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Sample records for eukaryotic protein kinases

  1. Evolutionary Ancestry of Eukaryotic Protein Kinases and Choline Kinases*

    PubMed Central

    Lai, Shenshen; Safaei, Javad

    2016-01-01

    The reversible phosphorylation of proteins catalyzed by protein kinases in eukaryotes supports an important role for eukaryotic protein kinases (ePKs) in the emergence of nucleated cells in the third superkingdom of life. Choline kinases (ChKs) could also be critical in the early evolution of eukaryotes, because of their function in the biosynthesis of phosphatidylcholine, which is unique to eukaryotic membranes. However, the genomic origins of ePKs and ChKs are unclear. The high degeneracy of protein sequences and broad expansion of ePK families have made this fundamental question difficult to answer. In this study, we identified two class-I aminoacyl-tRNA synthetases with high similarities to consensus amino acid sequences of human protein-serine/threonine kinases. Comparisons of primary and tertiary structures supported that ePKs and ChKs evolved from a common ancestor related to glutaminyl aminoacyl-tRNA synthetases, which may have been one of the key factors in the successful of emergence of ancient eukaryotic cells from bacterial colonies. PMID:26742849

  2. Genomic analysis of the eukaryotic protein kinase superfamily: a perspective

    PubMed Central

    Hanks, Steven K

    2003-01-01

    Protein kinases with a conserved catalytic domain make up one of the largest 'superfamilies' of eukaryotic proteins and play many key roles in biology and disease. Efforts to identify and classify all the members of the eukaryotic protein kinase superfamily have recently culminated in the mining of essentially complete human genome data. PMID:12734000

  3. Structural and evolutionary divergence of eukaryotic protein kinases in Apicomplexa.

    PubMed

    Talevich, Eric; Mirza, Amar; Kannan, Natarajan

    2011-11-02

    The Apicomplexa constitute an evolutionarily divergent phylum of protozoan pathogens responsible for widespread parasitic diseases such as malaria and toxoplasmosis. Many cellular functions in these medically important organisms are controlled by protein kinases, which have emerged as promising drug targets for parasitic diseases. However, an incomplete understanding of how apicomplexan kinases structurally and mechanistically differ from their host counterparts has hindered drug development efforts to target parasite kinases. We used the wealth of sequence data recently made available for 15 apicomplexan species to identify the kinome of each species and quantify the evolutionary constraints imposed on each family of apicomplexan kinases. Our analysis revealed lineage-specific adaptations in selected families, namely cyclin-dependent kinase (CDK), calcium-dependent protein kinase (CDPK) and CLK/LAMMER, which have been identified as important in the pathogenesis of these organisms. Bayesian analysis of selective constraints imposed on these families identified the sequence and structural features that most distinguish apicomplexan protein kinases from their homologs in model organisms and other eukaryotes. In particular, in a subfamily of CDKs orthologous to Plasmodium falciparum crk-5, the activation loop contains a novel PTxC motif which is absent from all CDKs outside Apicomplexa. Our analysis also suggests a convergent mode of regulation in a subset of apicomplexan CDPKs and mammalian MAPKs involving a commonly conserved arginine in the αC helix. In all recognized apicomplexan CLKs, we find a set of co-conserved residues involved in substrate recognition and docking that are distinct from metazoan CLKs. We pinpoint key conserved residues that can be predicted to mediate functional differences from eukaryotic homologs in three identified kinase families. We discuss the structural, functional and evolutionary implications of these lineage-specific variations and

  4. Structural and evolutionary divergence of eukaryotic protein kinases in Apicomplexa

    PubMed Central

    2011-01-01

    Background The Apicomplexa constitute an evolutionarily divergent phylum of protozoan pathogens responsible for widespread parasitic diseases such as malaria and toxoplasmosis. Many cellular functions in these medically important organisms are controlled by protein kinases, which have emerged as promising drug targets for parasitic diseases. However, an incomplete understanding of how apicomplexan kinases structurally and mechanistically differ from their host counterparts has hindered drug development efforts to target parasite kinases. Results We used the wealth of sequence data recently made available for 15 apicomplexan species to identify the kinome of each species and quantify the evolutionary constraints imposed on each family of apicomplexan kinases. Our analysis revealed lineage-specific adaptations in selected families, namely cyclin-dependent kinase (CDK), calcium-dependent protein kinase (CDPK) and CLK/LAMMER, which have been identified as important in the pathogenesis of these organisms. Bayesian analysis of selective constraints imposed on these families identified the sequence and structural features that most distinguish apicomplexan protein kinases from their homologs in model organisms and other eukaryotes. In particular, in a subfamily of CDKs orthologous to Plasmodium falciparum crk-5, the activation loop contains a novel PTxC motif which is absent from all CDKs outside Apicomplexa. Our analysis also suggests a convergent mode of regulation in a subset of apicomplexan CDPKs and mammalian MAPKs involving a commonly conserved arginine in the αC helix. In all recognized apicomplexan CLKs, we find a set of co-conserved residues involved in substrate recognition and docking that are distinct from metazoan CLKs. Conclusions We pinpoint key conserved residues that can be predicted to mediate functional differences from eukaryotic homologs in three identified kinase families. We discuss the structural, functional and evolutionary implications of these

  5. Crystal Structures of Two Aminoglycoside Kinases Bound with a Eukaryotic Protein Kinase Inhibitor

    PubMed Central

    Hwang, Jiyoung; Berghuis, Albert M.

    2011-01-01

    Antibiotic resistance is recognized as a growing healthcare problem. To address this issue, one strategy is to thwart the causal mechanism using an adjuvant in partner with the antibiotic. Aminoglycosides are a class of clinically important antibiotics used for the treatment of serious infections. Their usefulness has been compromised predominantly due to drug inactivation by aminoglycoside-modifying enzymes, such as aminoglycoside phosphotransferases or kinases. These kinases are structurally homologous to eukaryotic Ser/Thr and Tyr protein kinases and it has been shown that some can be inhibited by select protein kinase inhibitors. The aminoglycoside kinase, APH(3′)-IIIa, can be inhibited by CKI-7, an ATP-competitive inhibitor for the casein kinase 1. We have determined that CKI-7 is also a moderate inhibitor for the atypical APH(9)-Ia. Here we present the crystal structures of CKI-7-bound APH(3′)-IIIa and APH(9)-Ia, the first structures of a eukaryotic protein kinase inhibitor in complex with bacterial kinases. CKI-7 binds to the nucleotide-binding pocket of the enzymes and its binding alters the conformation of the nucleotide-binding loop, the segment homologous to the glycine-rich loop in eurkaryotic protein kinases. Comparison of these structures with the CKI-7-bound casein kinase 1 reveals features in the binding pockets that are distinct in the bacterial kinases and could be exploited for the design of a bacterial kinase specific inhibitor. Our results provide evidence that an inhibitor for a subset of APHs can be developed in order to curtail resistance to aminoglycosides. PMID:21573013

  6. Small molecule modulators of eukaryotic initiation factor 2α kinases, the key regulators of protein synthesis.

    PubMed

    Joshi, Manali; Kulkarni, Abhijeet; Pal, Jayanta K

    2013-11-01

    Eukaryotic initiation factor 2 alpha kinases (eIF-2α kinases) are key mediators of stress response in cells. In mammalian cells, there are four eIF-2α kinases, namely HRI (Heme-Regulated Inhibitor), PKR (RNA-dependent Protein Kinase), PERK (PKR-like ER Kinase) and GCN2 (General Control Non-derepressible 2). These kinases get activated during diverse cytoplasmic stress conditions and phosphorylate the alpha-subunit of eIF2, leading to global protein synthesis inhibition. Therefore, eIF-2α kinases play a vital role in various cellular processes such as proliferation, differentiation, apoptosis and cell signaling. Deregulation of eIF-2α kinases and protein synthesis has been linked to numerous pathological conditions such as certain cancers, anemia and neurodegenerative disorders. Thus, modulation of these kinases by small molecules holds a great therapeutic promise. In this review we have compiled the available information on inhibitors and activators of these four eIF-2α kinases. The review concludes with a note on the selectivity issue of currently available modulators and future perspectives for the design of specific small molecule probes.

  7. Eukaryotic-like protein kinases in the prokaryotes and the myxobacterial kinome

    PubMed Central

    Pérez, J.; Castañeda-García, A.; Jenke-Kodama, H.; Müller, R.; Muñoz-Dorado, J.

    2008-01-01

    Ser/Thr/Tyr kinases, which together comprise a major class of regulatory proteins in eukaryotes, were not believed to play an important role in prokaryotes until recently. However, our analysis of 626 prokaryotic genomes reveals that eukaryotic-like protein kinases (ELKs) are found in nearly two-thirds of the sequenced strains. We have identified 2697 ELKs, most of which are encoded by multicellular strains of the phyla Proteobacteria (Myxococcales), Actinobacteria, Cyanobacteria, and Chloroflexi, and 2 Acidobacteria and 1 Planctomycetes. Astonishingly, 7 myxobacterial strains together encode 892 ELKs, with 4 of the strains exhibiting a genomic ELK density similar to that observed in eukaryotes. Most myxobacterial ELKs show a modular organization in which the kinase domain is located at the N terminus. The C-terminal portion of the ELKs is highly diverse and often contains sequences with similarity to characterized domains, most of them involved in signaling mechanisms or in protein–protein interactions. However, many of these architectures are unique to the myxobacteria, an observation that suggests that this group exploits sophisticated and novel signal transduction systems. Phylogenetic reconstruction using the kinase domains revealed many orthologous sequence pairs and a huge number of gene duplications that probably occurred after speciation. Furthermore, studies of the microsynteny in the ELK-encoding regions reveal only low levels of synteny among Myxococcus xanthus, Plesiocystis pacifica, and Sorangium cellulosum. However, extensive similarities between M. xanthus, Stigmatella aurantiaca, and 3 Anaeromyxobacter strains were observed, indicating that they share regulatory pathways involving various ELKs. PMID:18836084

  8. Protein PknE, a novel transmembrane eukaryotic-like serine/threonine kinase from Mycobacterium tuberculosis.

    PubMed

    Molle, Virginie; Girard-Blanc, Christine; Kremer, Laurent; Doublet, Patricia; Cozzone, Alain J; Prost, Jean-François

    2003-09-05

    Protein PknE from Mycobacterium tuberculosis has been overproduced and purified, and its biochemical properties have been analyzed. This protein is shown to be a eukaryotic-like (Hanks'-type) protein kinase with a structural organization similar to that of membrane-bound eukaryotic sensor serine/threonine kinases. It consists of a N-terminal catalytic domain located in the cytoplasm, linked via a single transmembrane-spanning region to an extracellular C-terminal domain. The full-length enzyme, as well as the cytosolic domain alone, can autophosphorylate on serine and threonine residues. Such autokinase activity requires the presence of a lysine residue at position 45 in subdomain II, which is known to be essential also for eukaryotic kinase activity. Involvement of PknE in the transduction of external signals into the cytosol of bacteria is proposed.

  9. Decoding the Interactions Regulating the Active State Mechanics of Eukaryotic Protein Kinases

    PubMed Central

    Meharena, Hiruy S.; Fan, Xiaorui; Ahuja, Lalima G.; Keshwani, Malik M.; McClendon, Christopher L.; Chen, Angela M.; Adams, Joseph A.; Taylor, Susan S.

    2016-01-01

    Eukaryotic protein kinases regulate most cellular functions by phosphorylating targeted protein substrates through a highly conserved catalytic core. In the active state, the catalytic core oscillates between open, intermediate, and closed conformations. Currently, the intramolecular interactions that regulate the active state mechanics are not well understood. Here, using cAMP-dependent protein kinase as a representative model coupled with biochemical, biophysical, and computational techniques, we define a set of highly conserved electrostatic and hydrophobic interactions working harmoniously to regulate these mechanics. These include the previously identified salt bridge between a lysine from the β3-strand and a glutamate from the αC-helix as well as an electrostatic interaction between the phosphorylated activation loop and αC-helix and an ensemble of hydrophobic residues of the Regulatory spine and Shell. Moreover, for over three decades it was thought that the highly conserved β3-lysine was essential for phosphoryl transfer, but our findings show that the β3-lysine is not required for phosphoryl transfer but is essential for the active state mechanics. PMID:27902690

  10. Protein kinases of the human malaria parasite Plasmodium falciparum: the kinome of a divergent eukaryote

    PubMed Central

    Ward, Pauline; Equinet, Leila; Packer, Jeremy; Doerig, Christian

    2004-01-01

    Background Malaria, caused by the parasitic protist Plasmodium falciparum, represents a major public health problem in the developing world. The P. falciparum genome has been sequenced, which provides new opportunities for the identification of novel drug targets. Eukaryotic protein kinases (ePKs) form a large family of enzymes with crucial roles in most cellular processes; hence malarial ePKS represent potential drug targets. We report an exhaustive analysis of the P. falciparum genomic database (PlasmoDB) aimed at identifying and classifying all ePKs in this organism. Results Using a variety of bioinformatics tools, we identified 65 malarial ePK sequences and constructed a phylogenetic tree to position these sequences relative to the seven established ePK groups. Predominant features of the tree were: (i) that several malarial sequences did not cluster within any of the known ePK groups; (ii) that the CMGC group, whose members are usually involved in the control of cell proliferation, had the highest number of malarial ePKs; and (iii) that no malarial ePK clustered with the tyrosine kinase (TyrK) or STE groups, pointing to the absence of three-component MAPK modules in the parasite. A novel family of 20 ePK-related sequences was identified and called FIKK, on the basis of a conserved amino acid motif. The FIKK family seems restricted to Apicomplexa, with 20 members in P. falciparum and just one member in some other Apicomplexan species. Conclusion The considerable phylogenetic distance between Apicomplexa and other Eukaryotes is reflected by profound divergences between the kinome of malaria parasites and that of yeast or mammalian cells. PMID:15479470

  11. Intrauterine growth restriction inhibits expression of eukaryotic elongation factor 2 kinase, a regulator of protein translation.

    PubMed

    McKnight, Robert A; Yost, Christian C; Zinkhan, Erin K; Fu, Qi; Callaway, Christopher W; Fung, Camille M

    2016-08-01

    Nutrient deprivation suppresses protein synthesis by blocking peptide elongation. Transcriptional upregulation and activation of eukaryotic elongation factor 2 kinase (eEF2K) blocks peptide elongation by phosphorylating eukaryotic elongation factor 2. Previous studies examining placentas from intrauterine growth restricted (IUGR) newborn infants show decreased eEF2K expression and activity despite chronic nutrient deprivation. However, the effect of IUGR on hepatic eEF2K expression in the fetus is unknown. We, therefore, examined the transcriptional regulation of hepatic eEF2K gene expression in a Sprague-Dawley rat model of IUGR. We found decreased hepatic eEF2K mRNA and protein levels in IUGR offspring at birth compared with control, consistent with previous placental observations. Furthermore, the CpG island within the eEF2K promoter demonstrated increased methylation at a critical USF 1/2 transcription factor binding site. In vitro methylation of this binding site caused near complete loss of eEF2K promoter activity, designating this promoter as methylation sensitive. The eEF2K promotor in IUGR offspring also lost the protective histone covalent modifications associated with unmethylated CGIs. In addition, the +1 nucleosome was displaced 3' and RNA polymerase loading was reduced at the IUGR eEF2K promoter. Our findings provide evidence to explain why IUGR-induced chronic nutrient deprivation does not result in the upregulation of eEF2K gene transcription. Copyright © 2016 the American Physiological Society.

  12. [Mode of action of cyclic amp in prokaryotes and eukaryotes, CAP and cAMP-dependent protein kinases].

    PubMed

    de Gunzburg, J

    1985-06-01

    cAMP is an ubiquitous compound which is involved in the regulation of many biological processes. In bacteria such as E. coli, cAMP mediates the activation of catabolic operons via the CAP protein. The CAP-cAMP complex, whose tridimensional structure has recently been established, binds to the promoter regions of catabolic operons at a specific site, and activates their transcription by inducing RNA polymerase to bind and initiate transcription at the correct site. Various phenomenons including protein-protein interactions or CAP-induced DNA bending or kinking could be involved in the process of forming the open transcription complex. In eukaryotes, cAMP activates cAMP dependent protein kinases which covalently modify proteins by phosphorylation on serine or threonine residues. The catalytically inactive holoenzyme is generally a tetramer containing two regulatory subunits, each capable of binding two molecules of cAMP, and two catalytic subunits. In mammalian cells, two types of cAMP dependent protein kinases (I and II) can be distinguished on the basis of their regulatory subunits; their relative proportion varies from tissue to tissue. Binding of cAMP to the regulatory subunits induces the dissociation of the holoenzyme and releases the free and active catalytic subunits. Phosphorylation of proteins occurs at sequences containing two basic residues in the vicinity of the phosphorylated serine or threonine. A heat-stable protein, present in most eukaryotic cells, specifically interacts with the catalytic subunit and inhibits its activity. The amino-acid sequence of cAMP dependent protein kinases has recently been determined. It is interesting to note that the domains responsible for cAMP binding by the regulatory subunits of mammalian cAMP dependent protein kinases and CAP share important sequence homologies. The same phenomenon is observed concerning the domain responsible for ATP binding to the catalytic subunit of cAMP dependent protein kinases and that of

  13. A calmodulin-dependent protein kinase from lower eukaryote Physarum polycephalum.

    PubMed

    Nakamura, Akio; Hanyuda, Yuki; Okagaki, Tuyoshi; Takagi, Takashi; Kohama, Kazuhiro

    2005-03-25

    A full-length cDNA coding a calmodulin (CaM)-dependent protein kinase gene was cloned from Physarum plasmodia poly(A)-RNA by polymerase chain reaction with the oligonucleotide primers that were designed after the amino acid sequence of highly conserved regions of myosin light-chain kinase. Sequence analysis of the cDNA revealed that this Physarum kinase was a 42,519-Da protein with an ATP-binding domain, Ser/Thr kinase active site signature, and CaM-binding domain. Expression of the cDNA in Escherichia coli demonstrated that the Physarum kinase in the presence of Ca2+ and CaM phosphorylated the recombinant phosphorylatable light chain (PLc) of Physarum myosin II. The peptide analysis after proteolysis of the phosphorylated PLc indicated that Ser 18 was phosphorylated. The site was confirmed by the failure of phosphorylation of PLc, the Ser 18 of which was replaced by Ala. The physiological role of the kinase will be discussed with special reference to the 55-kDa kinase, which had been previously purified from Physarum plasmodia for phosphorylated PLc.

  14. A conserved Glu-Arg salt bridge connects co-evolved motifs that define the eukaryote protein kinase fold

    PubMed Central

    Yang, Jie; Wu, Jian; Steichen, Jon M.; Kornev, Alexandr P.; Deal, Michael S.; Li, Sheng; Sankaran, Banumathi; Woods, Virgil L.; Taylor, Susan S.

    2012-01-01

    Eukaryotic protein kinases (EPK)feature two co-evolved structural segments, the Activation segment which starts with the Asp-Phe-Gly (DFG) and ends with the Ala-Pro-Glu (APE) motifs, and the helical GHI-subdomain that comprises αG-αH-αI helices. Eukaryotic-like kinases have a much shorter Activation segment and lack the GHI-subdomain. They thus lack the conserved salt bridge interaction between the APE Glu and an Arg from the GHI-subdomain, a hallmark signature of EPKs. Although the conservation of this salt bridge in EPKs is well known and its implication in diseases has been illustrated by polymorphism analysis, its function has not been carefully studied. In this work, we use murine cAMP dependent protein kinase (PKA) as the model enzyme (Glu208 and Arg280) to examine the role of these two residues. We showed that Ala replacement of either residue caused a 40–120 fold decrease in catalytic efficiency of the enzyme due to an increase in Km(ATP) and a decrease in kcat. Crystal structures, as well as solution studies, also demonstratethat this ion pair contributes to the hydrophobic network and stability of the enzyme. We show that mutation of either Glu or Arg to Ala renders bothmutant proteins less effective substrates for upstream kinase phosphoinositide dependent kinase 1. We propose that the Glu208-Arg280 pair serves as a center hub of connectivity between these two structurally conserved elements in EPKs. Mutations of either residue disrupt communication not only between the two segments but also within the rest of the molecule leading to altered catalytic activity and enzyme regulation. PMID:22138346

  15. Bacterial eukaryotic type serine-threonine protein kinases: from structural biology to targeted anti-infective drug design.

    PubMed

    Danilenko, Valery N; Osolodkin, Dmitry I; Lakatosh, Sergey A; Preobrazhenskaya, Maria N; Shtil, Alexander A

    2011-01-01

    Signaling through protein kinases is an evolutionary conserved, widespread language of biological regulation. The eukaryotic type serine-threonine protein kinases (STPKs) found in normal human microbiote and in pathogenic bacteria play a key role in regulation of microbial survival, virulence and pathogenicity. Therefore, down-regulation of bacterial STPKs emerges as an attractive approach to cure infections. In this review we focused on actinobacterial STPKs to demonstrate that these enzymes can be used for crystal structure studies, modeling of 3D structure, construction of test systems and design of novel chemical libraries of low molecule as weight inhibitors. In particular, the prototypic pharmacological antagonists of Mycobacterium tuberculosis STPKs are perspective for development of a novel generation of drugs to combat the socially important disease. These inhibitors may modulate both actinobacterial and host STPKs and trigger programmed death of pathogenic bacteria.

  16. Role of Mitogen-Activated Protein Kinase Sty1 in Regulation of Eukaryotic Initiation Factor 2α Kinases in Response to Environmental Stress in Schizosaccharomyces pombe▿

    PubMed Central

    Berlanga, Juan José; Rivero, Damariz; Martín, Ruth; Herrero, Saturnino; Moreno, Sergio; de Haro, César

    2010-01-01

    The mitogen-activated protein kinase (MAPK) Sty1 is essential for the regulation of transcriptional responses that promote cell survival in response to different types of environmental stimuli in Schizosaccharomyces pombe. In fission yeast, three distinct eukaryotic initiation factor 2α (eIF2α) kinases, two mammalian HRI-related protein kinases (Hri1 and Hri2) and the Gcn2 ortholog, regulate protein synthesis in response to cellular stress conditions. In this study, we demonstrate that both Hri1 and Hri2 exhibited an autokinase activity, specifically phosphorylated eIF2α, and functionally replaced the endogenous Saccharomyces cerevisiae Gcn2. We further show that Gcn2, but not Hri1 or Hri2, is activated early after exposure to hydrogen peroxide and methyl methanesulfonate (MMS). Cells lacking Gcn2 exhibit a later activation of Hri2. The activated MAPK Sty1 negatively regulates Gcn2 and Hri2 activities under oxidative stress but not in response to MMS. In contrast, Hri2 is the primary activated eIF2α kinase in response to heat shock. In this case, the activation of Sty1 appears to be transitory and does not contribute to the modulation of the eIF2α kinase stress pathway. In strains lacking Hri2, a type 2A protein phosphatase is activated soon after heat shock to reduce eIF2α phosphorylation. Finally, the MAPK Sty1, but not the eIF2α kinases, is essential for survival upon oxidative stress or heat shock, but not upon MMS treatment. These findings point to a regulatory coordination between the Sty1 MAPK and eIF2α kinase pathways for a particular range of stress responses. PMID:19880757

  17. Fludarabine resistance mediated by aminoglycoside-3'-phosphotransferase-IIa and the structurally related eukaryotic cAMP-dependent protein kinase.

    PubMed

    Sánchez-Carrera, Dámaso; Bravo-Navas, Sara; Cabezón, Elena; Arechaga, Ignacio; Cabezas, Matilde; Yáñez, Lucrecia; Pipaón, Carlos

    2017-04-03

    While working with G418-resistant stably transfected cells, we realized the neomycin resistance gene (NeoR), which encodes the aminoglycoside-3'-phosphotransferase-IIa [APH(3')-IIa], also confers resistance to the nucleoside analog fludarabine. Fludarabine is a cytostatic drug widely used in the treatment of hematologic and solid tumors as well as in the conditioning of patients before transplantation of hematopoietic progenitors. We present evidence that NeoR-transfected cells do not incorporate fludarabine, thus avoiding DNA damage caused by the drug, evidenced by a lack of FANCD2 monoubiquitination and impaired apoptosis. A screening of other nucleoside analogs revealed that APH(3')-IIa only protects against ATP purine analogs. Moreover, APH(3')-IIa ATPase activity is inhibited by fludarabine monophosphate, suggesting that APH(3')-IIa blocks fludarabine incorporation into DNA by dephosphorylating its active fludarabine triphosphate form. Furthermore, overexpression of the catalytic subunit of the eukaryotic kinase PKA, which is structurally related to APHs, also provides resistance to fludarabine, anticipating its putative utility as a response marker to the drug. Our results preclude the use of Neo marker plasmids in the study of purine analogs and unveils a new resistance mechanism against these chemotherapeuticals.-Sánchez-Carrera, D., Bravo-Navas, S., Cabezón, E., Arechaga, I., Cabezas, M., Yáñez, L., Pipaón, C. Fludarabine resistance mediated by aminoglycoside-3'-phosphotransferase-IIa and the structurally related eukaryotic cAMP-dependent protein kinase.

  18. A mammalian homologue of GCN2 protein kinase important for translational control by phosphorylation of eukaryotic initiation factor-2alpha.

    PubMed Central

    Sood, R; Porter, A C; Olsen, D A; Cavener, D R; Wek, R C

    2000-01-01

    A family of protein kinases regulates translation in response to different cellular stresses by phosphorylation of the alpha subunit of eukaryotic initiation factor-2 (eIF-2alpha). In yeast, an eIF-2alpha kinase, GCN2, functions in translational control in response to amino acid starvation. It is thought that uncharged tRNA that accumulates during amino acid limitation binds to sequences in GCN2 homologous to histidyl-tRNA synthetase (HisRS) enzymes, leading to enhanced kinase catalytic activity. Given that starvation for amino acids also stimulates phosphorylation of eIF-2alpha in mammalian cells, we searched for and identified a GCN2 homologue in mice. We cloned three different cDNAs encoding mouse GCN2 isoforms, derived from a single gene, that vary in their amino-terminal sequences. Like their yeast counterpart, the mouse GCN2 isoforms contain HisRS-related sequences juxtaposed to the kinase catalytic domain. While GCN2 mRNA was found in all mouse tissues examined, the isoforms appear to be differentially expressed. Mouse GCN2 expressed in yeast was found to inhibit growth by hyperphosphorylation of eIF-2alpha, requiring both the kinase catalytic domain and the HisRS-related sequences. Additionally, lysates prepared from yeast expressing mGCN2 were found to phosphorylate recombinant eIF-2alpha substrate. Mouse GCN2 activity in both the in vivo and in vitro assays required the presence of serine-51, the known regulatory phosphorylation site in eIF-2alpha. Together, our studies identify a new mammalian eIF-2alpha kinase, GCN2, that can mediate translational control. PMID:10655230

  19. Eukaryotic elongation factor 2 kinase regulates the synthesis of microtubule-related proteins in neurons.

    PubMed

    Kenney, Justin W; Genheden, Maja; Moon, Kyung-Mee; Wang, Xuemin; Foster, Leonard J; Proud, Christopher G

    2016-01-01

    Modulation of the elongation phase of protein synthesis is important for numerous physiological processes in both neurons and other cell types. Elongation is primarily regulated via eukaryotic elongation factor 2 kinase (eEF2K). However, the consequence of altering eEF2K activity on the synthesis of specific proteins is largely unknown. Using both pharmacological and genetic manipulations of eEF2K combined with two protein-labeling techniques, stable isotope labeling of amino acids in cell culture and bio-orthogonal non-canonical amino acid tagging, we identified a subset of proteins whose synthesis is sensitive to inhibition of eEF2K in murine primary cortical neurons. Gene ontology (GO) analyses indicated that processes related to microtubules are particularly sensitive to eEF2K inhibition. Our findings suggest that eEF2K likely contributes to neuronal function by regulating the synthesis of microtubule-related proteins. Modulation of the elongation phase of protein synthesis is important for numerous physiological processes in neurons. Here, using labeling of new proteins coupled with proteomic techniques in primary cortical neurons, we find that the synthesis of microtubule-related proteins is up-regulated by inhibition of elongation. This suggests that translation elongation is a key regulator of cytoskeletal dynamics in neurons.

  20. Interaction of plant chimeric calcium/calmodulin-dependent protein kinase with a homolog of eukaryotic elongation factor-1alpha

    NASA Technical Reports Server (NTRS)

    Wang, W.; Poovaiah, B. W.

    1999-01-01

    A chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) was previously cloned and characterized in this laboratory. To investigate the biological functions of CCaMK, the yeast two-hybrid system was used to isolate genes encoding proteins that interact with CCaMK. One of the cDNA clones obtained from the screening (LlEF-1alpha1) has high similarity with the eukaryotic elongation factor-1alpha (EF-1alpha). CCaMK phosphorylated LlEF-1alpha1 in a Ca2+/calmodulin-dependent manner. The phosphorylation site for CCaMK (Thr-257) was identified by site-directed mutagenesis. Interestingly, Thr-257 is located in the putative tRNA-binding region of LlEF-1alpha1. An isoform of Ca2+-dependent protein kinase (CDPK) phosphorylated multiple sites of LlEF-1alpha1 in a Ca2+-dependent but calmodulin-independent manner. Unlike CDPK, CCaMK phosphorylated only one site, and this site is different from CDPK phosphorylation sites. This suggests that the phosphorylation of EF-1alpha by these two kinases may have different functional significance. Although the phosphorylation of LlEF-1alpha1 by CCaMK is Ca2+/calmodulin-dependent, in vitro binding assays revealed that CCaMK binds to LlEF-1alpha1 in a Ca2+-independent manner. This was further substantiated by coimmunoprecipitation of CCaMK and EF-1alpha using the protein extract from lily anthers. Dissociation of CCaMK from EF-1alpha by Ca2+ and phosphorylation of EF-1alpha by CCaMK in a Ca2+/calmodulin-dependent manner suggests that these interactions may play a role in regulating the biological functions of EF-1alpha.

  1. Interaction of plant chimeric calcium/calmodulin-dependent protein kinase with a homolog of eukaryotic elongation factor-1alpha

    NASA Technical Reports Server (NTRS)

    Wang, W.; Poovaiah, B. W.

    1999-01-01

    A chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) was previously cloned and characterized in this laboratory. To investigate the biological functions of CCaMK, the yeast two-hybrid system was used to isolate genes encoding proteins that interact with CCaMK. One of the cDNA clones obtained from the screening (LlEF-1alpha1) has high similarity with the eukaryotic elongation factor-1alpha (EF-1alpha). CCaMK phosphorylated LlEF-1alpha1 in a Ca2+/calmodulin-dependent manner. The phosphorylation site for CCaMK (Thr-257) was identified by site-directed mutagenesis. Interestingly, Thr-257 is located in the putative tRNA-binding region of LlEF-1alpha1. An isoform of Ca2+-dependent protein kinase (CDPK) phosphorylated multiple sites of LlEF-1alpha1 in a Ca2+-dependent but calmodulin-independent manner. Unlike CDPK, CCaMK phosphorylated only one site, and this site is different from CDPK phosphorylation sites. This suggests that the phosphorylation of EF-1alpha by these two kinases may have different functional significance. Although the phosphorylation of LlEF-1alpha1 by CCaMK is Ca2+/calmodulin-dependent, in vitro binding assays revealed that CCaMK binds to LlEF-1alpha1 in a Ca2+-independent manner. This was further substantiated by coimmunoprecipitation of CCaMK and EF-1alpha using the protein extract from lily anthers. Dissociation of CCaMK from EF-1alpha by Ca2+ and phosphorylation of EF-1alpha by CCaMK in a Ca2+/calmodulin-dependent manner suggests that these interactions may play a role in regulating the biological functions of EF-1alpha.

  2. Molecular Basis of the Activity and the Regulation of the Eukaryotic-like S/T Protein Kinase PknG from Mycobacterium tuberculosis.

    PubMed

    Lisa, María-Natalia; Gil, Magdalena; André-Leroux, Gwénaëlle; Barilone, Nathalie; Durán, Rosario; Biondi, Ricardo M; Alzari, Pedro M

    2015-06-02

    Tuberculosis remains one of the world's deadliest human diseases, with a high prevalence of antibiotic-resistant Mycobacterium tuberculosis (Mtb) strains. A molecular understanding of processes underlying regulation and adaptation of bacterial physiology may provide novel avenues for the development of antibiotics with unconventional modes of action. Here, we focus on the multidomain S/T protein kinase PknG, a soluble enzyme that controls central metabolism in Actinobacteria and has been linked to Mtb infectivity. Our biochemical and structural studies reveal how different motifs and domains flanking the catalytic core regulate substrate selectivity without significantly affecting the intrinsic kinase activity, whereas a rubredoxin-like domain is shown to downregulate catalysis through specific intramolecular interactions that modulate access to a profound substrate-binding site. Our findings provide the basis for the selective and specific inhibition of PknG, and open new questions about regulation of related bacterial and eukaryotic protein kinases.

  3. Possible roles of protein kinase A in cell motility and excystation of the early diverging eukaryote Giardia lamblia.

    PubMed

    Abel, E S; Davids, B J; Robles, L D; Loflin, C E; Gillin, F D; Chakrabarti, R

    2001-03-30

    Since little is known of how the primitive protozoan parasite, Giardia lamblia, senses and responds to its changing environment, we characterized a giardial protein kinase A (gPKA) catalytic subunit with unusual subcellular localization. Sequence analysis of the 1080-base pair open reading frame shows 48% amino acid identity with the cyclic AMP-dependent kinase from Euglena gracilis. Northern analysis indicated a 1.28- kilobase pair transcript at relatively constant concentrations during growth and encystation. gPKA is autophosphorylated, although amino acid residues corresponding to Thr-197 and Ser-338 of human protein kinase A (PKA) that are important for autophosphorylation are absent. Kinetic analysis of the recombinant PKA showed that ATP and magnesium are preferred over GTP and manganese. Kinase activity of the native PKA has also been detected in crude extracts using kemptide as a substrate. A myristoylated PKA inhibitor, amide 14-22, inhibited excystation with an IC(50) of 3 microm, suggesting an important role of gPKA during differentiation from the dormant cyst form into the active trophozoite. gPKA localizes independently of cell density to the eight flagellar basal bodies between the two nuclei together with centrin, a basal body/centrosome-specific protein. However, localization of gPKA to marginal plates along the intracellular portions of the anterior and caudal pairs of flagella was evident only at low cell density and higher endogenous cAMP concentrations or after refeeding with fresh medium. These data suggest an important role of PKA in trophozoite motility during vegetative growth and the cellular activation of excystation.

  4. Eukaryotic-like Ser/Thr Protein Kinases SpkC/F/K Are Involved in Phosphorylation of GroES in the Cyanobacterium Synechocystis

    PubMed Central

    Zorina, Anna; Stepanchenko, Natalia; Novikova, Galina V.; Sinetova, Maria; Panichkin, Vladimir B.; Moshkov, Igor E.; Zinchenko, Vladislav V.; Shestakov, Sergey V.; Suzuki, Iwane; Murata, Norio; Los, Dmitry A.

    2011-01-01

    Serine/threonine protein kinases (STPKs) are the major participants in intracellular signal transduction in eukaryotes, such as yeasts, fungi, plants, and animals. Genome sequences indicate that these kinases are also present in prokaryotes, such as cyanobacteria. However, their roles in signal transduction in prokaryotes remain poorly understood. We have attempted to identify the roles of STPKs in response to heat stress in the prokaryotic cyanobacterium Synechocystis sp. PCC 6803, which has 12 genes for STPKs. Each gene was individually inactivated to generate a gene-knockout library of STPKs. We applied in vitro Ser/Thr protein phosphorylation and phosphoproteomics and identified the methionyl-tRNA synthetase, large subunit of RuBisCO, 6-phosphogluconate dehydrogenase, translation elongation factor Tu, heat-shock protein GrpE, and small chaperonin GroES as the putative targets for Ser/Thr phosphorylation. The expressed and purified GroES was used as an external substrate to screen the protein extracts of the individual mutants for their Ser/Thr kinase activities. The mutants that lack one of the three protein kinases, SpkC, SpkF, and SpkK, were unable to phosphorylate GroES in vitro, suggesting possible interactions between them towards their substrate. Complementation of the mutated SpkC, SpkF, and SpkK leads to the restoration of the ability of cells to phosphorylate the GroES. This suggests that these three STPKs are organized in a sequential order or a cascade and they work one after another to finally phosphorylate the GroES. PMID:21551175

  5. Eukaryote-like Ser/Thr protein kinase PrkA modulates sporulation via regulating the transcriptional factor σK in Bacillus subtilis

    PubMed Central

    Yan, Jinyuan; Zou, Wei; Fang, Juan; Huang, Xiaowei; Gao, Feng; He, Zeying; Zhang, Keqin; Zhao, Ninghui

    2015-01-01

    Protein kinase A (PrkA), also known as AMP-activated protein kinase, functions as a serine/threonine protein kinase (STPK), has been shown to be involved in a variety of important biologic processes, including pathogenesis of many important diseases in mammals. However, the biological functions of PrkA are less known in prokaryote cells. Here, we explored the function of PrkA as well as its underlying molecular mechanisms using the model bacterium Bacillus subtilis168. When PrkA is inhibited by 9-β-D-arabinofuranosyladenine (ara-A) in the wild type strain or deleted in the ΔprkA mutant strain, we observed sporulation defects in B. subtilis 168, suggesting that PrkA functions as a sporulation-related protein. Transcriptional analysis using the lacZ reporter gene demonstrated that deletion of prkA significantly reduced the expression of the transcriptional factor σK and its downstream genes. Complementation of sigK gene in prkA knockout mutant partially rescued the phenotype of ΔprkA, further supporting the hypothesis that the decreased σK expression should be one of the reasons for the sporulation defect resulting from prkA disruption. Finally, our data confirmed that Hpr (ScoC) negatively controlled the expression of transcriptional factor σK, and thus PrkA accelerated sporulation and the expression of σK by suppression of Hpr (ScoC). Taken together, our study discovered a novel function of the eukaryotic-like STPK PrkA in spore development as well as its underlying molecular mechanism in B. subtilis. PMID:25983726

  6. Identification of autophosphorylation sites in eukaryotic elongation factor-2 kinase

    PubMed Central

    Pyr Dit Ruys, Sébastien; Wang, Xuemin; Smith, Ewan M.; Herinckx, Gaëtan; Hussain, Nusrat; Rider, Mark H.; Vertommen, Didier; Proud, Christopher G.

    2012-01-01

    eEF2K [eEF2 (eukaryotic elongation factor 2) kinase] phosphorylates and inactivates the translation elongation factor eEF2. eEF2K is not a member of the main eukaryotic protein kinase superfamily, but instead belongs to a small group of so-called α-kinases. The activity of eEF2K is normally dependent upon Ca2+ and calmodulin. eEF2K has previously been shown to undergo autophosphorylation, the stoichiometry of which suggested the existence of multiple sites. In the present study we have identified several autophosphorylation sites, including Thr348, Thr353, Ser366 and Ser445, all of which are highly conserved among vertebrate eEF2Ks. We also identified a number of other sites, including Ser78, a known site of phosphorylation, and others, some of which are less well conserved. None of the sites lies in the catalytic domain, but three affect eEF2K activity. Mutation of Ser78, Thr348 and Ser366 to a non-phosphorylatable alanine residue decreased eEF2K activity. Phosphorylation of Thr348 was detected by immunoblotting after transfecting wild-type eEF2K into HEK (human embryonic kidney)-293 cells, but not after transfection with a kinase-inactive construct, confirming that this is indeed a site of autophosphorylation. Thr348 appears to be constitutively autophosphorylated in vitro. Interestingly, other recent data suggest that the corresponding residue in other α-kinases is also autophosphorylated and contributes to the activation of these enzymes [Crawley, Gharaei, Ye, Yang, Raveh, London, Schueler-Furman, Jia and Cote (2011) J. Biol. Chem. 286, 2607–2616]. Ser366 phosphorylation was also detected in intact cells, but was still observed in the kinase-inactive construct, demonstrating that this site is phosphorylated not only autocatalytically but also in trans by other kinases. PMID:22216903

  7. AMP-activated Protein Kinase Up-regulates Mitogen-activated Protein (MAP) Kinase-interacting Serine/Threonine Kinase 1a-dependent Phosphorylation of Eukaryotic Translation Initiation Factor 4E.

    PubMed

    Zhu, Xiaoqing; Dahlmans, Vivian; Thali, Ramon; Preisinger, Christian; Viollet, Benoit; Voncken, J Willem; Neumann, Dietbert

    2016-08-12

    AMP-activated protein kinase (AMPK) is a molecular energy sensor that acts to sustain cellular energy balance. Although AMPK is implicated in the regulation of a multitude of ATP-dependent cellular processes, exactly how these processes are controlled by AMPK as well as the identity of AMPK targets and pathways continues to evolve. Here we identify MAP kinase-interacting serine/threonine protein kinase 1a (MNK1a) as a novel AMPK target. Specifically, we show AMPK-dependent Ser(353) phosphorylation of the human MNK1a isoform in cell-free and cellular systems. We show that AMPK and MNK1a physically interact and that in vivo MNK1a-Ser(353) phosphorylation requires T-loop phosphorylation, in good agreement with a recently proposed structural regulatory model of MNK1a. Our data suggest a physiological role for MNK1a-Ser(353) phosphorylation in regulation of the MNK1a kinase, which correlates with increased eIF4E phosphorylation in vitro and in vivo. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. IMPACT is a developmentally regulated protein in neurons that opposes the eukaryotic initiation factor 2α kinase GCN2 in the modulation of neurite outgrowth.

    PubMed

    Roffé, Martín; Hajj, Glaucia N M; Azevedo, Hátylas F; Alves, Viviane S; Castilho, Beatriz A

    2013-04-12

    The product of the mouse Imprinted and Ancient gene, IMPACT, is preferentially expressed in neurons. We have previously shown that IMPACT overexpression inhibits the activation of the protein kinase GCN2, which signals amino acid starvation. GCN2 phosphorylates the α-subunit of eukaryotic translation initiation factor 2 (eIF2α), resulting in inhibition of general protein synthesis but increased translation of specific messages, such as ATF4. GCN2 is also involved in the regulation of neuronal functions, controlling synaptic plasticity, memory, and feeding behavior. We show here that IMPACT abundance increases during differentiation of neurons and neuron-like N2a cells, whereas GCN2 displays lowered activation levels. Upon differentiation, IMPACT associates with translating ribosomes, enhances translation initiation, and down-regulates the expression of ATF4. We further show that endogenous IMPACT promotes neurite outgrowth whereas GCN2 is a strong inhibitor of spontaneous neuritogenesis. Together, these results uncover the participation of the GCN2-IMPACT module of translational regulation in a highly controlled step in the development of the nervous system.

  9. Conserved initiator proteins in eukaryotes.

    PubMed

    Gavin, K A; Hidaka, M; Stillman, B

    1995-12-08

    The origin recognition complex (ORC), a multisubunit protein identified in Saccharomyces cerevisiae, binds to chromosomal replicators and is required for the initiation of cellular DNA replication. Complementary DNAs (cDNAs) encoding proteins related to the two largest subunits of ORC were cloned from various eukaryotes. The cDNAs encoding proteins related to S. cerevisiae Orc1p were cloned from the budding yeast Kluyveromyces lactis, the fission yeast Schizosaccharomyces pombe, and human cells. These proteins show similarity to regulators of the S and M phases of the cell cycle. Genetic analysis of orc1+ from S. pombe reveals that it is essential for cell viability. The cDNAs encoding proteins related to S. cerevisiae Orc2p were cloned from Arabidopsis thaliana, Caenorhabditis elegans, and human cells. The human ORC-related proteins interact in vivo to form a complex. These studies studies suggest that ORC subunits are conserved and that the role of ORC is a general feature of eukaryotic DNA replication.

  10. Induction of Apoptosis by Double-Stranded-RNA-Dependent Protein Kinase (PKR) Involves the α Subunit of Eukaryotic Translation Initiation Factor 2 and NF-κB

    PubMed Central

    Gil, Jesús; Alcamí, José; Esteban, Mariano

    1999-01-01

    The double-stranded (ds) RNA-dependent protein kinase (PKR) is a key mediator of antiviral effects of interferon (IFN) and an active player in apoptosis induced by different stimuli. The translation initiation factor eIF-2α (α subunit of eukaryotic translation initiation factor 2) and IκBα, the inhibitor of the transcription factor NF-κB, have been proposed as downstream mediators of PKR effects. To evaluate the involvement of NF-κB and eIF-2α in the induction of apoptosis by PKR, we have used vaccinia virus (VV) recombinants that inducibly express PKR concomitantly with a dominant negative mutant of eIF-2α or a repressor form of IκBα. We found that while expression of PKR by a VV vector resulted in extensive inhibition of protein synthesis and induction of apoptosis, coexpression of PKR with a dominant negative mutant of eIF-2α (Ser-51→Ala) reversed both the PKR-mediated translational block and PKR-induced apoptosis. Coexpression of PKR with a repressor form of IκBα (Ser-32,36-Ala) also leads to the inhibition of apoptosis by abolishing NF-κB induction, while translation remains blocked. Treating cells with two different proteasome inhibitors which block IκBα degradation, prevented PKR-induced apoptosis, supporting results from coexpression studies. Biochemical analysis and transient assays revealed that PKR expression by a VV vector induced NF-κB binding and transactivation. In addition, upregulation of Fas mRNA transcription occurred during PKR activation. Our findings provide direct evidence for the involvement of eIF-2α and NF-κB in the induction of apoptosis by PKR. PMID:10373514

  11. Genetic interactions of yeast eukaryotic translation initiation factor 5A (eIF5A) reveal connections to poly(A)-binding protein and protein kinase C signaling.

    PubMed Central

    Valentini, Sandro R; Casolari, Jason M; Oliveira, Carla C; Silver, Pamela A; McBride, Anne E

    2002-01-01

    The highly conserved eukaryotic translation initiation factor eIF5A has been proposed to have various roles in the cell, from translation to mRNA decay to nuclear protein export. To further our understanding of this essential protein, three temperature-sensitive alleles of the yeast TIF51A gene have been characterized. Two mutant eIF5A proteins contain mutations in a proline residue at the junction between the two eIF5A domains and the third, strongest allele encodes a protein with a single mutation in each domain, both of which are required for the growth defect. The stronger tif51A alleles cause defects in degradation of short-lived mRNAs, supporting a role for this protein in mRNA decay. A multicopy suppressor screen revealed six genes, the overexpression of which allows growth of a tif51A-1 strain at high temperature; these genes include PAB1, PKC1, and PKC1 regulators WSC1, WSC2, and WSC3. Further results suggest that eIF5A may also be involved in ribosomal synthesis and the WSC/PKC1 signaling pathway for cell wall integrity or related processes. PMID:11861547

  12. Redox Regulation of Protein Kinases

    PubMed Central

    Truong, Thu H.; Carroll, Kate S.

    2015-01-01

    Protein kinases represent one of the largest families of genes found in eukaryotes. Kinases mediate distinct cellular processes ranging from proliferation, differentiation, survival, and apoptosis. Ligand-mediated activation of receptor kinases can lead to the production of endogenous H2O2 by membrane-bound NADPH oxidases. In turn, H2O2 can be utilized as a secondary messenger in signal transduction pathways. This review presents an overview of the molecular mechanisms involved in redox regulation of protein kinases and its effects on signaling cascades. In the first half, we will focus primarily on receptor tyrosine kinases (RTKs), whereas the latter will concentrate on downstream non-receptor kinases involved in relaying stimulant response. Select examples from the literature are used to highlight the functional role of H2O2 regarding kinase activity, as well as the components involved in H2O2 production and regulation during cellular signaling. In addition, studies demonstrating direct modulation of protein kinases by H2O2 through cysteine oxidation will be emphasized. Identification of these redox-sensitive residues may help uncover signaling mechanisms conserved within kinase subfamilies. In some cases, these residues can even be exploited as targets for the development of new therapeutics. Continued efforts in this field will further basic understanding of kinase redox regulation, and delineate the mechanisms involved in physiologic and pathological H2O2 responses. PMID:23639002

  13. Complementing the Eukaryotic Protein Interactome

    PubMed Central

    Pesch, Robert; Zimmer, Ralf

    2013-01-01

    Protein interaction networks are important for the understanding of regulatory mechanisms, for the explanation of experimental data and for the prediction of protein functions. Unfortunately, most interaction data is available only for model organisms. As a possible remedy, the transfer of interactions to organisms of interest is common practice, but it is not clear when interactions can be transferred from one organism to another and, thus, the confidence in the derived interactions is low. Here, we propose to use a rich set of features to train Random Forests in order to score transferred interactions. We evaluated the transfer from a range of eukaryotic organisms to S. cerevisiae using orthologs. Directly transferred interactions to S. cerevisiae are on average only 24% consistent with the current S. cerevisiae interaction network. By using commonly applied filter approaches the transfer precision can be improved, but at the cost of a large decrease in the number of transferred interactions. Our Random Forest approach uses various features derived from both the target and the source network as well as the ortholog annotations to assign confidence values to transferred interactions. Thereby, we could increase the average transfer consistency to 85%, while still transferring almost 70% of all correctly transferable interactions. We tested our approach for the transfer of interactions to other species and showed that our approach outperforms competing methods for the transfer of interactions to species where no experimental knowledge is available. Finally, we applied our predictor to score transferred interactions to 83 targets species and we were able to extend the available interactome of B. taurus, M. musculus and G. gallus with over 40,000 interactions each. Our transferred interaction networks are publicly available via our web interface, which allows to inspect and download transferred interaction sets of different sizes, for various species, and at specified

  14. Protein Kinases and Addiction

    PubMed Central

    Lee, Anna M.; Messing, Robert O.

    2011-01-01

    Although drugs of abuse have different chemical structures and interact with different protein targets, all appear to usurp common neuronal systems that regulate reward and motivation. Addiction is a complex disease that is thought to involve drug-induced changes in synaptic plasticity due to alterations in cell signaling, gene transcription, and protein synthesis. Recent evidence suggests that drugs of abuse interact with and change a common network of signaling pathways that include a subset of specific protein kinases. The best studied of these kinases are reviewed here and include extracellular signal-regulated kinase, cAMP-dependent protein kinase, cyclin-dependent protein kinase 5, protein kinase C, calcium/calmodulin-dependent protein kinase II, and Fyn tyrosine kinase. These kinases have been implicated in various aspects of drug addiction including acute drug effects, drug self-administration, withdrawal, reinforcement, sensitization, and tolerance. Identifying protein kinase substrates and signaling pathways that contribute to the addicted state may provide novel approaches for new pharma-cotherapies to treat drug addiction. PMID:18991950

  15. Bacterial proteins pinpoint a single eukaryotic root

    PubMed Central

    Derelle, Romain; Torruella, Guifré; Klimeš, Vladimír; Brinkmann, Henner; Kim, Eunsoo; Vlček, Čestmír; Lang, B. Franz; Eliáš, Marek

    2015-01-01

    The large phylogenetic distance separating eukaryotic genes and their archaeal orthologs has prevented identification of the position of the eukaryotic root in phylogenomic studies. Recently, an innovative approach has been proposed to circumvent this issue: the use as phylogenetic markers of proteins that have been transferred from bacterial donor sources to eukaryotes, after their emergence from Archaea. Using this approach, two recent independent studies have built phylogenomic datasets based on bacterial sequences, leading to different predictions of the eukaryotic root. Taking advantage of additional genome sequences from the jakobid Andalucia godoyi and the two known malawimonad species (Malawimonas jakobiformis and Malawimonas californiana), we reanalyzed these two phylogenomic datasets. We show that both datasets pinpoint the same phylogenetic position of the eukaryotic root that is between “Unikonta” and “Bikonta,” with malawimonad and collodictyonid lineages on the Unikonta side of the root. Our results firmly indicate that (i) the supergroup Excavata is not monophyletic and (ii) the last common ancestor of eukaryotes was a biflagellate organism. Based on our results, we propose to rename the two major eukaryotic groups Unikonta and Bikonta as Opimoda and Diphoda, respectively. PMID:25646484

  16. Role of eukaryotic-like serine/threonine kinases in bacterial cell division and morphogenesis.

    PubMed

    Manuse, Sylvie; Fleurie, Aurore; Zucchini, Laure; Lesterlin, Christian; Grangeasse, Christophe

    2016-01-01

    Bacteria possess a repertoire of versatile protein kinases modulating diverse aspects of their physiology by phosphorylating proteins on various amino acids including histidine, cysteine, aspartic acid, arginine, serine, threonine and tyrosine. One class of membrane serine/threonine protein kinases possesses a catalytic domain sharing a common fold with eukaryotic protein kinases and an extracellular mosaic domain found in bacteria only, named PASTA for 'Penicillin binding proteins And Serine/Threonine kinase Associated'. Over the last decade, evidence has been accumulating that these protein kinases are involved in cell division, morphogenesis and developmental processes in Firmicutes and Actinobacteria. However, observations differ from one species to another suggesting that a general mechanism of activation of their kinase activity is unlikely and that species-specific regulation of cell division is at play. In this review, we survey the latest research on the structural aspects and the cellular functions of bacterial serine/threonine kinases with PASTA motifs to illustrate the diversity of the regulatory mechanisms controlling bacterial cell division and morphogenesis.

  17. Teaching resources. Protein kinases.

    PubMed

    Caplan, Avrom

    2005-02-22

    This Teaching Resource provides lecture notes and slides for a class covering the structure and function of protein kinases and is part of the course "Cell Signaling Systems: A Course for Graduate Students." The lecture begins with a discussion of the genomics and evolutionary relationships among kinases and then proceeds to describe the structure-function relationships of specific kinases, the molecular mechanisms underlying substrate specificity, and selected issues in regulation of kinase activity.

  18. Energizing eukaryotic cell-free protein synthesis with glucose metabolism.

    PubMed

    Anderson, Mark J; Stark, Jessica C; Hodgman, C Eric; Jewett, Michael C

    2015-07-08

    Eukaryotic cell-free protein synthesis (CFPS) is limited by the dependence on costly high-energy phosphate compounds and exogenous enzymes to power protein synthesis (e.g., creatine phosphate and creatine kinase, CrP/CrK). Here, we report the ability to use glucose as a secondary energy substrate to regenerate ATP in a Saccharomyces cerevisiae crude extract CFPS platform. We observed synthesis of 3.64±0.35 μg mL(-1) active luciferase in batch reactions with 16 mM glucose and 25 mM phosphate, resulting in a 16% increase in relative protein yield (μg protein/$ reagents) compared to the CrP/CrK system. Our demonstration provides the foundation for development of cost-effective eukaryotic CFPS platforms.

  19. An Asymmetrically Balanced Organization of Kinases versus Phosphatases across Eukaryotes Determines Their Distinct Impacts

    PubMed Central

    Shemesh, Netta; Ziv-Ukelson, Michal; Ben-Zvi, Anat

    2017-01-01

    Protein phosphorylation underlies cellular response pathways across eukaryotes and is governed by the opposing actions of phosphorylating kinases and de-phosphorylating phosphatases. While kinases and phosphatases have been extensively studied, their organization and the mechanisms by which they balance each other are not well understood. To address these questions we performed quantitative analyses of large-scale 'omics' datasets from yeast, fly, plant, mouse and human. We uncovered an asymmetric balance of a previously-hidden scale: Each organism contained many different kinase genes, and these were balanced by a small set of highly abundant phosphatase proteins. Kinases were much more responsive to perturbations at the gene and protein levels. In addition, kinases had diverse scales of phenotypic impact when manipulated. Phosphatases, in contrast, were stable, highly robust and flatly organized, with rather uniform impact downstream. We validated aspects of this organization experimentally in nematode, and supported additional aspects by theoretic analysis of the dynamics of protein phosphorylation. Our analyses explain the empirical bias in the protein phosphorylation field toward characterization and therapeutic targeting of kinases at the expense of phosphatases. We show quantitatively and broadly that this is not only a historical bias, but stems from wide-ranging differences in their organization and impact. The asymmetric balance between these opposing regulators of protein phosphorylation is also common to opposing regulators of two other post-translational modification systems, suggesting its fundamental value. PMID:28135269

  20. Expanding the archaellum regulatory network - the eukaryotic protein kinases ArnC and ArnD influence motility of Sulfolobus acidocaldarius.

    PubMed

    Hoffmann, Lena; Schummer, Andreas; Reimann, Julia; Haurat, Maria F; Wilson, Amanda J; Beeby, Morgan; Warscheid, Bettina; Albers, Sonja-V

    2017-02-01

    Expression of the archaellum, the archaeal-type IV pilus-like rotating motility structure is upregulated under nutrient limitation. This is controlled by a network of regulators, called the archaellum regulatory network (arn). Several of the components of this network in Sulfolobus acidocaldarius can be phosphorylated, and the deletion of the phosphatase PP2A results in strongly increased motility during starvation, indicating a role for phosphorylation in the regulation of motility. Analysis of the motility of different protein kinase deletion strains revealed that deletion of saci_0965, saci_1181, and saci_1193 resulted in reduced motility, whereas the deletion of saci_1694 resulted in hypermotility. Here ArnC (Saci_1193) and ArnD (Saci_1694) are characterized. Purified ArnC and ArnD phosphorylate serine and threonine residues in the C-terminus of the repressor ArnB. arnC is upregulated in starvation medium, whereas arnD is constitutively expressed. However, while differences in the expression and levels of flaB were observed in the ΔarnD strain during growth under rich conditions, under nutrient limiting conditions the ΔarnC and ΔarnD strains showed no large differences in the expression levels of the archaellum or of the studied regulators. This suggests that next to the regulation via the archaellum regulatory network additional regulatory mechanisms of expression and/or activity of the archaellum exist.

  1. Activation of NADPH oxidase 4 in the endoplasmic reticulum promotes cardiomyocyte autophagy and survival during energy stress through the protein kinase RNA-activated-like endoplasmic reticulum kinase/eukaryotic initiation factor 2α/activating transcription factor 4 pathway.

    PubMed

    Sciarretta, Sebastiano; Zhai, Peiyong; Shao, Dan; Zablocki, Daniela; Nagarajan, Narayani; Terada, Lance S; Volpe, Massimo; Sadoshima, Junichi

    2013-11-08

    Autophagy is an essential survival mechanism during energy stress in the heart. Oxidative stress is activated by energy stress, but its role in mediating autophagy is poorly understood. NADPH oxidase (Nox) 4 is an enzyme that generates reactive oxygen species (ROS) at intracellular membranes. Whether Nox4 acts as a sensor of energy stress to mediate activation of autophagy is unknown. We investigated whether Nox4 is involved in the regulation of autophagy and cell survival during energy stress in cardiomyocytes. Production of ROS in cardiomyocytes was increased during glucose deprivation (GD) in a Nox4-dependent manner. Protein levels and the ROS-producing activity of Nox4 were increased in the endoplasmic reticulum (ER), but not in mitochondria, in response to GD. Selective knockdown of Nox4, but not Nox2, or selective reduction of ROS in the ER with ER-targeted catalase, but not mitochondria-targeted perioxiredoxin 3, abrogated GD-induced autophagy. Nox4 promoted autophagy during GD through activation of the protein kinase RNA-activated-like ER kinase pathway by suppression of prolyl hydroxylase 4. The decrease in cell survival during GD in the presence of Nox4 knockdown was rescued by reactivation of autophagy by Atg7 overexpression, indicating that the effect of Nox4 on cell survival is critically mediated through regulation of autophagy. Nox4 was activated during fasting and prolonged ischemia in the mouse heart, where Nox4 is also required for autophagy activation and cardioprotection. Nox4 critically mediates autophagy in response to energy stress in cardiomyocytes by eliciting ROS in the ER and stimulating the protein kinase RNA-activated-like ER kinase signaling pathway.

  2. A framework for classification of prokaryotic protein kinases.

    PubMed

    Tyagi, Nidhi; Anamika, Krishanpal; Srinivasan, Narayanaswamy

    2010-05-26

    Overwhelming majority of the Serine/Threonine protein kinases identified by gleaning archaeal and eubacterial genomes could not be classified into any of the well known Hanks and Hunter subfamilies of protein kinases. This is owing to the development of Hanks and Hunter classification scheme based on eukaryotic protein kinases which are highly divergent from their prokaryotic homologues. A large dataset of prokaryotic Serine/Threonine protein kinases recognized from genomes of prokaryotes have been used to develop a classification framework for prokaryotic Ser/Thr protein kinases. We have used traditional sequence alignment and phylogenetic approaches and clustered the prokaryotic kinases which represent 72 subfamilies with at least 4 members in each. Such a clustering enables classification of prokaryotic Ser/Thr kinases and it can be used as a framework to classify newly identified prokaryotic Ser/Thr kinases. After series of searches in a comprehensive sequence database we recognized that 38 subfamilies of prokaryotic protein kinases are associated to a specific taxonomic level. For example 4, 6 and 3 subfamilies have been identified that are currently specific to phylum proteobacteria, cyanobacteria and actinobacteria respectively. Similarly subfamilies which are specific to an order, sub-order, class, family and genus have also been identified. In addition to these, we also identify organism-diverse subfamilies. Members of these clusters are from organisms of different taxonomic levels, such as archaea, bacteria, eukaryotes and viruses. Interestingly, occurrence of several taxonomic level specific subfamilies of prokaryotic kinases contrasts with classification of eukaryotic protein kinases in which most of the popular subfamilies of eukaryotic protein kinases occur diversely in several eukaryotes. Many prokaryotic Ser/Thr kinases exhibit a wide variety of modular organization which indicates a degree of complexity and protein-protein interactions in the

  3. A Framework for Classification of Prokaryotic Protein Kinases

    PubMed Central

    Tyagi, Nidhi; Anamika, Krishanpal; Srinivasan, Narayanaswamy

    2010-01-01

    Background Overwhelming majority of the Serine/Threonine protein kinases identified by gleaning archaeal and eubacterial genomes could not be classified into any of the well known Hanks and Hunter subfamilies of protein kinases. This is owing to the development of Hanks and Hunter classification scheme based on eukaryotic protein kinases which are highly divergent from their prokaryotic homologues. A large dataset of prokaryotic Serine/Threonine protein kinases recognized from genomes of prokaryotes have been used to develop a classification framework for prokaryotic Ser/Thr protein kinases. Methodology/Principal Findings We have used traditional sequence alignment and phylogenetic approaches and clustered the prokaryotic kinases which represent 72 subfamilies with at least 4 members in each. Such a clustering enables classification of prokaryotic Ser/Thr kinases and it can be used as a framework to classify newly identified prokaryotic Ser/Thr kinases. After series of searches in a comprehensive sequence database we recognized that 38 subfamilies of prokaryotic protein kinases are associated to a specific taxonomic level. For example 4, 6 and 3 subfamilies have been identified that are currently specific to phylum proteobacteria, cyanobacteria and actinobacteria respectively. Similarly subfamilies which are specific to an order, sub-order, class, family and genus have also been identified. In addition to these, we also identify organism-diverse subfamilies. Members of these clusters are from organisms of different taxonomic levels, such as archaea, bacteria, eukaryotes and viruses. Conclusion/Significance Interestingly, occurrence of several taxonomic level specific subfamilies of prokaryotic kinases contrasts with classification of eukaryotic protein kinases in which most of the popular subfamilies of eukaryotic protein kinases occur diversely in several eukaryotes. Many prokaryotic Ser/Thr kinases exhibit a wide variety of modular organization which indicates a

  4. Protein acetylation in archaea, bacteria, and eukaryotes.

    PubMed

    Soppa, Jörg

    2010-09-16

    Proteins can be acetylated at the alpha-amino group of the N-terminal amino acid (methionine or the penultimate amino acid after methionine removal) or at the epsilon-amino group of internal lysines. In eukaryotes the majority of proteins are N-terminally acetylated, while this is extremely rare in bacteria. A variety of studies about N-terminal acetylation in archaea have been reported recently, and it was revealed that a considerable fraction of proteins is N-terminally acetylated in haloarchaea and Sulfolobus, while this does not seem to apply for methanogenic archaea. Many eukaryotic proteins are modified by differential internal acetylation, which is important for a variety of processes. Until very recently, only two bacterial proteins were known to be acetylation targets, but now 125 acetylation sites are known for E. coli. Knowledge about internal acetylation in archaea is extremely limited; only two target proteins are known, only one of which--Alba--was used to study differential acetylation. However, indications accumulate that the degree of internal acetylation of archaeal proteins might be underestimated, and differential acetylation has been shown to be essential for the viability of haloarchaea. Focused proteomic approaches are needed to get an overview of the extent of internal protein acetylation in archaea.

  5. Autophagy and protein kinase RNA-like endoplasmic reticulum kinase (PERK)/eukaryotic initiation factor 2 alpha kinase (eIF2α) pathway protect ovarian cancer cells from metformin-induced apoptosis.

    PubMed

    Moon, Hee-Sun; Kim, Boyun; Gwak, HyeRan; Suh, Dong Hoon; Song, Yong Sang

    2016-04-01

    Metformin, an oral biguanide for the treatment of type II diabetes, has been shown to have anticancer effects in ovarian cancer. Energy starvation induced by metformin causes endoplasmic reticulum stress-mediated unfolded protein response (UPR) and autophagy. UPR and autophagy act as a survival or death mechanism in cells. In this study, we observed that metformin-induced apoptosis was relieved by autophagy and the PERK/eIF2α pathway in ovarian cancer cells, but not in peripheral blood mononuclear cells (PBMC) or 'normal' ovarian surface epithelial cells (OSE). Increased PARP cleavage and increased LC3B-II with ATG5-ATG12 complex suggested the induction of apoptosis and autophagy, respectively, in metformin-treated ovarian cancer cells. Accumulation of acidic vacuoles in the cytoplasm and downregulation of p62 further supported late-stage autophagy. Interestingly, metformin induced interdependent activation between autophagy and the UPR, especially the PERK/eIF2α pathway. Inhibition of autophagy-induced PERK inhibition, and vice versa, were demonstrated using small molecular inhibitors (PERK inhibitor I, GSK2606414; autophagy inhibitor, 3-MA, and BafA1). Moreover, autophagy and PERK activation protected ovarian cancer cells against metformin-induced apoptosis. Metformin treatment in the presence of inhibitors of PERK and autophagy, however, had no cytotoxic effects on OSE or PBMC. In conclusion, these results suggest that inhibition of autophagy and PERK can enhance the selective anticancer effects of metformin on ovarian cancer cells. © 2015 Wiley Periodicals, Inc.

  6. Conserved herpesvirus protein kinases

    PubMed Central

    Gershburg, Edward; Pagano, Joseph S.

    2008-01-01

    Conserved herpesviral protein kinases (CHPKs) are a group of enzymes conserved throughout all subfamilies of Herpesviridae. Members of this group are serine/threonine protein kinases that are likely to play a conserved role in viral infection by interacting with common host cellular and viral factors; however along with a conserved role, individual kinases may have unique functions in the context of viral infection in such a way that they are only partially replaceable even by close homologues. Recent studies demonstrated that CHPKs are crucial for viral infection and suggested their involvement in regulation of numerous processes at various infection steps (primary infection, nuclear egress, tegumentation), although the mechanisms of this regulation remain unknown. Notwithstanding, recent advances in discovery of new CHPK targets, and studies of CHPK knockout phenotypes have raised their attractiveness as targets for antiviral therapy. A number of compounds have been shown to inhibit the activity of human cytomegalovirus (HCMV)-encoded UL97 protein kinase and exhibit a pronounced antiviral effect, although the same compounds are inactive against Epstein-Barr Virus (EBV)-encoded protein kinase BGLF4, illustrating the fact that low homology between the members of this group complicates development of compounds targeting the whole group, and suggesting that individualized, structure-based inhibitor design will be more effective. Determination of CHPK structures will greatly facilitate this task. PMID:17881303

  7. Involvement of the Eukaryote-Like Kinase-Phosphatase System and a Protein That Interacts with Penicillin-Binding Protein 5 in Emergence of Cephalosporin Resistance in Cephalosporin-Sensitive Class A Penicillin-Binding Protein Mutants in Enterococcus faecium

    PubMed Central

    Desbonnet, Charlene; Tait-Kamradt, Amelia; Garcia-Solache, Monica; Dunman, Paul; Coleman, Jeffrey; Arthur, Michel

    2016-01-01

    ABSTRACT The intrinsic resistance of Enterococcus faecium to ceftriaxone and cefepime (here referred to as “cephalosporins”) is reliant on the presence of class A penicillin-binding proteins (Pbps) PbpF and PonA. Mutants lacking these Pbps exhibit cephalosporin susceptibility that is reversible by exposure to penicillin and by selection on cephalosporin-containing medium. We selected two cephalosporin-resistant mutants (Cro1 and Cro2) of class A Pbp-deficient E. faecium CV598. Genome analysis revealed changes in the serine-threonine kinase Stk in Cro1 and a truncation in the associated phosphatase StpA in Cro2 whose respective involvements in resistance were confirmed in separate complementation experiments. In an additional effort to identify proteins linked to cephalosporin resistance, we performed tandem affinity purification using Pbp5 as bait in penicillin-exposed E. faecium; these experiments yielded a protein designated Pbp5-associated protein (P5AP). Transcription of the P5AP gene was increased after exposure to penicillin in wild-type strains and in Cro2 and suppressed in Cro2 complemented with the wild-type stpA. Transformation of class A Pbp-deficient strains with the plasmid-carried P5AP gene conferred cephalosporin resistance. These data suggest that Pbp5-associated cephalosporin resistance in E. faecium devoid of typical class A Pbps is related to the presence of P5AP, whose expression is influenced by the activity of the serine-threonine phosphatase/kinase system. PMID:27048803

  8. Comparative analysis of human and bovine protein kinases reveals unique relationship and functional diversity.

    PubMed

    Kabir, Nuzhat N; Kazi, Julhash U

    2011-10-01

    Reversible protein phosphorylation by protein kinases and phosphatases is a common event in various cellular processes. The eukaryotic protein kinase superfamily, which is one of the largest superfamilies of eukaryotic proteins, plays several roles in cell signaling and diseases. We identified 482 eukaryotic protein kinases and 39 atypical protein kinases in the bovine genome, by searching publicly accessible genetic-sequence databases. Bovines have 512 putative protein kinases, each orthologous to a human kinase. Whereas orthologous kinase pairs are, on an average, 90.6% identical, orthologous kinase catalytic domain pairs are, on an average, 95.9% identical at the amino acid level. This bioinformatic study of bovine protein kinases provides a suitable framework for further characterization of their functional and structural properties.

  9. AMP-activated protein kinase--an archetypal protein kinase cascade?

    PubMed

    Hardie, D G; MacKintosh, R W

    1992-10-01

    Mammalian AMP-activated protein kinase is the central component of a protein kinase cascade which inactivates three key enzymes involved in the synthesis or release of free fatty acids and cholesterol inside the cell. The kinase cascade is activated by elevation of AMP, and perhaps also by fatty acid and cholesterol metabolites. The system may fulfil a protective function, preventing damage caused by depletion of ATP or excessive intracellular release of free lipids, a type of stress response. Recent evidence suggests that it may have been in existence for at least a billion years, since a very similar protein kinase cascade is present in higher plants. This system therefore represents an early eukaryotic protein kinase cascade, which is unique in that it is regulated by intracellular metabolites rather than extracellular signals or cell cycle events.

  10. Mechanism and regulation of eukaryotic protein synthesis.

    PubMed Central

    Merrick, W C

    1992-01-01

    This review presents a description of the numerous eukaryotic protein synthesis factors and their apparent sequential utilization in the processes of initiation, elongation, and termination. Additionally, the rare use of reinitiation and internal initiation is discussed, although little is known biochemically about these processes. Subsequently, control of translation is addressed in two different settings. The first is the global control of translation, which is effected by protein phosphorylation. The second is a series of specific mRNAs for which there is a direct and unique regulation of the synthesis of the gene product under study. Other examples of translational control are cited but not discussed, because the general mechanism for the regulation is unknown. Finally, as is often seen in an active area of investigation, there are several observations that cannot be readily accommodated by the general model presented in the first part of the review. Alternate explanations and various lines of experimentation are proposed to resolve these apparent contradictions. PMID:1620067

  11. Leishmania MAP kinases--familiar proteins in an unusual context.

    PubMed

    Wiese, Martin

    2007-08-01

    Mitogen-activated protein kinases are well-known mediators of signal transduction of higher eukaryotes regulating important processes like proliferation, differentiation, stress response and apoptosis. In Leishmania, the typical three-tiered module of MAP kinase signal transduction pathways is present. However, typical activators like cell surface receptors and substrates such as RNA polymerase II transcription factors are missing. Here, I describe the set of 15 putative mitogen-activated protein kinases encoded in the Leishmania genome and discuss their potential function.

  12. Structural relationship between a bacterial developmental protein and eukaryotic PP2C protein phosphatases.

    PubMed

    Adler, E; Donella-Deana, A; Arigoni, F; Pinna, L A; Stragler, P

    1997-01-01

    Bacillus subtilis SpoIIE is a Ser protein phosphatase whose action on the phosphoprotein SpoIIAA triggers the cell type-specific activation of a sporulation transcription factor. Here we report that SpoIIE displays sequence similarity to the PP2C family of eukaryotic Ser/Thr protein phosphatases, and that residues common to these proteins are required for the function of both SpoIIE and TPD1, a yeast PP2C. These findings suggest that SpoIIE and the PP2C protein phosphatases are structurally related, and reveal a striking formal similarity between the SpoIIAA regulatory circuit and that of mammalian mitochondrial pyruvate dehydrogenase. This similarity may reflect an evolutionarily conserved mechanism of biological regulation based on the interplay of His protein kinase-like Ser kinases and PP2C-like protein phosphatases.

  13. Evolutionary history of phosphatidylinositol- 3-kinases: ancestral origin in eukaryotes and complex duplication patterns.

    PubMed

    Philippon, Héloïse; Brochier-Armanet, Céline; Perrière, Guy

    2015-10-19

    Phosphatidylinositol-3-kinases (PI3Ks) are a family of eukaryotic enzymes modifying phosphoinositides in phosphatidylinositols-3-phosphate. Located upstream of the AKT/mTOR signalling pathway, PI3Ks activate secondary messengers of extracellular signals. They are involved in many critical cellular processes such as cell survival, angiogenesis and autophagy. PI3K family is divided into three classes, including 14 human homologs. While class II enzymes are composed of a single catalytic subunit, class I and III also contain regulatory subunits. Here we present an in-depth phylogenetic analysis of all PI3K proteins. We confirmed that PI3K catalytic subunits form a monophyletic group, whereas regulatory subunits form three distinct groups. The phylogeny of the catalytic subunits indicates that they underwent two major duplications during their evolutionary history: the most ancient arose in the Last Eukaryotic Common Ancestor (LECA) and led to the emergence of class III and class I/II, while the second - that led to the separation between class I and II - occurred later, in the ancestor of Unikonta (i.e., the clade grouping Amoebozoa, Fungi, and Metazoa). These two major events were followed by many lineage specific duplications in particular in vertebrates, but also in various protist lineages. Major loss events were also detected in Vidiriplantae and Fungi. For the regulatory subunits, we identified homologs of class III in all eukaryotic groups indicating that, for this class, both the catalytic and the regulatory subunits were presents in LECA. In contrast, homologs of the regulatory class I have a more recent origin. The phylogenetic analysis of the PI3K shed a new light on the evolutionary history of these enzymes. We found that LECA already contained a PI3K class III composed of a catalytic and a regulatory subunit. Absence of class II regulatory subunits and the recent origin of class I regulatory subunits is puzzling given that the class I/II catalytic subunit

  14. An X-ray structural study of pyruvate dehydrogenase kinase: A eukaryotic serine kinase with a prokaryotic histidine-kinase fold

    NASA Astrophysics Data System (ADS)

    Steussy, Calvin Nicklaus, Jr.

    2001-07-01

    Pyruvate Dehydrogenase Kinase is an enzyme that controls the flow of glucose through the eukaryotic cell and contributes to the pathology of diabetes mellitus. Early work on this kinase demonstrated that it has an amino acid sequence much like bacterial histidine kinases, but an activity similar to that of modern serine/threonine kinases. This project utilized the techniques of X-ray crystallography to determine molecular structure of pyruvate dehydrogenase kinase, isozyme 2. The structure was phased using selenium substituted for sulfur in methionine residues, and data at multiple wavelengths was collected at the National Synchrotron Light Source, Brookhaven National Laboratories. PDK 2 was found to fold into a two-domain monomer that forms a dimer through two beta sheets in the C-terminal domain. The N-terminal domain is an alpha-helical bundle while the C-terminal domain is an alpha/beta sandwich. The fold of the C-terminal domain is very similar to that of the prokaryotic histidine kinases, indicating that they share a common ancestor. The catalytic mechanism, however, has evolved to use general base catalysis to activate the serine substrate, rather than the direct nucleophilic attack by the imidazole sidechain used in the prokaryotic kinases. Thus, the structure of the protein echoes its prokaryotic ancestor, while the chemical mechanism has adapted to a serine substrate. The electrostatic surface of PDK2 leads to the suggestion that the lipoyl domain of the pyruvate dehydrogenase kinase, an important associated structure, may bind in the cleft formed between the N- and C-terminal domains. In addition, a network of hydrogen bonds directly connects the nucleotide binding pocket to the dimer interface, suggesting that there may be some interaction between dimer formation and ATP binding or ADP release.

  15. Eukaryotic Elongation Factor 2 Kinase Activity Is Controlled by Multiple Inputs from Oncogenic Signaling

    PubMed Central

    Wang, Xuemin; Regufe da Mota, Sergio; Liu, Rui; Moore, Claire E.; Xie, Jianling; Lanucara, Francesco; Agarwala, Usha; Pyr dit Ruys, Sébastien; Vertommen, Didier; Rider, Mark H.; Eyers, Claire E.

    2014-01-01

    Eukaryotic elongation factor 2 kinase (eEF2K), an atypical calmodulin-dependent protein kinase, phosphorylates and inhibits eEF2, slowing down translation elongation. eEF2K contains an N-terminal catalytic domain, a C-terminal α-helical region and a linker containing several regulatory phosphorylation sites. eEF2K is expressed at high levels in certain cancers, where it may act to help cell survival, e.g., during nutrient starvation. However, it is a negative regulator of protein synthesis and thus cell growth, suggesting that cancer cells may possess mechanisms to inhibit eEF2K under good growth conditions, to allow protein synthesis to proceed. We show here that the mTORC1 pathway and the oncogenic Ras/Raf/MEK/extracellular signal-regulated kinase (ERK) pathway cooperate to restrict eEF2K activity. We identify multiple sites in eEF2K whose phosphorylation is regulated by mTORC1 and/or ERK, including new ones in the linker region. We demonstrate that certain sites are phosphorylated directly by mTOR or ERK. Our data reveal that glycogen synthase kinase 3 signaling also regulates eEF2 phosphorylation. In addition, we show that phosphorylation sites remote from the N-terminal calmodulin-binding motif regulate the phosphorylation of N-terminal sites that control CaM binding. Mutations in the former sites, which occur in cancer cells, cause the activation of eEF2K. eEF2K is thus regulated by a network of oncogenic signaling pathways. PMID:25182533

  16. Diversity, classification and function of the plant protein kinase superfamily

    PubMed Central

    Lehti-Shiu, Melissa D.; Shiu, Shin-Han

    2012-01-01

    Eukaryotic protein kinases belong to a large superfamily with hundreds to thousands of copies and are components of essentially all cellular functions. The goals of this study are to classify protein kinases from 25 plant species and to assess their evolutionary history in conjunction with consideration of their molecular functions. The protein kinase superfamily has expanded in the flowering plant lineage, in part through recent duplications. As a result, the flowering plant protein kinase repertoire, or kinome, is in general significantly larger than other eukaryotes, ranging in size from 600 to 2500 members. This large variation in kinome size is mainly due to the expansion and contraction of a few families, particularly the receptor-like kinase/Pelle family. A number of protein kinases reside in highly conserved, low copy number families and often play broadly conserved regulatory roles in metabolism and cell division, although functions of plant homologues have often diverged from their metazoan counterparts. Members of expanded plant kinase families often have roles in plant-specific processes and some may have contributed to adaptive evolution. Nonetheless, non-adaptive explanations, such as kinase duplicate subfunctionalization and insufficient time for pseudogenization, may also contribute to the large number of seemingly functional protein kinases in plants. PMID:22889912

  17. Diversity, classification and function of the plant protein kinase superfamily.

    PubMed

    Lehti-Shiu, Melissa D; Shiu, Shin-Han

    2012-09-19

    Eukaryotic protein kinases belong to a large superfamily with hundreds to thousands of copies and are components of essentially all cellular functions. The goals of this study are to classify protein kinases from 25 plant species and to assess their evolutionary history in conjunction with consideration of their molecular functions. The protein kinase superfamily has expanded in the flowering plant lineage, in part through recent duplications. As a result, the flowering plant protein kinase repertoire, or kinome, is in general significantly larger than other eukaryotes, ranging in size from 600 to 2500 members. This large variation in kinome size is mainly due to the expansion and contraction of a few families, particularly the receptor-like kinase/Pelle family. A number of protein kinases reside in highly conserved, low copy number families and often play broadly conserved regulatory roles in metabolism and cell division, although functions of plant homologues have often diverged from their metazoan counterparts. Members of expanded plant kinase families often have roles in plant-specific processes and some may have contributed to adaptive evolution. Nonetheless, non-adaptive explanations, such as kinase duplicate subfunctionalization and insufficient time for pseudogenization, may also contribute to the large number of seemingly functional protein kinases in plants.

  18. A set of ligation-independent in vitro translation vectors for eukaryotic protein production

    PubMed Central

    Bardóczy, Viola; Géczi, Viktória; Sawasaki, Tatsuya; Endo, Yaeta; Mészáros, Tamás

    2008-01-01

    Background The last decade has brought the renaissance of protein studies and accelerated the development of high-throughput methods in all aspects of proteomics. Presently, most protein synthesis systems exploit the capacity of living cells to translate proteins, but their application is limited by several factors. A more flexible alternative protein production method is the cell-free in vitro protein translation. Currently available in vitro translation systems are suitable for high-throughput robotic protein production, fulfilling the requirements of proteomics studies. Wheat germ extract based in vitro translation system is likely the most promising method, since numerous eukaryotic proteins can be cost-efficiently synthesized in their native folded form. Although currently available vectors for wheat embryo in vitro translation systems ensure high productivity, they do not meet the requirements of state-of-the-art proteomics. Target genes have to be inserted using restriction endonucleases and the plasmids do not encode cleavable affinity purification tags. Results We designed four ligation independent cloning (LIC) vectors for wheat germ extract based in vitro protein translation. In these constructs, the RNA transcription is driven by T7 or SP6 phage polymerase and two TEV protease cleavable affinity tags can be added to aid protein purification. To evaluate our improved vectors, a plant mitogen activated protein kinase was cloned in all four constructs. Purification of this eukaryotic protein kinase demonstrated that all constructs functioned as intended: insertion of PCR fragment by LIC worked efficiently, affinity purification of translated proteins by GST-Sepharose or MagneHis particles resulted in high purity kinase, and the affinity tags could efficiently be removed under different reaction conditions. Furthermore, high in vitro kinase activity testified of proper folding of the purified protein. Conclusion Four newly designed in vitro translation

  19. 14-3-3 proteins: regulators of numerous eukaryotic proteins.

    PubMed

    van Heusden, G Paul H

    2005-09-01

    14-3-3 proteins form a family of highly conserved proteins capable of binding to more than 200 different mostly phosphorylated proteins. They are present in all eukaryotic organisms investigated, often in multiple isoforms, up to 13 in some plants. 14-3-3 binding partners are involved in almost every cellular process and 14-3-3 proteins play a key role in these processes. 14-3-3 proteins interact with products encoded by oncogenes, with filament forming proteins involved in Alzheimer'ss disease and many other proteins related to human diseases. Disturbance of the interactions with 14-3-3 proteins may lead to diseases like cancer and the neurological Miller-Dieker disease. The molecular consequences of 14-3-3 binding are diverse and only partly understood. Binding of a protein to a 14-3-3 protein may result in stabilization of the active or inactive phosphorylated form of the protein, to a conformational alteration leading to activation or inhibition, to a different subcellular localization or to the interaction with other proteins. Currently genome- and proteome-wide studies are contributing to a wider knowledge of this important family of proteins.

  20. Rational Redesign of a Functional Protein Kinase-Substrate Interaction

    PubMed Central

    2017-01-01

    Eukaryotic protein kinases typically phosphorylate substrates in the context of specific sequence motifs, contributing to specificity essential for accurate signal transmission. Protein kinases recognize their target sequences through complementary interactions within the active site cleft. As a step toward the construction of orthogonal kinase signaling systems, we have re-engineered the protein kinase Pim1 to alter its phosphorylation consensus sequence. Residues in the Pim1 catalytic domain interacting directly with a critical arginine residue in the substrate were substituted to produce a kinase mutant that instead accommodates a hydrophobic residue. We then introduced a compensating mutation into a Pim1 substrate, the pro-apoptotic protein BAD, to reconstitute phosphorylation both in vitro and in living cells. Coexpression of the redesigned kinase with its substrate in cells protected them from apoptosis. Such orthogonal kinase–substrate pairs provide tools to probe the functional consequences of specific phosphorylation events in living cells and to design synthetic signaling pathways. PMID:28314095

  1. The Sec translocon mediated protein transport in prokaryotes and eukaryotes.

    PubMed

    Denks, Kärt; Vogt, Andreas; Sachelaru, Ilie; Petriman, Narcis-Adrian; Kudva, Renuka; Koch, Hans-Georg

    2014-01-01

    Protein transport via the Sec translocon represents an evolutionary conserved mechanism for delivering cytosolically-synthesized proteins to extra-cytosolic compartments. The Sec translocon has a three-subunit core, termed Sec61 in Eukaryotes and SecYEG in Bacteria. It is located in the endoplasmic reticulum of Eukaryotes and in the cytoplasmic membrane of Bacteria where it constitutes a channel that can be activated by multiple partner proteins. These partner proteins determine the mechanism of polypeptide movement across the channel. During SRP-dependent co-translational targeting, the ribosome threads the nascent protein directly into the Sec channel. This pathway is in Bacteria mainly dedicated for membrane proteins but in Eukaryotes also employed by secretory proteins. The alternative pathway, leading to post-translational translocation across the Sec translocon engages an ATP-dependent pushing mechanism by the motor protein SecA in Bacteria and a ratcheting mechanism by the lumenal chaperone BiP in Eukaryotes. Protein transport and biogenesis is also assisted by additional proteins at the lateral gate of SecY/Sec61α and in the lumen of the endoplasmic reticulum or in the periplasm of bacterial cells. The modular assembly enables the Sec complex to transport a vast array of substrates. In this review we summarize recent biochemical and structural information on the prokaryotic and eukaryotic Sec translocons and we describe the remarkably complex interaction network of the Sec complexes.

  2. MAPKAP kinase-2; a novel protein kinase activated by mitogen-activated protein kinase.

    PubMed Central

    Stokoe, D; Campbell, D G; Nakielny, S; Hidaka, H; Leevers, S J; Marshall, C; Cohen, P

    1992-01-01

    A novel protein kinase, which was only active when phosphorylated by the mitogen-activated protein kinase (MAP kinase), has been purified 85,000-fold to homogeneity from rabbit skeletal muscle. This MAP kinase activated protein kinase, termed MAPKAP kinase-2, was distinguished from S6 kinase-II (MAPKAP kinase-1) by its response to inhibitors, lack of phosphorylation of S6 peptides and amino acid sequence. MAPKAP kinase-2 phosphorylated glycogen synthase at Ser7 and the equivalent serine (*) in the peptide KKPLNRTLS*VASLPGLamide whose sequence is similar to the N terminus of glycogen synthase. MAPKAP kinase-2 was resolved into two monomeric species of apparent molecular mass 60 and 53 kDa that had similar specific activities and substrate specificities. Peptide sequences of the 60 and 53 kDa species were identical, indicating that they are either closely related isoforms or derived from the same gene. MAP kinase activated the 60 and 53 kDa forms of MAPKAP kinase-2 by phosphorylating the first threonine residue in the sequence VPQTPLHTSR. Furthermore, Mono Q chromatography of extracts from rat phaeochromocytoma and skeletal muscle demonstrated that two MAP kinase isoforms (p42mapk and p44mapk) were the only enzymes in these cells that were capable of reactivating MAPKAP kinase-2. These results indicate that MAP kinase activates at least two distinct protein kinases, suggesting that it represents a point at which the growth factor-stimulated protein kinase cascade bifurcates. Images PMID:1327754

  3. Mitogen-activated protein kinase cascades in Vitis vinifera

    PubMed Central

    Çakır, Birsen; Kılıçkaya, Ozan

    2015-01-01

    Protein phosphorylation is one of the most important mechanisms to control cellular functions in response to external and endogenous signals. Mitogen-activated protein kinases (MAPK) are universal signaling molecules in eukaryotes that mediate the intracellular transmission of extracellular signals resulting in the induction of appropriate cellular responses. MAPK cascades are composed of four protein kinase modules: MAPKKK kinases (MAPKKKKs), MAPKK kinases (MAPKKKs), MAPK kinases (MAPKKs), and MAPKs. In plants, MAPKs are activated in response to abiotic stresses, wounding, and hormones, and during plant pathogen interactions and cell division. In this report, we performed a complete inventory of MAPK cascades genes in Vitis vinifera, the whole genome of which has been sequenced. By comparison with MAPK, MAPK kinases, MAPK kinase kinases and MAPK kinase kinase kinase kinase members of Arabidopsis thaliana, we revealed the existence of 14 MAPKs, 5 MAPKKs, 62 MAPKKKs, and 7 MAPKKKKs in Vitis vinifera. We identified orthologs of V. vinifera putative MAPKs in different species, and ESTs corresponding to members of MAPK cascades in various tissues. This work represents the first complete inventory of MAPK cascades in V. vinifera and could help elucidate the biological and physiological functions of these proteins in V. vinifera. PMID:26257761

  4. Horizontal transfer of bacterial polyphosphate kinases to eukaryotes: implications for the ice age and land colonisation.

    PubMed

    Whitehead, Michael P; Hooley, Paul; W Brown, Michael R

    2013-06-05

    Studies of online database(s) showed that convincing examples of eukaryote PPKs derived from bacteria type PPK1 and PPK2 enzymes are rare and currently confined to a few simple eukaryotes. These enzymes probably represent several separate horizontal transfer events. Retention of such sequences may be an advantage for tolerance to stresses such as desiccation or nutrient depletion for simple eukaryotes that lack more sophisticated adaptations available to multicellular organisms. We propose that the acquisition of encoding sequences for these enzymes by horizontal transfer enhanced the ability of early plants to colonise the land. The improved ability to sequester and release inorganic phosphate for carbon fixation by photosynthetic algae in the ocean may have accelerated or even triggered global glaciation events. There is some evidence for DNA sequences encoding PPKs in a wider range of eukaryotes, notably some invertebrates, though it is unclear that these represent functional genes.Polyphosphate (poly P) is found in all cells, carrying out a wide range of essential roles. Studied mainly in prokaryotes, the enzymes responsible for synthesis of poly P in eukaryotes (polyphosphate kinases PPKs) are not well understood. The best characterised enzyme from bacteria known to catalyse the formation of high molecular weight polyphosphate from ATP is PPK1 which shows some structural similarity to phospholipase D. A second bacterial PPK (PPK2) resembles thymidylate kinase. Recent reports have suggested a widespread distribution of these bacteria type enzymes in eukaryotes. On - line databases show evidence for the presence of genes encoding PPK1 in only a limited number of eukaryotes. These include the photosynthetic eukaryotes Ostreococcus tauri, O. lucimarinus, Porphyra yezoensis, Cyanidioschyzon merolae and the moss Physcomitrella patens, as well as the amoeboid symbiont Capsaspora owczarzaki and the non-photosynthetic eukaryotes Dictyostelium (3 species

  5. Autophosphorylation in the Activation Loop Is Required for Full Kinase Activity In Vivo of Human and Yeast Eukaryotic Initiation Factor 2α Kinases PKR and GCN2

    PubMed Central

    Romano, Patrick R.; Garcia-Barrio, Minerva T.; Zhang, Xiaolong; Wang, Qizhi; Taylor, Deborah R.; Zhang, Fan; Herring, Christopher; Mathews, Michael B.; Qin, Jun; Hinnebusch, Alan G.

    1998-01-01

    The human double-stranded RNA-dependent protein kinase (PKR) is an important component of the interferon response to virus infection. The activation of PKR is accompanied by autophosphorylation at multiple sites, including one in the N-terminal regulatory region (Thr-258) that is required for full kinase activity. Several protein kinases are activated by phosphorylation in the region between kinase subdomains VII and VIII, referred to as the activation loop. We show that Thr-446 and Thr-451 in the PKR activation loop are required in vivo and in vitro for high-level kinase activity. Mutation of either residue to Ala impaired translational control by PKR in yeast cells and COS1 cells and led to tumor formation in mice. These mutations also impaired autophosphorylation and eukaryotic initiation factor 2 subunit α (eIF2α) phosphorylation by PKR in vitro. Whereas the Ala-446 substitution substantially reduced PKR function, the mutant kinase containing Ala-451 was completely inactive. PKR specifically phosphorylated Thr-446 and Thr-451 in synthetic peptides in vitro, and mass spectrometry analysis of PKR phosphopeptides confirmed that Thr-446 is an autophosphorylation site in vivo. Substitution of Glu-490 in subdomain X of PKR partially restored kinase activity when combined with the Ala-451 mutation. This finding suggests that the interaction between subdomain X and the activation loop, described previously for MAP kinase, is a regulatory feature conserved in PKR. We found that the yeast eIF2α kinase GCN2 autophosphorylates at Thr-882 and Thr-887, located in the activation loop at exactly the same positions as Thr-446 and Thr-451 in PKR. Thr-887 was more critically required than was Thr-882 for GCN2 kinase activity, paralleling the relative importance of Thr-446 and Thr-451 in PKR. These results indicate striking similarities between GCN2 and PKR in the importance of autophosphorylation and the conserved Thr residues in the activation loop. PMID:9528799

  6. Eukaryotic LYR Proteins Interact with Mitochondrial Protein Complexes

    PubMed Central

    Angerer, Heike

    2015-01-01

    In eukaryotic cells, mitochondria host ancient essential bioenergetic and biosynthetic pathways. LYR (leucine/tyrosine/arginine) motif proteins (LYRMs) of the Complex1_LYR-like superfamily interact with protein complexes of bacterial origin. Many LYR proteins function as extra subunits (LYRM3 and LYRM6) or novel assembly factors (LYRM7, LYRM8, ACN9 and FMC1) of the oxidative phosphorylation (OXPHOS) core complexes. Structural insights into complex I accessory subunits LYRM6 and LYRM3 have been provided by analyses of EM and X-ray structures of complex I from bovine and the yeast Yarrowia lipolytica, respectively. Combined structural and biochemical studies revealed that LYRM6 resides at the matrix arm close to the ubiquinone reduction site. For LYRM3, a position at the distal proton-pumping membrane arm facing the matrix space is suggested. Both LYRMs are supposed to anchor an acyl-carrier protein (ACPM) independently to complex I. The function of this duplicated protein interaction of ACPM with respiratory complex I is still unknown. Analysis of protein-protein interaction screens, genetic analyses and predicted multi-domain LYRMs offer further clues on an interaction network and adaptor-like function of LYR proteins in mitochondria. PMID:25686363

  7. Kinase Pathway Database: An Integrated Protein-Kinase and NLP-Based Protein-Interaction Resource

    PubMed Central

    Koike, Asako; Kobayashi, Yoshiyuki; Takagi, Toshihisa

    2003-01-01

    Protein kinases play a crucial role in the regulation of cellular functions. Various kinds of information about these molecules are important for understanding signaling pathways and organism characteristics. We have developed the Kinase Pathway Database, an integrated database involving major completely sequenced eukaryotes. It contains the classification of protein kinases and their functional conservation, ortholog tables among species, protein–protein, protein–gene, and protein–compound interaction data, domain information, and structural information. It also provides an automatic pathway graphic image interface. The protein, gene, and compound interactions are automatically extracted from abstracts for all genes and proteins by natural-language processing (NLP).The method of automatic extraction uses phrase patterns and the GENA protein, gene, and compound name dictionary, which was developed by our group. With this database, pathways are easily compared among species using data with more than 47,000 protein interactions and protein kinase ortholog tables. The database is available for querying and browsing at http://kinasedb.ontology.ims.u-tokyo.ac.jp/. PMID:12799355

  8. Neuronal migration and protein kinases

    PubMed Central

    Ohshima, Toshio

    2015-01-01

    The formation of the six-layered structure of the mammalian cortex via the inside-out pattern of neuronal migration is fundamental to neocortical functions. Extracellular cues such as Reelin induce intracellular signaling cascades through the protein phosphorylation. Migrating neurons also have intrinsic machineries to regulate cytoskeletal proteins and adhesion properties. Protein phosphorylation regulates these processes. Moreover, the balance between phosphorylation and dephosphorylation is modified by extracellular cues. Multipolar-bipolar transition, radial glia-guided locomotion and terminal translocation are critical steps of radial migration of cortical pyramidal neurons. Protein kinases such as Cyclin-dependent kinase 5 (Cdk5) and c-Jun N-terminal kinases (JNKs) involve these steps. In this review, I shall give an overview the roles of protein kinases in neuronal migration. PMID:25628530

  9. The protein kinase C family.

    PubMed

    Azzi, A; Boscoboinik, D; Hensey, C

    1992-09-15

    Protein kinase C represents a structurally homologous group of proteins similar in size, structure and mechanism of activation. They can modulate the biological function of proteins in a rapid and reversible manner. Protein kinase C participates in one of the major signal transduction systems triggered by the external stimulation of cells by various ligands including hormones, neurotransmitters and growth factors. Hydrolysis of membrane inositol phospholipids by phospholipase C or of phosphatidylcholine, generates sn-1,2-diacylglycerol, considered the physiological activator of this kinase. Other agents, such as arachidonic acid, participate in the activation of some of these proteins. Activation of protein kinase C by phorbol esters and related compounds is not physiological and may be responsible, at least in part, for their tumor-promoting activity. The cellular localization of the different calcium-activated protein kinases, their substrate and activator specificity are dissimilar and thus their role in signal transduction is unlike. A better understanding of the exact cellular function of the different protein kinase C isoenzymes requires the identification and characterization of their physiological substrates.

  10. Ancient diversification of eukaryotic MCM DNA replication proteins

    PubMed Central

    Liu, Yuan; Richards, Thomas A; Aves, Stephen J

    2009-01-01

    Background Yeast and animal cells require six mini-chromosome maintenance proteins (Mcm2-7) for pre-replication complex formation, DNA replication initiation and DNA synthesis. These six individual MCM proteins form distinct heterogeneous subunits within a hexamer which is believed to form the replicative helicase and which associates with the essential but non-homologous Mcm10 protein during DNA replication. In contrast Archaea generally only possess one MCM homologue which forms a homohexameric MCM helicase. In some eukaryotes Mcm8 and Mcm9 paralogues also appear to be involved in DNA replication although their exact roles are unclear. Results We used comparative genomics and phylogenetics to reconstruct the diversification of the eukaryotic Mcm2-9 gene family, demonstrating that Mcm2-9 were formed by seven gene duplication events before the last common ancestor of the eukaryotes. Mcm2-7 protein paralogues were present in all eukaryote genomes studied suggesting that no gene loss or functional replacements have been tolerated during the evolutionary diversification of eukaryotes. Mcm8 and 9 are widely distributed in eukaryotes and group together on the MCM phylogenetic tree to the exclusion of all other MCM paralogues suggesting co-ancestry. Mcm8 and Mcm9 are absent in some taxa, including Trichomonas and Giardia, and appear to have been secondarily lost in some fungi and some animals. The presence and absence of Mcm8 and 9 is concordant in all taxa sampled with the exception of Drosophila species. Mcm10 is present in most eukaryotes sampled but shows no concordant pattern of presence or absence with Mcm8 or 9. Conclusion A multifaceted and heterogeneous Mcm2-7 hexamer evolved during the early evolution of the eukaryote cell in parallel with numerous other acquisitions in cell complexity and prior to the diversification of extant eukaryotes. The conservation of all six paralogues throughout the eukaryotes suggests that each Mcm2-7 hexamer component has an

  11. Eukaryote-like serine/threonine kinases and phosphatases in bacteria.

    PubMed

    Pereira, Sandro F F; Goss, Lindsie; Dworkin, Jonathan

    2011-03-01

    Genomic studies have revealed the presence of Ser/Thr kinases and phosphatases in many bacterial species, although their physiological roles have largely been unclear. Here we review bacterial Ser/Thr kinases (eSTKs) that show homology in their catalytic domains to eukaryotic Ser/Thr kinases and their partner phosphatases (eSTPs) that are homologous to eukaryotic phosphatases. We first discuss insights into the enzymatic mechanism of eSTK activation derived from structural studies on both the ligand-binding and catalytic domains. We then turn our attention to the identified substrates of eSTKs and eSTPs for a number of species and to the implications of these findings for understanding their physiological roles in these organisms.

  12. A family of human cdc2-related protein kinases.

    PubMed Central

    Meyerson, M; Enders, G H; Wu, C L; Su, L K; Gorka, C; Nelson, C; Harlow, E; Tsai, L H

    1992-01-01

    The p34cdc2 protein kinase is known to regulate important transitions in the eukaryotic cell cycle. We have identified 10 human protein kinases based on their structural relation to p34cdc2. Seven of these kinases are novel and the products of five share greater than 50% amino acid sequence identity with p34cdc2. The seven novel genes are broadly expressed in human cell lines and tissues with each displaying some cell type or tissue specificity. The cdk3 gene, like cdc2 and cdk2, can complement cdc28 mutants of Saccharomyces cerevisiae, suggesting that all three of these protein kinases can play roles in the regulation of the mammalian cell cycle. The identification of a large family of cdc2-related kinases opens the possibility of combinatorial regulation of the cell cycle together with the emerging large family of cyclins. Images PMID:1639063

  13. CUL3 and protein kinases

    PubMed Central

    Metzger, Thibaud; Kleiss, Charlotte; Sumara, Izabela

    2013-01-01

    Posttranslational mechanisms drive fidelity of cellular processes. Phosphorylation and ubiquitination of substrates represent very common, covalent, posttranslational modifications and are often co-regulated. Phosphorylation may play a critical role both by directly regulating E3-ubiquitin ligases and/or by ensuring specificity of the ubiquitination substrate. Importantly, many kinases are not only critical regulatory components of these pathways but also represent themselves the direct ubiquitination substrates. Recent data suggest the role of CUL3-based ligases in both proteolytic and non-proteolytic regulation of protein kinases. Our own recent study identified the mitotic kinase PLK1 as a direct target of the CUL3 E3-ligase complex containing BTB-KELCH adaptor protein KLHL22.1 In this study, we aim at gaining mechanistic insights into CUL3-mediated regulation of the substrates, in particular protein kinases, by analyzing mechanisms of interaction between KLHL22 and PLK1. We find that kinase activity of PLK1 is redundant for its targeting for CUL3-ubiquitination. Moreover, CUL3/KLHL22 may contact 2 distinct motifs within PLK1 protein, consistent with the bivalent mode of substrate targeting found in other CUL3-based complexes. We discuss these findings in the context of the existing knowledge on other protein kinases and substrates targeted by CUL3-based E3-ligases. PMID:24067371

  14. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Linn, Anning

    1996-01-01

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK.

  15. Prokaryotic and eukaryotic integral membrane proteins have similar architecture.

    PubMed

    Gaur, Rajneesh Kumar; Natekar, Girija Arun

    2010-03-01

    Integral membrane proteins constitute a major constituent of lipid bilayer both in prokaryotes and eukaryotes. The statistical analysis was carried out to determine the bias in amino acid distribution between prokaryotic and eukaryotic integral membrane proteins (pIntMPs and eIntMPs). Our results indicate that both pIntMPs and eIntMPs demonstrate the striking similarity in amino acid distribution in their transmembrane and extramembranous region. pIntMPs have relatively greater functional importance for Gly and Asn in comparison to eIntMPs.

  16. Phosphorylation of the Yeast Choline Kinase by Protein Kinase C

    PubMed Central

    Choi, Mal-Gi; Kurnov, Vladlen; Kersting, Michael C.; Sreenivas, Avula; Carman, George M.

    2005-01-01

    The Saccharomyces cerevisiae CKI1-encoded choline kinase catalyzes the committed step in phosphatidylcholine synthesis via the Kennedy pathway. The enzyme is phosphorylated on multiple serine residues, and some of this phosphorylation is mediated by protein kinase A. In this work, we examined the hypothesis that choline kinase is also phosphorylated by protein kinase C. Using choline kinase as a substrate, protein kinase C activity was dose- and time-dependent, and dependent on the concentrations of choline kinase (Km = 27 μg/ml) and ATP (Km = 15 μM). This phosphorylation, which occurred on a serine residue, was accompanied by a 1.6-fold stimulation of choline kinase activity. The synthetic peptide SRSSS25QRRHS (Vmax/Km = 17.5 mM-1 μmol min-1 mg-1) that contains the protein kinase C motif for Ser25 was a substrate for protein kinase C. A Ser25 to Ala (S25A) mutation in choline kinase resulted in a 60% decrease in protein kinase C phosphorylation of the enzyme. Phosphopeptide mapping analysis of the S25A mutant enzyme confirmed that Ser25 was a protein kinase C target site. In vivo, the S25A mutation correlated with a decrease (55%) in phosphatidylcholine synthesis via the Kennedy pathway whereas an S25D phosphorylation site mimic correlated with an increase (44%) in phosphatidylcholine synthesis. Whereas the S25A (protein kinase C site) mutation did not affect the phosphorylation of choline kinase by protein kinase A, the S30A (protein kinase A site) mutation caused a 46% reduction in enzyme phosphorylation by protein kinase C. A choline kinase synthetic peptide (SQRRHS30LTRQ) containing Ser30 was a substrate (Vmax/Km = 3.0 mM−1 μmol min−1 mg−1) for protein kinase C. Comparison of phosphopeptide maps of the wild type and S30A mutant choline kinase enzymes phosphorylated by protein kinase C confirmed that Ser30 was also a target site for protein kinase C. PMID:15919656

  17. Oncoprotein protein kinase

    DOEpatents

    Karin, M.; Hibi, M.; Lin, A.

    1997-02-25

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE is disclosed. The polypeptide has serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences. The method of detection of JNK is also provided. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites. 44 figs.

  18. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    2004-03-16

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  19. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Lin, Anning

    1999-11-30

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD or 55 kD as determined by reducing SDS-PAGE, having serine and theonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  20. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    1998-01-01

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  1. Oncoprotein protein kinase

    DOEpatents

    Davis, Roger; Derijard, Benoit; Karin, Michael; Hibi, Masahiko; Lin, Anning

    2005-01-25

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  2. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning; Davis, Roger; Derijard, Benoit

    2003-02-04

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  3. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    1997-01-01

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  4. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning; Davis, Roger; Derijard, Benoit

    2005-03-08

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  5. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    1999-01-01

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD or 55 kD as determined by reducing SDS-PAGE, having serine and theonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  6. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    1997-01-01

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  7. Evolution of DNA replication protein complexes in eukaryotes and Archaea.

    PubMed

    Chia, Nicholas; Cann, Isaac; Olsen, Gary J

    2010-06-02

    The replication of DNA in Archaea and eukaryotes requires several ancillary complexes, including proliferating cell nuclear antigen (PCNA), replication factor C (RFC), and the minichromosome maintenance (MCM) complex. Bacterial DNA replication utilizes comparable proteins, but these are distantly related phylogenetically to their archaeal and eukaryotic counterparts at best. While the structures of each of the complexes do not differ significantly between the archaeal and eukaryotic versions thereof, the evolutionary dynamic in the two cases does. The number of subunits in each complex is constant across all taxa. However, they vary subtly with regard to composition. In some taxa the subunits are all identical in sequence, while in others some are homologous rather than identical. In the case of eukaryotes, there is no phylogenetic variation in the makeup of each complex-all appear to derive from a common eukaryotic ancestor. This is not the case in Archaea, where the relationship between the subunits within each complex varies taxon-to-taxon. We have performed a detailed phylogenetic analysis of these relationships in order to better understand the gene duplications and divergences that gave rise to the homologous subunits in Archaea. This domain level difference in evolution suggests that different forces have driven the evolution of DNA replication proteins in each of these two domains. In addition, the phylogenies of all three gene families support the distinctiveness of the proposed archaeal phylum Thaumarchaeota.

  8. Evolution of DNA Replication Protein Complexes in Eukaryotes and Archaea

    PubMed Central

    Chia, Nicholas; Cann, Isaac; Olsen, Gary J.

    2010-01-01

    Background The replication of DNA in Archaea and eukaryotes requires several ancillary complexes, including proliferating cell nuclear antigen (PCNA), replication factor C (RFC), and the minichromosome maintenance (MCM) complex. Bacterial DNA replication utilizes comparable proteins, but these are distantly related phylogenetically to their archaeal and eukaryotic counterparts at best. Methodology/Principal Findings While the structures of each of the complexes do not differ significantly between the archaeal and eukaryotic versions thereof, the evolutionary dynamic in the two cases does. The number of subunits in each complex is constant across all taxa. However, they vary subtly with regard to composition. In some taxa the subunits are all identical in sequence, while in others some are homologous rather than identical. In the case of eukaryotes, there is no phylogenetic variation in the makeup of each complex—all appear to derive from a common eukaryotic ancestor. This is not the case in Archaea, where the relationship between the subunits within each complex varies taxon-to-taxon. We have performed a detailed phylogenetic analysis of these relationships in order to better understand the gene duplications and divergences that gave rise to the homologous subunits in Archaea. Conclusion/Significance This domain level difference in evolution suggests that different forces have driven the evolution of DNA replication proteins in each of these two domains. In addition, the phylogenies of all three gene families support the distinctiveness of the proposed archaeal phylum Thaumarchaeota. PMID:20532250

  9. Unveiling the Novel Dual Specificity Protein Kinases in Bacillus anthracis

    PubMed Central

    Arora, Gunjan; Sajid, Andaleeb; Arulanandh, Mary Diana; Singhal, Anshika; Mattoo, Abid R.; Pomerantsev, Andrei P.; Leppla, Stephen H.; Maiti, Souvik; Singh, Yogendra

    2012-01-01

    Dual specificity protein kinases (DSPKs) are unique enzymes that can execute multiple functions in the cell, which are otherwise performed exclusively by serine/threonine and tyrosine protein kinases. In this study, we have characterized the protein kinases Bas2152 (PrkD) and Bas2037 (PrkG) from Bacillus anthracis. Transcriptional analyses of these kinases showed that they are expressed in all phases of growth. In a serendipitous discovery, both kinases were found to be DSPKs. PrkD was found to be similar to the eukaryotic dual specificity Tyr phosphorylation-regulated kinase class of dual specificity kinases, which autophosphorylates on Ser, Thr, and Tyr residues and phosphorylates Ser and Thr residues on substrates. PrkG was found to be a bona fide dual specificity protein kinase that mediates autophosphorylation and substrate phosphorylation on Ser, Thr, and Tyr residues. The sites of phosphorylation in both of the kinases were identified through mass spectrometry. Phosphorylation on Tyr residues regulates the kinase activity of PrkD and PrkG. PrpC, the only known Ser/Thr protein phosphatase, was also found to possess dual specificity. Genistein, a known Tyr kinase inhibitor, was found to inhibit the activities of PrkD and PrkG and affect the growth of B. anthracis cells, indicating a possible role of these kinases in cell growth and development. In addition, the glycolytic enzyme pyruvate kinase was found to be phosphorylated by PrkD on Ser and Thr residues but not by PrkG. Thus, this study provides the first evidence of DSPKs in B. anthracis that belong to different classes and have different modes of regulation. PMID:22711536

  10. Auto-phosphorylation Represses Protein Kinase R Activity.

    PubMed

    Wang, Die; de Weerd, Nicole A; Willard, Belinda; Polekhina, Galina; Williams, Bryan R G; Sadler, Anthony J

    2017-03-10

    The central role of protein kinases in controlling disease processes has spurred efforts to develop pharmaceutical regulators of their activity. A rational strategy to achieve this end is to determine intrinsic auto-regulatory processes, then selectively target these different states of kinases to repress their activation. Here we investigate auto-regulation of the innate immune effector protein kinase R, which phosphorylates the eukaryotic initiation factor 2α to inhibit global protein translation. We demonstrate that protein kinase R activity is controlled by auto-inhibition via an intra-molecular interaction. Part of this mechanism of control had previously been reported, but was then controverted. We account for the discrepancy and extend our understanding of the auto-inhibitory mechanism by identifying that auto-inhibition is paradoxically instigated by incipient auto-phosphorylation. Phosphor-residues at the amino-terminus instigate an intra-molecular interaction that enlists both of the N-terminal RNA-binding motifs of the protein with separate surfaces of the C-terminal kinase domain, to co-operatively inhibit kinase activation. These findings identify an innovative mechanism to control kinase activity, providing insight for strategies to better regulate kinase activity.

  11. Auto-phosphorylation Represses Protein Kinase R Activity

    PubMed Central

    Wang, Die; de Weerd, Nicole A.; Willard, Belinda; Polekhina, Galina; Williams, Bryan R. G.; Sadler, Anthony J.

    2017-01-01

    The central role of protein kinases in controlling disease processes has spurred efforts to develop pharmaceutical regulators of their activity. A rational strategy to achieve this end is to determine intrinsic auto-regulatory processes, then selectively target these different states of kinases to repress their activation. Here we investigate auto-regulation of the innate immune effector protein kinase R, which phosphorylates the eukaryotic initiation factor 2α to inhibit global protein translation. We demonstrate that protein kinase R activity is controlled by auto-inhibition via an intra-molecular interaction. Part of this mechanism of control had previously been reported, but was then controverted. We account for the discrepancy and extend our understanding of the auto-inhibitory mechanism by identifying that auto-inhibition is paradoxically instigated by incipient auto-phosphorylation. Phosphor-residues at the amino-terminus instigate an intra-molecular interaction that enlists both of the N-terminal RNA-binding motifs of the protein with separate surfaces of the C-terminal kinase domain, to co-operatively inhibit kinase activation. These findings identify an innovative mechanism to control kinase activity, providing insight for strategies to better regulate kinase activity. PMID:28281686

  12. Overexpression of membrane proteins from higher eukaryotes in yeasts.

    PubMed

    Emmerstorfer, Anita; Wriessnegger, Tamara; Hirz, Melanie; Pichler, Harald

    2014-09-01

    Heterologous expression and characterisation of the membrane proteins of higher eukaryotes is of paramount interest in fundamental and applied research. Due to the rather simple and well-established methods for their genetic modification and cultivation, yeast cells are attractive host systems for recombinant protein production. This review provides an overview on the remarkable progress, and discusses pitfalls, in applying various yeast host strains for high-level expression of eukaryotic membrane proteins. In contrast to the cell lines of higher eukaryotes, yeasts permit efficient library screening methods. Modified yeasts are used as high-throughput screening tools for heterologous membrane protein functions or as benchmark for analysing drug-target relationships, e.g., by using yeasts as sensors. Furthermore, yeasts are powerful hosts for revealing interactions stabilising and/or activating membrane proteins. We also discuss the stress responses of yeasts upon heterologous expression of membrane proteins. Through co-expression of chaperones and/or optimising yeast cultivation and expression strategies, yield-optimised hosts have been created for membrane protein crystallography or efficient whole-cell production of fine chemicals.

  13. Endoplasmic reticulum stress induced by tunicamycin increases resistin messenger ribonucleic acid through the pancreatic endoplasmic reticulum eukaryotic initiation factor 2α kinase-activating transcription factor 4-CAAT/enhancer binding protein-α homologous protein pathway in THP-1 human monocytes.

    PubMed

    Hamada, Junpei; Onuma, Hiroshi; Ochi, Fumihiro; Hirai, Hiroki; Takemoto, Koji; Miyoshi, Akiko; Matsushita, Manami; Kadota, Yuko; Ohashi, Jun; Kawamura, Ryoichi; Takata, Yasunori; Nishida, Wataru; Hashida, Seiichi; Ishii, Eiichi; Osawa, Haruhiko

    2016-05-01

    Resistin, secreted from adipocytes, causes insulin resistance in mice. In humans, the resistin gene is mainly expressed in monocytes and macrophages. Tunicamycin is known to induce endoplasmic reticulum (ER) stress, and reduce resistin gene expression in 3T3-L1 mouse adipocytes. The aim of the present study was to examine whether ER stress affects resistin gene expression in human monocytes. The relationship between resistin messenger ribonucleic acid (mRNA) and ER stress markers mRNA was analyzed by reverse transcription polymerase chain reaction in isolated monocytes of 30 healthy volunteers. The effect of endotoxin/lipopolysaccharides or tunicamycin on resistin gene expression was analyzed in THP-1 human monocytes. Signaling pathways leading to resistin mRNA were assessed by the knockdown using small interfering RNA or overexpression of key molecules involved in unfolded protein response. Resistin mRNA was positively associated with immunoglobulin heavy chain-binding protein (BiP) or CAAT/enhancer binding protein-α homologous protein (CHOP) mRNA in human isolated monocytes. In THP-1 cells, lipopolysaccharides increased mRNA of BiP, pancreatic endoplasmic reticulum eukaryotic initiation factor 2α kinase (PERK) and CHOP, as well as resistin. Tunicamycin also increased resistin mRNA. This induction appeared to be dose- and time-dependent. Tunicamycin-induced resistin mRNA was inhibited by chemical chaperone, 4-phenylbutyric acid. The knockdown of either PERK, activating transcription factor 4 (ATF4) or CHOP reduced tunicamycin-induced resistin mRNA. Conversely, overexpression of ATF4 or CHOP increased resistin mRNA. Endoplasmic reticulum stress induced by tunicamycin increased resistin mRNA through the PERK-ATF4-CHOP pathway in THP-1 human monocytes. ER stress could lead to insulin resistance through enhanced resistin gene expression in human monocytes.

  14. Survey, analysis and genetic organization of genes encoding eukaryotic-like signaling proteins on a cyanobacterial genome.

    PubMed Central

    Zhang, C C; Gonzalez, L; Phalip, V

    1998-01-01

    Bacteria usually use two-component systems for signal transduction, while eukaryotic organisms employ Ser/Thr and Tyr kinases and phosphatases for the same purpose. Many prokaryotes turn out to harbor Ser/Thr and Tyr kinases, Ser/Thr and Tyr phosphatases, and their accessory components as well. The sequence determination of the genome of the cyanobacterium Synechocystis sp. strain PCC 6803 offers the possibility to survey the extent of such molecules in a prokaryotic organism. This cyanobacterium possesses seven Ser/Thr kinases, seven Ser/Thr and Tyr phosphatases, one protein kinase interacting protein, one protein kinase regulatory subunit and several WD40-repeat-containing proteins. The majority of the protein phosphatases presented in this study were previously reported as hypothetical proteins. We analyze here the structure and genetic organization of these ORFs in the hope of providing a guidance for their functional analysis. Unlike their eukaryotic counterparts, many of these genes are clustered on the chromosome, and this genetic organization offers the opportunity to study their possible interaction. In several cases, genes of two-component transducers are found within the same cluster as those encoding a Ser/Thr kinase or a Ser/Thr phosphatase; the implication for signal transduction mechanism will be discussed. PMID:9685474

  15. Degradation of Activated Protein Kinases by Ubiquitination

    PubMed Central

    Lu, Zhimin; Hunter, Tony

    2009-01-01

    Protein kinases are important regulators of intracellular signal transduction pathways and play critical roles in diverse cellular functions. Once a protein kinase is activated, its activity is subsequently downregulated through a variety of mechanisms. Accumulating evidence indicates that the activation of protein kinases commonly initiates their downregulation via the ubiquitin/proteasome pathway. Failure to regulate protein kinase activity or expression levels can cause human diseases. PMID:19489726

  16. Modification of Bacterial Effector Proteins Inside Eukaryotic Host Cells.

    PubMed

    Popa, Crina M; Tabuchi, Mitsuaki; Valls, Marc

    2016-01-01

    Pathogenic bacteria manipulate their hosts by delivering a number of virulence proteins -called effectors- directly into the plant or animal cells. Recent findings have shown that such effectors can suffer covalent modifications inside the eukaryotic cells. Here, we summarize the recent reports where effector modifications by the eukaryotic machinery have been described. We restrict our focus on proteins secreted by the type III or type IV systems, excluding other bacterial toxins. We describe the known examples of effectors whose enzymatic activity is triggered by interaction with plant and animal cell factors, including GTPases, E2-Ubiquitin conjugates, cyclophilin and thioredoxins. We focus on the structural interactions with these factors and their influence on effector function. We also review the described examples of host-mediated post-translational effector modifications which are required for proper subcellular location and function. These host-specific covalent modifications include phosphorylation, ubiquitination, SUMOylation, and lipidations such as prenylation, fatty acylation and phospholipid binding.

  17. Modification of Bacterial Effector Proteins Inside Eukaryotic Host Cells

    PubMed Central

    Popa, Crina M.; Tabuchi, Mitsuaki; Valls, Marc

    2016-01-01

    Pathogenic bacteria manipulate their hosts by delivering a number of virulence proteins -called effectors- directly into the plant or animal cells. Recent findings have shown that such effectors can suffer covalent modifications inside the eukaryotic cells. Here, we summarize the recent reports where effector modifications by the eukaryotic machinery have been described. We restrict our focus on proteins secreted by the type III or type IV systems, excluding other bacterial toxins. We describe the known examples of effectors whose enzymatic activity is triggered by interaction with plant and animal cell factors, including GTPases, E2-Ubiquitin conjugates, cyclophilin and thioredoxins. We focus on the structural interactions with these factors and their influence on effector function. We also review the described examples of host-mediated post-translational effector modifications which are required for proper subcellular location and function. These host-specific covalent modifications include phosphorylation, ubiquitination, SUMOylation, and lipidations such as prenylation, fatty acylation and phospholipid binding. PMID:27489796

  18. Molecular cloning of plant transcripts encoding protein kinase homologs.

    PubMed Central

    Lawton, M A; Yamamoto, R T; Hanks, S K; Lamb, C J

    1989-01-01

    Oligonucleotides, corresponding to conserved regions of animal protein-serine/threonine kinases, were used to isolate cDNAs encoding plant homologs in the dicot bean (Phaseolus vulgaris L.) and the monocot rice (Oryzae sativa L.). The C-terminal regions of the deduced polypeptides encoded by the bean (PVPK-1) and rice (G11A) cDNAs, prepared from mRNAs of suspension cultures and leaves, respectively, contain features characteristic of the catalytic domains of eukaryotic protein-serine/threonine kinases, indicating that these cDNAs encode plant protein kinases. The putative catalytic domains are most closely related to cyclic nucleotide-dependent protein kinases and the protein kinase C family, suggesting the plant homologs may likewise transduce extracellular signals. However, outside these domains, PVPK-1 and G11A exhibit no homology either to each other or to regulatory domains of other protein kinases, indicating the plant homologs are modulated by other signals. PVPK-1 corresponds to a 2.4-kb transcript in suspension cultured bean cells. Southern blots of genomic DNA indicate that PVPK-1 and G11A correspond to single copy genes that form part of a family of related plant sequences. Images PMID:2541432

  19. Expression and purification of active protein kinases from wheat germ extracts.

    PubMed

    Sonkoly, Boglárka; Bardóczy, Viola; Mészáros, Tamás

    2011-01-01

    In vitro functional studies of eukaryotic kinases are often constrained by the availability of pure and -enzymatically active kinase of interest. Though numerous proteins have been synthesized by cell-based systems, in vivo production of properly folded, eukaryotic proteins remains a challenging task. Current wheat-germ-based cell-free in vitro translation systems present a plausible alternative for protein synthesis since majority of eukaryotic proteins could be obtained in their native folded form with general protocols. The use of special in vitro translation vectors with ligation-independent cloning sites and cleavable affinity tags eliminates further bottlenecks of the protein producing procedure and makes this system a reasonable method for simultaneous generation of active kinases.

  20. Cadmium activates a mitogen-activated protein kinase gene and MBP kinases in rice.

    PubMed

    Yeh, Chuan-Ming; Hsiao, Lin-June; Huang, Hao-Jen

    2004-09-01

    Mitogen-activated protein kinase (MAPK) pathways are modules involved in the transduction of extracellular signals to intracellular targets in all eukaryotes. In plants, it has been evidenced that MAPKs play a role in the signaling of biotic and abiotic stresses, plant hormones, and cell cycle cues. However, the effect of heavy metals on plant MAPKs has not been well examined. The Northern blot analysis of OsMAPK mRNA levels has shown that only OsMAPK2, but not OsMAPK3 and OsMAPK4, expressed in suspension-cultured cells in response to 100-400 microM Cd treatments. The OsMAPK2 transcripts increased within 12 h upon 400 microM Cd treatment. In addition, we found that 42- and 50-kDa MBP kinases were significantly activated by Cd treatment in rice suspension-cultured cells. And 40-, 42-, 50- and 64-kDa MBP kinases were activated in rice roots. Furthermore, GSH inhibits Cd-induced 40-kDa MBP kinase activation. By immunoblot analysis and immunoprecipitation followed by in-gel kinase assay, we confirmed that Cd-activated 42-kDa MBP kinase is a MAP kinase. Our results suggest that a MAP kinase cascade may function in the Cd-signalling pathway in rice.

  1. Protein N-myristoylation in Escherichia coli: reconstitution of a eukaryotic protein modification in bacteria.

    PubMed Central

    Duronio, R J; Jackson-Machelski, E; Heuckeroth, R O; Olins, P O; Devine, C S; Yonemoto, W; Slice, L W; Taylor, S S; Gordon, J I

    1990-01-01

    Protein N-myristoylation refers to the covalent attachment of a myristoyl group (C14:0), via amide linkage, to the NH2-terminal glycine residue of certain cellular and viral proteins. Myristoyl-CoA:protein N-myristoyltransferase (NMT) catalyzes this cotranslational modification. We have developed a system for studying the substrate requirements and biological effects of protein N-myristoylation as well as NMT structure-activity relationships. Expression of the yeast NMT1 gene in Escherichia coli, a bacterium that has no endogenous NMT activity, results in production of the intact 53-kDa NMT polypeptide as well as a truncated polypeptide derived from proteolytic removal of its NH2-terminal 39 amino acids. Each E. coli-synthesized NMT species has fatty acid and peptide substrate specificities that are indistinguishable from those of NMT recovered from Saccharomyces cerevisiae, suggesting that the NH2-terminal domain of this enzyme is not required for its catalytic activity. By using a dual plasmid system, N-myristoylation of a mammalian protein was reconstituted in E. coli by simultaneous expression of the yeast NMT1 gene and a murine cDNA encoding the catalytic (C) subunit of cAMP-dependent protein kinase (PK-A). The fatty acid specificity of N-myristoylation was preserved in this system: [9,10(n)-3H]myristate but not [9,10(n)3H]palmitate was efficiently linked to Gly-1 of the C subunit. [13,14(n)-3H]10-Propoxydecanoic acid, a heteroatom-containing analog of myristic acid with reduced hydrophobicity but similar chain length, was an effective alternative substrate for NMT that also could be incorporated into the C subunit of PK-A. Such analogs have recently been shown to inhibit replication of certain retroviruses that depend upon linkage of a myristoyl group to their gag polyprotein precursors (e.g., the Pr55gag of human immunodeficiency virus type 1). A major advantage of the bacterial system over eukaryotic systems is the absence of endogenous NMT and substrates

  2. Phosphorylation of spore coat proteins by a family of atypical protein kinases

    PubMed Central

    Nguyen, Kim B.; Sreelatha, Anju; Durrant, Eric S.; Lopez-Garrido, Javier; Muszewska, Anna; Dudkiewicz, Małgorzata; Grynberg, Marcin; Yee, Samantha; Pogliano, Kit; Tomchick, Diana R.; Pawłowski, Krzysztof; Dixon, Jack E.; Tagliabracci, Vincent S.

    2016-01-01

    The modification of proteins by phosphorylation occurs in all life forms and is catalyzed by a large superfamily of enzymes known as protein kinases. We recently discovered a family of secretory pathway kinases that phosphorylate extracellular proteins. One member, family with sequence similarity 20C (Fam20C), is the physiological Golgi casein kinase. While examining distantly related protein sequences, we observed low levels of identity between the spore coat protein H (CotH), and the Fam20C-related secretory pathway kinases. CotH is a component of the spore in many bacterial and eukaryotic species, and is required for efficient germination of spores in Bacillus subtilis; however, the mechanism by which CotH affects germination is unclear. Here, we show that CotH is a protein kinase. The crystal structure of CotH reveals an atypical protein kinase-like fold with a unique mode of ATP binding. Examination of the genes neighboring cotH in B. subtilis led us to identify two spore coat proteins, CotB and CotG, as CotH substrates. Furthermore, we show that CotH-dependent phosphorylation of CotB and CotG is required for the efficient germination of B. subtilis spores. Collectively, our results define a family of atypical protein kinases and reveal an unexpected role for protein phosphorylation in spore biology. PMID:27185916

  3. Phosphorylation of spore coat proteins by a family of atypical protein kinases

    SciTech Connect

    Nguyen, Kim B.; Sreelatha, Anju; Durrant, Eric S.; Lopez-Garrido, Javier; Muszewska, Anna; Dudkiewicz, Małgorzata; Grynberg, Marcin; Yee, Samantha; Pogliano, Kit; Tomchick, Diana R.; Pawłowski, Krzysztof; Dixon, Jack E.; Tagliabracci, Vincent S.

    2016-05-16

    The modification of proteins by phosphorylation occurs in all life forms and is catalyzed by a large superfamily of enzymes known as protein kinases. We recently discovered a family of secretory pathway kinases that phosphorylate extracellular proteins. One member, family with sequence similarity 20C (Fam20C), is the physiological Golgi casein kinase. While examining distantly related protein sequences, we observed low levels of identity between the spore coat protein H (CotH), and the Fam20C-related secretory pathway kinases. CotH is a component of the spore in many bacterial and eukaryotic species, and is required for efficient germination of spores in Bacillus subtilis; however, the mechanism by which CotH affects germination is unclear. In this paper, we show that CotH is a protein kinase. The crystal structure of CotH reveals an atypical protein kinase-like fold with a unique mode of ATP binding. Examination of the genes neighboring cotH in B. subtilis led us to identify two spore coat proteins, CotB and CotG, as CotH substrates. Furthermore, we show that CotH-dependent phosphorylation of CotB and CotG is required for the efficient germination of B. subtilis spores. Finally and collectively, our results define a family of atypical protein kinases and reveal an unexpected role for protein phosphorylation in spore biology.

  4. Phosphorylation of spore coat proteins by a family of atypical protein kinases

    DOE PAGES

    Nguyen, Kim B.; Sreelatha, Anju; Durrant, Eric S.; ...

    2016-05-16

    The modification of proteins by phosphorylation occurs in all life forms and is catalyzed by a large superfamily of enzymes known as protein kinases. We recently discovered a family of secretory pathway kinases that phosphorylate extracellular proteins. One member, family with sequence similarity 20C (Fam20C), is the physiological Golgi casein kinase. While examining distantly related protein sequences, we observed low levels of identity between the spore coat protein H (CotH), and the Fam20C-related secretory pathway kinases. CotH is a component of the spore in many bacterial and eukaryotic species, and is required for efficient germination of spores in Bacillus subtilis;more » however, the mechanism by which CotH affects germination is unclear. In this paper, we show that CotH is a protein kinase. The crystal structure of CotH reveals an atypical protein kinase-like fold with a unique mode of ATP binding. Examination of the genes neighboring cotH in B. subtilis led us to identify two spore coat proteins, CotB and CotG, as CotH substrates. Furthermore, we show that CotH-dependent phosphorylation of CotB and CotG is required for the efficient germination of B. subtilis spores. Finally and collectively, our results define a family of atypical protein kinases and reveal an unexpected role for protein phosphorylation in spore biology.« less

  5. Phosphorylation of spore coat proteins by a family of atypical protein kinases

    SciTech Connect

    Nguyen, Kim B.; Sreelatha, Anju; Durrant, Eric S.; Lopez-Garrido, Javier; Muszewska, Anna; Dudkiewicz, Małgorzata; Grynberg, Marcin; Yee, Samantha; Pogliano, Kit; Tomchick, Diana R.; Pawłowski, Krzysztof; Dixon, Jack E.; Tagliabracci, Vincent S.

    2016-05-16

    The modification of proteins by phosphorylation occurs in all life forms and is catalyzed by a large superfamily of enzymes known as protein kinases. We recently discovered a family of secretory pathway kinases that phosphorylate extracellular proteins. One member, family with sequence similarity 20C (Fam20C), is the physiological Golgi casein kinase. While examining distantly related protein sequences, we observed low levels of identity between the spore coat protein H (CotH), and the Fam20C-related secretory pathway kinases. CotH is a component of the spore in many bacterial and eukaryotic species, and is required for efficient germination of spores in Bacillus subtilis; however, the mechanism by which CotH affects germination is unclear. In this paper, we show that CotH is a protein kinase. The crystal structure of CotH reveals an atypical protein kinase-like fold with a unique mode of ATP binding. Examination of the genes neighboring cotH in B. subtilis led us to identify two spore coat proteins, CotB and CotG, as CotH substrates. Furthermore, we show that CotH-dependent phosphorylation of CotB and CotG is required for the efficient germination of B. subtilis spores. Finally and collectively, our results define a family of atypical protein kinases and reveal an unexpected role for protein phosphorylation in spore biology.

  6. Degradation of newly synthesized polypeptides by ribosome-associated RACK1/c-Jun N-terminal kinase/eukaryotic elongation factor 1A2 complex.

    PubMed

    Gandin, Valentina; Gutierrez, Gustavo J; Brill, Laurence M; Varsano, Tal; Feng, Yongmei; Aza-Blanc, Pedro; Au, Qingyan; McLaughlan, Shannon; Ferreira, Tiago A; Alain, Tommy; Sonenberg, Nahum; Topisirovic, Ivan; Ronai, Ze'ev A

    2013-07-01

    Folding of newly synthesized polypeptides (NSPs) into functional proteins is a highly regulated process. Rigorous quality control ensures that NSPs attain their native fold during or shortly after completion of translation. Nonetheless, signaling pathways that govern the degradation of NSPs in mammals remain elusive. We demonstrate that the stress-induced c-Jun N-terminal kinase (JNK) is recruited to ribosomes by the receptor for activated protein C kinase 1 (RACK1). RACK1 is an integral component of the 40S ribosome and an adaptor for protein kinases. Ribosome-associated JNK phosphorylates the eukaryotic translation elongation factor 1A isoform 2 (eEF1A2) on serines 205 and 358 to promote degradation of NSPs by the proteasome. These findings establish a role for a RACK1/JNK/eEF1A2 complex in the quality control of NSPs in response to stress.

  7. Signals fly when kinases meet Rho-of-plants (ROP) small G-proteins.

    PubMed

    Fehér, Attila; Lajkó, Dézi Bianka

    2015-08-01

    Rho-type small GTP-binding plant proteins function as two-state molecular switches in cellular signalling. There is accumulating evidence that Rho-of-plants (ROP) signalling is positively controlled by plant receptor kinases, through the ROP guanine nucleotide exchange factor proteins. These signalling modules regulate cell polarity, cell shape, hormone responses, and pathogen defence, among other things. Other ROP-regulatory proteins might also be subjected to protein phosphorylation by cellular kinases (e.g., mitogen-activated protein kinases or calcium-dependent protein kinases), in order to integrate various cellular signalling pathways with ROP GTPase-dependent processes. In contrast to the role of kinases in upstream ROP regulation, much less is known about the potential link between ROP GTPases and downstream kinase signalling. In other eukaryotes, Rho-type G-protein-activated kinases are widespread and have a key role in many cellular processes. Recent data indicate the existence of structurally different ROP-activated kinases in plants, but their ROP-dependent biological functions still need to be validated. In addition to these direct interactions, ROPs may also indirectly control the activity of mitogen-activated protein kinases or calcium-dependent protein kinases. These kinases may therefore function as upstream as well as downstream kinases in ROP-mediated signalling pathways, such as the phosphatidylinositol monophosphate kinases involved in cell polarity establishment. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  8. Genome-wide analysis of eukaryote thaumatin-like proteins (TLPs) with an emphasis on poplar

    PubMed Central

    2011-01-01

    Background Plant inducible immunity includes the accumulation of a set of defense proteins during infection called pathogenesis-related (PR) proteins, which are grouped into families termed PR-1 to PR-17. The PR-5 family is composed of thaumatin-like proteins (TLPs), which are responsive to biotic and abiotic stress and are widely studied in plants. TLPs were also recently discovered in fungi and animals. In the poplar genome, TLPs are over-represented compared with annual species and their transcripts strongly accumulate during stress conditions. Results Our analysis of the poplar TLP family suggests that the expansion of this gene family was followed by diversification, as differences in expression patterns and predicted properties correlate with phylogeny. In particular, we identified a clade of poplar TLPs that cluster to a single 350 kb locus of chromosome I and that are up-regulated by poplar leaf rust infection. A wider phylogenetic analysis of eukaryote TLPs - including plant, animal and fungi sequences - shows that TLP gene content and diversity increased markedly during land plant evolution. Mapping the reported functions of characterized TLPs to the eukaryote phylogenetic tree showed that antifungal or glycan-lytic properties are widespread across eukaryote phylogeny, suggesting that these properties are shared by most TLPs and are likely associated with the presence of a conserved acidic cleft in their 3D structure. Also, we established an exhaustive catalog of TLPs with atypical architectures such as small-TLPs, TLP-kinases and small-TLP-kinases, which have potentially developed alternative functions (such as putative receptor kinases for pathogen sensing and signaling). Conclusion Our study, based on the most recent plant genome sequences, provides evidence for TLP gene family diversification during land plant evolution. We have shown that the diverse functions described for TLPs are not restricted to specific clades but seem to be universal among

  9. Genome-wide analysis of eukaryote thaumatin-like proteins (TLPs) with an emphasis on poplar.

    PubMed

    Petre, Benjamin; Major, Ian; Rouhier, Nicolas; Duplessis, Sébastien

    2011-02-15

    Plant inducible immunity includes the accumulation of a set of defense proteins during infection called pathogenesis-related (PR) proteins, which are grouped into families termed PR-1 to PR-17. The PR-5 family is composed of thaumatin-like proteins (TLPs), which are responsive to biotic and abiotic stress and are widely studied in plants. TLPs were also recently discovered in fungi and animals. In the poplar genome, TLPs are over-represented compared with annual species and their transcripts strongly accumulate during stress conditions. Our analysis of the poplar TLP family suggests that the expansion of this gene family was followed by diversification, as differences in expression patterns and predicted properties correlate with phylogeny. In particular, we identified a clade of poplar TLPs that cluster to a single 350 kb locus of chromosome I and that are up-regulated by poplar leaf rust infection. A wider phylogenetic analysis of eukaryote TLPs - including plant, animal and fungi sequences - shows that TLP gene content and diversity increased markedly during land plant evolution. Mapping the reported functions of characterized TLPs to the eukaryote phylogenetic tree showed that antifungal or glycan-lytic properties are widespread across eukaryote phylogeny, suggesting that these properties are shared by most TLPs and are likely associated with the presence of a conserved acidic cleft in their 3D structure. Also, we established an exhaustive catalog of TLPs with atypical architectures such as small-TLPs, TLP-kinases and small-TLP-kinases, which have potentially developed alternative functions (such as putative receptor kinases for pathogen sensing and signaling). Our study, based on the most recent plant genome sequences, provides evidence for TLP gene family diversification during land plant evolution. We have shown that the diverse functions described for TLPs are not restricted to specific clades but seem to be universal among eukaryotes, with some exceptions

  10. The protein activator of protein kinase R, PACT/RAX, negatively regulates protein kinase R during mouse anterior pituitary development.

    PubMed

    Dickerman, Benjamin K; White, Christine L; Kessler, Patricia M; Sadler, Anthony J; Williams, Bryan R G; Sen, Ganes C

    2015-12-01

    The murine double-stranded RNA-binding protein termed protein kinase R (PKR)-associated protein X (RAX) and the human homolog, protein activator of PKR (PACT), were originally characterized as activators of PKR. Mice deficient in RAX show reproductive and developmental defects, including reduced body size, craniofacial defects and anterior pituitary hypoplasia. As these defects are not observed in PKR-deficient mice, the phenotype has been attributed to PKR-independent activities of RAX. Here we further investigated the involvement of PKR in the physiological function of RAX, by generating rax(-/-) mice deficient in PKR, or carrying a kinase-inactive mutant of PKR (K271R) or an unphosphorylatable mutant of the PKR substrate eukaryotic translation initiation factor 2 α subunit (eIF2α) (S51A). Ablating PKR expression rescued the developmental and reproductive deficiencies in rax(-/-) mice. Generating rax(-/-) mice with a kinase-inactive mutant of PKR resulted in similar rescue, confirming that the rax(-/-) defects are PKR dependent; specifically that the kinase activity of PKR was required for these defects. Moreover, generating rax(-/-) mice that were heterozygous for an unphosphorylatable mutant eIF2α provides partial rescue of the rax(-/-) defect, consistent with mutation of one copy of the Eif2s1 gene. These observations were further investigated in vitro by reducing RAX expression in anterior pituitary cells, resulting in increased PKR activity and induction of the PKR-regulated cyclin-dependent kinase inhibitor p21(WAF1/CIP1). These results demonstrate that PKR kinase activity is required for onset of the rax(-/-) phenotype, implying an unexpected function for RAX as a negative regulator of PKR in the context of postnatal anterior pituitary tissue, and identify a critical role for the regulation of PKR activity for normal development.

  11. Protein-responsive ribozyme switches in eukaryotic cells

    PubMed Central

    Kennedy, Andrew B.; Vowles, James V.; d'Espaux, Leo; Smolke, Christina D.

    2014-01-01

    Genetic devices that directly detect and respond to intracellular concentrations of proteins are important synthetic biology tools, supporting the design of biological systems that target, respond to or alter specific cellular states. Here, we develop ribozyme-based devices that respond to protein ligands in two eukaryotic hosts, yeast and mammalian cells, to regulate the expression of a gene of interest. Our devices allow for both gene-ON and gene-OFF response upon sensing the protein ligand. As part of our design process, we describe an in vitro characterization pipeline for prescreening device designs to identify promising candidates for in vivo testing. The in vivo gene-regulatory activities in the two types of eukaryotic cells correlate with in vitro cleavage activities determined at different physiologically relevant magnesium concentrations. Finally, localization studies with the ligand demonstrate that ribozyme switches respond to ligands present in the nucleus and/or cytoplasm, providing new insight into their mechanism of action. By extending the sensing capabilities of this important class of gene-regulatory device, our work supports the implementation of ribozyme-based devices in applications requiring the detection of protein biomarkers. PMID:25274734

  12. Eukaryotic protein glycosylation: a primer for histochemists and cell biologists.

    PubMed

    Corfield, Anthony

    2017-02-01

    Proteins undergo co- and posttranslational modifications, and their glycosylation is the most frequent and structurally variegated type. Histochemically, the detection of glycan presence has first been performed by stains. The availability of carbohydrate-specific tools (lectins, monoclonal antibodies) has revolutionized glycophenotyping, allowing monitoring of distinct structures. The different types of protein glycosylation in Eukaryotes are described. Following this educational survey, examples where known biological function is related to the glycan structures carried by proteins are given. In particular, mucins and their glycosylation patterns are considered as instructive proof-of-principle case. The tissue and cellular location of glycoprotein biosynthesis and metabolism is reviewed, with attention to new findings in goblet cells. Finally, protein glycosylation in disease is documented, with selected examples, where aberrant glycan expression impacts on normal function to let disease pathology become manifest. The histological applications adopted in these studies are emphasized throughout the text.

  13. Eukaryote-specific extensions in ribosomal proteins of the small subunit: Structure and function

    PubMed Central

    Ghosh, Arnab; Komar, Anton A

    2015-01-01

    High-resolution structures of yeast ribosomes have improved our understanding of the architecture and organization of eukaryotic rRNA and proteins, as well as eukaryote-specific extensions present in some conserved ribosomal proteins. Despite this progress, assignment of specific functions to individual proteins and/or eukaryote-specific protein extensions remains challenging. It has been suggested that eukaryote-specific extensions of conserved proteins from the small ribosomal subunit may facilitate eukaryote-specific reactions in the initiation phase of protein synthesis. This review summarizes emerging data describing the structural and functional significance of eukaryote-specific extensions of conserved small ribosomal subunit proteins, particularly their possible roles in recruitment and spatial organization of eukaryote-specific initiation factors. PMID:26779416

  14. Dual activators of Protein Kinase R (PKR) and Protein Kinase R Like Kinase (PERK) Identify Common and Divergent Catalytic Targets

    PubMed Central

    Ming, Jie; Sun, Hong; Cao, Peng; Fusco, Dahlene N.; Chung, Raymond T.; Chorev, Michael; Jin, Qi; Aktas, Bertal H.

    2013-01-01

    Chemical genetics has evolved into a powerful tool for studying gene function in normal- and patho-biology. PKR and PERK, two eukaryotic translation initiation factor 2 alpha (eIF2α) kinases, play critical roles in maintenance of cellular hemostasis, metabolic stability, and anti-viral defenses. Both kinases interact with and phosphorylate additional substrates including tumor suppressor p53 and nuclear protein 90. Loss of function of both kinases has been studied by reverse genetics and recently identified inhibitors. In contrast, activating probes for studying the role of catalytic activity of these kinases are not available. We identified a 3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-5,7-dihydroxy-4H-chromen-4-one (DHBDC) as specific dual activator of PKR and PERK by screening a chemical library of 20,000 small molecules in a dual luciferase surrogate eIF2α phosphorylation assay. We present here extensive biological characterization and preliminary structure-activity relationship of DHBDC, which phosphorylate eIF2α by activating PKR and PERK but no other eIF2α kinases. These agents also activate downstream effectors of eIF2α phosphorylation; inducing CHOP and suppressing cyclin D1 expression and inhibiting cancer cell proliferation, all in a manner dependent on PKR and PERK. Consistent with the role of eIF2α phosphorylation in viral infection, DHBDC inhibits proliferation of human hepatitis C virus. Finally, DHBDC induces phosphorylation of Ikβα, and activates NF-κB pathway. Surprisingly, activation of NF-κB pathway is dependent on PERK but independent of PKR activity. These data indicate that DHBDC is an invaluable probe for elucidating the role of PKR and PERK in normal- and patho-biology. PMID:23784735

  15. Protein Kinase Mitogen-activated Protein Kinase Kinase Kinase Kinase 4 (MAP4K4) Promotes Obesity-induced Hyperinsulinemia.

    PubMed

    Roth Flach, Rachel J; Danai, Laura V; DiStefano, Marina T; Kelly, Mark; Menendez, Lorena Garcia; Jurczyk, Agata; Sharma, Rohit B; Jung, Dae Young; Kim, Jong Hun; Kim, Jason K; Bortell, Rita; Alonso, Laura C; Czech, Michael P

    2016-07-29

    Previous studies revealed a paradox whereby mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) acted as a negative regulator of insulin sensitivity in chronically obese mice, yet systemic deletion of Map4k4 did not improve glucose tolerance. Here, we report markedly reduced glucose-responsive plasma insulin and C-peptide levels in whole body Map4k4-depleted mice (M4K4 iKO) as well as an impaired first phase of insulin secretion from islets derived from M4K4 iKO mice ex vivo After long-term high fat diet (HFD), M4K4 iKO mice pancreata also displayed reduced β cell mass, fewer proliferating β cells and reduced islet-specific gene mRNA expression compared with controls, although insulin content was normal. Interestingly, the reduced plasma insulin in M4K4 iKO mice exposed to chronic (16 weeks) HFD was not observed in response to acute HFD challenge or short term treatment with the insulin receptor antagonist S961. Furthermore, the improved insulin sensitivity in obese M4K4 iKO mice was abrogated by high exogenous insulin over the course of a euglycemic clamp study, indicating that hypoinsulinemia promotes insulin sensitivity in chronically obese M4K4 iKO mice. These results demonstrate that protein kinase Map4k4 drives obesity-induced hyperinsulinemia and insulin resistance in part by promoting insulin secretion from β cells in mice. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Protein Kinase Mitogen-activated Protein Kinase Kinase Kinase Kinase 4 (MAP4K4) Promotes Obesity-induced Hyperinsulinemia*

    PubMed Central

    Roth Flach, Rachel J.; Danai, Laura V.; DiStefano, Marina T.; Kelly, Mark; Menendez, Lorena Garcia; Jurczyk, Agata; Sharma, Rohit B.; Jung, Dae Young; Kim, Jong Hun; Kim, Jason K.; Bortell, Rita; Alonso, Laura C.; Czech, Michael P.

    2016-01-01

    Previous studies revealed a paradox whereby mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) acted as a negative regulator of insulin sensitivity in chronically obese mice, yet systemic deletion of Map4k4 did not improve glucose tolerance. Here, we report markedly reduced glucose-responsive plasma insulin and C-peptide levels in whole body Map4k4-depleted mice (M4K4 iKO) as well as an impaired first phase of insulin secretion from islets derived from M4K4 iKO mice ex vivo. After long-term high fat diet (HFD), M4K4 iKO mice pancreata also displayed reduced β cell mass, fewer proliferating β cells and reduced islet-specific gene mRNA expression compared with controls, although insulin content was normal. Interestingly, the reduced plasma insulin in M4K4 iKO mice exposed to chronic (16 weeks) HFD was not observed in response to acute HFD challenge or short term treatment with the insulin receptor antagonist S961. Furthermore, the improved insulin sensitivity in obese M4K4 iKO mice was abrogated by high exogenous insulin over the course of a euglycemic clamp study, indicating that hypoinsulinemia promotes insulin sensitivity in chronically obese M4K4 iKO mice. These results demonstrate that protein kinase Map4k4 drives obesity-induced hyperinsulinemia and insulin resistance in part by promoting insulin secretion from β cells in mice. PMID:27226575

  17. Immunochemical characterization of rat brain protein kinase

    SciTech Connect

    Huang, K.P.; Huang, F.L.

    1986-11-05

    Polyclonal antibodies against rat brain protein kinase C (the Ca/sup 2 +//phospholipid-dependent enzyme) were raised in goat. These antibodies can neutralize completely the kinase activity in purified enzyme preparation as well as that in the crude homogenate. Immunoblot analysis of the purified and the crude protein kinase C preparations revealed a major immunoreactive band of 80 kDa. The antibodies also recognize the same enzyme from other rat tissues. Neuronal tissues (cerebral cortex, cerebellum, hypothalamus, and retina) and lymphoid organs (thymus and spleen) were found to be enriched in protein kinase C, whereas lung, kidney, liver, heart, and skeletal muscle contained relatively low amounts of this kinase. Limited proteolysis of the purified rat brain protein kinase C with trypsin results in an initial degradation of the kinase into two major fragments of 48 and 38 kDa. Both fragments are recognized by the antibodies. However, further digestion of the 48-kDa fragment to 45 kDa and the 38-kDa fragment to 33 kDa causes a loss of the immunoreactivity. Upon incubation of the cerebellar extract with Ca/sup 2 +/, the 48-kDa fragment was also identified as a major proteolytic product of protein kinase C. Proteolytic degradation of protein kinase C converts the Ca/sup 2 +//phospholipid-dependent kinase to an independent form without causing a large impairment of the binding of (/sup 3/H)phorbol 12,13-dibutyrate. The two major proteolytic fragments were separated by ion exchange chromatography and one of them (45-48 kDa) was identified as a protein kinase and the other (33-38 kDa) as a phorbol ester-binding protein. These results demonstrate that rat brain protein kinase C is composed of two functionally distinct units, namely, a protein kinase and a Ca/sup 2 +/-independent/phospholipid-dependent phorbol ester-binding protein.

  18. Selenocystamine improves protein accumulation in chloroplasts of eukaryotic green algae.

    PubMed

    Ferreira-Camargo, Livia S; Tran, Miller; Beld, Joris; Burkart, Michael D; Mayfield, Stephen P

    2015-12-01

    Eukaryotic green algae have become an increasingly popular platform for recombinant proteins production. In particular, Chlamydomonas reinhardtii, has garnered increased attention for having the necessary biochemical machinery to produce vaccines, human antibodies and next generation cancer targeting immunotoxins. While it has been shown that chloroplasts contain chaperones, peptidyl prolylisomerases and protein disulfide isomerases that facilitate these complex proteins folding and assembly, little has been done to determine which processes serve as rate-limiting steps for protein accumulation. In other expression systems, as Escherichia coli, Chinese hamster ovary cells, and insect cells, recombinant protein accumulation can be hampered by cell's inability to fold the target polypeptide into the native state, resulting in aggregation and degradation. To determine if chloroplasts' ability to oxidize proteins that require disulfide bonds into a stable conformation is a rate-limiting step of protein accumulation, three recombinant strains, each expressing a different recombinant protein, were analyzed. These recombinant proteins included fluorescent GFP, a reporter containing no disulfide bonds; Gaussia princeps luciferase, a luminescent reporter containing disulfide bonds; and an immunotoxin, an antibody-fusion protein containing disulfide bonds. Each strain was analyzed for its ability to accumulate proteins when supplemented with selenocystamine, a small molecule capable of catalyzing the formation of disulfide bonds. Selenocystamine supplementation led to an increase in luciferase and immunotoxin but not GFP accumulation. These results demonstrated that selenocystamine can increase the accumulation of proteins containing disulfide bonds and suggests that a rate-limiting step in chloroplast protein accumulation is the disulfide bonds formation in recombinant proteins native structure.

  19. Pathway illuminated: visualizing protein kinase C signaling.

    PubMed

    Violin, Jonathan D; Newton, Alexandra C

    2003-12-01

    Protein kinase C has been at the center of cell signaling since the discovery 25 years ago that it transduces signals that promote phospholipid hydrolysis. In recent years, the use of genetically encoded fluorescent reporters has enabled studies of the regulation of protein kinase C signaling in living cells. Advances in imaging techniques have unveiled unprecedented detail of the signal processing mechanics of protein kinase C, from the second messengers calcium and diacylglycerol that regulate protein kinase C activity, to the locations and kinetics of different protein kinase C isozymes, to the spatial and temporal dynamics of substrate phosphorylation by this key enzyme. This review discusses how fluorescence imaging studies have illuminated the fidelity with which protein kinase C transduces rapidly changing extracellular information into intracellular phosphorylation signals.

  20. The haem-regulated eukaryotic initiation factor 2alpha kinase: a molecular indicator of lead-toxicity anaemia in rabbits.

    PubMed

    Anand, Sanjay; Pal, Jayanta K

    2002-08-01

    The haem-regulated eukaryotic initiation factor 2alpha kinase, also called the haem-regulated inhibitor (HRI), has been shown to increase in the peripheral blood cells as a function of drug-induced anaemia in rabbits, suggesting that it could be a molecular indicator of drug-induced anaemia [Anand and Pal (1997) J. Biosci. 22, 287-298]. In the present investigation, we have determined the expression of HRI during lead-induced anaemia in rabbits. The level of anaemia has been determined by routine procedures such as reticulocyte count, haemoglobin content and packed cell volume. These values were compared with the results obtained for a quantitative Western blot of HRI in the blood cell lysates of drug- and lead-induced anaemic rabbits. These results indicate that HRI could be used as a molecular marker for lead-induced anaemia since a progressive increase in HRI levels could be detected as a function of the time of lead exposure. In order to understand the role of stress proteins, heat-shock protein (Hsp) 70 and Hsp90, in inducing anaemia during lead exposure, levels of Hsp70 and Hsp90, and their interaction with HRI, have been determined. Increased levels of these proteins and their intermolecular complexes with HRI suggest their role in regulating protein synthesis during lead-induced anaemia. These observations further reiterate the use of HRI as a potential indicator for drug- and heavy-metal-induced anaemia in humans.

  1. Regulation of transcription by eukaryotic-like serine-threonine kinases and phosphatases in Gram-positive bacterial pathogens

    PubMed Central

    Wright, David P; Ulijasz, Andrew T

    2014-01-01

    Bacterial eukaryotic-like serine threonine kinases (eSTKs) and serine threonine phosphatases (eSTPs) have emerged as important signaling elements that are indispensable for pathogenesis. Differing considerably from their histidine kinase counterparts, few eSTK genes are encoded within the average bacterial genome, and their targets are pleiotropic in nature instead of exclusive. The growing list of important eSTK/P substrates includes proteins involved in translation, cell division, peptidoglycan synthesis, antibiotic tolerance, resistance to innate immunity and control of virulence factors. Recently it has come to light that eSTK/Ps also directly modulate transcriptional machinery in many microbial pathogens. This novel form of regulation is now emerging as an additional means by which bacteria can alter their transcriptomes in response to host-specific environmental stimuli. Here we focus on the ability of eSTKs and eSTPs in Gram-positive bacterial pathogens to directly modulate transcription, the known mechanistic outcomes of these modifications, and their roles as an added layer of complexity in controlling targeted RNA synthesis to enhance virulence potential. PMID:25603430

  2. Protein kinase CK2: a newcomer in the 'druggable kinome'.

    PubMed

    Pagano, M A; Cesaro, L; Meggio, F; Pinna, L A

    2006-12-01

    The acronym CK2 (derived from the misnomer 'casein kinase' 2) denotes one of the most pleiotropic members of the eukaryotic protein kinase superfamily, characterized by an acidic consensus sequence in which a carboxylic acid (or pre-phosphorylated) side chain at position n+3 relative to the target serine/threonine residue plays a crucial role. The latest repertoire of CK2 substrates includes approx. 300 proteins, but the analysis of available phosphopeptide databases from different sources suggests that CK2 alone may be responsible for the generation of a much larger proportion (10-20%) of the eukaryotic phosphoproteome. Although for the time being CK2 is not included among protein kinases whose inhibitors are in clinical practice or in advanced clinical trials, evidence is accumulating that elevated CK2 constitutive activity co-operates to induce a number of pathological conditions, including cancer, infectious diseases, neurodegeneration and cardiovascular pathologies. The development and usage of cell-permeant, selective inhibitors discloses a scenario whereby CK2 plays a global anti-apoptotic role, which under special circumstances may lead to untimely and pathogenic cell survival.

  3. Interactions of Bacterial Proteins with Host Eukaryotic Ubiquitin Pathways

    PubMed Central

    Perrett, Charlotte Averil; Lin, David Yin-Wei; Zhou, Daoguo

    2011-01-01

    Ubiquitination is a post-translational modification in which one or more 76 amino acid polypeptide ubiquitin molecules are covalently linked to the lysine residues of target proteins. Ubiquitination is the main pathway for protein degradation that governs a variety of eukaryotic cellular processes, including the cell-cycle, vesicle trafficking, antigen presentation, and signal transduction. Not surprisingly, aberrations in the system have been implicated in the pathogenesis of many diseases including inflammatory and neurodegenerative disorders. Recent studies have revealed that viruses and bacterial pathogens exploit the host ubiquitination pathways to gain entry and to aid their survival/replication inside host cells. This review will summarize recent developments in understanding the biochemical and structural mechanisms utilized by bacterial pathogens to interact with the host ubiquitination pathways. PMID:21772834

  4. Two versatile eukaryotic vectors permitting epitope tagging, radiolabelling and nuclear localisation of expressed proteins.

    PubMed

    Georgiev, O; Bourquin, J P; Gstaiger, M; Knoepfel, L; Schaffner, W; Hovens, C

    1996-02-12

    Two versatile eukaryotic expression vectors have been developed which permit the production of an epitope-tagged cDNA insert by transient transfection in mammalian cells or by in vitro transcription-translation. The first vector, pCATCH, can be used to clone cDNA inserts in three different frames via eight unique restriction sites in a multiple cloning site (MCS) located downstream from both the FLAG epitope and the specific heart muscle kinase phosphorylation site, conferring the possibility of in vitro radiolabelling. A specific protease cleavage site enables the removal of the FLAG epitope, simplifying affinity purification of recombinant CATCH proteins. pCATCH possesses stop codons in all three reading frames at the 3' terminal end of the MCS. A derivate of this vector, pCATCH-NLS, was constructed by incorporating an SV40 nuclear localisation signal upstream from the MCS, for directed localisation of the tagged proteins.

  5. Protein-tyrosine phosphorylation interaction network in Bacillus subtilis reveals new substrates, kinase activators and kinase cross-talk

    PubMed Central

    Shi, Lei; Pigeonneau, Nathalie; Ventroux, Magali; Derouiche, Abderahmane; Bidnenko, Vladimir; Mijakovic, Ivan; Noirot-Gros, Marie-Françoise

    2014-01-01

    Signal transduction in eukaryotes is generally transmitted through phosphorylation cascades that involve a complex interplay of transmembrane receptors, protein kinases, phosphatases and their targets. Our previous work indicated that bacterial protein-tyrosine kinases and phosphatases may exhibit similar properties, since they act on many different substrates. To capture the complexity of this phosphorylation-based network, we performed a comprehensive interactome study focused on the protein-tyrosine kinases and phosphatases in the model bacterium Bacillus subtilis. The resulting network identified many potential new substrates of kinases and phosphatases, some of which were experimentally validated. Our study highlighted the role of tyrosine and serine/threonine kinases and phosphatases in DNA metabolism, transcriptional control and cell division. This interaction network reveals significant crosstalk among different classes of kinases. We found that tyrosine kinases can bind to several modulators, transmembrane or cytosolic, consistent with a branching of signaling pathways. Most particularly, we found that the division site regulator MinD can form a complex with the tyrosine kinase PtkA and modulate its activity in vitro. In vivo, it acts as a scaffold protein which anchors the kinase at the cell pole. This network highlighted a role of tyrosine phosphorylation in the spatial regulation of the Z-ring during cytokinesis. PMID:25374563

  6. Eukaryotic damaged DNA-binding proteins: DNA repair proteins or transcription factors?

    SciTech Connect

    Protic, M.

    1994-12-31

    Recognition and removal of structural defects in the genome, caused by diverse physical and chemical agents, are among the most important cell functions. Proteins that recognize and bind to modified DNA, and thereby initiate damage-induced recovery processes, have been identified in prokaryotic and eukaryotic cells. Damaged DNA-binding (DDB) proteins from prokaryotes are either DNA repair enzymes or noncatalytic subunits of larger DNA repair complexes that participate in excision repair, or in recombinational repair and SOS-mutagenesis. Although the methods employed may not have allowed detection of all eukaryotic DDB proteins and identification of their functions, it appears that during evolution cells have developed a wide array of DDB proteins that can discriminate among the diversity of DNA conformations found in the eukaryotic nucleus, as well as a gene-sharing feature found in DDB proteins that also act as transcription factors.

  7. Requirement for kinase-induced conformational change in eukaryotic initiation factor 2alpha (eIF2alpha) restricts phosphorylation of Ser51.

    PubMed

    Dey, Madhusudan; Velyvis, Algirdas; Li, John J; Chiu, Elaine; Chiovitti, David; Kay, Lewis E; Sicheri, Frank; Dever, Thomas E

    2011-03-15

    As phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) on Ser51 inhibits protein synthesis, cells restrict this phosphorylation to the antiviral protein kinase PKR and related eIF2α kinases. In the crystal structure of the PKR-eIF2α complex, the C-terminal lobe of the kinase contacts eIF2α on a face remote from Ser51, leaving Ser51 ∼ 20 Å from the kinase active site. PKR mutations that cripple the eIF2α-binding site impair phosphorylation; here, we identify mutations in eIF2α that restore Ser51 phosphorylation by PKR with a crippled substrate-binding site. These eIF2α mutations either disrupt a hydrophobic network that restricts the position of Ser51 or alter a linkage between the PKR-docking region and the Ser51 loop. We propose that the protected state of Ser51 in free eIF2α prevents promiscuous phosphorylation and the attendant translational regulation by heterologous kinases, whereas docking of eIF2α on PKR induces a conformational change that regulates the degree of Ser51 exposure and thus restricts phosphorylation to the proper kinases.

  8. Cellular reprogramming through mitogen-activated protein kinases

    PubMed Central

    Lee, Justin; Eschen-Lippold, Lennart; Lassowskat, Ines; Böttcher, Christoph; Scheel, Dierk

    2015-01-01

    Mitogen-activated protein kinase (MAPK) cascades are conserved eukaryote signaling modules where MAPKs, as the final kinases in the cascade, phosphorylate protein substrates to regulate cellular processes. While some progress in the identification of MAPK substrates has been made in plants, the knowledge on the spectrum of substrates and their mechanistic action is still fragmentary. In this focused review, we discuss the biological implications of the data in our original paper (Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana; Frontiers in Plant Science 5: 554) in the context of related research. In our work, we mimicked in vivo activation of two stress-activated MAPKs, MPK3 and MPK6, through transgenic manipulation of Arabidopsis thaliana and used phosphoproteomics analysis to identify potential novel MAPK substrates. Here, we plotted the identified putative MAPK substrates (and downstream phosphoproteins) as a global protein clustering network. Based on a highly stringent selection confidence level, the core networks highlighted a MAPK-induced cellular reprogramming at multiple levels of gene and protein expression—including transcriptional, post-transcriptional, translational, post-translational (such as protein modification, folding, and degradation) steps, and also protein re-compartmentalization. Additionally, the increase in putative substrates/phosphoproteins of energy metabolism and various secondary metabolite biosynthesis pathways coincides with the observed accumulation of defense antimicrobial substances as detected by metabolome analysis. Furthermore, detection of protein networks in phospholipid or redox elements suggests activation of downstream signaling events. Taken in context with other studies, MAPKs are key regulators that reprogram cellular events to orchestrate defense signaling in eukaryotes. PMID:26579181

  9. Deciphering Protein Kinase Specificity through Large-Scale Analysis of Yeast Phosphorylation Site Motifs

    PubMed Central

    Mok, Janine; Kim, Philip M.; Lam, Hugo Y. K.; Piccirillo, Stacy; Zhou, Xiuqiong; Jeschke, Grace R.; Sheridan, Douglas L.; Parker, Sirlester A.; Desai, Ved; Jwa, Miri; Cameroni, Elisabetta; Niu, Hengyao; Good, Matthew; Remenyi, Attila; Ma, Jia-Lin Nianhan; Sheu, Yi-Jun; Sassi, Holly E.; Sopko, Richelle; Chan, Clarence S. M.; De Virgilio, Claudio; Hollingsworth, Nancy M.; Lim, Wendell A.; Stern, David F.; Stillman, Bruce; Andrews, Brenda J.; Gerstein, Mark B.; Snyder, Michael; Turk, Benjamin E.

    2010-01-01

    Phosphorylation is a universal mechanism for regulating cell behavior in eukaryotes. Although protein kinases are known to target short linear sequence motifs on their substrates, the rules for kinase substrate recognition are not completely understood. We used a rapid peptide screening approach to determine consensus phosphorylation site motifs targeted by 61 of the 122 kinases in Saccharomyces cerevisae. Correlation of these motifs with kinase primary sequence has uncovered previously unappreciated rules for determining specificity within the kinase family, including a residue determining P−3 Arg specificity among members of the CMGC group of kinases. Furthermore, computational scanning of the yeast proteome enabled the prediction of thousands of new kinase-substrate relationships. We experimentally verified several candidate substrates of the Prk1 family of kinases in vitro and in vivo, and we identified a protein substrate of the kinase Vhs1. Together, these results elucidate how kinase catalytic domains recognize their phosphorylation targets and suggest general avenues for the identification of new kinase substrates across eukaryotes. PMID:20159853

  10. Deep conservation of human protein tandem repeats within the eukaryotes.

    PubMed

    Schaper, Elke; Gascuel, Olivier; Anisimova, Maria

    2014-05-01

    Tandem repeats (TRs) are a major element of protein sequences in all domains of life. They are particularly abundant in mammals, where by conservative estimates one in three proteins contain a TR. High generation-scale duplication and deletion rates were reported for nucleic TR units. However, it is not known whether protein TR units can also be frequently lost or gained providing a source of variation for rapid adaptation of protein function, or alternatively, tend to have conserved TR unit configurations over long evolutionary times. To obtain a systematic picture, we performed a proteome-wide analysis of the mode of evolution for human protein TRs. For this purpose, we propose a novel method for the detection of orthologous TRs based on circular profile hidden Markov models. For all detected TRs, we reconstructed bispecies TR unit phylogenies across 61 eukaryotes ranging from human to yeast. Moreover, we performed additional analyses to correlate functional and structural annotations of human TRs with their mode of evolution. Surprisingly, we find that the vast majority of human TRs are ancient, with TR unit number and order preserved intact since distant speciation events. For example, ≥ 61% of all human TRs have been strongly conserved at least since the root of all mammals, approximately 300 Ma. Further, we find no human protein TR that shows evidence for strong recent duplications and deletions. The results are in contrast to the high generation-scale mutability of nucleic TRs. Presumably, most protein TRs fold into stable and conserved structures that are indispensable for the function of the TR-containing protein. All of our data and results are available for download from http://www.atgc-montpellier.fr/TRE.

  11. Macroevolutionary trends of atomic composition and related functional group proportion in eukaryotic and prokaryotic proteins.

    PubMed

    Zhang, Yu-Juan; Yang, Chun-Lin; Hao, You-Jin; Li, Ying; Chen, Bin; Wen, Jian-Fan

    2014-01-25

    To fully explore the trends of atomic composition during the macroevolution from prokaryote to eukaryote, five atoms (oxygen, sulfur, nitrogen, carbon, hydrogen) and related functional groups in prokaryotic and eukaryotic proteins were surveyed and compared. Genome-wide analysis showed that eukaryotic proteins have more oxygen, sulfur and nitrogen atoms than prokaryotes do. Clusters of Orthologous Groups (COG) analysis revealed that oxygen, sulfur, carbon and hydrogen frequencies are higher in eukaryotic proteins than in their prokaryotic orthologs. Furthermore, functional group analysis demonstrated that eukaryotic proteins tend to have higher proportions of sulfhydryl, hydroxyl and acylamino, but lower of sulfide and carboxyl. Taken together, an apparent trend of increase was observed for oxygen and sulfur atoms in the macroevolution; the variation of oxygen and sulfur compositions and their related functional groups in macroevolution made eukaryotic proteins carry more useful functional groups. These results will be helpful for better understanding the functional significances of atomic composition evolution.

  12. Overcoming Resistance to Inhibitors of the Akt Protein Kinase by Modulation of the Pim Kinase Pathway

    DTIC Science & Technology

    2014-10-01

    kinase . This grant proposal will explore the resistance to small molecule AKT protein kinase inhibitors mediated by the... molecule AKT protein kinase inhibitors is potentially mediated by the Pim-1 protein kinase , and that unique Pim protein kinase inhibitors that can in...application is essential for the development of this combined chemotherapeutic strategy. 15. SUBJECT TERMS Small Molecule AKT Inhibitors ,

  13. Prokaryotic ancestry of eukaryotic protein networks mediating innate immunity and apoptosis.

    PubMed

    Dunin-Horkawicz, Stanislaw; Kopec, Klaus O; Lupas, Andrei N

    2014-04-03

    Protein domains characteristic of eukaryotic innate immunity and apoptosis have many prokaryotic counterparts of unknown function. By reconstructing interactomes computationally, we found that bacterial proteins containing these domains are part of a network that also includes other domains not hitherto associated with immunity. This network is connected to the network of prokaryotic signal transduction proteins, such as histidine kinases and chemoreceptors. The network varies considerably in domain composition and degree of paralogy, even between strains of the same species, and its repetitive domains are often amplified recently, with individual repeats sharing up to 100% sequence identity. Both phenomena are evidence of considerable evolutionary pressure and thus compatible with a role in the "arms race" between host and pathogen. In order to investigate the relationship of this network to its eukaryotic counterparts, we performed a cluster analysis of organisms based on a census of its constituent domains across all fully sequenced genomes. We obtained a large central cluster of mainly unicellular organisms, from which multicellular organisms radiate out in two main directions. One is taken by multicellular bacteria, primarily cyanobacteria and actinomycetes, and plants form an extension of this direction, connected via the basal, unicellular cyanobacteria. The second main direction is taken by animals and fungi, which form separate branches with a common root in the α-proteobacteria of the central cluster. This analysis supports the notion that the innate immunity networks of eukaryotes originated from their endosymbionts and that increases in the complexity of these networks accompanied the emergence of multicellularity. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

  14. The classification, structure and functioning of Ago proteins in Eukaryotes.

    PubMed

    Poterala, Aleksandra; Rzeszowska-Wolny, Joanna

    2016-09-28

    Ago proteins are members of the highly specialized and conserved Argonaute family, primarily responsible for regulation of gene expression. As a part of RNA-induced silencing complexes (RISCs) Ago proteins are responsible for binding a short RNA and cleavage/inhibition of translation of target mRNAs. Phosphorylation may work as the switch between those two functions, but the role of magnesium ion concentration is also taken into consideration. Recent reports indicate that Ago proteins can interact with an mRNA and cause inhibition of translation without the participation of a short RNA. As key elements in RNA interference processes, Ago proteins are an important and intensively exploited area of research. Furthermore, these proteins are involved in the repair of DNA double-strand breaks by homologous recombination, modifications of chromatin, and alternative splicing. Their role in the cell cycle and senescence is also being studied. In addition, Ago expression is tissue-specific, which potentially may be used for diagnostic purposes. Understanding the mechanisms of Ago functioning is therefore crucial for understanding many cellular processes. The following article presents a detailed description of the Ago proteins including their post-translational modifications, recent data and hypotheses concerning their interactions with short RNAs and mRNAs as well as the mechanisms of siRNA/miRNA sorting into individual members of the Ago subfamily, and their role in eukaryotic cells. The latest classification of Ago proteins within the Argonaute family based on evolutionary studies and their possible interactions with DNA are also described.

  15. Protein kinase biochemistry and drug discovery.

    PubMed

    Schwartz, Phillip A; Murray, Brion W

    2011-12-01

    Protein kinases are fascinating biological catalysts with a rapidly expanding knowledge base, a growing appreciation in cell regulatory control, and an ascendant role in successful therapeutic intervention. To better understand protein kinases, the molecular underpinnings of phosphoryl group transfer, protein phosphorylation, and inhibitor interactions are examined. This analysis begins with a survey of phosphate group and phosphoprotein properties which provide context to the evolutionary selection of phosphorylation as a central mechanism for biological regulation of most cellular processes. Next, the kinetic and catalytic mechanisms of protein kinases are examined with respect to model aqueous systems to define the elements of catalysis. A brief structural biology overview further delves into the molecular basis of catalysis and regulation of catalytic activity. Concomitant with a prominent role in normal physiology, protein kinases have important roles in the disease state. To facilitate effective kinase drug discovery, classic and emerging approaches for characterizing kinase inhibitors are evaluated including biochemical assay design, inhibitor mechanism of action analysis, and proper kinetic treatment of irreversible inhibitors. As the resulting protein kinase inhibitors can modulate intended and unintended targets, profiling methods are discussed which can illuminate a more complete range of an inhibitor's biological activities to enable more meaningful cellular studies and more effective clinical studies. Taken as a whole, a wealth of protein kinase biochemistry knowledge is available, yet it is clear that a substantial extent of our understanding in this field remains to be discovered which should yield many new opportunities for therapeutic intervention.

  16. Purine inhibitors of protein kinases, G proteins and polymerases

    DOEpatents

    Gray, Nathanael S.; Schultz, Peter; Kim, Sung-Hou; Meijer, Laurent

    2001-07-03

    The present invention relates to purine analogs that inhibit, inter alia, protein kinases, G-proteins and polymerases. In addition, the present invention relates to methods of using such purine analogs to inhibit protein kinases, G-proteins, polymerases and other cellular processes and to treat cellular proliferative diseases.

  17. Archaeal protein kinases and protein phosphatases: insights from genomics and biochemistry.

    PubMed Central

    Kennelly, Peter J

    2003-01-01

    Protein phosphorylation/dephosphorylation has long been considered a recent addition to Nature's regulatory arsenal. Early studies indicated that this molecular regulatory mechanism existed only in higher eukaryotes, suggesting that protein phosphorylation/dephosphorylation had emerged to meet the particular signal-transduction requirements of multicellular organisms. Although it has since become apparent that simple eukaryotes and even bacteria are sites of protein phosphorylation/dephosphorylation, the perception widely persists that this molecular regulatory mechanism emerged late in evolution, i.e. after the divergence of the contemporary phylogenetic domains. Only highly developed cells, it was reasoned, could afford the high 'overhead' costs inherent in the acquisition of dedicated protein kinases and protein phosphatases. The advent of genome sequencing has provided an opportunity to exploit Nature's phylogenetic diversity as a vehicle for critically examining this hypothesis. In tracing the origins and evolution of protein phosphorylation/dephosphorylation, the members of the Archaea, the so-called 'third domain of life', will play a critical role. Whereas several studies have demonstrated that archaeal proteins are subject to modification by covalent phosphorylation, relatively little is known concerning the identities of the proteins affected, the impact on their functional properties, or the enzymes that catalyse these events. However, examination of several archaeal genomes has revealed the widespread presence of several ostensibly 'eukaryotic' and 'bacterial' protein kinase and protein phosphatase paradigms. Similar findings of 'phylogenetic trespass' in members of the Eucarya (eukaryotes) and the Bacteria suggest that this versatile molecular regulatory mechanism emerged at an unexpectedly early point in development of 'life as we know it'. PMID:12444920

  18. Cyclic-GMP-dependent protein kinase inhibits the Ras/Mitogen-activated protein kinase pathway.

    PubMed

    Suhasini, M; Li, H; Lohmann, S M; Boss, G R; Pilz, R B

    1998-12-01

    Agents which increase the intracellular cyclic GMP (cGMP) concentration and cGMP analogs inhibit cell growth in several different cell types, but it is not known which of the intracellular target proteins of cGMP is (are) responsible for the growth-suppressive effects of cGMP. Using baby hamster kidney (BHK) cells, which are deficient in cGMP-dependent protein kinase (G-kinase), we show that 8-(4-chlorophenylthio)guanosine-3', 5'-cyclic monophosphate and 8-bromoguanosine-3',5'-cyclic monophosphate inhibit cell growth in cells stably transfected with a G-kinase Ibeta expression vector but not in untransfected cells or in cells transfected with a catalytically inactive G-kinase. We found that the cGMP analogs inhibited epidermal growth factor (EGF)-induced activation of mitogen-activated protein (MAP) kinase and nuclear translocation of MAP kinase in G-kinase-expressing cells but not in G-kinase-deficient cells. Ras activation by EGF was not impaired in G-kinase-expressing cells treated with cGMP analogs. We show that activation of G-kinase inhibited c-Raf kinase activation and that G-kinase phosphorylated c-Raf kinase on Ser43, both in vitro and in vivo; phosphorylation of c-Raf kinase on Ser43 uncouples the Ras-Raf kinase interaction. A mutant c-Raf kinase with an Ala substitution for Ser43 was insensitive to inhibition by cGMP and G-kinase, and expression of this mutant kinase protected cells from inhibition of EGF-induced MAP kinase activity by cGMP and G-kinase, suggesting that Ser43 in c-Raf is the major target for regulation by G-kinase. Similarly, B-Raf kinase was not inhibited by G-kinase; the Ser43 phosphorylation site of c-Raf is not conserved in B-Raf. Activation of G-kinase induced MAP kinase phosphatase 1 expression, but this occurred later than the inhibition of MAP kinase activation. Thus, in BHK cells, inhibition of cell growth by cGMP analogs is strictly dependent on G-kinase and G-kinase activation inhibits the Ras/MAP kinase pathway (i) by

  19. The Role of Mitogen-Activated Protein Kinase-Activated Protein Kinases (MAPKAPKs) in Inflammation

    PubMed Central

    Moens, Ugo; Kostenko, Sergiy; Sveinbjørnsson, Baldur

    2013-01-01

    Mitogen-activated protein kinase (MAPK) pathways are implicated in several cellular processes including proliferation, differentiation, apoptosis, cell survival, cell motility, metabolism, stress response and inflammation. MAPK pathways transmit and convert a plethora of extracellular signals by three consecutive phosphorylation events involving a MAPK kinase kinase, a MAPK kinase, and a MAPK. In turn MAPKs phosphorylate substrates, including other protein kinases referred to as MAPK-activated protein kinases (MAPKAPKs). Eleven mammalian MAPKAPKs have been identified: ribosomal-S6-kinases (RSK1-4), mitogen- and stress-activated kinases (MSK1-2), MAPK-interacting kinases (MNK1-2), MAPKAPK-2 (MK2), MAPKAPK-3 (MK3), and MAPKAPK-5 (MK5). The role of these MAPKAPKs in inflammation will be reviewed. PMID:24705157

  20. Rapid Identification of Protein Kinase Phosphorylation Site Motifs Using Combinatorial Peptide Libraries.

    PubMed

    Miller, Chad J; Turk, Benjamin E

    2016-01-01

    Eukaryotic protein kinases phosphorylate substrates at serine, threonine, and tyrosine residues that fall within the context of short sequence motifs. Knowing the phosphorylation site motif for a protein kinase facilitates designing substrates for kinase assays and mapping phosphorylation sites in protein substrates. Here, we describe an arrayed peptide library protocol for rapidly determining kinase phosphorylation consensus sequences. This method uses a set of peptide mixtures in which each of the 20 amino acid residues is systematically substituted at nine positions surrounding a central site of phosphorylation. Peptide mixtures are arrayed in multiwell plates and analyzed by radiolabel assay with the kinase of interest. The preferred sequence is determined from the relative rate of phosphorylation of each peptide in the array. Consensus peptides based on these sequences typically serve as efficient and specific kinase substrates for high-throughput screening or incorporation into biosensors.

  1. A eukaryotic LOV-histidine kinase with circadian clock function in the picoalga Ostreococcus.

    PubMed

    Djouani-Tahri, El-Batoul; Christie, John M; Sanchez-Ferandin, Sophie; Sanchez, Frédéric; Bouget, François-Yves; Corellou, Florence

    2011-02-01

    The marine environment has unique properties of light transmission, with an attenuation of long wavelengths within the first meters of the water column. Marine organisms have therefore evolved specific blue-light receptors such as aureochromes to absorb shorter-wavelength light. Here, we identify and characterize a light, oxygen, or voltage sensing (LOV) containing histidine kinase (LOV-HK) that functions as a new class of eukaryotic blue-light receptor in the pico-phytoplanktonic cell Ostreococcus tauri. This LOV-HK is related to the large family of LOV-HKs found in prokaryotes. Phylogenetic analysis indicates that the LOV domains from LOV-HKs, including O. tauri LOV-HK, and phototropins (phot; plant and green algal LOV serine/threonine kinases) have different evolutionary histories. Photochemical analysis shows that the LOV domain of LOV-HK binds a flavin cofactor and absorbs blue light with a fast photocycle compared with its prokaryotic counterparts. Ostreococcus tauri LOV-HK expression is induced by blue light and is under circadian control. Further, both overexpression and downregulation of LOV-HK result in arrhythmia of the circadian reporter CCA1:Luc under constant blue light. In contrast, photochemical inactivation of O. tauri LOV-HK is without effect, demonstrating its importance for function of the circadian clock under blue light. Overexpression/downregulation of O. tauriLOV-HK alters CCA1 rhythmicity under constant red light, irrespective of LOV-HK's photochemical reactivity, suggesting that O. tauri LOV-HK also participates in regulation of the circadian clock independent of its blue-light-sensing property. Molecular characterization of O. tauri LOV-HK demonstrates that this type of photoreceptor family is not limited to prokaryotes. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

  2. Evolution of an intricate J-protein network driving protein disaggregation in eukaryotes

    PubMed Central

    Nillegoda, Nadinath B; Stank, Antonia; Malinverni, Duccio; Alberts, Niels; Szlachcic, Anna; Barducci, Alessandro; De Los Rios, Paolo; Wade, Rebecca C; Bukau, Bernd

    2017-01-01

    Hsp70 participates in a broad spectrum of protein folding processes extending from nascent chain folding to protein disaggregation. This versatility in function is achieved through a diverse family of J-protein cochaperones that select substrates for Hsp70. Substrate selection is further tuned by transient complexation between different classes of J-proteins, which expands the range of protein aggregates targeted by metazoan Hsp70 for disaggregation. We assessed the prevalence and evolutionary conservation of J-protein complexation and cooperation in disaggregation. We find the emergence of a eukaryote-specific signature for interclass complexation of canonical J-proteins. Consistently, complexes exist in yeast and human cells, but not in bacteria, and correlate with cooperative action in disaggregation in vitro. Signature alterations exclude some J-proteins from networking, which ensures correct J-protein pairing, functional network integrity and J-protein specialization. This fundamental change in J-protein biology during the prokaryote-to-eukaryote transition allows for increased fine-tuning and broadening of Hsp70 function in eukaryotes. DOI: http://dx.doi.org/10.7554/eLife.24560.001 PMID:28504929

  3. Evolution of an intricate J-protein network driving protein disaggregation in eukaryotes.

    PubMed

    Nillegoda, Nadinath B; Stank, Antonia; Malinverni, Duccio; Alberts, Niels; Szlachcic, Anna; Barducci, Alessandro; De Los Rios, Paolo; Wade, Rebecca C; Bukau, Bernd

    2017-05-15

    Hsp70 participates in a broad spectrum of protein folding processes extending from nascent chain folding to protein disaggregation. This versatility in function is achieved through a diverse family of J-protein cochaperones that select substrates for Hsp70. Substrate selection is further tuned by transient complexation between different classes of J-proteins, which expands the range of protein aggregates targeted by metazoan Hsp70 for disaggregation. We assessed the prevalence and evolutionary conservation of J-protein complexation and cooperation in disaggregation. We find the emergence of a eukaryote-specific signature for interclass complexation of canonical J-proteins. Consistently, complexes exist in yeast and human cells, but not in bacteria, and correlate with cooperative action in disaggregation in vitro. Signature alterations exclude some J-proteins from networking, which ensures correct J-protein pairing, functional network integrity and J-protein specialization. This fundamental change in J-protein biology during the prokaryote-to-eukaryote transition allows for increased fine-tuning and broadening of Hsp70 function in eukaryotes.

  4. Graph theoretic analysis of protein interaction networks of eukaryotes

    NASA Astrophysics Data System (ADS)

    Goh, K.-I.; Kahng, B.; Kim, D.

    2005-11-01

    Owing to the recent progress in high-throughput experimental techniques, the datasets of large-scale protein interactions of prototypical multicellular species, the nematode worm Caenorhabditis elegans and the fruit fly Drosophila melanogaster, have been assayed. The datasets are obtained mainly by using the yeast hybrid method, which contains false-positive and false-negative simultaneously. Accordingly, while it is desirable to test such datasets through further wet experiments, here we invoke recent developed network theory to test such high-throughput datasets in a simple way. Based on the fact that the key biological processes indispensable to maintaining life are conserved across eukaryotic species, and the comparison of structural properties of the protein interaction networks (PINs) of the two species with those of the yeast PIN, we find that while the worm and yeast PIN datasets exhibit similar structural properties, the current fly dataset, though most comprehensively screened ever, does not reflect generic structural properties correctly as it is. The modularity is suppressed and the connectivity correlation is lacking. Addition of interologs to the current fly dataset increases the modularity and enhances the occurrence of triangular motifs as well. The connectivity correlation function of the fly, however, remains distinct under such interolog additions, for which we present a possible scenario through an in silico modeling.

  5. Septin-Associated Protein Kinases in the Yeast Saccharomyces cerevisiae

    PubMed Central

    Perez, Adam M.; Finnigan, Gregory C.; Roelants, Françoise M.; Thorner, Jeremy

    2016-01-01

    Septins are a family of eukaryotic GTP-binding proteins that associate into linear rods, which, in turn, polymerize end-on-end into filaments, and further assemble into other, more elaborate super-structures at discrete subcellular locations. Hence, septin-based ensembles are considered elements of the cytoskeleton. One function of these structures that has been well-documented in studies conducted in budding yeast Saccharomyces cerevisiae is to serve as a scaffold that recruits regulatory proteins, which dictate the spatial and temporal control of certain aspects of the cell division cycle. In particular, septin-associated protein kinases couple cell cycle progression with cellular morphogenesis. Thus, septin-containing structures serve as signaling platforms that integrate a multitude of signals and coordinate key downstream networks required for cell cycle passage. This review summarizes what we currently understand about how the action of septin-associated protein kinases and their substrates control information flow to drive the cell cycle into and out of mitosis, to regulate bud growth, and especially to direct timely and efficient execution of cytokinesis and cell abscission. Thus, septin structures represent a regulatory node at the intersection of many signaling pathways. In addition, and importantly, the activities of certain septin-associated protein kinases also regulate the state of organization of the septins themselves, creating a complex feedback loop. PMID:27847804

  6. Conformational characterization of human eukaryotic initiation factor 2alpha: a single tryptophan protein.

    PubMed

    Sreejith, R K; Yadav, Viveka Nand; Varshney, Nishant K; Berwal, Sunil K; Suresh, C G; Gaikwad, Sushama M; Pal, Jayanta K

    2009-12-11

    The alpha-subunit of the human eukaryotic initiation factor 2 (heIF2alpha), a GTP binding protein, plays a major role in the initiation of protein synthesis. During various cytoplasmic stresses, eIF2alpha gets phosphorylated by eIF2alpha-specific kinases resulting in inhibition of protein synthesis. The cloned and over expressed heIF2alpha, a protein with a single tryptophan (trp) residue was examined for its conformational characteristics using steady-state and time-resolved tryptophan fluorescence, circular dichroism (CD) and hydrophobic dye binding. The steady-state fluorescence spectrum, fluorescence lifetimes (tau(1)=1.13ns and tau(2)=4.74ns) and solute quenching studies revealed the presence of trp conformers in hydrophobic and differential polar environment at any given time. Estimation of the alpha-helix and beta-sheet content showed: (i) more compact structure at pH 2.0, (ii) distorted alpha-helix and rearranged beta-sheet in presence of 4M guanidine hydrochloride and (iii) retention of more than 50% ordered structure at 95 degrees C. Hydrophobic dye binding to the protein with loosened tertiary structure was observed at pH 2.0 indicating the existence of a molten globule-like structure. These observations indicate the inherent structural stability of the protein under various denaturing conditions.

  7. Novel leucine rich repeat domains in proteins from unicellular eukaryotes and bacteria.

    PubMed

    Miyashita, Hiroki; Kuroki, Yoshio; Matsushima, Norio

    2014-03-01

    Leucine rich repeats (LRRs) are present in over 20,000 proteins from viruses to eukaryotes. Two to sixty-two LRRs occur in tandem. Each repeat is typically 20-30 residues long and can be divided into an HCS (Highly conserved segment) and a VS (Variable segment). The HCS part consists of an eleven or a twelve residue stretch, LxxLxLxxNx(x/-)L, in which "L" is Leu, Ile, Val, or Phe, "N" is Asn, Thr, Ser, or Cys, "x" is a non-conserved residue, and "-" is a possible deletion site. Eight classes have been recognized. However, there are many unclassified or unrecognized LRRs. Here we performed to search novel LRRs using protein sequence database. The novel LRR domains are present over three hundred proteins, which include fungal ECM33 protein and Monosiga brevicollis LRR receptor kinase, from unicellular eukaryotes and bacteria. The HCS part is clearly different from that of the known LRRs and consists of a twelve or a thirteen residue stretch, VxGx(L/F)x(L/C)xxNx(x/-)L, that is characterized by the addition of Gly between the first conserved Val and the second conserved Leu. The novel LRRs identified here form a new family. The novel LRR domains were classified into four classes. The VS parts of the two classes are consistent with those of known, normal "SDS22-like" and "IRREKO" classes, while the other two classes have unique VS parts. The structures, functions, and evolution of the novel LRR domains and their proteins are described. The present results should stimulate various experimental studies.

  8. The Eukaryote-Like Serine/Threonine Kinase STK Regulates the Growth and Metabolism of Zoonotic Streptococcus suis

    PubMed Central

    Zhang, Chunyan; Sun, Wen; Tan, Meifang; Dong, Mengmeng; Liu, Wanquan; Gao, Ting; Li, Lu; Xu, Zhuofei; Zhou, Rui

    2017-01-01

    Like eukaryotes, bacteria express one or more serine/threonine kinases (STKs) that initiate diverse signaling networks. The STK from Streptococcus suis is encoded by a single-copy stk gene, which is crucial in stress response and virulence. To further understand the regulatory mechanism of STK in S. suis, a stk deletion strain (Δstk) and its complementary strain (CΔstk) were constructed to systematically decode STK characteristics by applying whole transcriptome RNA sequencing (RNA-Seq) and phosphoproteomic analysis. Numerous genes were differentially expressed in Δstk compared with the wild-type parental strain SC-19, including 320 up-regulated and 219 down-regulated genes. Particularly, 32 virulence-associated genes (VAGs) were significantly down-regulated in Δstk. Seven metabolic pathways relevant to bacterial central metabolism and translation are significantly repressed in Δstk. Phosphoproteomic analysis further identified 12 phosphoproteins that exhibit differential phosphorylation in Δstk. These proteins are associated with cell growth and division, glycolysis, and translation. Consistently, phenotypic assays confirmed that the Δstk strain displayed deficient growth and attenuated pathogenicity. Thus, STK is a central regulator that plays an important role in cell growth and division, as well as S. suis metabolism. PMID:28326294

  9. The SH2 domain-containing proteins in 21 species establish the provenance and scope of phosphotyrosine signaling in eukaryotes.

    PubMed

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

    2011-12-06

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

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

    PubMed Central

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

    2014-01-01

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

  11. Eukaryotic expression vectors containing genes encoding plant proteins for killing of cancer cells.

    PubMed

    Glinka, Elena M

    2013-12-01

    Gene therapy has attracted attention for its potential to specifically and efficiently target cancer cells with minimal toxicity to normal cells. At present, it offers a promising direction for the treatment of cancer patients. Numerous vectors have been engineered for the sole purpose of killing cancer cells, and some have successfully suppressed malignant tumours. Many plant proteins have anticancer properties; consequently, genes encoding some of these proteins are being used to design constructs for the inhibition of multiplying cancer cells. Data addressing the function of vectors harbouring genes specifically encoding ricin, saporin, lunasin, linamarase, and tomato thymidine kinase 1 under the control of different promoters are summarised here. Constructs employing genes to encode cytotoxic proteins as well as constructs employing genes of enzymes that convert a nontoxic prodrug into a toxic drug are considered here. Generation of eukaryotic expression vectors containing genes encoding plant proteins for killing of cancer cells may permit the broadening of cancer gene therapy strategy, particularly because of the specific mode of action of anticancer plant proteins. Copyright © 2013 Elsevier Ltd. All rights reserved.

  12. Eukaryotic Initiation Factor 6, an evolutionarily conserved regulator of ribosome biogenesis and protein translation

    SciTech Connect

    Guo, Jianjun; Jin, Zhaoqing; Yang, Xiaohan; Li, Jian-Feng; Chen, Jay

    2011-01-01

    We recently identified Receptor for Activated C Kinase 1 (RACK1) as one of the molecular links between abscisic acid (ABA) signaling and its regulation on protein translation. Moreover, we identified Eukaryotic Initiation Factor 6 (eIF6) as an interacting partner of RACK1. Because the interaction between RACK1 and eIF6 in mammalian cells is known to regulate the ribosome assembly step of protein translation initiation, it was hypothesized that the same process of protein translation in Arabidopsis is also regulated by RACK1 and eIF6. In this article, we analyzed the amino acid sequences of eIF6 in different species from different lineages and discovered some intriguing differences in protein phosphorylation sites that may contribute to its action in ribosome assembly and biogenesis. In addition, we discovered that, distinct from non-plant organisms in which eIF6 is encoded by a single gene, all sequenced plant genomes contain two or more copies of eIF6 genes. While one copy of plant eIF6 is expressed ubiquitously and might possess the conserved function in ribosome biogenesis and protein translation, the other copy seems to be only expressed in specific organs and therefore may have gained some new functions. We proposed some important studies that may help us better understand the function of eIF6 in plants.

  13. Reexamining opportunities for therapeutic protein production in eukaryotic microorganisms.

    PubMed

    Matthews, Catherine B; Wright, Chapman; Kuo, Angel; Colant, Noelle; Westoby, Matthew; Love, J Christopher

    2017-11-01

    Antibodies are an important class of therapeutics and are predominantly produced in Chinese Hamster Ovary (CHO) cell lines. While this manufacturing platform is sufficiently productive to supply patient populations of currently approved therapies, it is unclear whether or not the current CHO platform can address two significant areas of need: affordable access to biologics for patients around the globe and production of unprecedented quantities needed for very large populations of patients. Novel approaches to recombinant protein production for therapeutic biologic products may be needed, and might be enabled by non-mammalian expression systems and recent advances in bioengineering. Eukaryotic microorganisms such as fungi, microalgae, and protozoa offer the potential to produce high-quality antibodies in large quantities. In this review, we lay out the current understanding of a wide range of species and evaluate based on theoretical considerations which are best poised to deliver a step change in cost of manufacturing and volumetric productivity within the next decade.Related article: http://onlinelibrary.wiley.com/doi/10.1002/bit.26383/full. © 2017 Wiley Periodicals, Inc.

  14. ProteinHistorian: tools for the comparative analysis of eukaryote protein origin.

    PubMed

    Capra, John A; Williams, Alexander G; Pollard, Katherine S

    2012-01-01

    The evolutionary history of a protein reflects the functional history of its ancestors. Recent phylogenetic studies identified distinct evolutionary signatures that characterize proteins involved in cancer, Mendelian disease, and different ontogenic stages. Despite the potential to yield insight into the cellular functions and interactions of proteins, such comparative phylogenetic analyses are rarely performed, because they require custom algorithms. We developed ProteinHistorian to make tools for performing analyses of protein origins widely available. Given a list of proteins of interest, ProteinHistorian estimates the phylogenetic age of each protein, quantifies enrichment for proteins of specific ages, and compares variation in protein age with other protein attributes. ProteinHistorian allows flexibility in the definition of protein age by including several algorithms for estimating ages from different databases of evolutionary relationships. We illustrate the use of ProteinHistorian with three example analyses. First, we demonstrate that proteins with high expression in human, compared to chimpanzee and rhesus macaque, are significantly younger than those with human-specific low expression. Next, we show that human proteins with annotated regulatory functions are significantly younger than proteins with catalytic functions. Finally, we compare protein length and age in many eukaryotic species and, as expected from previous studies, find a positive, though often weak, correlation between protein age and length. ProteinHistorian is available through a web server with an intuitive interface and as a set of command line tools; this allows biologists and bioinformaticians alike to integrate these approaches into their analysis pipelines. ProteinHistorian's modular, extensible design facilitates the integration of new datasets and algorithms. The ProteinHistorian web server, source code, and pre-computed ages for 32 eukaryotic genomes are freely available under

  15. ProteinHistorian: Tools for the Comparative Analysis of Eukaryote Protein Origin

    PubMed Central

    Capra, John A.; Williams, Alexander G.; Pollard, Katherine S.

    2012-01-01

    The evolutionary history of a protein reflects the functional history of its ancestors. Recent phylogenetic studies identified distinct evolutionary signatures that characterize proteins involved in cancer, Mendelian disease, and different ontogenic stages. Despite the potential to yield insight into the cellular functions and interactions of proteins, such comparative phylogenetic analyses are rarely performed, because they require custom algorithms. We developed ProteinHistorian to make tools for performing analyses of protein origins widely available. Given a list of proteins of interest, ProteinHistorian estimates the phylogenetic age of each protein, quantifies enrichment for proteins of specific ages, and compares variation in protein age with other protein attributes. ProteinHistorian allows flexibility in the definition of protein age by including several algorithms for estimating ages from different databases of evolutionary relationships. We illustrate the use of ProteinHistorian with three example analyses. First, we demonstrate that proteins with high expression in human, compared to chimpanzee and rhesus macaque, are significantly younger than those with human-specific low expression. Next, we show that human proteins with annotated regulatory functions are significantly younger than proteins with catalytic functions. Finally, we compare protein length and age in many eukaryotic species and, as expected from previous studies, find a positive, though often weak, correlation between protein age and length. ProteinHistorian is available through a web server with an intuitive interface and as a set of command line tools; this allows biologists and bioinformaticians alike to integrate these approaches into their analysis pipelines. ProteinHistorian's modular, extensible design facilitates the integration of new datasets and algorithms. The ProteinHistorian web server, source code, and pre-computed ages for 32 eukaryotic genomes are freely available under

  16. Methyl labeling and TROSY NMR spectroscopy of proteins expressed in the eukaryote Pichia pastoris.

    PubMed

    Clark, Lindsay; Zahm, Jacob A; Ali, Rustam; Kukula, Maciej; Bian, Liangqiao; Patrie, Steven M; Gardner, Kevin H; Rosen, Michael K; Rosenbaum, Daniel M

    2015-07-01

    (13)C Methyl TROSY NMR spectroscopy has emerged as a powerful method for studying the dynamics of large systems such as macromolecular assemblies and membrane proteins. Specific (13)C labeling of aliphatic methyl groups and perdeuteration has been limited primarily to proteins expressed in E. coli, preventing studies of many eukaryotic proteins of physiological and biomedical significance. We demonstrate the feasibility of efficient (13)C isoleucine δ1-methyl labeling in a deuterated background in an established eukaryotic expression host, Pichia pastoris, and show that this method can be used to label the eukaryotic protein actin, which cannot be expressed in bacteria. This approach will enable NMR studies of previously intractable targets.

  17. Substrate thiophosphorylation by Arabidopsis mitogen-activated protein kinases.

    PubMed

    Leissing, Franz; Nomoto, Mika; Bocola, Marco; Schwaneberg, Ulrich; Tada, Yasuomi; Conrath, Uwe; Beckers, Gerold J M

    2016-02-24

    Mitogen-activated protein kinase (MPK) cascades are important to cellular signaling in eukaryotes. They regulate growth, development and the response to environmental challenges. MPK cascades function via reversible phosphorylation of cascade components, MEKK, MEK, and MPK, but also by MPK substrate phosphorylation. Using mass spectrometry, we previously identified many in vivo MPK3 and MPK6 substrates in Arabidopsis thaliana, and we disclosed their phosphorylation sites. We verified phosphorylation of several of our previously identified MPK3/6 substrates using a nonradioactive in vitro labeling assay. We engineered MPK3, MPK4, and MPK6 to accept bio-orthogonal ATPγS analogs for thiophosphorylating their appropriate substrate proteins. Subsequent alkylation of the thiophosphorylated amino acid residue(s) allows immunodetection using thiophosphate ester-specific antibodies. Site-directed mutagenesis of amino acids confirmed the protein substrates' site-specific phosphorylation by MPK3 and MPK6. A combined assay with MPK3, MPK6, and MPK4 revealed substrate specificity of the individual kinases. Our work demonstrates that the in vitro-labeling assay represents an effective, specific and highly sensitive test for determining kinase-substrate relationships.

  18. Novel protein kinase C inhibitors: alpha-terthiophene derivatives.

    PubMed

    Kim, D S; Ashendel, C L; Zhou, Q; Chang, C T; Lee, E S; Chang, C J

    1998-10-06

    A series of alpha-terthiophene derivatives were prepared and their protein kinase C inhibitory activity were evaluated. The aldehyde derivatives were most potent inhibitors (IC50 < 1 microM). alpha-Terthiophene monoaldehyde was inactive in the inhibitions of protein kinase A, mitogen activated protein kinase and protein tyrosine kinase.

  19. Purine inhibitors of protein kinases, G proteins and polymerases

    DOEpatents

    Gray, Nathanael S.; Schultz, Peter; Kim, Sung-Hou; Meijer, Laurent

    2004-10-12

    The present invention relates to 2-N-substituted 6-(4-methoxybenzylamino)-9-isopropylpurines that inhibit, inter alia, protein kinases, G-proteins and polymerases. In addition, the present invention relates to methods of using such 2-N-substituted 6-(4-methoxybenzylamino)-9-isopropylpurines to inhibit protein kinases, G-proteins, polymerases and other cellular processes and to treat cellular proliferative diseases.

  20. A general procedure for the production of antibody reagents against eukaryotic ribosomal proteins.

    PubMed

    Dieci, Giorgio; Bottarelli, Lorena; Ottonello, Simone

    2005-08-01

    Despite recent progress in the structural and functional analysis of bacterial and archaeal ribosomes, the structure and biogenesis of eukaryotic ribosomes still awaits a detailed characterization. Ribosomal protein-specific antibodies would be valuable tools for such studies, but their production is commonly hindered by the poor expression and solubility of eukaryotic ribosomal proteins in E. coli. We report here an improved general procedure for the over-production of recombinant eukaryotic ribosomal proteins and for the generation of the corresponding polyclonal antibodies. The specificity and sensitivity of detection of the antibodies produced by this procedure are documented.

  1. Protein kinase C-associated kinase (PKK), a novel membrane-associated, ankyrin repeat-containing protein kinase.

    PubMed

    Chen, L; Haider, K; Ponda, M; Cariappa, A; Rowitch, D; Pillai, S

    2001-06-15

    A novel murine membrane-associated protein kinase, PKK (protein kinase C-associated kinase), was cloned on the basis of its physical association with protein kinase Cbeta (PKCbeta). The regulated expression of PKK in mouse embryos is consistent with a role for this kinase in early embryogenesis. The human homolog of PKK has over 90% identity to its murine counterpart, has been localized to chromosome 21q22.3, and is identical to the PKCdelta-interacting kinase, DIK (Bahr, C., Rohwer, A., Stempka, L., Rincke, G., Marks, F., and Gschwendt, M. (2000) J. Biol. Chem. 275, 36350-36357). PKK comprises an N-terminal kinase domain and a C-terminal region containing 11 ankyrin repeats. PKK exhibits protein kinase activity in vitro and associates with cellular membranes. PKK exists in three discernible forms at steady state: an underphosphorylated form of 100 kDa; a soluble, cytosolic, phosphorylated form of 110 kDa; and a phosphorylated, detergent-insoluble form of 112 kDa. PKK is initially synthesized as an underphosphorylated soluble 100-kDa protein that is quantitatively converted to a detergent-soluble 110-kDa form. This conversion requires an active catalytic domain. Although PKK physically associates with PKCbeta, it does not phosphorylate this PKC isoform. However, PKK itself may be phosphorylated by PKCbeta. PKK represents a developmentally regulated protein kinase that can associate with membranes. The functional significance of its association with PKCbeta remains to be ascertained.

  2. Dynamics driven allostery in protein kinases

    PubMed Central

    Kornev, Alexandr P.; Taylor, Susan S.

    2015-01-01

    Protein kinases have very dynamic structures and their functionality strongly depends on their dynamic state. Active kinases reveal a dynamic pattern with residues clustering into semirigid communities that move in µs-ms timescale. Previously detected hydrophobic spines serve as connectors between communities. Communities do not follow the traditional subdomain structure of the kinase core or its secondary structure elements. Instead they are organized around main functional units. Integration of the communities depends on the assembly of the hydrophobic spine and phosphorylation of the activation loop. Single mutations can significantly disrupt the dynamic infrastructure and thereby interfere with long distance allosteric signaling that propagates throughout the whole molecule. Dynamics is proposed to be the underlying mechanism for allosteric regulation in protein kinases. PMID:26481499

  3. Expanding the Kinome World: A New Protein Kinase Family Widely Conserved in Bacteria.

    PubMed

    Nguyen, Hien-Anh; El Khoury, Takla; Guiral, Sébastien; Laaberki, Maria-Halima; Candusso, Marie-Pierre; Galisson, Frédéric; Foucher, Anne-Emmanuelle; Kesraoui, Salsabil; Ballut, Lionel; Vallet, Sylvain; Orelle, Cédric; Zucchini, Laure; Martin, Juliette; Page, Adeline; Attieh, Jihad; Aghajari, Nushin; Grangeasse, Christophe; Jault, Jean-Michel

    2017-10-13

    Fine tuning of signaling pathways is essential for cells to cope with sudden environmental variations. This delicate balance is maintained in particular by protein kinases that control the activity of target proteins by reversible phosphorylation. In addition to homologous eukaryotic enzymes, bacteria have evolved some specific Ser/Thr/Tyr protein kinases without any structural resemblance to their eukaryotic counterparts. Here, we show that a previously identified family of ATPases, broadly conserved among bacteria, is in fact a new family of protein kinases with a Ser/Thr/Tyr kinase activity. A prototypic member of this family, YdiB from Bacillus subtilis, is able to autophosphorylate and to phosphorylate a surrogate substrate, the myelin basic protein. Two crystal structures of YdiB were solved (1.8 and 2.0Å) that display a unique ATP-binding fold unrelated to known protein kinases, although a conserved HxD motif is reminiscent of that found in Hanks-type protein kinases. The effect of mutations of conserved residues further highlights the unique nature of this new protein kinase family that we name ubiquitous bacterial kinase. We investigated the cellular role of YdiB and showed that a ∆ydiB mutant was more sensitive to paraquat treatment than the wild type, with ~13% of cells with an aberrant morphology. In addition, YdiE, which is known to participate with both YdiC and YdiB in an essential chemical modification of some specific tRNAs, is phosphorylated in vitro by YdiB. These results expand the boundaries of the bacterial kinome and support the involvement of YdiB in protein translation and resistance to oxidative stress in B. subtilis. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Oncoprotein protein kinase antibody kit

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    2008-12-23

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  5. Targeting Plasmodium falciparum protein kinases with adenosine analogue-oligoarginine conjugates.

    PubMed

    Lavogina, Darja; Budu, Alexandre; Enkvist, Erki; Hopp, Christine S; Baker, David A; Langsley, Gordon; Garcia, Celia R S; Uri, Asko

    2014-03-01

    During the last decade, a vast number of inhibitors, ligands and fluorescent probes have evolved for mammalian protein kinases; however, the suitability of these compounds for studies of evolutionarily divergent eukaryotes has mostly been left beyond the scope of research. Here, we examined whether adenosine analogue-oligoarginine conjugates that had been extensively characterized as efficient inhibitors of the human protein kinases are applicable for targeting Plasmodium protein kinases. We demonstrated that ARCs were not only able to bind to and inhibit a representative member of Plasmodium falciparum kinome (cGMP-dependent protein kinase) in biochemical assay, but also affected the general phosphorylation levels in parasites released from the infected red blood cells upon saponin treatment. These findings urge advantaging of already existing biochemical tools, whose initially generic, but intrinsically "tunable" selectivity profiles could be used for dissection of signaling pathways outside the initially defined group of biological targets. Copyright © 2014 Elsevier Inc. All rights reserved.

  6. Potential involvement of extracellular signal-regulated kinase 1 and 2 in encystation of a primitive eukaryote, Giardia lamblia. Stage-specific activation and intracellular localization.

    PubMed

    Ellis, John G; Davila, Monica; Chakrabarti, Ratna

    2003-01-17

    Mitogen-activated protein kinase (MAPK) pathways are major signaling systems by which eukaryotic cells convert environmental cues to intracellular events such as proliferation and differentiation. We have identified Giardia lamblia homologues of two members of the MAPK family ERK1 and ERK2. Functional characterization of giardial ERK1 and ERK2 revealed that both kinases were expressed in trophozoites and encysting cells as 44- and 41-kDa polypeptides, respectively, and were catalytically active. Analysis of the kinetic parameters of the recombinant proteins showed that ERK2 is approximately 5 times more efficient than ERK1 in phosphorylating myelin basic protein as a substrate, although the phosphorylating efficiency of the native ERK1 and ERK2 appeared to be the same. Immunofluorescence analysis of the subcellular localization of ERK1 and ERK2 in trophozoites showed ERK1 staining mostly in the median body and in the outer edges of the adhesive disc and ERK2 staining in the nuclei and in the caudal flagella. Our study also showed a noticeable change in the subcellular distribution of ERK2 during encystation, which became more punctate and mostly cytoplasmic, but no significant change in the ERK1 localization at any time during encystation. Interestingly, both ERK1 and ERK2 enzymes exhibited a significantly reduced kinase activity during encystation reaching a minimum at 24 h, except for an initial approximately 2.5-fold increase in the ERK1 activity at 2 h, which resumed back to the normal levels at 48 h despite no apparent change in the expression level of either one of these kinases in encysting cells. A reduced concentration of the phosphorylated ERK1 and ERK2 was also evident in these cells at 24 h. Our study suggests a functional distinction between ERK1 and ERK2 and that these kinases may play a critical role in trophozoite differentiation into cysts.

  7. A class of selective antibacterials derived from a protein kinase inhibitor pharmacophore

    PubMed Central

    Miller, J. Richard; Dunham, Steve; Mochalkin, Igor; Banotai, Craig; Bowman, Matthew; Buist, Susan; Dunkle, Bill; Hanna, Debra; Harwood, H. James; Huband, Michael D.; Karnovsky, Alla; Kuhn, Michael; Limberakis, Chris; Liu, Jia Y.; Mehrens, Shawn; Mueller, W. Thomas; Narasimhan, Lakshmi; Ogden, Adam; Ohren, Jeff; Prasad, J. V. N. Vara; Shelly, John A.; Skerlos, Laura; Sulavik, Mark; Thomas, V. Hayden; VanderRoest, Steve; Wang, LiAnn; Wang, Zhigang; Whitton, Amy; Zhu, Tong; Stover, C. Kendall

    2009-01-01

    As the need for novel antibiotic classes to combat bacterial drug resistance increases, the paucity of leads resulting from target-based antibacterial screening of pharmaceutical compound libraries is of major concern. One explanation for this lack of success is that antibacterial screening efforts have not leveraged the eukaryotic bias resulting from more extensive chemistry efforts targeting eukaryotic gene families such as G protein-coupled receptors and protein kinases. Consistent with a focus on antibacterial target space resembling these eukaryotic targets, we used whole-cell screening to identify a series of antibacterial pyridopyrimidines derived from a protein kinase inhibitor pharmacophore. In bacteria, the pyridopyrimidines target the ATP-binding site of biotin carboxylase (BC), which catalyzes the first enzymatic step of fatty acid biosynthesis. These inhibitors are effective in vitro and in vivo against fastidious Gram-negative pathogens including Haemophilus influenzae. Although the BC active site has architectural similarity to those of eukaryotic protein kinases, inhibitor binding to the BC ATP-binding site is distinct from the protein kinase-binding mode, such that the inhibitors are selective for bacterial BC. In summary, we have discovered a promising class of potent antibacterials with a previously undescribed mechanism of action. In consideration of the eukaryotic bias of pharmaceutical libraries, our findings also suggest that pursuit of a novel inhibitor leads for antibacterial targets with active-site structural similarity to known human targets will likely be more fruitful than the traditional focus on unique bacterial target space, particularly when structure-based and computational methodologies are applied to ensure bacterial selectivity. PMID:19164768

  8. A class of selective antibacterials derived from a protein kinase inhibitor pharmacophore.

    PubMed

    Miller, J Richard; Dunham, Steve; Mochalkin, Igor; Banotai, Craig; Bowman, Matthew; Buist, Susan; Dunkle, Bill; Hanna, Debra; Harwood, H James; Huband, Michael D; Karnovsky, Alla; Kuhn, Michael; Limberakis, Chris; Liu, Jia Y; Mehrens, Shawn; Mueller, W Thomas; Narasimhan, Lakshmi; Ogden, Adam; Ohren, Jeff; Prasad, J V N Vara; Shelly, John A; Skerlos, Laura; Sulavik, Mark; Thomas, V Hayden; VanderRoest, Steve; Wang, LiAnn; Wang, Zhigang; Whitton, Amy; Zhu, Tong; Stover, C Kendall

    2009-02-10

    As the need for novel antibiotic classes to combat bacterial drug resistance increases, the paucity of leads resulting from target-based antibacterial screening of pharmaceutical compound libraries is of major concern. One explanation for this lack of success is that antibacterial screening efforts have not leveraged the eukaryotic bias resulting from more extensive chemistry efforts targeting eukaryotic gene families such as G protein-coupled receptors and protein kinases. Consistent with a focus on antibacterial target space resembling these eukaryotic targets, we used whole-cell screening to identify a series of antibacterial pyridopyrimidines derived from a protein kinase inhibitor pharmacophore. In bacteria, the pyridopyrimidines target the ATP-binding site of biotin carboxylase (BC), which catalyzes the first enzymatic step of fatty acid biosynthesis. These inhibitors are effective in vitro and in vivo against fastidious gram-negative pathogens including Haemophilus influenzae. Although the BC active site has architectural similarity to those of eukaryotic protein kinases, inhibitor binding to the BC ATP-binding site is distinct from the protein kinase-binding mode, such that the inhibitors are selective for bacterial BC. In summary, we have discovered a promising class of potent antibacterials with a previously undescribed mechanism of action. In consideration of the eukaryotic bias of pharmaceutical libraries, our findings also suggest that pursuit of a novel inhibitor leads for antibacterial targets with active-site structural similarity to known human targets will likely be more fruitful than the traditional focus on unique bacterial target space, particularly when structure-based and computational methodologies are applied to ensure bacterial selectivity.

  9. A class of selective antibacterials derived from a protein kinase inhibitor pharmacophore

    SciTech Connect

    Miller, J. Richard; Dunham, Steve; Mochalkin, Igor; Banotai, Craig; Bowman, Matthew; Buist, Susan; Dunkle, Bill; Hanna, Debra; Harwood, H. James; Huband, Michael D.; Karnovsky, Alla; Kuhn, Michael; Limberakis, Chris; Liu, Jia Y.; Mehrens, Shawn; Mueller, W. Thomas; Narasimhan, Lakshmi; Ogden, Adam; Ohren, Jeff; Prasad, J.V.N. Vara; Shelly, John A.; Skerlos, Laura; Sulavik, Mark; Thomas, V. Hayden; VanderRoest, Steve; Wang, LiAnn; Wang, Zhigang; Whitton, Amy; Zhu, Tong; Stover, C. Kendall

    2009-06-25

    As the need for novel antibiotic classes to combat bacterial drug resistance increases, the paucity of leads resulting from target-based antibacterial screening of pharmaceutical compound libraries is of major concern. One explanation for this lack of success is that antibacterial screening efforts have not leveraged the eukaryotic bias resulting from more extensive chemistry efforts targeting eukaryotic gene families such as G protein-coupled receptors and protein kinases. Consistent with a focus on antibacterial target space resembling these eukaryotic targets, we used whole-cell screening to identify a series of antibacterial pyridopyrimidines derived from a protein kinase inhibitor pharmacophore. In bacteria, the pyridopyrimidines target the ATP-binding site of biotin carboxylase (BC), which catalyzes the first enzymatic step of fatty acid biosynthesis. These inhibitors are effective in vitro and in vivo against fastidious Gram-negative pathogens including Haemophilus influenzae. Although the BC active site has architectural similarity to those of eukaryotic protein kinases, inhibitor binding to the BC ATP-binding site is distinct from the protein kinase-binding mode, such that the inhibitors are selective for bacterial BC. In summary, we have discovered a promising class of potent antibacterials with a previously undescribed mechanism of action. In consideration of the eukaryotic bias of pharmaceutical libraries, our findings also suggest that pursuit of a novel inhibitor leads for antibacterial targets with active-site structural similarity to known human targets will likely be more fruitful than the traditional focus on unique bacterial target space, particularly when structure-based and computational methodologies are applied to ensure bacterial selectivity.

  10. Haemophilus ducreyi LspA proteins are tyrosine phosphorylated by macrophage-encoded protein tyrosine kinases.

    PubMed

    Deng, Kaiping; Mock, Jason R; Greenberg, Steven; van Oers, Nicolai S C; Hansen, Eric J

    2008-10-01

    The LspA proteins (LspA1 and LspA2) of Haemophilus ducreyi are necessary for this pathogen to inhibit the phagocytic activity of macrophage cell lines, an event that can be correlated with a reduction in the level of active Src family protein tyrosine kinases (PTKs) in these eukaryotic cells. During studies investigating this inhibitory mechanism, it was discovered that the LspA proteins themselves were tyrosine phosphorylated after wild-type H. ducreyi cells were incubated with macrophages. LspA proteins in cell-free concentrated H. ducreyi culture supernatant fluid could also be tyrosine phosphorylated by macrophages. This ability to tyrosine phosphorylate the LspA proteins was not limited to immune cell lineages but could be accomplished by both HeLa and COS-7 cells. Kinase inhibitor studies with macrophages demonstrated that the Src family PTKs were required for this tyrosine phosphorylation activity. In silico methods and site-directed mutagenesis were used to identify EPIYG and EPVYA motifs in LspA1 that contained tyrosines that were targets for phosphorylation. A total of four tyrosines could be phosphorylated in LspA1, with LspA2 containing eight predicted tyrosine phosphorylation motifs. Purified LspA1 fusion proteins containing either the EPIYG or EPVYA motifs were shown to be phosphorylated by purified Src PTK in vitro. Macrophage lysates could also tyrosine phosphorylate the LspA proteins and an LspA1 fusion protein via a mechanism that was dependent on the presence of both divalent cations and ATP. Several motifs known to interact with or otherwise affect eukaryotic kinases were identified in the LspA proteins.

  11. Phylogenetic distributions and histories of proteins involved in anaerobic pyruvate metabolism in eukaryotes.

    PubMed

    Hug, Laura A; Stechmann, Alexandra; Roger, Andrew J

    2010-02-01

    Protists that live in low oxygen conditions often oxidize pyruvate to acetate via anaerobic ATP-generating pathways. Key enzymes that commonly occur in these pathways are pyruvate:ferredoxin oxidoreductase (PFO) and [FeFe]-hydrogenase (H(2)ase) as well as the associated [FeFe]-H(2)ase maturase proteins HydE, HydF, and HydG. Determining the origins of these proteins in eukaryotes is of key importance to understanding the origins of anaerobic energy metabolism in microbial eukaryotes. We conducted a comprehensive search for genes encoding these proteins in available whole genomes and expressed sequence tag data from diverse eukaryotes. Our analyses of the presence/absence of eukaryotic PFO, [FeFe]-H(2)ase, and H(2)ase maturase sequences across eukaryotic diversity reveal orthologs of these proteins encoded in the genomes of a variety of protists previously not known to contain them. Our phylogenetic analyses revealed: 1) extensive lateral gene transfers of both PFO and [FeFe]-H(2)ase in eubacteria, 2) decreased support for the monophyly of eukaryote PFO domains, and 3) that eukaryotic [FeFe]-H(2)ases are not monophyletic. Although there are few eukaryote [FeFe]-H(2)ase maturase orthologs characterized, phylogenies of these proteins do recover eukaryote monophyly, although a consistent eubacterial sister group for eukaryotic homologs could not be determined. An exhaustive search for these five genes in diverse genomes from two representative eubacterial groups, the Clostridiales and the alpha-proteobacteria, shows that although these enzymes are nearly universally present within the former group, they are very rare in the latter. No alpha-proteobacterial genome sequenced to date encodes all five proteins. Molecular phylogenies and the extremely restricted distribution of PFO, [FeFe]-H(2)ases, and their associated maturases within the alpha-proteobacteria do not support a mitochondrial origin for these enzymes in eukaryotes. However, the unexpected prevalence of PFO

  12. Protein kinase C is essential for viability of the rice blast fungus M agnaporthe oryzae

    PubMed Central

    Penn, Tina J.; Wood, Mark E.; Soanes, Darren M.; Csukai, Michael; Corran, Andrew John

    2015-01-01

    Summary Protein kinase C constitutes a family of serine–threonine kinases found in all eukaryotes and implicated in a wide range of cellular functions, including regulation of cell growth, cellular differentiation and immunity. Here, we present three independent lines of evidence which indicate that protein kinase C is essential for viability of M agnaporthe oryzae. First, all attempts to generate a target deletion of PKC 1, the single copy protein kinase C‐encoding gene, proved unsuccessful. Secondly, conditional gene silencing of PKC 1 by RNA interference led to severely reduced growth of the fungus, which was reversed by targeted deletion of the Dicer2‐encoding gene, MDL 2. Finally, selective kinase inhibition of protein kinase C by targeted allelic replacement with an analogue‐sensitive PKC 1AS allele led to specific loss of fungal viability in the presence of the PP1 inhibitor. Global transcriptional profiling following selective PKC inhibition identified significant changes in gene expression associated with cell wall re‐modelling, autophagy, signal transduction and secondary metabolism. When considered together, these results suggest protein kinase C is essential for growth and development of M . oryzae with extensive downstream targets in addition to the cell integrity pathway. Targeting protein kinase C signalling may therefore prove an effective means of controlling rice blast disease. PMID:26192090

  13. Non-degradative Ubiquitination of Protein Kinases

    PubMed Central

    Ball, K. Aurelia; Johnson, Jeffrey R.; Lewinski, Mary K.; Guatelli, John; Verschueren, Erik; Krogan, Nevan J.; Jacobson, Matthew P.

    2016-01-01

    Growing evidence supports other regulatory roles for protein ubiquitination in addition to serving as a tag for proteasomal degradation. In contrast to other common post-translational modifications, such as phosphorylation, little is known about how non-degradative ubiquitination modulates protein structure, dynamics, and function. Due to the wealth of knowledge concerning protein kinase structure and regulation, we examined kinase ubiquitination using ubiquitin remnant immunoaffinity enrichment and quantitative mass spectrometry to identify ubiquitinated kinases and the sites of ubiquitination in Jurkat and HEK293 cells. We find that, unlike phosphorylation, ubiquitination most commonly occurs in structured domains, and on the kinase domain, ubiquitination is concentrated in regions known to be important for regulating activity. We hypothesized that ubiquitination, like other post-translational modifications, may alter the conformational equilibrium of the modified protein. We chose one human kinase, ZAP-70, to simulate using molecular dynamics with and without a monoubiquitin modification. In Jurkat cells, ZAP-70 is ubiquitinated at several sites that are not sensitive to proteasome inhibition and thus may have other regulatory roles. Our simulations show that ubiquitination influences the conformational ensemble of ZAP-70 in a site-dependent manner. When monoubiquitinated at K377, near the C-helix, the active conformation of the ZAP-70 C-helix is disrupted. In contrast, when monoubiquitinated at K476, near the kinase hinge region, an active-like ZAP-70 C-helix conformation is stabilized. These results lead to testable hypotheses that ubiquitination directly modulates kinase activity, and that ubiquitination is likely to alter structure, dynamics, and function in other protein classes as well. PMID:27253329

  14. Protein Kinase A: A Master Kinase of Granulosa Cell Differentiation

    PubMed Central

    Puri, Pawan; Little-Ihrig, Lynda; Chandran, Uma; Law, Nathan C.; Hunzicker-Dunn, Mary; Zeleznik, Anthony J.

    2016-01-01

    Activation of protein kinase A (PKA) by follicle stimulating hormone (FSH) transduces the signal that drives differentiation of ovarian granulosa cells (GCs). An unresolved question is whether PKA is sufficient to initiate the complex program of GC responses to FSH. We compared signaling pathways and gene expression profiles of GCs stimulated with FSH or expressing PKA-CQR, a constitutively active mutant of PKA. Both FSH and PKA-CQR stimulated the phosphorylation of proteins known to be involved in GC differentiation including CREB, ß-catenin, AKT, p42/44 MAPK, GAB2, GSK-3ß, FOXO1, and YAP. In contrast, FSH stimulated the phosphorylation of p38 MAP kinase but PKA-CQR did not. Microarray analysis revealed that 85% of transcripts that were up-regulated by FSH were increased to a comparable extent by PKA-CQR and of the transcripts that were down-regulated by FSH, 76% were also down-regulated by PKA-CQR. Transcripts regulated similarly by FSH and PKA-CQR are involved in steroidogenesis and differentiation, while transcripts more robustly up-regulated by PKA-CQR are involved in ovulation. Thus, PKA, under the conditions of our experimental approach appears to function as a master upstream kinase that is sufficient to initiate the complex pattern of intracellular signaling pathway and gene expression profiles that accompany GC differentiation. PMID:27324437

  15. A comprehensive evolutionary classification of proteins encoded in complete eukaryotic genomes

    PubMed Central

    Koonin, Eugene V; Fedorova, Natalie D; Jackson, John D; Jacobs, Aviva R; Krylov, Dmitri M; Makarova, Kira S; Mazumder, Raja; Mekhedov, Sergei L; Nikolskaya, Anastasia N; Rao, B Sridhar; Rogozin, Igor B; Smirnov, Sergei; Sorokin, Alexander V; Sverdlov, Alexander V; Vasudevan, Sona; Wolf, Yuri I; Yin, Jodie J; Natale, Darren A

    2004-01-01

    Background Sequencing the genomes of multiple, taxonomically diverse eukaryotes enables in-depth comparative-genomic analysis which is expected to help in reconstructing ancestral eukaryotic genomes and major events in eukaryotic evolution and in making functional predictions for currently uncharacterized conserved genes. Results We examined functional and evolutionary patterns in the recently constructed set of 5,873 clusters of predicted orthologs (eukaryotic orthologous groups or KOGs) from seven eukaryotic genomes: Caenorhabditis elegans, Drosophila melanogaster, Homo sapiens, Arabidopsis thaliana, Saccharomyces cerevisiae, Schizosaccharomyces pombe and Encephalitozoon cuniculi. Conservation of KOGs through the phyletic range of eukaryotes strongly correlates with their functions and with the effect of gene knockout on the organism's viability. The approximately 40% of KOGs that are represented in six or seven species are enriched in proteins responsible for housekeeping functions, particularly translation and RNA processing. These conserved KOGs are often essential for survival and might approximate the minimal set of essential eukaryotic genes. The 131 single-member, pan-eukaryotic KOGs we identified were examined in detail. For around 20 that remained uncharacterized, functions were predicted by in-depth sequence analysis and examination of genomic context. Nearly all these proteins are subunits of known or predicted multiprotein complexes, in agreement with the balance hypothesis of evolution of gene copy number. Other KOGs show a variety of phyletic patterns, which points to major contributions of lineage-specific gene loss and the 'invention' of genes new to eukaryotic evolution. Examination of the sets of KOGs lost in individual lineages reveals co-elimination of functionally connected genes. Parsimonious scenarios of eukaryotic genome evolution and gene sets for ancestral eukaryotic forms were reconstructed. The gene set of the last common ancestor of

  16. Prediction of Protein Kinase-Ligand Interactions through 2.5D Kinochemometrics.

    PubMed

    Bosc, Nicolas; Wroblowski, Berthold; Meyer, Christophe; Bonnet, Pascal

    2017-01-23

    So far, 518 protein kinases have been identified in the human genome. They share a common mechanism of protein phosphorylation and are involved in many critical biological processes of eukaryotic cells. Deregulation of the kinase phosphorylation function induces severe illnesses such as cancer, diabetes, or inflammatory diseases. Many actors in the pharmaceutical domain have made significant efforts to design potent and selective protein kinase inhibitors as new potential drugs. Because the ATP binding site is highly conserved in the protein kinase family, the design of selective inhibitors remains a challenge and has negatively impacted the progression of drug candidates to late-stage clinical development. The work presented here adopts a 2.5D kinochemometrics (KCM) approach, derived from proteochemometrics (PCM), in which protein kinases are depicted by a novel 3D descriptor and the ligands by 2D fingerprints. We demonstrate in two examples that the protein descriptor successfully classified protein kinases based on their group membership and their Asp-Phe-Gly (DFG) conformation. We also compared the performance of our models with those obtained from a full 2D KCM model and QSAR models. In both cases, the internal validation of the models demonstrated good capabilities to distinguish "active" from "inactive" protein kinase-ligand pairs. However, the external validation performed on two independent data sets showed that the two statistical models tended to overestimate the number of "inactive" pairs.

  17. An inducible and secreted eukaryote-like serine/threonine kinase of Salmonella enterica serovar Typhi promotes intracellular survival and pathogenesis.

    PubMed

    Theeya, Nagaraja; Ta, Atri; Das, Sayan; Mandal, Rahul S; Chakrabarti, Oishee; Chakrabarti, Saikat; Ghosh, Amar N; Das, Santasabuj

    2015-02-01

    Eukaryote-like serine/threonine kinases (eSTKs) constitute an important family of bacterial virulence factors. Genome analysis had predicted putative eSTKs in Salmonella enterica serovar Typhi, although their functional characterization and the elucidation of their role in pathogenesis are still awaited. We show here that the primary sequence and secondary structure of the t4519 locus of Salmonella Typhi Ty2 have all the signatures of eukaryotic superfamily kinases. t4519 encodes a ∼39-kDa protein (T4519), which shows serine/threonine kinase activities in vitro. Recombinant T4519 (rT4519) is autophosphorylated and phosphorylates the universal substrate myelin basic protein. Infection of macrophages results in decreased viability of the mutant (Ty2Δt4519) strain, which is reversed by gene complementation. Moreover, reactive oxygen species produced by the macrophages signal to the bacteria to induce T4519, which is translocated to the host cell cytoplasm. That T4519 may target a host substrate(s) is further supported by the activation of host cellular signaling pathways and the induction of cytokines/chemokines. Finally, the role of T4519 in the pathogenesis of Salmonella Typhi is underscored by the significantly decreased mortality of mice infected with the Ty2Δt4519 strain and the fact that the competitive index of this strain for causing systemic infection is 0.25% that of the wild-type strain. This study characterizes the first eSTK of Salmonella Typhi and demonstrates its role in promoting phagosomal survival of the bacteria within macrophages, which is a key determinant of pathogenesis. This, to the best of our knowledge, is the first study to describe the essential role of eSTKs in the in vivo pathogenesis of Salmonella spp.

  18. Eukaryotic DNA replication: Orchestrated action of multi-subunit protein complexes.

    PubMed

    Kang, Sukhyun; Kang, Mi-Sun; Ryu, Eunjin; Myung, Kyungjae

    2017-05-01

    Genome duplication is an essential process to preserve genetic information between generations. The eukaryotic cell cycle is composed of functionally distinct phases: G1, S, G2, and M. One of the key replicative proteins that participate at every stage of DNA replication is the Mcm2-7 complex, a replicative helicase. In the G1 phase, inactive Mcm2-7 complexes are loaded on the replication origins by replication-initiator proteins, ORC and Cdc6. Two kinases, S-CDK and DDK, convert the inactive origin-loaded Mcm2-7 complex to an active helicase, the CMG complex in the S phase. The activated CMG complex begins DNA unwinding and recruits enzymes essential for DNA synthesis to assemble a replisome at the replication fork. After completion of DNA synthesis, the inactive CMG complex on the replicated DNA is removed from chromatin to terminate DNA replication. In this review, we will discuss the structure, function, and regulation of the molecular machines involved in each step of DNA replication. Copyright © 2017. Published by Elsevier B.V.

  19. Conservation and Variability of Synaptonemal Complex Proteins in Phylogenesis of Eukaryotes

    PubMed Central

    Grishaeva, Tatiana M.; Bogdanov, Yuri F.

    2014-01-01

    The problems of the origin and evolution of meiosis include the enigmatic variability of the synaptonemal complexes (SCs) which, being morphology similar, consist of different proteins in different eukaryotic phyla. Using bioinformatics methods, we monitored all available eukaryotic proteomes to find proteins similar to known SC proteins of model organisms. We found proteins similar to SC lateral element (LE) proteins and possessing the HORMA domain in the majority of the eukaryotic taxa and assume them the most ancient among all SC proteins. Vertebrate LE proteins SYCP2, SYCP3, and SC65 proved to have related proteins in many invertebrate taxa. Proteins of SC central space are most evolutionarily variable. It means that different protein-protein interactions can exist to connect LEs. Proteins similar to the known SC proteins were not found in Euglenophyta, Chrysophyta, Charophyta, Xanthophyta, Dinoflagellata, and primitive Coelomata. We conclude that different proteins whose common feature is the presence of domains with a certain conformation are involved in the formation of the SC in different eukaryotic phyla. This permits a targeted search for orthologs of the SC proteins using phylogenetic trees. Here we consider example of phylogenetic trees for protozoans, fungi, algae, mosses, and flowering plants. PMID:25147749

  20. Conservation and variability of synaptonemal complex proteins in phylogenesis of eukaryotes.

    PubMed

    Grishaeva, Tatiana M; Bogdanov, Yuri F

    2014-01-01

    The problems of the origin and evolution of meiosis include the enigmatic variability of the synaptonemal complexes (SCs) which, being morphology similar, consist of different proteins in different eukaryotic phyla. Using bioinformatics methods, we monitored all available eukaryotic proteomes to find proteins similar to known SC proteins of model organisms. We found proteins similar to SC lateral element (LE) proteins and possessing the HORMA domain in the majority of the eukaryotic taxa and assume them the most ancient among all SC proteins. Vertebrate LE proteins SYCP2, SYCP3, and SC65 proved to have related proteins in many invertebrate taxa. Proteins of SC central space are most evolutionarily variable. It means that different protein-protein interactions can exist to connect LEs. Proteins similar to the known SC proteins were not found in Euglenophyta, Chrysophyta, Charophyta, Xanthophyta, Dinoflagellata, and primitive Coelomata. We conclude that different proteins whose common feature is the presence of domains with a certain conformation are involved in the formation of the SC in different eukaryotic phyla. This permits a targeted search for orthologs of the SC proteins using phylogenetic trees. Here we consider example of phylogenetic trees for protozoans, fungi, algae, mosses, and flowering plants.

  1. Protein knockouts in living eukaryotes using deGradFP and green fluorescent protein fusion targets.

    PubMed

    Caussinus, Emmanuel; Kanca, Oguz; Affolter, Markus

    2013-09-24

    This unit describes deGradFP (degrade Green Fluorescent Protein), an easy-to-implement protein knockout method applicable in any eukaryotic genetic system. Depleting a protein in order to study its function in a living organism is usually achieved at the gene level (genetic mutations) or at the RNA level (RNA interference and morpholinos). However, any system that acts upstream of the proteic level depends on the turnover rate of the existing target protein, which can be extremely slow. In contrast, deGradFP is a fast method that directly depletes GFP fusion proteins. In particular, deGradFP is able to counteract maternal effects in embryos and causes early and fast onset loss-of-function phenotypes of maternally contributed proteins.

  2. Inhibitory activity for the interferon-induced protein kinase is associated with the reovirus serotype 1 sigma 3 protein.

    PubMed Central

    Imani, F; Jacobs, B L

    1988-01-01

    In this report we demonstrate that reovirus serotype 1-infected cells contain an inhibitor of the interferon-induced, double-stranded RNA (dsRNA)-dependent protein kinase. We provide evidence that suggests that the virus-encoded sigma 3 protein is likely responsible for this kinase inhibitory activity. We could not detect activation of the dsRNA-dependent protein kinase in extracts prepared from either interferon-treated or untreated reovirus serotype 1-infected mouse L cells under conditions that led to activation of the kinase in extracts prepared from either interferon-treated or untreated, uninfected cells. Extracts from reovirus-infected cells blocked activation of kinase in extracts from interferon-treated cells when the two were mixed prior to assay. The kinase inhibitory activity in extracts of reovirus-infected cells could be overcome by adding approximately 100-fold excess of dsRNA over the amount required to activate kinase in extracts of uninfected cells. Kinase inhibitory activity in extracts of interferon-treated, virus-infected cells could be overcome with somewhat less dsRNA (approximately 10-fold excess). Most of the inhibitory activity in the extracts could be removed by adsorption with immobilized anti-reovirus sigma 3 serum or immobilized dsRNA, suggesting that the dsRNA-binding sigma 3 protein is necessary for kinase inhibitory activity. Purified sigma 3 protein, when added to reaction mixtures containing partially purified kinase, inhibited enzyme activation. Control of activation of this kinase, which can modify eukaryotic protein synthesis initiation factor 2, may be relevant to the sensitivity of reovirus replication to treatment of cells with interferon and to the shutoff of host protein synthesis in reovirus-infected cells. Images PMID:2460857

  3. Conformational transitions of the catalytic domain of heme-regulated eukaryotic initiation factor 2α kinase, a key translational regulatory molecule.

    PubMed

    Sreejith, R K; Suresh, C G; Bhosale, Siddharth H; Bhavnani, Varsha; Kumar, Avinash; Gaikwad, Sushama M; Pal, Jayanta K

    2012-01-01

    In mammalian cells, the heme-regulated inhibitor (HRI) plays a critical role in the regulation of protein synthesis at the initiation step through phosphorylation of α-subunit of the eukaryotic initiation factor 2 (eIF2). In this study we have cloned and performed biophysical characterization of the kinase catalytic domain (KD) of rabbit HRI. The KD described here comprises kinase 1, the kinase insertion domain (KI) and kinase 2. We report here the existence of an active and stable monomer of HRI (KD). The HRI (KD) containing three tryptophan residues was examined for its conformational transitions occurring under various denaturing conditions using steady-state and time-resolved tryptophan fluorescence, circular dichroism (CD) and hydrophobic dye binding. The parameter A and phase diagram analysis revealed multi-state unfolding and existence of three stable intermediates during guanidine hydrochloride (Gdn-HCl) induced unfolding of HRI (KD). The protein treated with 6 M Gdn-HCl showed collisional and static mechanism of acrylamide quenching and the constants (K(sv) = 3.08 M(-1) and K(s)= 5.62 M(-1)) were resolved using time resolved fluorescence titration. Based on pH, guanidine hydrochloride and temperature mediated transitions, HRI (KD) appears to exemplify a rigid molten globule-like intermediate with compact secondary structure, altered tertiary structure and exposed hydrophobic patches at pH 3.0. The results indicate the inherent structural stability of HRI (KD), a member of the class of stress response proteins.

  4. How Do Protein Kinases Take a Selfie (Autophosphorylate)?

    PubMed

    Beenstock, Jonah; Mooshayef, Navit; Engelberg, David

    2016-11-01

    Eukaryotic protein kinases (EPKs) control most biological processes and play central roles in many human diseases. To become catalytically active, EPKs undergo conversion from an inactive to an active conformation, an event that depends upon phosphorylation of their activation loop. Intriguingly, EPKs can use their own catalytic activity to achieve this critical phosphorylation. In other words, paradoxically, EPKs catalyze autophosphorylation when supposedly in their inactive state. This indicates the existence of another important conformation that specifically permits autophosphorylation at the activation loop, which in turn imposes adoption of the active conformation. This can be considered a prone-to-autophosphorylate conformation. Recent findings suggest that in prone-to-autophosphorylate conformations catalytic motifs are aligned allosterically, by dimerization or by regulators, and support autophosphorylation in cis or trans. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Crystal structure of the protein At3g01520, a eukaryotic universal stress protein-like protein from Arabidopsis thaliana in complex with AMP.

    PubMed

    Kim, Do Jin; Bitto, Eduard; Bingman, Craig A; Kim, Hyun-Jung; Han, Byung Woo; Phillips, George N

    2015-07-01

    Members of the universal stress protein (USP) family are conserved in a phylogenetically diverse range of prokaryotes, fungi, protists, and plants and confer abilities to respond to a wide range of environmental stresses. Arabidopsis thaliana contains 44 USP domain-containing proteins, and USP domain is found either in a small protein with unknown physiological function or in an N-terminal portion of a multi-domain protein, usually a protein kinase. Here, we report the first crystal structure of a eukaryotic USP-like protein encoded from the gene At3g01520. The crystal structure of the protein At3g01520 was determined by the single-wavelength anomalous dispersion method and refined to an R factor of 21.8% (Rfree = 26.1%) at 2.5 Å resolution. The crystal structure includes three At3g01520 protein dimers with one AMP molecule bound to each protomer, comprising a Rossmann-like α/β overall fold. The bound AMP and conservation of residues in the ATP-binding loop suggest that the protein At3g01520 also belongs to the ATP-binding USP subfamily members. © 2015 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.

  6. Problem-Solving Test: "In Vitro" Protein Kinase A Reaction

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2009-01-01

    Phosphorylation of proteins by protein kinases is an important mechanism in the regulation of protein activity. Among hundreds of protein kinases present in human cells, PKA, the first kinase discovered, belongs to the most important and best characterized group of these enzymes. The author presents an experiment that analyzes the "in vitro"…

  7. Problem-Solving Test: "In Vitro" Protein Kinase A Reaction

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2009-01-01

    Phosphorylation of proteins by protein kinases is an important mechanism in the regulation of protein activity. Among hundreds of protein kinases present in human cells, PKA, the first kinase discovered, belongs to the most important and best characterized group of these enzymes. The author presents an experiment that analyzes the "in vitro"…

  8. Thymidylate kinase of Mycobacterium tuberculosis: A chimera sharing properties common to eukaryotic and bacterial enzymes

    PubMed Central

    Munier-Lehmann, HÉLÈNE; Chaffotte, Alain; Pochet, Sylvie; Labesse, Gilles

    2001-01-01

    We have overexpressed in Escherichia coli the thymidylate kinase of Mycobacterium tuberculosis (TMPKmt). Biochemical and physico-chemical characterization of TMPKmt revealed distinct structural and catalytic features when compared to its counterpart from yeast (TMPKy) or E. coli (TMPKec). Denaturation of the dimeric TMPKmt by urea under equilibrium conditions was studied by intrinsic fluorescence and circular dichroism (CD) spectroscopy. It suggested a three-state unfolding mechanism with a monomeric intermediate. On the other hand, 3′-azido-3′-deoxythymidine monophosphate (AZT-MP), which is substrate for TMPKy and TMPKec acts as a potent competitive inhibitor for TMPKmt. We propose a structural model of TMPKmt in which the overall fold described in TMPKy and TMPKec is conserved and slight differences at the level of primary and 3D-structure explain strong variations in the phosphorylation rate of substrate analogs. According to the model, we synthesized dTMP analogs acting either as substrates or specific inhibitors of TMPKmt. This approach based on slight structural differences among similar proteins could be applied to other essential enzymes for the design of new species-specific antimicrobials. PMID:11369858

  9. Thymidylate kinase of Mycobacterium tuberculosis: a chimera sharing properties common to eukaryotic and bacterial enzymes.

    PubMed

    Munier-Lehmann, H; Chaffotte, A; Pochet, S; Labesse, G

    2001-06-01

    We have overexpressed in Escherichia coli the thymidylate kinase of Mycobacterium tuberculosis (TMPKmt). Biochemical and physico-chemical characterization of TMPKmt revealed distinct structural and catalytic features when compared to its counterpart from yeast (TMPKy) or E. coli (TMPKec). Denaturation of the dimeric TMPKmt by urea under equilibrium conditions was studied by intrinsic fluorescence and circular dichroism (CD) spectroscopy. It suggested a three-state unfolding mechanism with a monomeric intermediate. On the other hand, 3'-azido-3'-deoxythymidine monophosphate (AZT-MP), which is substrate for TMPKy and TMPKec acts as a potent competitive inhibitor for TMPKMT: We propose a structural model of TMPKmt in which the overall fold described in TMPKy and TMPKec is conserved and slight differences at the level of primary and 3D-structure explain strong variations in the phosphorylation rate of substrate analogs. According to the model, we synthesized dTMP analogs acting either as substrates or specific inhibitors of TMPKMT: This approach based on slight structural differences among similar proteins could be applied to other essential enzymes for the design of new species-specific antimicrobials.

  10. Mycobacterium tuberculosis Serine/Threonine Protein Kinases

    PubMed Central

    PRISIC, SLADJANA; HUSSON, ROBERT N.

    2014-01-01

    The Mycobacterium tuberculosis genome encodes 11 serine/threonine protein kinases (STPKs). A similar number of two-component systems are also present, indicating that these two signal transduction mechanisms are both important in the adaptation of this bacterial pathogen to its environment. The M. tuberculosis phosphoproteome includes hundreds of Ser- and Thr-phosphorylated proteins that participate in all aspects of M. tuberculosis biology, supporting a critical role for the STPKs in regulating M. tuberculosis physiology. Nine of the STPKs are receptor type kinases, with an extracytoplasmic sensor domain and an intracellular kinase domain, indicating that these kinases transduce external signals. Two other STPKs are cytoplasmic and have regulatory domains that sense changes within the cell. Structural analysis of some of the STPKs has led to advances in our understanding of the mechanisms by which these STPKs are activated and regulated. Functional analysis has provided insights into the effects of phosphorylation on the activity of several proteins, but for most phosphoproteins the role of phosphorylation in regulating function is unknown. Major future challenges include characterizing the functional effects of phosphorylation for this large number of phosphoproteins, identifying the cognate STPKs for these phosphoproteins, and determining the signals that the STPKs sense. Ultimately, combining these STPK-regulated processes into larger, integrated regulatory networks will provide deeper insight into M. tuberculosis adaptive mechanisms that contribute to tuberculosis pathogenesis. Finally, the STPKs offer attractive targets for inhibitor development that may lead to new therapies for drug-susceptible and drug-resistant tuberculosis. PMID:25429354

  11. A retroviral-derived peptide phosphorylates protein kinase D/protein kinase Cmu involving phospholipase C and protein kinase C.

    PubMed

    Luangwedchakarn, Voravich; Day, Noorbibi K; Hitchcock, Remi; Brown, Pam G; Lerner, Danica L; Rucker, Rajivi P; Cianciolo, George J; Good, Robert A; Haraguchi, Soichi

    2003-05-01

    CKS-17, a synthetic peptide representing a unique amino acid motif which is highly conserved in retroviral transmembrane proteins and other immunoregulatory proteins, induces selective immunomodulatory functions, both in vitro and in vivo, and activates intracellular signaling molecules such as cAMP and extracellular signal-regulated kinases. In the present study, using Jurkat T-cells, we report that CKS-17 phosphorylates protein kinase D (PKD)/protein kinase C (PKC) mu. Total cell extracts from CKS-17-stimulated Jurkat cells were immunoblotted with an anti-phospho-PKCmu antibody. The results show that CKS-17 significantly phosphorylates PKD/PKCmu in a dose- and time-dependent manner. Treatment of cells with the PKC inhibitors GF 109203X and Ro 31-8220, which do not act directly on PKD/PKCmu, attenuates CKS-17-induced phosphorylation of PKD/PKCmu. In contrast, the selective protein kinase A inhibitor H-89 does not reverse the action of CKS-17. Furthermore, a phospholipase C (PLC) selective inhibitor, U-73122, completely blocks the phosphorylation of PKD/PKCmu by CKS-17 while a negative control U-73343 does not. In addition, substitution of lysine for arginine residues in the CKS-17 sequence completely abrogates the ability of CKS-17 to phosphorylate PKD/PKCmu. These results clearly indicate that CKS-17 phosphorylates PKD/PKCmu through a PLC- and PKC-dependent mechanism and that arginine residues play an essential role in this activity of CKS-17, presenting a novel modality of the retroviral peptide CKS-17 and molecular interaction of this compound with target cells.

  12. Loss of the eukaryotic initiation factor 2α kinase general control nonderepressible 2 protects mice from pressure overload-induced congestive heart failure without affecting ventricular hypertrophy.

    PubMed

    Lu, Zhongbing; Xu, Xin; Fassett, John; Kwak, Dongmin; Liu, Xiaoyu; Hu, Xinli; Wang, Huan; Guo, Haipeng; Xu, Dachun; Yan, Shuo; McFalls, Edward O; Lu, Fei; Bache, Robert J; Chen, Yingjie

    2014-01-01

    In response to several stresses, including nutrient deprivation, general control nonderepressible 2 kinase (GCN2) attenuates mRNA translation by phosphorylating eukaryotic initiation factor 2α(Ser51). Energy starvation is known to exacerbate congestive heart failure, and eukaryotic initiation factor 2α(Ser51) phosphorylation is increased in the failing heart. However, the effect of GCN2 during the evolution of congestive heart failure has not been tested. In this study, we examined the influence of GCN2 expression in response to a cardiac stress by inducing chronic pressure overload with transverse aortic constriction in wild-type and GCN2 knockout mice. Under basal conditions, GCN2 knockout mice had normal left ventricular structure and function, but after transverse aortic constriction, they demonstrated less contractile dysfunction, less increase in lung weight, less increase in lung inflammation and vascular remodeling, and less myocardial apoptosis and fibrosis compared with wild-type mice, despite an equivalent degree of left ventricular hypertrophy. As expected, GCN2 knockout attenuated transverse aortic constriction-induced cardiac eukaryotic initiation factor 2α(Ser51) phosphorylation and preserved sarcoplasmic reticulum Ca(2+) ATPase expression compared with wild-type mice. Interestingly, the expression of the antiapoptotic protein Bcl-2 was significantly elevated in GCN2 knockout hearts, whereas in isolated neonatal cardiomyocytes, selective knockdown of GCN2 increased Bcl-2 protein expression and enhanced myocyte resistance to an apoptotic stress. Collectively, our data support the notion that GCN2 impairs the ventricular adaptation to chronic pressure overload by reducing Bcl-2 expression and increasing cardiomyocyte susceptibility to apoptotic stimuli. Our findings suggest that strategies to reduce GCN2 activity in cardiac tissue may be a novel approach to attenuate congestive heart failure development.

  13. Eukaryotic elongation factor 2 is a prognostic marker and its kinase a potential therapeutic target in HCC.

    PubMed

    Pott, Leona L; Hagemann, Sascha; Reis, Henning; Lorenz, Kristina; Bracht, Thilo; Herold, Thomas; Skryabin, Boris V; Megger, Dominik A; Kälsch, Julia; Weber, Frank; Sitek, Barbara; Baba, Hideo A

    2017-02-14

    Hepatocellular carcinoma is a cancer with increasing incidence and largely refractory to current anticancer drugs. Since Sorafenib, a multikinase inhibitor has shown modest efficacy in advanced hepatocellular carcinoma additional treatments are highly needed. Protein phosphorylation via kinases is an important post-translational modification to regulate cell homeostasis including proliferation and apoptosis. Therefore kinases are valuable targets in cancer therapy. To this end we performed 2D differential gel electrophoresis and mass spectrometry analysis of phosphoprotein-enriched lysates of tumor and corresponding non-tumorous liver samples to detect differentially abundant phosphoproteins to screen for novel kinases as potential drug targets. We identified 34 differentially abundant proteins in phosphoprotein enriched lysates. Expression and distribution of the candidate protein eEF2 and its phosphorylated isoform was validated immunohistochemically on 78 hepatocellular carcinoma and non-tumorous tissue samples. Validation showed that total eEF2 and phosphorylated eEF2 at threonine 56 are prognostic markers for overall survival of HCC-patients. The activity of the regulating eEF2 kinase, compared between tumor and non-tumorous tissue lysates by in vitro kinase assays, is more than four times higher in tumor tissues. Functional analyzes regarding eEF2 kinase were performed in JHH5 cells with CRISPR/Cas9 mediated eEF2 kinase knock out. Proliferation and growth is decreased in eEF2 kinase knock out cells. eEF2 and phosphorylated eEF2 are prognostic markers for survival of hepatocellular carcinoma patients and the regulating eEF2 kinase is a potential drug target for tumor therapy.

  14. Silencing of EEF2K (eukaryotic elongation factor-2 kinase) reveals AMPK-ULK1-dependent autophagy in colon cancer cells.

    PubMed

    Xie, Chuan-Ming; Liu, Xiao-Yu; Sham, Kathy W Y; Lai, Josie M Y; Cheng, Christopher H K

    2014-09-01

    EEF2K (eukaryotic elongation factor-2 kinase), also known as Ca (2+)/calmodulin-dependent protein kinase III, functions in downregulating peptide chain elongation through inactivation of EEF2 (eukaryotic translation elongation factor 2). Currently, there is a limited amount of information on the promotion of autophagic survival by EEF2K in breast and glioblastoma cell lines. However, the precise role of EEF2K in carcinogenesis as well as the underlying mechanism involved is still poorly understood. In this study, contrary to the reported autophagy-promoting activity of EEF2K in certain cancer cells, EEF2K is shown to negatively regulate autophagy in human colon cancer cells as indicated by the increase of LC3-II levels, the accumulation of LC3 dots per cell, and the promotion of autophagic flux in EEF2K knockdown cells. EEF2K negatively regulates cell viability, clonogenicity, cell proliferation, and cell size in colon cancer cells. Autophagy induced by EEF2K silencing promotes cell survival and does not potentiate the anticancer efficacy of the AKT inhibitor MK-2206. In addition, autophagy induced by silencing of EEF2K is attributed to induction of protein synthesis and activation of the AMPK-ULK1 pathway, independent of the suppression of MTOR activity and ROS generation. Knockdown of AMPK or ULK1 significantly abrogates EEF2K silencing-induced increase of LC3-II levels, accumulation of LC3 dots per cell as well as cell proliferation in colon cancer cells. In conclusion, silencing of EEF2K promotes autophagic survival via activation of the AMPK-ULK1 pathway in colon cancer cells. This finding suggests that upregulation of EEF2K activity may constitute a novel approach for the treatment of human colon cancer.

  15. A Hypothesis for DNA Viruses as the Origin of Eukaryotic Replication Proteins

    PubMed Central

    Villarreal, Luis P.; DeFilippis, Victor R.

    2000-01-01

    The eukaryotic replicative DNA polymerases are similar to those of large DNA viruses of eukaryotic and bacterial T4 phages but not to those of eubacteria. We develop and examine the hypothesis that DNA virus replication proteins gave rise to those of eukaryotes during evolution. We chose the DNA polymerase from phycodnavirus (which infects microalgae) as the basis of this analysis, as it represents a virus of a primitive eukaryote. We show that it has significant similarity with replicative DNA polymerases of eukaryotes and certain of their large DNA viruses. Sequence alignment confirms this similarity and establishes the presence of highly conserved domains in the polymerase amino terminus. Subsequent reconstruction of a phylogenetic tree indicates that these algal viral DNA polymerases are near the root of the clade containing all eukaryotic DNA polymerase delta members but that this clade does not contain the polymerases of other DNA viruses. We consider arguments for the polarity of this relationship and present the hypothesis that the replication genes of DNA viruses gave rise to those of eukaryotes and not the reverse direction. PMID:10888648

  16. Differential regulation of rice mitogen activated protein kinase kinase (MKK) by abiotic stress.

    PubMed

    Kumar, Kundan; Rao, Kudupudi Prabhakara; Sharma, Pallavi; Sinha, Alok Krishna

    2008-10-01

    Mitogen activated protein kinase cascade plays a crucial role in various biotic and abiotic stresses, hormones, cell division and developmental processes. MAP kinase kinase being integral part of this cascade performs an important function of integrating upstream signals to mitogen activated protein kinase for further appropriate cellular responses. We here report cloning of five MAP kinase kinase members from Oryza sativa indica cultivar var. Pusa Basmati 1, namely MAP kinase kinases 1, 3, 4, 6 and 10-2. All these members, except MKK10-2 possess fully canonical motif structures of MAP kinase kinase. The deduced amino acid sequence showed changes at certain position within japonica and indica variety of rice. Analysis of transcript regulation by quantitative real time PCR revealed that these five members are differentially regulated by cold, heat, salinity and drought stresses. MAP kinase kinases 4 and 6 are strongly regulated by cold and salt stresses while MAP kinase kinase 1 is regulated by salt and drought stresses. MAP kinase kinase 10-2 is regulated only by cold stress. The study provides the indication of involvement of specific MAP kinase kinase in different abiotic stress signaling and also possible cross talks that exist during the signaling processes.

  17. Methyl labeling and TROSY NMR spectroscopy of proteins expressed in the eukaryote Pichia pastoris

    PubMed Central

    Clark, Lindsay; Zahm, Jacob A.; Ali, Rustam; Kukula, Maciej; Bian, Liangqiao; Patrie, Steven M.; Gardner, Kevin H.; Rosen, Michael K.; Rosenbaum, Daniel M.

    2015-01-01

    13C Methyl TROSY NMR spectroscopy has emerged as a powerful method for studying the dynamics of large systems such as macromolecular assemblies and membrane proteins. Specific 13C labeling of aliphatic methyl groups and perdeuteration has been limited primarily to proteins expressed in E. coli, preventing studies of many eukaryotic proteins of physiological and biomedical significance. We demonstrate the feasibility of efficient 13C isoleucine δ1-methyl labeling in a deuterated background in an established eukaryotic expression host, Pichia pastoris, and show that this method can be used to label the eukaryotic protein actin, which cannot be expressed in bacteria. This approach will enable NMR studies of previously intractable targets. PMID:26025061

  18. Crystal Structure of the Protein Kinase Domain of Yeast AMP-Activated Protein Kinase Snf1

    SciTech Connect

    Rudolph,M.; Amodeo, G.; Bai, Y.; Tong, L.

    2005-01-01

    AMP-activated protein kinase (AMPK) is a master metabolic regulator, and is an important target for drug development against diabetes, obesity, and other diseases. AMPK is a hetero-trimeric enzyme, with a catalytic ({alpha}) subunit, and two regulatory ({beta} and {gamma}) subunits. Here we report the crystal structure at 2.2 Angstrom resolution of the protein kinase domain (KD) of the catalytic subunit of yeast AMPK (commonly known as SNF1). The Snf1-KD structure shares strong similarity to other protein kinases, with a small N-terminal lobe and a large C-terminal lobe. Two negative surface patches in the structure may be important for the recognition of the substrates of this kinase.

  19. Heart failure-specific changes in protein kinase signalling.

    PubMed

    Lorenz, Kristina; Stathopoulou, Konstantina; Schmid, Evelyn; Eder, Petra; Cuello, Friederike

    2014-06-01

    Among the myriad of molecular alterations occurring in heart failure development, aggravation of the disease is often attributed to global or local changes in protein kinase activity, thus making protein kinases attractive targets for therapeutic intervention. Since protein kinases do not only have maladaptive roles, but also contribute to the physiological integrity of cells, it is a challenging task to circumvent undesired inhibition of protein kinase activity. Identification of posttranslational modifications and/or protein-protein interactions that are exclusively apparent under pathophysiological conditions provides exciting information for alternative non-kinase inhibitory treatment strategies that eliminate maladaptive functions of a protein kinase, but preserve the beneficial ones. Here, we focus on the disease-specific regulation of a number of protein kinases, namely, Ca(2+)/calmodulin-dependent protein kinase II isoform δ (CaMKIIδ), G protein-coupled receptor kinase 2 (GRK2), extracellular signal-regulated kinase 1 and 2 (ERK1/2), protein kinase D (PKD) and protein kinase C isoform β2 (PKCβ2), which are embedded in complex signal transduction pathways implicated in heart failure development, and discuss potential avenues for novel treatment strategies to combat heart disease.

  20. Tv-RIO1 - an atypical protein kinase from the parasitic nematode Trichostrongylus vitrinus.

    PubMed

    Hu, Min; Laronde-Leblanc, Nicole; Sternberg, Paul W; Gasser, Robin B

    2008-09-22

    Protein kinases are key enzymes that regulate a wide range of cellular processes, including cell-cycle progression, transcription, DNA replication and metabolic functions. These enzymes catalyse the transfer of phosphates to serine, threonine and tyrosine residues, thus playing functional roles in reversible protein phosphorylation. There are two main groups, namely eukaryotic protein kinases (ePKs) and atypical protein kinases (aPKs); RIO kinases belong to the latter group. While there is some information about RIO kinases and their roles in animals, nothing is known about them in parasites. This is the first study to characterise a RIO1 kinase from any parasite. A full-length cDNA (Tv-rio-1) encoding a RIO1 protein kinase (Tv-RIO1) was isolated from the economically important parasitic nematode Trichostrongylus vitrinus (Order Strongylida). The uninterrupted open reading frame (ORF) of 1476 nucleotides encoded a protein of 491 amino acids, containing the characteristic RIO1 motif LVHADLSEYNTL. Tv-rio-1 was transcribed at the highest level in the third-stage larva (L3), and a higher level in adult females than in males. Comparison with homologues from other organisms showed that protein Tv-RIO1 had significant homology to related proteins from a range of metazoans and plants. Amino acid sequence identity was most pronounced in the ATP-binding motif, active site and metal binding loop. Phylogenetic analyses of selected amino acid sequence data revealed Tv-RIO1 to be most closely related to the proteins in the species of Caenorhabditis. A structural model of Tv-RIO1 was constructed and compared with the published crystal structure of RIO1 of Archaeoglobus fulgidus (Af-Rio1). This study provides the first insights into the RIO1 protein kinases of nematodes, and a foundation for further investigations into the biochemical and functional roles of this molecule in biological processes in parasitic nematodes.

  1. Inferring protein function from genomic sequence: Giardia lamblia expresses a phosphatidylinositol kinase-related kinase similar to yeast and mammalian TOR.

    PubMed

    Morrison, Hilary G; Zamora, Gus; Campbell, Robert K; Sogin, Mitchell L

    2002-12-01

    Functional assays of genes have historically led to insights about the activities of a protein or protein cascade. However, the rapid expansion of genomic and proteomic information for a variety of diverse taxa is an alternative and powerful means of predicting function by comparing the enzymes and metabolic pathways used by different organisms. As part of the Giardia lamblia genome sequencing project, we routinely survey the complement of predicted proteins and compare those found in this putatively early diverging eukaryote with those of prokaryotes and more recently evolved eukaryotic lineages. Such comparisons reveal the minimal composition of conserved metabolic pathways, suggest which proteins may have been acquired by lateral transfer, and, by their absence, hint at functions lost in the transition from a free-living to a parasitic lifestyle. Here, we describe the use of bioinformatic approaches to investigate the complement and conservation of proteins in Giardia involved in the regulation of translation. We compare an FK506 binding protein homologue and phosphatidylinositol kinase-related kinase present in Giardia to those found in other eukaryotes for which complete genomic sequence data are available. Our investigation of the Giardia genome suggests that PIK-related kinases are of ancient origin and are highly conserved.

  2. A-kinase Anchoring Protein 79/150 Recruits Protein Kinase C to Phosphorylate Roundabout Receptors.

    PubMed

    Samelson, Bret K; Gore, Bryan B; Whiting, Jennifer L; Nygren, Patrick J; Purkey, Alicia M; Colledge, Marcie; Langeberg, Lorene K; Dell'Acqua, Mark L; Zweifel, Larry S; Scott, John D

    2015-05-29

    Anchoring proteins direct protein kinases and phosphoprotein phosphatases toward selected substrates to control the efficacy, context, and duration of neuronal phosphorylation events. The A-kinase anchoring protein AKAP79/150 interacts with protein kinase A (PKA), protein kinase C (PKC), and protein phosphatase 2B (calcineurin) to modulate second messenger signaling events. In a mass spectrometry-based screen for additional AKAP79/150 binding partners, we have identified the Roundabout axonal guidance receptor Robo2 and its ligands Slit2 and Slit3. Biochemical and cellular approaches confirm that a linear sequence located in the cytoplasmic tail of Robo2 (residues 991-1070) interfaces directly with sites on the anchoring protein. Parallel studies show that AKAP79/150 interacts with the Robo3 receptor in a similar manner. Immunofluorescent staining detects overlapping expression patterns for murine AKAP150, Robo2, and Robo3 in a variety of brain regions, including hippocampal region CA1 and the islands of Calleja. In vitro kinase assays, peptide spot array mapping, and proximity ligation assay staining approaches establish that human AKAP79-anchored PKC selectively phosphorylates the Robo3.1 receptor subtype on serine 1330. These findings imply that anchored PKC locally modulates the phosphorylation status of Robo3.1 in brain regions governing learning and memory and reward. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Evolutionarily conserved binding of translationally controlled tumor protein to eukaryotic elongation factor 1B.

    PubMed

    Wu, Huiwen; Gong, Weibin; Yao, Xingzhe; Wang, Jinfeng; Perrett, Sarah; Feng, Yingang

    2015-04-03

    Translationally controlled tumor protein (TCTP) is an abundant protein that is highly conserved in eukaryotes. However, its primary function is still not clear. Human TCTP interacts with the metazoan-specific eukaryotic elongation factor 1Bδ (eEF1Bδ) and inhibits its guanine nucleotide exchange factor (GEF) activity, but the structural mechanism remains unknown. The interaction between TCTP and eEF1Bδ was investigated by NMR titration, structure determination, paramagnetic relaxation enhancement, site-directed mutagenesis, isothermal titration calorimetry, and HADDOCK docking. We first demonstrated that the catalytic GEF domain of eEF1Bδ is not responsible for binding to TCTP but rather a previously unnoticed central acidic region (CAR) domain in eEF1Bδ. The mutagenesis data and the structural model of the TCTP-eEF1Bδ CAR domain complex revealed the key binding residues. These residues are highly conserved in eukaryotic TCTPs and in eEF1B GEFs, including the eukaryotically conserved eEF1Bα, implying the interaction may be conserved in all eukaryotes. Interactions were confirmed between TCTP and the eEF1Bα CAR domain for human, fission yeast, and unicellular photosynthetic microalgal proteins, suggesting that involvement in protein translation through the conserved interaction with eEF1B represents a primary function of TCTP. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Evolutionarily Conserved Binding of Translationally Controlled Tumor Protein to Eukaryotic Elongation Factor 1B*

    PubMed Central

    Wu, Huiwen; Gong, Weibin; Yao, Xingzhe; Wang, Jinfeng; Perrett, Sarah; Feng, Yingang

    2015-01-01

    Translationally controlled tumor protein (TCTP) is an abundant protein that is highly conserved in eukaryotes. However, its primary function is still not clear. Human TCTP interacts with the metazoan-specific eukaryotic elongation factor 1Bδ (eEF1Bδ) and inhibits its guanine nucleotide exchange factor (GEF) activity, but the structural mechanism remains unknown. The interaction between TCTP and eEF1Bδ was investigated by NMR titration, structure determination, paramagnetic relaxation enhancement, site-directed mutagenesis, isothermal titration calorimetry, and HADDOCK docking. We first demonstrated that the catalytic GEF domain of eEF1Bδ is not responsible for binding to TCTP but rather a previously unnoticed central acidic region (CAR) domain in eEF1Bδ. The mutagenesis data and the structural model of the TCTP-eEF1Bδ CAR domain complex revealed the key binding residues. These residues are highly conserved in eukaryotic TCTPs and in eEF1B GEFs, including the eukaryotically conserved eEF1Bα, implying the interaction may be conserved in all eukaryotes. Interactions were confirmed between TCTP and the eEF1Bα CAR domain for human, fission yeast, and unicellular photosynthetic microalgal proteins, suggesting that involvement in protein translation through the conserved interaction with eEF1B represents a primary function of TCTP. PMID:25635048

  5. Protein Kinase C Isozyme in Mammary Carcinogenesis.

    DTIC Science & Technology

    1996-10-01

    11 A B Clone 72 Clone 34 AKAP 86 Clone 35H CInn 72 lonn 4 P 95 lone 351 9. 5J 4.4- 688 431 Clone 45 Clone 35F Clone 64 Annexln 1 Clone 45 Clone 35F...purified antibodies directed against a variety of PKC substrates and an A-kinase anchoring protein, AKAP 95. (B) PolyA+ mRNAs isolated from confluent cell

  6. Mitochondrial protein import under kinase surveillance

    PubMed Central

    Opalińska, Magdalena; Meisinger, Chris

    2014-01-01

    Despite the simplicity of the yeast Saccharomyces cerevisiae, its basic cellular machinery tremendously mirrors that of higher eukaryotic counterparts. Thus, this unicellular organism turned out to be an invaluable model system to study the countless mechanisms that govern life of the cell. Recently, it has also enabled the deciphering of signalling pathways that control flux of mitochondrial proteins to the organelle according to metabolic requirements. For decades mitochondria were considered autonomous organelles that are only partially incorporated into cellular signalling networks. Consequently, only little has been known about the role of reversible phosphorylation as a meaningful mechanism that orchestrates mitochondrial biology accordingly to cellular needs. Therefore, research in this direction has been vastly neglected. However, findings over the past few years have changed this view and new exciting fields in mitochondrial biology have emerged. Here, we summarize recent discoveries in the yeast model system that point towards a vital role of reversible phosphorylation in regulation of mitochondrial protein import. PMID:28357222

  7. Global Analysis of Serine-Threonine Protein Kinase Genes in Neurospora crassa ▿ †

    PubMed Central

    Park, Gyungsoon; Servin, Jacqueline A.; Turner, Gloria E.; Altamirano, Lorena; Colot, Hildur V.; Collopy, Patrick; Litvinkova, Liubov; Li, Liande; Jones, Carol A.; Diala, Fitz-Gerald; Dunlap, Jay C.; Borkovich, Katherine A.

    2011-01-01

    Serine/threonine (S/T) protein kinases are crucial components of diverse signaling pathways in eukaryotes, including the model filamentous fungus Neurospora crassa. In order to assess the importance of S/T kinases to Neurospora biology, we embarked on a global analysis of 86 S/T kinase genes in Neurospora. We were able to isolate viable mutants for 77 of the 86 kinase genes. Of these, 57% exhibited at least one growth or developmental phenotype, with a relatively large fraction (40%) possessing a defect in more than one trait. S/T kinase knockouts were subjected to chemical screening using a panel of eight chemical treatments, with 25 mutants exhibiting sensitivity or resistance to at least one chemical. This brought the total percentage of S/T mutants with phenotypes in our study to 71%. Mutants lacking apg-1, an S/T kinase required for autophagy in other organisms, possessed the greatest number of phenotypes, with defects in asexual and sexual growth and development and in altered sensitivity to five chemical treatments. We showed that NCU02245/stk-19 is required for chemotropic interactions between female and male cells during mating. Finally, we demonstrated allelism between the S/T kinase gene NCU00406 and velvet (vel), encoding a p21-activated protein kinase (PAK) gene important for asexual and sexual growth and development in Neurospora. PMID:21965514

  8. Mechanisms of regulation of SNF1/AMPK/SnRK1 protein kinases

    PubMed Central

    Crozet, Pierre; Margalha, Leonor; Confraria, Ana; Rodrigues, Américo; Martinho, Cláudia; Adamo, Mattia; Elias, Carlos A.; Baena-González, Elena

    2014-01-01

    The SNF1 (sucrose non-fermenting 1)-related protein kinases 1 (SnRKs1) are the plant orthologs of the budding yeast SNF1 and mammalian AMPK (AMP-activated protein kinase). These evolutionarily conserved kinases are metabolic sensors that undergo activation in response to declining energy levels. Upon activation, SNF1/AMPK/SnRK1 kinases trigger a vast transcriptional and metabolic reprograming that restores energy homeostasis and promotes tolerance to adverse conditions, partly through an induction of catabolic processes and a general repression of anabolism. These kinases typically function as a heterotrimeric complex composed of two regulatory subunits, β and γ, and an α-catalytic subunit, which requires phosphorylation of a conserved activation loop residue for activity. Additionally, SNF1/AMPK/SnRK1 kinases are controlled by multiple mechanisms that have an impact on kinase activity, stability, and/or subcellular localization. Here we will review current knowledge on the regulation of SNF1/AMPK/SnRK1 by upstream components, post-translational modifications, various metabolites, hormones, and others, in an attempt to highlight both the commonalities of these essential eukaryotic kinases and the divergences that have evolved to cope with the particularities of each one of these systems. PMID:24904600

  9. Mechanisms of regulation of SNF1/AMPK/SnRK1 protein kinases.

    PubMed

    Crozet, Pierre; Margalha, Leonor; Confraria, Ana; Rodrigues, Américo; Martinho, Cláudia; Adamo, Mattia; Elias, Carlos A; Baena-González, Elena

    2014-01-01

    The SNF1 (sucrose non-fermenting 1)-related protein kinases 1 (SnRKs1) are the plant orthologs of the budding yeast SNF1 and mammalian AMPK (AMP-activated protein kinase). These evolutionarily conserved kinases are metabolic sensors that undergo activation in response to declining energy levels. Upon activation, SNF1/AMPK/SnRK1 kinases trigger a vast transcriptional and metabolic reprograming that restores energy homeostasis and promotes tolerance to adverse conditions, partly through an induction of catabolic processes and a general repression of anabolism. These kinases typically function as a heterotrimeric complex composed of two regulatory subunits, β and γ, and an α-catalytic subunit, which requires phosphorylation of a conserved activation loop residue for activity. Additionally, SNF1/AMPK/SnRK1 kinases are controlled by multiple mechanisms that have an impact on kinase activity, stability, and/or subcellular localization. Here we will review current knowledge on the regulation of SNF1/AMPK/SnRK1 by upstream components, post-translational modifications, various metabolites, hormones, and others, in an attempt to highlight both the commonalities of these essential eukaryotic kinases and the divergences that have evolved to cope with the particularities of each one of these systems.

  10. Atomic structure of the KEOPS complex: an ancient protein kinase-containing molecular machine

    PubMed Central

    Mao, Daniel Y.L.; Neculai, Dante; Downey, Michael; Orlicky, Stephen; Haffani, Yosr Z.; Ceccarelli, Derek F.; Ho, Jenny S.L.; Szilard, Rachel K.; Zhang, Wei; Ho, Cynthia S.; Wan, Leo; Fares, Christophe; Rumpel, Sigrun; Kurinov, Igor; Arrowsmith, Cheryl H.; Durocher, Daniel; Sicheri, Frank

    2011-01-01

    SUMMARY Kae1 is a universally conserved ATPase and part of the essential gene set in bacteria. In archaea and eukaryotes, Kae1 is embedded within the protein kinase-containing KEOPS complex. Mutation of KEOPS subunits in yeast leads to striking telomere and transcription defects but the exact biochemical function of KEOPS is not known. As a first step to elucidating its function, we solved the atomic structure of archaea-derived KEOPS complexes involving Kae1, Bud32, Pcc1 and Cgi121 subunits. Our studies suggest that Kae1 is regulated at two levels by the primordial protein kinase Bud32, which is itself regulated by Cgi121. Moreover, Pcc1 appears to function as a dimerization module, perhaps suggesting that KEOPS may be a processive molecular machine. Lastly, as Bud32 lacks the conventional substrate-recognition infrastructure of eukaryotic protein kinases including an activation segment, Bud32 may provide a glimpse of the evolutionary history of the protein kinase family. PMID:18951093

  11. Atomic structure of the KEOPS complex: an ancient protein kinase-containing molecular machine

    SciTech Connect

    Mao, D.Y.; Neculai, D.; Downey, M.; Orlicky, S.; Haffani, Y.Z.; Ceccarelli, D.F.; Ho, J.S.; Szilard, R.K.; Zhang, W.; Ho, C.S.; Wan, L.; Fares, C.; Rumpel, S.; Kurinov, I.; Arrowsmith, C.H.; Durocher, D.; Sicheri, F.

    2009-03-27

    Kae1 is a universally conserved ATPase and part of the essential gene set in bacteria. In archaea and eukaryotes, Kae1 is embedded within the protein kinase-containing KEOPS complex. Mutation of KEOPS subunits in yeast leads to striking telomere and transcription defects, but the exact biochemical function of KEOPS is not known. As a first step to elucidating its function, we solved the atomic structure of archaea-derived KEOPS complexes involving Kae1, Bud32, Pcc1, and Cgi121 subunits. Our studies suggest that Kae1 is regulated at two levels by the primordial protein kinase Bud32, which is itself regulated by Cgi121. Moreover, Pcc1 appears to function as a dimerization module, perhaps suggesting that KEOPS may be a processive molecular machine. Lastly, as Bud32 lacks the conventional substrate-recognition infrastructure of eukaryotic protein kinases including an activation segment, Bud32 may provide a glimpse of the evolutionary history of the protein kinase family.

  12. Differential AMP-activated Protein Kinase (AMPK) Recognition Mechanism of Ca2+/Calmodulin-dependent Protein Kinase Kinase Isoforms.

    PubMed

    Fujiwara, Yuya; Kawaguchi, Yoshinori; Fujimoto, Tomohito; Kanayama, Naoki; Magari, Masaki; Tokumitsu, Hiroshi

    2016-06-24

    Ca(2+)/calmodulin-dependent protein kinase kinase β (CaMKKβ) is a known activating kinase for AMP-activated protein kinase (AMPK). In vitro, CaMKKβ phosphorylates Thr(172) in the AMPKα subunit more efficiently than CaMKKα, with a lower Km (∼2 μm) for AMPK, whereas the CaMKIα phosphorylation efficiencies by both CaMKKs are indistinguishable. Here we found that subdomain VIII of CaMKK is involved in the discrimination of AMPK as a native substrate by measuring the activities of various CaMKKα/CaMKKβ chimera mutants. Site-directed mutagenesis analysis revealed that Leu(358) in CaMKKβ/Ile(322) in CaMKKα confer, at least in part, a distinct recognition of AMPK but not of CaMKIα.

  13. Inhibition of eukaryotic translation by tetratricopeptide-repeat proteins of Orientia tsutsugamushi.

    PubMed

    Bang, Sunyoung; Min, Chan-Ki; Ha, Na-Young; Choi, Myung-Sik; Kim, Ik-Sang; Kim, Yeon-Sook; Cho, Nam-Hyuk

    2016-02-01

    Orientia tsutsugamushi, an obligate intracellular bacterium, is the causative agent of scrub typhus. The genome of Orientia tsutsugamushi has revealed multiple ORFs encoding tetratricopeptide-repeat (TPR) proteins. The TPR protein family has been shown to be involved in a diverse spectrum of cellular functions such as cell cycle control, transcription, protein transport, and protein folding, especially in eukaryotic cells. However, little is known about the function of the TPR proteins in O. tsutsugamushi. To investigate the potential role of TPR proteins in host-pathogen interaction, two oriential TPR proteins were expressed in E. coli and applied for GSTpull down assay. DDX3, a DEAD-box containing RNA helicase, was identified as a specific eukaryotic target of the TPR proteins. Since the RNA helicase is involved in multiple RNA-modifying processes such as initiation of translation reaction, we performed in vitro translation assay in the presence of GST-TPR fusion proteins by using rabbit reticulocyte lysate system. The TPR proteins inhibited in vitro translation of a reporter luciferase in a dose dependent manner whereas the GST control proteins did not. These results suggested TPR proteins of O. tsutsugamushi might be involved in the modulation of eukaryotic translation through the interaction with DDX3 RNA helicase after secretion into host cytoplasm.

  14. Conservation of coevolving protein interfaces bridges prokaryote-eukaryote homologies in the twilight zone.

    PubMed

    Rodriguez-Rivas, Juan; Marsili, Simone; Juan, David; Valencia, Alfonso

    2016-12-27

    Protein-protein interactions are fundamental for the proper functioning of the cell. As a result, protein interaction surfaces are subject to strong evolutionary constraints. Recent developments have shown that residue coevolution provides accurate predictions of heterodimeric protein interfaces from sequence information. So far these approaches have been limited to the analysis of families of prokaryotic complexes for which large multiple sequence alignments of homologous sequences can be compiled. We explore the hypothesis that coevolution points to structurally conserved contacts at protein-protein interfaces, which can be reliably projected to homologous complexes with distantly related sequences. We introduce a domain-centered protocol to study the interplay between residue coevolution and structural conservation of protein-protein interfaces. We show that sequence-based coevolutionary analysis systematically identifies residue contacts at prokaryotic interfaces that are structurally conserved at the interface of their eukaryotic counterparts. In turn, this allows the prediction of conserved contacts at eukaryotic protein-protein interfaces with high confidence using solely mutational patterns extracted from prokaryotic genomes. Even in the context of high divergence in sequence (the twilight zone), where standard homology modeling of protein complexes is unreliable, our approach provides sequence-based accurate information about specific details of protein interactions at the residue level. Selected examples of the application of prokaryotic coevolutionary analysis to the prediction of eukaryotic interfaces further illustrate the potential of this approach.

  15. MAP kinase activator from insulin-stimulated skeletal muscle is a protein threonine/tyrosine kinase.

    PubMed Central

    Nakielny, S; Cohen, P; Wu, J; Sturgill, T

    1992-01-01

    A 'MAP kinase activator' was purified several thousand-fold from insulin-stimulated rabbit skeletal muscle, which resembled the 'activator' from nerve growth factor-stimulated PC12 cells in that it could be inactivated by incubation with protein phosphatase 2A, but not by protein tyrosine phosphatases and its apparent molecular mass was 45-50 kDa. In the presence of MgATP, 'MAP kinase activator' converted the normal 'wild-type' 42 kDa MAP kinase from an inactive dephosphorylated form to the fully active diphosphorylated species. Phosphorylation occurred on the same threonine and tyrosine residues which are phosphorylated in vivo in response to growth factors or phorbol esters. A mutant MAP kinase produced by changing a lysine at the active centre to arginine was phosphorylated in an identical manner by the 'MAP kinase activator', but no activity was generated. The results demonstrate that 'MAP kinase activator' is a protein kinase (MAP kinase kinase) and not a protein that stimulates the autophosphorylation of MAP kinase. MAP kinase kinase is the first established example of a protein kinase that can phosphorylate an exogenous protein on threonine as well as tyrosine residues. Images PMID:1318193

  16. Cell cycle regulation by the NEK family of protein kinases.

    PubMed

    Fry, Andrew M; O'Regan, Laura; Sabir, Sarah R; Bayliss, Richard

    2012-10-01

    Genetic screens for cell division cycle mutants in the filamentous fungus Aspergillus nidulans led to the discovery of never-in-mitosis A (NIMA), a serine/threonine kinase that is required for mitotic entry. Since that discovery, NIMA-related kinases, or NEKs, have been identified in most eukaryotes, including humans where eleven genetically distinct proteins named NEK1 to NEK11 are expressed. Although there is no evidence that human NEKs are essential for mitotic entry, it is clear that several NEK family members have important roles in cell cycle control. In particular, NEK2, NEK6, NEK7 and NEK9 contribute to the establishment of the microtubule-based mitotic spindle, whereas NEK1, NEK10 and NEK11 have been implicated in the DNA damage response. Roles for NEKs in other aspects of mitotic progression, such as chromatin condensation, nuclear envelope breakdown, spindle assembly checkpoint signalling and cytokinesis have also been proposed. Interestingly, NEK1 and NEK8 also function within cilia, the microtubule-based structures that are nucleated from basal bodies. This has led to the current hypothesis that NEKs have evolved to coordinate microtubule-dependent processes in both dividing and non-dividing cells. Here, we review the functions of the human NEKs, with particular emphasis on those family members that are involved in cell cycle control, and consider their potential as therapeutic targets in cancer.

  17. Pyrrolopyridine inhibitors of mitogen-activated protein kinase-activated protein kinase 2 (MK-2).

    PubMed

    Anderson, David R; Meyers, Marvin J; Vernier, William F; Mahoney, Matthew W; Kurumbail, Ravi G; Caspers, Nicole; Poda, Gennadiy I; Schindler, John F; Reitz, David B; Mourey, Robert J

    2007-05-31

    A new class of potent kinase inhibitors selective for mitogen-activated protein kinase-activated protein kinase 2 (MAPKAP-K2 or MK-2) for the treatment of rheumatoid arthritis has been prepared and evaluated. These inhibitors have IC50 values as low as 10 nM against the target and have good selectivity profiles against a number of kinases including CDK2, ERK, JNK, and p38. These MK-2 inhibitors have been shown to suppress TNFalpha production in U397 cells and to be efficacious in an acute inflammation model. The structure-activity relationships of this series, the selectivity for MK-2 and their activity in both in vitro and in vivo models are discussed. The observed selectivity is discussed with the aid of an MK-2/inhibitor crystal structure.

  18. Protein kinase activity of the insulin receptor.

    PubMed Central

    Gammeltoft, S; Van Obberghen, E

    1986-01-01

    exogenous protein and peptide substrates on tyrosine residues, a reaction which is insulin-sensitive, Mn2+-dependent and specific for ATP. Tyrosine phosphorylation of the beta-subunit activates receptor kinase activity, and dephosphorylation with alkaline phosphatase deactivates the kinase. In intact cells or impure receptor preparations, a serine kinase is also activated by insulin. The cellular role of two kinase activities associated with the insulin receptor is not known, but we propose that the tyrosine- and serine-specific kinases mediate insulin actions on metabolism and growth either through dual-signalling or sequential pathways.(ABSTRACT TRUNCATED AT 400 WORDS) Images Fig. 3. Fig. 5. PMID:3017297

  19. Real-time protein kinase assay.

    PubMed

    Sun, Hongye; Low, Karen E; Woo, Sam; Noble, Richard L; Graham, Ronald J; Connaughton, Sonia S; Gee, Melissa A; Lee, Linda G

    2005-04-01

    We report a novel, real-time fluorogenic kinase assay. The peptide substrates are synthesized with a fluorescent dye and a hydrocarbon tail. The substrate self-assembles into micelles, increasing the local concentration of the dye and quenching its fluorescence. Upon phosphorylation, the fluorescence intensity increases 4-6-fold due to micelle reorganization. Both dynamic light scattering data and cryoelectron microscope images show that the size and the shape of the phosphopeptide micelles are significantly different from micelles of substrate peptide. The system provides a robust fluorescence increase in a real-time protein kinase assay. Unlike other fluorogenic systems, the fluorophore may be distant from the serine, threonine, or tyrosine that is phosphorylated. Assays for several kinases, including PKA, PKC, p38, MAPKAP K2, akt, Erk1, and src-family kinases, have been developed. IC(50) values of inhibitors for PKC betaII determined with this technology are consistent with published values. The utility of this assay to high-throughput screening was demonstrated with Sigma's LOPAC library, a collection of 640 compounds with known biological activities, and satisfactory results were obtained.

  20. Thiopental Inhibits Global Protein Synthesis by Repression of Eukaryotic Elongation Factor 2 and Protects from Hypoxic Neuronal Cell Death

    PubMed Central

    Schwer, Christian I.; Lehane, Cornelius; Guelzow, Timo; Zenker, Simone; Strosing, Karl M.; Spassov, Sashko; Erxleben, Anika; Heimrich, Bernd; Buerkle, Hartmut; Humar, Matjaz

    2013-01-01

    Ischemic and traumatic brain injury is associated with increased risk for death and disability. The inhibition of penumbral tissue damage has been recognized as a target for therapeutic intervention, because cellular injury evolves progressively upon ATP-depletion and loss of ion homeostasis. In patients, thiopental is used to treat refractory intracranial hypertension by reducing intracranial pressure and cerebral metabolic demands; however, therapeutic benefits of thiopental-treatment are controversially discussed. In the present study we identified fundamental neuroprotective molecular mechanisms mediated by thiopental. Here we show that thiopental inhibits global protein synthesis, which preserves the intracellular energy metabolite content in oxygen-deprived human neuronal SK-N-SH cells or primary mouse cortical neurons and thus ameliorates hypoxic cell damage. Sensitivity to hypoxic damage was restored by pharmacologic repression of eukaryotic elongation factor 2 kinase. Translational inhibition was mediated by calcium influx, activation of the AMP-activated protein kinase, and inhibitory phosphorylation of eukaryotic elongation factor 2. Our results explain the reduction of cerebral metabolic demands during thiopental treatment. Cycloheximide also protected neurons from hypoxic cell death, indicating that translational inhibitors may generally reduce secondary brain injury. In conclusion our study demonstrates that therapeutic inhibition of global protein synthesis protects neurons from hypoxic damage by preserving energy balance in oxygen-deprived cells. Molecular evidence for thiopental-mediated neuroprotection favours a positive clinical evaluation of barbiturate treatment. The chemical structure of thiopental could represent a pharmacologically relevant scaffold for the development of new organ-protective compounds to ameliorate tissue damage when oxygen availability is limited. PMID:24167567

  1. Protein Scaffolds in MAP Kinase Signalling

    PubMed Central

    Brown, Matthew D.; Sacks, David B.

    2009-01-01

    The mitogen-activated protein kinase (MAPK) pathway allows cells to interpret external signals and respond in an appropriate way. Diverse cellular functions, ranging from differentiation and proliferation to migration and inflammation, are regulated by MAPK signalling. Therefore, cells have developed mechanisms by which this single pathway modulates numerous cellular responses from a wide range of activating factors. This specificity is achieved by several mechanisms, including temporal and spatial control of MAPK signalling components. Key to this control are protein scaffolds, which are multidomain proteins that interact with components of the MAPK cascade in order to assemble signalling complexes. Studies conducted on different scaffolds, in different biological systems, have shown that scaffolds exert substantial control over MAPK signalling, influencing the signal intensity, time course and, importantly, the cellular responses. Protein scaffolds, therefore, are integral elements in the modulation of the MAPK network in fundamental physiological processes. PMID:19091303

  2. Functional eukaryotic nuclear localization signals are widespread in terminal proteins of bacteriophages.

    PubMed

    Redrejo-Rodríguez, Modesto; Muñoz-Espín, Daniel; Holguera, Isabel; Mencía, Mario; Salas, Margarita

    2012-11-06

    A number of prokaryotic proteins have been shown to contain nuclear localization signals (NLSs), although its biological role remains sometimes unclear. Terminal proteins (TPs) of bacteriophages prime DNA replication and become covalently linked to the genome ends. We predicted NLSs within the TPs of bacteriophages from diverse families and hosts and, indeed, the TPs of Φ29, Nf, PRD1, Bam35, and Cp-1, out of seven TPs tested, were found to localize to the nucleus when expressed in mammalian cells. Detailed analysis of Φ29 TP led us to identify a bona fide NLS within residues 1-37. Importantly, gene delivery into the eukaryotic nucleus is enhanced by the presence of Φ29 TP attached to the 5' DNA ends. These findings show a common feature of TPs from diverse bacteriophages targeting the eukaryotic nucleus and suggest a possible common function by facilitating the horizontal transfer of genes between prokaryotes and eukaryotes.

  3. Functional eukaryotic nuclear localization signals are widespread in terminal proteins of bacteriophages

    PubMed Central

    Redrejo-Rodríguez, Modesto; Muñoz-Espín, Daniel; Holguera, Isabel; Mencía, Mario; Salas, Margarita

    2012-01-01

    A number of prokaryotic proteins have been shown to contain nuclear localization signals (NLSs), although its biological role remains sometimes unclear. Terminal proteins (TPs) of bacteriophages prime DNA replication and become covalently linked to the genome ends. We predicted NLSs within the TPs of bacteriophages from diverse families and hosts and, indeed, the TPs of Φ29, Nf, PRD1, Bam35, and Cp-1, out of seven TPs tested, were found to localize to the nucleus when expressed in mammalian cells. Detailed analysis of Φ29 TP led us to identify a bona fide NLS within residues 1–37. Importantly, gene delivery into the eukaryotic nucleus is enhanced by the presence of Φ29 TP attached to the 5′ DNA ends. These findings show a common feature of TPs from diverse bacteriophages targeting the eukaryotic nucleus and suggest a possible common function by facilitating the horizontal transfer of genes between prokaryotes and eukaryotes. PMID:23091024

  4. Membrane protein insertion: mixing eukaryotic and prokaryotic concepts.

    PubMed

    Schleiff, Enrico; Soll, Jürgen

    2005-11-01

    Proteins are translocated across or inserted into membranes by machines that are composed of soluble and membrane-anchored subunits. The molecular action of these machines and their evolutionary origin are at present the focus of intense research. For instance, our understanding of the mode of insertion of beta-barrel membrane proteins into the outer membrane of endosymbiotically derived organelles has increased rapidly during the past few years. In particular, the identification of the Omp85/YaeT-involving pathways in Neisseria meningitidis, Escherichia coli and cyanobacteria, and homologues of Omp85/YaeT in chloroplasts and mitochondria, has provided new clues about the ancestral beta-barrel protein insertion pathway. This review focuses on recent advances in the elucidation of the evolutionarily conserved concepts that underlie the translocation and insertion of beta-barrel membrane proteins.

  5. Identification of a dual-specificity protein phosphatase that inactivates a MAP kinase from Arabidopsis

    NASA Technical Reports Server (NTRS)

    Gupta, R.; Huang, Y.; Kieber, J.; Luan, S.; Evans, M. L. (Principal Investigator)

    1998-01-01

    Mitogen-activated protein kinases (MAPKs) play a key role in plant responses to stress and pathogens. Activation and inactivation of MAPKs involve phosphorylation and dephosphorylation on both threonine and tyrosine residues in the kinase domain. Here we report the identification of an Arabidopsis gene encoding a dual-specificity protein phosphatase capable of hydrolysing both phosphoserine/threonine and phosphotyrosine in protein substrates. This enzyme, designated AtDsPTP1 (Arabidopsis thaliana dual-specificity protein tyrosine phosphatase), dephosphorylated and inactivated AtMPK4, a MAPK member from the same plant. Replacement of a highly conserved cysteine by serine abolished phosphatase activity of AtDsPTP1, indicating a conserved catalytic mechanism of dual-specificity protein phosphatases from all eukaryotes.

  6. Identification of a dual-specificity protein phosphatase that inactivates a MAP kinase from Arabidopsis

    NASA Technical Reports Server (NTRS)

    Gupta, R.; Huang, Y.; Kieber, J.; Luan, S.; Evans, M. L. (Principal Investigator)

    1998-01-01

    Mitogen-activated protein kinases (MAPKs) play a key role in plant responses to stress and pathogens. Activation and inactivation of MAPKs involve phosphorylation and dephosphorylation on both threonine and tyrosine residues in the kinase domain. Here we report the identification of an Arabidopsis gene encoding a dual-specificity protein phosphatase capable of hydrolysing both phosphoserine/threonine and phosphotyrosine in protein substrates. This enzyme, designated AtDsPTP1 (Arabidopsis thaliana dual-specificity protein tyrosine phosphatase), dephosphorylated and inactivated AtMPK4, a MAPK member from the same plant. Replacement of a highly conserved cysteine by serine abolished phosphatase activity of AtDsPTP1, indicating a conserved catalytic mechanism of dual-specificity protein phosphatases from all eukaryotes.

  7. Endothelial Mitogen-Activated Protein Kinase Kinase Kinase Kinase 4 Is Critical for Lymphatic Vascular Development and Function

    PubMed Central

    Guo, Chang-An; Danai, Laura V.; Yawe, Joseph C.; Gujja, Sharvari; Edwards, Yvonne J. K.

    2016-01-01

    The molecular mechanisms underlying lymphatic vascular development and function are not well understood. Recent studies have suggested a role for endothelial cell (EC) mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) in developmental angiogenesis and atherosclerosis. Here, we show that constitutive loss of EC Map4k4 in mice causes postnatal lethality due to chylothorax, suggesting that Map4k4 is required for normal lymphatic vascular function. Mice constitutively lacking EC Map4k4 displayed dilated lymphatic capillaries, insufficient lymphatic valves, and impaired lymphatic flow; furthermore, primary ECs derived from these animals displayed enhanced proliferation compared with controls. Yeast 2-hybrid analyses identified the Ras GTPase-activating protein Rasa1, a known regulator of lymphatic development and lymphatic endothelial cell fate, as a direct interacting partner for Map4k4. Map4k4 silencing in ECs enhanced basal Ras and extracellular signal-regulated kinase (Erk) activities, and primary ECs lacking Map4k4 displayed enhanced lymphatic EC marker expression. Taken together, these results reveal that EC Map4k4 is critical for lymphatic vascular development by regulating EC quiescence and lymphatic EC fate. PMID:27044870

  8. Sequence alignment and homology threading reveals prokaryotic and eukaryotic proteins similar to lactose permease.

    PubMed

    Kasho, Vladimir N; Smirnova, Irina N; Kaback, H Ronald

    2006-05-12

    Certain prokaryotic transport proteins similar to the lactose permease of Escherichia coli (LacY) have been identified by BLAST searches from available genomic databanks. These proteins exhibit conservation of amino acid residues that participate in sugar binding and H(+) translocation in LacY. Homology threading of prokaryotic transporters based on the X-ray structure of LacY (PDB ID: 1PV7) and sequence similarities reveals a common overall fold for sugar transporters belonging to the Major Facilitator Superfamily (MFS) and suggest new targets for study. Evolution-based searches for sequence similarities also identify eukaryotic proteins bearing striking resemblance to MFS sugar transporters. Like LacY, the eukaryotic proteins are predicted to have 12 transmembrane domains (TMDs), and many of the irreplaceable residues for sugar binding and H(+) translocation in LacY appear to be largely conserved. The overall size of the eukaryotic homologs is about twice that of prokaryotic permeases with longer N and C termini and loops between TMDs III-IV and VI-VII. The human gene encoding protein FLJ20160 consists of six exons located on more than 60,000 bp of DNA sequences and requires splicing to produce mature mRNA. Cellular localization predictions suggest membrane insertion with possible proteolysis at the N terminus, and expression studies with the human protein FJL20160 demonstrate membrane insertion in both E.coli and Pichia pastoris. Widespread expression of the eukaryotic sugar transport candidates suggests an important role in cellular metabolism, particularly in brain and tumors. Homology is observed in the TMDs of both the eukaryotic and prokaryotic proteins that contain residues involved in sugar binding and H(+) translocation in LacY.

  9. Redox-Assisted Protein Folding Systems in Eukaryotic Parasites

    PubMed Central

    Haque, Saikh Jaharul; Majumdar, Tanmay

    2012-01-01

    Abstract Significance: The cysteine (Cys) residues of proteins play two fundamentally important roles. They serve as sites of post-translational redox modifications as well as influence the conformation of the protein through the formation of disulfide bonds. Recent Advances: Redox-related and redox-associated protein folding in protozoan parasites has been found to be a major mode of regulation, affecting myriad aspects of the parasitic life cycle, host-parasite interactions, and the disease pathology. Available genome sequences of various parasites have begun to complement the classical biochemical and enzymological studies of these processes. In this article, we summarize the reversible Cys disulfide (S-S) bond formation in various classes of strategically important parasitic proteins, and its structural consequence and functional relevance. Critical Issues: Molecular mechanisms of folding remain under-studied and often disconnected from functional relevance. Future Directions: The clinical benefit of redox research will require a comprehensive characterization of the various isoforms and paralogs of the redox enzymes and their concerted effect on the structure and function of the specific parasitic client proteins. Antioxid. Redox Signal. 17, 674–683. PMID:22122448

  10. Glycogen Synthase Kinase 3β Interaction Protein Functions as an A-kinase Anchoring Protein*

    PubMed Central

    Hundsrucker, Christian; Skroblin, Philipp; Christian, Frank; Zenn, Hans-Michael; Popara, Viola; Joshi, Mangesh; Eichhorst, Jenny; Wiesner, Burkhard; Herberg, Friedrich W.; Reif, Bernd; Rosenthal, Walter; Klussmann, Enno

    2010-01-01

    A-kinase anchoring proteins (AKAPs) include a family of scaffolding proteins that target protein kinase A (PKA) and other signaling proteins to cellular compartments and thereby confine the activities of the associated proteins to distinct regions within cells. AKAPs bind PKA directly. The interaction is mediated by the dimerization and docking domain of regulatory subunits of PKA and the PKA-binding domain of AKAPs. Analysis of the interactions between the dimerization and docking domain and various PKA-binding domains yielded a generalized motif allowing the identification of AKAPs. Our bioinformatics and peptide array screening approaches based on this signature motif identified GSKIP (glycogen synthase kinase 3β interaction protein) as an AKAP. GSKIP directly interacts with PKA and GSK3β (glycogen synthase kinase 3β). It is widely expressed and facilitates phosphorylation and thus inactivation of GSK3β by PKA. GSKIP contains the evolutionarily conserved domain of unknown function 727. We show here that this domain of GSKIP and its vertebrate orthologues binds both PKA and GSK3β and thereby provides a mechanism for the integration of PKA and GSK3β signaling pathways. PMID:20007971

  11. Photoinduced structural changes to protein kinase A

    NASA Astrophysics Data System (ADS)

    Rozinek, Sarah C.; Thomas, Robert J.; Brancaleon, Lorenzo

    2014-03-01

    The importance of porphyrins in organisms is underscored by the ubiquitous biological and biochemical functions that are mediated by these compounds and by their potential biomedical and biotechnological applications. Protoporphyrin IX (PPIX) is the precursor to heme and has biomedical applications such as its use as a photosensitizer in phototherapy and photodetection of cancer. Among other applications, our group has demonstrated that low-irradiance exposure to laser irradiation of PPIX, Fe-PPIX, or meso-tetrakis (4-sulfonatophenyl) porphyrin (TSPP) non-covalently docked to a protein causes conformational changes in the polypeptide. Such approach can have remarkable consequences in the study of protein structure/function relationship and can be used to prompt non-native protein properties. Therefore we have investigated protein kinase A (PKA), a more relevant protein model towards the photo-treatment of cancer. PKA's enzymatic functions are regulated by the presence of cyclic adenosine monophosphate for intracellular signal transduction involved in, among other things, stimulation of transcription, tumorigenesis in Carney complex and migration of breast carcinoma cells. Since phosphorylation is a necessary step in some cancers and inflammatory diseases, inhibiting the protein kinase, and therefore phosphorylation, may serve to treat these diseases. Changes in absorption, steady-state fluorescence, and fluorescence lifetime indicate: 1) both TSPP and PPIX non-covalently bind to PKA where they maintain photoreactivity; 2) absorptive photoproduct formation occurs only when PKA is bound to TSPP and irradiated; and 3) PKA undergoes secondary structural changes after irradiation with either porphyrin bound. These photoinduced changes could affect the protein's enzymatic and signaling capabilities.

  12. Interacting factors and cellular localization of SR protein-specific kinase Dsk1

    SciTech Connect

    Tang, Zhaohua; Luca, Maria; Taggart-Murphy, Laura; Portillio, Jessica; Chang, Cathey; Guven, Ayse; Lin, Ren-Jang; Murray, Johanne; Carr, Antony

    2012-10-01

    Schizosaccharomyces pombe Dsk1 is an SR protein-specific kinase (SRPK), whose homologs have been identified in every eukaryotic organism examined. Although discovered as a mitotic regulator with protein kinase activity toward SR splicing factors, it remains largely unknown about what and how Dsk1 contributes to cell cycle and pre-mRNA splicing. In this study, we investigated the Dsk1 function by determining interacting factors and cellular localization of the kinase. Consistent with its reported functions, we found that pre-mRNA processing and cell cycle factors are prominent among the proteins co-purified with Dsk1. The identification of these factors led us to find Rsd1 as a novel Dsk1 substrate, as well as the involvement of Dsk1 in cellular distribution of poly(A){sup +} RNA. In agreement with its role in nuclear events, we also found that Dsk1 is mainly localized in the nucleus during G{sub 2} phase and at mitosis. Furthermore, we revealed the oscillation of Dsk1 protein in a cell cycle-dependent manner. This paper marks the first comprehensive analysis of in vivo Dsk1-associated proteins in fission yeast. Our results reflect the conserved role of SRPK family in eukaryotic organisms, and provide information about how Dsk1 functions in pre-mRNA processing and cell-division cycle.

  13. Purification of eukaryotic translation factors from wheat germ for reconstitution of protein synthesis.

    PubMed

    Nagano, Hikaru; Sugihara, Shouhei; Takagi, Hisanori; Ogasawara, Tomio; Endo, Yaeta; Takai, Kazuyuki

    2008-01-01

    The wheat germ cell-free protein synthesis is a powerful and versatile method for preparation of proteins based on the accumulated DNA sequence information. As the cell extract used for it contains many factors that are unknown or do not directly involve in protein synthesis, details of the translation reaction is yet to be understood. Therefore, we have decided to try reconstitution of protein synthesis, which would be useful for better understanding of the mechanisms supporting eukaryotic protein synthesis and translational regulation and probably applicable to synthetic biology. In the present study, we fractionated an extract from crude wheat germ according to published protocols to obtain the fractions containing the eukaryotic elongation factors (eEFs) 1A, 1B, and 2. The eEF1A and eEF2 fractions supported polyphenylalanine synthesis.

  14. Global analysis of the glycoproteome in Saccharomyces cerevisiae reveals new roles for protein glycosylation in eukaryotes

    PubMed Central

    Kung, Li A; Tao, Sheng-Ce; Qian, Jiang; Smith, Michael G; Snyder, Michael; Zhu, Heng

    2009-01-01

    To further understand the roles of protein glycosylation in eukaryotes, we globally identified glycan-containing proteins in yeast. A fluorescent lectin binding assay was developed and used to screen protein microarrays containing over 5000 proteins purified from yeast. A total of 534 yeast proteins were identified that bound either Concanavalin A (ConA) or Wheat-Germ Agglutinin (WGA); 406 of them were novel. Among the novel glycoproteins, 45 were validated by mobility shift upon treatment with EndoH and PNGase F, thereby extending the number of validated yeast glycoproteins to 350. In addition to many components of the secretory pathway, we identified other types of proteins, such as transcription factors and mitochondrial proteins. To further explore the role of glycosylation in mitochondrial function, the localization of four mitochondrial proteins was examined in the presence and absence of tunicamycin, an inhibitor of N-linked protein glycosylation. For two proteins, localization to the mitochondria is diminished upon tunicamycin treatment, indicating that protein glycosylation is important for protein function. Overall, our studies greatly extend our understanding of protein glycosylation in eukaryotes through the cataloguing of glycoproteins, and describe a novel role for protein glycosylation in mitochondrial protein function and localization. PMID:19756047

  15. Malaria Protein Kinase CK2 (PfCK2) Shows Novel Mechanisms of Regulation

    PubMed Central

    Graciotti, Michele; Alam, Mahmood; Solyakov, Lev; Schmid, Ralf; Burley, Glenn; Bottrill, Andrew R.; Doerig, Christian; Cullis, Paul; Tobin, Andrew B.

    2014-01-01

    Casein kinase 2 (protein kinase CK2) is a conserved eukaryotic serine/theronine kinase with multiple substrates and roles in the regulation of cellular processes such as cellular stress, cell proliferation and apoptosis. Here we report a detailed analysis of the Plasmodium falciparum CK2, PfCK2, demonstrating that this kinase, like the mammalian orthologue, is a dual specificity kinase able to phosphorylate at both serine and tyrosine. However, unlike the human orthologue that is auto-phosphorylated on tyrosine within the activation loop, PfCK2 shows no activation loop auto-phosphorylation but rather is auto-phosphorylated at threonine 63 within subdomain I. Phosphorylation at this site in PfCK2 is shown here to regulate the intrinsic kinase activity of PfCK2. Furthermore, we generate an homology model of PfCK2 in complex with the known selective protein kinase CK2 inhibitor, quinalizarin, and in so doing identify key co-ordinating residues in the ATP binding pocket that could aid in designing selective inhibitors to PfCK2. PMID:24658579

  16. Six Subgroups and Extensive Recent Duplications Characterize the Evolution of the Eukaryotic Tubulin Protein Family

    PubMed Central

    Findeisen, Peggy; Mühlhausen, Stefanie; Dempewolf, Silke; Hertzog, Jonny; Zietlow, Alexander; Carlomagno, Teresa; Kollmar, Martin

    2014-01-01

    Tubulins belong to the most abundant proteins in eukaryotes providing the backbone for many cellular substructures like the mitotic and meiotic spindles, the intracellular cytoskeletal network, and the axonemes of cilia and flagella. Homologs have even been reported for archaea and bacteria. However, a taxonomically broad and whole-genome-based analysis of the tubulin protein family has never been performed, and thus, the number of subfamilies, their taxonomic distribution, and the exact grouping of the supposed archaeal and bacterial homologs are unknown. Here, we present the analysis of 3,524 tubulins from 504 species. The tubulins formed six major subfamilies, α to ζ. Species of all major kingdoms of the eukaryotes encode members of these subfamilies implying that they must have already been present in the last common eukaryotic ancestor. The proposed archaeal homologs grouped together with the bacterial TubZ proteins as sister clade to the FtsZ proteins indicating that tubulins are unique to eukaryotes. Most species contained α- and/or β-tubulin gene duplicates resulting from recent branch- and species-specific duplication events. This shows that tubulins cannot be used for constructing species phylogenies without resolving their ortholog–paralog relationships. The many gene duplicates and also the independent loss of the δ-, ε-, or ζ-tubulins, which have been shown to be part of the triplet microtubules in basal bodies, suggest that tubulins can functionally substitute each other. PMID:25169981

  17. Protein Kinases in Mammary Gland Development and Carcinogenesis

    DTIC Science & Technology

    1999-09-01

    differ among CaM kinase family members include their subcellular localization , regulation by autophosphorylation, and regulation by other proteins. In...addition, CaM kinases have unique amino- and carboxyl- terminal domains that contribute to kinase-specific differences in subcellular localization ...chromosomal localization of Punc, a calcium/calmodulin-dependent protein kinase, (Submitted). 14. Hennings, H., Glick, A., Lowry, D., Krsmanovic, L

  18. Conservation of coevolving protein interfaces bridges prokaryote–eukaryote homologies in the twilight zone

    PubMed Central

    Rodriguez-Rivas, Juan; Marsili, Simone; Juan, David; Valencia, Alfonso

    2016-01-01

    Protein–protein interactions are fundamental for the proper functioning of the cell. As a result, protein interaction surfaces are subject to strong evolutionary constraints. Recent developments have shown that residue coevolution provides accurate predictions of heterodimeric protein interfaces from sequence information. So far these approaches have been limited to the analysis of families of prokaryotic complexes for which large multiple sequence alignments of homologous sequences can be compiled. We explore the hypothesis that coevolution points to structurally conserved contacts at protein–protein interfaces, which can be reliably projected to homologous complexes with distantly related sequences. We introduce a domain-centered protocol to study the interplay between residue coevolution and structural conservation of protein–protein interfaces. We show that sequence-based coevolutionary analysis systematically identifies residue contacts at prokaryotic interfaces that are structurally conserved at the interface of their eukaryotic counterparts. In turn, this allows the prediction of conserved contacts at eukaryotic protein–protein interfaces with high confidence using solely mutational patterns extracted from prokaryotic genomes. Even in the context of high divergence in sequence (the twilight zone), where standard homology modeling of protein complexes is unreliable, our approach provides sequence-based accurate information about specific details of protein interactions at the residue level. Selected examples of the application of prokaryotic coevolutionary analysis to the prediction of eukaryotic interfaces further illustrate the potential of this approach. PMID:27965389

  19. Protein kinase C alpha-dependent phosphorylation of Golgi proteins.

    PubMed

    Radau, B; Otto, A; Müller, E C; Westermann, P

    2000-07-01

    Golgi-enriched membranes were phosphorylated in order to understand the mechanism for protein kinase C (PKC) regulation of exocytic vesicle formation at the trans-Golgi network. Two of the main PKC substrates were identified as MARCKS and Mac-MARCKS by two-dimensional electrophoresis (2-DE) and mass spectrometric sequencing. Annexin IV and profilin I, two other Golgi-associated proteins--although known as in vitro PKC substrates--were not phosphorylated in the Golgi-bound state.

  20. Molecular dynamics reveal a novel kinase-substrate interface that regulates protein translation.

    PubMed

    Liu, Ming S; Wang, Die; Morimoto, Hiroyuki; Yim, Howard C H; Irving, Aaron T; Williams, Bryan R G; Sadler, Anthony J

    2014-12-01

    A key control point in gene expression is the initiation of protein translation, with a universal stress response being constituted by inhibitory phosphorylation of the eukaryotic initiation factor 2α (eIF2α). In humans, four kinases sense diverse physiological stresses to regulate eIF2α to control cell differentiation, adaptation, and survival. Here we develop a computational molecular model of eIF2α and one of its kinases, the protein kinase R, to simulate the dynamics of their interaction. Predictions generated by coarse-grained dynamics simulations suggest a novel mode of action. Experimentation substantiates these predictions, identifying a previously unrecognized interface in the protein complex, which is constituted by dynamic residues in both eIF2α and its kinases that are crucial to regulate protein translation. These findings call for a reinterpretation of the current mechanism of action of the eIF2α kinases and demonstrate the value of conducting computational analysis to evaluate protein function. © The Author (2014). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.

  1. Phosphorylation Reaction in cAPK Protein Kinase - Free Energy Quantum Mechanic/Molecular Mechanics Simulations.

    SciTech Connect

    Valiev, Marat; Yang, Jie; Adams, Joseph; Taylor, Susan S.; Weare, John H.

    2007-11-29

    Protein kinases catalyze the transfer of the γ-phosphoryl group from ATP, a key regulatory process governing signalling pathways in eukaryotic cells. The structure of the active site in these enzymes is highly conserved implying common catalytic mechanism. In this work we investigate the reaction process in cAPK protein kinase (PKA) using a combined quantum mechanics and molecular mechanics approach. The novel computational features of our work include reaction pathway determination with nudged elastic band methodology and calculation of free energy profiles of the reaction process taking into account finite temperature fluctuations of the protein environment. We find that the transfer of the γ-phosphoryl group in the protein environment is an exothermic reaction with the reaction barrier of 15 kcal/mol.

  2. The presence of GPI-linked protein(s) in an archaeobacterium, Sulfolobus acidocaldarius, closely related to eukaryotes.

    PubMed

    Kobayashi, T; Nishizaki, R; Ikezawa, H

    1997-02-11

    GPI-anchored proteins are distributed ubiquitously in eukaryotes, but not in procaryotes. By metabolic-labeling of Sulfolobus acidocaldarius cells, 14C-radiolabeled precursors of GPI and caldarchaetidylinositol were incorporated into 120, 143 and 185 kDa proteins. The 185 kDa protein was specifically solubilized by bacterial phosphatidylinositol-specific phospholipase C. Therefore, Sulfolobus proved to contain at least one GPI-anchored proteins.

  3. Solution structure of an archaeal DNA binding protein with an eukaryotic zinc finger fold.

    PubMed

    Guillière, Florence; Danioux, Chloé; Jaubert, Carole; Desnoues, Nicole; Delepierre, Muriel; Prangishvili, David; Sezonov, Guennadi; Guijarro, J Iñaki

    2013-01-01

    While the basal transcription machinery in archaea is eukaryal-like, transcription factors in archaea and their viruses are usually related to bacterial transcription factors. Nevertheless, some of these organisms show predicted classical zinc fingers motifs of the C2H2 type, which are almost exclusively found in proteins of eukaryotes and most often associated with transcription regulators. In this work, we focused on the protein AFV1p06 from the hyperthermophilic archaeal virus AFV1. The sequence of the protein consists of the classical eukaryotic C2H2 motif with the fourth histidine coordinating zinc missing, as well as of N- and C-terminal extensions. We showed that the protein AFV1p06 binds zinc and solved its solution structure by NMR. AFV1p06 displays a zinc finger fold with a novel structure extension and disordered N- and C-termini. Structure calculations show that a glutamic acid residue that coordinates zinc replaces the fourth histidine of the C2H2 motif. Electromobility gel shift assays indicate that the protein binds to DNA with different affinities depending on the DNA sequence. AFV1p06 is the first experimentally characterised archaeal zinc finger protein with a DNA binding activity. The AFV1p06 protein family has homologues in diverse viruses of hyperthermophilic archaea. A phylogenetic analysis points out a common origin of archaeal and eukaryotic C2H2 zinc fingers.

  4. [Cloning of human bone morphogenetic protein-2 gene and the construction of its eukaryotic expression vector].

    PubMed

    Zhou, Nuo; Huang, Xuan-ping; Liao, Ni; Wei, Shan-liang; Liang, Fei-xin; Mai, Hua-ming

    2007-10-01

    To clone human bone morphogenetic protein-2 (hBMP2) gene and construct its eukaryotic expression vector pcDNA3.1 -hBMP2. Human BMP2 gene was amplified by RT-PCR method from human osteosarcoma cells and constructed into eukaryotic expression vector pcDNA3.1-hBMP2. The gene in the vector pcDNA3.1-hBMP2 was identified by PCR amplification, enzyme digestion and DNA sequencing. The cloned DNA was confirmed to be hBMP-2 gene. In this study, hBMP2 gene is successfully cloned and its eukaryotic expression vector pcDNA3.1-hBMP2 is constructed, which provides the foundation of using BMP2 gene therapy to accelerate new bone formation in distraction osteogenesis.

  5. Protein Kinases in Zucchini (Characterization of Calcium-Requiring Plasma Membrane Kinases).

    PubMed Central

    Verhey, S. D.; Gaiser, J. C.; Lomax, T. L.

    1993-01-01

    Using an in situ phosphorylation assay with zucchini (Cucurbita pepo L. cv Dark Green) seedling tissue, we have identified numerous polypeptides that are capable of acting as protein kinases. Total protein preparations from different organs contain different kinase profiles, but all are within the range of 55 to 70 kD. At least four kinases are associated with highly purified plasma membranes from etiolated zucchini hypocotyls. The major phosphorylated polypeptides from plasma membranes range in apparent molecular mass from 58 to 68 kD. The plasma membrane kinases are activated by micromolar concentrations of calcium and phosphorylate serine, and, to a lesser extent, threonine residues. These characteristics are similar to those of a soluble calcium-dependent protein kinase that has been purified to homogeneity from soybean suspension cultures. Three of the zucchini plasma membrane kinases share antigenic epitopes with the soluble soybean kinase. The presence of kinase activity at different apparent molecular masses may be indicative of separate kinases with similar characteristics. The zucchini hypocotyl protein kinases are not removed from plasma membrane vesicles by 0.5 M NaCl/5 mM ethylenediaminetetraacetate or by detergent concentrations below the critical micelle concentration of two types of detergent. This indicates that the plasma membrane protein kinases are tightly associated with the membrane in zucchini seedlings. PMID:12231949

  6. Identification of eukaryotic peptide deformylases reveals universality of N-terminal protein processing mechanisms.

    PubMed

    Giglione, C; Serero, A; Pierre, M; Boisson, B; Meinnel, T

    2000-11-01

    The N-terminal protein processing pathway is an essential mechanism found in all organisms. However, it is widely believed that deformylase, a key enzyme involved in this process in bacteria, does not exist in eukaryotes, thus making it a target for antibacterial agents such as actinonin. In an attempt to define this process in higher eukaryotes we have used Arabidopsis thaliana as a model organism. Two deformylase cDNAs, the first identified in any eukaryotic system, and six distinct methionine aminopeptidase cDNAs were cloned. The corresponding proteins were characterized in vivo and in vitro. Methionine aminopeptidases were found in the cytoplasm and in the organelles, while deformylases were localized in the organelles only. Our work shows that higher plants have a much more complex machinery for methionine removal than previously suspected. We were also able to identify deformylase homologues from several animals and clone the corresponding cDNA from human cells. Our data provide the first evidence that lower and higher eukaryotes, as well as bacteria, share a similar N-terminal protein processing machinery, indicating universality of this system.

  7. Phosphoregulators: Protein Kinases and Protein Phosphatases of Mouse

    PubMed Central

    Forrest, Alistair R.R.; Ravasi, Timothy; Taylor, Darrin; Huber, Thomas; Hume, David A.; Grimmond, Sean

    2003-01-01

    With the completion of the human and mouse genome sequences, the task now turns to identifying their encoded transcripts and assigning gene function. In this study, we have undertaken a computational approach to identify and classify all of the protein kinases and phosphatases present in the mouse gene complement. A nonredundant set of these sequences was produced by mining Ensembl gene predictions and publicly available cDNA sequences with a panel of InterPro domains. This approach identified 561 candidate protein kinases and 162 candidate protein phosphatases. This cohort was then analyzed using TribeMCL protein sequence similarity clustering followed by CLUSTALV alignment and hierarchical tree generation. This approach allowed us to (1) distinguish between true members of the protein kinase and phosphatase families and enzymes of related biochemistry, (2) determine the structure of the families, and (3) suggest functions for previously uncharacterized members. The classifications obtained by this approach were in good agreement with previous schemes and allowed us to demonstrate domain associations with a number of clusters. Finally, we comment on the complementary nature of cDNA and genome-based gene detection and the impact of the FANTOM2 transcriptome project. PMID:12819143

  8. The P3 domain of eukaryotic RNases P/MRP: making a protein-rich RNA-based enzyme.

    PubMed

    Perederina, Anna; Krasilnikov, Andrey S

    2010-01-01

    Nuclear Ribonuclease (RNase) P is a universal essential RNA-based enzyme made of a catalytic RNA component and a protein part; eukaryotic RNase P is closely related to a universal eukaryotic ribonucleoprotein RNase MRP. The protein part of the eukaryotic RNases P/MRP is dramatically more complex than that in bacterial and archaeal RNases P. The increase in the complexity of the protein part in eukaryotic RNases P/MRP was accompanied by the appearance of a novel structural element in the RNA component: an essential and phylogenetically conserved helix-loop-helix P3 RNA domain. The crystal structure of the P3 RNA domain in a complex with protein components Pop6 and Pop7 has been recently solved. Here we discuss the most salient structural features of the P3 domain as well as its possible role in the evolutionary transition to the protein-rich eukaryotic RNases P/MRP.

  9. Intra-plastid protein trafficking: how plant cells adapted prokaryotic mechanisms to the eukaryotic condition.

    PubMed

    Celedon, Jose M; Cline, Kenneth

    2013-02-01

    Protein trafficking and localization in plastids involve a complex interplay between ancient (prokaryotic) and novel (eukaryotic) translocases and targeting machineries. During evolution, ancient systems acquired new functions and novel translocation machineries were developed to facilitate the correct localization of nuclear encoded proteins targeted to the chloroplast. Because of its post-translational nature, targeting and integration of membrane proteins posed the biggest challenge to the organelle to avoid aggregation in the aqueous compartments. Soluble proteins faced a different kind of problem since some had to be transported across three membranes to reach their destination. Early studies suggested that chloroplasts addressed these issues by adapting ancient-prokaryotic machineries and integrating them with novel-eukaryotic systems, a process called 'conservative sorting'. In the last decade, detailed biochemical, genetic, and structural studies have unraveled the mechanisms of protein targeting and localization in chloroplasts, suggesting a highly integrated scheme where ancient and novel systems collaborate at different stages of the process. In this review we focus on the differences and similarities between chloroplast ancestral translocases and their prokaryotic relatives to highlight known modifications that adapted them to the eukaryotic situation. This article is part of a Special Issue entitled: Protein Import and Quality Control in Mitochondria and Plastids. Copyright © 2012 Elsevier B.V. All rights reserved.

  10. Protein kinase C activity in boar sperm.

    PubMed

    Teijeiro, J M; Marini, P E; Bragado, M J; Garcia-Marin, L J

    2017-03-01

    Male germ cells undergo different processes within the female reproductive tract to successfully fertilize the oocyte. These processes are triggered by different extracellular stimuli leading to activation of protein phosphorylation. Protein kinase C (PKC) is a key regulatory enzyme in signal transduction mechanisms involved in many cellular processes. Studies in boar sperm demonstrated a role for PKC in the intracellular signaling involved in motility and cellular volume regulation. Experiments using phorbol 12-myristate 13-acetate (PMA) showed increases in the Serine/Threonine phosphorylation of substrates downstream of PKC in boar sperm. In order to gain knowledge about those cellular processes regulated by PKC, we evaluate the effects of PMA on boar sperm motility, lipid organization of plasma membrane, integrity of acrosome membrane and sperm agglutination. Also, we investigate the crosstalk between PKA and PKC intracellular pathways in spermatozoa from this species. The results presented here reveal a participation of PKC in sperm motility regulation and membrane fluidity changes, which is probably associated to acrosome reaction and to agglutination. Also, we show the existence of a hierarchy in the kinases pathway. Previous works on boar sperm suggest a pathway in which PKA is positioned upstream to PKC and this new results support such model.

  11. Molecular evolution of translin superfamily proteins within the genomes of eubacteria, archaea and eukaryotes.

    PubMed

    Gupta, Gagan D; Kale, Avinash; Kumar, Vinay

    2012-12-01

    Translin and its interacting partner protein, TRAX, are members of the translin superfamily. These proteins are involved in mRNA regulation and in promoting RISC activity by removing siRNA passenger strand cleavage products, and have been proposed to play roles in DNA repair and recombination. Both homomeric translin and heteromeric translin-TRAX complex bind to ssDNA and RNA; however, the heteromeric complex is a key activator in siRNA-mediated silencing in human and drosophila. The residues critical for RNase activity of the complex reside in TRAX sequence. Both translin and TRAX are well conserved in eukaryotes. In present work, a single translin superfamily protein is detected in Chloroflexi eubacteria, in the known phyla of archaea and in some unicellular eukaryotes. The prokaryotic proteins essentially share unique sequence motifs with eukaryotic TRAX, while the proteins possessing both the unique sequences and conserved indels of TRAX or translin can be identified from protists. Intriguingly, TRAX protein in all the known genomes of extant Chloroflexi share high sequence similarity and conserved indels with the archaeal protein, suggesting occurrence of TRAX at least at the time of Chloroflexi divergence as well as evolutionary relationship between Chloroflexi and archaea. The mirror phylogeny in phylogenetic tree, constructed using diverse translin and TRAX sequences, indicates gene duplication event leading to evolution of translin in unicellular eukaryotes, prior to divergence of multicellular eukayrotes. Since Chloroflexi has been debated to be near the last universal common ancestor, the present analysis indicates that TRAX may be useful to understand the tree of life.

  12. IRES-Mediated Translation of Membrane Proteins and Glycoproteins in Eukaryotic Cell-Free Systems

    PubMed Central

    Brödel, Andreas K.; Sonnabend, Andrei; Roberts, Lisa O.; Stech, Marlitt; Wüstenhagen, Doreen A.; Kubick, Stefan

    2013-01-01

    Internal ribosome entry site (IRES) elements found in the 5′ untranslated region of mRNAs enable translation initiation in a cap-independent manner, thereby representing an alternative to cap-dependent translation in cell-free protein expression systems. However, IRES function is largely species-dependent so their utility in cell-free systems from different species is rather limited. A promising approach to overcome these limitations would be the use of IRESs that are able to recruit components of the translation initiation apparatus from diverse origins. Here, we present a solution to this technical problem and describe the ability of a number of viral IRESs to direct efficient protein expression in different eukaryotic cell-free expression systems. The IRES from the intergenic region (IGR) of the Cricket paralysis virus (CrPV) genome was shown to function efficiently in four different cell-free systems based on lysates derived from cultured Sf21, CHO and K562 cells as well as wheat germ. Our results suggest that the CrPV IGR IRES-based expression vector is universally applicable for a broad range of eukaryotic cell lysates. Sf21, CHO and K562 cell-free expression systems are particularly promising platforms for the production of glycoproteins and membrane proteins since they contain endogenous microsomes that facilitate the incorporation of membrane-spanning proteins and the formation of post-translational modifications. We demonstrate the use of the CrPV IGR IRES-based expression vector for the enhanced synthesis of various target proteins including the glycoprotein erythropoietin and the membrane proteins heparin-binding EGF-like growth factor receptor as well as epidermal growth factor receptor in the above mentioned eukaryotic cell-free systems. CrPV IGR IRES-mediated translation will facilitate the development of novel eukaryotic cell-free expression platforms as well as the high-yield synthesis of desired proteins in already established systems. PMID

  13. ARC3, a chloroplast division factor, is a chimera of prokaryotic FtsZ and part of eukaryotic phosphatidylinositol-4-phosphate 5-kinase.

    PubMed

    Shimada, Hiroshi; Koizumi, Masato; Kuroki, Kouta; Mochizuki, Mariko; Fujimoto, Hitoshi; Ohta, Hiroyuki; Masuda, Tatsuru; Takamiya, Ken-ichiro

    2004-08-01

    The arc3 (accumulation and replication of chloroplast) mutant of Arabidopsis thaliana has a small number of abnormally large chloroplasts in the cell, suggesting that chloroplast division is arrested in the mutant and ARC3 has an important role in the initiation of chloroplast division. To elucidate the role of ARC3, first we identified the ARC3 gene, and determined the location of ARC3 protein during chloroplast division because the localization and spatial orientation of such division factors are vital for correct chloroplast division. Sequencing analysis showed that ARC3 was a fusion of the prokaryotic FtsZ and part of the eukaryotic phosphatidylinositol-4-phosphate 5-kinase (PIP5K) genes. The PIP5K-homologous region of ARC3 had no catalytic domain but a membrane-occupation-and-recognition-nexus (MORN) repeat motif. Immunofluorescence microscopy, Western blotting analysis and in vitro chloroplast import and protease protection assays revealed that ARC3 protein was soluble, and located on the outer surface of the chloroplast in a ring-like structure at the early stage of chloroplast division. Prokaryotes have one FtsZ as a gene for division but have no ARC3 counterparts, the chimera of FtsZ and PIP5K, suggesting that the ARC3 gene might have been generated from FtsZ as another division factor during the evolution of chloroplast by endosymbiosis.

  14. The Roles of Protein Kinases in Learning and Memory

    ERIC Educational Resources Information Center

    Giese, Karl Peter; Mizuno, Keiko

    2013-01-01

    In the adult mammalian brain, more than 250 protein kinases are expressed, but only a few of these kinases are currently known to enable learning and memory. Based on this information it appears that learning and memory-related kinases either impact on synaptic transmission by altering ion channel properties or ion channel density, or regulate…

  15. The Roles of Protein Kinases in Learning and Memory

    ERIC Educational Resources Information Center

    Giese, Karl Peter; Mizuno, Keiko

    2013-01-01

    In the adult mammalian brain, more than 250 protein kinases are expressed, but only a few of these kinases are currently known to enable learning and memory. Based on this information it appears that learning and memory-related kinases either impact on synaptic transmission by altering ion channel properties or ion channel density, or regulate…

  16. Protein-mediated transbilayer movement of lipids in eukaryotes and prokaryotes: the relevance of ABC transporters.

    PubMed

    Tannert, Astrid; Pohl, Antje; Pomorski, Thomas; Herrmann, Andreas

    2003-09-01

    Lipid distribution across cellular membranes is regulated by specific membrane proteins controlling transbilayer movement of lipids. Flippases facilitate flip-flop of lipids and allow them to equilibrate between the two membrane leaflets independent of ATP. Distinct P-Type-ATPases transport specific lipids unidirectionally across the membrane at the expense of ATP. A group of ATP-dependent lipid transporters, the ATP-binding cassette (ABC) transporter family, was identified in studies originally related to multidrug resistance (MDR) in cancer cells. Meanwhile, lipid transport activity has been shown for full and half size ABC proteins in eukaryotic and prokaryotic cells. This activity may not only modify the organisation of lipids in membranes, but could also be of significant consequence for cell homeostasis. The various types of lipid movement mediating proteins and their cellular localisation in eukaryotes and prokaryotes are reviewed.

  17. Quantification of DNA-associated proteins inside eukaryotic cells using single-molecule localization microscopy

    PubMed Central

    Etheridge, Thomas J.; Boulineau, Rémi L.; Herbert, Alex; Watson, Adam T.; Daigaku, Yasukazu; Tucker, Jem; George, Sophie; Jönsson, Peter; Palayret, Matthieu; Lando, David; Laue, Ernest; Osborne, Mark A.; Klenerman, David; Lee, Steven F.; Carr, Antony M.

    2014-01-01

    Development of single-molecule localization microscopy techniques has allowed nanometre scale localization accuracy inside cells, permitting the resolution of ultra-fine cell structure and the elucidation of crucial molecular mechanisms. Application of these methodologies to understanding processes underlying DNA replication and repair has been limited to defined in vitro biochemical analysis and prokaryotic cells. In order to expand these techniques to eukaryotic systems, we have further developed a photo-activated localization microscopy-based method to directly visualize DNA-associated proteins in unfixed eukaryotic cells. We demonstrate that motion blurring of fluorescence due to protein diffusivity can be used to selectively image the DNA-bound population of proteins. We designed and tested a simple methodology and show that it can be used to detect changes in DNA binding of a replicative helicase subunit, Mcm4, and the replication sliding clamp, PCNA, between different stages of the cell cycle and between distinct genetic backgrounds. PMID:25106872

  18. Optimizing eukaryotic cell hosts for protein production through systems biotechnology and genome-scale modeling.

    PubMed

    Gutierrez, Jahir M; Lewis, Nathan E

    2015-07-01

    Eukaryotic cell lines, including Chinese hamster ovary cells, yeast, and insect cells, are invaluable hosts for the production of many recombinant proteins. With the advent of genomic resources, one can now leverage genome-scale computational modeling of cellular pathways to rationally engineer eukaryotic host cells. Genome-scale models of metabolism include all known biochemical reactions occurring in a specific cell. By describing these mathematically and using tools such as flux balance analysis, the models can simulate cell physiology and provide targets for cell engineering that could lead to enhanced cell viability, titer, and productivity. Here we review examples in which metabolic models in eukaryotic cell cultures have been used to rationally select targets for genetic modification, improve cellular metabolic capabilities, design media supplementation, and interpret high-throughput omics data. As more comprehensive models of metabolism and other cellular processes are developed for eukaryotic cell culture, these will enable further exciting developments in cell line engineering, thus accelerating recombinant protein production and biotechnology in the years to come. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. The Ancient Gamete Fusogen HAP2 Is a Eukaryotic Class II Fusion Protein.

    PubMed

    Fédry, Juliette; Liu, Yanjie; Péhau-Arnaudet, Gérard; Pei, Jimin; Li, Wenhao; Tortorici, M Alejandra; Traincard, François; Meola, Annalisa; Bricogne, Gérard; Grishin, Nick V; Snell, William J; Rey, Félix A; Krey, Thomas

    2017-02-23

    Sexual reproduction is almost universal in eukaryotic life and involves the fusion of male and female haploid gametes into a diploid cell. The sperm-restricted single-pass transmembrane protein HAP2-GCS1 has been postulated to function in membrane merger. Its presence in the major eukaryotic taxa-animals, plants, and protists (including important human pathogens like Plasmodium)-suggests that many eukaryotic organisms share a common gamete fusion mechanism. Here, we report combined bioinformatic, biochemical, mutational, and X-ray crystallographic studies on the unicellular alga Chlamydomonas reinhardtii HAP2 that reveal homology to class II viral membrane fusion proteins. We further show that targeting the segment corresponding to the fusion loop by mutagenesis or by antibodies blocks gamete fusion. These results demonstrate that HAP2 is the gamete fusogen and suggest a mechanism of action akin to viral fusion, indicating a way to block Plasmodium transmission and highlighting the impact of virus-cell genetic exchanges on the evolution of eukaryotic life. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  20. In Silico Resurrection of the Major Vault Protein Suggests It Is Ancestral in Modern Eukaryotes

    PubMed Central

    Daly, Toni K.; Sutherland-Smith, Andrew J.; Penny, David

    2013-01-01

    Vaults are very large oligomeric ribonucleoproteins conserved among a variety of species. The rat vault 3D structure shows an ovoid oligomeric particle, consisting of 78 major vault protein monomers, each of approximately 861 amino acids. Vaults are probably the largest ribonucleoprotein structures in eukaryote cells, being approximately 70 nm in length with a diameter of 40 nm—the size of three ribosomes and with a lumen capacity of 50 million Å3. We use both protein sequences and inferred ancestral sequences for in silico virtual resurrection of tertiary and quaternary structures to search for vaults in a wide variety of eukaryotes. We find that the vault’s phylogenetic distribution is widespread in eukaryotes, but is apparently absent in some notable model organisms. Our conclusion from the distribution of vaults is that they were present in the last eukaryote common ancestor but they have apparently been lost from a number of groups including fungi, insects, and probably plants. Our approach of inferring ancestral 3D and quaternary structures is expected to be useful generally. PMID:23887922

  1. In silico resurrection of the major vault protein suggests it is ancestral in modern eukaryotes.

    PubMed

    Daly, Toni K; Sutherland-Smith, Andrew J; Penny, David

    2013-01-01

    Vaults are very large oligomeric ribonucleoproteins conserved among a variety of species. The rat vault 3D structure shows an ovoid oligomeric particle, consisting of 78 major vault protein monomers, each of approximately 861 amino acids. Vaults are probably the largest ribonucleoprotein structures in eukaryote cells, being approximately 70 nm in length with a diameter of 40 nm--the size of three ribosomes and with a lumen capacity of 50 million Å(3). We use both protein sequences and inferred ancestral sequences for in silico virtual resurrection of tertiary and quaternary structures to search for vaults in a wide variety of eukaryotes. We find that the vault's phylogenetic distribution is widespread in eukaryotes, but is apparently absent in some notable model organisms. Our conclusion from the distribution of vaults is that they were present in the last eukaryote common ancestor but they have apparently been lost from a number of groups including fungi, insects, and probably plants. Our approach of inferring ancestral 3D and quaternary structures is expected to be useful generally.

  2. Intra-plastid protein trafficking; how plant cells adapted prokaryotic mechanisms to the eukaryotic condition

    PubMed Central

    Celedon, Jose M.; Cline, Kenneth

    2012-01-01

    Protein trafficking and localization in plastids involves a complex interplay between ancient (prokaryotic) and novel (eukaryotic) translocases and targeting machineries. During evolution, ancient systems acquired new functions and novel translocation machineries were developed to facilitate the correct localization of nuclear encoded proteins targeted to the chloroplast. Because of its post-translational nature, targeting and integration of membrane proteins posed the biggest challenge to the organelle to avoid aggregation in the aqueous compartments. Soluble proteins faced a different kind of problem since some had to be transported across three membranes to reach their destination. Early studies suggested that chloroplasts addressed these issues by adapting ancient-prokaryotic machineries and integrating them with novel-eukaryotic systems, a process called ‘conservative sorting’. In the last decade, detailed biochemical, genetic, and structural studies have unraveled the mechanisms of protein targeting and localization in chloroplasts, suggesting a highly integrated scheme where ancient and novel systems collaborate at different stages of the process. In this review we focus on the differences and similarities between chloroplast ancestral translocases and their prokaryotic relatives to highlight known modifications that adapted them to the eukaryotic situation. PMID:22750312

  3. Constraints and consequences of the emergence of amino acid repeats in eukaryotic proteins.

    PubMed

    Chavali, Sreenivas; Chavali, Pavithra L; Chalancon, Guilhem; de Groot, Natalia Sanchez; Gemayel, Rita; Latysheva, Natasha S; Ing-Simmons, Elizabeth; Verstrepen, Kevin J; Balaji, Santhanam; Babu, M Madan

    2017-09-01

    Proteins with amino acid homorepeats have the potential to be detrimental to cells and are often associated with human diseases. Why, then, are homorepeats prevalent in eukaryotic proteomes? In yeast, homorepeats are enriched in proteins that are essential and pleiotropic and that buffer environmental insults. The presence of homorepeats increases the functional versatility of proteins by mediating protein interactions and facilitating spatial organization in a repeat-dependent manner. During evolution, homorepeats are preferentially retained in proteins with stringent proteostasis, which might minimize repeat-associated detrimental effects such as unregulated phase separation and protein aggregation. Their presence facilitates rapid protein divergence through accumulation of amino acid substitutions, which often affect linear motifs and post-translational-modification sites. These substitutions may result in rewiring protein interaction and signaling networks. Thus, homorepeats are distinct modules that are often retained in stringently regulated proteins. Their presence facilitates rapid exploration of the genotype-phenotype landscape of a population, thereby contributing to adaptation and fitness.

  4. Prokaryotic and eukaryotic DNA helicases. Essential molecular motor proteins for cellular machinery.

    PubMed

    Tuteja, Narendra; Tuteja, Renu

    2004-05-01

    DNA helicases are ubiquitous molecular motor proteins which harness the chemical free energy of ATP hydrolysis to catalyze the unwinding of energetically stable duplex DNA, and thus play important roles in nearly all aspects of nucleic acid metabolism, including replication, repair, recombination, and transcription. They break the hydrogen bonds between the duplex helix and move unidirectionally along the bound strand. All helicases are also translocases and DNA-dependent ATPases. Most contain conserved helicase motifs that act as an engine to power DNA unwinding. All DNA helicases share some common properties, including nucleic acid binding, NTP binding and hydrolysis, and unwinding of duplex DNA in the 3' to 5' or 5' to 3' direction. The minichromosome maintenance (Mcm) protein complex (Mcm4/6/7) provides a DNA-unwinding function at the origin of replication in all eukaryotes and may act as a licensing factor for DNA replication. The RecQ family of helicases is highly conserved from bacteria to humans and is required for the maintenance of genome integrity. They have also been implicated in a variety of human genetic disorders. Since the discovery of the first DNA helicase in Escherichia coli in 1976, and the first eukaryotic one in the lily in 1978, a large number of these enzymes have been isolated from both prokaryotic and eukaryotic systems, and the number is still growing. In this review we cover the historical background of DNA helicases, helicase assays, biochemical properties, prokaryotic and eukaryotic DNA helicases including Mcm proteins and the RecQ family of helicases. The properties of most of the known DNA helicases from prokaryotic and eukaryotic systems, including viruses and bacteriophages, are summarized in tables.

  5. Membrane-associated protein kinase activities in the developing mesocarp of grape berry.

    PubMed

    Shen, Yuan-Yue; Duan, Chang-Qing; Liang, Xiao-E; Zhang, Da-Peng

    2004-01-01

    Fruit development is a process involving various signals and gene expression. Protein phosphorylation catalyzed by protein kinases is known to play a key role in eukaryotic cell signalling and so may be involved in the regulation of fruit development. Using the method of exogenous substrate phosphorylation, we characterised the calcium-dependent and calmodulin-independent protein kinase (CDPK) activity and the myelin basic protein (MBP)-phosphoralating activity that could be due to a mitogen-activated protein kinase (MAPK)-like activity in the developing mesocarp of grape berry. The CDPK activity was shown to be predominantly localised in the plasma membrane, while the MAPK-like activity was predominantly associated with endomembranes. The assays of bivalent cation requirement showed that Mn2+ could to a certain extent replace Mg2+ in the incubation system for the protein kinase activities. Both CDPK and MAPK-like activities were resistant to heat treatment. The activities of the two enzymes were fruit developmental stage-specific with the highest activities of both enzymes in the lag growth phase before the ripening stage, suggesting strongly the important roles of the detected CDPK and MAPK-like activities in the fruit development.

  6. HRR25, a putative protein kinase from budding yeast: Association with repair of damaged DNA

    SciTech Connect

    Hoekstra, M.F.; Ou, A.C.; DeMaggio, A.J.; Burbee, D.G. ); Liskay, R.M. ); Heffron, F. )

    1991-08-30

    In simple eukaryotes, protein kinases regulate mitotic and meiotic cell cycles, the response to polypeptide pheromones, and the initiation of nuclear DNA synthesis. The protein HRR25 from the budding yeast Saccharomyces cerevisiae was defined by the mutation hrr25-1. This mutation resulted in sensitivity to continuous expression of the HO double-strand endonuclease, to methyl methanesulfonate, and to x-irradiation. Homozygotes of hrr25-1 were unable to sporulate and disruption and deletion of HRR25 interfered with mitotic and meiotic cell division. Sequence analysis revealed two distinctive regions in the protein. The NH{sub 2}-terminus of HRR25 contains the hallmark features of protein kinases, whereas the COOH-terminus is rich in proline and glutamine. Mutations in HRR25 at conserved residues found in all protein kinases inactivated the gene, and these mutants exhibited the hrr25 null phenotypes. Taken together, the hrr25 mutant phenotypes and the features of the gene product indicate that HRR25 is a distinctive member of the protein kinase superfamily.

  7. Protein kinase CK2 triggers cytosolic zinc signaling pathways by phosphorylation of zinc channel ZIP7.

    PubMed

    Taylor, Kathryn M; Hiscox, Stephen; Nicholson, Robert I; Hogstrand, Christer; Kille, Peter

    2012-02-07

    The transition element zinc, which has recently been identified as an intracellular second messenger, has been implicated in various signaling pathways, including those leading to cell proliferation. Zinc channels of the ZIP (ZRT1- and IRT1-like protein) family [also known as solute carrier family 39A (SLC39A)] transiently increase the cytosolic free zinc (Zn(2+)) concentration in response to extracellular signals. We show that phosphorylation of evolutionarily conserved residues in endoplasmic reticulum zinc channel ZIP7 is associated with the gated release of Zn(2+) from intracellular stores, leading to activation of tyrosine kinases and the phosphorylation of AKT and extracellular signal-regulated kinases 1 and 2. Through pharmacological manipulation, proximity ligation assay, and mutagenesis, we identified protein kinase CK2 as the kinase responsible for ZIP7 activation. Together, the present results show that transition element channels in eukaryotes can be activated posttranslationally by phosphorylation, as part of a cell signaling cascade. Our study links the regulated release of zinc from intracellular stores to phosphorylation of kinases involved in proliferative responses and cell migration, suggesting a functional role for ZIP7 and zinc signals in these events. The connection with proliferation and migration, as well as the activation of ZIP7 by CK2, a kinase that is antiapoptotic and promotes cell division, suggests that ZIP7 may provide a target for anticancer drug development.

  8. The nuts and bolts of AGC protein kinases.

    PubMed

    Pearce, Laura R; Komander, David; Alessi, Dario R

    2010-01-01

    The AGC kinase subfamily of protein kinases contains 60 members, including PKA, PKG and PKC. The family comprises some intensely examined protein kinases (such as Akt, S6K, RSK, MSK, PDK1 and GRK) as well as many less well-studied enzymes (such as SGK, NDR, LATS, CRIK, SGK494, PRKX, PRKY and MAST). Research has shed new light onto the architecture and regulatory mechanisms of these kinases. In addition, AGC kinases mediate diverse and important cellular functions, and their mutation and/or dysregulation contributes to the pathogenesis of many human diseases, including cancer and diabetes.

  9. Protein kinase A contributes to the negative control of Snf1 protein kinase in Saccharomyces cerevisiae.

    PubMed

    Barrett, LaKisha; Orlova, Marianna; Maziarz, Marcin; Kuchin, Sergei

    2012-02-01

    Snf1 protein kinase regulates responses to glucose limitation and other stresses. Snf1 activation requires phosphorylation of its T-loop threonine by partially redundant upstream kinases (Sak1, Tos3, and Elm1). Under favorable conditions, Snf1 is turned off by Reg1-Glc7 protein phosphatase. The reg1 mutation causes increased Snf1 activation and slow growth. To identify new components of the Snf1 pathway, we searched for mutations that, like snf1, suppress reg1 for the slow-growth phenotype. In addition to mutations in genes encoding known pathway components (SNF1, SNF4, and SAK1), we recovered "fast" mutations, designated fst1 and fst2. Unusual morphology of the mutants in the Σ1278b strains employed here helped us identify fst1 and fst2 as mutations in the RasGAP genes IRA1 and IRA2. Cells lacking Ira1, Ira2, or Bcy1, the negative regulatory subunit of cyclic AMP (cAMP)-dependent protein kinase A (PKA), exhibited reduced Snf1 pathway activation. Conversely, Snf1 activation was elevated in cells lacking the Gpr1 sugar receptor, which contributes to PKA signaling. We show that the Snf1-activating kinase Sak1 is phosphorylated in vivo on a conserved serine (Ser1074) within an ideal PKA motif. However, this phosphorylation alone appears to play only a modest role in regulation, and Sak1 is not the only relevant target of the PKA pathway. Collectively, our results suggest that PKA, which integrates multiple regulatory inputs, could contribute to Snf1 regulation under various conditions via a complex mechanism. Our results also support the view that, like its mammalian counterpart, AMP-activated protein kinase (AMPK), yeast Snf1 participates in metabolic checkpoint control that coordinates growth with nutrient availability.

  10. BDNF stimulation of protein synthesis in cortical neurons requires the MAP kinase-interacting kinase MNK1.

    PubMed

    Genheden, Maja; Kenney, Justin W; Johnston, Harvey E; Manousopoulou, Antigoni; Garbis, Spiros D; Proud, Christopher G

    2015-01-21

    Although the MAP kinase-interacting kinases (MNKs) have been known for >15 years, their roles in the regulation of protein synthesis have remained obscure. Here, we explore the involvement of the MNKs in brain-derived neurotrophic factor (BDNF)-stimulated protein synthesis in cortical neurons from mice. Using a combination of pharmacological and genetic approaches, we show that BDNF-induced upregulation of protein synthesis requires MEK/ERK signaling and the downstream kinase, MNK1, which phosphorylates eukaryotic initiation factor (eIF) 4E. Translation initiation is mediated by the interaction of eIF4E with the m(7)GTP cap of mRNA and with eIF4G. The latter interaction is inhibited by the interactions of eIF4E with partner proteins, such as CYFIP1, which acts as a translational repressor. We find that BDNF induces the release of CYFIP1 from eIF4E, and that this depends on MNK1. Finally, using a novel combination of BONCAT and SILAC, we identify a subset of proteins whose synthesis is upregulated by BDNF signaling via MNK1 in neurons. Interestingly, this subset of MNK1-sensitive proteins is enriched for functions involved in neurotransmission and synaptic plasticity. Additionally, we find significant overlap between our subset of proteins whose synthesis is regulated by MNK1 and those encoded by known FMRP-binding mRNAs. Together, our data implicate MNK1 as a key component of BDNF-mediated translational regulation in neurons. Copyright © 2015 Genheden et al.

  11. KinasePhos: a web tool for identifying protein kinase-specific phosphorylation sites.

    PubMed

    Huang, Hsien-Da; Lee, Tzong-Yi; Tzeng, Shih-Wei; Horng, Jorng-Tzong

    2005-07-01

    KinasePhos is a novel web server for computationally identifying catalytic kinase-specific phosphorylation sites. The known phosphorylation sites from public domain data sources are categorized by their annotated protein kinases. Based on the profile hidden Markov model, computational models are learned from the kinase-specific groups of the phosphorylation sites. After evaluating the learned models, the model with highest accuracy was selected from each kinase-specific group, for use in a web-based prediction tool for identifying protein phosphorylation sites. Therefore, this work developed a kinase-specific phosphorylation site prediction tool with both high sensitivity and specificity. The prediction tool is freely available at http://KinasePhos.mbc.nctu.edu.tw/.

  12. Auxin efflux by PIN-FORMED proteins is activated by two different protein kinases, D6 PROTEIN KINASE and PINOID.

    PubMed

    Zourelidou, Melina; Absmanner, Birgit; Weller, Benjamin; Barbosa, Inês C R; Willige, Björn C; Fastner, Astrid; Streit, Verena; Port, Sarah A; Colcombet, Jean; de la Fuente van Bentem, Sergio; Hirt, Heribert; Kuster, Bernhard; Schulze, Waltraud X; Hammes, Ulrich Z; Schwechheimer, Claus

    2014-06-19

    The development and morphology of vascular plants is critically determined by synthesis and proper distribution of the phytohormone auxin. The directed cell-to-cell distribution of auxin is achieved through a system of auxin influx and efflux transporters. PIN-FORMED (PIN) proteins are proposed auxin efflux transporters, and auxin fluxes can seemingly be predicted based on the--in many cells--asymmetric plasma membrane distribution of PINs. Here, we show in a heterologous Xenopus oocyte system as well as in Arabidopsis thaliana inflorescence stems that PIN-mediated auxin transport is directly activated by D6 PROTEIN KINASE (D6PK) and PINOID (PID)/WAG kinases of the Arabidopsis AGCVIII kinase family. At the same time, we reveal that D6PKs and PID have differential phosphosite preferences. Our study suggests that PIN activation by protein kinases is a crucial component of auxin transport control that must be taken into account to understand auxin distribution within the plant.

  13. Understanding and exploiting substrate recognition by protein kinases.

    PubMed

    Turk, Benjamin E

    2008-02-01

    Protein kinases play a virtually universal role in cellular regulation and are emerging as an important class of new drug targets, yet the cellular functions of most human kinases largely remain obscure. Aspects of substrate recognition common to all kinases in the ATP nucleotide binding site have been exploited in the generation of analog-specific mutants for exploring kinase function and discovering novel protein substrates. Likewise, understanding interactions with the protein substrate, which differ substantially between kinases, can also help to identify substrates and to produce tools for studying kinase pathways, including fluorescent biosensors. Principles of kinase substrate recognition are particularly valuable in guiding bioinformatics and phosphoproteomics approaches that impact our understanding of signaling pathways and networks on a global scale.

  14. CDPKs are dual-specificity protein kinases and tyrosine autophosphorylation attenuates kinase activity

    USDA-ARS?s Scientific Manuscript database

    Calcium-dependent protein kinases (CDPKs or CPKs) are classified as serine/threonine protein kinases but we made the surprising observation that soybean CDPK' and several Arabidopsis isoforms (AtCPK4 and AtCPK34) could also autophosphorylate on tyrosine residues. In studies with His6-GmCDPK', we ide...

  15. The initiation of eukaryotic and prokaryotic protein synthesis: a selective accessibility and multisubstrate enzyme reaction.

    PubMed

    Nakamoto, Tokumasa

    2007-11-15

    An extension of our unique accessibility hypothesis for the initiation of protein synthesis is proposed following a review of the initiation of protein synthesis. The E. coli model initiation sequence generated by computer from 68 initiation sequences and the eukaryotic consensus initiation sequence derived by non-computer analysis of 211 initiation sequences do not contain a specific base in any position; they are only assigned preferred bases. The initiation site, in other words, is a varied sequence of preferred bases and its sequence is non-unique. This indicates that the ribosomal recognition of the initiation site may be the result of multiple interactions that are cooperative and cumulative and typical of multisubstrate enzymes. Because of this characteristic, the model of multisubstrate enzymes with broad substrate specificity is proposed as a paradigm for the initiation of protein synthesis. As predicted by this model, changes in the leader and downstream sequences that improve the agreement with the preferred base sequence do indeed enhance the rate of protein synthesis. The eukaryotic/prokaryotic hybrid studies show a considerable overlap in the specificities of the two groups of ribosomes. The scanning of the mRNA from the 5'-end postulated by the scanning hypothesis is not a necessary step since eukaryotic ribosomes are able to bind to internal mRNA sites and initiate synthesis. Our unique accessibility hypothesis, which is extended by coupling cooperative and cumulative specificity in ribosomal function, is referred to for brevity as the cumulative specificity hypothesis. The hypothesis actually postulates a selective accessibility and cooperative-cumulative specificity mechanism; it is able to account for the behavior of both eukaryotic and prokaryotic initiation of protein synthesis. From another perspective, the hypothesis can be regarded as providing a mechanism that enables ribosomes to recognize the IS in the absence of a unique initiation

  16. Crystal structure of the MID-PIWI lobe of a eukaryotic Argonaute protein

    PubMed Central

    Boland, Andreas; Huntzinger, Eric; Schmidt, Steffen; Izaurralde, Elisa; Weichenrieder, Oliver

    2011-01-01

    Argonaute proteins (AGOs) are essential effectors in RNA-mediated gene silencing pathways. They are characterized by a bilobal architecture, in which one lobe contains the N-terminal and PAZ domains and the other contains the MID and PIWI domains. Here, we present the first crystal structure of the MID-PIWI lobe from a eukaryotic AGO, the Neurospora crassa QDE-2 protein. Compared to prokaryotic AGOs, the domain orientation is conserved, indicating a conserved mode of nucleic acid binding. The PIWI domain shows an adaptable surface loop next to a eukaryote-specific α-helical insertion, which are both likely to contact the PAZ domain in a conformation-dependent manner to sense the functional state of the protein. The MID-PIWI interface is hydrophilic and buries residues that were previously thought to participate directly in the allosteric regulation of guide RNA binding. The interface includes the binding pocket for the guide RNA 5′ end, and residues from both domains contribute to binding. Accordingly, micro-RNA (miRNA) binding is particularly sensitive to alteration in the MID-PIWI interface in Drosophila melanogaster AGO1 in vivo. The structure of the QDE-2 MID-PIWI lobe provides molecular and mechanistic insight into eukaryotic AGOs and has significant implications for understanding the role of these proteins in silencing. PMID:21646546

  17. Heterotrimeric G-protein shuttling via Gip1 extends the dynamic range of eukaryotic chemotaxis

    PubMed Central

    Kamimura, Yoichiro; Miyanaga, Yukihiro; Ueda, Masahiro

    2016-01-01

    Chemotactic eukaryote cells can sense chemical gradients over a wide range of concentrations via heterotrimeric G-protein signaling; however, the underlying wide-range sensing mechanisms are only partially understood. Here we report that a novel regulator of G proteins, G protein-interacting protein 1 (Gip1), is essential for extending the chemotactic range of Dictyostelium cells. Genetic disruption of Gip1 caused severe defects in gradient sensing and directed cell migration at high but not low concentrations of chemoattractant. Also, Gip1 was found to bind and sequester G proteins in cytosolic pools. Receptor activation induced G-protein translocation to the plasma membrane from the cytosol in a Gip1-dependent manner, causing a biased redistribution of G protein on the membrane along a chemoattractant gradient. These findings suggest that Gip1 regulates G-protein shuttling between the cytosol and the membrane to ensure the availability and biased redistribution of G protein on the membrane for receptor-mediated chemotactic signaling. This mechanism offers an explanation for the wide-range sensing seen in eukaryotic chemotaxis. PMID:27044073

  18. Identification of intracellular receptor proteins for activated protein kinase C.

    PubMed Central

    Mochly-Rosen, D; Khaner, H; Lopez, J

    1991-01-01

    Protein kinase C (PKC) translocates from the cytosol to the particulate fraction on activation. This activation-induced translocation of PKC is thought to reflect PKC binding to the membrane lipids. However, immunological and biochemical data suggest that PKC may bind to proteins in the cytoskeletal elements in the particulate fraction and in the nuclei. Here we describe evidence for the presence of intracellular receptor proteins that bind activated PKC. Several proteins from the detergent-insoluble material of the particulate fraction bound PKC in the presence of phosphatidylserine and calcium; binding was further increased with the addition of diacylglycerol. Binding of PKC to two of these proteins was concentration-dependent, saturable, and specific, suggesting that these binding proteins are receptors for activated C-kinase, termed here "RACKs." PKC binds to RACKs via a site on PKC distinct from the substrate binding site. We suggest that binding to RACKs may play a role in activation-induced translocation of PKC. Images PMID:1850844

  19. Identification and Analysis of Mitogen-Activated Protein Kinase (MAPK) Cascades in Fragaria vesca

    PubMed Central

    Zhou, Heying; Ren, Suyue; Han, Yuanfang; Zhang, Qing; Qin, Ling; Xing, Yu

    2017-01-01

    Mitogen-activated protein kinase (MAPK) cascades are highly conserved signaling modules in eukaryotes, including yeasts, plants and animals. MAPK cascades are responsible for protein phosphorylation during signal transduction events, and typically consist of three protein kinases: MAPK, MAPK kinase, and MAPK kinase kinase. In this current study, we identified a total of 12 FvMAPK, 7 FvMAPKK, 73 FvMAPKKK, and one FvMAPKKKK genes in the recently published Fragaria vesca genome sequence. This work reported the classification, annotation and phylogenetic evaluation of these genes and an assessment of conserved motifs and the expression profiling of members of the gene family were also analyzed here. The expression profiles of the MAPK and MAPKK genes in different organs and fruit developmental stages were further investigated using quantitative real-time reverse transcription PCR (qRT-PCR). Finally, the MAPK and MAPKK expression patterns in response to hormone and abiotic stresses (salt, drought, and high and low temperature) were investigated in fruit and leaves of F. vesca. The results provide a platform for further characterization of the physiological and biochemical functions of MAPK cascades in strawberry. PMID:28805715

  20. Structural insight into nucleotide recognition by human death-associated protein kinase

    SciTech Connect

    McNamara, Laurie K.; Watterson, D.M.; Brunzelle, Joseph S.

    2009-06-01

    Death-associated protein kinase (DAPK) is a member of the Ca{sup 2+}/calmodulin-regulated family of serine/threonine protein kinases. The role of the kinase activity of DAPK in eukaryotic cell apoptosis and the ability of bioavailable DAPK inhibitors to rescue neuronal death after brain injury have made it a drug-discovery target for neurodegenerative disorders. In order to understand the recognition of nucleotides by DAPK and to gain insight into DAPK catalysis, the crystal structure of human DAPK was solved in complex with ADP and Mg{sup 2+} at 1.85 {angstrom} resolution. ADP is a product of the kinase reaction and product release is considered to be the rate-limiting step of protein kinase catalytic cycles. The structure of DAPK-ADP-Mg{sup 2+} was compared with a newly determined DAPK-AMP-PNP-Mg{sup 2+} structure and the previously determined apo DAPK structure (PDB code 1 jks). The comparison shows that nucleotide-induced changes are localized to the glycine-rich loop region of DAPK.

  1. Structural investigation of protein kinase C inhibitors.

    PubMed

    Barak, D; Shibata, M; Rein, R

    1991-01-01

    The phospholipid and Ca2+ dependent protein kinase (PKC) plays an essential role in a variety of cellular events. Inhibition of PKC was shown to arrest growth in tumor cell cultures making it a target for possible antitumor therapy. Calphostins are potent inhibitors of PKC with high affinity for the enzyme regulatory site. Structural characteristics of calphostins, which confer the inhibitory activity, are investigated by comparing their optimized structures with the existing models for PKC activation. The resulting model of inhibitory activity assumes interaction with two out of the three electrostatic interaction sites postulated for activators. The model shows two sites of hydrophobic interaction and enables the inhibitory activity of gossypol to be accounted for.

  2. Protein kinase C isozymes and addiction.

    PubMed

    Olive, M Foster; Messing, Robert O

    2004-04-01

    Protein kinase C (PKC) has long been recognized an important family of enzymes that regulate numerous aspects of neuronal signal transduction, neurotransmitter synthesis, release and reuptake, receptor and ion channel function, neuronal excitability, development, and gene expression. Much evidence has implicated PKCs in the effects of several drugs of abuse, and in behavioral responses to these drugs. The present review summarizes the effects of both acute and chronic exposure to various drugs of abuse on individual PKC isozymes in the brain. In addition, we summarize recent studies utilizing mice with targeted deletions of the genes for PKCgamma and PKCepsilon. These studies suggest that individual PKC isozymes play a role in the development of drug dependence and addiction.

  3. Structural investigation of protein kinase C inhibitors

    NASA Technical Reports Server (NTRS)

    Barak, D.; Shibata, M.; Rein, R.

    1991-01-01

    The phospholipid and Ca2+ dependent protein kinase (PKC) plays an essential role in a variety of cellular events. Inhibition of PKC was shown to arrest growth in tumor cell cultures making it a target for possible antitumor therapy. Calphostins are potent inhibitors of PKC with high affinity for the enzyme regulatory site. Structural characteristics of calphostins, which confer the inhibitory activity, are investigated by comparing their optimized structures with the existing models for PKC activation. The resulting model of inhibitory activity assumes interaction with two out of the three electrostatic interaction sites postulated for activators. The model shows two sites of hydrophobic interaction and enables the inhibitory activity of gossypol to be accounted for.

  4. Rab protein evolution and the history of the eukaryotic endomembrane system.

    PubMed

    Brighouse, Andrew; Dacks, Joel B; Field, Mark C

    2010-10-01

    Spectacular increases in the quantity of sequence data genome have facilitated major advances in eukaryotic comparative genomics. By exploiting homology with classical model organisms, this makes possible predictions of pathways and cellular functions currently impossible to address in intractable organisms. Echoing realization that core metabolic processes were established very early following evolution of life on earth, it is now emerging that many eukaryotic cellular features, including the endomembrane system, are ancient and organized around near-universal principles. Rab proteins are key mediators of vesicle transport and specificity, and via the presence of multiple paralogues, alterations in interaction specificity and modification of pathways, contribute greatly to the evolution of complexity of membrane transport. Understanding system-level contributions of Rab proteins to evolutionary history provides insight into the multiple processes sculpting cellular transport pathways and the exciting challenges that we face in delving further into the origins of membrane trafficking specificity.

  5. Rab protein evolution and the history of the eukaryotic endomembrane system

    PubMed Central

    Brighouse, Andrew; Dacks, Joel B.

    2010-01-01

    Spectacular increases in the quantity of sequence data genome have facilitated major advances in eukaryotic comparative genomics. By exploiting homology with classical model organisms, this makes possible predictions of pathways and cellular functions currently impossible to address in intractable organisms. Echoing realization that core metabolic processes were established very early following evolution of life on earth, it is now emerging that many eukaryotic cellular features, including the endomembrane system, are ancient and organized around near-universal principles. Rab proteins are key mediators of vesicle transport and specificity, and via the presence of multiple paralogues, alterations in interaction specificity and modification of pathways, contribute greatly to the evolution of complexity of membrane transport. Understanding system-level contributions of Rab proteins to evolutionary history provides insight into the multiple processes sculpting cellular transport pathways and the exciting challenges that we face in delving further into the origins of membrane trafficking specificity. PMID:20582450

  6. Association of eukaryotic DNA topoisomerase I with nucleosomes and chromosomal proteins.

    PubMed Central

    Javaherian, K; Liu, L F

    1983-01-01

    A DNA topoisomerase activity is found to be associated with the nucleosomes released by the Staphylococcal nuclease digestion of HeLa nuclei. Such an association is found to be salt dependent. A number of criteria have established that this DNA topoisomerase activity is due to HeLa topo I (Liu, L. F. and Miller, K. G. (1980) Proc. Natl. Acad. Sci. USA 78, 3489-3491). A similar association has been demonstrated from the in vitro studies using purified mononucleosomes and eukaryotic DNA topoisomerase I. Nonhistone HMG proteins and histone H1 are found to stimulate topoisomerase activity in vitro and form tight complexes with eukaryotic DNA topoisomerase I. The intimate interactions of topoisomerase I with chromosomal proteins and nucleosomes may be an essential feature of the topoisomerase function in vivo. Images PMID:6298726

  7. Crystal structure of the homology domain of the eukaryotic DNA replication proteins Sld3/Treslin.

    PubMed

    Itou, Hiroshi; Muramatsu, Sachiko; Shirakihara, Yasuo; Araki, Hiroyuki

    2014-09-02

    The initiation of eukaryotic chromosomal DNA replication requires the formation of an active replicative helicase at the replication origins of chromosomal DNA. Yeast Sld3 and its metazoan counterpart Treslin are the hub proteins mediating protein associations critical for the helicase formation. Here, we show the crystal structure of the central domain of Sld3 that is conserved in Sld3/Treslin family of proteins. The domain consists of two segments with 12 helices and is sufficient to bind to Cdc45, the essential helicase component. The structure model of the Sld3-Cdc45 complex, which is crucial for the formation of the active helicase, is proposed.

  8. AKAP-Lbc nucleates a protein kinase D activation scaffold.

    PubMed

    Carnegie, Graeme K; Smith, F Donelson; McConnachie, George; Langeberg, Lorene K; Scott, John D

    2004-09-24

    The transmission of cellular signals often proceeds through multiprotein complexes where enzymes are positioned in proximity to their upstream activators and downstream substrates. In this report we demonstrate that the A-kinase anchoring protein AKAP-Lbc assembles an activation complex for the lipid-dependent enzyme protein kinase D (PKD). Using a combination of biochemical, enzymatic, and immunofluorescence techniques, we show that the anchoring protein contributes to PKD activation in two ways: it recruits an upstream kinase PKCeta and coordinates PKA phosphorylation events that release activated protein kinase D. Thus, AKAP-Lbc synchronizes PKA and PKC activities in a manner that leads to the activation of a third kinase. This configuration illustrates the utility of kinase anchoring as a mechanism to constrain the action of broad-spectrum enzymes.

  9. Phylogenetic Diversity of NTT Nucleotide Transport Proteins in Free-Living and Parasitic Bacteria and Eukaryotes

    PubMed Central

    Major, Peter; Embley, T. Martin

    2017-01-01

    Plasma membrane-located nucleotide transport proteins (NTTs) underpin the lifestyle of important obligate intracellular bacterial and eukaryotic pathogens by importing energy and nucleotides from infected host cells that the pathogens can no longer make for themselves. As such their presence is often seen as a hallmark of an intracellular lifestyle associated with reductive genome evolution and loss of primary biosynthetic pathways. Here, we investigate the phylogenetic distribution of NTT sequences across the domains of cellular life. Our analysis reveals an unexpectedly broad distribution of NTT genes in both host-associated and free-living prokaryotes and eukaryotes. We also identify cases of within-bacteria and bacteria-to-eukaryote horizontal NTT transfer, including into the base of the oomycetes, a major clade of parasitic eukaryotes. In addition to identifying sequences that retain the canonical NTT structure, we detected NTT gene fusions with HEAT-repeat and cyclic nucleotide binding domains in Cyanobacteria, pathogenic Chlamydiae and Oomycetes. Our results suggest that NTTs are versatile functional modules with a much wider distribution and a broader range of potential roles than has previously been appreciated. PMID:28164241

  10. Insights into the evolution and domain structure of Ataxin-2 proteins across eukaryotes.

    PubMed

    Jiménez-López, Domingo; Guzmán, Plinio

    2014-07-15

    Ataxin-2 is an evolutionarily conserved protein first identified in humans as responsible for spinocerebellar ataxia type 2 (SCA2). The molecular basis of SCA2 is the expansion of a polyglutamine tract in Ataxin-2, encoding a Lsm domain that may bind RNA and a PAM2 motif that enables interaction with the poly (A) binding protein. Although the association with SCA2 has been verified, a detailed molecular function for Ataxin-2 has not been established. We have undertaken a survey of Ataxin-2 proteins across all eukaryotic domains. In eukaryotes, except for vertebrates and land plants, a single ortholog was identified. Notably, with the exception of birds, two Ataxin-2 genes exist in vertebrates. Expansion was observed in land plants and a novel class lacking the LsmAD domain was identified. Large polyQ tracts appear limited to primates and insects of the orders Hymenoptera and Diptera. A common feature across Ataxin-2 orthologs is the presence of proline-rich motifs, formerly described in the human protein. Our analysis provides valuable information on the evolution and domain structure of Ataxin-2 proteins. Proline-rich motifs that may mediate protein interactions are widespread in Ataxin-2 proteins, but expansion of polyglutamine tracts associated with spinocerebellar ataxia type 2, is present only in primates, as well as some insects. Our analysis of Ataxin-2 proteins provides also a source to examine orthologs in a number of different species.

  11. Insights into the evolution and domain structure of ataxin-2 proteins across eukaryotes

    PubMed Central

    2014-01-01

    Background Ataxin-2 is an evolutionarily conserved protein first identified in humans as responsible for spinocerebellar ataxia type 2 (SCA2). The molecular basis of SCA2 is the expansion of a polyglutamine tract in Ataxin-2, encoding a Lsm domain that may bind RNA and a PAM2 motif that enables interaction with the poly (A) binding protein. Although the association with SCA2 has been verified, a detailed molecular function for Ataxin-2 has not been established. Results We have undertaken a survey of Ataxin-2 proteins across all eukaryotic domains. In eukaryotes, except for vertebrates and land plants, a single ortholog was identified. Notably, with the exception of birds, two Ataxin-2 genes exist in vertebrates. Expansion was observed in land plants and a novel class lacking the LsmAD domain was identified. Large polyQ tracts appear limited to primates and insects of the orders Hymenoptera and Diptera. A common feature across Ataxin-2 orthologs is the presence of proline-rich motifs, formerly described in the human protein. Conclusion Our analysis provides valuable information on the evolution and domain structure of Ataxin-2 proteins. Proline-rich motifs that may mediate protein interactions are widespread in Ataxin-2 proteins, but expansion of polyglutamine tracts associated with spinocerebellar ataxia type 2, is present only in primates, as well as some insects. Our analysis of Ataxin-2 proteins provides also a source to examine orthologs in a number of different species. PMID:25027299

  12. Cryo-EM of dynamic protein complexes in eukaryotic DNA replication.

    PubMed

    Sun, Jingchuan; Yuan, Zuanning; Bai, Lin; Li, Huilin

    2017-01-01

    DNA replication in Eukaryotes is a highly dynamic process that involves several dozens of proteins. Some of these proteins form stable complexes that are amenable to high-resolution structure determination by cryo-EM, thanks to the recent advent of the direct electron detector and powerful image analysis algorithm. But many of these proteins associate only transiently and flexibly, precluding traditional biochemical purification. We found that direct mixing of the component proteins followed by 2D and 3D image sorting can capture some very weakly interacting complexes. Even at 2D average level and at low resolution, EM images of these flexible complexes can provide important biological insights. It is often necessary to positively identify the feature-of-interest in a low resolution EM structure. We found that systematically fusing or inserting maltose binding protein (MBP) to selected proteins is highly effective in these situations. In this chapter, we describe the EM studies of several protein complexes involved in the eukaryotic DNA replication over the past decade or so. We suggest that some of the approaches used in these studies may be applicable to structural analysis of other biological systems. © 2016 The Protein Society.

  13. Prediction of 492 human protein kinase substrate specificities.

    PubMed

    Safaei, Javad; Maňuch, Ján; Gupta, Arvind; Stacho, Ladislav; Pelech, Steven

    2011-10-14

    Complex intracellular signaling networks monitor diverse environmental inputs to evoke appropriate and coordinated effector responses. Defective signal transduction underlies many pathologies, including cancer, diabetes, autoimmunity and about 400 other human diseases. Therefore, there is high impetus to define the composition and architecture of cellular communications networks in humans. The major components of intracellular signaling networks are protein kinases and protein phosphatases, which catalyze the reversible phosphorylation of proteins. Here, we have focused on identification of kinase-substrate interactions through prediction of the phosphorylation site specificity from knowledge of the primary amino acid sequence of the catalytic domain of each kinase. The presented method predicts 488 different kinase catalytic domain substrate specificity matrices in 478 typical and 4 atypical human kinases that rely on both positive and negative determinants for scoring individual phosphosites for their suitability as kinase substrates. This represents a marked advancement over existing methods such as those used in NetPhorest (179 kinases in 76 groups) and NetworKIN (123 kinases), which consider only positive determinants for kinase substrate prediction. Comparison of our predicted matrices with experimentally-derived matrices from about 9,000 known kinase-phosphosite substrate pairs revealed a high degree of concordance with the established preferences of about 150 well studied protein kinases. Furthermore for many of the better known kinases, the predicted optimal phosphosite sequences were more accurate than the consensus phosphosite sequences inferred by simple alignment of the phosphosites of known kinase substrates. Application of this improved kinase substrate prediction algorithm to the primary structures of over 23, 000 proteins encoded by the human genome has permitted the identification of about 650, 000 putative phosphosites, which are posted on the

  14. Eukaryotic RNase P RNA mediates cleavage in the absence of protein.

    PubMed

    Kikovska, Ema; Svärd, Staffan G; Kirsebom, Leif A

    2007-02-13

    The universally conserved ribonucleoprotein RNase P is involved in the processing of tRNA precursor transcripts. RNase P consists of one RNA and, depending on its origin, a variable number of protein subunits. Catalytic activity of the RNA moiety so far has been demonstrated only for bacterial and some archaeal RNase P RNAs but not for their eukaryotic counterparts. Here, we show that RNase P RNAs from humans and the lower eukaryote Giardia lamblia mediate cleavage of four tRNA precursors and a model RNA hairpin loop substrate in the absence of protein. Compared with bacterial RNase P RNA, the rate of cleavage (k(obs)) was five to six orders of magnitude lower, whereas the affinity for the substrate (appK(d)) was reduced approximately 20- to 50-fold. We conclude that the RNA-based catalytic activity of RNase P has been preserved during evolution. This finding opens previously undescribed ways to study the role of the different proteins subunits of eukaryotic RNase P.

  15. Bacterial SET domain proteins and their role in eukaryotic chromatin modification

    PubMed Central

    Alvarez-Venegas, Raúl

    2014-01-01

    It has been shown by many researchers that SET-domain containing proteins modify chromatin structure and, as expected, genes coding for SET-domain containing proteins have been found in all eukaryotic genomes sequenced to date. However, during the last years, a great number of bacterial genomes have been sequenced and an important number of putative genes involved in histone post-translational modifications (histone PTMs) have been identified in many bacterial genomes. Here, I aim at presenting an overview of SET domain genes that have been identified in numbers of bacterial genomes based on similarity to SET domains of eukaryotic histone methyltransferases. I will argue in favor of the hypothesis that SET domain genes found in extant bacteria are of bacterial origin. Then, I will focus on the available information on pathogen and symbiont SET-domain containing proteins and their targets in eukaryotic organisms, and how such histone methyltransferases allow a pathogen to inhibit transcriptional activation of host defense genes. PMID:24765100

  16. Structural and Functional Characterization of the Protein Kinase Mps1 in Arabidopsis thaliana

    PubMed Central

    de Oliveira, Eduardo Alves Gamosa; Romeiro, Nelilma Correia; Ribeiro, Elane da Silva; Santa-Catarina, Claudete; Oliveira, Antônia Elenir Amâncio; Silveira, Vanildo; de Souza Filho, Gonçalo Apolinário; Venancio, Thiago Motta; Cruz, Marco Antônio Lopes

    2012-01-01

    In eukaryotes, protein kinases catalyze the transfer of a gamma-phosphate from ATP (or GTP) to specific amino acids in protein targets. In plants, protein kinases have been shown to participate in signaling cascades driving responses to environmental stimuli and developmental processes. Plant meristems are undifferentiated tissues that provide the major source of cells that will form organs throughout development. However, non-dividing specialized cells can also dedifferentiate and re-initiate cell division if exposed to appropriate conditions. Mps1 (Monopolar spindle) is a dual-specificity protein kinase that plays a critical role in monitoring the accuracy of chromosome segregation in the mitotic checkpoint mechanism. Although Mps1 functions have been clearly demonstrated in animals and fungi, its role in plants is so far unclear. Here, using structural and biochemical analyses here we show that Mps1 has highly similar homologs in many plant genomes across distinct lineages (e.g. AtMps1 in Arabidopsis thaliana). Several structural features (i.e. catalytic site, DFG motif and threonine triad) are clearly conserved in plant Mps1 kinases. Structural and sequence analysis also suggest that AtMps1 interact with other cell cycle proteins, such as Mad2 and MAPK1. By using a very specific Mps1 inhibitor (SP600125) we show that compromised AtMps1 activity hampers the development of A. thaliana seedlings in a dose-dependent manner, especially in secondary roots. Moreover, concomitant administration of the auxin IAA neutralizes the AtMps1 inhibition phenotype, allowing secondary root development. These observations let us to hypothesize that AtMps1 might be a downstream regulator of IAA signaling in the formation of secondary roots. Our results indicate that Mps1 might be a universal component of the Spindle Assembly Checkpoint machinery across very distant lineages of eukaryotes. PMID:23049844

  17. Protein-protein interactions of tandem affinity purification-tagged protein kinases in rice.

    PubMed

    Rohila, Jai S; Chen, Mei; Chen, Shuo; Chen, Johann; Cerny, Ronald; Dardick, Chris; Canlas, Patrick; Xu, Xia; Gribskov, Michael; Kanrar, Siddhartha; Zhu, Jian-Kang; Ronald, Pamela; Fromm, Michael E

    2006-04-01

    Forty-one rice cDNAs encoding protein kinases were fused to the tandem affinity purification (TAP) tag and expressed in transgenic rice plants. The TAP-tagged kinases and interacting proteins were purified from the T1 progeny of the transgenic rice plants and identified by mass spectrometry. Ninety-five percent of the TAP-tagged kinases were recovered. Fifty-six percent of the TAP-tagged kinases were found to interact with other rice proteins. A number of these interactions were consistent with known protein complexes found in other species, validating the TAP-tag method in rice plants. Phosphorylation sites were identified on four of the kinases that interacted with either 14-3-3 proteins or cyclins.

  18. Reusable amperometric biosensor for measuring protein tyrosine kinase activity.

    PubMed

    Wang, Chung-Liang; Wei, Lan-Yi; Yuan, Chiun-Jye; Hwang, Kuo Chu

    2012-01-17

    This work presents a simple, low-cost and reusable label-free method for detecting protein tyrosine kinase activity using a tyrosinase-based amperometric biosensor (tyrosine kinase biosensor). This method is based on the observation that phosphorylation can block the tyrosinase-catalyzed oxidation of tyrosine or tyrosyl residue in peptides. Therefore, the activity of p60c-src protein tyrosine kinase (Src) on the developed tyrosine kinase biosensor could be quickly determined when its specific peptide substrate, p60c-src substrate I, was used. The tyrosine kinase biosensor was highly sensitive to the activity of Src with a linear dynamic range of 1.9-237.6 U/mL and the lowest detection limit of 0.23 U/mL. Interestingly, the tyrosine kinase activity can be measured using the developed tyrosine kinase biosensor repetitively without regeneration. The inhibitory effect of various kinase inhibitors on the Src activity could be determined on the tyrosine kinase biosensor. Src-specific inhibitors, PP2 and Src inhibitor I, effectively suppressed Src activity, whereas PD153035, an inhibitor of the epidermal growth factor receptor, was ineffective. Staurosporine, a universal kinase inhibitor, inhibited Src activity in an ATP concentration-dependent manner. These results suggests that the activities of tyrosine kinases and their behaviors toward various reagents can be effectively measured using the developed tyrosine kinase biosensor.

  19. Comprehensive Characterization of AMP-Activated Protein Kinase Catalytic Domain by Top-Down Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Yu, Deyang; Peng, Ying; Ayaz-Guner, Serife; Gregorich, Zachery R.; Ge, Ying

    2016-02-01

    AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that is essential in regulating energy metabolism in all eukaryotic cells. It is a heterotrimeric protein complex composed of a catalytic subunit (α) and two regulatory subunits (β and γ). C-terminal truncation of AMPKα at residue 312 yielded a protein that is active upon phosphorylation of Thr172 in the absence of β and γ subunits, which is refered to as the AMPK catalytic domain and commonly used to substitute for the AMPK heterotrimeric complex in in vitro kinase assays. However, a comprehensive characterization of the AMPK catalytic domain is lacking. Herein, we expressed a His-tagged human AMPK catalytic domin (denoted as AMPKΔ) in E. coli, comprehensively characterized AMPKΔ in its basal state and after in vitro phosphorylation using top-down mass spectrometry (MS), and assessed how phosphorylation of AMPKΔ affects its activity. Unexpectedly, we found that bacterially-expressed AMPKΔ was basally phosphorylated and localized the phosphorylation site to the His-tag. We found that AMPKΔ had noticeable basal activity and was capable of phosphorylating itself and its substrates without activating phosphorylation at Thr172. Moreover, our data suggested that Thr172 is the only site phosphorylated by its upstream kinase, liver kinase B1, and that this phosphorylation dramatically increases the kinase activity of AMPKΔ. Importantly, we demonstrated that top-down MS in conjunction with in vitro phosphorylation assay is a powerful approach for monitoring phosphorylation reaction and determining sequential order of phosphorylation events in kinase-substrate systems.

  20. Comprehensive Characterization of AMP-activated Protein Kinase Catalytic Domain by Top-down Mass Spectrometry

    PubMed Central

    Yu, Deyang; Peng, Ying; Ayaz-Guner, Serife; Gregorich, Zachery R.; Ge, Ying

    2015-01-01

    AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that is essential in regulating energy metabolism in all eukaryotic cells. It is a heterotrimeric protein complex composed of a catalytic subunit (α) and two regulatory subunits (β and γ. C-terminal truncation of AMPKα at residue 312 yielded a protein that is active upon phosphorylation of Thr172 in the absence of β and γ subunits, which is refered to as the AMPK catalytic domain and commonly used to substitute for the AMPK heterotrimeric complex in in vitro kinase assays. However, a comprehensive characterization of the AMPK catalytic domain is lacking. Herein, we expressed a His-tagged human AMPK catalytic domin (denoted as AMPKΔ) in E. coli, comprehensively characterized AMPKΔ in its basal state and after in vitro phosphorylation using top-down mass spectrometry (MS), and assessed how phosphorylation of AMPKΔ affects its activity. Unexpectedly, we found that bacterially-expressed AMPKΔ was basally phosphorylated and localized the phosphorylation site to the His-tag. We found that AMPKΔ has noticeable basal activity and was capable of phosphorylating itself and its substrates without activating phosphorylation at Thr172. Moreover, our data suggested that Thr172 is the only site phosphorylated by its upstream kinase, liver kinase B1, and that this phosphorylation dramatically increases the kinase activity of AMPKΔ. Importantly, we demonstrated that top-down MS in conjunction with in vitro phosphorylation assay is a powerful approach for monitoring phosphorylation reaction and determining sequential order of phosphorylation events in kinase-substrate systems. PMID:26489410

  1. Prostaglandin E2 negatively regulates AMP-activated protein kinase via protein kinase A signaling pathway.

    PubMed

    Funahashi, Koji; Cao, Xia; Yamauchi, Masako; Kozaki, Yasuko; Ishiguro, Naoki; Kambe, Fukushi

    2009-01-01

    We investigated possible involvement of prostaglandin (PG) E2 in regulation of AMP-activated protein kinase (AMPK). When osteoblastic MG63 cells were cultured in serum-deprived media, Thr-172 phosphorylation of AMPK alpha-subunit was markedly increased. Treatment of the cells with PGE2 significantly reduced the phosphorylation. Ser-79 phosphorylation of acetyl-CoA carboxylase, a direct target for AMPK, was also reduced by PGE2. On the other hand, PGE2 reciprocally increased Ser-485 phosphorylation of the alpha-subunit that could be associated with inhibition of AMPK activity. These effects of PGE2 were mimicked by PGE2 receptor EP2 and EP4 agonists and forskolin, but not by EP1 and EP3 agonists, and the effects were suppressed by an adenylate cyclase inhibitor SQ22536 and a protein kinase A inhibitor H89. Additionally, the PGE2 effects were duplicated in primary calvarial osteoblasts. Together, the present study demonstrates that PGE2 negatively regulates AMPK activity via activation of protein kinase A signaling pathway.

  2. Protein Kinase A Subunit α Catalytic and A Kinase Anchoring Protein 79 in Human Placental Mitochondria.

    PubMed

    Ma, Maggie Pui Chi; Thomson, Murray

    2012-01-01

    Components of protein phosphorylation signalling systems have been discovered in mitochondria and it has been proposed that these molecules modulate processes including oxidative phosphorylation, apoptosis and steroidogenesis. We used electrophoresis and Western blots probed with specific antibodies to protein kinase A α catalytic subunit (PKAα Cat) and A kinase anchoring protein of approximately 79 kDa molecular weight (AKAP79) to demonstrate the presence of these two proteins in human placental mitochondria. Heavy mitochondria characteristic of cytotrophoblast were separated from light mitochondria characteristic of syncytiotrophoblast by centrifugation. PKAα Cat and AKAP79 were present in both heavy and light mitochondria with no significant difference in concentration. Sucrose density gradient separation of submitochondrial fractions indicated PKAα Cat is located predominantly in the outer membrane whereas AKAP79 is present mainly in the contact site fractions. These data indicate that PKAα Cat is present in the cytoplasm, nucleus and mitochondria of placental cells. AKAP79 is also present in human placental mitochondria but there may be anchoring proteins other than AKAP79 responsible for fixing PKA to the outer membrane. PKA may play roles in mitochondrial protein phosphorylation systems in both cytotrophoblast and syncytiotrophoblast.

  3. A-kinase anchoring proteins: scaffolding proteins in the heart

    PubMed Central

    Dodge-Kafka, Kimberly L.; Li, Jinliang; Kapiloff, Michael S.

    2011-01-01

    The pleiotropic cyclic nucleotide cAMP is the primary second messenger responsible for autonomic regulation of cardiac inotropy, chronotropy, and lusitropy. Under conditions of prolonged catecholaminergic stimulation, cAMP also contributes to the induction of both cardiac myocyte hypertrophy and apoptosis. The formation of localized, multiprotein complexes that contain different combinations of cAMP effectors and regulatory enzymes provides the architectural infrastructure for the specialization of the cAMP signaling network. Scaffolds that bind protein kinase A are called “A-kinase anchoring proteins” (AKAPs). In this review, we discuss recent advances in our understanding of how PKA is compartmentalized within the cardiac myocyte by AKAPs and how AKAP complexes modulate cardiac function in both health and disease. PMID:21856912

  4. Methylglyoxal Activates the Target of Rapamycin Complex 2-Protein Kinase C Signaling Pathway in Saccharomyces cerevisiae

    PubMed Central

    Nomura, Wataru

    2015-01-01

    Methylglyoxal is a typical 2-oxoaldehyde derived from glycolysis. We show here that methylglyoxal activates the Pkc1-Mpk1 mitogen-activated protein (MAP) kinase cascade in a target of rapamycin complex 2 (TORC2)-dependent manner in the budding yeast Saccharomyces cerevisiae. We demonstrate that TORC2 phosphorylates Pkc1 at Thr1125 and Ser1143. Methylglyoxal enhanced the phosphorylation of Pkc1 at Ser1143, which transmitted the signal to the downstream Mpk1 MAP kinase cascade. We found that the phosphorylation status of Pkc1T1125 affected the phosphorylation of Pkc1 at Ser1143, in addition to its protein levels. Methylglyoxal activated mammalian TORC2 signaling, which, in turn, phosphorylated Akt at Ser473. Our results suggest that methylglyoxal is a conserved initiator of TORC2 signaling among eukaryotes. PMID:25624345

  5. Mitogen-activated protein kinase cascades in signaling plant growth and development.

    PubMed

    Xu, Juan; Zhang, Shuqun

    2015-01-01

    Mitogen-activated protein kinase (MAPK) cascades are ubiquitous signaling modules in eukaryotes. Early research of plant MAPKs has been focused on their functions in immunity and stress responses. Recent studies reveal that they also play essential roles in plant growth and development downstream of receptor-like protein kinases (RLKs). With only a limited number of MAPK components, multiple functional pathways initiated from different receptors often share the same MAPK components or even a complete MAPK cascade. In this review, we discuss how MAPK cascades function as molecular switches in response to spatiotemporal-specific ligand-receptor interactions and the availability of downstream substrates. In addition, we discuss other possible mechanisms governing the functional specificity of plant MAPK cascades, a question central to our understanding of MAPK functions.

  6. Methylglyoxal activates the target of rapamycin complex 2-protein kinase C signaling pathway in Saccharomyces cerevisiae.

    PubMed

    Nomura, Wataru; Inoue, Yoshiharu

    2015-04-01

    Methylglyoxal is a typical 2-oxoaldehyde derived from glycolysis. We show here that methylglyoxal activates the Pkc1-Mpk1 mitogen-activated protein (MAP) kinase cascade in a target of rapamycin complex 2 (TORC2)-dependent manner in the budding yeast Saccharomyces cerevisiae. We demonstrate that TORC2 phosphorylates Pkc1 at Thr(1125) and Ser(1143). Methylglyoxal enhanced the phosphorylation of Pkc1 at Ser(1143), which transmitted the signal to the downstream Mpk1 MAP kinase cascade. We found that the phosphorylation status of Pkc1(T1125) affected the phosphorylation of Pkc1 at Ser(1143), in addition to its protein levels. Methylglyoxal activated mammalian TORC2 signaling, which, in turn, phosphorylated Akt at Ser(473). Our results suggest that methylglyoxal is a conserved initiator of TORC2 signaling among eukaryotes.

  7. Replicative Functions of Minute Virus of Mice NS1 Protein Are Regulated In Vitro by Phosphorylation through Protein Kinase C

    PubMed Central

    Nüesch, Jürg P. F.; Dettwiler, Sabine; Corbau, Romuald; Rommelaere, Jean

    1998-01-01

    NS1, the major nonstructural protein of the parvovirus minute virus of mice, is a multifunctional phosphoprotein which is involved in cytotoxicity, transcriptional regulation, and initiation of viral DNA replication. For coordination of these various functions during virus propagation, NS1 has been proposed to be regulated by posttranslational modifications, in particular phosphorylation. Recent in vitro studies (J. P. F. Nüesch, R. Corbau, P. Tattersall, and J. Rommelaere, J. Virol. 72:8002–8012, 1998) provided evidence that distinct NS1 activities, notably the intrinsic helicase function, are modulated by the phosphorylation state of the protein. In order to study the dependence of the initiation of viral DNA replication on NS1 phosphorylation and to identify the protein kinases involved, we established an in vitro replication system that is devoid of endogenous protein kinases and is based on plasmid substrates containing the minimal left-end origins of replication. Cellular components necessary to drive NS1-dependent rolling-circle replication (RCR) were freed from endogenous serine/threonine protein kinases by affinity chromatography, and the eukaryotic DNA polymerases were replaced by the bacteriophage T4 DNA polymerase. While native NS1 (NS1P) supported RCR under these conditions, dephosphorylated NS1 (NS1O) was impaired. Using fractionated HeLa cell extracts, we identified two essential protein components which are able to phosphorylate NS1O, are enriched in protein kinase C (PKC), and, when present together, reactivate NS1O for replication. One of these components, containing atypical PKC, was sufficient to restore NS1O helicase activity. The requirement of NS1O reactivation for characteristic PKC cofactors such as Ca2+/phosphatidylserine or phorbol esters strongly suggests the involvement of this protein kinase family in regulation of NS1 replicative functions in vitro. PMID:9811734

  8. DNA unwinding proteins of an eukaryotic organism. Final report, July 1977-April 1981

    SciTech Connect

    Patel, G.L.

    1982-06-01

    A rat liver protein, which preferentially binds in vitro to single-stranded DNA, and destabilizes the DNA double helix to promote its unwinding or melting was investigated. Proteins of this class isolated from viral, prokaryotic, and eukaryotic sources have been referred to as DNA melting, unwinding, helix-destabilizing, or single-strand binding proteins. The rat liver protein that we have purified and studied here is referred to as a helix-destabilizing protein (HDP). The specific objectives proposed were to: (1) purify and characterize physico-chemically one or more rat liver HDP; (2) prepare specific anti-HDP sera for immunological approaches; (3) characterize the HDP interaction with DNA; and (4) study the HDP metabolism in normal and regenerating liver. A novel conceptual contribution was made with regards to the biological roles of this class of proteins. Activities and accomplishments during this entire contract period are summarized. (ERB)

  9. Protein Kinase Activity of Phosphoinositide 3-Kinase Regulates Cytokine-Dependent Cell Survival

    PubMed Central

    Green, Benjamin D.; Barry, Emma F.; Ma, Yuefang; Woodcock, Joanna; Fitter, Stephen; Zannettino, Andrew C. W.; Pitson, Stuart M.; Hughes, Timothy P.; Lopez, Angel F.; Shepherd, Peter R.; Wei, Andrew H.; Ekert, Paul G.; Guthridge, Mark A.

    2013-01-01

    The dual specificity protein/lipid kinase, phosphoinositide 3-kinase (PI3K), promotes growth factor-mediated cell survival and is frequently deregulated in cancer. However, in contrast to canonical lipid-kinase functions, the role of PI3K protein kinase activity in regulating cell survival is unknown. We have employed a novel approach to purify and pharmacologically profile protein kinases from primary human acute myeloid leukemia (AML) cells that phosphorylate serine residues in the cytoplasmic portion of cytokine receptors to promote hemopoietic cell survival. We have isolated a kinase activity that is able to directly phosphorylate Ser585 in the cytoplasmic domain of the interleukin 3 (IL-3) and granulocyte macrophage colony stimulating factor (GM-CSF) receptors and shown it to be PI3K. Physiological concentrations of cytokine in the picomolar range were sufficient for activating the protein kinase activity of PI3K leading to Ser585 phosphorylation and hemopoietic cell survival but did not activate PI3K lipid kinase signaling or promote proliferation. Blockade of PI3K lipid signaling by expression of the pleckstrin homology of Akt1 had no significant impact on the ability of picomolar concentrations of cytokine to promote hemopoietic cell survival. Furthermore, inducible expression of a mutant form of PI3K that is defective in lipid kinase activity but retains protein kinase activity was able to promote Ser585 phosphorylation and hemopoietic cell survival in the absence of cytokine. Blockade of p110α by RNA interference or multiple independent PI3K inhibitors not only blocked Ser585 phosphorylation in cytokine-dependent cells and primary human AML blasts, but also resulted in a block in survival signaling and cell death. Our findings demonstrate a new role for the protein kinase activity of PI3K in phosphorylating the cytoplasmic tail of the GM-CSF and IL-3 receptors to selectively regulate cell survival highlighting the importance of targeting such pathways in

  10. CDPKs are dual-specificity protein kinases and tyrosine autophosphorylation attenuates kinase activity.

    PubMed

    Oh, Man-Ho; Wu, Xia; Kim, Hyoung Seok; Harper, Jeffrey F; Zielinski, Raymond E; Clouse, Steven D; Huber, Steven C

    2012-11-30

    Although calcium-dependent protein kinases (CDPKs or CPKs) are classified as serine/threonine protein kinases, autophosphorylation on tyrosine residues was observed for soybean CDPKβ and several Arabidopsis isoforms (AtCPK4 and AtCPK34). We identified Ser-8, Thr-17, Tyr-24 (in the kinase domain), Ser-304, and Ser-358 as autophosphorylation sites of His(6)-GmCDPKβ. Overall autophosphorylation increased kinase activity with synthetic peptides, but autophosphorylation of Tyr-24 appears to attenuate kinase activity based on studies with the Y24F directed mutant. While much remains to be done, it is clear that several CDPKs are dual-specificity kinases, which raises the possibility that phosphotyrosine signaling may play a role in Ca(2+)/CDPK-mediated processes. Published by Elsevier B.V.

  11. Rac-1 and Raf-1 kinases, components of distinct signaling pathways, activate myotonic dystrophy protein kinase

    NASA Technical Reports Server (NTRS)

    Shimizu, M.; Wang, W.; Walch, E. T.; Dunne, P. W.; Epstein, H. F.

    2000-01-01

    Myotonic dystrophy protein kinase (DMPK) is a serine-threonine protein kinase encoded by the myotonic dystrophy (DM) locus on human chromosome 19q13.3. It is a close relative of other kinases that interact with members of the Rho family of small GTPases. We show here that the actin cytoskeleton-linked GTPase Rac-1 binds to DMPK, and coexpression of Rac-1 and DMPK activates its transphosphorylation activity in a GTP-sensitive manner. DMPK can also bind Raf-1 kinase, the Ras-activated molecule of the MAP kinase pathway. Purified Raf-1 kinase phosphorylates and activates DMPK. The interaction of DMPK with these distinct signals suggests that it may play a role as a nexus for cross-talk between their respective pathways and may partially explain the remarkable pleiotropy of DM.

  12. The Origin and Evolution of G Protein-Coupled Receptor Kinases

    PubMed Central

    Mushegian, Arcady; Gurevich, Vsevolod V.; Gurevich, Eugenia V.

    2012-01-01

    G protein-coupled receptor (GPCR) kinases (GRKs) play key role in homologous desensitization of GPCRs. GRKs phosphorylate activated receptors, promoting high affinity binding of arrestins, which precludes G protein coupling. Direct binding to active GPCRs activates GRKs, so that they selectively phosphorylate only the activated form of the receptor regardless of the accessibility of the substrate peptides within it and their Ser/Thr-containing sequence. Mammalian GRKs were classified into three main lineages, but earlier GRK evolution has not been studied. Here we show that GRKs emerged at the early stages of eukaryotic evolution via an insertion of a kinase similar to ribosomal protein S6 kinase into a loop in RGS domain. GRKs in Metazoa fall into two clades, one including GRK2 and GRK3, and the other consisting of all remaining GRKs, split into GRK1-GRK7 lineage and GRK4-GRK5-GRK6 lineage in vertebrates. One representative of each of the two ancient clades is found as early as placozoan Trichoplax adhaerens. Several protists, two oomycetes and unicellular brown algae have one GRK-like protein, suggesting that the insertion of a kinase domain into the RGS domain preceded the origin of Metazoa. The two GRK families acquired distinct structural units in the N- and C-termini responsible for membrane recruitment and receptor association. Thus, GRKs apparently emerged before animals and rapidly expanded in true Metazoa, most likely due to the need for rapid signalling adjustments in fast-moving animals. PMID:22442725

  13. Resveratrol Inhibits Cardiac Hypertrophy via AMP-activated Protein Kinase and Akt*

    PubMed Central

    Chan, Anita Y. M.; Dolinsky, Vernon W.; Soltys, Carrie-Lynn M.; Viollet, Benoit; Baksh, Shairaz; Light, Peter E.; Dyck, Jason R. B.

    2008-01-01

    Whereas studies involving animal models of cardiovascular disease demonstrated that resveratrol is able to inhibit hypertrophic growth, the mechanisms involved have not been elucidated. Because studies in cells other than cardiomyocytes revealed that AMP-activated protein kinase (AMPK) and Akt are affected by resveratrol, we hypothesized that resveratrol prevents cardiac myocyte hypertrophy via these two kinase systems. Herein, we demonstrate that resveratrol reduces phenylephrine-induced protein synthesis and cell growth in rat cardiac myocytes via alterations of intracellular pathways involved in controlling protein synthesis (p70S6 kinase and eukaryotic elongation factor-2). Additionally, we demonstrate that resveratrol negatively regulates the calcineurin-nuclear factor of activated T cells pathway thus modifying a critical component of the transcriptional mechanism involved in pathological cardiac hypertrophy. Our data also indicate that these effects of resveratrol are mediated via AMPK activation and Akt inhibition, and in the case of AMPK, is dependent on the presence of the AMPK kinase, LKB1. Taken together, our data suggest that resveratrol exerts anti-hypertrophic effects by activating AMPK via LKB1 and inhibiting Akt, thus suppressing protein synthesis and gene transcription. PMID:18562309

  14. Resveratrol inhibits cardiac hypertrophy via AMP-activated protein kinase and Akt.

    PubMed

    Chan, Anita Y M; Dolinsky, Vernon W; Soltys, Carrie-Lynn M; Viollet, Benoit; Baksh, Shairaz; Light, Peter E; Dyck, Jason R B

    2008-08-29

    Whereas studies involving animal models of cardiovascular disease demonstrated that resveratrol is able to inhibit hypertrophic growth, the mechanisms involved have not been elucidated. Because studies in cells other than cardiomyocytes revealed that AMP-activated protein kinase (AMPK) and Akt are affected by resveratrol, we hypothesized that resveratrol prevents cardiac myocyte hypertrophy via these two kinase systems. Herein, we demonstrate that resveratrol reduces phenylephrine-induced protein synthesis and cell growth in rat cardiac myocytes via alterations of intracellular pathways involved in controlling protein synthesis (p70S6 kinase and eukaryotic elongation factor-2). Additionally, we demonstrate that resveratrol negatively regulates the calcineurin-nuclear factor of activated T cells pathway thus modifying a critical component of the transcriptional mechanism involved in pathological cardiac hypertrophy. Our data also indicate that these effects of resveratrol are mediated via AMPK activation and Akt inhibition, and in the case of AMPK, is dependent on the presence of the AMPK kinase, LKB1. Taken together, our data suggest that resveratrol exerts anti-hypertrophic effects by activating AMPK via LKB1 and inhibiting Akt, thus suppressing protein synthesis and gene transcription.

  15. Association of protein kinase Cmu with type II phosphatidylinositol 4-kinase and type I phosphatidylinositol-4-phosphate 5-kinase.

    PubMed

    Nishikawa, K; Toker, A; Wong, K; Marignani, P A; Johannes, F J; Cantley, L C

    1998-09-04

    Protein kinase Cmu (PKCmu), also named protein kinase D, is an unusual member of the PKC family that has a putative transmembrane domain and pleckstrin homology domain. This enzyme has a substrate specificity distinct from other PKC isoforms (Nishikawa, K., Toker, A., Johannes, F. J., Songyang, Z., and Cantley, L. C. (1997) J. Biol. Chem. 272, 952-960), and its mechanism of regulation is not yet clear. Here we show that PKCmu forms a complex in vivo with a phosphatidylinositol 4-kinase and a phosphatidylinositol-4-phosphate 5-kinase. A region of PKCmu between the amino-terminal transmembrane domain and the pleckstrin homology domain is shown to be involved in the association with the lipid kinases. Interestingly, a kinase-dead point mutant of PKCmu failed to associate with either lipid kinase activity, indicating that autophosphorylation may be required to expose the lipid kinase interaction domain. Furthermore, the subcellular distribution of the PKCmu-associated lipid kinases to the particulate fraction depends on the presence of the amino-terminal region of PKCmu including the predicted transmembrane region. These results suggest a novel model in which the non-catalytic region of PKCmu acts as a scaffold for assembly of enzymes involved in phosphoinositide synthesis at specific membrane locations.

  16. Prefabrication of a ribosomal protein subcomplex essential for eukaryotic ribosome formation

    PubMed Central

    Peña, Cohue; Schütz, Sabina; Fischer, Ute; Chang, Yiming; Panse, Vikram G

    2016-01-01

    Spatial clustering of ribosomal proteins (r-proteins) through tertiary interactions is a striking structural feature of the eukaryotic ribosome. However, the functional importance of these intricate inter-connections, and how they are established is currently unclear. Here, we reveal that a conserved ATPase, Fap7, organizes interactions between neighboring r-proteins uS11 and eS26 prior to their delivery to the earliest ribosome precursor, the 90S. In vitro, uS11 only when bound to Fap7 becomes competent to recruit eS26 through tertiary contacts found between these r-proteins on the mature ribosome. Subsequently, Fap7 ATPase activity unloads the uS11:eS26 subcomplex onto its rRNA binding site, and therefore ensures stoichiometric integration of these r-proteins into the 90S. Fap7-depletion in vivo renders uS11 susceptible to proteolysis, and precludes eS26 incorporation into the 90S. Thus, prefabrication of a native-like r-protein subcomplex drives efficient and accurate construction of the eukaryotic ribosome. DOI: http://dx.doi.org/10.7554/eLife.21755.001 PMID:27929371

  17. Isotope labeling of eukaryotic membrane proteins in yeast for solid-state NMR.

    PubMed

    Fan, Ying; Emami, Sanaz; Munro, Rachel; Ladizhansky, Vladimir; Brown, Leonid S

    2015-01-01

    Solid-state NMR (ssNMR) is a rapidly developing technique for exploring structure and dynamics of membrane proteins, but its progress is hampered by its low sensitivity. Despite the latest technological advances, routine ssNMR experiments still require several milligrams of isotopically labeled protein. While production of bacterial membrane proteins on this scale is usually feasible, obtaining such quantities of eukaryotic membrane proteins is often impossible or extremely costly. We have demonstrated that, by using isotopic labeling in yeast Pichia pastoris, one can inexpensively produce milligram quantities of doubly labeled functional samples, which yield multidimensional ssNMR spectra of high resolution suitable for detailed structural investigation. This was achieved by combining protocols of economical isotope labeling of soluble proteins previously used for solution NMR with protocols of expression of eukaryotic membrane proteins successfully employed for other methods. We review two cases of such isotope labeling, of fungal rhodopsin from Leptosphaeria maculans and human aquaporin-1. © 2015 Elsevier Inc. All rights reserved.

  18. Protein kinase C phosphorylates AMP-activated protein kinase α1 Ser487

    PubMed Central

    Heathcote, Helen R.; Mancini, Sarah J.; Strembitska, Anastasiya; Jamal, Kunzah; Reihill, James A.; Palmer, Timothy M.; Gould, Gwyn W.; Salt, Ian P.

    2016-01-01

    The key metabolic regulator, AMP-activated protein kinase (AMPK), is reported to be down-regulated in metabolic disorders, but the mechanisms are poorly characterised. Recent studies have identified phosphorylation of the AMPKα1/α2 catalytic subunit isoforms at Ser487/491, respectively, as an inhibitory regulation mechanism. Vascular endothelial growth factor (VEGF) stimulates AMPK and protein kinase B (Akt) in cultured human endothelial cells. As Akt has been demonstrated to be an AMPKα1 Ser487 kinase, the effect of VEGF on inhibitory AMPK phosphorylation in cultured primary human endothelial cells was examined. Stimulation of endothelial cells with VEGF rapidly increased AMPKα1 Ser487 phosphorylation in an Akt-independent manner, without altering AMPKα2 Ser491 phosphorylation. In contrast, VEGF-stimulated AMPKα1 Ser487 phosphorylation was sensitive to inhibitors of protein kinase C (PKC) and PKC activation using phorbol esters or overexpression of PKC-stimulated AMPKα1 Ser487 phosphorylation. Purified PKC and Akt both phosphorylated AMPKα1 Ser487 in vitro with similar efficiency. PKC activation was associated with reduced AMPK activity, as inhibition of PKC increased AMPK activity and phorbol esters inhibited AMPK, an effect lost in cells expressing mutant AMPKα1 Ser487Ala. Consistent with a pathophysiological role for this modification, AMPKα1 Ser487 phosphorylation was inversely correlated with insulin sensitivity in human muscle. These data indicate a novel regulatory role of PKC to inhibit AMPKα1 in human cells. As PKC activation is associated with insulin resistance and obesity, PKC may underlie the reduced AMPK activity reported in response to overnutrition in insulin-resistant metabolic and vascular tissues. PMID:27784766

  19. The shape of things to come: an emerging role for protein kinase CK2 in the regulation of cell morphology and the cytoskeleton.

    PubMed

    Canton, David A; Litchfield, David W

    2006-03-01

    Protein kinase CK2 is a highly conserved, pleiotropic, protein serine/threonine kinase that is essential for life in eukaryotes. CK2 has been implicated in diverse cellular processes such as cell cycle regulation, circadian rhythms, apoptosis, transformation and tumorigenesis. In addition, there is increasing evidence that CK2 is involved in the maintenance of cell morphology and cell polarity, and in the regulation of the actin and tubulin cytoskeletons. Accordingly, this review will highlight published evidence in experimental models ranging from yeast to mammals documenting the emerging roles of protein kinase CK2 in the regulation of cell polarity, cell morphology and the cytoskeleton.

  20. Transphosphorylation of E. coli proteins during production of recombinant protein kinases provides a robust system to characterize kinase specificity

    USDA-ARS?s Scientific Manuscript database

    Protein kinase specificity is of fundamental importance to pathway regulation and signal transduction. Here, we report a convenient system to monitor the activity and specificity of recombinant protein kinases expressed in E.coli. We apply this to the study of the cytoplasmic domain of the plant rec...

  1. Protein domains correlate strongly with exons in multiple eukaryotic genomes--evidence of exon shuffling?

    PubMed

    Liu, Mingyi; Grigoriev, Andrei

    2004-09-01

    We conducted a multi-genome analysis correlating protein domain organization with the exon-intron structure of genes in nine eukaryotic genomes. We observed a significant correlation between the borders of exons and domains on a genomic scale for both invertebrates and vertebrates. In addition, we found that the more complex organisms displayed consistently stronger exon-domain correlation, with substantially more significant correlations detected in vertebrates compared with invertebrates. Our observations concur with the principles of exon shuffling theory, including the prediction of predominantly symmetric phase of introns flanking the borders of correlating exons. These results suggest that extensive exon shuffling events during evolution significantly contributed to the shaping of eukaryotic proteomes.

  2. Phylogenetic analysis of eukaryotes using heat-shock protein Hsp90.

    PubMed

    Stechmann, Alexandra; Cavalier-Smith, Thomas

    2003-10-01

    Most eukaryote molecular phylogenies have been based on small-subunit ribosomal RNA as its database includes the most species, but serious problems have been encountered that can make these trees misleading. More recent studies using concatenated protein sequences have increased the data per organism, reducing misleading signals from a single sequence, but taxon sampling is limited. To increase the database of protein-coding genes we sequenced the cytosolic form of heat-shock protein Hsp90 from a broad variety of previously unsampled eukaryote groups: protozoan flagellates (phyla Choanozoa, Apusozoa, Cercozoa) and all three groups of chromists (Cryptophyta, Heterokonta, Haptophyta). Gamma-corrected distance trees robustly show three groups: bacterial sequences are sister to all eukaryote sequences, which are cleanly subdivided into the cytosolic sequences and a clade comprising the chloroplast and endoplasmic reticulum (ER) Hsp90 sequences. The eukaryote cytosolic sequences comprise a robust opisthokont clade (animals/Choanozoa/fungi), a bikont clade, and an amoebozoan branch. However their topology is not robust. When the cytosolic sequences are rooted using only the ER/ chloroplast clade as outgroup the amoebozoan Dictyostelium is sister to the opisthokonts forming a unikont clade in the distance tree. Congruence of this tree with that for concatenated mitochondrial proteins suggests that the root of the eukaryote tree is between unikonts and bikonts. Gamma-corrected maximum likelihood analyses of cytosolic sequences alone (519 unambiguously aligned amino acid positions) show bikonts as a clade, as do least-squares distance trees, but with other distance methods and parsimony the sole amoebozoan species branches weakly within bikonts. Choanozoa are clearly sisters to animals. Some major bikont groups (e.g. green plants, alveolates, Euglenozoa) are consistently recovered, but others (e.g. discicristates, chromalveolates) appear only in some trees; the backbone of

  3. Activation of cGMP-dependent protein kinase by protein kinase C.

    PubMed

    Hou, Yali; Lascola, Judith; Dulin, Nickolai O; Ye, Richard D; Browning, Darren D

    2003-05-09

    The cGMP-dependent protein kinases (PKG) are emerging as important components of mainstream signal transduction pathways. Nitric oxide-induced cGMP formation by stimulation of soluble guanylate cyclase is generally accepted as being the most widespread mechanism underlying PKG activation. In the present study, PKG was found to be a target for phorbol 12-myristate 13-acetate (PMA)-responsive protein kinase C (PKC). PKG1alpha became phosphorylated in HEK-293 cells stimulated with PMA and also in vitro using purified components. PKC-dependent phosphorylation was found to activate PKG as measured by phosphorylation of vasodilator-stimulated phosphoprotein, and by in vitro kinase assays. Although there are 11 potential PKC substrate recognition sites in PKG1alpha, threonine 58 was examined due to its proximity to the pseudosubstrate domain. Antibodies generated against the phosphorylated form of this region were used to demonstrate phosphorylation in response to PMA treatment of the cells with kinetics similar to vasodilator-stimulated phosphoprotein phosphorylation. A phospho-mimetic mutation at this site (T58E) generated a partially activated PKG that was more sensitive to cGMP levels. A phospho-null mutation (T58A) revealed that this residue is important but not sufficient for PKG activation by PKC. Taken together, these findings outline a novel signal transduction pathway that links PKC stimulation with cyclic nucleotide-independent activation of PKG.

  4. This Déjà Vu Feeling—Analysis of Multidomain Protein Evolution in Eukaryotic Genomes

    PubMed Central

    Zmasek, Christian M.; Godzik, Adam

    2012-01-01

    Evolutionary innovation in eukaryotes and especially animals is at least partially driven by genome rearrangements and the resulting emergence of proteins with new domain combinations, and thus potentially novel functionality. Given the random nature of such rearrangements, one could expect that proteins with particularly useful multidomain combinations may have been rediscovered multiple times by parallel evolution. However, existing reports suggest a minimal role of this phenomenon in the overall evolution of eukaryotic proteomes. We assembled a collection of 172 complete eukaryotic genomes that is not only the largest, but also the most phylogenetically complete set of genomes analyzed so far. By employing a maximum parsimony approach to compare repertoires of Pfam domains and their combinations, we show that independent evolution of domain combinations is significantly more prevalent than previously thought. Our results indicate that about 25% of all currently observed domain combinations have evolved multiple times. Interestingly, this percentage is even higher for sets of domain combinations in individual species, with, for instance, 70% of the domain combinations found in the human genome having evolved independently at least once in other species. We also show that previous, much lower estimates of this rate are most likely due to the small number and biased phylogenetic distribution of the genomes analyzed. The process of independent emergence of identical domain combination is widespread, not limited to domains with specific functional categories. Besides data from large-scale analyses, we also present individual examples of independent domain combination evolution. The surprisingly large contribution of parallel evolution to the development of the domain combination repertoire in extant genomes has profound consequences for our understanding of the evolution of pathways and cellular processes in eukaryotes and for comparative functional genomics. PMID

  5. Cdc37 Promotes the Stability of Protein Kinases Cdc28 and Cak1

    PubMed Central

    Farrell, Alison; Morgan, David O.

    2000-01-01

    In the budding yeast Saccharomyces cerevisiae, Cdc37 is required for the productive formation of Cdc28-cyclin complexes. The cdc37-1 mutant arrests at Start with low levels of Cdc28 protein, which is predominantly unphosphorylated at Thr169, fails to bind cyclin, and has little protein kinase activity. We show here that Cdc28 and not cyclin is specifically defective in the cdc37-1 mutant and that Cdc37 likely does not act as an assembly factor for Cdc28-cyclin complex formation. We have also found that the levels and activity of the protein kinase Cak1 are significantly reduced in the cdc37-1 mutant. Pulse-chase analysis indicates that Cdc28 and Cak1 proteins are both destabilized when Cdc37 function is absent during but not after translation. In addition, Cdc37 promotes the production of Cak1, but not that of Cdc28, when coexpressed in insect cells. We conclude that budding yeast Cdc37, like its higher eukaryotic homologs, promotes the physical integrity of multiple protein kinases, perhaps by virtue of a cotranslational role in protein folding. PMID:10629030

  6. Protein translocation across the eukaryotic endoplasmic reticulum and bacterial plasma membranes.

    PubMed

    Rapoport, Tom A

    2007-11-29

    A decisive step in the biosynthesis of many proteins is their partial or complete translocation across the eukaryotic endoplasmic reticulum membrane or the prokaryotic plasma membrane. Most of these proteins are translocated through a protein-conducting channel that is formed by a conserved, heterotrimeric membrane-protein complex, the Sec61 or SecY complex. Depending on channel binding partners, polypeptides are moved by different mechanisms: the polypeptide chain is transferred directly into the channel by the translating ribosome, a ratcheting mechanism is used by the endoplasmic reticulum chaperone BiP, and a pushing mechanism is used by the bacterial ATPase SecA. Structural, genetic and biochemical data show how the channel opens across the membrane, releases hydrophobic segments of membrane proteins laterally into lipid, and maintains the membrane barrier for small molecules.

  7. Regulation of tomato Prf by Pto-like protein kinases.

    PubMed

    Mucyn, Tatiana S; Wu, Ai-Jiuan; Balmuth, Alexi L; Arasteh, Julia Maryam; Rathjen, John P

    2009-04-01

    Tomato Prf encodes a nucleotide-binding domain shared by Apaf-1, certain R proteins, and CED-4 fused to C-terminal leucine-rich repeats (NBARC-LRR) protein that is required for bacterial immunity to Pseudomonas syringae and sensitivity to the organophosphate fenthion. The signaling pathways involve two highly related protein kinases. Pto kinase mediates direct recognition of the bacterial effector proteins AvrPto or AvrPtoB. Fen kinase is required for fenthion sensitivity and recognition of bacterial effectors related to AvrPtoB. The role of Pto and its association with Prf has been characterized but Fen is poorly described. We show that, similar to Pto, Fen requires N-myristoylation and kinase activity for signaling and interacts with the N-terminal domain of Prf. Thus, the mechanisms of activation of Prf by the respective protein kinases are similar. Prf-Fen interaction is underlined by coregulatory mechanisms in which Prf negatively regulates Fen, most likely by controlling kinase activity. We further characterized negative regulation of Prf by Pto, and show that regulation is mediated by the previously described negative regulatory patch. Remarkably, the effectors released negative regulation of Prf in a manner dependent on Pto kinase activity. The data suggest a model in which Prf associates generally with Pto-like kinases in tightly regulated complexes, which are activated by effector-mediated disruption of negative regulation. Release of negative regulation may be a general feature of activation of NBARC-LRR proteins by cognate effectors.

  8. The essential vertebrate ABCE1 protein interacts with eukaryotic initiation factors.

    PubMed

    Chen, Zhang-Qun; Dong, Jinsheng; Ishimura, Akihiko; Daar, Ira; Hinnebusch, Alan G; Dean, Michael

    2006-03-17

    The ABCE1 gene is a member of the ATP-binding cassette (ABC) multigene family and is composed of two nucleotide binding domains and an N-terminal Fe-S binding domain. The ABCE1 gene encodes a protein originally identified for its inhibition of ribonuclease L, a nuclease induced by interferon in mammalian cells. The protein is also required for the assembly of the HIV and SIV gag polypeptides. However, ABCE1 is one of the most highly conserved proteins and is found in one or two copies in all characterized eukaryotes and archaea. Yeast ABCE1/RLI1 is essential to cell division and interacts with translation initiation factors in the assembly of the pre-initiation complex. We show here that the human ABCE1 protein is essential for in vitro and in vivo translation of mRNA and that it binds to eIF2alpha and eIF5. Inhibition of the Xenopus ABCE1 arrests growth at the gastrula stage of development, consistent with a block in translation. The human ABCE1 gene contains 16 introns, and the extremely high degree of amino acid identity allows the evolution of its introns to be examined throughout eukaryotes. The demonstration that ABCE1 plays a role in vertebrate translation initiation extends the known functions of this highly conserved protein. Translation is a highly regulated process important to development and pathologies such as cancer, making ABCE1 a potential target for therapeutics. The evolutionary analysis supports a model in which an ancestral eukaryote had large number of introns and that many of these introns were lost in non-vertebrate lineages.

  9. Identification and characterization of a novel sucrose-non-fermenting protein kinase/AMP-activated protein kinase-related protein kinase, SNARK.

    PubMed Central

    Lefebvre, D L; Bai, Y; Shahmolky, N; Sharma, M; Poon, R; Drucker, D J; Rosen, C F

    2001-01-01

    Subtraction hybridization after the exposure of keratinocytes to ultraviolet radiation identified a differentially expressed cDNA that encodes a protein of 630 amino acid residues possessing significant similarity to the catalytic domain of the sucrose-non-fermenting protein kinase (SNF1)/AMP-activated protein kinase (AMPK) family of serine/threonine protein kinases. Northern blotting and reverse-transcriptase-mediated PCR demonstrated that mRNA transcripts for the SNF1/AMPK-related kinase (SNARK) were widely expressed in rodent tissues. The SNARK gene was localized to human chromosome 1q32 by fluorescent in situ hybridization. SNARK was translated in vitro to yield a single protein band of approx. 76 kDa; Western analysis of transfected baby hamster kidney (BHK) cells detected two SNARK-immunoreactive bands of approx. 76-80 kDa. SNARK was capable of autophosphorylation in vitro; immunoprecipitated SNARK exhibited phosphotransferase activity with the synthetic peptide substrate HMRSAMSGLHLVKRR (SAMS) as a kinase substrate. SNARK activity was significantly increased by AMP and 5-amino-4-imidazolecarboxamide riboside (AICAriboside) in rat keratinocyte cells, implying that SNARK might be activated by an AMPK kinase-dependent pathway. Furthermore, glucose deprivation increased SNARK activity 3-fold in BHK fibroblasts. These findings identify SNARK as a glucose- and AICAriboside-regulated member of the AMPK-related gene family that represents a new candidate mediator of the cellular response to metabolic stress. PMID:11284715

  10. Protein kinase A phosphorylation modulates transport of the polypyrimidine tract-binding protein

    PubMed Central

    Xie, Jiuyong; Lee, Ji-Ann; Kress, Tracy L.; Mowry, Kimberly L.; Black, Douglas L.

    2003-01-01

    The heterogeneous nuclear ribonucleoprotein particle (hnRNP) proteins play important roles in mRNA processing in eukaryotes, but little is known about how they are regulated by cellular signaling pathways. The polypyrimidine-tract binding protein (PTB, or hnRNP I) is an important regulator of alternative pre-mRNA splicing, of viral RNA translation, and of mRNA localization. Here we show that the nucleo-cytoplasmic transport of PTB is regulated by the 3′,5′-cAMP-dependent protein kinase (PKA). PKA directly phosphorylates PTB on conserved Ser-16, and PKA activation in PC12 cells induces Ser-16 phosphorylation. PTB carrying a Ser-16 to alanine mutation accumulates normally in the nucleus. However, export of this mutant protein from the nucleus is greatly reduced in heterokaryon shuttling assays. Conversely, hyperphosphorylation of PTB by coexpression with the catalytic subunit of PKA results in the accumulation of PTB in the cytoplasm. This accumulation is again specifically blocked by the S16A mutation. Similarly, in Xenopus oocytes, the phospho-Ser-16-PTB is restricted to the cytoplasm, whereas the non-Ser-16-phosphorylated PTB is nuclear. Thus, direct PKA phosphorylation of PTB at Ser-16 modulates the nucleo-cytoplasmic distribution of PTB. This phosphorylation likely plays a role in the cytoplasmic function of PTB. PMID:12851456

  11. VZV ORF47 serine protein kinase and its viral substrates.

    PubMed

    Kenyon, Teri K; Grose, Charles

    2010-01-01

    ORF47, a serine protein kinase of varicella-zoster virus (VZV) and homolog of herpes simplex virus UL13, is an interesting modulator of VZV pathogenesis. This chapter summarizes research showing that ORF47 protein kinase activity, by virtue of phosphorylation of or binding to various viral substrates, regulates VZV proteins during all phases of viral infection and has a pronounced effect on the trafficking of gE, the predominant VZV glycoprotein, which in turn is critical for cell-to-cell spread of the virus. Casein kinase II, an ubiquitous cellular protein kinase, recognizes a similar but less stringent phosphorylation consensus sequence and can partially compensate for lack of ORF47 activity in VZV-infected cells. Differences between the phosphorylation consensus sites of the viral and cellular kinases are outlined in detail.

  12. Binding of exogenous brain protein kinase C to liver nuclei

    SciTech Connect

    Misra, U.K.; Wolf, M.; Besterman, J.; Cuatrecasas, P.; Sahyoun, N.

    1986-05-01

    Protein kinase C is found both in the cytosol and bound to membranes. Binding of the enzyme to plasma membranes is controlled by calcium whereas enzyme activators regulate both its membrane binding and enzyme catalysis. Activation of protein kinase C has been implicated in several regulatory processes including gene expression. Accordingly, the possibility of direct interaction of protein kinase C with the nucleus was examined utilizing /sup 3/H-PDBu binding to detect the enzyme. Purified protein kinase C from rat brain could bind to purified rat liver nuclei at 4/sup 0/C or at 21/sup 0/C, and the reaction was completed by 20 min. The binding was linearly dependent on protein kinase C concentration and required free Ca/sup 2 +/ with a K/sub m/sub app// of 0.5 ..mu..M. Chelation of Ca/sup 2 +/ with EGTA resulted in a rapid dissociation of protein kinase C from the nuclei. Differential extraction experiments suggested that about 50% of the enzyme was bound to chromatin and 25% was associated with the nuclear matrix. Moreover, protein kinase C bound to nuclei was able to phosphorylate several endogenous nuclear substrates, including chromatin proteins, in a Ca/sup 2 +/ phosphatidyl serine dependent reaction.

  13. Baculovirus protein PK2 subverts eIF2α kinase function by mimicry of its kinase domain C-lobe

    PubMed Central

    Li, John J.; Cao, Chune; Fixsen, Sarah M.; Young, Janet M.; Bando, Hisanori; Elde, Nels C.; Katsuma, Susumu; Dever, Thomas E.; Sicheri, Frank

    2015-01-01

    Phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) by eIF2α family kinases is a conserved mechanism to limit protein synthesis under specific stress conditions. The baculovirus-encoded protein PK2 inhibits eIF2α family kinases in vivo, thereby increasing viral fitness. However, the precise mechanism by which PK2 inhibits eIF2α kinase function remains an enigma. Here, we probed the mechanism by which PK2 inhibits the model eIF2α kinase human RNA-dependent protein kinase (PKR) as well as native insect eIF2α kinases. Although PK2 structurally mimics the C-lobe of a protein kinase domain and possesses the required docking infrastructure to bind eIF2α, we show that PK2 directly binds the kinase domain of PKR (PKRKD) but not eIF2α. The PKRKD–PK2 interaction requires a 22-residue N-terminal extension preceding the globular PK2 body that we term the “eIF2α kinase C-lobe mimic” (EKCM) domain. The functional insufficiency of the N-terminal extension of PK2 implicates a role for the adjacent EKCM domain in binding and inhibiting PKR. Using a genetic screen in yeast, we isolated PK2-activating mutations that cluster to a surface of the EKCM domain that in bona fide protein kinases forms the catalytic cleft through sandwiching interactions with a kinase N-lobe. Interaction assays revealed that PK2 associates with the N- but not the C-lobe of PKRKD. We propose an inhibitory model whereby PK2 engages the N-lobe of an eIF2α kinase domain to create a nonfunctional pseudokinase domain complex, possibly through a lobe-swapping mechanism. Finally, we show that PK2 enhances baculovirus fitness in insect hosts by targeting the endogenous insect heme-regulated inhibitor (HRI)–like eIF2α kinase. PMID:26216977

  14. Developing irreversible inhibitors of the protein kinase cysteinome

    PubMed Central

    Liu, Qingsong; Sabnis, Yogesh; Zhao, Zheng; Zhang, Tinghu; Buhrlage, Sara J.; Jones, Lyn H.; Gray, Nathanael S.

    2013-01-01

    Protein kinases are a large family of approximately 530 highly conserved enzymes that transfer a γ-phosphate group from ATP to a variety of amino acid residues such as tyrosine, serine and threonine which serves as a ubiquitous mechanism for cellular signal transduction. The clinical success of a number of kinase-directed drugs and the frequent observation of disease causing mutations in protein kinases suggest that a large number of kinases may represent therapeutically relevant targets. To-date the majority of clinical and preclinical kinase inhibitors are ATP-competitive, non-covalent inhibitors that achieve selectivity through recognition of unique features of particular protein kinases. Recently there has been renewed interest in the development of irreversible inhibitors that form covalent bonds with cysteine or other nucleophilic residues in the ATP-binding pocket. Irreversible kinase inhibitors have a number of potential advantages including prolonged pharmacodynamics, suitability for rational design, high potency and ability to validate pharmacological specificity through mutation of the reactive cysteine residue. Here we review recent efforts to develop cysteine-targeted irreversible protein kinase inhibitors and discuss their modes of recognizing the ATP-binding pocket and their biological activity profiles. In addition, we provided an informatics assessment of the potential ‘kinase-cysteinome’ and discuss strategies for the efficient development of new covalent inhibitors. PMID:23438744

  15. Serine/Threonine Kinase 3-Phosphoinositide-Dependent Protein Kinase-1 (PDK1) as a Key Regulator of Cell Migration and Cancer Dissemination

    PubMed Central

    Di Blasio, Laura; Gagliardi, Paolo A.; Puliafito, Alberto; Primo, Luca

    2017-01-01

    Dissecting the cellular signaling that governs the motility of eukaryotic cells is one of the fundamental tasks of modern cell biology, not only because of the large number of physiological processes in which cell migration is crucial, but even more so because of the pathological ones, in particular tumor invasion and metastasis. Cell migration requires the coordination of at least four major processes: polarization of intracellular signaling, regulation of the actin cytoskeleton and membrane extension, focal adhesion and integrin signaling and contractile forces generation and rear retraction. Among the molecular components involved in the regulation of locomotion, the phosphatidylinositol-3-kinase (PI3K) pathway has been shown to exert fundamental role. A pivotal node of such pathway is represented by the serine/threonine kinase 3-phosphoinositide-dependent protein kinase-1 (PDPK1 or PDK1). PDK1, and the majority of its substrates, belong to the AGC family of kinases (related to cAMP-dependent protein kinase 1, cyclic Guanosine monophosphate-dependent protein kinase and protein kinase C), and control a plethora of cellular processes, downstream either to PI3K or to other pathways, such as RAS GTPase-MAPK (mitogen-activated protein kinase). Interestingly, PDK1 has been demonstrated to be crucial for the regulation of each step of cell migration, by activating several proteins such as protein kinase B/Akt (PKB/Akt), myotonic dystrophy-related CDC42-binding kinases alpha (MRCKα), Rho associated coiled-coil containing protein kinase 1 (ROCK1), phospholipase C gamma 1 (PLCγ1) and β3 integrin. Moreover, PDK1 regulates cancer cell invasion as well, thus representing a possible target to prevent cancer metastasis in human patients. The aim of this review is to summarize the various mechanisms by which PDK1 controls the cell migration process, from cell polarization to actin cytoskeleton and focal adhesion regulation, and finally, to discuss the evidence supporting a

  16. Resolution of thylakoid polyphenol oxidase and a protein kinase

    SciTech Connect

    Race, H.L.; Davenport, J.W.; Hind, G.

    1995-12-31

    The predominant protein kinase activity in octylglucoside (OG) extracts of spinach thylakoids has been attributed to a 64-kDa protein, tp64. Recent work calls into question the relation between tp64 and protein kinase activity, which were fractionated apart using fluid phase IEF and hydroxylapatite chromatography. Hind et al. sequenced tp64 from the cDNA and showed it to be a polyphenol oxidase (PPO) homolog. Its transit peptide indicates a location for the mature protein within the thylakoid lumen, where there is presumably no ATP and where it is remote from the presumed kinase substrates: the stromally exposed regions of integral PS-II membrane proteins. Here the authors suggest that the kinase is a 64-kDa protein distinct from tp64.

  17. Drosophila Protein Kinase CK2: Genetics, Regulatory Complexity and Emerging Roles during Development

    PubMed Central

    Bandyopadhyay, Mohna; Arbet, Scott; Bishop, Clifton P.; Bidwai, Ashok P.

    2016-01-01

    CK2 is a Ser/Thr protein kinase that is highly conserved amongst all eukaryotes. It is a well-known oncogenic kinase that regulates vital cell autonomous functions and animal development. Genetic studies in the fruit fly Drosophila are providing unique insights into the roles of CK2 in cell signaling, embryogenesis, organogenesis, neurogenesis, and the circadian clock, and are revealing hitherto unknown complexities in CK2 functions and regulation. Here, we review Drosophila CK2 with respect to its structure, subunit diversity, potential mechanisms of regulation, developmental abnormalities linked to mutations in the gene encoding CK2 subunits, and emerging roles in multiple aspects of eye development. We examine the Drosophila CK2 “interaction map” and the eye-specific “transcriptome” databases, which raise the prospect that this protein kinase has many additional targets in the developing eye. We discuss the possibility that CK2 functions during early retinal neurogenesis in Drosophila and mammals bear greater similarity than has been recognized, and that this conservation may extend to other developmental programs. Together, these studies underscore the immense power of the Drosophila model organism to provide new insights and avenues to further investigate developmentally relevant targets of this protein kinase. PMID:28036067

  18. Functional Analysis of Protein Kinase CK2 of the Human Malaria Parasite Plasmodium falciparum▿ †

    PubMed Central

    Holland, Zoë; Prudent, Renaud; Reiser, Jean-Baptiste; Cochet, Claude; Doerig, Christian

    2009-01-01

    Protein kinase CK2 (casein kinase 2) is a eukaryotic serine/threonine protein kinase with multiple substrates and roles in diverse cellular processes, including differentiation, proliferation, and translation. The mammalian holoenzyme consists of two catalytic alpha or alpha′ subunits and two regulatory beta subunits. We report the identification and characterization of a Plasmodium falciparum CK2α orthologue, PfCK2α, and two PfCK2β orthologues, PfCK2β1 and PfCK2β2. Recombinant PfCK2α possesses protein kinase activity, exhibits similar substrate and cosubstrate preferences to those of CK2α subunits from other organisms, and interacts with both of the PfCK2β subunits in vitro. Gene disruption experiments show that the presence of PfCK2α is crucial to asexual blood stage parasites and thereby validate the enzyme as a possible drug target. PfCK2α is amenable to inhibitor screening, and we report differential susceptibility between the human and P. falciparum CK2α enzymes to a small molecule inhibitor. Taken together, our data identify PfCK2α as a potential target for antimalarial chemotherapeutic intervention. PMID:19114502

  19. Protein kinase C as a tumor suppressor.

    PubMed

    Newton, Alexandra C

    2017-05-02

    Protein kinase C (PKC) has historically been considered an oncoprotein. This stems in large part from the discovery in the early 1980s that PKC is directly activated by tumor-promoting phorbol esters. Yet three decades of clinical trials using PKC inhibitors in cancer therapies not only failed, but in some cases worsened patient outcome. Why has targeting PKC in cancer eluded successful therapies? Recent studies looking at the disease for insight provide an explanation: cancer-associated mutations in PKC are generally loss-of-function (LOF), supporting an unexpected function as tumor suppressors. And, contrasting with LOF mutations in cancer, germline mutations that enhance the activity of some PKC isozymes are associated with degenerative diseases such as Alzheimer's disease. This review provides a background on the diverse mechanisms that ensure PKC is only active when, where, and for the appropriate duration needed and summarizes recent findings converging on a paradigm reversal: PKC family members generally function by suppressing, rather than promoting, survival signaling. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. The specificities of protein kinase inhibitors: an update.

    PubMed Central

    Bain, Jenny; McLauchlan, Hilary; Elliott, Matthew; Cohen, Philip

    2003-01-01

    We have previously examined the specificities of 28 commercially available compounds, reported to be relatively selective inhibitors of particular serine/threonine-specific protein kinases [Davies, Reddy, Caivano and Cohen (2000) Biochem. J. 351, 95-105]. In the present study, we have extended this analysis to a further 14 compounds. Of these, indirubin-3'-monoxime, SP 600125, KT 5823 and ML-9 were found to inhibit a number of protein kinases and conclusions drawn from their use in cell-based assays are likely to be erroneous. Kenpaullone, Alsterpaullone, Purvalanol, Roscovitine, pyrazolopyrimidine 1 (PP1), PP2 and ML-7 were more specific, but still inhibited two or more protein kinases with similar potency. Our results suggest that the combined use of Roscovitine and Kenpaullone may be useful for identifying substrates and physiological roles of cyclin-dependent protein kinases, whereas the combined use of Kenpaullone and LiCl may be useful for identifying substrates and physiological roles of glycogen synthase kinase 3. The combined use of SU 6656 and either PP1 or PP2 may be useful for identifying substrates of Src family members. Epigallocatechin 3-gallate, one of the main polyphenolic constituents of tea, inhibited two of the 28 protein kinases in the panel, dual-specificity, tyrosine-phosphorylated and regulated kinase 1A (DYRK1A; IC(50)=0.33 microM) and p38-regulated/activated kinase (PRAK; IC(50)=1.0 microM). PMID:12534346

  1. Protein kinase C-associated kinase can activate NFkappaB in both a kinase-dependent and a kinase-independent manner.

    PubMed

    Moran, Stewart T; Haider, Khaleda; Ow, Yongkai; Milton, Peter; Chen, Luojing; Pillai, Shiv

    2003-06-13

    Protein kinase C-associated kinase (PKK, also known as RIP4/DIK) activates NFkappaB when overexpressed in cell lines and is required for keratinocyte differentiation in vivo. However, very little is understood about the factors upstream of PKK or how PKK activates NFkappaB. Here we show that certain catalytically inactive mutants of PKK can activate NFkappaB, although to a lesser degree than wild type PKK. The deletion of specific domains of wild type PKK diminishes the ability of this enzyme to activate NFkappaB; the same deletions made on a catalytically inactive PKK background completely ablate NFkappaB activation. PKK may be phosphorylated by two specific mitogen-activated protein kinase kinase kinases, MEKK2 and MEKK3, and this interaction may in part be mediated through a critical activation loop residue, Thr184. Catalytically inactive PKK mutants that block phorbol ester-induced NFkappaB activation do not interfere with, but unexpectedly enhance, the activation of NFkappaB by these two mitogen-activated protein kinase kinase kinases. Taken together, these data indicate that PKK may function in both a kinase-dependent as well as a kinase-independent manner to activate NFkappaB.

  2. The major architects of chromatin: architectural proteins in bacteria, archaea and eukaryotes.

    PubMed

    Luijsterburg, Martijn S; White, Malcolm F; van Driel, Roel; Dame, Remus Th

    2008-01-01

    The genomic DNA of all organisms across the three kingdoms of life needs to be compacted and functionally organized. Key players in these processes are DNA supercoiling, macromolecular crowding and architectural proteins that shape DNA by binding to it. The architectural proteins in bacteria, archaea and eukaryotes generally do not exhibit sequence or structural conservation especially across kingdoms. Instead, we propose that they are functionally conserved. Most of these proteins can be classified according to their architectural mode of action: bending, wrapping or bridging DNA. In order for DNA transactions to occur within a compact chromatin context, genome organization cannot be static. Indeed chromosomes are subject to a whole range of remodeling mechanisms. In this review, we discuss the role of (i) DNA supercoiling, (ii) macromolecular crowding and (iii) architectural proteins in genome organization, as well as (iv) mechanisms used to remodel chromosome structure and to modulate genomic activity. We conclude that the underlying mechanisms that shape and remodel genomes are remarkably similar among bacteria, archaea and eukaryotes.

  3. Functions of Ribosomal Proteins in Assembly of Eukaryotic Ribosomes In Vivo

    PubMed Central

    2016-01-01

    The proteome of cells is synthesized by ribosomes, complex ribonucleoproteins that in eukaryotes contain 79–80 proteins and four ribosomal RNAs (rRNAs) more than 5,400 nucleotides long. How these molecules assemble together and how their assembly is regulated in concert with the growth and proliferation of cells remain important unanswered questions. Here, we review recently emerging principles to understand how eukaryotic ribosomal proteins drive ribosome assembly in vivo. Most ribosomal proteins assemble with rRNA cotranscriptionally; their association with nascent particles is strengthened as assembly proceeds. Each subunit is assembled hierarchically by sequential stabilization of their subdomains. The active sites of both subunits are constructed last, perhaps to prevent premature engagement of immature ribosomes with active subunits. Late-assembly intermediates undergo quality-control checks for proper function. Mutations in ribosomal proteins that affect mostly late steps lead to ribosomopathies, diseases that include a spectrum of cell type–specific disorders that often transition from hypoproliferative to hyperproliferative growth. PMID:25706898

  4. Functions of ribosomal proteins in assembly of eukaryotic ribosomes in vivo.

    PubMed

    de la Cruz, Jesús; Karbstein, Katrin; Woolford, John L

    2015-01-01

    The proteome of cells is synthesized by ribosomes, complex ribonucleoproteins that in eukaryotes contain 79-80 proteins and four ribosomal RNAs (rRNAs) more than 5,400 nucleotides long. How these molecules assemble together and how their assembly is regulated in concert with the growth and proliferation of cells remain important unanswered questions. Here, we review recently emerging principles to understand how eukaryotic ribosomal proteins drive ribosome assembly in vivo. Most ribosomal proteins assemble with rRNA cotranscriptionally; their association with nascent particles is strengthened as assembly proceeds. Each subunit is assembled hierarchically by sequential stabilization of their subdomains. The active sites of both subunits are constructed last, perhaps to prevent premature engagement of immature ribosomes with active subunits. Late-assembly intermediates undergo quality-control checks for proper function. Mutations in ribosomal proteins that affect mostly late steps lead to ribosomopathies, diseases that include a spectrum of cell type-specific disorders that often transition from hypoproliferative to hyperproliferative growth.

  5. Advanced method for high-throughput expression of mutated eukaryotic membrane proteins in Saccharomyces cerevisiae

    SciTech Connect

    Ito, Keisuke; Sugawara, Taishi; Shiroishi, Mitsunori; Tokuda, Natsuko; Kurokawa, Azusa; Misaka, Takumi; Makyio, Hisayoshi; Yurugi-Kobayashi, Takami; Shimamura, Tatsuro; Nomura, Norimichi; Murata, Takeshi; Abe, Keiko; Iwata, So

    2008-07-11

    Crystallization of eukaryotic membrane proteins is a challenging, iterative process. The protein of interest is often modified in an attempt to improve crystallization and diffraction results. To accelerate this process, we took advantage of a GFP-fusion yeast expression system that uses PCR to direct homologous recombination and gene cloning. We explored the possibility of employing more than one PCR fragment to introduce various mutations in a single step, and found that when up to five PCR fragments were co-transformed into yeast, the recombination frequency was maintained as the number of fragments was increased. All transformants expressed the model membrane protein, while the resulting plasmid from each clone contained the designed mutations only. Thus, we have demonstrated a technique allowing the expression of mutant membrane proteins within 5 days, combining a GFP-fusion expression system and yeast homologous recombination.

  6. Pkg2, a Novel Transmembrane Protein Ser/Thr Kinase of Streptomyces granaticolor

    PubMed Central

    Nádvorník, Richard; Vomastek, Tomáš; Janeček, Jiří; Techniková, Zuzana; Branny, Pavel

    1999-01-01

    A 4.2-kb SphI-BamHI fragment of chromosomal DNA from Streptomyces granaticolor was cloned and shown to encode a protein with significant sequence similarity to the eukaryotic protein serine/threonine kinases. It consists of 701 amino acids and in the N-terminal part contains all conserved catalytic domains of protein kinases. The C-terminal domain of Pkg2 contains seven tandem repeats of 11 or 12 amino acids with similarity to the tryptophan-docking motif known to stabilize a symmetrical three-dimensional structure called a propeller structure. The pkg2 gene was overexpressed in Escherichia coli, and the gene product (Pkg2) has been found to be autophosphorylated at serine and threonine residues. The N- and C-terminal parts of Pkg2 are separated with a hydrophobic stretch of 21 amino acids which translocated a PhoA fusion protein into the periplasm. Thus, Pkg2 is the first transmembrane protein serine/threonine kinase described for streptomycetes. Replacement of the pkg2 gene by the spectinomycin resistance gene resulted in changes in the morphology of aerial hyphae. PMID:9864307

  7. Activation of fat cell adenylate cyclase by protein kinase C

    SciTech Connect

    Naghshineh, S.; Noguchi, M.; Huang, K.P.; Londos, C.

    1986-05-01

    Purified protein kinase C (C-kinase) from guinea pig pancreas and rat brain stimulated adenylate cyclase activity in purified rat adipocyte membranes. Cyclase stimulation occurred over 100 to 1000 mU/ml of C-kinase activity, required greater than 10 ..mu..M calcium, proceeded without a lag, was not readily reversible, and required no exogenous phospholipid. Moreover, C-kinase inhibitors, such as chlorpromazine and palmitoyl carnitine, inhibited selectively adenylate cyclase which was activated by C-kinase and calcium. Depending on assay conditions, 10 nM 12-0-tetradecanoylphorbol-13-acetate (TPA) either enhanced or was required for kinase action on cyclase. Also, TPA plus calcium promoted the quantitative association of C-kinase with membranes. Adenylate cyclase activation by C-kinase was seen both in the presence and absence of exogenous GTP, indicating that the kinase effect does not result from an action on the GTP-binding, inhibitory regulatory component (N/sub i/) of the cyclase system. Moreover, the kinase effect was seen in the presence of non-phosphorylating ATP analogs, such as AppNHp and AppCH/sub 2/p, suggesting that the effects of C-kinase described herein may result from association with, rather than phosphorylation of, adenylate cyclase.

  8. Protein and DNA Modifications: Evolutionary Imprints of Bacterial Biochemical Diversification and Geochemistry on the Provenance of Eukaryotic Epigenetics

    PubMed Central

    Aravind, L.; Burroughs, A. Maxwell; Zhang, Dapeng; Iyer, Lakshminarayan M.

    2014-01-01

    Epigenetic information, which plays a major role in eukaryotic biology, is transmitted by covalent modifications of nuclear proteins (e.g., histones) and DNA, along with poorly understood processes involving cytoplasmic/secreted proteins and RNAs. The origin of eukaryotes was accompanied by emergence of a highly developed biochemical apparatus for encoding, resetting, and reading covalent epigenetic marks in proteins such as histones and tubulins. The provenance of this apparatus remained unclear until recently. Developments in comparative genomics show that key components of eukaryotic epigenetics emerged as part of the extensive biochemical innovation of secondary metabolism and intergenomic/interorganismal conflict systems in prokaryotes, particularly bacteria. These supplied not only enzymatic components for encoding and removing epigenetic modifications, but also readers of some of these marks. Diversification of these prokaryotic systems and subsequently eukaryotic epigenetics appear to have been considerably influenced by the great oxygenation event in the Earth’s history. PMID:24984775

  9. The Link between Protein Kinase CK2 and Atypical Kinase Rio1

    PubMed Central

    Kubiński, Konrad; Masłyk, Maciej

    2017-01-01

    The atypical kinase Rio1 is widespread in many organisms, ranging from Archaebacteria to humans, and is an essential factor in ribosome biogenesis. Little is known about the protein substrates of the enzyme and small-molecule inhibitors of the kinase. Protein kinase CK2 was the first interaction partner of Rio1, identified in yeast cells. The enzyme from various sources undergoes CK2-mediated phosphorylation at several sites and this modification regulates the activity of Rio1. The aim of this review is to present studies of the relationship between the two different kinases, with respect to CK2-mediated phosphorylation of Rio1, regulation of Rio1 activity, and similar susceptibility of the kinases to benzimidazole inhibitors. PMID:28178206

  10. [The role of Gilgamesh protein kinase in Drosophila melanogaster spermatogenesis].

    PubMed

    Nerusheva, O O; Dorogova, N V; Gubanova, N V; Omel'ianchuk, L V

    2008-09-01

    The cellular function of the gilgamesh mutation (89B9-12) of casein kinase gene in Drosophila spermatogenesis was studied. It was demonstrated that the sterility resulting from this mutation is connected with the abnormalities in spermatid individualization. A phylogenetic study of the protein sequences of casein kinases 1 from various organisms was conducted. The Gilgamesh protein was shown to be phylogenetically closer to the cytoplasmic casein kinase family, represented by the YCK3, YCK2, and YCK1 proteins of Saccharomyces cerevisiae and animal gamma-casein kinases. It is known that these yeast casein kinases are involved in vesicular trafficking, which, in turn, is related in its genetic control to the cell membrane remodeling during spermatid individualization. Thus, the data of phylogenetic analysis fit well the results obtained by studying the mutation phenotype.

  11. Cloning of Giardia lamblia heat shock protein HSP70 homologs: implications regarding origin of eukaryotic cells and of endoplasmic reticulum.

    PubMed Central

    Gupta, R S; Aitken, K; Falah, M; Singh, B

    1994-01-01

    The genes for two different 70-kDa heat shock protein (HSP70) homologs have been cloned and sequenced from the protozoan Giardia lamblia. On the basis of their sequence features, one of these genes corresponds to the cytoplasmic form of HSP70. The second gene, on the basis of its characteristic N-terminal hydrophobic signal sequence and C-terminal endoplasmic reticulum (ER) retention sequence (Lys-Asp-Glu-Leu), is the equivalent of ER-resident GRP78 or the Bip family of proteins. Phylogenetic trees based on HSP70 sequences show that G. lamblia homologs show the deepest divergence among eukaryotic species. The identification of a GRP78 or Bip homolog in G. lamblia strongly suggests the existence of ER in this ancient eukaryote. Detailed phylogenetic analyses of HSP70 sequences by boot-strap neighbor-joining and maximum-parsimony methods show that the cytoplasmic and ER homologs form distinct subfamilies that evolved from a common eukaryotic ancestor by gene duplication that occurred very early in the evolution of eukaryotic cells. It is postulated that because of the essential "molecular chaperone" function of these proteins in translocation of other proteins across membranes, duplication of their genes accompanied the evolution of ER or nucleus in the eukaryotic cell ancestor. The presence in all eukaryotic cytoplasmic HSP70 homologs (including the cognate, heat-induced, and ER forms) of a number of autapomorphic sequence signatures that are not present in any prokaryotic or organellar homologs provides strong evidence regarding the monophyletic nature of eukaryotic lineage. Further, all eukaryotic HSP70 homologs share in common with the Gram-negative group of eubacteria a number of sequence features that are not present in any archaebacterium or Gram-positive bacterium, indicating their evolution from this group of organisms. Some implications of these findings regarding the evolution of eukaryotic cells and ER are discussed. Images PMID:8159675

  12. Ranking Gene Ontology terms for predicting non-classical secretory proteins in eukaryotes and prokaryotes.

    PubMed

    Huang, Wen-Lin

    2012-11-07

    Protein secretion is an important biological process for both eukaryotes and prokaryotes. Several sequence-based methods mainly rely on utilizing various types of complementary features to design accurate classifiers for predicting non-classical secretory proteins. Gene Ontology (GO) terms are increasing informative in predicting protein functions. However, the number of used GO terms is often very large. For example, there are 60,020 GO terms used in the prediction method Euk-mPLoc 2.0 for subcellular localization. This study proposes a novel approach to identify a small set of m top-ranked GO terms served as the only type of input features to design a support vector machine (SVM) based method Sec-GO to predict non-classical secretory proteins in both eukaryotes and prokaryotes. To evaluate the Sec-GO method, two existing methods and their used datasets are adopted for performance comparisons. The Sec-GO method using m=436 GO terms yields an independent test accuracy of 96.7% on mammalian proteins, much better than the existing method SPRED (82.2%) which uses frequencies of tri-peptides and short peptides, secondary structure, and physicochemical properties as input features of a random forest classifier. Furthermore, when applying to Gram-positive bacterial proteins, the Sec-GO with m=158 GO terms has a test accuracy of 94.5%, superior to NClassG+ (90.0%) which uses SVM with several feature types, comprising amino acid composition, di-peptides, physicochemical properties and the position specific weighting matrix. Analysis of the distribution of secretory proteins in a GO database indicates the percentage of the non-classical secretory proteins annotated by GO is larger than that of classical secretory proteins in both eukaryotes and prokaryotes. Of the m top-ranked GO features, the top-four GO terms are all annotated by such subcellular locations as GO:0005576 (Extracellular region). Additionally, the method Sec-GO is easily implemented and its web tool of

  13. Autophosphorylating protein kinase activity in titin-like arthropod projectin.

    PubMed

    Maroto, M; Vinós, J; Marco, R; Cervera, M

    1992-03-20

    The function of the high molecular weight structural proteins from muscle, namely vertebrate titin, arthropod projectin and nematode twitchin, remains to be established. Using a simple method for the purification of projectin from crayfish and Drosophila melanogaster, a polyclonal antibody has been raised against crayfish projectin, and shown to immunocrossreact with Drosophila projectin but not with rat titin. In this study, evidence is presented that projectin and twitchin may share functional protein kinase domains, indicating a possible relationship between them. Projectin has a serine/threonine protein kinase activity. This supports the relationship with twitchin since, in sequence analysis of the latter, a protein-kinase-like domain has been found. Moreover, projectin is capable of autophosphorylation in vitro. These kinase activities imply regulatory functions for this group of proteins, extending its previously assumed structural role in the sarcomere. We also show here that projectin is phosphorylated in vivo at serine residues, as described for titin.

  14. Protein kinase CK2 triggers cytosolic zinc signaling pathways by phosphorylation of zinc channel ZIP7

    PubMed Central

    Taylor, Kathryn M.; Hiscox, Stephen; Nicholson, Robert I.; Hogstrand, Christer; Kille, Peter

    2012-01-01

    The transition element zinc, which has recently been identified as an intracellular second messenger, has been implicated in various signaling pathways, including those leading to cell proliferation. Zinc channels of the ZIP protein family (Solute Carrier Family 39A, SLC39A) transiently increase the cytosolic free zinc (Zn2+) concentration in response to extracellular signals. Here, we show that phosphorylation of evolutionarily conserved residues in zinc transporter ZIP7 is associated with the gated release of Zn2+ from intracellular stores, leading to activation of tyrosine kinases and the phosphorylation of AKT and extracellular signal-regulated kinases 1 and 2 (ERK1/2). Through pharmacological manipulation, proximity assay, and mutagenesis, we identified CK2 as the kinase responsible for ZIP7 activation. Together, the present results show that eukaryotic transition element channels can be activated post-translationally by phosphorylation eliciting a cell signaling cascade. Our study links the regulated release of zinc from intracellular stores to phosphorylation of kinases involved in proliferative responses and cell migration, suggesting a functional role for ZIP7 and zinc signals for these events which are characteristic of cancerous cells. Furthermore, the interaction of ZIP7 with CK2, a kinase that is antiapoptotoc and promotes cell division, highlights the potential for ZIP7 as a target for anti-cancer drug development. PMID:22317921

  15. Dietary Methionine Restriction Regulates Liver Protein Synthesis and Gene Expression Independently of Eukaryotic Initiation Factor 2 Phosphorylation in Mice.

    PubMed

    Pettit, Ashley P; Jonsson, William O; Bargoud, Albert R; Mirek, Emily T; Peelor, Frederick F; Wang, Yongping; Gettys, Thomas W; Kimball, Scot R; Miller, Benjamin F; Hamilton, Karyn L; Wek, Ronald C; Anthony, Tracy G

    2017-06-01

    Background: The phosphorylation of eukaryotic initiation factor 2 (p-eIF2) during dietary amino acid insufficiency reduces protein synthesis and alters gene expression via the integrated stress response (ISR).Objective: We explored whether a Met-restricted (MR) diet activates the ISR to reduce body fat and regulate protein balance.Methods: Male and female mice aged 3-6 mo with either whole-body deletion of general control nonderepressible 2 (Gcn2) or liver-specific deletion of protein kinase R-like endoplasmic reticulum kinase (Perk) alongside wild-type or floxed control mice were fed an obesogenic diet sufficient in Met (0.86%) or an MR (0.12% Met) diet for ≤5 wk. Ala enrichment with deuterium was measured to calculate protein synthesis rates. The guanine nucleotide exchange factor activity of eIF2B was measured alongside p-eIF2 and hepatic mRNA expression levels at 2 d and 5 wk. Metabolic phenotyping was conducted at 4 wk, and body composition was measured throughout. Results were evaluated with the use of ANOVA (P < 0.05).Results: Feeding an MR diet for 2 d did not increase hepatic p-eIF2 or reduce eIF2B activity in wild-type or Gcn2(-/-) mice, yet many genes transcriptionally regulated by the ISR were altered in both strains in the same direction and amplitude. Feeding an MR diet for 5 wk increased p-eIF2 and reduced eIF2B activity in wild-type but not Gcn2(-/-) mice, yet ISR-regulated genes altered in both strains similarly. Furthermore, the MR diet reduced mixed and cytosolic but not mitochondrial protein synthesis in both the liver and skeletal muscle regardless of Gcn2 status. Despite the similarities between strains, the MR diet did not increase energy expenditure or reduce body fat in Gcn2(-/-) mice. Finally, feeding the MR diet to mice with Perk deleted in the liver increased hepatic p-eIF2 and altered body composition similar to floxed controls.Conclusions: Hepatic activation of the ISR resulting from an MR diet does not require p-eIF2. Gcn2 status

  16. RAF protein-serine/threonine kinases: Structure and regulation

    SciTech Connect

    Roskoski, Robert

    2010-08-27

    Research highlights: {yields} The formation of unique side-to-side RAF dimers is required for full kinase activity. {yields} RAF kinase inhibitors block MEK activation in cells containing oncogenic B-RAF. {yields} RAF kinase inhibitors can lead to the paradoxical increase in RAF kinase activity. -- Abstract: A-RAF, B-RAF, and C-RAF are a family of three protein-serine/threonine kinases that participate in the RAS-RAF-MEK-ERK signal transduction cascade. This cascade participates in the regulation of a large variety of processes including apoptosis, cell cycle progression, differentiation, proliferation, and transformation to the cancerous state. RAS mutations occur in 15-30% of all human cancers, and B-RAF mutations occur in 30-60% of melanomas, 30-50% of thyroid cancers, and 5-20% of colorectal cancers. Activation of the RAF kinases requires their interaction with RAS-GTP along with dephosphorylation and also phosphorylation by SRC family protein-tyrosine kinases and other protein-serine/threonine kinases. The formation of unique side-to-side RAF dimers is required for full kinase activity. RAF kinase inhibitors are effective in blocking MEK1/2 and ERK1/2 activation in cells containing the oncogenic B-RAF Val600Glu activating mutation. RAF kinase inhibitors lead to the paradoxical increase in RAF kinase activity in cells containing wild-type B-RAF and wild-type or activated mutant RAS. C-RAF plays a key role in this paradoxical increase in downstream MEK-ERK activation.

  17. An Aminopyridazine Inhibitor of Death Associated Protein Kinase Attenuates Hypoxia-Ischemia Induced Brain Damage

    SciTech Connect

    Velentza, A.V.; Wainwright, M.S.; Zasadzki, M.; Mirzoeva, S.; Haiech, J.; Focia, P.J.; Egli, M.; Watterson, D.M.

    2010-03-08

    Death associated protein kinase (DAPK) is a calcium and calmodulin regulated enzyme that functions early in eukaryotic programmed cell death, or apoptosis. To validate DAPK as a potential drug discovery target for acute brain injury, the first small molecule DAPK inhibitor was synthesized and tested in vivo. A single injection of the aminopyridazine-based inhibitor administered 6 h after injury attenuated brain tissue or neuronal biomarker loss measured, respectively, 1 week and 3 days later. Because aminopyridazine is a privileged structure in neuropharmacology, we determined the high-resolution crystal structure of a binary complex between the kinase domain and a molecular fragment of the DAPK inhibitor. The co-crystal structure describes a structural basis for interaction and provides a firm foundation for structure-assisted design of lead compounds with appropriate molecular properties for future drug development.

  18. Interplay of noncoding RNAs, mRNAs, and proteins during the growth of eukaryotic cells

    SciTech Connect

    Zhdanov, V. P.

    2010-10-15

    Numerous biological functions of noncoding RNAs (ncRNAs) in eukaryotic cells are based primarily on their ability to pair with target mRNAs and then either to prevent translation or to result in rapid degradation of the mRNA-ncRNA complex. Using a general model describing this scenario, we show that ncRNAs may help to maintain constant mRNA and protein concentrations during the growth of cells. The possibility of observation of this effect on the global scale is briefly discussed.

  19. Toxin effect on protein biosynthesis in eukaryotic cells: a simple kinetic model.

    PubMed

    Skakauskas, Vladas; Katauskis, Pranas; Skvortsov, Alex; Gray, Peter

    2015-03-01

    A model for toxin inhibition of protein synthesis inside eukaryotic cells is presented. Mitigation of this effect by introduction of an antibody is also studied. Antibody and toxin (ricin) initially are delivered outside the cell. The model describes toxin internalization from the extracellular into the intracellular domain, its transport to the endoplasmic reticulum (ER) and the cleavage inside the ER into the RTA and RTB chains, the release of RTA into the cytosol, inactivation (depurination) of ribosomes, and the effect on translation. The model consists of a set of ODEs which are solved numerically. Numerical results are illustrated by figures and discussed. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Phosphorylation of the Drosophila transient receptor potential ion channel is regulated by the phototransduction cascade and involves several protein kinases and phosphatases.

    PubMed

    Voolstra, Olaf; Bartels, Jonas-Peter; Oberegelsbacher, Claudia; Pfannstiel, Jens; Huber, Armin

    2013-01-01

    Protein phosphorylation plays a cardinal role in regulating cellular processes in eukaryotes. Phosphorylation of proteins is controlled by protein kinases and phosphatases. We previously reported the light-dependent phosphorylation of the Drosophila transient receptor potential (TRP) ion channel at multiple sites. TRP generates the receptor potential upon stimulation of the photoreceptor cell by light. An eye-enriched protein kinase C (eye-PKC) has been implicated in the phosphorylation of TRP by in vitro studies. Other kinases and phosphatases of TRP are elusive. Using phosphospecific antibodies and mass spectrometry, we here show that phosphorylation of most TRP sites depends on the phototransduction cascade and the activity of the TRP ion channel. A candidate screen to identify kinases and phosphatases provided in vivo evidence for an involvement of eye-PKC as well as other kinases and phosphatases in TRP phosphorylation.

  1. Hsp90 Binds and Regulates the Ligand-Inducible α Subunit of Eukaryotic Translation Initiation Factor Kinase Gcn2

    PubMed Central

    Donzé, Olivier; Picard, Didier

    1999-01-01

    The protein kinase Gcn2 stimulates translation of the yeast transcription factor Gcn4 upon amino acid starvation. Using genetic and biochemical approaches, we show that Gcn2 is regulated by the molecular chaperone Hsp90 in budding yeast Saccharomyces cerevisiae. Specifically, we found that (i) several Hsp90 mutant strains exhibit constitutive expression of a GCN4-lacZ reporter plasmid; (ii) Gcn2 and Hsp90 form a complex in vitro as well as in vivo; (iii) the specific inhibitors of Hsp90, geldanamycin and macbecin I, enhance the association of Gcn2 with Hsp90 and inhibit its kinase activity in vitro; (iv) in vivo, macbecin I strongly reduces the levels of Gcn2; (v) in a strain expressing the temperature-sensitive Hsp90 mutant G170D, both the accumulation and activity of Gcn2 are abolished at the restrictive temperature; and (vi) the Hsp90 cochaperones Cdc37, Sti1, and Sba1 are required for the response to amino acid starvation. Taken together, these data identify Gcn2 as a novel target for Hsp90, which plays a crucial role for the maturation and regulation of Gcn2. PMID:10567567

  2. An elaborate classification of SNARE proteins sheds light on the conservation of the eukaryotic endomembrane system.

    PubMed

    Kloepper, Tobias H; Kienle, C Nickias; Fasshauer, Dirk

    2007-09-01

    Proteins of the SNARE (soluble N-ethylmalemide-sensitive factor attachment protein receptor) family are essential for the fusion of transport vesicles with an acceptor membrane. Despite considerable sequence divergence, their mechanism of action is conserved: heterologous sets assemble into membrane-bridging SNARE complexes, in effect driving membrane fusion. Within the cell, distinct functional SNARE units are involved in different trafficking steps. These functional units are conserved across species and probably reflect the conservation of the particular transport step. Here, we have systematically analyzed SNARE sequences from 145 different species and have established a highly accurate classification for all SNARE proteins. Principally, all SNAREs split into four basic types, reflecting their position in the four-helix bundle complex. Among these four basic types, we established 20 SNARE subclasses that probably represent the original repertoire of a eukaryotic cenancestor. This repertoire has been modulated independently in different lines of organisms. Our data are in line with the notion that the ur-eukaryotic cell was already equipped with the various compartments found in contemporary cells. Possibly, the development of these compartments is closely intertwined with episodes of duplication and divergence of a prototypic SNARE unit.

  3. Purification and characterization of a casein kinase 2-type protein kinase from pea nuclei

    NASA Technical Reports Server (NTRS)

    Li, H.; Roux, S. J.

    1992-01-01

    Almost all the polyamine-stimulated protein kinase activity associated with the chromatin fraction of nuclei purified from etiolated pea (Pisum sativum L.) plumules is present in a single enzyme that can be extracted from chromatin by 0.35 molar NaCl. This protein kinase can be further purified over 2000-fold by salt fractionation and anion-exchange and casein-agarose column chromatography, after which it is more than 90% pure. The purified kinase has a specific activity of about 650 nanomoles per minute per milligram protein in the absence of polyamines, with either ATP or GTP as phosphoryl donor. Spermidine can stimulate its activity fourfold, with half-maximal activation at about 2 millimolar. Spermine and putrescine also stimulate activity, although somewhat less effectively. This kinase has a tetrameric alpha 2 beta 2 structure with a native molecular weight of 130,000, and subunit molecular weights of 36,000 for the catalytic subunit (alpha) and 29,000 for the regulatory subunit (beta). In western blot analyses, only the alpha subunit reacts strongly with polyclonal antibodies to a Drosophila casein kinase II. The pea kinase can use casein and phosvitin as artificial substrates, phosphorylating both the serine and threonine residues of casein. It has a pH optimum near 8.0, a Vmax of 1.5 micromoles per minute per milligram protein, and a Km for ATP of approximately 75 micromolar. Its activity can be almost completely inhibited by heparin at 5 micrograms per milliliter, but is relatively insensitive to concentrations of staurosporine, K252a, and chlorpromazine that strongly antagonize Ca(2+) -regulated protein kinases. These results are discussed in relation to recent findings that casein kinase 2-type kinases may phosphorylate trans-acting factors that bind to light-regulated promoters in plants.

  4. Identification of positive-acting domains in GCN2 protein kinase required for translational activation of GCN4 expression

    SciTech Connect

    Wek, R.C.; Ramirez, M.; Jackson, B.M.; Hinnebusch, A.G. )

    1990-06-01

    GCN4 is a transcriptional activator of amino acid-biosynthetic genes in the yeast {ital Saccharomyces cerevisiae}. GCN2, a translational activator of {ital GCN4} expression, contains a domain homologous to the catalytic subunit of eukaryotic protein kinases. Substitution of a highly conserved lysine residue in the kinase domain abolished GCN2 regulatory function in vivo and its ability to autophosphorylate in vitro, indicating that GCN2 acts as a protein kinase in stimulating {ital GCN4} expression. Elevated {ital GCN2} gene dosage led to depression of {ital GCN4} under nonstarvation conditions; however, the authors found that {ital GCN2} mRNA and protein levels did not increase in wild-type cells in response to amino acid starvation. Therefore, it appears that GCN2 protein kinase function is stimulated postranslationally in amino acid-starved cells. Three dominant-constitutive {ital GCN2} point mutations were isolated that led to derepressed {ital GCN4} expression under nonstarvation conditions. Two of the {ital GCN2}(Con) mutations mapped in the kinase domain itself. The third mapped just downstream from a carboxyl-terminal segment homologous to histidyl-tRNA synthetase (HisRS), which the authors suggest might function to detect uncharged tRNA in amino acid-starved cells and activate the adjacent protein kinase moiety.

  5. A feature analysis of lower solubility proteins in three eukaryotic systems.

    PubMed

    Albu, Razvan F; Chan, Gerard T; Zhu, Mang; Wong, Eric T C; Taghizadeh, Farnaz; Hu, Xiaoke; Mehran, Arya E; Johnson, James D; Gsponer, Jörg; Mayor, Thibault

    2015-04-06

    Because misfolded and damaged proteins can form potentially harmful aggregates, all living organisms have evolved a wide variety of quality control mechanisms. However, the timely clearance of aggregation-prone species may not always be achieved, potentially leading to the accumulation of low solubility proteins. At the same time, promiscuity, which can be a driving force for aggregation, is also important to the functionality of certain proteins which have a large number of interaction partners. Considerable efforts have been made towards characterizing why some proteins appear to be more aggregation-prone than others. In this study, we analyze the features of proteins which precipitate following centrifugation in unstressed yeast cells, human SH-SY5Y cells and mouse brain tissue. By normalizing for protein abundance, we devised an approach whereby lower solubility proteins are reliably identified. Our findings indicate that these tend to be longer, low abundance proteins, which contain fewer hydrophobic amino acids. Furthermore, low solubility proteins also contain more low complexity and disordered regions. Overall, we observed an increase in features that link low solubility proteins to functional aggregates. Our results indicate that lower solubility proteins from three biologically distinct model systems share several common traits, shedding light on potentially universal solubility determinants. We set up a novel approach to identify lower solubility proteins in unstressed cells by comparing precipitated proteins with those that remain soluble after centrifugation. By analyzing three eukaryotic model systems in parallel, we were able to identify traits which cross the species barrier, as well as species-specific characteristics. Notably, our analyses revealed a number of primary and secondary structural features that set apart lower solubility proteins, a number of which connected them to a greater potential for promiscuity. This article is part of a Special

  6. Protein tyrosine kinase and mitogen-activated protein kinase signalling pathways contribute to differences in heterophil-mediated innate immune responsiveness between two lines of broilers

    USDA-ARS?s Scientific Manuscript database

    Protein tyrosine phosphorylation mediates signal transduction of cellular processes, with protein tyrosine kinases (PTKs) regulating virtually all signaling events. The mitogen-activated protein kinase (MAPK) super-family consists of three conserved pathways that convert receptor activation into ce...

  7. Comparative analysis of eukaryotic-type protein phosphatases in two Streptomyces genomes

    SciTech Connect

    Shi, Liang; Zhang, Weiwen

    2004-07-01

    Summary - Inspection of the genomes of Streptomyces coelicolor and S. avermitilis reveals that each contains 55 putative eukaryotic-type protein phosphatases (PPs), the largest number ever identified from any single prokaryotic organism. Unlike most other prokaryotic genomes, that have only one or two super-families of protein phosphatases, the Streptomyces genomes possess 4 different super-families of protein phosphatases: 2 PPPs and 2 LMWPTPs in each species, 49 PPMs and 2 CPTPs in S. coelicolor, and 48 PPMs and 3 CPTPs in S. avermitilis. Sixty four percent of the PPs found in S. coelicolor have orthologs in S. avermitilis, indicating that they originated from a common ancestor and may be involved in the regulation of more conversed metabolic activities...

  8. Protein kinase C activators inhibit capillary endothelial cell growth

    SciTech Connect

    Doctrow, S.R.

    1986-05-01

    Phorbol 12,13-dibutyrate (PDBu) binds specifically to bovine capillary endothelial (BCE) cells (K/sub d/ = 8nM) and inhibits the proliferation (K/sub 50/ = 6 +/- 4 nM). Under similar conditions, PDBu does not inhibit the growth of bovine aortic endothelial or smooth muscle cells. PDBu markedly attenuates the response of BCE cells to purified human hepatoma-derived growth factor which, in the absence of PDBu, stimulates BCE cell growth by about 3-fold. Several observations suggest that the inhibition of BCE cell growth by PDBu is mediated by protein kinase C: (1) different phorbol compounds inhibit BCE cell growth according to the relative potencies as protein kinase C activators (12-tetradecanoylphorbol 13-acetate > PDBu >> phorbol 12,13-diacetate >>>..beta..-phorbol; ..cap alpha..-phorbol 12,13-didecanoate). (2) Specific binding of PDBu to BCE cells is displaced by sn-1,2-dioctanoylglycerol (diC/sub 8/), a protein kinase C activator and an analog of the putative second messenger activating this kinase in vivo. The weak protein kinase C activator, sn-1,2-dibutyrylglycerol, does not affect PDBu binding. (3) A cytosolic extract from BCE cells contains a Ca/sup 2 +//phosphatidylserine-dependent kinase that is activated by diC/sub 8/ and PDBu, but not by ..beta..-phorbol. These results support a role for protein kinase C in suppressing capillary endothelial cell growth and may therefore have implications in the intracellular regulation of angiogenesis.

  9. Interaction of SNF1 Protein Kinase with Its Activating Kinase Sak1▿

    PubMed Central

    Liu, Yang; Xu, Xinjing; Carlson, Marian

    2011-01-01

    The Saccharomyces cerevisiae SNF1 protein kinase, a member of the SNF1/AMP-activated protein kinase (AMPK) family, is activated by three kinases, Sak1, Tos3, and Elm1, which phosphorylate the Snf1 catalytic subunit on Thr-210 in response to glucose limitation and other stresses. Sak1 is the primary Snf1-activating kinase and is associated with Snf1 in a complex. Here we examine the interaction of Sak1 with SNF1. We report that Sak1 coimmunopurifies with the Snf1 catalytic subunit from extracts of both glucose-replete and glucose-limited cultures and that interaction occurs independently of the phosphorylation state of Snf1 Thr-210, Snf1 catalytic activity, and other SNF1 subunits. Sak1 interacts with the Snf1 kinase domain, and nonconserved sequences C terminal to the Sak1 kinase domain mediate interaction with Snf1 and augment the phosphorylation and activation of Snf1. The Sak1 C terminus is modified in response to glucose depletion, dependent on SNF1 activity. Replacement of the C terminus of Elm1 (or Tos3) with that of Sak1 enhanced the ability of the Elm1 kinase domain to interact with and phosphorylate Snf1. These findings indicate that the C terminus of Sak1 confers its function as the primary Snf1-activating kinase and suggest that the physical association of Sak1 with SNF1 facilitates responses to environmental change. PMID:21216941

  10. Shedding light on the expansion and diversification of the Cdc48 protein family during the rise of the eukaryotic cell.

    PubMed

    Kienle, Nickias; Kloepper, Tobias H; Fasshauer, Dirk

    2016-10-18

    A defining feature of eukaryotic cells is the presence of various distinct membrane-bound compartments with different metabolic roles. Material exchange between most compartments occurs via a sophisticated vesicle trafficking system. This intricate cellular architecture of eukaryotes appears to have emerged suddenly, about 2 billion years ago, from much less complex ancestors. How the eukaryotic cell acquired its internal complexity is poorly understood, partly because no prokaryotic precursors have been found for many key factors involved in compartmentalization. One exception is the Cdc48 protein family, which consists of several distinct classical ATPases associated with various cellular activities (AAA+) proteins with two consecutive AAA domains. Here, we have classified the Cdc48 family through iterative use of hidden Markov models and tree building. We found only one type, Cdc48, in prokaryotes, although a set of eight diverged members that function at distinct subcellular compartments were retrieved from eukaryotes and were probably present in the last eukaryotic common ancestor (LECA). Pronounced changes in sequence and domain structure during the radiation into the LECA set are delineated. Moreover, our analysis brings to light lineage-specific losses and duplications that often reflect important biological changes. Remarkably, we also found evidence for internal duplications within the LECA set that probably occurred during the rise of the eukaryotic cell. Our analysis corroborates the idea that the diversification of the Cdc48 family is closely intertwined with the development of the compartments of the eukaryotic cell.

  11. How protein kinases co-ordinate mitosis in animal cells.

    PubMed

    Ma, Hoi Tang; Poon, Randy Y C

    2011-04-01

    Mitosis is associated with profound changes in cell physiology and a spectacular surge in protein phosphorylation. To accomplish these, a remarkably large portion of the kinome is involved in the process. In the present review, we will focus on classic mitotic kinases, such as cyclin-dependent kinases, Polo-like kinases and Aurora kinases, as well as more recently characterized players such as NIMA (never in mitosis in Aspergillus nidulans)-related kinases, Greatwall and Haspin. Together, these kinases co-ordinate the proper timing and fidelity of processes including centrosomal functions, spindle assembly and microtubule-kinetochore attachment, as well as sister chromatid separation and cytokinesis. A recurrent theme of the mitotic kinase network is the prevalence of elaborated feedback loops that ensure bistable conditions. Sequential phosphorylation and priming phosphorylation on substrates are also frequently employed. Another important concept is the role of scaffolds, such as centrosomes for protein kinases during mitosis. Elucidating the entire repertoire of mitotic kinases, their functions, regulation and interactions is critical for our understanding of normal cell growth and in diseases such as cancers.

  12. Sensing of energy and nutrients by AMP-activated protein kinase.

    PubMed

    Hardie, D Grahame

    2011-04-01

    AMP-activated protein kinase (AMPK) is a cellular energy sensor that exists in almost all eukaryotes. Genetic studies in lower eukaryotes suggest that the ancestral role of AMPK was in response to starvation for a carbon source and that AMPK is involved in life-span extension in response to caloric restriction. In mammals, AMPK is activated by an increasing cellular AMP:ATP ratio (which signifies a decrease in energy) caused by metabolic stresses that interfere with ATP production (eg, hypoxia) or that accelerate ATP consumption (eg, muscle contraction). Because glucose deprivation can increase the AMP:ATP ratio, AMPK can also act as a glucose sensor. AMPK activation occurs by a dual mechanism that involves allosteric activation and phosphorylation by upstream kinases. Once activated, AMPK switches on catabolic pathways that generate ATP (eg, the uptake and oxidation of glucose and fatty acids and mitochondrial biogenesis) while switching off ATP-consuming, anabolic pathways (eg, the synthesis of lipids, glucose, glycogen, and proteins). In addition to the acute effects via direct phosphorylation of metabolic enzymes, AMPK has longer-term effects by regulating transcription. These features make AMPK an ideal drug target in the treatment of metabolic disorders such as insulin resistance and type 2 diabetes. The antidiabetic drug metformin (which is derived from an herbal remedy) works in part by activating AMPK, whereas many xenobiotics or "nutraceuticals," including resveratrol, quercetin, and berberine, are also AMPK activators. Most of these agents activate AMPK because they inhibit mitochondrial function.

  13. Regulatory Crosstalk by Protein Kinases on CFTR Trafficking and Activity

    PubMed Central

    Farinha, Carlos M.; Swiatecka-Urban, Agnieszka; Brautigan, David L.; Jordan, Peter

    2016-01-01

    Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a member of the ATP binding cassette (ABC) transporter superfamily that functions as a cAMP-activated chloride ion channel in fluid-transporting epithelia. There is abundant evidence that CFTR activity (i.e., channel opening and closing) is regulated by protein kinases and phosphatases via phosphorylation and dephosphorylation. Here, we review recent evidence for the role of protein kinases in regulation of CFTR delivery to and retention in the plasma membrane. We review this information in a broader context of regulation of other transporters by protein kinases because the overall functional output of transporters involves the integrated control of both their number at the plasma membrane and their specific activity. While many details of the regulation of intracellular distribution of CFTR and other transporters remain to be elucidated, we hope that this review will motivate research providing new insights into how protein kinases control membrane transport to impact health and disease. PMID:26835446

  14. Regulatory crosstalk by protein kinases on CFTR trafficking and activity

    NASA Astrophysics Data System (ADS)

    Farinha, Carlos Miguel; Swiatecka-Urban, Agnieszka; Brautigan, David; Jordan, Peter

    2016-01-01

    Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a member of the ATP binding cassette (ABC) transporter superfamily that functions as a cAMP-activated chloride ion channel in fluid-transporting epithelia. There is abundant evidence that CFTR activity (i.e. channel opening and closing) is regulated by protein kinases and phosphatases via phosphorylation and dephosphorylation. Here, we review recent evidence for the role of protein kinases in regulation of CFTR delivery to and retention in the plasma membrane. We review this information in a broader context of regulation of other transporters by protein kinases because the overall functional output of transporters involves the integrated control of both their number at the plasma membrane and their specific activity. While many details of the regulation of intracellular distribution of CFTR and other transporters remain to be elucidated, we hope that this review will motivate research providing new insights into how protein kinases control membrane transport to impact health and disease.

  15. Characterization of microtubule-associated protein 1-associated protein kinases from rat brain.

    PubMed

    Fujii, T; Watanabe, M; Nakamura, A

    1996-01-01

    The microtubule-associated protein (MAP) 1 preparation, MAP1A and 1B, obtained from rat brain microtubules was associated with protein kinases that were insensitive to cAMP, cGMP, calcium, calcium/calmodulin and calcium/phosphatidylserine. The fractionation of highly purified MAP1 by phosphocellulose chromatography revealed that protein kinase activity to phosphorylate phosvitin was separated into three major peaks (MAP1 kinases A, B and C). MAP1 was recovered in the MAP1 kinase A fraction and phosphorylated by the contained kinase. MAP1 kinase A is a novel protein kinase that is remarkably activated by poly-L-lysine and poly-L-arginine, but very insensitive to heparin among the kinases. Photoaffinity labeling using [alpha-32P]8-azido ATP indicated that the 65 kDa polypeptide is identified as an ATP-binding protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the highly purified MAP1 and MAP1 kinase A fractions. MAP1 kinases B and C may be identified as casein kinase I- and II-like kinases. The present results show that MAP1 is associated with at least three kinases and provide an insight for understanding thoroughly the MAP1-mediated microtubule functions.

  16. Prolyl hydroxylase-dependent modulation of eukaryotic elongation factor 2 activity and protein translation under acute hypoxia.

    PubMed

    Romero-Ruiz, Antonio; Bautista, Lucía; Navarro, Virginia; Heras-Garvín, Antonio; March-Díaz, Rosana; Castellano, Antonio; Gómez-Díaz, Raquel; Castro, María J; Berra, Edurne; López-Barneo, José; Pascual, Alberto

    2012-03-16

    Early adaptive responses to hypoxia are essential for cell survival, but their nature and underlying mechanisms are poorly known. We have studied the post-transcriptional changes in the proteome of mammalian cells elicited by acute hypoxia and found that phosphorylation of eukaryotic elongation factor 2 (eEF2), a ribosomal translocase whose phosphorylation inhibits protein synthesis, is under the precise and reversible control of O(2) tension. Upon exposure to hypoxia, phosphorylation of eEF2 at Thr(56) occurred rapidly (<15 min) and resulted in modest translational arrest, a fundamental homeostatic response to hypoxia that spares ATP and thus facilitates cell survival. Acute inhibitory eEF2 phosphorylation occurred without ATP depletion or AMP kinase activation. Furthermore, eEF2 phosphorylation was mimicked by prolyl hydroxylase (PHD) inhibition with dimethyloxalylglycine or by selective PHD2 siRNA silencing but was independent of hypoxia-inducible factor α stabilization. Moreover, overexpression of PHD2 blocked hypoxic accumulation of phosphorylated eEF2. Therefore, our findings suggest that eEF2 phosphorylation status (and, as a consequence, translation rate) is controlled by PHD2 activity. They unravel a novel pathway for cell adaptation to hypoxia that could have pathophysiologic relevance in tissue ischemia and cancer.

  17. Automated production of functional membrane proteins using eukaryotic cell-free translation systems.

    PubMed

    Quast, Robert B; Kortt, Oliver; Henkel, Jörg; Dondapati, Srujan K; Wüstenhagen, Doreen A; Stech, Marlitt; Kubick, Stefan

    2015-06-10

    Due to their high abundance and pharmacological relevance there is a growing demand for the efficient production of functional membrane proteins. In this context, cell-free protein synthesis represents a valuable alternative that allows for the high-throughput synthesis of functional membrane proteins. Here, we demonstrate the potential of our cell-free protein synthesis system, based on lysates from cultured Spodoptera frugiperda 21 cells, to produce pro- and eukaryotic membrane proteins with individual topological characteristics in an automated fashion. Analytical techniques, including confocal laser scanning microscopy, fluorescence detection of eYFP fusion proteins in a microplate reader and in-gel fluorescence of statistically incorporated fluorescent amino acid derivatives were employed. The reproducibility of our automated synthesis approach is underlined by coefficients of variation below 7.2%. Moreover, the functionality of the cell-free synthesized potassium channel KcsA was analyzed electrophysiologically. Finally, we expanded our cell-free membrane protein synthesis system by an orthogonal tRNA/synthetase pair for the site-directed incorporation of p-Azido-l-phenylalanine based on stop codon suppression. Incorporation was optimized by performance of a two-dimensional screening with different Mg(2+) and lysate concentrations. Subsequently, the selective modification of membrane proteins with incorporated p-Azido-l-phenylalanine was exemplified by Staudinger ligation with a phosphine-based fluorescence dye. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  18. Highly hydrophilic proteins in prokaryotes and eukaryotes are common during conditions of water deficit.

    PubMed

    Garay-Arroyo, A; Colmenero-Flores, J M; Garciarrubio, A; Covarrubias, A A

    2000-02-25

    The late embryogenesis abundant (LEA) proteins are plant proteins that are synthesized at the onset of desiccation in maturing seeds and in vegetative organs exposed to water deficit. Here, we show that most LEA proteins are comprised in a more widespread group, which we call "hydrophilins." The defining characteristics of hydrophilins are high glycine content (>6%) and a high hydrophilicity index (>1.0). By data base searching, we show that this criterion selectively differentiates most known LEA proteins as well as additional proteins from different taxons. We found that within the genomes of Escherichia coli and Saccharomyces cerevisiae, only 5 and 12 proteins, respectively, meet our criterion. Despite their deceivingly loose definition, hydrophilins usually represent <0.2% of the proteins of a genome. Additionally, we demonstrate that the criterion that defines hydrophilins seems to be an excellent predictor of responsiveness to hyperosmosis since most of the genes encoding these proteins in E. coli and S. cerevisiae are induced by osmotic stress. Evidence for the participation of one of the E. coli hydrophilins in the adaptive response to hyperosmotic conditions is presented. Apparently, hydrophilins represent analogous adaptations to a common problem in such diverse taxons as prokaryotes and eukaryotes.

  19. Physiological roles of the Ca2+/CaM-dependent protein kinase cascade in health and disease.

    PubMed

    Colomer, J; Means, A R

    2007-01-01

    Numerous hormones, growth factors and physiological processes cause a rise in cytosolic Ca2+, which is translated into meaningful cellular responses by interacting with a large number of Ca2(+)-binding proteins. The Ca2(+)-binding protein that is most pervasive in mediating these responses is calmodulin (CaM), which acts as a primary receptor for Ca2+ in all eukaryotic cells. In turn, Ca2+/CaM functions as an allosteric activator of a host of enzymatic proteins including a considerable number of protein kinases. The topic of this review is to discuss the physiological roles of a sub-set of these protein kinases which can function in cells as a Ca2+/CaM-dependent kinase signaling cascade. The cascade was originally believed to consist of a CaM kinase kinase that phosphorylates and activates one of two CaM kinases, CaMKI or CaMKIV. The unusual aspect of this cascade is that both the kinase kinase and the kinase require the binding of Ca2+/CaM for activation. More recently, one of the CaM kinase kinases has been found to activate another important enzyme, the AMP-dependent protein kinase so the concept of the CaM kinase cascade must be expanded. A CaM kinase cascade is important for many normal physiological processes that when misregulated can lead to a variety of disease states. These processes include: cell proliferation and apoptosis that may conspire in the genesis of cancer; neuronal growth and function related to brain development, synaptic plasticity as well as memory formation and maintenance; proper function of the immune system including the inflammatory response, activation of T lymphocytes and hematopoietic stem cell maintenance; and the central control of energy balance that, when altered, can lead to obesity and diabetes. Although the study of the CaM-dependent kinase cascades is still in its infancy continued analysis of the pathways regulated by these Ca2(+)-initiated signaling cascades holds considerable promise for the future of disease

  20. Eukaryotic G Protein Signaling Evolved to Require G Protein–Coupled Receptors for Activation

    PubMed Central

    Bradford, William; Buckholz, Adam; Morton, John; Price, Collin; Jones, Alan M.; Urano, Daisuke

    2016-01-01

    Although bioinformatic analysis of the increasing numbers of diverse genome sequences and amount of functional data has provided insight into the evolution of signaling networks, bioinformatics approaches have limited application for understanding the evolution of highly divergent protein families. We used biochemical analyses to determine the in vitro properties of selected divergent components of the heterotrimeric guanine nucleotide–binding protein (G protein) signaling network to investigate signaling network evolution. In animals, G proteins are activated by cell-surface seven-transmembrane (7TM) receptors, which are named G protein–coupled receptors (GPCRs) and function as guanine nucleotide exchange factors (GEFs). In contrast, the plant G protein is intrinsically active, and a 7TM protein terminates G protein activity by functioning as a guanosine triphosphatase–activating protein (GAP). We showed that ancient regulation of the G protein active state is GPCR-independent and “self-activating,” a property that is maintained in Bikonts, one of the two fundamental evolutionary clades containing eukaryotes, whereas G proteins of the other clade, the Unikonts, evolved from being GEF-independent to being GEF-dependent. Self-activating G proteins near the base of the Eukaryota are controlled by 7TM-GAPs, suggesting that the ancestral regulator of G protein activation was a GAP-functioning receptor, not a GEF-functioning GPCR. Our findings indicate that the GPCR paradigm describes a recently evolved network architecture found in a relatively small group of Eukaryota and suggest that the evolution of signaling network architecture is constrained by the availability of molecules that control the activation state of nexus proteins. PMID:23695163

  1. Regulated protein kinases and phosphatases in cell cycle decisions.

    PubMed

    Novak, Bela; Kapuy, Orsolya; Domingo-Sananes, Maria Rosa; Tyson, John J

    2010-12-01

    Many aspects of cell physiology are controlled by protein kinases and phosphatases, which together determine the phosphorylation state of targeted substrates. Some of these target proteins are themselves kinases or phosphatases or other components of a regulatory network characterized by feedback and feed-forward loops. In this review we describe some common regulatory motifs involving kinases, phosphatases, and their substrates, focusing particularly on bistable switches involved in cellular decision processes. These general principles are applied to cell cycle transitions, with special emphasis on the roles of regulated phosphatases in orchestrating progression from one phase to the next of the DNA replication-division cycle.

  2. Regulated protein kinases and phosphatases in cell cycle decisions

    PubMed Central

    Novak, Bela; Kapuy, Orsolya; Domingo-Sananes, Maria Rosa; Tyson, John J

    2013-01-01

    Many aspects of cell physiology are controlled by protein kinases and phosphatases, which together determine the phosphorylation state of targeted substrates. Some of these target proteins are themselves kinases or phosphatases or other components of a regulatory network characterized by feedback and feed-forward loops. In this review we describe some common regulatory motifs involving kinases, phosphatases, and their substrates, focusing particularly on bistable switches involved in cellular decision processes. These general principles are applied to cell cycle transitions, with special emphasis on the roles of regulated phosphatases in orchestrating progression from one phase to the next of the DNA replication-division cycle. PMID:20678910

  3. Fluorescent sensors of protein kinases: from basics to biomedical applications.

    PubMed

    Nhu Ngoc Van, Thi; Morris, May C

    2013-01-01

    Protein kinases constitute a major class of enzymes underlying essentially all biological processes. These enzymes present similar structural folds, yet their mechanism of action and of regulation vary largely, as well as their substrate specificity and their subcellular localization. Classical approaches to study the function/activity of protein kinases rely on radioactive endpoint assays, which do not allow for characterization of their dynamic activity in their native environment. The development of fluorescent biosensors has provided a whole new avenue for studying protein kinase behavior and regulation in living cells in real time with high spatial and temporal resolution. Two major classes of biosensors have been developed: genetically encoded single-chain fluorescence resonance energy transfer biosensors and peptide/protein biosensors coupled to small synthetic fluorophores which are sensitive to changes in their environment. In this review, we discuss the developments in fluorescent biosensor technology related to protein kinase sensing and the different strategies employed to monitor protein kinase activity, conformation, or relative abundance, as well as kinase regulation and subcellular dynamics in living cells. Moreover, we discuss their application in biomedical settings, for diagnostics and therapeutics, to image disease progression and monitor response to therapeutics, in drug discovery programs, for high-throughput screening assays, for postscreen characterization of drug candidates, and for clinical evaluation of novel drugs. Copyright © 2013 Elsevier Inc. All rights reserved.

  4. PKR and GCN2 kinases and guanine nucleotide exchange factor eukaryotic translation initiation factor 2B (eIF2B) recognize overlapping surfaces on eIF2alpha.

    PubMed

    Dey, Madhusudan; Trieselmann, Bruce; Locke, Emily G; Lu, Jingfang; Cao, Chune; Dar, Arvin C; Krishnamoorthy, Thanuja; Dong, Jinsheng; Sicheri, Frank; Dever, Thomas E

    2005-04-01

    Four stress-responsive protein kinases, including GCN2 and PKR, phosphorylate eukaryotic translation initiation factor 2alpha (eIF2alpha) on Ser51 to regulate general and gene-specific protein synthesis. Phosphorylated eIF2 is an inhibitor of its guanine nucleotide exchange factor, eIF2B. Mutations that block translational regulation were isolated throughout the N-terminal OB-fold domain in Saccharomyces cerevisiae eIF2alpha, including those at residues flanking Ser51 and around 20 A away in the conserved motif K79GYID83. Any mutation at Glu49 or Asp83 blocked translational regulation; however, only a subset of these mutations impaired Ser51 phosphorylation. Substitution of Ala for Asp83 eliminated phosphorylation by GCN2 and PKR both in vivo and in vitro, establishing the critical contributions of remote residues to kinase-substrate recognition. In contrast, mutations that blocked translational regulation but not Ser51 phosphorylation impaired the binding of eIF2B to phosphorylated eIF2alpha. Thus, two structurally distinct effectors of eIF2 function, eIF2alpha kinases and eIF2B, have evolved to recognize the same surface and overlapping determinants on eIF2alpha.

  5. The protein activator of protein kinase R, PACT/RAX, negatively regulates protein kinase R during mouse anterior pituitary development

    PubMed Central

    Dickerman, Benjamin K.; White, Christine L.; Kessler, Patricia M.; Sadler, Anthony J.; Williams, Bryan R.G.; Sen, Ganes C.

    2015-01-01

    The murine double-stranded RNA-binding protein RAX and the human homolog PACT were originally characterized as activators of protein kinase R (PKR). Mice deficient in RAX show reproductive and developmental defects, including reduced body size, craniofacial defects and anterior pituitary hypoplasia. As these defects are not observed in PKR-deficient mice, the phenotype has been attributed to PKR-independent activities of RAX. Here we further investigated the involvement of PKR in the physiological function of RAX, by generating rax−/− mice deficient in PKR, or carrying a kinase-inactive mutant of PKR (K271R) or an unphosphorylatable mutant of the PKR substrate eIF2α (S51A). Ablating PKR expression rescued the developmental and reproductive deficiencies in rax−/− mice. Generating rax−/− mice with a kinase-inactive mutant of PKR resulted in similar rescue, confirming that the rax−/− defects are PKR dependent; specifically that the kinase activity of PKR was required for these defects. Moreover, generating rax−/− mice that were heterozygous for an unphosphorylatable mutant eIF2α provides partial rescue of the rax−/− defect, consistent with mutation of one copy of the Eif2s1 gene. These observations were further investigated in vitro by reducing RAX expression in anterior pituitary cells, resulting in increased PKR activity and induction of the PKR-regulated cyclin-dependent kinase inhibitor p21WAF1/CIP1. These results demonstrate that PKR kinase activity is required for onset of the rax−/− phenotype, implying an unexpected function for RAX as a negative regulator of PKR in the context of postnatal anterior pituitary tissue, and identify a critical role for the regulation of PKR activity for normal development. PMID:26414443

  6. Casein kinase 1δ-dependent Wee1 protein degradation.

    PubMed

    Penas, Clara; Ramachandran, Vimal; Simanski, Scott; Lee, Choogon; Madoux, Franck; Rahaim, Ronald J; Chauhan, Ruchi; Barnaby, Omar; Schurer, Stephan; Hodder, Peter; Steen, Judith; Roush, William R; Ayad, Nagi G

    2014-07-04

    Eukaryotic mitotic entry is controlled by Cdk1, which is activated by the Cdc25 phosphatase and inhibited by Wee1 tyrosine kinase, a target of the ubiquitin proteasome pathway. Here we use a reporter of Wee1 degradation, K328M-Wee1-luciferase, to screen a kinase-directed chemical library. Hit profiling identified CK1δ-dependent Wee1 degradation. Small-molecule CK1δ inhibitors specifically disrupted Wee1 destruction and arrested HeLa cell proliferation. Pharmacological inhibition, siRNA knockdown, or conditional deletion of CK1δ also reduced Wee1 turnover. Thus, these studies define a previously unappreciated role for CK1δ in controlling the cell cycle.

  7. Universal distribution of mutational effects on protein stability, uncoupling of protein robustness from sequence evolution and distinct evolutionary modes of prokaryotic and eukaryotic proteins

    PubMed Central

    Faure, Guilhem; Koonin, Eugene V.

    2016-01-01

    Robustness to destabilizing effects of mutations is thought of as a key factor of protein evolution. The connections between two measures of robustness, the relative core size and the computationally estimated effect of mutations on protein stability (ΔΔG), protein abundance and the selection pressure on protein-coding genes (dN/dS) were analyzed for the organisms with a large number of available protein structures including four eukaryotes, two bacteria and one archaeon. The distribution of the effects of mutations in the core on protein stability is universal and indistinguishable in eukaryotes and bacteria, centered at slightly destabilizing amino acid replacements, and with a heavy tail of more strongly destabilizing replacements. The distribution of mutational effects in the hyperthermophilic archaeon Thermococcus gammatolerans is significantly shifted toward strongly destabilizing replacements which is indicative of stronger constraints that are imposed on proteins in hyperthermophiles. The median effect of mutations is strongly, positively correlated with the relative core size, in evidence of the congruence between the two measures of protein robustness. However, both measures show only limited correlations to the expression level and selection pressure on protein-coding genes. Thus, the degree of robustness reflected in the universal distribution of mutational effects appears to be a fundamental, ancient feature of globular protein folds whereas the observed variations are largely neutral and uncoupled from short term protein evolution. A weak anticorrelation between protein core size and selection pressure is observed only for surface residues in prokaryotes but a stronger anticorrelation is observed for all residues in eukaryotic proteins. This substantial difference between proteins of prokaryotes and eukaryotes is likely to stem from the demonstrable higher compactness of prokaryotic proteins. PMID:25927823

  8. Universal distribution of mutational effects on protein stability, uncoupling of protein robustness from sequence evolution and distinct evolutionary modes of prokaryotic and eukaryotic proteins

    NASA Astrophysics Data System (ADS)

    Faure, Guilhem; Koonin, Eugene V.

    2015-05-01

    Robustness to destabilizing effects of mutations is thought of as a key factor of protein evolution. The connections between two measures of robustness, the relative core size and the computationally estimated effect of mutations on protein stability (ΔΔG), protein abundance and the selection pressure on protein-coding genes (dN/dS) were analyzed for the organisms with a large number of available protein structures including four eukaryotes, two bacteria and one archaeon. The distribution of the effects of mutations in the core on protein stability is universal and indistinguishable in eukaryotes and bacteria, centered at slightly destabilizing amino acid replacements, and with a heavy tail of more strongly destabilizing replacements. The distribution of mutational effects in the hyperthermophilic archaeon Thermococcus gammatolerans is significantly shifted toward strongly destabilizing replacements which is indicative of stronger constraints that are imposed on proteins in hyperthermophiles. The median effect of mutations is strongly, positively correlated with the relative core size, in evidence of the congruence between the two measures of protein robustness. However, both measures show only limited correlations to the expression level and selection pressure on protein-coding genes. Thus, the degree of robustness reflected in the universal distribution of mutational effects appears to be a fundamental, ancient feature of globular protein folds whereas the observed variations are largely neutral and uncoupled from short term protein evolution. A weak anticorrelation between protein core size and selection pressure is observed only for surface residues in prokaryotes but a stronger anticorrelation is observed for all residues in eukaryotic proteins. This substantial difference between proteins of prokaryotes and eukaryotes is likely to stem from the demonstrable higher compactness of prokaryotic proteins.

  9. Universal distribution of mutational effects on protein stability, uncoupling of protein robustness from sequence evolution and distinct evolutionary modes of prokaryotic and eukaryotic proteins.

    PubMed

    Faure, Guilhem; Koonin, Eugene V

    2015-04-30

    Robustness to destabilizing effects of mutations is thought of as a key factor of protein evolution. The connections between two measures of robustness, the relative core size and the computationally estimated effect of mutations on protein stability (ΔΔG), protein abundance and the selection pressure on protein-coding genes (dN/dS) were analyzed for the organisms with a large number of available protein structures including four eukaryotes, two bacteria and one archaeon. The distribution of the effects of mutations in the core on protein stability is universal and indistinguishable in eukaryotes and bacteria, centered at slightly destabilizing amino acid replacements, and with a heavy tail of more strongly destabilizing replacements. The distribution of mutational effects in the hyperthermophilic archaeon Thermococcus gammatolerans is significantly shifted toward strongly destabilizing replacements which is indicative of stronger constraints that are imposed on proteins in hyperthermophiles. The median effect of mutations is strongly, positively correlated with the relative core size, in evidence of the congruence between the two measures of protein robustness. However, both measures show only limited correlations to the expression level and selection pressure on protein-coding genes. Thus, the degree of robustness reflected in the universal distribution of mutational effects appears to be a fundamental, ancient feature of globular protein folds whereas the observed variations are largely neutral and uncoupled from short term protein evolution. A weak anticorrelation between protein core size and selection pressure is observed only for surface residues in prokaryotes but a stronger anticorrelation is observed for all residues in eukaryotic proteins. This substantial difference between proteins of prokaryotes and eukaryotes is likely to stem from the demonstrable higher compactness of prokaryotic proteins.

  10. The C-terminal tail of protein kinase D2 and protein kinase D3 regulates their intracellular distribution

    SciTech Connect

    Papazyan, Romeo; Rozengurt, Enrique; Rey, Osvaldo . E-mail: orey@mednet.ucla.edu

    2006-04-14

    We generated a set of GFP-tagged chimeras between protein kinase D2 (PKD2) and protein kinase D3 (PKD3) to examine in live cells the contribution of their C-terminal region to their intracellular localization. We found that the catalytic domain of PKD2 and PKD3 can localize to the nucleus when expressed without other kinase domains. However, when the C-terminal tail of PKD2 was added to its catalytic domain, the nuclear localization of the resulting protein was inhibited. In contrast, the nuclear localization of the CD of PKD3 was not inhibited by its C-terminal tail. Furthermore, the exchange of the C-terminal tail of PKD2 and PKD3 in the full-length proteins was sufficient to exchange their intracellular localization. Collectively, these data demonstrate that the short C-terminal tail of these kinases plays a critical role in determining their cytoplasmic/nuclear localization.

  11. Protein Kinases and Parkinson’s Disease

    PubMed Central

    Mehdi, Syed Jafar; Rosas-Hernandez, Hector; Cuevas, Elvis; Lantz, Susan M.; Barger, Steven W.; Sarkar, Sumit; Paule, Merle G.; Ali, Syed F.; Imam, Syed Z.

    2016-01-01

    Currently, the lack of new drug candidates for the treatment of major neurological disorders such as Parkinson’s disease has intensified the search for drugs that can be repurposed or repositioned for such treatment. Typically, the search focuses on drugs that have been approved and are used clinically for other indications. Kinase inhibitors represent a family of popular molecules for the treatment and prevention of various cancers, and have emerged as strong candidates for such repurposing because numerous serine/threonine and tyrosine kinases have been implicated in the pathobiology of Parkinson’s disease. This review focuses on various kinase-dependent pathways associated with the expression of Parkinson’s disease pathology, and evaluates how inhibitors of these pathways might play a major role as effective therapeutic molecules. PMID:27657053

  12. Nonconsensus Protein Binding to Repetitive DNA Sequence Elements Significantly Affects Eukaryotic Genomes

    PubMed Central

    Barber-Zucker, Shiran; Gordân, Raluca; Lukatsky, David B.

    2015-01-01

    Recent genome-wide experiments in different eukaryotic genomes provide an unprecedented view of transcription factor (TF) binding locations and of nucleosome occupancy. These experiments revealed that a large fraction of TF binding events occur in regions where only a small number of specific TF binding sites (TFBSs) have been detected. Furthermore, in vitro protein-DNA binding measurements performed for hundreds of TFs indicate that TFs are bound with wide range of affinities to different DNA sequences that lack known consensus motifs. These observations have thus challenged the classical picture of specific protein-DNA binding and strongly suggest the existence of additional recognition mechanisms that affect protein-DNA binding preferences. We have previously demonstrated that repetitive DNA sequence elements characterized by certain symmetries statistically affect protein-DNA binding preferences. We call this binding mechanism nonconsensus protein-DNA binding in order to emphasize the point that specific consensus TFBSs do not contribute to this effect. In this paper, using the simple statistical mechanics model developed previously, we calculate the nonconsensus protein-DNA binding free energy for the entire C. elegans and D. melanogaster genomes. Using the available chromatin immunoprecipitation followed by sequencing (ChIP-seq) results on TF-DNA binding preferences for ~100 TFs, we show that DNA sequences characterized by low predicted free energy of nonconsensus binding have statistically higher experimental TF occupancy and lower nucleosome occupancy than sequences characterized by high free energy of nonconsensus binding. This is in agreement with our previous analysis performed for the yeast genome. We suggest therefore that nonconsensus protein-DNA binding assists the formation of nucleosome-free regions, as TFs outcompete nucleosomes at genomic locations with enhanced nonconsensus binding. In addition, here we perform a new, large-scale analysis using

  13. Nonconsensus Protein Binding to Repetitive DNA Sequence Elements Significantly Affects Eukaryotic Genomes.

    PubMed

    Afek, Ariel; Cohen, Hila; Barber-Zucker, Shiran; Gordân, Raluca; Lukatsky, David B

    2015-08-01

    Recent genome-wide experiments in different eukaryotic genomes provide an unprecedented view of transcription factor (TF) binding locations and of nucleosome occupancy. These experiments revealed that a large fraction of TF binding events occur in regions where only a small number of specific TF binding sites (TFBSs) have been detected. Furthermore, in vitro protein-DNA binding measurements performed for hundreds of TFs indicate that TFs are bound with wide range of affinities to different DNA sequences that lack known consensus motifs. These observations have thus challenged the classical picture of specific protein-DNA binding and strongly suggest the existence of additional recognition mechanisms that affect protein-DNA binding preferences. We have previously demonstrated that repetitive DNA sequence elements characterized by certain symmetries statistically affect protein-DNA binding preferences. We call this binding mechanism nonconsensus protein-DNA binding in order to emphasize the point that specific consensus TFBSs do not contribute to this effect. In this paper, using the simple statistical mechanics model developed previously, we calculate the nonconsensus protein-DNA binding free energy for the entire C. elegans and D. melanogaster genomes. Using the available chromatin immunoprecipitation followed by sequencing (ChIP-seq) results on TF-DNA binding preferences for ~100 TFs, we show that DNA sequences characterized by low predicted free energy of nonconsensus binding have statistically higher experimental TF occupancy and lower nucleosome occupancy than sequences characterized by high free energy of nonconsensus binding. This is in agreement with our previous analysis performed for the yeast genome. We suggest therefore that nonconsensus protein-DNA binding assists the formation of nucleosome-free regions, as TFs outcompete nucleosomes at genomic locations with enhanced nonconsensus binding. In addition, here we perform a new, large-scale analysis using

  14. Genome-Wide Identification, Evolution, and Co-expression Network Analysis of Mitogen-Activated Protein Kinase Kinase Kinases in Brachypodium distachyon

    PubMed Central

    Feng, Kewei; Liu, Fuyan; Zou, Jinwei; Xing, Guangwei; Deng, Pingchuan; Song, Weining; Tong, Wei; Nie, Xiaojun

    2016-01-01

    Mitogen-activated protein kinase (MAPK) cascades are the conserved and universal signal transduction modules in all eukaryotes, which play the vital roles in plant growth, development, and in response to multiple stresses. In this study, we used bioinformatics methods to identify 86 MAPKKK protein encoded by 73 MAPKKK genes in Brachypodium. Phylogenetic analysis of MAPKKK family from Arabidopsis, rice, and Brachypodium has classified them into three subfamilies, of which 28 belonged to MEKK, 52 to Raf, and 6 to ZIK subfamily, respectively. Conserved protein motif, exon-intron organization, and splicing intron phase in kinase domains supported the evolutionary relationships inferred from the phylogenetic analysis. And gene duplication analysis suggested the chromosomal segment duplication happened before the divergence of the rice and Brachypodium, while all of three tandem duplicated gene pairs happened after their divergence. We further demonstrated that the MAPKKKs have evolved under strong purifying selection, implying the conservation of them. The splicing transcripts expression analysis showed that the splicesome translating longest protein tended to be adopted. Furthermore, the expression analysis of BdMAPKKKs in different organs and development stages as well as heat, virus and drought stresses revealed that the MAPKKK genes were involved in various signaling pathways. And the circadian analysis suggested there were 41 MAPKKK genes in Brachypodium showing cycled expression in at least one condition, of which seven MAPKKK genes expressed in all conditions and the promoter analysis indicated these genes possessed many cis-acting regulatory elements involved in circadian and light response. Finally, the co-expression network of MAPK, MAPKK, and MAPKKK in Brachypodium was constructed using 144 microarray and RNA-seq datasets, and ten potential MAPK cascades pathway were predicted. To conclude, our study provided the important information for evolutionary and

  15. Targeting protein kinases in central nervous system disorders

    PubMed Central

    Chico, Laura K.; Van Eldik, Linda J.; Watterson, D. Martin

    2010-01-01

    Protein kinases are a growing drug target class in disorders in peripheral tissues, but the development of kinase-targeted therapies for central nervous system (CNS) diseases remains a challenge, largely owing to issues associated specifically with CNS drug discovery. However, several candidate therapeutics that target CNS protein kinases are now in various stages of preclinical and clinical development. We review candidate compounds and discuss selected CNS protein kinases that are emerging as important therapeutic targets. In addition, we analyse trends in small-molecule properties that correlate with key challenges in CNS drug discovery, such as blood–brain barrier penetrance and cytochrome P450-mediated metabolism, and discuss the potential of future approaches that will integrate molecular-fragment expansion with pharmacoinformatics to address these challenges. PMID:19876042

  16. Genomic reduction and evolution of novel genetic membranes and protein-targeting machinery in eukaryote-eukaryote chimaeras (meta-algae).

    PubMed Central

    Cavalier-Smith, T

    2003-01-01

    Chloroplasts originated just once, from cyanobacteria enslaved by a biciliate protozoan to form the plant kingdom (green plants, red and glaucophyte algae), but subsequently, were laterally transferred to other lineages to form eukaryote-eukaryote chimaeras or meta-algae. This process of secondary symbiogenesis (permanent merger of two phylogenetically distinct eukaryote cells) has left remarkable traces of its evolutionary role in the more complex topology of the membranes surrounding all non-plant (meta-algal) chloroplasts. It took place twice, soon after green and red algae diverged over 550 Myr ago to form two independent major branches of the eukaryotic tree (chromalveolates and cabozoa), comprising both meta-algae and numerous secondarily non-photosynthetic lineages. In both cases, enslavement probably began by evolving a novel targeting of endomembrane vesicles to the perialgal vacuole to implant host porter proteins for extracting photosynthate. Chromalveolates arose by such enslavement of a unicellular red alga and evolution of chlorophyll c to form the kingdom Chromista and protozoan infrakingdom Alveolata, which diverged from the ancestral chromalveolate chimaera. Cabozoa arose when the common ancestor of euglenoids and cercozoan chlorarachnean algae enslaved a tetraphyte green alga with chlorophyll a and b. I suggest that in cabozoa the endomembrane vesicles originally budded from the Golgi, whereas in chromalveolates they budded from the endoplasmic reticulum (ER) independently of Golgi-targeted vesicles, presenting a potentially novel target for drugs against alveolate Sporozoa such as malaria parasites and Toxoplasma. These hypothetical ER-derived vesicles mediated fusion of the perialgal vacuole and rough ER (RER) in the ancestral chromist, placing the former red alga within the RER lumen. Subsequently, this chimaera diverged to form cryptomonads, which retained the red algal nucleus as a nucleomorph (NM) with approximately 464 protein-coding genes

  17. Genomic reduction and evolution of novel genetic membranes and protein-targeting machinery in eukaryote-eukaryote chimaeras (meta-algae).

    PubMed

    Cavalier-Smith, T

    2003-01-29

    Chloroplasts originated just once, from cyanobacteria enslaved by a biciliate protozoan to form the plant kingdom (green plants, red and glaucophyte algae), but subsequently, were laterally transferred to other lineages to form eukaryote-eukaryote chimaeras or meta-algae. This process of secondary symbiogenesis (permanent merger of two phylogenetically distinct eukaryote cells) has left remarkable traces of its evolutionary role in the more complex topology of the membranes surrounding all non-plant (meta-algal) chloroplasts. It took place twice, soon after green and red algae diverged over 550 Myr ago to form two independent major branches of the eukaryotic tree (chromalveolates and cabozoa), comprising both meta-algae and numerous secondarily non-photosynthetic lineages. In both cases, enslavement probably began by evolving a novel targeting of endomembrane vesicles to the perialgal vacuole to implant host porter proteins for extracting photosynthate. Chromalveolates arose by such enslavement of a unicellular red alga and evolution of chlorophyll c to form the kingdom Chromista and protozoan infrakingdom Alveolata, which diverged from the ancestral chromalveolate chimaera. Cabozoa arose when the common ancestor of euglenoids and cercozoan chlorarachnean algae enslaved a tetraphyte green alga with chlorophyll a and b. I suggest that in cabozoa the endomembrane vesicles originally budded from the Golgi, whereas in chromalveolates they budded from the endoplasmic reticulum (ER) independently of Golgi-targeted vesicles, presenting a potentially novel target for drugs against alveolate Sporozoa such as malaria parasites and Toxoplasma. These hypothetical ER-derived vesicles mediated fusion of the perialgal vacuole and rough ER (RER) in the ancestral chromist, placing the former red alga within the RER lumen. Subsequently, this chimaera diverged to form cryptomonads, which retained the red algal nucleus as a nucleomorph (NM) with approximately 464 protein-coding genes

  18. Phosphorylation of the Kinase Interaction Motif in Mitogen-activated Protein (MAP) Kinase Phosphatase-4 Mediates Cross-talk between Protein Kinase A and MAP Kinase Signaling Pathways*

    PubMed Central

    Dickinson, Robin J.; Delavaine, Laurent; Cejudo-Marín, Rocío; Stewart, Graeme; Staples, Christopher J.; Didmon, Mark P.; Trinidad, Antonio Garcia; Alonso, Andrés; Pulido, Rafael; Keyse, Stephen M.

    2011-01-01

    MAP kinase phosphatase 4 (DUSP9/MKP-4) plays an essential role during placental development and is one of a subfamily of three closely related cytoplasmic dual-specificity MAPK phosphatases, which includes the ERK-specific enzymes DUSP6/MKP-3 and DUSP7/MKP-X. However, unlike DUSP6/MKP-3, DUSP9/MKP-4 also inactivates the p38α MAP kinase both in vitro and in vivo. Here we demonstrate that inactivation of both ERK1/2 and p38α by DUSP9/MKP-4 is mediated by a conserved arginine-rich kinase interaction motif located within the amino-terminal non-catalytic domain of the protein. Furthermore, DUSP9/MKP-4 is unique among these cytoplasmic MKPs in containing a conserved PKA consensus phosphorylation site 55RRXSer-58 immediately adjacent to the kinase interaction motif. DUSP9/MKP-4 is phosphorylated on Ser-58 by PKA in vitro, and phosphorylation abrogates the binding of DUSP9/MKP-4 to both ERK2 and p38α MAP kinases. In addition, although mutation of Ser-58 to either alanine or glutamic acid does not affect the intrinsic catalytic activity of DUSP9/MKP-4, phospho-mimetic (Ser-58 to Glu) substitution inhibits both the interaction of DUSP9/MKP-4 with ERK2 and p38α in vivo and its ability to dephosphorylate and inactivate these MAP kinases. Finally, the use of a phospho-specific antibody demonstrates that endogenous DUSP9/MKP-4 is phosphorylated on Ser-58 in response to the PKA agonist forskolin and is also modified in placental tissue. We conclude that DUSP9/MKP-4 is a bona fide target of PKA signaling and that attenuation of DUSP9/MKP-4 function can mediate cross-talk between the PKA pathway and MAPK signaling through both ERK1/2 and p38α in vivo. PMID:21908610

  19. Lead-induced upregulation of the heme-regulated eukaryotic initiation factor 2alpha kinase is compromised by hemin in human K562 cells.

    PubMed

    Sarkar, Angshuman; Kulkarni, Abhijeet; Chattopadhyay, Samit; Mogare, Devraj; Sharma, Kiran K; Singh, Kamini; Pal, Jayanta K

    2005-12-30

    Expression and kinase activity of the heme-regulated-eIF-2alpha kinase or -inhibitor (HRI) are induced during cytoplasmic stresses leading to inhibition of protein synthesis. Using a reporter construct with HRI promoter, we have determined the promoter activity during heat-shock and lead toxicity in human K562 cells. These two conditions induced HRI promoter activity by 2- to 3-fold. Contrary to this, hemin, a suppressor of HRI kinase activity, downregulated HRI promoter activity and stimulated hemoglobin synthesis. Interestingly, when hemin-treated cells were transfected and exposed to lead, hemin compromised lead-effect substantially by downregulating HRI promoter activity, HRI transcription and HRI kinase activity. These results together suggest that heme signaling in relation to translation regulation is not only restricted to the cytoplasm (modulating HRI kinase activity) alone but it also spans to the nucleus modulating HRI expression. Hemin may thus be useful for alleviation of stress-induced inhibition of protein synthesis.

  20. Phosphorylation of lipid metabolic enzymes by yeast protein kinase C requires phosphatidylserine and diacylglycerol.

    PubMed

    Dey, Prabuddha; Su, Wen-Min; Han, Gil-Soo; Carman, George M

    2017-04-01

    Protein kinase C in Saccharomyces cerevisiae, i.e., Pkc1, is an enzyme that plays an important role in signal transduction and the regulation of lipid metabolic enzymes. Pkc1 is structurally similar to its counterparts in higher eukaryotes, but its requirement of phosphatidylserine (PS) and diacylglycerol (DAG) for catalytic activity has been unclear. In this work, we examined the role of these lipids in Pkc1 activity with protein and peptide substrates. In agreement with previous findings, yeast Pkc1 did not require PS and DAG for its activity on the peptide substrates derived from lipid metabolic proteins such as Pah1 [phosphatidate (PA) phosphatase], Nem1 (PA phosphatase phosphatase), and Spo7 (protein phosphatase regulatory subunit). However, the lipids were required for Pkc1 activity on the protein substrates Pah1, Nem1, and Spo7. Compared with DAG, PS had a greater effect on Pkc1 activity, and its dose-dependent interaction with the protein kinase was shown by the liposome binding assay. The Pkc1-mediated degradation of Pah1 was attenuated in the cho1Δ mutant, which is deficient in PS synthase, supporting the notion that the phospholipid regulates Pkc1 activity in vivo. Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.

  1. Protein Phylogenies and Signature Sequences: A Reappraisal of Evolutionary Relationships among Archaebacteria, Eubacteria, and Eukaryotes

    PubMed Central

    Gupta, Radhey S.

    1998-01-01

    The presence of shared conserved insertion or deletions (indels) in protein sequences is a special type of signature sequence that shows considerable promise for phylogenetic inference. An alternative model of microbial evolution based on the use of indels of conserved proteins and the morphological features of prokaryotic organisms is proposed. In this model, extant archaebacteria and gram-positive bacteria, which have a simple, single-layered cell wall structure, are termed monoderm prokaryotes. They are believed to be descended from the most primitive organisms. Evidence from indels supports the view that the archaebacteria probably evolved from gram-positive bacteria, and I suggest that this evolution occurred in response to antibiotic selection pressures. Evidence is presented that diderm prokaryotes (i.e., gram-negative bacteria), which have a bilayered cell wall, are derived from monoderm prokaryotes. Signature sequences in different proteins provide a means to define a number of different taxa within prokaryotes (namely, low G+C and high G+C gram-positive, Deinococcus-Thermus, cyanobacteria, chlamydia-cytophaga related, and two different groups of Proteobacteria) and to indicate how they evolved from a common ancestor. Based on phylogenetic information from indels in different protein sequences, it is hypothesized that all eukaryotes, including amitochondriate and aplastidic organisms, received major gene contributions from both an archaebacterium and a gram-negative eubacterium. In this model, the ancestral eukaryotic cell is a chimera that resulted from a unique fusion event between the two separate groups of prokaryotes followed by integration of their genomes. PMID:9841678

  2. The N-terminal region of eukaryotic translation initiation factor 5A signals to nuclear localization of the protein

    SciTech Connect

    Parreiras-e-Silva, Lucas T.; Gomes, Marcelo D.; Oliveira, Eduardo B.; Costa-Neto, Claudio M.

    2007-10-19

    The eukaryotic translation initiation factor 5A (eIF5A) is a ubiquitous protein of eukaryotic and archaeal organisms which undergoes hypusination, a unique post-translational modification. We have generated a polyclonal antibody against murine eIF5A, which in immunocytochemical assays in B16-F10 cells revealed that the endogenous protein is preferentially localized to the nuclear region. We therefore analyzed possible structural features present in eIF5A proteins that could be responsible for that characteristic. Multiple sequence alignment analysis of eIF5A proteins from different eukaryotic and archaeal organisms showed that the former sequences have an extended N-terminal segment. We have then performed in silico prediction analyses and constructed different truncated forms of murine eIF5A to verify any possible role that the N-terminal extension might have in determining the subcellular localization of the eIF5A in eukaryotic organisms. Our results indicate that the N-terminal extension of the eukaryotic eIF5A contributes in signaling this protein to nuclear localization, despite of bearing no structural similarity with classical nuclear localization signals.

  3. Flagellated algae protein evolution suggests the prevalence of lineage-specific rules governing evolutionary rates of eukaryotic proteins.

    PubMed

    Chang, Ting-Yan; Liao, Ben-Yang

    2013-01-01

    Understanding the general rules governing the rate of protein evolution is fundamental to evolutionary biology. However, attempts to address this issue in yeasts and mammals have revealed considerable differences in the relative importance of determinants for protein evolutionary rates. This phenomenon was previously explained by the fact that yeasts and mammals are different in many cellular and genomic properties. Flagellated algae species have several cellular and genomic characteristics that are intermediate between yeasts and mammals. Using partial correlation analyses on the evolution of 6,921 orthologous proteins from Chlamydomonas reinhardtii and Volvox carteri, we examined factors influencing evolutionary rates of proteins in flagellated algae. Previous studies have shown that mRNA abundance and gene compactness are strong determinants for protein evolutionary rates in yeasts and mammals, respectively. We show that both factors also influence algae protein evolution with mRNA abundance having a larger impact than gene compactness on the rates of algae protein evolution. More importantly, among all the factors examined, coding sequence (CDS) length has the strongest (positive) correlation with protein evolutionary rates. This correlation between CDS length and the rates of protein evolution is not due to alignment-related issues or domain density. These results suggest no simple and universal rules governing protein evolutionary rates across different eukaryotic lineages. Instead, gene properties influence the rate of protein evolution in a lineage-specific manner.

  4. G protein-coupled receptor kinases: more than just kinases and not only for GPCRs

    PubMed Central

    Gurevich, Eugenia V.; Tesmer, John J. G.; Mushegian, Arcady; Gurevich, Vsevolod V.

    2011-01-01

    G protein-coupled receptor (GPCR) kinases (GRKs) are best known for their role in homologous desensitization of GPCRs. GRKs phosphorylate activated receptors and promote high affinity binding of arrestins, which precludes G protein coupling. GRKs have a multidomain structure, with the kinase domain inserted into a loop of a regulator of G protein signaling homology domain. Unlike many other kinases, GRKs do not need to be phosphorylated in their activation loop to achieve an activated state. Instead, they are directly activated by docking with active GPCRs. In this manner they are able to selectively phosphorylate Ser/Thr residues on only the activated form of the receptor, unlike related kinases such as protein kinase A. GRKs also phosphorylate a variety of non-GPCR substrates and regulate several signaling pathways via direct interactions with other proteins in a phosphorylation-independent manner. Multiple GRK subtypes are present in virtually every animal cell, with the highest expression levels found in neurons, with their extensive and complex signal regulation. Insufficient or excessive GRK activity was implicated in a variety of human disorders, ranging from heart failure to depression to Parkinson’s disease. As key regulators of GPCR-dependent and -independent signaling pathways, GRKs are emerging drug targets and promising molecular tools for therapy. Targeted modulation of expression and/or of activity of several GRK isoforms for therapeutic purposes was recently validated in cardiac disorders and Parkinson’s disease. PMID:21903131

  5. A Novel Mode of Protein Kinase Inhibition Exploiting Hydrophobic Motifs of Autoinhibited Kinases

    PubMed Central

    Eathiraj, Sudharshan; Palma, Rocio; Hirschi, Marscha; Volckova, Erika; Nakuci, Enkeleda; Castro, Jennifer; Chen, Chang-Rung; Chan, Thomas C. K.; France, Dennis S.; Ashwell, Mark A.

    2011-01-01

    Protein kinase inhibitors with enhanced selectivity can be designed by optimizing binding interactions with less conserved inactive conformations because such inhibitors will be less likely to compete with ATP for binding and therefore may be less impacted by high intracellular concentrations of ATP. Analysis of the ATP-binding cleft in a number of inactive protein kinases, particularly in the autoinhibited conformation, led to the identification of a previously undisclosed non-polar region in this cleft. This ATP-incompatible hydrophobic region is distinct from the previously characterized hydrophobic allosteric back pocket, as well as the main pocket. Generalized hypothetical models of inactive kinases were constructed and, for the work described here, we selected the fibroblast growth factor receptor (FGFR) tyrosine kinase family as a case study. Initial optimization of a FGFR2 inhibitor identified from a library of commercial compounds was guided using structural information from the model. We describe the inhibitory characteristics of this compound in biophysical, biochemical, and cell-based assays, and have characterized the binding mode using x-ray crystallographic studies. The results demonstrate, as expected, that these inhibitors prevent activation of the autoinhibited conformation, retain full inhibitory potency in the presence of physiological concentrations of ATP, and have favorable inhibitory activity in cancer cells. Given the widespread regulation of kinases by autoinhibitory mechanisms, the approach described herein provides a new paradigm for the discovery of inhibitors by targeting inactive conformations of protein kinases. PMID:21454610

  6. Structural insight into nucleotide recognition by human death-associated protein kinase

    SciTech Connect

    McNamara, Laurie K.; Watterson, D. Martin; Brunzelle, Joseph S.

    2009-03-01

    The crystal structures of DAPK–ADP–Mg{sup 2+} and DAPK–AMP-PNP–Mg{sup 2+} complexes were determined at 1.85 and 2.00 Å resolution, respectively. Comparison of the two nucleotide-bound states with apo DAPK revealed localized changes in the glycine-rich loop region that were indicative of a transition from a more open state to a more closed state on binding of the nucleotide substrate and to an intermediate state with the bound nucleotide product. Death-associated protein kinase (DAPK) is a member of the Ca{sup 2+}/calmodulin-regulated family of serine/threonine protein kinases. The role of the kinase activity of DAPK in eukaryotic cell apoptosis and the ability of bioavailable DAPK inhibitors to rescue neuronal death after brain injury have made it a drug-discovery target for neurodegenerative disorders. In order to understand the recognition of nucleotides by DAPK and to gain insight into DAPK catalysis, the crystal structure of human DAPK was solved in complex with ADP and Mg{sup 2+} at 1.85 Å resolution. ADP is a product of the kinase reaction and product release is considered to be the rate-limiting step of protein kinase catalytic cycles. The structure of DAPK–ADP–Mg{sup 2+} was compared with a newly determined DAPK–AMP-PNP–Mg{sup 2+} structure and the previously determined apo DAPK structure (PDB code http://scripts.iucr.org/cgi-bin/cr.cgi?rm). The comparison shows that nucleotide-induced changes are localized to the glycine-rich loop region of DAPK.

  7. dsRNA-Dependent Protein Kinase PKR and its Role in Stress, Signaling and HCV Infection

    PubMed Central

    Dabo, Stéphanie; Meurs, Eliane F.

    2012-01-01

    The double-stranded RNA-dependent protein kinase PKR plays multiple roles in cells, in response to different stress situations. As a member of the interferon (IFN)‑Stimulated Genes, PKR was initially recognized as an actor in the antiviral action of IFN, due to its ability to control translation, through phosphorylation, of the alpha subunit of eukaryotic initiation factor 2 (eIF2α). As such, PKR participates in the generation of stress granules, or autophagy and a number of viruses have designed strategies to inhibit its action. However, PKR deficient mice resist most viral infections, indicating that PKR may play other roles in the cell other than just acting as an antiviral agent. Indeed, PKR regulates several signaling pathways, either as an adapter protein and/or using its kinase activity. Here we review the role of PKR as an eIF2α kinase, its participation in the regulation of the NF-κB, p38MAPK and insulin pathways, and we focus on its role during infection with the hepatitis C virus (HCV). PKR binds the HCV IRES RNA, cooperates with some functions of the HCV core protein and may represent a target for NS5A or E2. Novel data points out for a role of PKR as a pro-HCV agent, both as an adapter protein and as an eIF2α-kinase, and in cooperation with the di-ubiquitin-like protein ISG15. Developing pharmaceutical inhibitors of PKR may help in resolving some viral infections as well as stress-related damages. PMID:23202496

  8. Induction of viral, 7-methyl-guanosine cap-independent translation and oncolysis by mitogen-activated protein kinase-interacting kinase-mediated effects on the serine/arginine-rich protein kinase.

    PubMed

    Brown, Michael C; Bryant, Jeffrey D; Dobrikova, Elena Y; Shveygert, Mayya; Bradrick, Shelton S; Chandramohan, Vidyalakshmi; Bigner, Darell D; Gromeier, Matthias

    2014-11-01

    Protein synthesis, the most energy-consuming process in cells, responds to changing physiologic priorities, e.g., upon mitogen- or stress-induced adaptations signaled through the mitogen-activated protein kinases (MAPKs). The prevailing status of protein synthesis machinery is a viral pathogenesis factor, particularly for plus-strand RNA viruses, where immediate translation of incoming viral RNAs shapes host-virus interactions. In this study, we unraveled signaling pathways centered on the ERK1/2 and p38α MAPK-interacting kinases MNK1/2 and their role in controlling 7-methyl-guanosine (m(7)G) "cap"-independent translation at enterovirus type 1 internal ribosomal entry sites (IRESs). Activation of Raf-MEK-ERK1/2 signals induced viral IRES-mediated translation in a manner dependent on MNK1/2. This effect was not due to MNK's known functions as eukaryotic initiation factor (eIF) 4G binding partner or eIF4E(S209) kinase. Rather, MNK catalytic activity enabled viral IRES-mediated translation/host cell cytotoxicity through negative regulation of the Ser/Arg (SR)-rich protein kinase (SRPK). Our investigations suggest that SRPK activity is a major determinant of type 1 IRES competency, host cell cytotoxicity, and viral proliferation in infected cells. We are targeting unfettered enterovirus IRES activity in cancer with PVSRIPO, the type 1 live-attenuated poliovirus (PV) (Sabin) vaccine containing a human rhinovirus type 2 (HRV2) IRES. A phase I clinical trial of PVSRIPO with intratumoral inoculation in patients with recurrent glioblastoma (GBM) is showing early promise. Viral translation proficiency in infected GBM cells is a core requirement for the antineoplastic efficacy of PVSRIPO. Therefore, it is critically important to understand the mechanisms controlling viral cap-independent translation in infected host cells. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  9. Structures of Eukaryotic Ribosomal Stalk Proteins and Its Complex with Trichosanthin, and Their Implications in Recruiting Ribosome-Inactivating Proteins to the Ribosomes

    PubMed Central

    Choi, Andrew K. H.; Wong, Eddie C. K.; Lee, Ka-Ming; Wong, Kam-Bo

    2015-01-01

    Ribosome-inactivating proteins (RIP) are RNA N-glycosidases that inactivate ribosomes by specifically depurinating a conserved adenine residue at the α-sarcin/ricin loop of 28S rRNA. Recent studies have pointed to the involvement of the C-terminal domain of the eukaryotic stalk proteins in facilitating the toxic action of RIPs. This review highlights how structural studies of eukaryotic stalk proteins provide insights into the recruitment of RIPs to the ribosomes. Since the C-terminal domain of eukaryotic stalk proteins is involved in specific recognition of elongation factors and some eukaryote-specific RIPs (e.g., trichosanthin and ricin), we postulate that these RIPs may have evolved to hijack the translation-factor-recruiting function of ribosomal stalk in reaching their target site of rRNA. PMID:25723321

  10. Structures of eukaryotic ribosomal stalk proteins and its complex with trichosanthin, and their implications in recruiting ribosome-inactivating proteins to the ribosomes.

    PubMed

    Choi, Andrew K H; Wong, Eddie C K; Lee, Ka-Ming; Wong, Kam-Bo

    2015-02-25

    Ribosome-inactivating proteins (RIP) are RNA N-glycosidases that inactivate ribosomes by specifically depurinating a conserved adenine residue at the α-sarcin/ricin loop of 28S rRNA. Recent studies have pointed to the involvement of the C-terminal domain of the eukaryotic stalk proteins in facilitating the toxic action of RIPs. This review highlights how structural studies of eukaryotic stalk proteins provide insights into the recruitment of RIPs to the ribosomes. Since the C-terminal domain of eukaryotic stalk proteins is involved in specific recognition of elongation factors and some eukaryote-specific RIPs (e.g., trichosanthin and ricin), we postulate that these RIPs may have evolved to hijack the translation-factor-recruiting function of ribosomal stalk in reaching their target site of rRNA.

  11. Diacylglycerol kinase is phosphorylated in vivo upon stimulation of the epidermal growth factor receptor and serine/threonine kinases, including protein kinase C-epsilon.

    PubMed Central

    Schaap, D; van der Wal, J; van Blitterswijk, W J; van der Bend, R L; Ploegh, H L

    1993-01-01

    In signal transduction, diacylglycerol (DG) kinase attenuates levels of the second messenger DG by converting it to phosphatidic acid. A previously cloned full-length human 86 kDa DG kinase cDNA was expressed as fusion protein in Escherichia coli, to aid in the generation of DG-kinase-specific monoclonal antibodies suitable for immunoprecipitation experiments. To investigate whether phosphorylation of DG kinase is a possible mechanism for its regulation, COS-7 cells were transiently transfected with the DG kinase cDNA and phosphorylation of the expressed DG kinase was induced by various stimuli. Activation of both cyclic AMP-dependent protein kinase and protein kinase C (PKC) resulted in phosphorylation of DG kinase on serine residues in vivo, and both kinases induced this phosphorylation within the same tryptic phosphopeptide, suggesting that they may exert similar control over DG kinase. No phosphorylation was observed upon ionomycin treatment, intended to activate Ca2+/calmodulin-dependent kinases. Co-transfections of DG kinase with either PKC-alpha or PKC-epsilon cDNA revealed that both protein kinases, when stimulated, are able to phosphorylate DG kinase. For PKC-epsilon, DG kinase is the first in vivo substrate identified. Stimulation with epidermal growth factor (EGF) of COS-7 cells transfected with both DG kinase and EGF-receptor cDNA results mainly in phosphorylation of DG kinase on tyrosine. Since the EGF receptor has an intrinsic tyrosine kinase activity, this finding implies that DG kinase may be a direct substrate for the activated EGF receptor. Images Figure 2 Figure 3 Figure 4 PMID:7679574

  12. The quaternary structure of the eukaryotic DNA replication proteins Sld7 and Sld3.

    PubMed

    Itou, Hiroshi; Shirakihara, Yasuo; Araki, Hiroyuki

    2015-08-01

    The initiation of eukaryotic chromosomal DNA replication requires the formation of an active replicative helicase at the replication origins of chromosomes. Yeast Sld3 and its metazoan counterpart treslin are the hub proteins mediating protein associations critical for formation of the helicase. The Sld7 protein interacts with Sld3, and the complex formed is thought to regulate the function of Sld3. Although Sld7 is a non-essential DNA replication protein that is found in only a limited range of yeasts, its depletion slowed the growth of cells and caused a delay in the S phase. Recently, the Mdm2-binding protein was found to bind to treslin in humans, and its depletion causes defects in cells similar to the depletion of Sld7 in yeast, suggesting their functional relatedness and importance during the initiation step of DNA replication. Here, the crystal structure of Sld7 in complex with Sld3 is presented. Sld7 comprises two structural domains. The N-terminal domain of Sld7 binds to Sld3, and the C-terminal domains connect two Sld7 molecules in an antiparallel manner. The quaternary structure of the Sld3-Sld7 complex shown from the crystal structures appears to be suitable to activate two helicase molecules loaded onto replication origins in a head-to-head manner.

  13. Evolution of a domain conserved in microtubule-associated proteins of eukaryotes

    PubMed Central

    Rajangam, Alex S; Yang, Hongqian; Teeri, Tuula T; Arvestad, Lars

    2008-01-01

    The microtubule network, the major organelle of the eukaryotic cytoskeleton, is involved in cell division and differentiation but also with many other cellular functions. In plants, microtubules seem to be involved in the ordered deposition of cellulose microfibrils by a so far unknown mechanism. Microtubule-associated proteins (MAP) typically contain various domains targeting or binding proteins with different functions to microtubules. Here we have investigated a proposed microtubule-targeting domain, TPX2, first identified in the Kinesin-like protein 2 in Xenopus. A TPX2 containing microtubule binding protein, PttMAP20, has been recently identified in poplar tissues undergoing xylogenesis. Furthermore, the herbicide 2,6-dichlorobenzonitrile (DCB), which is a known inhibitor of cellulose synthesis, was shown to bind specifically to PttMAP20. It is thus possible that PttMAP20 may have a role in coupling cellulose biosynthesis and the microtubular networks in poplar secondary cell walls. In order to get more insight into the occurrence, evolution and potential functions of TPX2-containing proteins we have carried out bioinformatic analysis for all genes so far found to encode TPX2 domains with special reference to poplar PttMAP20 and its putative orthologs in other plants. PMID:21918606

  14. Coupling phosphoryl transfer and substrate interactions in protein kinases.

    PubMed

    Lieser, Scot A; Aubol, Brandon E; Wong, Lilly; Jennings, Patricia A; Adams, Joseph A

    2005-12-30

    Protein kinases control cell signaling events through the ATP-dependent phosphorylation of serine, threonine and tyrosine residues in protein targets. The recognition of these protein substrates by the kinases relies on two principal factors: proper subcellular co-localization and molecular interactions between the kinase and substrate. In this review, we will focus on the kinetic role of the latter in conveying favorable substrate recognition. Using rapid mixing technologies, we demonstrate that the intrinsic thermodynamic affinities of two protein substrates for their respective kinases (Csk with Src and Sky1p with Npl3) are weak compared to their apparent affinities measured in traditional steady-state kinetic assays (i.e.--Km < Kd). The source of the high apparent affinities rests in a very fast and highly favorable phosphoryl transfer step that serves as a clamp for substrate recognition. In this mechanism, both Csk and Sky1p utilize this step to draw the substrate toward product, thereby, converting a high Kd into a low Km. We propose that this one form of substrate recognition employed by protein kinases is advantageous since it simultaneously facilitates high apparent substrate affinity and fast protein turnover.

  15. The Evolutionary History of MAPL (Mitochondria-Associated Protein Ligase) and Other Eukaryotic BAM/GIDE Domain Proteins.

    PubMed

    Wideman, Jeremy G; Moore, Blake P

    2015-01-01

    MAPL (mitochondria-associated protein ligase, also called MULAN/GIDE/MUL1) is a multifunctional mitochondrial outer membrane protein found in human cells that contains a unique BAM (beside a membrane) domain and a C-terminal RING-finger domain. MAPL has been implicated in several processes that occur in animal cells such as NF-kB activation, innate immunity and antiviral signaling, suppression of PINK1/parkin defects, mitophagy in skeletal muscle, and caspase-dependent apoptosis. Previous studies demonstrated that the BAM domain is present in diverse organisms in which most of these processes do not occur, including plants, archaea, and bacteria. Thus the conserved function of MAPL and its BAM domain remains an open question. In order to gain insight into its conserved function, we investigated the evolutionary origins of MAPL by searching for homologues in predicted proteomes of diverse eukaryotes. We show that MAPL proteins with a conserved BAM-RING architecture are present in most animals, protists closely related to animals, a single species of fungus, and several multicellular plants and related green algae. Phylogenetic analysis demonstrated that eukaryotic MAPL proteins originate from a common ancestor and not from independent horizontal gene transfers from bacteria. We also determined that two independent duplications of MAPL occurred, one at the base of multicellular plants and another at the base of vertebrates. Although no other eukaryote genome examined contained a verifiable MAPL orthologue, BAM domain-containing proteins were identified in the protists Bigelowiella natans and Ectocarpus siliculosis. Phylogenetic analyses demonstrated that these proteins are more closely related to prokaryotic BAM proteins and therefore likely arose from independent horizontal gene transfers from bacteria. We conclude that MAPL proteins with BAM-RING architectures have been present in the holozoan and viridiplantae lineages since their very beginnings. Our work paves

  16. Cloning and purification of protein kinase CK2 recombinant alpha and beta subunits from the Mediterranean fly Ceratitis capitata.

    PubMed

    Kouyanou-Koutsoukou, Sophia; Baier, Andrea; Kolaitis, Regina-Maria; Maniatopoulou, Evanthia; Thanopoulou, Konstantina; Szyszka, Ryszard

    2011-10-01

    The Mediterranean fruit fly Ceratitis capitata is an insect capable of wreaking extensive damage to a wide range of fruit crops. Protein kinase CK2 is a ubiquitous Ser/Thr kinase that is highly conserved among eukaryotes; it is a heterotetramer composed of two catalytic (α) and a dimer of regulatory (β) subunits. We present here the construction of the cDNA molecules of the CK2α and CK2β subunits from the medfly C. capitata by the 5'/3' RACE and RT-PCR methods, respectively. CcCK2α catalytic subunit presents the characteristic and conserved features of a typical protein kinase, similar to the regulatory CcCK2β subunit, that also possess the conserved features of regulatory CK2β subunits, as revealed by comparison of their predicted amino acid sequences with other eukaryotic species. The recombinant CcCK2α and CcCK2β proteins were purified by affinity chromatography to homogeneity, after overexpression in Escherichia coli. CcCK2α is capable to utilize GTP and its activity and is inhibited by polyanions and stimulated by polycations in phosphorylation assays, using purified acidic ribosomal protein P1 as a substrate.

  17. Genome-wide survey of putative Serine/Threonine protein kinases in cyanobacteria

    PubMed Central

    Zhang, Xiaowen; Zhao, Fangqing; Guan, Xiangyu; Yang, Yu; Liang, Chengwei; Qin, Song

    2007-01-01

    Background Serine/threonine kinases (STKs) have been found in an increasing number of prokaryotes, showing important roles in signal transduction that supplement the well known role of two-component system. Cyanobacteria are photoautotrophic prokaryotes able to grow in a wide range of ecological environments, and their signal transduction systems are important in adaptation to the environment. Sequence information from several cyanobacterial genomes offers a unique opportunity to conduct a comprehensive comparative analysis of this kinase family. In this study, we extracted information regarding Ser/Thr kinases from 21 species of sequenced cyanobacteria and investigated their diversity, conservation, domain structure, and evolution. Results 286 putative STK homologues were identified. STKs are absent in four Prochlorococcus strains and one marine Synechococcus strain and abundant in filamentous nitrogen-fixing cyanobacteria. Motifs and invariant amino acids typical in eukaryotic STKs were conserved well in these proteins, and six more cyanobacteria- or bacteria-specific conserved residues were found. These STK proteins were classified into three major families according to their domain structures. Fourteen types and a total of 131 additional domains were identified, some of which are reported to participate in the recognition of signals or substrates. Cyanobacterial STKs show rather complicated phylogenetic relationships that correspond poorly with phylogenies based on 16S rRNA and those based on additional domains. Conclusion The number of STK genes in different cyanobacteria is the result of the genome size, ecophysiology, and physiological properties of the organism. Similar conserved motifs and amino acids indicate that cyanobacterial STKs make use of a similar catalytic mechanism as eukaryotic STKs. Gene gain-and-loss is significant during STK evolution, along with domain shuffling and insertion. This study has established an overall framework of sequence

  18. Extracellular cAMP-dependent protein kinase (ECPKA) in melanoma.

    PubMed

    Kita, Tsunekazu; Goydos, James; Reitman, Elena; Ravatn, Roald; Lin, Yong; Shih, Wei-Chung; Kikuchi, Yoshihiro; Chin, Khew-Voon

    2004-05-28

    Melanoma is one of the fastest rising malignancies in the United States. When detected early, primary melanomas are curable through surgery. However, despite significant improvements in diagnosis and surgical, local and systemic therapy, mortality rate in metastatic melanoma remains high. Furthermore, genetic alterations associated with the development and stepwise progression of melanoma, are still unclear. Previous reports show that the catalytic kinase subunit of the cAMP-dependent protein kinase is secreted by tumor cells and can be detected in the serum of cancer patients. We examine in this report the clinical significance of this secreted C subunit kinase termed extracellular protein kinase (ECPKA) in melanoma patients. Our results showed the presence of ECPKA activity in the serum of melanoma patients and correlate with the appearance and size of the tumor. Most importantly, surgical removal of melanoma causes a precipitous decrease in ECPKA activity in the sera of patients, suggesting that ECPKA may be a novel predictive marker in melanoma.

  19. Protein kinase CK2 in development and differentiation

    PubMed Central

    Götz, Claudia; Montenarh, Mathias

    2017-01-01

    Among the human kinomes, protein kinase CK2 (formerly termed casein kinase II) is considered to be essential, as it is implicated in the regulation of various cellular processes. Experiments with pharmacological inhibitors of the kinase activity of CK2 provide evidence that CK2 is essential for development and differentiation. Therefore, the present review addresses the role of CK2 during embryogenesis, neuronal, adipogenic, osteogenic and myogenic differentiation in established model cell lines, and in embryonic, neural and mesenchymal stem cells. CK2 kinase activity appears to be essential in the early stages of differentiation, as CK2 inhibition at early time points generally prevents differentiation. In addition, the present review reports on target proteins of CK2 in embryogenesis and differentiation. PMID:28357063

  20. Allosteric activation of apicomplexan calcium-dependent protein kinases

    SciTech Connect

    Ingram, Jessica R.; Knockenhauer, Kevin E.; Markus, Benedikt M.; Mandelbaum, Joseph; Ramek, Alexander; Shan, Yibing; Shaw, David E.; Schwartz, Thomas U.; Ploegh, Hidde L.; Lourido, Sebastian

    2015-08-24

    Calcium-dependent protein kinases (CDPKs) comprise the major group of Ca2+-regulated kinases in plants and protists. It has long been assumed that CDPKs are activated, like other Ca2+-regulated kinases, by derepression of the kinase domain (KD). However, we found that removal of the autoinhibitory domain from Toxoplasma gondii CDPK1 is not sufficient for kinase activation. From a library of heavy chain-only antibody fragments (VHHs), we isolated an antibody (1B7) that binds TgCDPK1 in a conformation-dependent manner and potently inhibits it. We uncovered the molecular basis for this inhibition by solving the crystal structure of the complex and simulating, through molecular dynamics, the effects of 1B7–kinase interactions. In contrast to other Ca2+-regulated kinases, the regulatory domain of TgCDPK1 plays a dual role, inhibiting or activating the kinase in response to changes in Ca2+ concentrations. We propose that the regulatory domain of TgCDPK1 acts as a molecular splint to stabilize the otherwise inactive KD. This dependence on allosteric stabilization reveals a novel susceptibility in this important class of parasite enzymes.

  1. Allosteric activation of apicomplexan calcium-dependent protein kinases

    DOE PAGES

    Ingram, Jessica R.; Knockenhauer, Kevin E.; Markus, Benedikt M.; ...

    2015-08-24

    Calcium-dependent protein kinases (CDPKs) comprise the major group of Ca2+-regulated kinases in plants and protists. It has long been assumed that CDPKs are activated, like other Ca2+-regulated kinases, by derepression of the kinase domain (KD). However, we found that removal of the autoinhibitory domain from Toxoplasma gondii CDPK1 is not sufficient for kinase activation. From a library of heavy chain-only antibody fragments (VHHs), we isolated an antibody (1B7) that binds TgCDPK1 in a conformation-dependent manner and potently inhibits it. We uncovered the molecular basis for this inhibition by solving the crystal structure of the complex and simulating, through molecular dynamics,more » the effects of 1B7–kinase interactions. In contrast to other Ca2+-regulated kinases, the regulatory domain of TgCDPK1 plays a dual role, inhibiting or activating the kinase in response to changes in Ca2+ concentrations. We propose that the regulatory domain of TgCDPK1 acts as a molecular splint to stabilize the otherwise inactive KD. This dependence on allosteric stabilization reveals a novel susceptibility in this important class of parasite enzymes.« less

  2. Allosteric activation of apicomplexan calcium-dependent protein kinases

    PubMed Central

    Ingram, Jessica R.; Knockenhauer, Kevin E.; Markus, Benedikt M.; Mandelbaum, Joseph; Ramek, Alexander; Shan, Yibing; Shaw, David E.; Schwartz, Thomas U.; Ploegh, Hidde L.; Lourido, Sebastian

    2015-01-01

    Calcium-dependent protein kinases (CDPKs) comprise the major group of Ca2+-regulated kinases in plants and protists. It has long been assumed that CDPKs are activated, like other Ca2+-regulated kinases, by derepression of the kinase domain (KD). However, we found that removal of the autoinhibitory domain from Toxoplasma gondii CDPK1 is not sufficient for kinase activation. From a library of heavy chain-only antibody fragments (VHHs), we isolated an antibody (1B7) that binds TgCDPK1 in a conformation-dependent manner and potently inhibits it. We uncovered the molecular basis for this inhibition by solving the crystal structure of the complex and simulating, through molecular dynamics, the effects of 1B7–kinase interactions. In contrast to other Ca2+-regulated kinases, the regulatory domain of TgCDPK1 plays a dual role, inhibiting or activating the kinase in response to changes in Ca2+ concentrations. We propose that the regulatory domain of TgCDPK1 acts as a molecular splint to stabilize the otherwise inactive KD. This dependence on allosteric stabilization reveals a novel susceptibility in this important class of parasite enzymes. PMID:26305940

  3. Homology modeling of yeast cyclin-dependent protein kinase

    NASA Astrophysics Data System (ADS)

    Selwyne, R. A.; Kholmurodov, Kh. T.; Koltovaya, N. A.

    2007-07-01

    The important functions that CDKs perform in cell division and cell cycle regulation made central protein kinase of Saccharomyces cerevisiae CDC28 a target model for structural and functional analysis. The 3D models of CDC28 protein kinase using molecular modeling techniques will enlarge our understanding of the phosphorylation mechanism and the structural changes of mutant kinases. The structural template for S. cerevisiae CDC28 was identified from PDB (Protein Databank) using BLASTP (basic local alignment search tool for proteins). Template-target alignments were generated for homology modeling and checked manually for errors. The models were then generated using MODELLER and validated using PROCHECK followed by energy minimization and molecular dynamics calculations in AMBER force field.

  4. Light-assisted small molecule screening against protein kinases

    PubMed Central

    Inglés-Prieto, Álvaro; Reichhart, Eva; Muellner, Markus K.; Nowak, Matthias; Nijman, Sebastian M.; Grusch, Michael; Janovjak, Harald

    2015-01-01

    High-throughput live-cell screens are intricate elements of systems biology studies and drug discovery pipelines. Here, we demonstrate an optogenetics-assisted method that obviates the addition of chemical activators and reporters, reduces the number of operational steps and increases information content in a cell-based small molecule screen against human protein kinases including an orphan receptor tyrosine kinase. This blueprint for all-optical screening can be adapted to many drug targets and cellular processes. PMID:26457372

  5. Evaluation of the enzyme activity of protozoan protein kinases by using an in vitro kinase assay.

    PubMed

    Kato, Kentaro

    2016-10-01

    The life cycles of parasites are more complicated than those of other biological species. Protein kinases (PKs) encoded by parasites are the main triggers of life stage conversions. Phosphorylation by cellular PKs regulates important cellular processes, and the protozoan genome contains many PKs. Some PK inhibitors inhibit specific parasite life cycle event. In this report, I present a practical approach to expressing and purifying protozoan PKs by using a wheat germ cell-free protein synthesis system and I assess the phosphorylation activities of protozoan PKs by using an in vitro kinase assay. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  6. An efficient sliding window strategy for accurate location of eukaryotic protein coding regions.

    PubMed

    Rao, Nini; Lei, Xu; Guo, Jianxiu; Huang, Hao; Ren, Zhenglong

    2009-04-01

    The sliding window is one of important factors that seriously affect the accuracy of coding region prediction and location for the methods based on power spectrum technique. It is very difficult to select the appropriate sliding step and the window length for different organisms. In this study, a novel sliding window strategy is proposed on the basis of power spectrum analysis for the accurate location of eukaryotic protein coding regions. The proposed sliding window strategy is very simple and the sliding step of window is changeable. Our tests show that the average location error for the novel method is 12 bases. Compared with the previous location error of 54 bases using the fixed sliding step, the novel sliding window strategy increased the location accuracy greatly. Further, the consumed CPU time to run the novel strategy is much shorter than the strategy of the fixed length sliding step. So, the computational complexity for the novel method is decreased greatly.

  7. Biological Significance of Nuclear Localization of Mitogen-activated Protein Kinase Pmk1 in Fission Yeast*

    PubMed Central

    Sánchez-Mir, Laura; Franco, Alejandro; Madrid, Marisa; Vicente-Soler, Jero; Villar-Tajadura, M. Antonia; Soto, Teresa; Pérez, Pilar; Gacto, Mariano; Cansado, José

    2012-01-01

    Mitogen-activated protein kinase (MAPK) signaling pathways play a fundamental role in the response of eukaryotic cells to environmental changes. Also, much evidence shows that the stimulus-dependent nuclear targeting of this class of regulatory kinases is crucial for adequate regulation of distinct cellular events. In the fission yeast Schizosaccharomyces pombe, the cell integrity MAPK pathway, whose central element is the MAPK Pmk1, regulates multiple processes such as cell wall integrity, vacuole fusion, cytokinesis, and ionic homeostasis. In non-stressed cells Pmk1 is constitutively localized in both cytoplasm and nucleus, and its localization pattern appears unaffected by its activation status or in response to stress, thus questioning the biological significance of the presence of this MAPK into the nucleus. We have addressed this issue by characterizing mutants expressing Pmk1 versions excluded from the cell nucleus and anchored to the plasma membrane in different genetic backgrounds. Although nuclear Pmk1 partially regulates cell wall integrity at a transcriptional level, membrane-tethered Pmk1 performs many of the biological functions assigned to wild type MAPK like regulation of chloride homeostasis, vacuole fusion, and cellular separation. However, we found that down-regulation of nuclear Pmk1 by MAPK phosphatases induced by the stress activated protein kinase pathway is important for the fine modulation of extranuclear Pmk1 activity. These results highlight the importance of the control of MAPK activity at subcellular level. PMID:22685296

  8. Biological significance of nuclear localization of mitogen-activated protein kinase Pmk1 in fission yeast.

    PubMed

    Sánchez-Mir, Laura; Franco, Alejandro; Madrid, Marisa; Vicente-Soler, Jero; Villar-Tajadura, M Antonia; Soto, Teresa; Pérez, Pilar; Gacto, Mariano; Cansado, José

    2012-07-27

    Mitogen-activated protein kinase (MAPK) signaling pathways play a fundamental role in the response of eukaryotic cells to environmental changes. Also, much evidence shows that the stimulus-dependent nuclear targeting of this class of regulatory kinases is crucial for adequate regulation of distinct cellular events. In the fission yeast Schizosaccharomyces pombe, the cell integrity MAPK pathway, whose central element is the MAPK Pmk1, regulates multiple processes such as cell wall integrity, vacuole fusion, cytokinesis, and ionic homeostasis. In non-stressed cells Pmk1 is constitutively localized in both cytoplasm and nucleus, and its localization pattern appears unaffected by its activation status or in response to stress, thus questioning the biological significance of the presence of this MAPK into the nucleus. We have addressed this issue by characterizing mutants expressing Pmk1 versions excluded from the cell nucleus and anchored to the plasma membrane in different genetic backgrounds. Although nuclear Pmk1 partially regulates cell wall integrity at a transcriptional level, membrane-tethered Pmk1 performs many of the biological functions assigned to wild type MAPK like regulation of chloride homeostasis, vacuole fusion, and cellular separation. However, we found that down-regulation of nuclear Pmk1 by MAPK phosphatases induced by the stress activated protein kinase pathway is important for the fine modulation of extranuclear Pmk1 activity. These results highlight the importance of the control of MAPK activity at subcellular level.

  9. Toxoplasma gondii calcium-dependent protein kinase 1 is a target for selective kinase inhibitors

    PubMed Central

    Ojo, Kayode K; Larson, Eric T; Keyloun, Katelyn R; Castaneda, Lisa J; DeRocher, Amy E; Inampudi, Krishna K; Kim, Jessica E; Arakaki, Tracy L; Murphy, Ryan C; Zhang, Li; Napuli, Alberto J; Maly, Dustin J; Verlinde, Christophe LMJ; Buckner, Frederick S; Parsons, Marilyn; Hol, Wim GJ; Merritt, Ethan A; Van Voorhis, Wesley C

    2010-01-01

    New drugs are needed to treat toxoplasmosis. Toxoplasma gondii calcium-dependent protein kinases (TgCDPKs) are attractive targets because they are absent in mammals. We show that TgCDPK1 is inhibited by low nanomolar levels of bumped kinase inhibitors (BKIs), compounds designed to be inactive against mammalian kinases. Cocrystal structures of TgCDPK1 with BKIs confirm that the structural basis for selectivity is due to the unique glycine gatekeeper residue in the ATP-binding site at residue 128. We show that BKIs interfere with an early step in T. gondii infection of human cells in culture. Furthermore, we show that TgCDPK1 is the in vivo target of BKIs because T. gondii cells expressing a glycine to methionine gatekeeper mutant enzyme show significantly decreased sensitivity to this class of selective kinase inhibitors. Thus, design of selective TgCDPK1 inhibitors with low host toxicity may be achievable. PMID:20436472

  10. Protein Kinases of the Hippo Pathway: Regulation and Substrates

    PubMed Central

    Avruch, Joseph; Zhou, Dawang; Fitamant, Julien; Bardeesy, Nabeel; Mou, Fan; Barrufet, Laura Regué

    2012-01-01

    The “Hippo” signaling pathway has emerged as a major regulator of cell proliferation and survival in metazoans. The pathway, as delineated by genetic and biochemical studies in Drosophila, consists of a kinase cascade regulated by cell-cell contact and cell polarity that inhibits the transcriptional coactivator Yorkie and its proliferative, anti-differentiation, antiapoptotic transcriptional program. The core pathway components are the GC kinase Hippo, which phosphorylates the noncatalytic polypeptide Mats/Mob1 and, with the assistance of the scaffold protein Salvador, phosphorylates the ndr-family kinase Lats. In turn phospho-Lats, after binding to phospho-Mats, autoactivates and phosphorylates Yorkie, resulting in its nuclear exit. Hippo also uses the scaffold protein Furry and a different Mob protein to control another ndr-like kinase, the morphogenetic regulator Tricornered. Architecturally homologous kinase cascades consisting of a GC kinase, a Mob protein, a scaffolding polypeptide and an ndr-like kinase are well described in yeast; in S. cerevisiae e.g., the MEN pathway promotes mitotic exit whereas the RAM network, using a different GC kinase, Mob protein, scaffold and ndr-like kinase, regulates cell polarity and morphogenesis. In mammals, the Hippo orthologues Mst1 and Mst2 utilize the Salvador ortholog WW45/Sav1 and other scaffolds to regulate the kinases Lats1/Lats2 and ndr1/ndr2. As in Drosophila, murine Mst1/Mst2, in a redundant manner, negatively regulate the Yorkie ortholog YAP in the epithelial cells of the liver and gut; loss of both Mst1 and Mst2 results in hyperproliferation and tumorigenesis that can be largely negated by reduction or elimination of YAP. Despite this conservation, considerable diversification in pathway composition and regulation is already evident; in skin e.g., YAP phosphorylation is independent of Mst1Mst2 and Lats1Lats2. Moreover, in lymphoid cells, Mst1/Mst2, under the control of the Rap1 GTPase and independent of YAP

  11. Functional mapping of protein kinase A reveals its importance in adult Schistosoma mansoni motor activity.

    PubMed

    de Saram, Paulu S R; Ressurreição, Margarida; Davies, Angela J; Rollinson, David; Emery, Aidan M; Walker, Anthony J

    2013-01-01

    Cyclic AMP (cAMP)-dependent protein kinase/protein kinase A (PKA) is the major transducer of cAMP signalling in eukaryotic cells. Here, using laser scanning confocal microscopy and 'smart' anti-phospho PKA antibodies that exclusively detect activated PKA, we provide a detailed in situ analysis of PKA signalling in intact adult Schistosoma mansoni, a causative agent of debilitating human intestinal schistosomiasis. In both adult male and female worms, activated PKA was consistently found associated with the tegument, oral and ventral suckers, oesophagus and somatic musculature. In addition, the seminal vesicle and gynaecophoric canal muscles of the male displayed activated PKA whereas in female worms activated PKA localized to the ootype wall, the ovary, and the uterus particularly around eggs during expulsion. Exposure of live worms to the PKA activator forskolin (50 µM) resulted in striking PKA activation in the central and peripheral nervous system including at nerve endings at/near the tegument surface. Such neuronal PKA activation was also observed without forskolin treatment, but only in a single batch of worms. In addition, PKA activation within the central and peripheral nervous systems visibly increased within 15 min of worm-pair separation when compared to that observed in closely coupled worm pairs. Finally, exposure of adult worms to forskolin induced hyperkinesias in a time and dose dependent manner with 100 µM forskolin significantly increasing the frequency of gross worm movements to 5.3 times that of control worms (P≤0.001). Collectively these data are consistent with PKA playing a central part in motor activity and neuronal communication, and possibly interplay between these two systems in S. mansoni. This study, the first to localize a protein kinase when exclusively in an activated state in adult S. mansoni, provides valuable insight into the intricacies of functional protein kinase signalling in the context of whole schistosome physiology.

  12. FAM46 proteins are novel eukaryotic non-canonical poly(A) polymerases

    PubMed Central

    Kuchta, Krzysztof; Muszewska, Anna; Knizewski, Lukasz; Steczkiewicz, Kamil; Wyrwicz, Lucjan S.; Pawlowski, Krzysztof; Rychlewski, Leszek; Ginalski, Krzysztof

    2016-01-01

    FAM46 proteins, encoded in all known animal genomes, belong to the nucleotidyltransferase (NTase) fold superfamily. All four human FAM46 paralogs (FAM46A, FAM46B, FAM46C, FAM46D) are thought to be involved in several diseases, with FAM46C reported as a causal driver of multiple myeloma; however, their exact functions remain unknown. By using a combination of various bioinformatics analyses (e.g. domain architecture, cellular localization) and exhaustive literature and database searches (e.g. expression profiles, protein interactors), we classified FAM46 proteins as active non-canonical poly(A) polymerases, which modify cytosolic and/or nuclear RNA 3′ ends. These proteins may thus regulate gene expression and probably play a critical role during cell differentiation. A detailed analysis of sequence and structure diversity of known NTases possessing PAP/OAS1 SBD domain, combined with state-of-the-art comparative modelling, allowed us to identify potential active site residues responsible for catalysis and substrate binding. We also explored the role of single point mutations found in human cancers and propose that FAM46 genes may be involved in the development of other major malignancies including lung, colorectal, hepatocellular, head and neck, urothelial, endometrial and renal papillary carcinomas and melanoma. Identification of these novel enzymes taking part in RNA metabolism in eukaryotes may guide their further functional studies. PMID:27060136

  13. A small nucleolar RNP protein is required for pseudouridylation of eukaryotic ribosomal RNAs.

    PubMed Central

    Bousquet-Antonelli, C; Henry, Y; G'elugne, J P; Caizergues-Ferrer, M; Kiss, T

    1997-01-01

    Eukaryotic rRNAs possess numerous post-transcriptionally modified nucleotides. The most abundant modifications, 2'-O-ribose methylation and pseudouridylation, occur in the nucleolus during rRNA processing. The nucleolus contains a large number of small nucleolar RNAs (snoRNAs) most of which can be classified into two distinct families defined by conserved sequence boxes and common associated proteins. The C and D box-containing snoRNAs are associated with fibrillarin, and most of them function as guide RNAs in site-specific ribose methylation of rRNAs. The nucleolar function of the other class of snoRNAs, which share box H and ACA elements and are associated with a glycine- and arginine-rich nucleolar protein, Gar1p, remains elusive. Here we demonstrate that the yeast Saccharomyces cerevisiae Gar1 snoRNP protein plays an essential and specific role in the overall pseudouridylation of yeast rRNAs. These results establish a novel function for Gar1 protein and indicate that the box H/ACA snoRNAs, or at least a subset of these snoRNAs, function in the site-specific pseudouridylation of rRNAs. PMID:9303321

  14. N-glycosylation Triggers a Dual Selection Pressure in Eukaryotic Secretory Proteins.

    PubMed

    Medus, Máximo Lopez; Gomez, Gabriela E; Zacchi, Lucía F; Couto, Paula M; Labriola, Carlos A; Labanda, María S; Bielsa, Rodrigo Corti; Clérico, Eugenia M; Schulz, Benjamin L; Caramelo, Julio J

    2017-08-18

    Nearly one third of the eukaryotic proteome traverses the secretory pathway and most of these proteins are N-glycosylated in the lumen of the endoplasmic reticulum. N-glycans fulfill multiple structural and biological functions, and are crucial for productive folding of many glycoproteins. N-glycosylation involves the attachment of an oligosaccharide to selected asparagine residues in the sequence N-X-S/T (X ≠ P), a motif known as an N-glycosylation'sequon'. Mutations that create novel sequons can cause disease due to the destabilizing effect of a bulky N-glycan. Thus, an analogous process must have occurred during evolution, whenever ancestrally cytosolic proteins were recruited to the secretory pathway. Here, we show that during evolution N-glycosylation triggered a dual selection pressure on secretory pathway proteins: while sequons were positively selected in solvent exposed regions, they were almost completely eliminated from buried sites. This process is one of the sharpest evolutionary signatures of secretory pathway proteins, and was therefore critical for the evolution of an efficient secretory pathway.

  15. Sparse (13)C labelling for solid-state NMR studies of P. pastoris expressed eukaryotic seven-transmembrane proteins.

    PubMed

    Liu, Jing; Liu, Chang; Fan, Ying; Munro, Rachel A; Ladizhansky, Vladimir; Brown, Leonid S; Wang, Shenlin

    2016-05-01

    We demonstrate a novel sparse (13)C labelling approach for methylotrophic yeast P. pastoris expression system, towards solid-state NMR studies of eukaryotic membrane proteins. The labelling scheme was achieved by co-utilizing natural abundance methanol and specifically (13)C labelled glycerol as carbon sources in the expression medium. This strategy improves the spectral resolution by 1.5 fold, displays site-specific labelling patterns, and has advantages for collecting long-range distance restraints for structure determination of large eukaryotic membrane proteins by solid-state NMR.

  16. Evolutionary versatility of eukaryotic protein domains revealed by their bigram networks

    PubMed Central

    2011-01-01

    Background Protein domains are globular structures of independently folded polypeptides that exert catalytic or binding activities. Their sequences are recognized as evolutionary units that, through genome recombination, constitute protein repertoires of linkage patterns. Via mutations, domains acquire modified functions that contribute to the fitness of cells and organisms. Recent studies have addressed the evolutionary selection that may have shaped the functions of individual domains and the emergence of particular domain combinations, which led to new cellular functions in multi-cellular animals. This study focuses on modeling domain linkage globally and investigates evolutionary implications that may be revealed by novel computational analysis. Results A survey of 77 completely sequenced eukaryotic genomes implies a potential hierarchical and modular organization of biological functions in most living organisms. Domains in a genome or multiple genomes are modeled as a network of hetero-duplex covalent linkages, termed bigrams. A novel computational technique is introduced to decompose such networks, whereby the notion of domain "networking versatility" is derived and measured. The most and least "versatile" domains (termed "core domains" and "peripheral domains" respectively) are examined both computationally via sequence conservation measures and experimentally using selected domains. Our study suggests that such a versatility measure extracted from the bigram networks correlates with the adaptivity of domains during evolution, where the network core domains are highly adaptive, significantly contrasting the network peripheral domains. Conclusions Domain recombination has played a major part in the evolution of eukaryotes attributing to genome complexity. From a system point of view, as the results of selection and constant refinement, networks of domain linkage are structured in a hierarchical modular fashion. Domains with high degree of networking

  17. Phylogenetic analysis of eukaryotic NEET proteins uncovers a link between a key gene duplication event and the evolution of vertebrates

    NASA Astrophysics Data System (ADS)

    Inupakutika, Madhuri A.; Sengupta, Soham; Nechushtai, Rachel; Jennings, Patricia A.; Onuchic, Jose’ N.; Azad, Rajeev K.; Padilla, Pamela; Mittler, Ron

    2017-02-01

    NEET proteins belong to a unique family of iron-sulfur proteins in which the 2Fe-2S cluster is coordinated by a CDGSH domain that is followed by the “NEET” motif. They are involved in the regulation of iron and reactive oxygen metabolism, and have been associated with the progression of diabetes, cancer, aging and neurodegenerative diseases. Despite their important biological functions, the evolution and diversification of eukaryotic NEET proteins are largely unknown. Here we used the three members of the human NEET protein family (CISD1, mitoNEET; CISD2, NAF-1 or Miner 1; and CISD3, Miner2) as our guides to conduct a phylogenetic analysis of eukaryotic NEET proteins and their evolution. Our findings identified the slime mold Dictyostelium discoideum’s CISD proteins as the closest to the ancient archetype of eukaryotic NEET proteins. We further identified CISD3 homologs in fungi that were previously reported not to contain any NEET proteins, and revealed that plants lack homolog(s) of CISD3. Furthermore, our study suggests that the mammalian NEET proteins, mitoNEET (CISD1) and NAF-1 (CISD2), emerged via gene duplication around the origin of vertebrates. Our findings provide new insights into the classification and expansion of the NEET protein family, as well as offer clues to the diverged functions of the human mitoNEET and NAF-1 proteins.

  18. Phylogenetic analysis of eukaryotic NEET proteins uncovers a link between a key gene duplication event and the evolution of vertebrates

    PubMed Central

    Inupakutika, Madhuri A.; Sengupta, Soham; Nechushtai, Rachel; Jennings, Patricia A.; Onuchic, Jose’ N.; Azad, Rajeev K.; Padilla, Pamela; Mittler, Ron

    2017-01-01

    NEET proteins belong to a unique family of iron-sulfur proteins in which the 2Fe-2S cluster is coordinated by a CDGSH domain that is followed by the “NEET” motif. They are involved in the regulation of iron and reactive oxygen metabolism, and have been associated with the progression of diabetes, cancer, aging and neurodegenerative diseases. Despite their important biological functions, the evolution and diversification of eukaryotic NEET proteins are largely unknown. Here we used the three members of the human NEET protein family (CISD1, mitoNEET; CISD2, NAF-1 or Miner 1; and CISD3, Miner2) as our guides to conduct a phylogenetic analysis of eukaryotic NEET proteins and their evolution. Our findings identified the slime mold Dictyostelium discoideum’s CISD proteins as the closest to the ancient archetype of eukaryotic NEET proteins. We further identified CISD3 homologs in fungi that were previously reported not to contain any NEET proteins, and revealed that plants lack homolog(s) of CISD3. Furthermore, our study suggests that the mammalian NEET proteins, mitoNEET (CISD1) and NAF-1 (CISD2), emerged via gene duplication around the origin of vertebrates. Our findings provide new insights into the classification and expansion of the NEET protein family, as well as offer clues to the diverged functions of the human mitoNEET and NAF-1 proteins. PMID:28205535

  19. Actions of Rho family small G proteins and p21-activated protein kinases on mitogen-activated protein kinase family members.

    PubMed Central

    Frost, J A; Xu, S; Hutchison, M R; Marcus, S; Cobb, M H

    1996-01-01

    The mitogen-activated protein (MAP) kinases are a family of serine/threonine kinases that are regulated by distinct extracellular stimuli. The currently known members include extracellular signal-regulated protein kinase 1 (ERK1), ERK2, the c-Jun N-terminal kinase/stress-activated protein kinases (JNK/SAPKs), and p38 MAP kinases. We find that overexpression of the Ste20-related enzymes p21-activated kinase 1 (PAK1) and PAK2 in 293 cells is sufficient to activate JNK/SAPK and to a lesser extent p38 MAP kinase but not ERK2. Rat MAP/ERK kinase kinase 1 can stimulate the activity of each of these MAP kinases. Although neither activated Rac nor the PAKs stimulate ERK2 activity, overexpression of either dominant negative Rac2 or the N-terminal regulatory domain of PAK1 inhibits Ras-mediated activation of ERK2, suggesting a permissive role for Rac in the control of the ERK pathway. Furthermore, constitutively active Rac2, Cdc42hs, and RhoA synergize with an activated form of Raf to increase ERK2 activity. These findings reveal a previously unrecognized connection between Rho family small G proteins and the ERK pathway. PMID:8668187

  20. Bacterial-like PPP protein phosphatases: novel sequence alterations in pathogenic eukaryotes and peculiar features of bacterial sequence similarity.

    PubMed

    Kerk, David; Uhrig, R Glen; Moorhead, Greg B

    2013-01-01

    Reversible phosphorylation is a widespread modification affecting the great majority of eukaryotic cellular proteins, and whose effects influence nearly every cellular function. Protein phosphatases are increasingly recognized as exquisitely regulated contributors to these changes. The PPP (phosphoprotein phosphatase) family comprises enzymes, which catalyze dephosphorylation at serine and threonine residues. Nearly a decade ago, "bacterial-like" enzymes were recognized with similarity to proteins from various bacterial sources: SLPs (Shewanella-like phosphatases), RLPHs (Rhizobiales-like phosphatases), and ALPHs (ApaH-like phosphatases). A recent article from our laboratory appearing in Plant Physiology characterizes their extensive organismal distribution, abundance in plant species, predicted subcellular localization, motif organization, and sequence evolution. One salient observation is the distinct evolutionary trajectory followed by SLP genes and proteins in photosynthetic eukaryotes vs. animal and plant pathogens derived from photosynthetic ancestors. We present here a closer look at sequence data that emphasizes the distinctiveness of pathogen SLP proteins and that suggests that they might represent novel drug targets. A second observation in our original report was the high degree of similarity between the bacterial-like PPPs of eukaryotes and closely related proteins of the "eukaryotic-like" phyla Myxococcales and Planctomycetes. We here reflect on the possible implications of these observations and their importance for future research.

  1. Protein-protein interactions of tandem affinity purified protein kinases from rice.

    PubMed

    Rohila, Jai S; Chen, Mei; Chen, Shuo; Chen, Johann; Cerny, Ronald L; Dardick, Christopher; Canlas, Patrick; Fujii, Hiroaki; Gribskov, Michael; Kanrar, Siddhartha; Knoflicek, Lucas; Stevenson, Becky; Xie, Mingtang; Xu, Xia; Zheng, Xianwu; Zhu, Jian-Kang; Ronald, Pamela; Fromm, Michael E

    2009-08-19

    Eighty-eight rice (Oryza sativa) cDNAs encoding rice leaf expressed protein kinases (PKs) were fused to a Tandem Affinity Purification tag (TAP-tag) and expressed in transgenic rice plants. The TAP-tagged PKs and interacting proteins were purified from the T1 progeny of the transgenic rice plants and identified by tandem mass spectrometry. Forty-five TAP-tagged PKs were recovered in this study and thirteen of these were found to interact with other rice proteins with a high probability score. In vivo phosphorylated sites were found for three of the PKs. A comparison of the TAP-tagged data from a combined analysis of 129 TAP-tagged rice protein kinases with a concurrent screen using yeast two hybrid methods identified an evolutionarily new rice protein that interacts with the well conserved cell division cycle 2 (CDC2) protein complex.

  2. Protein-Protein Interactions of Tandem Affinity Purified Protein Kinases from Rice

    PubMed Central

    Rohila, Jai S.; Chen, Mei; Chen, Shuo; Chen, Johann; Cerny, Ronald L.; Dardick, Christopher; Canlas, Patrick; Fujii, Hiroaki; Gribskov, Michael; Kanrar, Siddhartha; Knoflicek, Lucas; Stevenson, Becky; Xie, Mingtang; Xu, Xia; Zheng, Xianwu; Zhu, Jian-Kang; Ronald, Pamela; Fromm, Michael E.

    2009-01-01

    Eighty-eight rice (Oryza sativa) cDNAs encoding rice leaf expressed protein kinases (PKs) were fused to a Tandem Affinity Purification tag (TAP-tag) and expressed in transgenic rice plants. The TAP-tagged PKs and interacting proteins were purified from the T1 progeny of the transgenic rice plants and identified by tandem mass spectrometry. Forty-five TAP-tagged PKs were recovered in this study and thirteen of these were found to interact with other rice proteins with a high probability score. In vivo phosphorylated sites were found for three of the PKs. A comparison of the TAP-tagged data from a combined analysis of 129 TAP-tagged rice protein kinases with a concurrent screen using yeast two hybrid methods identified an evolutionarily new rice protein that interacts with the well conserved cell division cycle 2 (CDC2) protein complex. PMID:19690613