical shell formation was much less in DAP12–/– mice. Immunohistochemistry revealed lower invasion of F4/80 positive monocytes and macrophages into the fracture hematoma in DAP12–/– mice. The expression levels of Col1a1, Col2a1 and Col10a1 in DAP12–/– mice increased and subsequently became higher than those in B6 mice. There was a decrease in the gene expression of Tnf during the early stages in DAP12–/– mice. Our results indicate that DAP12 deficiency impairs fracture healing, suggesting a significant role of DAP12 in the initial inflammatory response, bone remodeling and regeneration. PMID:26030755

  5. Adaptor protein 2–mediated endocytosis of the β-secretase BACE1 is dispensable for amyloid precursor protein processing

    PubMed Central

    Prabhu, Yogikala; Burgos, Patricia V.; Schindler, Christina; Farías, Ginny G.; Magadár, Javier G.; Bonifacino, Juan S.

    2012-01-01

    The β-site amyloid precursor protein (APP)–cleaving enzyme 1 (BACE1) is a transmembrane aspartyl protease that catalyzes the proteolytic processing of APP and other plasma membrane protein precursors. BACE1 cycles between the trans-Golgi network (TGN), the plasma membrane, and endosomes by virtue of signals contained within its cytosolic C-terminal domain. One of these signals is the DXXLL-motif sequence DISLL, which controls transport between the TGN and endosomes via interaction with GGA proteins. Here we show that the DISLL sequence is embedded within a longer [DE]XXXL[LI]-motif sequence, DDISLL, which mediates internalization from the plasma membrane by interaction with the clathrin-associated, heterotetrameric adaptor protein 2 (AP-2) complex. Mutation of this signal or knockdown of either AP-2 or clathrin decreases endosomal localization and increases plasma membrane localization of BACE1. Remarkably, internalization-defective BACE1 is able to cleave an APP mutant that itself cannot be delivered to endosomes. The drug brefeldin A reversibly prevents BACE1-catalyzed APP cleavage, ruling out that this reaction occurs in the endoplasmic reticulum (ER) or ER–Golgi intermediate compartment. Taken together, these observations support the notion that BACE1 is capable of cleaving APP in late compartments of the secretory pathway. PMID:22553349

  6. Clathrin binding by the adaptor Ent5 promotes late stages of clathrin coat maturation

    PubMed Central

    Hung, Chao-Wei; Duncan, Mara C.

    2016-01-01

    Clathrin is a ubiquitous protein that mediates membrane traffic at many locations. To function, clathrin requires clathrin adaptors that link it to transmembrane protein cargo. In addition to this cargo selection function, many adaptors also play mechanistic roles in the formation of the transport carrier. However, the full spectrum of these mechanistic roles is poorly understood. Here we report that Ent5, an endosomal clathrin adaptor in Saccharomyces cerevisiae, regulates the behavior of clathrin coats after the recruitment of clathrin. We show that loss of Ent5 disrupts clathrin-dependent traffic and prolongs the lifespan of endosomal structures that contain clathrin and other adaptors, suggesting a defect in coat maturation at a late stage. We find that the direct binding of Ent5 with clathrin is required for its role in coat behavior and cargo traffic. Surprisingly, the interaction of Ent5 with other adaptors is dispensable for coat behavior but not cargo traffic. These findings support a model in which Ent5 clathrin binding performs a mechanistic role in coat maturation, whereas Ent5 adaptor binding promotes cargo incorporation. PMID:26842894

  7. Germline-specific MATH-BTB substrate adaptor MAB1 regulates spindle length and nuclei identity in maize.

    PubMed

    Juranič, Martina; Srilunchang, Kanok-orn; Krohn, Nádia Graciele; Leljak-Levanic, Dunja; Sprunck, Stefanie; Dresselhaus, Thomas

    2012-12-01

    Germline and early embryo development constitute ideal model systems to study the establishment of polarity, cell identity, and asymmetric cell divisions (ACDs) in plants. We describe here the function of the MATH-BTB domain protein MAB1 that is exclusively expressed in the germ lineages and the zygote of maize (Zea mays). mab1 (RNA interference [RNAi]) mutant plants display chromosome segregation defects and short spindles during meiosis that cause insufficient separation and migration of nuclei. After the meiosis-to-mitosis transition, two attached nuclei of similar identity are formed in mab1 (RNAi) mutants leading to an arrest of further germline development. Transient expression studies of MAB1 in tobacco (Nicotiana tabacum) Bright Yellow-2 cells revealed a cell cycle-dependent nuclear localization pattern but no direct colocalization with the spindle apparatus. MAB1 is able to form homodimers and interacts with the E3 ubiquitin ligase component Cullin 3a (CUL3a) in the cytoplasm, likely as a substrate-specific adapter protein. The microtubule-severing subunit p60 of katanin was identified as a candidate substrate for MAB1, suggesting that MAB1 resembles the animal key ACD regulator Maternal Effect Lethal 26 (MEL-26). In summary, our findings provide further evidence for the importance of posttranslational regulation for asymmetric divisions and germline progression in plants and identified an unstable key protein that seems to be involved in regulating the stability of a spindle apparatus regulator(s). PMID:23250449

  8. Germline-Specific MATH-BTB Substrate Adaptor MAB1 Regulates Spindle Length and Nuclei Identity in Maize[W

    PubMed Central

    Juranić, Martina; Srilunchang, Kanok-orn; Krohn, Nádia Graciele; Leljak-Levanić, Dunja; Sprunck, Stefanie; Dresselhaus, Thomas

    2012-01-01

    Germline and early embryo development constitute ideal model systems to study the establishment of polarity, cell identity, and asymmetric cell divisions (ACDs) in plants. We describe here the function of the MATH-BTB domain protein MAB1 that is exclusively expressed in the germ lineages and the zygote of maize (Zea mays). mab1 (RNA interference [RNAi]) mutant plants display chromosome segregation defects and short spindles during meiosis that cause insufficient separation and migration of nuclei. After the meiosis-to-mitosis transition, two attached nuclei of similar identity are formed in mab1 (RNAi) mutants leading to an arrest of further germline development. Transient expression studies of MAB1 in tobacco (Nicotiana tabacum) Bright Yellow-2 cells revealed a cell cycle–dependent nuclear localization pattern but no direct colocalization with the spindle apparatus. MAB1 is able to form homodimers and interacts with the E3 ubiquitin ligase component Cullin 3a (CUL3a) in the cytoplasm, likely as a substrate-specific adapter protein. The microtubule-severing subunit p60 of katanin was identified as a candidate substrate for MAB1, suggesting that MAB1 resembles the animal key ACD regulator Maternal Effect Lethal 26 (MEL-26). In summary, our findings provide further evidence for the importance of posttranslational regulation for asymmetric divisions and germline progression in plants and identified an unstable key protein that seems to be involved in regulating the stability of a spindle apparatus regulator(s). PMID:23250449

  9. Cellular Heterogeneity During Embryonic Stem Cell Differentiation to Epiblast Stem Cells is Revealed by the ShcD/RaLP Adaptor Protein

    PubMed Central

    Turco, Margherita Y; Furia, Laura; Dietze, Anja; Fernandez Diaz, Luis; Ronzoni, Simona; Sciullo, Anna; Simeone, Antonio; Constam, Daniel; Faretta, Mario; Lanfrancone, Luisa

    2012-01-01

    The Shc family of adaptor proteins are crucial mediators of a plethora of receptors such as the tyrosine kinase receptors, cytokine receptors, and integrins that drive signaling pathways governing proliferation, differentiation, and migration. Here, we report the role of the newly identified family member, ShcD/RaLP, whose expression in vitro and in vivo suggests a function in embryonic stem cell (ESC) to epiblast stem cells (EpiSCs) transition. The transition from the naïve (ESC) to the primed (EpiSC) pluripotent state is the initial important step for ESCs to commit to differentiation and the mechanisms underlying this process are still largely unknown. Using a novel approach to simultaneously assess pluripotency, apoptosis, and proliferation by multiparameter flow cytometry, we show that ESC to EpiSC transition is a process involving a tight coordination between the modulation of the Oct4 expression, cell cycle progression, and cell death. We also describe, by high-content immunofluorescence analysis and time-lapse microscopy, the emergence of cells expressing caudal-related homeobox 2 (Cdx2) transcription factor during ESC to EpiSC transition. The use of the ShcD knockout ESCs allowed the unmasking of this process as they presented deregulated Oct4 modulation and an enrichment in Oct4-negative Cdx2-positive cells with increased MAPK/extracellular-regulated kinases 1/2 activation, within the differentiating population. Collectively, our data reveal ShcD as an important modulator in the switch of key pathway(s) involved in determining EpiSC identity. Stem Cells2012;30:2423–2436 PMID:22948967

  10. Mutations in the gene encoding the Sigma 2 subunit of the adaptor protein 1 complex, AP1S2, cause X-linked mental retardation.

    PubMed

    Tarpey, Patrick S; Stevens, Claire; Teague, Jon; Edkins, Sarah; O'Meara, Sarah; Avis, Tim; Barthorpe, Syd; Buck, Gemma; Butler, Adam; Cole, Jennifer; Dicks, Ed; Gray, Kristian; Halliday, Kelly; Harrison, Rachel; Hills, Katy; Hinton, Jonathon; Jones, David; Menzies, Andrew; Mironenko, Tatiana; Perry, Janet; Raine, Keiran; Richardson, David; Shepherd, Rebecca; Small, Alexandra; Tofts, Calli; Varian, Jennifer; West, Sofie; Widaa, Sara; Yates, Andy; Catford, Rachael; Butler, Julia; Mallya, Uma; Moon, Jenny; Luo, Ying; Dorkins, Huw; Thompson, Deborah; Easton, Douglas F; Wooster, Richard; Bobrow, Martin; Carpenter, Nancy; Simensen, Richard J; Schwartz, Charles E; Stevenson, Roger E; Turner, Gillian; Partington, Michael; Gecz, Jozef; Stratton, Michael R; Futreal, P Andrew; Raymond, F Lucy

    2006-12-01

    In a systematic sequencing screen of the coding exons of the X chromosome in 250 families with X-linked mental retardation (XLMR), we identified two nonsense mutations and one consensus splice-site mutation in the AP1S2 gene on Xp22 in three families. Affected individuals in these families showed mild-to-profound mental retardation. Other features included hypotonia early in life and delay in walking. AP1S2 encodes an adaptin protein that constitutes part of the adaptor protein complex found at the cytoplasmic face of coated vesicles located at the Golgi complex. The complex mediates the recruitment of clathrin to the vesicle membrane. Aberrant endocytic processing through disruption of adaptor protein complexes is likely to result from the AP1S2 mutations identified in the three XLMR-affected families, and such defects may plausibly cause abnormal synaptic development and function. AP1S2 is the first reported XLMR gene that encodes a protein directly involved in the assembly of endocytic vesicles. PMID:17186471

  11. Adaptors for radiation detectors

    DOEpatents

    Livesay, Ronald Jason

    2014-04-22

    Described herein are adaptors and other devices for radiation detectors that can be used to make accurate spectral measurements of both small and large bulk sources of radioactivity, such as building structures, soils, vessels, large equipment, and liquid bodies. Some exemplary devices comprise an adaptor for a radiation detector, wherein the adaptor can be configured to collimate radiation passing through the adapter from an external radiation source to the radiation detector and the adaptor can be configured to enclose a radiation source within the adapter to allow the radiation detector to measure radiation emitted from the enclosed radiation source.

  12. Adaptors for radiation detectors

    DOEpatents

    Livesay, Ronald Jason

    2015-07-28

    Described herein are adaptors and other devices for radiation detectors that can be used to make accurate spectral measurements of both small and large bulk sources of radioactivity, such as building structures, soils, vessels, large equipment, and liquid bodies. Some exemplary devices comprise an adaptor for a radiation detector, wherein the adaptor can be configured to collimate radiation passing through the adapter from an external radiation source to the radiation detector and the adaptor can be configured to enclose a radiation source within the adapter to allow the radiation detector to measure radiation emitted from the enclosed radiation source.

  13. Identification of Atg2 and ArfGAP1 as Candidate Genetic Modifiers of the Eye Pigmentation Phenotype of Adaptor Protein-3 (AP-3) Mutants in Drosophila melanogaster

    PubMed Central

    Rodriguez-Fernandez, Imilce A.; Dell’Angelica, Esteban C.

    2015-01-01

    The Adaptor Protein (AP)-3 complex is an evolutionary conserved, molecular sorting device that mediates the intracellular trafficking of proteins to lysosomes and related organelles. Genetic defects in AP-3 subunits lead to impaired biogenesis of lysosome-related organelles (LROs) such as mammalian melanosomes and insect eye pigment granules. In this work, we have performed a forward screening for genetic modifiers of AP-3 function in the fruit fly, Drosophila melanogaster. Specifically, we have tested collections of large multi-gene deletions–which together covered most of the autosomal chromosomes–to identify chromosomal regions that, when deleted in single copy, enhanced or ameliorated the eye pigmentation phenotype of two independent AP-3 subunit mutants. Fine-mapping led us to define two non-overlapping, relatively small critical regions within fly chromosome 3. The first critical region included the Atg2 gene, which encodes a conserved protein involved in autophagy. Loss of one functional copy of Atg2 ameliorated the pigmentation defects of mutants in AP-3 subunits as well as in two other genes previously implicated in LRO biogenesis, namely Blos1 and lightoid, and even increased the eye pigment content of wild-type flies. The second critical region included the ArfGAP1 gene, which encodes a conserved GTPase-activating protein with specificity towards GTPases of the Arf family. Loss of a single functional copy of the ArfGAP1 gene ameliorated the pigmentation phenotype of AP-3 mutants but did not to modify the eye pigmentation of wild-type flies or mutants in Blos1 or lightoid. Strikingly, loss of the second functional copy of the gene did not modify the phenotype of AP-3 mutants any further but elicited early lethality in males and abnormal eye morphology when combined with mutations in Blos1 and lightoid, respectively. These results provide genetic evidence for new functional links connecting the machinery for biogenesis of LROs with molecules implicated

  14. Impaired Lysosomal Integral Membrane Protein 2-dependent Peroxiredoxin 6 Delivery to Lamellar Bodies Accounts for Altered Alveolar Phospholipid Content in Adaptor Protein-3-deficient pearl Mice.

    PubMed

    Kook, Seunghyi; Wang, Ping; Young, Lisa R; Schwake, Michael; Saftig, Paul; Weng, Xialian; Meng, Ying; Neculai, Dante; Marks, Michael S; Gonzales, Linda; Beers, Michael F; Guttentag, Susan

    2016-04-15

    The Hermansky Pudlak syndromes (HPS) constitute a family of disorders characterized by oculocutaneous albinism and bleeding diathesis, often associated with lethal lung fibrosis. HPS results from mutations in genes of membrane trafficking complexes that facilitate delivery of cargo to lysosome-related organelles. Among the affected lysosome-related organelles are lamellar bodies (LB) within alveolar type 2 cells (AT2) in which surfactant components are assembled, modified, and stored. AT2 from HPS patients and mouse models of HPS exhibit enlarged LB with increased phospholipid content, but the mechanism underlying these defects is unknown. We now show that AT2 in the pearl mouse model of HPS type 2 lacking the adaptor protein 3 complex (AP-3) fails to accumulate the soluble enzyme peroxiredoxin 6 (PRDX6) in LB. This defect reflects impaired AP-3-dependent trafficking of PRDX6 to LB, because pearl mouse AT2 cells harbor a normal total PRDX6 content. AP-3-dependent targeting of PRDX6 to LB requires the transmembrane protein LIMP-2/SCARB2, a known AP-3-dependent cargo protein that functions as a carrier for lysosomal proteins in other cell types. Depletion of LB PRDX6 in AP-3- or LIMP-2/SCARB2-deficient mice correlates with phospholipid accumulation in lamellar bodies and with defective intraluminal degradation of LB disaturated phosphatidylcholine. Furthermore, AP-3-dependent LB targeting is facilitated by protein/protein interaction between LIMP-2/SCARB2 and PRDX6 in vitro and in vivo Our data provide the first evidence for an AP-3-dependent cargo protein required for the maturation of LB in AT2 and suggest that the loss of PRDX6 activity contributes to the pathogenic changes in LB phospholipid homeostasis found HPS2 patients. PMID:26907692

  15. The Adaptor Protein Myd88 Is a Key Signaling Molecule in the Pathogenesis of Irinotecan-Induced Intestinal Mucositis

    PubMed Central

    Wong, Deysi V. T.; Lima-Júnior, Roberto C. P.; Carvalho, Cibele B. M.; Borges, Vanessa F.; Wanderley, Carlos W. S.; Bem, Amanda X. C.; Leite, Caio A. V. G.; Teixeira, Maraiza A.; Batista, Gabriela L. P.; Silva, Rangel L.; Cunha, Thiago M.; Brito, Gerly A. C.; Almeida, Paulo R. C.; Cunha, Fernando Q.; Ribeiro, Ronaldo A.

    2015-01-01

    Intestinal mucositis is a common side effect of irinotecan-based anticancer regimens. Mucositis causes cell damage, bacterial/endotoxin translocation and production of cytokines including IL–1 and IL–18. These molecules and toll-like receptors (TLRs) activate a common signaling pathway that involves the Myeloid Differentiation adaptor protein, MyD88, whose role in intestinal mucositis is unknown. Then, we evaluated the involvement of TLRs and MyD88 in the pathogenesis of irinotecan-induced intestinal mucositis. MyD88-, TLR2- or TLR9-knockout mice and C57BL/6 (WT) mice were given either saline or irinotecan (75 mg/kg, i.p. for 4 days). On day 7, animal survival, diarrhea and bacteremia were assessed, and following euthanasia, samples of the ileum were obtained for morphometric analysis, myeloperoxidase (MPO) assay and measurement of pro-inflammatory markers. Irinotecan reduced the animal survival (50%) and induced a pronounced diarrhea, increased bacteremia, neutrophil accumulation in the intestinal tissue, intestinal damage and more than twofold increased expression of MyD88 (200%), TLR9 (400%), TRAF6 (236%), IL–1β (405%), IL–18 (365%), COX–2 (2,777%) and NF-κB (245%) in the WT animals when compared with saline-injected group (P<0.05). Genetic deletion of MyD88, TLR2 or TLR9 effectively controlled the signs of intestinal injury when compared with irinotecan-administered WT controls (P<0.05). In contrast to the MyD88-/- and TLR2-/- mice, the irinotecan-injected TLR9-/- mice showed a reduced survival, a marked diarrhea and an enhanced expression of IL–18 versus irinotecan-injected WT controls. Additionally, the expression of MyD88 was reduced in the TLR2-/- or TLR9-/- mice. This study shows a critical role of the MyD88-mediated TLR2 and TLR9 signaling in the pathogenesis of irinotecan-induced intestinal mucositis. PMID:26440613

  16. The μ Subunit of Arabidopsis Adaptor Protein-2 Is Involved in Effector-Triggered Immunity Mediated by Membrane-Localized Resistance Proteins.

    PubMed

    Hatsugai, Noriyuki; Hillmer, Rachel; Yamaoka, Shohei; Hara-Nishimura, Ikuko; Katagiri, Fumiaki

    2016-05-01

    Endocytosis has been suggested to be important in the cellular processes of plant immune responses. However, our understanding of its role during effector-triggered immunity (ETI) is still limited. We have previously shown that plant endocytosis, especially clathrin-coated vesicle formation at the plasma membrane, is mediated by the adaptor protein-2 (AP-2) complex and that loss of the μ subunit of AP-2 (AP2M) affects plant growth and floral organ development. Here, we report that AP2M is required for full-strength ETI mediated by the disease resistance (R) genes RPM1 and RPS2 in Arabidopsis. Reduced ETI was observed in an ap2m mutant plant, measured by growth of Pseudomonas syringae pv. tomato DC3000 strains carrying the corresponding effector genes avrRpm1 or avrRpt2 and by hypersensitive cell death response and defense gene expression triggered by these strains. In contrast, RPS4-mediated ETI and its associated immune responses were not affected by the ap2m mutation. While RPM1 and RPS2 are localized to the plasma membrane, RPS4 is localized to the cytoplasm and nucleus. Our results suggest that AP2M is involved in ETI mediated by plasma membrane-localized R proteins, possibly by mediating endocytosis of the immune receptor complex components from the plasma membrane. PMID:26828402

  17. A novel GTP-binding protein-adaptor protein complex responsible for export of Vangl2 from the trans Golgi network.

    PubMed

    Guo, Yusong; Zanetti, Giulia; Schekman, Randy

    2013-01-01

    Planar cell polarity (PCP) requires the asymmetric sorting of distinct signaling receptors to distal and proximal surfaces of polarized epithelial cells. We have examined the transport of one PCP signaling protein, Vangl2, from the trans Golgi network (TGN) in mammalian cells. Using siRNA knockdown experiments, we find that the GTP-binding protein, Arfrp1, and the clathrin adaptor complex 1 (AP-1) are required for Vangl2 transport from the TGN. In contrast, TGN export of Frizzled 6, which localizes to the opposing epithelial surface from Vangl2, does not depend on Arfrp1 or AP-1. Mutagenesis studies identified a YYXXF sorting signal in the C-terminal cytosolic domain of Vangl2 that is required for Vangl2 traffic and interaction with the μ subunit of AP-1. We propose that Arfrp1 exposes a binding site on AP-1 that recognizes the Vangl2 sorting motif for capture into a transport vesicle destined for the proximal surface of a polarized epithelial cell.DOI:http://dx.doi.org/10.7554/eLife.00160.001. PMID:23326640

  18. TIR domain-containing adaptor SARM is a late addition to the ongoing microbe–host dialog

    PubMed Central

    Zhang, Qing; Zmasek, Christian M.; Cai, Xiaohui; Godzik, Adam

    2011-01-01

    Toll/interleukin-1 receptor (TIR) domain-containing proteins play important roles in defense against pathogens in both animals and plants, connecting the immunity signaling pathways via a chain of specific protein–protein interactions. Among them is SARM, the only TIR domain-containing adaptor that can negatively regulate TLR signaling. By extensive phylogenetic analysis, we show here that SARM is closely related to bacterial proteins with TIR domains, suggesting that this family has a different evolutionary history from other animal TIR-containing adaptors, possibly emerging via a lateral gene transfer from bacteria to animals. We also show evidence of several similar, independent transfer events, none of which, however, survived in vertebrates. An evolutionary relationship between the animal SARM adaptor and bacterial proteins with TIR domains illustrates the possible role that bacterial TIR-containing proteins play in regulating eukaryotic immune responses and how this mechanism was possibly adapted by the eukaryotes themselves. PMID:21110998

  19. Structural and Functional Investigation of the Ag(+)/Cu(+) Binding Domains of the Periplasmic Adaptor Protein SilB from Cupriavidus metallidurans CH34.

    PubMed

    Urbina, Patricia; Bersch, Beate; De Angelis, Fabien; Derfoufi, Kheiro-Mouna; Prévost, Martine; Goormaghtigh, Erik; Vandenbussche, Guy

    2016-05-24

    Silver ion resistance in bacteria mainly relies on efflux systems, and notably on tripartite efflux complexes involving a transporter from the resistance-nodulation-cell division (RND) superfamily, such as the SilCBA system from Cupriavidus metallidurans CH34. The periplasmic adaptor protein SilB hosts two specific metal coordination sites, located in the N-terminal and C-terminal domains, respectively, that are believed to play a different role in the efflux mechanism and the trafficking of metal ions from the periplasm to the RND transporter. On the basis of the known domain structure of periplasmic adaptor proteins, we designed different protein constructs derived from SilB domains with either one or two metal binding sites per protein chain. ITC data acquired on proteins with single metal sites suggest a slightly higher affinity of Ag(+) for the N-terminal metal site, compared to that for the C-terminal one. Remarkably, via the study of a protein construct featuring both metal sites, nuclear magnetic resonance (NMR) and fluorescence spectroscopies concordantly show that the C-terminal site is saturated prior to the N-terminal one. The C-terminal binding site is supposed to transfer the metal ions to the RND protein, while the transport driven by this latter is activated upon binding of the metal ion to the N-terminal site. Our results suggest that the filling of the C-terminal metal site is a key prerequisite for preventing futile activation of the transport system. Exhaustive NMR studies reveal for the first time the structure and dynamics of the functionally important N-terminal domain connected to the membrane proximal domain as well as of its Ag(+) binding site. PMID:27145046

  20. Recombinant production of functional full-length and truncated human TRAM/TICAM-2 adaptor protein involved in Toll-like receptor and interferon signaling.

    PubMed

    Ullah, M Obayed; Valkov, Eugene; Ve, Thomas; Williams, Simon; Mas, Caroline; Mansell, Ashley; Kobe, Bostjan

    2015-02-01

    TRAM/TICAM-2 is used by Toll-like receptor 4 (TLR4) as a bridging adaptor during the mammalian innate immune response. It recruits TRIF, another TIR domain-containing adaptor protein, to TLR4 via TIR domain interactions, which leads to the activation of transcription factors responsible for the production of type-1 interferon and cytokines. The molecular mechanisms of these dual interactions mediated by the TRAM TIR domain are not clear. To understand the molecular basis of TIR:TIR domain interactions, structural and biochemical studies of TRAM TIR domain are necessary, and require a functional soluble protein. In this paper, we report a successful purification and characterization of full-length TRAM. Because full-length TRAM likely contains unstructured regions that may be disadvantageous for structural studies, we also carried out a systematic construct design to determine the boundaries of the TRAM TIR domain. The truncated TRAM constructs were designed based on secondary structure predictions and screened by small-scale expression. Selected constructs were subjected to biophysical analyses. We show that the expressed TRAM TIR domain is functional using in vitro GST pull-down assays that demonstrate a physical interaction with the TLR4 TIR domain. We further show, by site-directed mutagenesis, that the "BB loop" regions of both the TRAM TIR domain and the TLR4 TIR domain are crucial for this physical interaction. PMID:25306876

  1. Reduced photoreceptor death and improved retinal function during retinal degeneration in mice lacking innate immunity adaptor protein MyD88

    PubMed Central

    Syeda, Sarah; Patel, Amit K.; Lee, Tinthu; Hackam, Abigail S.

    2015-01-01

    The injury inflammatory response mediated by the innate immune system is an important contributor to neurodegeneration in the central nervous system (CNS) and retina. A major branch of the innate immune system is regulated by the Toll-like receptors (TLRs), which are receptors for endogenous damage associated molecules released from injured cells as well as pathogen-derived molecules, and interleukin-1 receptors (IL-1R), which are activated by IL-1α, IL-1β and IL-18 cytokines. TLRs and IL-1R are expressed on immune and non-immune cell types and act as first responders to cell damage, which results in tissue repair, or inflammation and apoptosis. Both TLR and IL-1R require the adaptor protein myeloid differentiation primary response gene 88 (MyD88) for signaling. Although inflammation is implicated in neuronal death in the retina, the role of MyD88-dependent TLR and IL-1R signaling in retinal degeneration is unknown. Therefore, the purpose of this study was to investigate the role of MyD88-mediated signaling in neuronal degeneration in the retinal degeneration 1 (rd1) mouse model, which exhibits a phenotype of rapid photoreceptor death and inflammation. To generate rd1 mice lacking the MyD88 gene, rd1 were bred with MyD88 knockout mice (MyD88-/-) for several generations to produce rd1/MyD88+/+ and rd1/MyD88-/- genotypes. Chemokine mRNA expression levels were analyzed by qRT-PCR, and recruitment of activated microglia was quantified by immunodetection of the IBA-1 protein. Retinal outer nuclear layer cell counts were performed to quantify photoreceptor degeneration, and retinal function was assessed using electroretinograms (ERG). Our results revealed that retinal expression of Ccl2, Ccl4, Ccl7 and Cxcl10 was reduced by 2 to 8-fold in rd1/MyD88-/- mice compared with rd1/MyD88+/+ mice (p<0.05), which coincided with attenuated microglial activation, higher numbers of photoreceptors and higher retina responses to photopic and scotopic stimuli. At later ages, rd1/MyD88

  2. Atypical regulation of G protein-coupled receptor intracellular trafficking by ubiquitination

    PubMed Central

    Dores, Michael R.; Trejo, JoAnn

    2014-01-01

    G protein-coupled receptor (GPCR) signaling is precisely regulated. After activation, GPCRs are desensitized, internalized and either recycled to the cell surface or sorted to lysosomes for degradation. The main route for GPCR lysosomal sorting requires ubiquitination and the endosomal-sorting complex required for transport (ESCRT). Four distinct ESCRT adaptor protein complexes act sequentially to bind and sort ubiquitinated cargo to lysosomes. Several studies now indicate that alternate pathways exist for GPCR lysosomal sorting that require only some components of the ESCRT and autophagy machinery. While direct GPCR ubiquitination is not required for alternate lysosomal sorting, new evidence suggests that ubiquitin may function indirectly to modulate adaptor protein activity. Here, we discuss the atypical regulation of GPCR lysosomal sorting by ubiquitination. PMID:24680429

  3. Adaptor protein-2 sigma subunit mutations causing familial hypocalciuric hypercalcaemia type 3 (FHH3) demonstrate genotype-phenotype correlations, codon bias and dominant-negative effects.

    PubMed

    Hannan, Fadil M; Howles, Sarah A; Rogers, Angela; Cranston, Treena; Gorvin, Caroline M; Babinsky, Valerie N; Reed, Anita A; Thakker, Clare E; Bockenhauer, Detlef; Brown, Rosalind S; Connell, John M; Cook, Jacqueline; Darzy, Ken; Ehtisham, Sarah; Graham, Una; Hulse, Tony; Hunter, Steven J; Izatt, Louise; Kumar, Dhavendra; McKenna, Malachi J; McKnight, John A; Morrison, Patrick J; Mughal, M Zulf; O'Halloran, Domhnall; Pearce, Simon H; Porteous, Mary E; Rahman, Mushtaqur; Richardson, Tristan; Robinson, Robert; Scheers, Isabelle; Siddique, Haroon; Van't Hoff, William G; Wang, Timothy; Whyte, Michael P; Nesbit, M Andrew; Thakker, Rajesh V

    2015-09-15

    The adaptor protein-2 sigma subunit (AP2σ2) is pivotal for clathrin-mediated endocytosis of plasma membrane constituents such as the calcium-sensing receptor (CaSR). Mutations of the AP2σ2 Arg15 residue result in familial hypocalciuric hypercalcaemia type 3 (FHH3), a disorder of extracellular calcium (Ca(2+) o) homeostasis. To elucidate the role of AP2σ2 in Ca(2+) o regulation, we investigated 65 FHH probands, without other FHH-associated mutations, for AP2σ2 mutations, characterized their functional consequences and investigated the genetic mechanisms leading to FHH3. AP2σ2 mutations were identified in 17 probands, comprising 5 Arg15Cys, 4 Arg15His and 8 Arg15Leu mutations. A genotype-phenotype correlation was observed with the Arg15Leu mutation leading to marked hypercalcaemia. FHH3 probands harboured additional phenotypes such as cognitive dysfunction. All three FHH3-causing AP2σ2 mutations impaired CaSR signal transduction in a dominant-negative manner. Mutational bias was observed at the AP2σ2 Arg15 residue as other predicted missense substitutions (Arg15Gly, Arg15Pro and Arg15Ser), which also caused CaSR loss-of-function, were not detected in FHH probands, and these mutations were found to reduce the numbers of CaSR-expressing cells. FHH3 probands had significantly greater serum calcium (sCa) and magnesium (sMg) concentrations with reduced urinary calcium to creatinine clearance ratios (CCCR) in comparison with FHH1 probands with CaSR mutations, and a calculated index of sCa × sMg/100 × CCCR, which was ≥ 5.0, had a diagnostic sensitivity and specificity of 83 and 86%, respectively, for FHH3. Thus, our studies demonstrate AP2σ2 mutations to result in a more severe FHH phenotype with genotype-phenotype correlations, and a dominant-negative mechanism of action with mutational bias at the Arg15 residue. PMID:26082470

  4. Adaptor protein Ste50p links the Ste11p MEKK to the HOG pathway through plasma membrane association

    PubMed Central

    Wu, Cunle; Jansen, Gregor; Zhang, Jianchun; Thomas, David Y.; Whiteway, Malcolm

    2006-01-01

    In a variety of yeast cellular pathways, the Ste50p protein regulates the kinase function of the mitogen extracellular signal-regulated kinase kinase (MEKK) Ste11p. Both Ste11p and Ste50p contain sterile α motif (SAM) domains; these are interchangeable, and can be replaced by other protein-interacting modules. Furthermore, the function of the Ras association (RA)-like domain of Ste50p can be mimicked by a plasma membrane recruiting signal, and direct plasma membrane targeting of Ste11p bypasses the requirement of Ste50p for Ste11p function. Thus the regulatory role of Ste50p requires both the N-terminal SAM domain to bind Ste11p and the C-terminal RA-like domain to direct kinase localization. We have identified Opy2p, an integral membrane protein that can interact with Ste50p, as a new component in the Sho1p–Ste11p/Ste50p signaling branch of the high-osmolarity glycerol (HOG) pathway. We propose that Opy2p can serve as a membrane anchor for the Ste50p/Ste11p module in the activation of the HOG pathway. PMID:16543225

  5. Structure of Staphylococcus aureus EsxA suggests a contribution to virulence by action as a transport chaperone and/or adaptor protein

    PubMed Central

    Sundaramoorthy, Ramasubramanian; Fyfe, Paul K.; Hunter, William N.

    2012-01-01

    Staphylococcus aureus pathogenesis depends on a specialized protein secretion system, ESX-1, that delivers a range of virulence factors to assist infectivity. We report the characterization of two such factors, EsxA and EsxB; small acidic dimeric proteins carrying a distinctive WXG motif. EsxA crystallized in triclinic and monoclinic forms and high-resolution structures were determined. The asymmetric unit of each crystal form is a dimer. The EsxA subunit forms an elongated cylindrical structure created from side-by-side α-helices linked with a hairpin bend formed by the WXG motif. Approximately 25% of the solvent accessible surface area of each subunit is involved in interactions, predominantly hydrophobic, with the partner subunit. Secondary structure predictions suggest that EsxB displays a similar structure. The WXG motif helps to create a shallow cleft at each end of the dimer, forming a short β-sheet-like feature with an N-terminal segment of the partner subunit. Structural and sequence comparisons, exploiting biological data on related proteins found in Mycobacteria tuberculosis suggest that this family of proteins may contribute to pathogenesis by transporting protein cargo through the ESX-1 system exploiting a C-terminal secretion signal and / or are capable of acting as adaptor proteins to facilitate interactions with host receptor proteins. PMID:18773907

  6. The exomer cargo adaptor structure reveals a novel GTPase-binding domain

    PubMed Central

    Paczkowski, Jon E; Richardson, Brian C; Strassner, Amanda M; Fromme, J Christopher

    2012-01-01

    Cargo adaptors control intracellular trafficking of transmembrane proteins by sorting them into membrane transport carriers. The COPI, COPII, and clathrin cargo adaptors are structurally well characterized, but other cargo adaptors remain poorly understood. Exomer is a specialized cargo adaptor that sorts specific proteins into trans-Golgi network (TGN)-derived vesicles in response to cellular signals. Exomer is recruited to the TGN by the Arf1 GTPase, a universally conserved trafficking regulator. Here, we report the crystal structure of a tetrameric exomer complex composed of two copies each of the Chs5 and Chs6 subunits. The structure reveals the FN3 and BRCT domains of Chs5, which together we refer to as the FBE domain (FN3–BRCT of exomer), project from the exomer core complex. The overall architecture of the FBE domain is reminiscent of the appendage domains of other cargo adaptors, although it exhibits a distinct topology. In contrast to appendage domains, which bind accessory factors, we show that the primary role of the FBE domain is to bind Arf1 for recruitment of exomer to membranes. PMID:23000721

  7. PLEKHM1 regulates autophagosome-lysosome fusion through HOPS complex and LC3/GABARAP proteins.

    PubMed

    McEwan, David G; Popovic, Doris; Gubas, Andrea; Terawaki, Seigo; Suzuki, Hironori; Stadel, Daniela; Coxon, Fraser P; Miranda de Stegmann, Diana; Bhogaraju, Sagar; Maddi, Karthik; Kirchof, Anja; Gatti, Evelina; Helfrich, Miep H; Wakatsuki, Soichi; Behrends, Christian; Pierre, Philippe; Dikic, Ivan

    2015-01-01

    The lysosome is the final destination for degradation of endocytic cargo, plasma membrane constituents, and intracellular components sequestered by macroautophagy. Fusion of endosomes and autophagosomes with the lysosome depends on the GTPase Rab7 and the homotypic fusion and protein sorting (HOPS) complex, but adaptor proteins that link endocytic and autophagy pathways with lysosomes are poorly characterized. Herein, we show that Pleckstrin homology domain containing protein family member 1 (PLEKHM1) directly interacts with HOPS complex and contains a LC3-interacting region (LIR) that mediates its binding to autophagosomal membranes. Depletion of PLEKHM1 blocks lysosomal degradation of endocytic (EGFR) cargo and enhances presentation of MHC class I molecules. Moreover, genetic loss of PLEKHM1 impedes autophagy flux upon mTOR inhibition and PLEKHM1 regulates clearance of protein aggregates in an autophagy- and LIR-dependent manner. PLEKHM1 is thus a multivalent endocytic adaptor involved in the lysosome fusion events controlling selective and nonselective autophagy pathways. PMID:25498145

  8. Control of protein function through regulated protein degradation: biotechnological and biomedical applications.

    PubMed

    Nagpal, Jyotsna; Tan, Ju Lin; Truscott, Kaye N; Heras, Begoña; Dougan, David A

    2013-01-01

    Targeted protein degradation is crucial for the correct function and maintenance of a cell. In bacteria, this process is largely performed by a handful of ATP-dependent machines, which generally consist of two components - an unfoldase and a peptidase. In some cases, however, substrate recognition by the protease may be regulated by specialized delivery factors (known as adaptor proteins). Our detailed understanding of how these machines are regulated to prevent uncontrolled degradation within a cell has permitted the identification of novel antimicrobials that dysregulate these machines, as well as the development of tunable degradation systems that have applications in biotechnology. Here, we focus on the physiological role of the ClpP peptidase in bacteria, its role as a novel antibiotic target and the use of protein degradation as a biotechnological approach to artificially control the expression levels of a protein of interest. PMID:23920496

  9. Syp1 is a conserved endocytic adaptor that contains domains involved in cargo selection and membrane tubulation

    SciTech Connect

    Reider, Amanda; Barker, Sarah L.; Mishra, Sanjay K.; Im, Young Jun; Maldonado-Báez, Lymarie; Hurley, James H.; Traub, Linton M.; Wendland, Beverly

    2010-10-28

    Internalization of diverse transmembrane cargos from the plasma membrane requires a similarly diverse array of specialized adaptors, yet only a few adaptors have been characterized. We report the identification of the muniscin family of endocytic adaptors that is conserved from yeast to human beings. Solving the structures of yeast muniscin domains confirmed the unique combination of an N-terminal domain homologous to the crescent-shaped membrane-tubulating EFC/F-BAR domains and a C-terminal domain homologous to cargo-binding {mu} homology domains ({mu}HDs). In vitro and in vivo assays confirmed membrane-tubulation activity for muniscin EFC/F-BAR domains. The {mu}HD domain has conserved interactions with the endocytic adaptor/scaffold Ede1/eps15, which influences muniscin localization. The transmembrane protein Mid2, earlier implicated in polarized Rho1 signalling, was identified as a cargo of the yeast adaptor protein. These and other data suggest a model in which the muniscins provide a combined adaptor/membrane-tubulation activity that is important for regulating endocytosis.

  10. Role of Scaffolding Proteins in the Regulation of TRPC-Dependent Calcium Entry.

    PubMed

    Constantin, Bruno

    2016-01-01

    Plasma membrane ion channels, and in particular TRPC channels need a specific membrane environment and association with scaffolding, signaling, and cytoskeleton proteins in order to play their important functional role. The molecular composition of TRPC channels is an important factor in determining channel activation mechanisms. TRPC proteins are incorporated in macromolecular complexes including several key Ca(2 +) signaling proteins as well as proteins involved in vesicle trafficking, cytoskeletal interactions, and scaffolding. Evidence has been provided for association of TRPC with calmodulin (CaM), IP3R, PMCA, Gq/11, RhoA, and a variety of scaffolding proteins. The interaction between TRPC channels with adaptor proteins, determines their mode of regulation as well as their cellular localization and function. Adaptor proteins do not display any enzymatic activity but act as scaffold for the building of signaling complexes. The scaffolding proteins are involved in the assembling of these Ca(2+) signaling complexes, the correct sub-cellular localization of protein partners, and the regulation of the TRPC channelosome. In particular, these proteins, via their multiple protein-protein interaction motifs, can interact with various ion channels involved in the transmembrane potential, and membrane excitability. Scaffolding proteins are key components for the functional organization of TRPC channelosomes that serves as a platform regulating slow Ca(2+) entry, spatially and temporally controlled [Ca(2+)]i signals and Ca(2+) -dependent cellular functions. PMID:27161237

  11. Protein Regulation in Signal Transduction.

    PubMed

    Lee, Michael J; Yaffe, Michael B

    2016-01-01

    SUMMARYCells must respond to a diverse, complex, and ever-changing mix of signals, using a fairly limited set of parts. Changes in protein level, protein localization, protein activity, and protein-protein interactions are critical aspects of signal transduction, allowing cells to respond highly specifically to a nearly limitless set of cues and also to vary the sensitivity, duration, and dynamics of the response. Signal-dependent changes in levels of gene expression and protein synthesis play an important role in regulation of protein levels, whereas posttranslational modifications of proteins regulate their degradation, localization, and functional interactions. Protein ubiquitylation, for example, can direct proteins to the proteasome for degradation or provide a signal that regulates their interactions and/or location within the cell. Similarly, protein phosphorylation by specific kinases is a key mechanism for augmenting protein activity and relaying signals to other proteins that possess domains that recognize the phosphorylated residues. PMID:27252361

  12. Human kidney anion exchanger 1 interacts with adaptor-related protein complex 1 {mu}1A (AP-1 mu1A)

    SciTech Connect

    Sawasdee, Nunghathai; Junking, Mutita; Ngaojanlar, Piengpaga; Sukomon, Nattakan; Ungsupravate, Duangporn; Limjindaporn, Thawornchai; Akkarapatumwong, Varaporn; Noisakran, Sansanee; Yenchitsomanus, Pa-thai

    2010-10-08

    Research highlights: {yields} Trafficking defect of kAE1 is a cause of dRTA but trafficking pathway of kAE1 has not been clearly described. {yields} Adaptor-related protein complex 1 {mu}1A (AP-1 mu1A) was firstly reported to interact with kAE1. {yields} The interacting site for AP-1 mu1A on Ct-kAE1 was found to be Y904DEV907, a subset of YXXO motif. {yields} AP-1 mu1A knockdown showed a marked reduction of kAE1 on the cell membrane and its accumulation in endoplasmic reticulum. {yields} AP-1 mu1A has a critical role in kAE1 trafficking to the plasma membrane. -- Abstract: Kidney anion exchanger 1 (kAE1) mediates chloride (Cl{sup -}) and bicarbonate (HCO{sub 3}{sup -}) exchange at the basolateral membrane of kidney {alpha}-intercalated cells. Impaired trafficking of kAE1 leads to defect of the Cl{sup -}/HCO{sub 3}{sup -} exchange at the basolateral membrane and failure of proton (H{sup +}) secretion at the apical membrane, causing a kidney disease - distal renal tubular acidosis (dRTA). To gain a better insight into kAE1 trafficking, we searched for proteins physically interacting with the C-terminal region of kAE1 (Ct-kAE1), which contains motifs crucial for intracellular trafficking, by a yeast two-hybrid (Y2H) system. An adaptor-related protein complex 1 {mu}1A (AP-1 mu1A) subunit was found to interact with Ct-kAE1. The interaction between either Ct-kAE1 or full-length kAE1 and AP-1 mu1A were confirmed in human embryonic kidney (HEK) 293T by co-immunoprecipitation, affinity co-purification, co-localization, yellow fluorescent protein (YFP)-based protein fragment complementation assay (PCA) and GST pull-down assay. The interacting site for AP-1 mu1A on Ct-kAE1 was found to be Y904DEV907, a subset of YXXO motif. Interestingly, suppression of endogenous AP-1 mu1A in HEK 293T by small interfering RNA (siRNA) decreased membrane localization of kAE1 and increased its intracellular accumulation, suggesting for the first time that AP-1 mu1A is involved in the kAE1

  13. The Ubiquitin-like Protein PLIC-2 Is a Negative Regulator of G Protein-coupled Receptor Endocytosis

    PubMed Central

    N'Diaye, Elsa-Noah; Hanyaloglu, Aylin C.; Kajihara, Kimberly K.; Puthenveedu, Manojkumar A.; Wu, Ping; von Zastrow, Mark

    2008-01-01

    The activity of many signaling receptors is regulated by their endocytosis via clathrin-coated pits (CCPs). For G protein-coupled receptors (GPCRs), recruitment of the adaptor protein arrestin to activated receptors is thought to be sufficient to drive GPCR clustering in CCPs and subsequent endocytosis. We have identified an unprecedented role for the ubiquitin-like protein PLIC-2 as a negative regulator of GPCR endocytosis. Protein Linking IAP to Cytoskeleton (PLIC)-2 overexpression delayed ligand-induced endocytosis of two GPCRs: the V2 vasopressin receptor and β-2 adrenergic receptor, without affecting endocytosis of the transferrin or epidermal growth factor receptor. The closely related isoform PLIC-1 did not affect receptor endocytosis. PLIC-2 specifically inhibited GPCR concentration in CCPs, without affecting membrane recruitment of arrestin-3 to activated receptors or its cellular levels. Depletion of cellular PLIC-2 accelerated GPCR endocytosis, confirming its regulatory function at endogenous levels. The ubiquitin-like domain of PLIC-2, a ligand for ubiquitin-interacting motifs (UIMs), was required for endocytic inhibition. Interestingly, the UIM-containing endocytic adaptors epidermal growth factor receptor protein substrate 15 and Epsin exhibited preferential binding to PLIC-2 over PLIC-1. This differential interaction may underlie PLIC-2 specific effect on GPCR endocytosis. Identification of a negative regulator of GPCR clustering reveals a new function of ubiquitin-like proteins and highlights a cellular requirement for exquisite regulation of receptor dynamics. PMID:18199683

  14. The Src homology and collagen A (ShcA) adaptor protein is required for the spatial organization of the costamere/Z-disk network during heart development.

    PubMed

    Mlih, Mohamed; Host, Lionel; Martin, Sophie; Niederhoffer, Nathalie; Monassier, Laurent; Terrand, Jérôme; Messaddeq, Nadia; Radke, Michael; Gotthardt, Michael; Bruban, Véronique; Kober, Frank; Bernard, Monique; Canet-Soulas, Emmanuelle; Abt-Jijon, Francisco; Boucher, Philippe; Matz, Rachel L

    2015-01-23

    Src homology and collagen A (ShcA) is an adaptor protein that binds to tyrosine kinase receptors. Its germ line deletion is embryonic lethal with abnormal cardiovascular system formation, and its role in cardiovascular development is unknown. To investigate its functional role in cardiovascular development in mice, ShcA was deleted in cardiomyocytes and vascular smooth muscle cells by crossing ShcA flox mice with SM22a-Cre transgenic mice. Conditional mutant mice developed signs of severe dilated cardiomyopathy, myocardial infarctions, and premature death. No evidence of a vascular contribution to the phenotype was observed. Histological analysis of the heart revealed aberrant sarcomeric Z-disk and M-band structures, and misalignments of T-tubules with Z-disks. We find that not only the ErbB3/Neuregulin signaling pathway but also the baroreceptor reflex response, which have been functionally associated, are altered in the mutant mice. We further demonstrate that ShcA interacts with Caveolin-1 and the costameric protein plasma membrane Ca(2+)/calmodulin-dependent ATPase (PMCA), and that its deletion leads to abnormal dystrophin signaling. Collectively, these results demonstrate that ShcA interacts with crucial proteins and pathways that link Z-disk and costamere. PMID:25488665

  15. The Src Homology and Collagen A (ShcA) Adaptor Protein Is Required for the Spatial Organization of the Costamere/Z-disk Network during Heart Development*

    PubMed Central

    Mlih, Mohamed; Host, Lionel; Martin, Sophie; Niederhoffer, Nathalie; Monassier, Laurent; Terrand, Jérôme; Messaddeq, Nadia; Radke, Michael; Gotthardt, Michael; Bruban, Véronique; Kober, Frank; Bernard, Monique; Canet-Soulas, Emmanuelle; Abt-Jijon, Francisco; Boucher, Philippe; Matz, Rachel L.

    2015-01-01

    Src homology and collagen A (ShcA) is an adaptor protein that binds to tyrosine kinase receptors. Its germ line deletion is embryonic lethal with abnormal cardiovascular system formation, and its role in cardiovascular development is unknown. To investigate its functional role in cardiovascular development in mice, ShcA was deleted in cardiomyocytes and vascular smooth muscle cells by crossing ShcA flox mice with SM22a-Cre transgenic mice. Conditional mutant mice developed signs of severe dilated cardiomyopathy, myocardial infarctions, and premature death. No evidence of a vascular contribution to the phenotype was observed. Histological analysis of the heart revealed aberrant sarcomeric Z-disk and M-band structures, and misalignments of T-tubules with Z-disks. We find that not only the ErbB3/Neuregulin signaling pathway but also the baroreceptor reflex response, which have been functionally associated, are altered in the mutant mice. We further demonstrate that ShcA interacts with Caveolin-1 and the costameric protein plasma membrane Ca2+/calmodulin-dependent ATPase (PMCA), and that its deletion leads to abnormal dystrophin signaling. Collectively, these results demonstrate that ShcA interacts with crucial proteins and pathways that link Z-disk and costamere. PMID:25488665

  16. Phosphorylation of Adaptor Protein Containing Pleckstrin Homology Domain, Phosphotyrosine Binding Domain, and Leucine Zipper Motif 1 (APPL1) at Ser430 Mediates Endoplasmic Reticulum (ER) Stress-induced Insulin Resistance in Hepatocytes*

    PubMed Central

    Liu, Meilian; Zhou, Lijun; Wei, Li; Villarreal, Ricardo; Yang, Xin; Hu, Derong; Riojas, Ramon A.; Holmes, Bekke M.; Langlais, Paul R.; Lee, Hakjoo; Dong, Lily Q.

    2012-01-01

    APPL1 is an adaptor protein that plays a critical role in regulating adiponectin and insulin signaling. However, how APPL1 is regulated under normal and pathological conditions remains largely unknown. In this study, we show that APPL1 undergoes phosphorylation at Ser430 and that this phosphorylation is enhanced in the liver of obese mice displaying insulin resistance. In cultured mouse hepatocytes, APPL1 phosphorylation at Ser430 is stimulated by phorbol 12-myristate 13-acetate, an activator of classic PKC isoforms, and by the endoplasmic reticulum (ER) stress inducer, thapsigargin. Overexpression of wild-type but not dominant negative PKCα increases APPL1 phosphorylation at Ser430 in mouse hepatocytes. In addition, suppressing PKCα expression by shRNA in hepatocytes reduces ER stress-induced APPL1 phosphorylation at Ser430 as well as the inhibitory effect of ER stress on insulin-stimulated Akt phosphorylation. Consistent with a negative regulatory role of APPL1 phosphorylation at Ser430 in insulin signaling, overexpression of APPL1S430D but not APPL1S430A impairs the potentiating effect of APPL1 on insulin-stimulated Akt phosphorylation at Thr308. Taken together, our results identify APPL1 as a novel target in ER stress-induced insulin resistance and PKCα as the kinase mediating ER stress-induced phosphorylation of APPL1 at Ser430. PMID:22685300

  17. Role of an adaptor protein Lin-7B in brain development: possible involvement in autism spectrum disorders.

    PubMed

    Mizuno, Makoto; Matsumoto, Ayumi; Hamada, Nanako; Ito, Hidenori; Miyauchi, Akihiko; Jimbo, Eriko F; Momoi, Mariko Y; Tabata, Hidenori; Yamagata, Takanori; Nagata, Koh-Ichi

    2015-01-01

    Using comparative genomic hybridization analysis for an autism spectrum disorder (ASD) patient, a 73-Kb duplication at 19q13.33 (nt. 49 562 755-49 635 956) including LIN7B and 5 other genes was detected. We then identified a novel frameshift mutation in LIN7B in another ASD patient. Since LIN7B encodes a scaffold protein essential for neuronal function, we analyzed the role of Lin-7B in the development of cerebral cortex. Acute knockdown of Lin-7B with in utero electroporation caused a delay in neuronal migration during corticogenesis. When Lin-7B was knocked down in cortical neurons in one hemisphere, their axons failed to extend efficiently into the contralateral hemisphere after leaving the corpus callosum. Meanwhile, enhanced expression of Lin-7B had no effects on both cortical neuron migration and axon growth. Notably, silencing of Lin-7B did not affect the proliferation of neuronal progenitors and stem cells. Taken together, Lin-7B was found to play a pivotal role in corticogenesis through the regulation of excitatory neuron migration and interhemispheric axon growth, while further analyses are required to directly link functional defects of Lin-7B to ASD pathophysiology. Lin-7 plays a pivotal role as a scaffold protein in synaptic development and plasticity. Based on genetic analyses we identified mutations in LIN-7B gene in some ASD (autism-spectrum disorder) patients. Functional defects in Lin-7B caused abnormal neuronal migration and interhemispheric axon growth during mouse brain development. Thus, functional deficiency in Lin-7B could be implicated in clinical phenotypes in some ASD patients through bringing about abnormal cortical architecture. PMID:25196215

  18. The psoriasis-associated D10N variant of the adaptor Act1 with impaired regulation by the molecular chaperone hsp90.

    PubMed

    Wang, Chenhui; Wu, Ling; Bulek, Katarzyna; Martin, Bradley N; Zepp, Jarod A; Kang, Zizhen; Liu, Caini; Herjan, Tomasz; Misra, Saurav; Carman, Julie A; Gao, Ji; Dongre, Ashok; Han, Shujie; Bunting, Kevin D; Ko, Jennifer S; Xiao, Hui; Kuchroo, Vijay K; Ouyang, Wenjun; Li, Xiaoxia

    2013-01-01

    Act1 is an essential adaptor in interleukin 17 (IL-17)-mediated signaling and is recruited to the receptor for IL-17 after stimulation with IL-17. Here we found that Act1 was a 'client' protein of the molecular chaperone hsp90. The D10N variant of Act1 (Act1(D10N)) that is linked to susceptibility to psoriasis was defective in its interaction with hsp90, which resulted in a global loss of Act1 function. Act1-deficient mice modeled the mechanistic link between loss of Act1 function and susceptibility to psoriasis. Although Act1 was necessary for IL-17-mediated inflammation, Act1-deficient mice had a hyperactive response of the T(H)17 subset of helper T cells and developed spontaneous IL-22-dependent skin inflammation. In the absence of IL-17 signaling, IL-22 was the main contributor to skin inflammation, which provides a molecular mechanism for the association of Act1(D10N) with psoriasis susceptibility. PMID:23202271

  19. Biophysical analysis of binding of WW domains of the YAP2 transcriptional regulator to PPXY motifs within WBP1 and WBP2 adaptors.

    PubMed

    McDonald, Caleb B; McIntosh, Samantha K N; Mikles, David C; Bhat, Vikas; Deegan, Brian J; Seldeen, Kenneth L; Saeed, Ali M; Buffa, Laura; Sudol, Marius; Nawaz, Zafar; Farooq, Amjad

    2011-11-01

    The YAP2 transcriptional regulator mediates a plethora of cellular functions, including the newly discovered Hippo tumor suppressor pathway, by virtue of its ability to recognize WBP1 and WBP2 signaling adaptors among a wide variety of other ligands. Herein, using isothermal titration calorimery and circular dichroism in combination with molecular modeling and molecular dynamics, we provide evidence that the WW1 and WW2 domains of YAP2 recognize various PPXY motifs within WBP1 and WBP2 in a highly promiscuous and subtle manner. Thus, although both WW domains strictly require the integrity of the consensus PPXY sequence, nonconsensus residues within and flanking this motif are not critical for high-affinity binding, implying that they most likely play a role in stabilizing the polyproline type II helical conformation of the PPXY ligands. Of particular interest is the observation that both WW domains bind to a PPXYXG motif with highest affinity, implicating a preference for a nonbulky and flexible glycine one residue to the C-terminal side of the consensus tyrosine. Importantly, a large set of residues within both WW domains and the PPXY motifs appear to undergo rapid fluctuations on a nanosecond time scale, suggesting that WW-ligand interactions are highly dynamic and that such conformational entropy may be an integral part of the reversible and temporal nature of cellular signaling cascades. Collectively, our study sheds light on the molecular determinants of a key WW-ligand interaction pertinent to cellular functions in health and disease. PMID:21981024

  20. Role of adaptor proteins and clathrin in the trafficking of human kidney anion exchanger 1 (kAE1) to the cell surface.

    PubMed

    Junking, Mutita; Sawasdee, Nunghathai; Duangtum, Natapol; Cheunsuchon, Boonyarit; Limjindaporn, Thawornchai; Yenchitsomanus, Pa-thai

    2014-07-01

    Kidney anion exchanger 1 (kAE1) plays an important role in acid-base homeostasis by mediating chloride/bicarbornate (Cl-/HCO3-) exchange at the basolateral membrane of α-intercalated cells in the distal nephron. Impaired intracellular trafficking of kAE1 caused by mutations of SLC4A1 encoding kAE1 results in kidney disease - distal renal tubular acidosis (dRTA). However, it is not known how the intracellular sorting and trafficking of kAE1 from trans-Golgi network (TGN) to the basolateral membrane occurs. Here, we studied the role of basolateral-related sorting proteins, including the mu1 subunit of adaptor protein (AP) complexes, clathrin and protein kinase D, on kAE1 trafficking in polarized and non-polarized kidney cells. By using RNA interference, co-immunoprecipitation, yellow fluorescent protein-based protein fragment complementation assays and immunofluorescence staining, we demonstrated that AP-1 mu1A, AP-3 mu1, AP-4 mu1 and clathrin (but not AP-1 mu1B, PKD1 or PKD2) play crucial roles in intracellular sorting and trafficking of kAE1. We also demonstrated colocalization of kAE1 and basolateral-related sorting proteins in human kidney tissues by double immunofluorescence staining. These findings indicate that AP-1 mu1A, AP-3 mu1, AP-4 mu1 and clathrin are required for kAE1 sorting and trafficking from TGN to the basolateral membrane of acid-secreting α-intercalated cells. PMID:24698155

  1. CRK proteins selectively regulate T cell migration into inflamed tissues

    PubMed Central

    Huang, Yanping; Clarke, Fiona; Karimi, Mobin; Roy, Nathan H.; Williamson, Edward K.; Okumura, Mariko; Mochizuki, Kazuhiro; Chen, Emily J.H.; Park, Tae-Ju; Debes, Gudrun F.; Zhang, Yi; Curran, Tom; Kambayashi, Taku; Burkhardt, Janis K.

    2015-01-01

    Effector T cell migration into inflamed sites greatly exacerbates tissue destruction and disease severity in inflammatory diseases, including graft-versus-host disease (GVHD). T cell migration into such sites depends heavily on regulated adhesion and migration, but the signaling pathways that coordinate these functions downstream of chemokine receptors are largely unknown. Using conditional knockout mice, we found that T cells lacking the adaptor proteins CRK and CRK-like (CRKL) exhibit reduced integrin-dependent adhesion, chemotaxis, and diapedesis. Moreover, these two closely related proteins exhibited substantial functional redundancy, as ectopic expression of either protein rescued defects in T cells lacking both CRK and CRKL. We determined that CRK proteins coordinate with the RAP guanine nucleotide exchange factor C3G and the adhesion docking molecule CASL to activate the integrin regulatory GTPase RAP1. CRK proteins were required for effector T cell trafficking into sites of inflammation, but not for migration to lymphoid organs. In a murine bone marrow transplantation model, the differential migration of CRK/CRKL-deficient T cells resulted in efficient graft-versus-leukemia responses with minimal GVHD. Together, the results from our studies show that CRK family proteins selectively regulate T cell adhesion and migration at effector sites and suggest that these proteins have potential as therapeutic targets for preventing GVHD. PMID:25621495

  2. Adaptor Protein Cerebral Cavernous Malformation 3 (CCM3) Mediates Phosphorylation of the Cytoskeletal Proteins Ezrin/Radixin/Moesin by Mammalian Ste20-4 to Protect Cells from Oxidative Stress*

    PubMed Central

    Fidalgo, Miguel; Guerrero, Ana; Fraile, María; Iglesias, Cristina; Pombo, Celia M.; Zalvide, Juan

    2012-01-01

    While studying the functions of CCM3/PDCD10, a gene encoding an adaptor protein whose mutation results in vascular malformations, we have found that it is involved in a novel response to oxidative stress that results in phosphorylation and activation of the ezrin/radixin/moesin (ERM) family of proteins. This phosphorylation protects cells from accidental cell death induced by oxidative stress. We also present evidence that ERM phosphorylation is performed by the GCKIII kinase Mst4, which is activated and relocated to the cell periphery after oxidative stress. The cellular levels of Mst4 and its activation after oxidative stress depend on the presence of CCM3, as absence of the latter impairs the phosphorylation of ERM proteins and enhances death of cells exposed to reactive oxygen species. These findings shed new light on the response of cells to oxidative stress and identify an important pathophysiological situation in which ERM proteins and their phosphorylation play a significant role. PMID:22291017

  3. Shugoshins: Tension-Sensitive Pericentromeric Adaptors Safeguarding Chromosome Segregation

    PubMed Central

    2014-01-01

    The shugoshin/Mei-S332 family are proteins that associate with the chromosomal region surrounding the centromere (the pericentromere) and that play multiple and distinct roles in ensuring the accuracy of chromosome segregation during both mitosis and meiosis. The underlying role of shugoshins appears to be to serve as pericentromeric adaptor proteins that recruit several different effectors to this region of the chromosome to regulate processes critical for chromosome segregation. Crucially, shugoshins undergo changes in their localization in response to the tension that is exerted on sister chromosomes by the forces of the spindle that will pull them apart. This has led to the idea that shugoshins provide a platform for activities required at the pericentromere only when sister chromosomes lack tension. Conversely, disassembly of the shugoshin pericentromeric platform may provide a signal that sister chromosomes are under tension. Here the functions and regulation of these important tension-sensitive pericentromeric proteins are discussed. PMID:25452306

  4. Overexpression of Isoforms of Nitric Oxide Synthase 1 Adaptor Protein, Encoded by a Risk Gene for Schizophrenia, Alters Actin Dynamics and Synaptic Function

    PubMed Central

    Hernandez, Kristina; Swiatkowski, Przemyslaw; Patel, Mihir V.; Liang, Chen; Dudzinski, Natasha R.; Brzustowicz, Linda M.; Firestein, Bonnie L.

    2016-01-01

    Proper communication between neurons depends upon appropriate patterning of dendrites and correct distribution and structure of spines. Schizophrenia is a neuropsychiatric disorder characterized by alterations in dendrite branching and spine density. Nitric oxide synthase 1 adaptor protein (NOS1AP), a risk gene for schizophrenia, encodes proteins that are upregulated in the dorsolateral prefrontal cortex (DLPFC) of individuals with schizophrenia. To elucidate the effects of NOS1AP overexpression observed in individuals with schizophrenia, we investigated changes in actin dynamics and spine development when a long (NOS1AP-L) or short (NOS1AP-S) isoform of NOS1AP is overexpressed. Increased NOS1AP-L protein promotes the formation of immature spines when overexpressed in rat cortical neurons from day in vitro (DIV) 14 to DIV 17 and reduces the amplitude of miniature excitatory postsynaptic currents (mEPSCs). In contrast, increased NOS1AP-S protein increases the rate of actin polymerization and the number of immature and mature spines, which may be attributed to a decrease in total Rac1 expression and a reduction in the levels of active cofilin. The increase in the number of mature spines by overexpression of NOS1AP-S is accompanied by an increase in the frequency of mEPSCs. Our findings show that overexpression of NOS1AP-L or NOS1AP-S alters the actin cytoskeleton and synaptic function. However, the mechanisms by which these isoforms induce these changes are distinct. These results are important for understanding how increased expression of NOS1AP isoforms can influence spine development and synaptic function. PMID:26869880

  5. Crystal structure of Src-like adaptor protein 2 reveals close association of SH3 and SH2 domains through β-sheet formation.

    PubMed

    Wybenga-Groot, Leanne E; McGlade, C Jane

    2013-12-01

    The Src-like adaptor proteins (SLAP/SLAP2) are key components of Cbl-dependent downregulation of antigen receptor, cytokine receptor, and receptor tyrosine kinase signaling in hematopoietic cells. SLAP and SLAP2 consist of adjacent SH3 and SH2 domains that are most similar in sequence to Src family kinases (SFKs). Notably, the SH3-SH2 connector sequence is significantly shorter in SLAP/SLAP2 than in SFKs. To understand the structural implication of a short SH3-SH2 connector sequence, we solved the crystal structure of a protein encompassing the SH3 domain, SH3-SH2 connector, and SH2 domain of SLAP2 (SLAP2-32). While both domains adopt typical folds, the short SH3-SH2 connector places them in close association. Strand βe of the SH3 domain interacts with strand βA of the SH2 domain, resulting in the formation of a continuous β sheet that spans the length of the protein. Disruption of the SH3/SH2 interface through mutagenesis decreases SLAP-32 stability in vitro, consistent with inter-domain binding being an important component of SLAP2 structure and function. The canonical peptide binding pockets of the SH3 and SH2 domains are fully accessible, in contrast to other protein structures that display direct interaction between SH3 and SH2 domains, in which either peptide binding surface is obstructed by the interaction. Our results reveal potential sites of novel interaction for SH3 and SH2 domains, and illustrate the adaptability of SH2 and SH3 domains in mediating interactions. As well, our results suggest that the SH3 and SH2 domains of SLAP2 function interdependently, with implications on their mode of substrate binding. PMID:24018043

  6. The Shc-related adaptor protein, Sck, forms a complex with the vascular-endothelial-growth-factor receptor KDR in transfected cells.

    PubMed Central

    Warner, A J; Lopez-Dee, J; Knight, E L; Feramisco, J R; Prigent, S A

    2000-01-01

    Despite much progress in recent years, the precise signalling events triggered by the vascular-endothelial-growth-factor (VEGF) receptors, fms-like tyrosine kinase (Flt1) and kinase insert domain-containing receptor (KDR), are incompletely defined. Results obtained when Flt1 and KDR are individually expressed in fibroblasts or porcine aortic endothelial cells have not been entirely consistent with those observed in other endothelial cells expressing both receptors endogenously. It has also been difficult to demonstrate VEGF-induced phosphorylation of Flt1, which has led to speculation that KDR may be the more important receptor for the mitogenic action of VEGF on endothelial cells. In an attempt to identify physiologically important effectors which bind to KDR, we have screened a yeast two-hybrid mouse embryo library with the cytoplasmic domain of KDR. Here we describe the identification of the adaptor protein, Shc-like protein (Sck), as a binding partner for KDR. We demonstrate that this interaction requires phosphorylation of KDR, and identify the binding site for the Src-homology 2 (SH2) domain as tyrosine-1175 of KDR. We have also shown that the SH2 domain of Sck, but not that of Src-homology collagen protein (Shc), can precipitate phosphorylated KDR from VEGF-stimulated porcine aortic endothelial cells expressing KDR, and that an N-terminally truncated Sck protein can associate with KDR, in a phosphorylation-dependent fashion, when co-expressed in human embryonic kidney 293 cells. Furthermore, we demonstrate that in the two-hybrid assay, both Shc and Sck SH2 domains can associate with the related receptor Flt1. PMID:10749680

  7. Overexpression of Isoforms of Nitric Oxide Synthase 1 Adaptor Protein, Encoded by a Risk Gene for Schizophrenia, Alters Actin Dynamics and Synaptic Function.

    PubMed

    Hernandez, Kristina; Swiatkowski, Przemyslaw; Patel, Mihir V; Liang, Chen; Dudzinski, Natasha R; Brzustowicz, Linda M; Firestein, Bonnie L

    2016-01-01

    Proper communication between neurons depends upon appropriate patterning of dendrites and correct distribution and structure of spines. Schizophrenia is a neuropsychiatric disorder characterized by alterations in dendrite branching and spine density. Nitric oxide synthase 1 adaptor protein (NOS1AP), a risk gene for schizophrenia, encodes proteins that are upregulated in the dorsolateral prefrontal cortex (DLPFC) of individuals with schizophrenia. To elucidate the effects of NOS1AP overexpression observed in individuals with schizophrenia, we investigated changes in actin dynamics and spine development when a long (NOS1AP-L) or short (NOS1AP-S) isoform of NOS1AP is overexpressed. Increased NOS1AP-L protein promotes the formation of immature spines when overexpressed in rat cortical neurons from day in vitro (DIV) 14 to DIV 17 and reduces the amplitude of miniature excitatory postsynaptic currents (mEPSCs). In contrast, increased NOS1AP-S protein increases the rate of actin polymerization and the number of immature and mature spines, which may be attributed to a decrease in total Rac1 expression and a reduction in the levels of active cofilin. The increase in the number of mature spines by overexpression of NOS1AP-S is accompanied by an increase in the frequency of mEPSCs. Our findings show that overexpression of NOS1AP-L or NOS1AP-S alters the actin cytoskeleton and synaptic function. However, the mechanisms by which these isoforms induce these changes are distinct. These results are important for understanding how increased expression of NOS1AP isoforms can influence spine development and synaptic function. PMID:26869880

  8. Human kidney anion exchanger 1 interacts with adaptor-related protein complex 1 μ1A (AP-1 mu1A).

    PubMed

    Sawasdee, Nunghathai; Junking, Mutita; Ngaojanlar, Piengpaga; Sukomon, Nattakan; Ungsupravate, Duangporn; Limjindaporn, Thawornchai; Akkarapatumwong, Varaporn; Noisakran, Sansanee; Yenchitsomanus, Pa-Thai

    2010-10-01

    Kidney anion exchanger 1 (kAE1) mediates chloride (Cl⁻) and bicarbonate (HCO₃⁻) exchange at the basolateral membrane of kidney α-intercalated cells. Impaired trafficking of kAE1 leads to defect of the Cl⁻/HCO₃⁻ exchange at the basolateral membrane and failure of proton (H+) secretion at the apical membrane, causing a kidney disease--distal renal tubular acidosis (dRTA). To gain a better insight into kAE1 trafficking, we searched for proteins physically interacting with the C-terminal region of kAE1 (Ct-kAE1), which contains motifs crucial for intracellular trafficking, by a yeast two-hybrid (Y2H) system. An adaptor-related protein complex 1 μ1A (AP-1 mu1A) subunit was found to interact with Ct-kAE1. The interaction between either Ct-kAE1 or full-length kAE1 and AP-1 mu1A were confirmed in human embryonic kidney (HEK) 293T by co-immunoprecipitation, affinity co-purification, co-localization, yellow fluorescent protein (YFP)-based protein fragment complementation assay (PCA) and GST pull-down assay. The interacting site for AP-1 mu1A on Ct-kAE1 was found to be Y904DEV907, a subset of YXXØ motif. Interestingly, suppression of endogenous AP-1 mu1A in HEK 293T by small interfering RNA (siRNA) decreased membrane localization of kAE1 and increased its intracellular accumulation, suggesting for the first time that AP-1 mu1A is involved in the kAE1 trafficking of kidney α-intercalated cells. PMID:20833140

  9. Differential Association of the Na+/H+ Exchanger Regulatory Factor (NHERF) Family of Adaptor Proteins with the Raft-and the Non-Raft Brush Border Membrane Fractions of NHE3

    PubMed Central

    Sultan, Ayesha; Luo, Min; Yu, Qin; Riederer, Brigitte; Xia, Weiliang; Chen, Mingmin; Lissner, Simone; Gessner, Johannes E.; Donowitz, Mark; Yun, C. Chris; deJonge, Hugo; Lamprecht, Georg; Seidler, Ursula

    2014-01-01

    Background/Aims Trafficking, brush border membrane (BBM) retention, and signal-specific regulation of the Na+/H+ exchanger NHE3 is regulated by the Na+/H+ Exchanger Regulatory Factor (NHERF) family of PDZ-adaptor proteins, which enable the formation of multiprotein complexes. It is unclear, however, what determines signal specificity of these NHERFs. Thus, we studied the association of NHE3, NHERF1 (EBP50), NHERF2 (E3KARP), and NHERF3 (PDZK1) with lipid rafts in murine small intestinal BBM. Methods Detergent resistant membranes (“lipid rafts”) were isolated by floatation of Triton X-incubated small intestinal BBM from a variety of knockout mouse strains in an Optiprep step gradient. Acid-activated NHE3 activity was measured fluorometrically in BCECF-loaded microdissected villi, or by assessment of CO2/HCO3− mediated increase in fluid absorption in perfused jejunal loops of anethetized mice. Results NHE3 was found to partially associate with lipid rafts in the native BBM, and NHE3 raft association had an impact on NHE3 transport activity and regulation in vivo. NHERF1, 2 and 3 were differentially distributed to rafts and non-rafts, with NHERF2 being most raft-associated and NHERF3 entirely non-raft associated. NHERF2 expression enhanced the localization of NHE3 to membrane rafts. The use of acid sphingomyelinase-deficient mice, which have altered membrane lipid as well as lipid raft composition, allowed us to test the validity of the lipid raft concept in vivo. Conclusions The differential association of the NHERFs with the raft-associated and the non-raft fraction of NHE3 in the brush border membrane is one component of the differential and signal-specific NHE3 regulation by the different NHERFs. PMID:24297041

  10. Loss of Apm1, the μ1 Subunit of the Clathrin-Associated Adaptor-Protein-1 Complex, Causes Distinct Phenotypes and Synthetic Lethality with Calcineurin Deletion in Fission Yeast

    PubMed Central

    Kita, Ayako; Sugiura, Reiko; Shoji, Hiromi; He, Yi; Deng, Lu; Lu, Yabin; Sio, Susie O.; Takegawa, Kaoru; Sakaue, Motoyoshi; Shuntoh, Hisato; Kuno, Takayoshi

    2004-01-01

    Calcineurin is a highly conserved regulator of Ca2+ signaling in eukaryotes. In fission yeast, calcineurin is not essential for viability but is required for cytokinesis and Cl- homeostasis. In a genetic screen for mutations that are synthetically lethal with calcineurin deletion, we isolated a mutant, cis1-1/apm1-1, an allele of the apm1+ gene that encodes a homolog of the mammalian μ1A subunit of the clathrin-associated adaptor protein-1 (AP-1) complex. The cis1-1/apm1-1 mutant as well as the apm1-deleted (Δapm1) cells showed distinct phenotypes: temperature sensitivity; tacrolimus (FK506) sensitivity; and pleiotropic defects in cytokinesis, cell integrity, and vacuole fusion. Electron micrographs revealed that Δapm1 cells showed large vesicular structures associated with Golgi stacks and accumulated post-Golgi secretory vesicles. Δapm1 cells also showed the massive accumulation of the exocytic v-SNARE Syb1 in the Golgi/endosomes and a reduced secretion of acid phosphatase. These phenotypes observed in apm1 mutations were accentuated upon temperature up-shift and FK506 treatment. Notably, Apm1-GFP localized to the Golgi/endosomes, the spindle pole bodies, and the medial region. These findings suggest a role for Apm1 associated with the Golgi/endosome function, thereby affecting various cellular processes, including secretion, cytokinesis, vacuole fusion, and cell integrity and also suggest that calcineurin is involved in these events. PMID:15047861

  11. Disruption of adaptor protein 2μ (AP-2μ) in cochlear hair cells impairs vesicle reloading of synaptic release sites and hearing.

    PubMed

    Jung, SangYong; Maritzen, Tanja; Wichmann, Carolin; Jing, Zhizi; Neef, Andreas; Revelo, Natalia H; Al-Moyed, Hanan; Meese, Sandra; Wojcik, Sonja M; Panou, Iliana; Bulut, Haydar; Schu, Peter; Ficner, Ralf; Reisinger, Ellen; Rizzoli, Silvio O; Neef, Jakob; Strenzke, Nicola; Haucke, Volker; Moser, Tobias

    2015-11-01

    Active zones (AZs) of inner hair cells (IHCs) indefatigably release hundreds of vesicles per second, requiring each release site to reload vesicles at tens per second. Here, we report that the endocytic adaptor protein 2μ (AP-2μ) is required for release site replenishment and hearing. We show that hair cell-specific disruption of AP-2μ slows IHC exocytosis immediately after fusion of the readily releasable pool of vesicles, despite normal abundance of membrane-proximal vesicles and intact endocytic membrane retrieval. Sound-driven postsynaptic spiking was reduced in a use-dependent manner, and the altered interspike interval statistics suggested a slowed reloading of release sites. Sustained strong stimulation led to accumulation of endosome-like vacuoles, fewer clathrin-coated endocytic intermediates, and vesicle depletion of the membrane-distal synaptic ribbon in AP-2μ-deficient IHCs, indicating a further role of AP-2μ in clathrin-dependent vesicle reformation on a timescale of many seconds. Finally, we show that AP-2 sorts its IHC-cargo otoferlin. We propose that binding of AP-2 to otoferlin facilitates replenishment of release sites, for example, via speeding AZ clearance of exocytosed material, in addition to a role of AP-2 in synaptic vesicle reformation. PMID:26446278

  12. Recruitment of the adaptor protein Nck to PECAM-1 couples oxidative stress to canonical NF-κB signaling and inflammation.

    PubMed

    Chen, Jie; Leskov, Igor L; Yurdagul, Arif; Thiel, Bonnie; Kevil, Christopher G; Stokes, Karen Y; Orr, A Wayne

    2015-02-24

    Oxidative stress stimulates nuclear factor κB (NF-κB) activation and NF-κB-dependent proinflammatory gene expression in endothelial cells during several pathological conditions, including ischemia/reperfusion injury. We found that the Nck family of adaptor proteins linked tyrosine kinase signaling to oxidative stress-induced activation of NF-κB through the classic IκB kinase-dependent pathway. Depletion of Nck prevented oxidative stress induced by exogenous hydrogen peroxide or hypoxia/reoxygenation injury from activating NF-κB in endothelial cells, increasing the abundance of the proinflammatory molecules ICAM-1 (intracellular adhesion molecule-1) and VCAM-1 (vascular cell adhesion molecule-1) and recruiting leukocytes. Nck depletion also attenuated endothelial cell expression of genes encoding proinflammatory factors but not those encoding antioxidants. Nck promoted oxidative stress-induced activation of NF-κB by coupling the tyrosine phosphorylation of PECAM-1 (platelet endothelial cell adhesion molecule-1) to the activation of p21-activated kinase, which mediates oxidative stress-induced NF-κB signaling. Consistent with this mechanism, treatment of mice subjected to ischemia/reperfusion injury in the cremaster muscle with a Nck inhibitory peptide blocked leukocyte adhesion and emigration and the accompanying vascular leak. Together, these data identify Nck as an important mediator of oxidative stress-induced inflammation and a potential therapeutic target for ischemia/reperfusion injury. PMID:25714462

  13. Adjuvanticity of the oil-in-water emulsion MF59 is independent of Nlrp3 inflammasome but requires the adaptor protein MyD88

    PubMed Central

    Seubert, Anja; Calabro, Samuele; Santini, Laura; Galli, Barbara; Genovese, Alessia; Valentini, Sara; Aprea, Susanna; Colaprico, Annalisa; D'Oro, Ugo; Giuliani, Marzia M.; Pallaoro, Michele; Pizza, Mariagrazia; O'Hagan, Derek T.; Wack, Andreas; Rappuoli, Rino; De Gregorio, Ennio

    2011-01-01

    Oil-in-water emulsions have been successfully used to increase the efficacy, immunogenicity, and cross-protection of human vaccines; however, their mechanism of action is still largely unknown. Nlrp3 inflammasome has been previously associated to the activity of alum, another adjuvant broadly used in human vaccines, and MyD88 adaptor protein is required for the adjuvanticity of most Toll-like receptor agonists. We compared the contribution of Nlrp3 and MyD88 to the adjuvanticity of alum, the oil-in-water emulsion MF59, and complete Freund's adjuvant in mice using a three-component vaccine against serogroup B Neisseria meningitidis (rMenB). Although the basal antibody responses to the nonadjuvanted rMenB vaccine were largely dependent on Nlrp3, the high-level antibody responses induced by alum, MF59, or complete Freund's adjuvant did not require Nlrp3. Surprisingly, we found that MF59 requires MyD88 to enhance bactericidal antibody responses to the rMenB vaccine. Because MF59 did not activate any of the Toll-like receptors in vitro, we propose that MF59 requires MyD88 for a Toll-like receptor-independent signaling pathway. PMID:21690334

  14. Recruitment of the adaptor protein Nck to PECAM-1 couples oxidant stress to canonical NF-κB signaling and inflammation

    PubMed Central

    Chen, Jie; Leskov, Igor L.; Yurdagul, Arif; Thiel, Bonnie; Kevil, Christopher G.; Stokes, Karen Y.; Orr, A. Wayne

    2015-01-01

    Oxidant stress drives nuclear factor κB (NF-κB) activation and NF-κB-dependent proinflammatory gene expression in endothelial cells during several pathological conditions, including ischemia/reperfusion injury. We showed that the Nck family of adaptor proteins linked tyrosine kinase signaling to oxidant stress-induced activation of NF-κB through the classic IκB kinase (IKK)-dependent pathway. Depletion of Nck prevented oxidant stress induced by exogenous peroxide or hypoxia/reoxygenation injury from triggering the activation of NF-κB in endothelial cells, increases in the abundance of the pro-inflammatory molecules ICAM-1 (intracellular adhesion molecule 1) and VCAM-1 (vascular cell adhesion molecule 1), and leukocyte recruitment. Nck depletion also attenuated endothelial cell expression of genes encoding proinflammatory factors, but not those encoding antioxidants. We further showed that Nck promoted oxidant stress-induced activation of NF-κB by coupling the tyrosine phosphorylation of platelet-endothelial cell adhesion molecule-1 (PECAM-1) to the activation of p21 activated kinase, which mediates oxidant stress-induced NF-κB signaling. Consistent with this model, treatment of mice subjected to ischemia/reperfusion injury in the cremaster muscle with a Nck inhibitory peptide inhibited leukocyte adhesion and emigration and the accompanying vascular leak. Together, these data identify Nck as an important mediator of oxidant stress-induced inflammation and a potential therapeutic target for ischemia/reperfusion injury. PMID:25714462

  15. Biophysical Analysis of the Binding of WW Domains of YAP2 Transcriptional Regulator to PPXY Motifs within WBP1 and WBP2 Adaptors

    PubMed Central

    McDonald, Caleb B.; McIntosh, Samantha K. N.; Mikles, David C.; Bhat, Vikas; Deegan, Brian J.; Seldeen, Kenneth L.; Saeed, Ali M.; Buffa, Laura; Sudol, Marius; Nawaz, Zafar; Farooq, Amjad

    2011-01-01

    YAP2 transcriptional regulator mediates a plethora of cellular functions, including the newly discovered Hippo tumor suppressor pathway, by virtue of its ability to recognize WBP1 and WBP2 signaling adaptors among a wide variety of other ligands. Herein, using isothermal titration calorimery (ITC) and circular dichroism (CD) in combination with molecular modeling (MM) and molecular dynamics (MD), we provide evidence that the WW1 and WW2 domains of YAP2 recognize various PPXY motifs within WBP1 and WBP2 in a highly promiscuous and subtle manner. Thus, although both WW domains strictly require the integrity of the consensus PPXY sequence, non-consensus residues within and flanking this motif are not critical for high-affinity binding, implying that they most likely play a role in stabilizing the polyproline type II (PPII) helical conformation of the PPXY ligands. Of particular interest is the observation that both WW domains bind to a PPXYXG motif with highest affinity, implicating a preference for a non-bulky and flexible glycine one-residue C-terminal to the consensus tyrosine. Importantly, a large set of residues within both WW domains and the PPXY motifs appear to undergo rapid fluctuations on a nanosecond time scale, arguing that WW-ligand interactions are highly dynamic and that such conformational entropy may be an integral part of the reversible and temporal nature of cellular signaling cascades. Collectively, our study sheds light on the molecular determinants of a key WW-ligand interaction pertinent to cellular functions in health and disease. PMID:21981024

  16. Myosin VI and its cargo adaptors – linking endocytosis and autophagy

    PubMed Central

    Tumbarello, David A.; Kendrick-Jones, John; Buss, Folma

    2013-01-01

    Summary The coordinated trafficking and tethering of membrane cargo within cells relies on the function of distinct cytoskeletal motors that are targeted to specific subcellular compartments through interactions with protein adaptors and phospholipids. The unique actin motor myosin VI functions at distinct steps during clathrin-mediated endocytosis and the early endocytic pathway – both of which are involved in cargo trafficking and sorting – through interactions with Dab2, GIPC, Tom1 and LMTK2. This multifunctional ability of myosin VI can be attributed to its cargo-binding tail region that contains two protein–protein interaction interfaces, a ubiquitin-binding motif and a phospholipid binding domain. In addition, myosin VI has been shown to be a regulator of the autophagy pathway, because of its ability to link the endocytic and autophagic pathways through interactions with the ESCRT-0 protein Tom1 and the autophagy adaptor proteins T6BP, NDP52 and optineurin. This function has been attributed to facilitating autophagosome maturation and subsequent fusion with the lysosome. Therefore, in this Commentary, we discuss the relationship between myosin VI and the different myosin VI adaptor proteins, particularly with regards to the spatial and temporal regulation that is required for the sorting of cargo at the early endosome, and their impact on autophagy. PMID:23781020

  17. The neuropilin-like protein ESDN regulates insulin signaling and sensitivity.

    PubMed

    Li, Xuan; Jung, Jae-Joon; Nie, Lei; Razavian, Mahmoud; Zhang, Jiasheng; Samuel, Varman; Sadeghi, Mehran M

    2016-05-01

    Insulin effects on cell metabolism, growth, and survival are mediated by its binding to, and activation of, insulin receptor. With increasing prevalence of insulin resistance and diabetes there is considerable interest in identifying novel regulators of insulin signal transduction. The transmembrane protein endothelial and smooth muscle cell-derived neuropilin-like protein (ESDN) is a novel regulator of vascular remodeling and angiogenesis. Here, we investigate a potential role of ESDN in insulin signaling, demonstrating that Esdn gene deletion promotes insulin-induced vascular smooth muscle cell proliferation and migration. This is associated with enhanced protein kinase B and mitogen-activated protein kinase activation as well as insulin receptor phosphorylation. Likewise, insulin signaling in the liver, muscle, and adipose tissue is enhanced in Esdn(-/-) mice, and these animals exhibit improved insulin sensitivity and glucose homeostasis in vivo. The effect of ESDN on insulin signaling is traced back to its interaction with insulin receptor, which alters the receptor interaction with regulatory adaptor protein-E3 ubiquitin ligase pairs, adaptor protein with pleckstrin homology and Src homology 2 domain-c-Cbl and growth factor receptor bound protein 10-neuronal precursor cell-expressed developmentally downregulated 4. In conclusion, our findings establish ESDN as an inhibitor of insulin receptor signal transduction through a novel regulatory mechanism. Loss of ESDN potentiates insulin's metabolic and mitotic effects and provides insights into a novel therapeutic avenue. PMID:26921437

  18. Parallel SCF Adaptor Capture Proteomics Reveals a Role for SCFFBXL17 in NRF2 Activation via BACH1 Repressor Turnover

    PubMed Central

    Tan, Meng-Kwang Marcus; Lim, Hui-Jun; Bennett, Eric J.; Shi, Yang; Harper, J. Wade

    2014-01-01

    Modular Cullin-RING E3 ubiquitin ligases (CRLs) use substrate binding adaptor proteins to specify target ubiquitylation. Many of the ~200 human CRL adaptor proteins remain poorly studied due to a shortage of efficient methods to identify biologically relevant substrates. Here, we report the development of Parallel Adaptor Capture (PAC) proteomics, and its use to systematically identify candidate targets for the leucine-rich repeat family of F-box proteins (FBXLs) that function with SKP1-CUL1-F-box protein (SCF) E3s. In validation experiments, we identify the unstudied F-box protein FBXL17 as a regulator of the NFR2 oxidative stress pathway. We demonstrate that FBXL17 controls the transcription of the NRF2 target HMOX1 via turnover of the transcriptional repressor BACH1 in the absence or presence of extrinsic oxidative stress. This work identifies a role for SCFFBXL17 in controlling the threshold for NRF2-dependent gene activation and provides a framework for elucidating the functions of CRL adaptor proteins. PMID:24035498

  19. Parallel SCF adaptor capture proteomics reveals a role for SCFFBXL17 in NRF2 activation via BACH1 repressor turnover.

    PubMed

    Tan, Meng-Kwang Marcus; Lim, Hui-Jun; Bennett, Eric J; Shi, Yang; Harper, J Wade

    2013-10-10

    Modular cullin-RING E3 ubiquitin ligases (CRLs) use substrate binding adaptor proteins to specify target ubiquitylation. Many of the ~200 human CRL adaptor proteins remain poorly studied due to a shortage of efficient methods to identify biologically relevant substrates. Here, we report the development of parallel adaptor capture (PAC) proteomics and its use to systematically identify candidate targets for the leucine-rich repeat family of F-box proteins (FBXLs) that function with SKP1-CUL1-F-box protein (SCF) E3s. In validation experiments, we identify the unstudied F-box protein FBXL17 as a regulator of the NFR2 oxidative stress pathway. We demonstrate that FBXL17 controls the transcription of the NRF2 target HMOX1 via turnover of the transcriptional repressor BACH1 in the absence or presence of extrinsic oxidative stress. This work identifies a role for SCF(FBXL17) in controlling the threshold for NRF2-dependent gene activation and provides a framework for elucidating the functions of CRL adaptor proteins. PMID:24035498

  20. Absence of the Adaptor Protein PEA-15 Is Associated with Altered Pattern of Th Cytokines Production by Activated CD4+ T Lymphocytes In Vitro, and Defective Red Blood Cell Alloimmune Response In Vivo

    PubMed Central

    Kerbrat, Stéphane; Vingert, Benoit; Junier, Marie-Pierre; Castellano, Flavia; Renault-Mihara, François; Dos Reis Tavares, Silvina; Surenaud, Mathieu; Noizat-Pirenne, France; Boczkowski, Jorge; Guellaën, Georges; Chneiweiss, Hervé; Le Gouvello, Sabine

    2015-01-01

    TCR-dependent and costimulation signaling, cell division, and cytokine environment are major factors driving cytokines expression induced by CD4+ T cell activation. PEA-15 15 (Protein Enriched in Astrocyte / 15kDa) is an adaptor protein that regulates death receptor-induced apoptosis and proliferation signaling by binding to FADD and relocating ERK1/2 to the cytosol, respectively. By using PEA-15-deficient mice, we examined the role of PEA-15 in TCR-dependent cytokine production in CD4+ T cells. TCR-stimulated PEA-15-deficient CD4+ T cells exhibited defective progression through the cell cycle associated with impaired expression of cyclin E and phosphoRb, two ERK1/2-dependent proteins of the cell cycle. Accordingly, expression of the division cycle-dependent cytokines IL-2 and IFNγ, a Th1 cytokine, was reduced in stimulated PEA-15-deficient CD4+ T cells. This was associated with abnormal subcellular compartmentalization of activated ERK1/2 in PEA-15-deficient T cells. Furthermore, in vitro TCR-dependent differentiation of naive CD4+ CD62L+ PEA-15-deficient T cells was associated with a lower production of the Th2 cytokine, IL-4, whereas expression of the Th17-associated molecule IL4I1 was enhanced. Finally, a defective humoral response was shown in PEA-15-deficient mice in a model of red blood cell alloimmunization performed with Poly IC, a classical adjuvant of Th1 response in vivo. Collectively, our data suggest that PEA-15 contributes to the specification of the cytokine pattern of activated Th cells, thus highlighting a potential new target to interfere with T cell functional polarization and subsequent immune response. PMID:26317969

  1. Absence of the Adaptor Protein PEA-15 Is Associated with Altered Pattern of Th Cytokines Production by Activated CD4+ T Lymphocytes In Vitro, and Defective Red Blood Cell Alloimmune Response In Vivo.

    PubMed

    Kerbrat, Stéphane; Vingert, Benoit; Junier, Marie-Pierre; Castellano, Flavia; Renault-Mihara, François; Dos Reis Tavares, Silvina; Surenaud, Mathieu; Noizat-Pirenne, France; Boczkowski, Jorge; Guellaën, Georges; Chneiweiss, Hervé; Le Gouvello, Sabine

    2015-01-01

    TCR-dependent and costimulation signaling, cell division, and cytokine environment are major factors driving cytokines expression induced by CD4(+) T cell activation. PEA-15 15 (Protein Enriched in Astrocyte / 15 kDa) is an adaptor protein that regulates death receptor-induced apoptosis and proliferation signaling by binding to FADD and relocating ERK1/2 to the cytosol, respectively. By using PEA-15-deficient mice, we examined the role of PEA-15 in TCR-dependent cytokine production in CD4(+) T cells. TCR-stimulated PEA-15-deficient CD4(+) T cells exhibited defective progression through the cell cycle associated with impaired expression of cyclin E and phosphoRb, two ERK1/2-dependent proteins of the cell cycle. Accordingly, expression of the division cycle-dependent cytokines IL-2 and IFNγ, a Th1 cytokine, was reduced in stimulated PEA-15-deficient CD4(+) T cells. This was associated with abnormal subcellular compartmentalization of activated ERK1/2 in PEA-15-deficient T cells. Furthermore, in vitro TCR-dependent differentiation of naive CD4(+) CD62L(+) PEA-15-deficient T cells was associated with a lower production of the Th2 cytokine, IL-4, whereas expression of the Th17-associated molecule IL4I1 was enhanced. Finally, a defective humoral response was shown in PEA-15-deficient mice in a model of red blood cell alloimmunization performed with Poly IC, a classical adjuvant of Th1 response in vivo. Collectively, our data suggest that PEA-15 contributes to the specification of the cytokine pattern of activated Th cells, thus highlighting a potential new target to interfere with T cell functional polarization and subsequent immune response. PMID:26317969

  2. Adaptor protein CRK induces epithelial–mesenchymal transition and metastasis of bladder cancer cells through HGF/c-Met feedback loop

    PubMed Central

    Matsumoto, Ryuji; Tsuda, Masumi; Wang, Lei; Maishi, Nako; Abe, Takashige; Kimura, Taichi; Tanino, Mishie; Nishihara, Hiroshi; Hida, Kyoko; Ohba, Yusuke; Shinohara, Nobuo; Nonomura, Katsuya; Tanaka, Shinya

    2015-01-01

    We have previously reported that an adaptor protein CRK, including CRK-I and CRK-II, plays essential roles in the malignant potential of various aggressive human cancers, suggesting the validity of targeting CRK in molecular targeted therapy of a wide range of cancers. Nevertheless, the role of CRK in human bladder cancer with marked invasion, characterized by distant metastasis and poor prognosis, remains obscure. In the present study, immunohistochemistry indicated a striking enhancement of CRK-I/-II, but not CRK-like, in human bladder cancer tissues compared to normal urothelium. We established CRK-knockdown bladder cancer cells using 5637 and UM-UC-3, which showed a significant decline in cell migration, invasion, and proliferation. It is noteworthy that an elimination of CRK conferred suppressed phosphorylation of c-Met and the downstream scaffold protein Gab1 in a hepatocyte growth factor-dependent and -independent manner. In epithelial–mesenchymal transition-related molecules, E-cadherin was upregulated by CRK elimination, whereas N-cadherin, vimentin, and Zeb1 were downregulated. A similar effect was observed following treatment with c-Met inhibitor SU11274. Depletion of CRK significantly decreased cell proliferation of 5637 and UM-UC-3, consistent with reduced activity of ERK. An orthotopic xenograft model with bioluminescent imaging revealed that CRK knockdown significantly attenuated not only tumor volume but also the number of circulating tumor cells, resulted in a complete abrogation of metastasis. Taken together, this evidence uncovered essential roles of CRK in invasive bladder cancer through the hepatocyte growth factor/c-Met/CRK feedback loop for epithelial–mesenchymal transition induction. Thus, CRK might be a potent molecular target in bladder cancer, particularly for preventing metastasis, leading to the resolution of clinically longstanding critical issues. PMID:25816892

  3. Pseudomonas aeruginosa ExoT Induces Atypical Anoikis Apoptosis in Target Host Cells by Transforming Crk Adaptor Protein into a Cytotoxin.

    PubMed

    Wood, Stephen; Goldufsky, Josef; Shafikhani, Sasha H

    2015-05-01

    Previously, we demonstrated that Pseudomonas aeruginosa ExoT induces potent apoptosis in host epithelial cells in a manner that primarily depends on its ADP-ribosyltransferase domain (ADPRT) activity. However, the mechanism underlying ExoT/ADPRT-induced apoptosis remains undetermined. We now report that ExoT/ADPRT disrupts focal adhesion sites, activates p38β and JNK, and interferes with integrin-mediated survival signaling; causing atypical anoikis. We show that ExoT/ADPRT-induced anoikis is mediated by the Crk adaptor protein. We found that Crk-/- knockout cells are significantly more resistant to ExoT-induced apoptosis, while Crk-/- cells complemented with Crk are rendered sensitive to ExoT-induced apoptosis. Moreover, a dominant negative (DN) mutant form of Crk phenocopies ExoT-induced apoptosis both kinetically and mechanistically. Crk is generally believed to be a component of focal adhesion (FA) and its role in cellular survival remains controversial in that it has been found to be either pro-survival or pro-apoptosis. Our data demonstrate that although Crk is recruited to FA sites, its function is likely not required for FA assembly or for survival per se. However, when modified by ExoT or by mutagenesis, it can be transformed into a cytotoxin that induces anoikis by disrupting FA sites and interfering with integrin survival signaling. To our knowledge, this is the first example whereby a bacterial toxin exerts its cytotoxicity by subverting the function of an innocuous host cellular protein and turning it against the host cell. PMID:26020630

  4. Baculovirus vectors expressing F proteins in combination with virus-induced signaling adaptor (VISA) molecules confer protection against respiratory syncytial virus infection.

    PubMed

    Zhang, Yuan; Qiao, Lei; Hu, Xiao; Zhao, Kang; Zhang, Yanwen; Chai, Feng; Pan, Zishu

    2016-01-01

    Baculovirus has been exploited for use as a novel vaccine vector. To investigate the feasibility and efficacy of recombinant baculoviruses (rBVs) expressing respiratory syncytial virus (RSV) fusion (F) proteins, four constructs (Bac-tF/64, Bac-CF, Bac-CF/tF64 and Bac-CF/tF64-VISA) were generated. Bac-tF64 displays the F ectodomain (tF) on the envelope of rBVs, whereas Bac-CF expresses full-length F protein in transduced mammalian cells. Bac-CF/tF64 not only displays tF on the envelope but also expresses F in cells. Bac-CF/tF64-VISA comprises Bac-CF/tF64 harboring the virus-induced signaling adaptor (VISA) gene. After administration to BALB/c mice, all four vectors elicited RSV neutralizing antibody (Ab), systemic Ab (IgG, IgG1, and IgG2a), and cytokine responses. Compared with Bac-tF64, mice inoculated with Bac-CF and Bac-CF/tF64 exhibited an increased mixed Th1/Th2 cytokine response, increased ratios of IgG2a/IgG1 antibody responses, and reduced immunopathology upon RSV challenge. Intriguingly, co-expression of VISA reduced Th2 cytokine (IL-4, IL-5, and IL-10) production induced by Bac-CF/tF64, thus relieving lung pathology upon a subsequent RSV challenge. Our results indicated that the Bac-CF/tF64 vector incorporated with the VISA molecule may provide an effective vaccine strategy for protection against RSV. PMID:26643933

  5. ScaC, an Adaptor Protein Carrying a Novel Cohesin That Expands the Dockerin-Binding Repertoire of the Ruminococcus flavefaciens 17 Cellulosome

    PubMed Central

    Rincón, Marco T.; Martin, Jennifer C.; Aurilia, Vincenzo; McCrae, Sheila I.; Rucklidge, Garry J.; Reid, Martin D.; Bayer, Edward A.; Lamed, Raphael; Flint, Harry J.

    2004-01-01

    A new gene, designated scaC and encoding a protein carrying a single cohesin, was identified in the cellulolytic rumen anaerobe Ruminococcus flavefaciens 17 as part of a gene cluster that also codes for the cellulosome structural components ScaA and ScaB. Phylogenetic analysis showed that the sequence of the ScaC cohesin is distinct from the sequences of other cohesins, including the sequences of R. flavefaciens ScaA and ScaB. The scaC gene product also includes at its C terminus a dockerin module that closely resembles those found in R. flavefaciens enzymes that bind to the cohesins of the primary ScaA scaffoldin. The putative cohesin domain and the C-terminal dockerin module were cloned and overexpressed in Escherichia coli as His6-tagged products (ScaC-Coh and ScaC-Doc, respectively). Affinity probing of protein extracts of R. flavefaciens 17 separated in one-dimensional and two-dimensional gels with recombinant cohesins from ScaC and ScaA revealed that two distinct subsets of native proteins interact with ScaC-Coh and ScaA-Coh. Furthermore, ScaC-Coh failed to interact with the recombinant dockerin module from the enzyme EndB that is recognized by ScaA cohesins. On the other hand, ScaC-Doc was shown to interact specifically with the recombinant cohesin domain from ScaA, and the ScaA-Coh probe was shown to interact with a native 29-kDa protein spot identified as ScaC by matrix-assisted laser desorption ionization—time of flight mass spectrometry. These results suggest that ScaC plays the role of an adaptor scaffoldin that is bound to ScaA via the ScaC dockerin module, which, via the distinctive ScaC cohesin, expands the range of proteins that can bind to the ScaA-based enzyme complex. PMID:15090497

  6. A Repressor Protein Complex Regulates Leaf Growth in Arabidopsis

    PubMed Central

    Gonzalez, Nathalie; Pauwels, Laurens; Baekelandt, Alexandra; De Milde, Liesbeth; Van Leene, Jelle; Besbrugge, Nienke; Heyndrickx, Ken S.; Pérez, Amparo Cuéllar; Durand, Astrid Nagels; De Clercq, Rebecca; Van De Slijke, Eveline; Vanden Bossche, Robin; Eeckhout, Dominique; Gevaert, Kris; Vandepoele, Klaas; De Jaeger, Geert; Goossens, Alain; Inzé, Dirk

    2015-01-01

    Cell number is an important determinant of final organ size. In the leaf, a large proportion of cells are derived from the stomatal lineage. Meristemoids, which are stem cell-like precursor cells, undergo asymmetric divisions, generating several pavement cells adjacent to the two guard cells. However, the mechanism controlling the asymmetric divisions of these stem cells prior to differentiation is not well understood. Here, we characterized PEAPOD (PPD) proteins, the only transcriptional regulators known to negatively regulate meristemoid division. PPD proteins interact with KIX8 and KIX9, which act as adaptor proteins for the corepressor TOPLESS. D3-type cyclin encoding genes were identified among direct targets of PPD2, being negatively regulated by PPDs and KIX8/9. Accordingly, kix8 kix9 mutants phenocopied PPD loss-of-function producing larger leaves resulting from increased meristemoid amplifying divisions. The identified conserved complex might be specific for leaf growth in the second dimension, since it is not present in Poaceae (grasses), which also lack the developmental program it controls. PMID:26232487

  7. Structural and functional insights into CARDs of zebrafish (Danio rerio) NOD1 and NOD2, and their interaction with adaptor protein RIP2.

    PubMed

    Maharana, Jitendra; Dehury, Budheswar; Sahoo, Jyoti Ranjan; Jena, Itishree; Bej, Aritra; Panda, Debashis; Sahoo, Bikash Ranjan; Patra, Mahesh Chandra; Pradhan, Sukanta Kumar

    2015-08-01

    Nucleotide-binding and oligomerization domain-containing protein 1 (NOD1) and NOD2 are cytosolic pattern-recognition receptors (PRRs) composed of an N-terminal caspase activation and recruitment domain (CARD), a central NACHT domain and C-terminal leucine-rich repeats (LRRs). They play a vital role in innate immune signaling by activating the NF-κB pathway via recognition of peptidoglycans by LRRs, and ATP-dependent self-oligomerization of NACHT followed by downstream signaling. After oligomerization, CARD/s play a crucial role in activating downstream signaling via the adaptor molecule, RIP2. Due to the inadequacy of experimental 3D structures of CARD/s of NOD2 and RIP2, and results from differential experimental setups, the RIP2-mediated CARD-CARD interaction has remained as a contradictory statement. We employed a combinatorial approach involving protein modeling, docking, molecular dynamics simulation, and binding free energy calculation to illuminate the molecular mechanism that shows the possible involvement of either the acidic or basic patch of zebrafish NOD1/2-CARD/a and RIP2-CARD in CARD-CARD interaction. Herein, we have hypothesized 'type-I' mode of CARD-CARD interaction in NOD1 and NOD2, where NOD1/2-CARD/a involve their acidic surfaces to interact with RIP2. Asp37 and Glu51 (of NOD1) and Arg477, Arg521 and Arg529 (of RIP2) were identified to be crucial for NOD1-RIP2 interaction. However, in NOD2-RIP2, Asp32 (of NOD2) and Arg477 and Arg521 (of RIP2) were anticipated to be significant for downstream signaling. Furthermore, we found that strong electrostatic contacts and salt bridges are crucial for protein-protein interactions. Altogether, our study has provided novel insights into the RIP2-mediated CARD-CARD interaction in zebrafish NOD1 and NOD2, which will be helpful to understand the molecular basis of the NOD1/2 signaling mechanism. PMID:26079944

  8. Rat and mouse CD94 associate directly with the activating transmembrane adaptor proteins DAP12 and DAP10 and activate NK cell cytotoxicity.

    PubMed

    Saether, Per C; Hoelsbrekken, Sigurd E; Fossum, Sigbjørn; Dissen, Erik

    2011-12-15

    Signaling by the CD94/NKG2 heterodimeric NK cell receptor family has been well characterized in the human but has remained unclear in the mouse and rat. In the human, the activating receptor CD94/NKG2C associates with DAP12 by an ionic bond between oppositely charged residues within the transmembrane regions of NKG2C and DAP12. The lysine residue responsible for DAP12 association is absent in rat and mouse NKG2C and -E, raising questions about signaling mechanisms in these species. As a possible substitute, rat and mouse NKG2C and -E contain an arginine residue in the transition between the transmembrane and stalk regions. In this article, we demonstrate that, similar to their human orthologs, NKG2A inhibits, whereas NKG2C activates, rat NK cells. Redirected lysis assays using NK cells transfected with a mutated NKG2C construct indicated that the activating function of CD94/NKG2C did not depend on the transmembrane/stalk region arginine residue. Flow cytometry and biochemical analysis demonstrated that both DAP12 and DAP10 can associate with rat CD94/NKG2C. Surprisingly, DAP12 and DAP10 did not associate with NKG2C but instead with CD94. These associations depended on a transmembrane lysine residue in CD94 that is unique to rodents. Thus, in the mouse and rat, the ability to bind activating adaptor proteins has been transferred from NKG2C/E to the CD94 chain as a result of mutation events in both chains. Remarkable from a phylogenetic perspective, this sheds new light on the evolution and function of the CD94/NKG2 receptor family. PMID:22084441

  9. Direct interactions of adaptor protein complexes 1 and 2 with the copper transporter ATP7A mediate its anterograde and retrograde trafficking

    PubMed Central

    Yi, Ling; Kaler, Stephen G.

    2015-01-01

    ATP7A is a P-type ATPase in which diverse mutations lead to X-linked recessive Menkes disease or occipital horn syndrome. Recently, two previously unknown ATP7A missense mutations, T994I and P1386S, were shown to cause an isolated distal motor neuropathy without clinical or biochemical features of other ATP7A disorders. These mutant alleles cause subtle defects in ATP7A intracellular trafficking, resulting in preferential plasma membrane localization compared with wild-type ATP7A. We reported previously that ATP7AP1386S causes unstable insertion of the eighth and final transmembrane segment, preventing proper position of the carboxyl-terminal tail in a proportion of mutant molecules. Here, we utilize this and other naturally occurring and engineered mutant ATP7A alleles to identify mechanisms of normal ATP7A trafficking. We show that adaptor protein (AP) complexes 1 and 2 physically interact with ATP7A and that binding is mediated in part by a carboxyl-terminal di-leucine motif. In contrast to other ATP7A missense mutations, ATP7AP1386S partially disturbs interactions with both APs, leading to abnormal axonal localization in transfected NSC-34 motor neurons and altered calcium-signaling following glutamate stimulation. Our results imply that AP-1 normally tethers ATP7A at the trans-Golgi network in the somatodendritic segments of motor neurons and that alterations affecting the ATP7A carboxyl-terminal tail induce release of the copper transporter to the axons or axonal membranes. The latter effects are intensified by diminished interaction with AP-2, impeding ATP7A retrograde trafficking. Taken together, these findings further illuminate the normal molecular mechanisms of ATP7A trafficking and suggest a pathophysiological basis for ATP7A-related distal motor neuropathy. PMID:25574028

  10. The Cytoskeletal Adaptor Protein Band 4.1B is Required for the Maintenance of Paranodal Axo-Glial Septate Junctions in Myelinated Axons

    PubMed Central

    Buttermore, Elizabeth D.; Dupree, Jeffrey L.; Cheng, JrGang; An, Xiuli; Tessarollo, Lino; Bhat, Manzoor A.

    2011-01-01

    Precise targeting and maintenance of axonal domains in myelinated axons is essential for saltatory conduction. Caspr and Caspr2, which localize at paranodal and juxtaparanodal domains, contain binding sites for the cytoskeletal adaptor protein 4.1B. The exact role of 4.1B in the organization and maintenance of axonal domains is still not clear. Here we report the generation and characterization of 4.1B null mice. We show that loss of 4.1B in the PNS results in mislocalization of Caspr at paranodes and destabilization of paranodal axo-glial septate junctions (AGSJs) as early as postnatal day 30. In the CNS, Caspr localization is progressively disrupted and ultrastructural analysis showed paranodal regions that were completely devoid of AGSJs, with axolemma separated from the myelin loops, and loops coming off the axolemma. Most importantly, our phenotypic analysis of previously generated 4.1B mutants, used in Horresh et al. (2010), showed that Caspr localization was not affected in the PNS, even after one year; and 4.1R was neither expressed, nor enriched at the paranodes. Furthermore, ultrastructural analysis of these 4.1B mutants showed destabilization of CNS AGSJs at about one year. We also discovered that the 4.1B locus is differentially expressed in the PNS and CNS, and generates multiple splice isoforms in the PNS, suggesting 4.1B may function differently in the PNS versus CNS. Together, our studies provide direct evidence that 4.1B plays a pivotal role in interactions between the paranodal AGSJs and axonal cytoskeleton, and that 4.1B is critically required for long-term maintenance of axonal domains in myelinated axons. PMID:21632923