Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The c-Mos serine/threonine protein kinase is an essential component of cytostatic factor (CSF), which is required for metaphase II arrest of eggs in vertebrates. Previously, we showed that c-Mos residue Ser-16 is phosphorylated in the ts110 Mo-MuSV-encoded Gag-Mos fusion protein. Here we provide evidence that Mos is phosphorylated at Ser-16 in transfected COS-1 cells. To investigate the role of this phosphorylation, Ser-16 was substituted with alanine or glutamic acid in full-length v-Mos (an Env-Mos fusion protein that contains 31 additional amino acids at the amino terminus of c-Mos), its mouse c-Mos equivalent version (v-Mos residues 32-374, hereafter referred to as Mos), and mouse c-Mos. Constructs expressing mutant versions of Mos were transfected into COS-1 and NIH3T3 cells in a transient and stable manner, respectively. Synthesis and proteolysis of Mos were evaluated by pulse-chase analysis of 35S-methionine-labeled proteins. Our findings indicate that the S16A mutant of Mos was highly unstable. It accumulated to approximately 10% of the level of wild-type Mos or its S16E mutant. In addition, the S16A mutation but not the S16E mutation inhibited Mos interaction with a cellular protein, p35, suggesting that phosphorylation at Ser-16 may promote Mos interaction with p35. As expected from its destabilizing effect, the S16A mutation caused a dramatic decrease in the cellular transforming activity of Mos (determined by soft-agar colony-formation assay with the stably transfected NIH3T3 cells), which is known to correlate with its CSF function. Efficient ubiquitin-mediated proteolysis of c-Mos requires proline as the second residue from the amino-terminus. In contrast to Mos, neither the stability nor protein kinase activity of v-Mos (in which c-Mos residue Pro-2 becomes Pro-33) was affected by the S16A mutation. To provide further proof that, similar to c-Mos, the S16A mutant is recognized by the proteolysis system through Pro-2, we show that the effect of the S16A mutation is reversed by the Pro-2-Ala mutation. Thus, our results indicate that Ser-16 has an important role in the regulation of c-Mos and that phosphorylation at Ser-16 may inhibit proteolysis of c-Mos.
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PMID:Evidence for an important role of serine 16 and its phosphorylation in the stabilization of c-Mos. 1043 36

The retinoblastoma tumor suppressor gene (Rb) has many functions within the cell including regulation of transcription, differentiation, apoptosis, and the cell cycle. Regulation of these functions is mediated by phosphorylation at as many as 16 cyclin-dependent kinase (CDK) phosphorylation sites in vivo. The contribution of these sites to the regulation of the various Rb functions is not well understood. To characterize the effect of phosphorylation at these sites, we systematically mutagenized the serines or threonines to glutamic acid. Thirty-five mutants with different combinations of modified phosphorylation sites were assayed for their ability to arrest the cell cycle and for their potential to induce differentiation. Only the most highly substituted mutants failed to arrest cell cycle progression. However, mutants with as few as four modified phosphorylation sites were unable to promote differentiation. Other mutants had increased activity in this assay. We conclude that modification of Rb phosphorylation sites can increase or decrease protein activity, that different Rb functions can be regulated independently by distinct combinations of sites, and that the effects of modification at any one site are context dependent.
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PMID:Glutamic acid mutagenesis of retinoblastoma protein phosphorylation sites has diverse effects on function. 1069 26

Activation of transcription factor NF-kappa B involves the signal-dependent degradation of basally phosphorylated inhibitors such as I kappa B alpha. In response to proinflammatory cytokines or mitogens, the transduction machinery has recently been characterized, but the activation mechanism upon oxidative stress remains unknown. In the present work, we provide several lines of evidence that NF-kappa B activation in a T lymphocytic cell line (EL4) by hydrogen peroxide (H2O2) did not involve phosphorylation of the serine residues 32 and 36 in the amino-terminal part of I kappa B alpha. Indeed, mutation of Ser32 and Ser36 blocked IL-1 beta- or PMA-induced NF-kappa B activation, but had no effect on its activation by H2O2. Although I kappa B alpha was phosphorylated upon exposure to H2O2, tyrosine residue 42 and the C-terminal PEST (proline-glutamic acid-serine-threonine) domain played an important role. Indeed, mutation of tyrosine 42 or serine/threonine residues of the PEST domain abolished NF-kappa B activation by H2O2, while it had no effect on activation by IL-1 beta or PMA-ionomycin. This H2O2-inducible phosphorylation was not dependent on I kappa B kinase activation, but could involve casein kinase II, because an inhibitor of this enzyme (5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole) blocks NF-kappa B activation. H2O2-induced I kappa B alpha phosphorylation was followed by its degradation by calpain proteases or through the proteasome. Taken together, our findings suggest that NF-kappa B activation by H2O2 involves a new mechanism that is totally distinct from those triggered by proinflammatory cytokines or mitogens.
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PMID:Crucial role of the amino-terminal tyrosine residue 42 and the carboxyl-terminal PEST domain of I kappa B alpha in NF-kappa B activation by an oxidative stress. 1075 28

A third calcium-dependent protein kinase (CDPK) gene has been isolated from the human malaria parasite Plasmodium falciparum by vectorette technology. The gene consists of five exons and four introns. The open reading frame resulting from removal of the four introns encodes a protein of 562 amino acid residues with a predicted molecular mass of 65.3 kDa. The encoded protein, termed PfCDPK3, consists of four distinct domains characteristic of a member of the CDPK family and displays the highest homology (46% identity and 69% similarity) to PfCDPK2, the second CDPK of P. falciparum. The N-terminal variable domain is rich in serine/threonine and lysine and contains multiple consensus phosphorylation sites for a range of protein kinases. The catalytic domain possesses all conserved motifs of the protein kinase family except for the highly conserved glutamic acid residue in subdomain VIII, which is replaced by a glutamine residue. The sequence of the junction domain comprising 31 amino acid residues is less conserved. The calmodulin-like regulatory domain contains four EF-hand calcium-binding motifs, each consisting of a loop of 12 amino acid residues which is flanked by two alpha-helices. Southern blotting of genomic DNA digests showed that the Pfcdpk3 gene is present as a single copy per haploid genome. A 2900 nucleotide transcript of this gene is expressed specifically in the sexual erythrocytic stage, indicating that PfCDPK3 is involved in sexual stage-specific events. It is proposed that PfCDPK3 may serve as a link between calcium and gametogenesis of P. falciparum.
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PMID:Sexual stage-specific expression of a third calcium-dependent protein kinase from Plasmodium falciparum. 1076 Jun 1

The association of the p27(Kip1) protein with cyclin and cyclin-dependent kinase complexes inhibits their kinase activities and contributes to the control of cell proliferation. The p27(Kip1) protein has now been shown to be phosphorylated in vivo, and this phosphorylation reduces the electrophoretic mobility of the protein. Substitution of Ser(10) with Ala (S10A) markedly reduced the extent of p27(Kip1) phosphorylation and prevented the shift in electrophoretic mobility. Phosphopeptide mapping and phosphoamino acid analysis revealed that phosphorylation at Ser(10) accounted for approximately 70% of the total phosphorylation of p27(Kip1), and the extent of phosphorylation at this site was approximately 25- and 75-fold greater than that at Ser(178) and Thr(187), respectively. The phosphorylation of p27(Kip1) was markedly reduced when the positions of Ser(10) and Pro(11) were reversed, suggesting that a proline-directed kinase is responsible for the phosphorylation of Ser(10). The extent of Ser(10) phosphorylation was markedly increased in cells in the G(0)-G(1) phase of the cell cycle compared with that apparent for cells in S or M phase. The p27(Kip1) protein phosphorylated at Ser(10) was significantly more stable than the unphosphorylated form. Furthermore, a mutant p27(Kip1) in which Ser(10) was replaced with glutamic acid in order to mimic the effect of Ser(10) phosphorylation exhibited a marked increase in stability both in vivo and in vitro compared with the wild-type or S10A mutant proteins. These results suggest that Ser(10) is the major site of phosphorylation of p27(Kip1) and that phosphorylation at this site, like that at Thr(187), contributes to regulation of p27(Kip1) stability.
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PMID:Phosphorylation at serine 10, a major phosphorylation site of p27(Kip1), increases its protein stability. 1083 86

The small G-protein Ras, a critical component in the signalling pathways regulating cell growth, is involved in the tonic upregulation of voltage-dependent calcium channels (VDCCs) in rat sensory neurones. To investigate which downstream effector(s) of Ras is involved in this process, a series of Ras mutant cDNAs were co-expressed with green fluorescent protein (GFP) in primary cultured rat dorsal root ganglion neurones (DRGs). Constitutively active V12Ras (glycine 12 to valine) markedly increased basal calcium current density by 41 % compared with control cells (GFP alone). In contrast, a farnesylation-defective mutant, V12S186Ras (cysteine 186 to serine; activates no downstream effectors), significantly reduced calcium current density by 47 %. Ras effector region mutants V12C40 (tyrosine 40 to cysteine; activates the p110 alpha-subunit of phosphatidylinositol 3-kinase) and V12G37 (glutamic acid 37 to glycine; activates Ral guanine nucleotide dissociation stimulator) had no significant effect on VDCC current. However, V12S35Ras (threonine 35 to serine; activates Raf-1 and the mitogen-activated protein kinase (MAPK) pathway) markedly increased basal calcium current density by 67 %, suggesting that Raf-1 activation is sufficient for Ras enhancement of calcium current in these cells. Raf-1 activates MEK (MAPK kinase) in the MAPK pathway, and the MEK inhibitor U0126 reduced calcium current by 45 % after 10-15 min, whereas the inactive analogue U0124 had no effect. This rapid time course for MEK inhibition suggests direct modulation of VDCCs via the Ras-MAPK pathway rather than gene expression-mediated effects. The relative proportions of omega-conotoxin GVIA- and nicardipine-sensitive N- ( approximately 40 %) and L- ( approximately 40 %) type currents were unaffected by either V12S35Ras expression or U0126 pre-treatment, suggesting that all components of calcium current in DRGs, are enhanced via this pathway.
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PMID:Regulation of voltage-dependent calcium channels in rat sensory neurones involves a Ras-mitogen-activated protein kinase pathway. 1099 May 31

Microtubule-associated protein 2 (MAP2) is a neuronal phosphoprotein that promotes net microtubule growth and actin cross-linking and bundling in vitro. Little is known about MAP2 regulation or its interaction with the cytoskeleton in vivo. Here we investigate the in vivo function of three specific sites of phosphorylation on MAP2. cAMP-dependent protein kinase activity disrupts the MAP2-microtubule interaction in living HeLa cells and promotes MAP2c localization to peripheral membrane ruffles enriched in actin. cAMP-dependent protein kinase phosphorylates serines within three KXGS motifs, one within each tubulin-binding repeat. These highly conserved motifs are also found in homologous proteins tau and MAP4. Phosphorylation at two of these sites was detected in brain tissue. Constitutive phosphorylation at these sites was mimicked by single, double, and triple mutations to glutamic acid. Biochemical and microscopy-based assays indicated that mutation of a single residue was adequate to disrupt the MAP2-microtubule interaction in HeLa cells. Double or triple point mutation promoted MAP2c localization to the actin cytoskeleton. Specific association between MAP2c and the actin cytoskeleton was demonstrated by retention of MAP2c-actin colocalization after detergent extraction. Specific phosphorylation states may enhance the interaction of MAP2 with the actin cytoskeleton, thereby providing a regulated mechanism for MAP2 function within distinct cytoskeletal domains.
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PMID:Phosphorylation-dependent localization of microtubule-associated protein MAP2c to the actin cytoskeleton. 1102 56

The c-myb gene encodes a transcription factor that is central to hematopoietic cell growth. Phosphorylation of c-Myb by casein kinase 2 (CK2) at serines 11 and 12 has been variously implicated in the regulation of DNA binding. However, it is unclear when c-Myb phosphorylation at serines 11 and 12 occurs during the cell cycle and how this is regulated. We generated specific antisera that recognize phosphoserines 11 and 12 of c-Myb. C-Myb protein levels, extent of CK2 phosphorylation and DNA binding were then monitored following mitogenic stimulus and passage through the cell cycle in normal peripheral T-cells and the T leukemia cell line CCRF-CEM. We found that endogenous c-Myb is constitutively phosphorylated at serines 11 and 12. The amount of phosphorylated c-Myb correlates with DNA binding activity in cycling CEM cells but not upon entry of T-cells into the cell cycle. Exogenous expression of c-Myb with substitutions of serines 11 and 12 with glutamic acid or alanine had no effect on the transactivation of a c-Myb responsive reporter. These data strongly suggest that c-Myb is constitutively phosphorylated on serines 11 and 12 by CK2 or like activity and is not regulated during the cell cycle.
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PMID:Constitutive c-Myb amino-terminal phosphorylation and DNA binding activity uncoupled during entry and passage through the cell cycle. 1131 25

The Arabidopsis thaliana somatic embryogenesis receptor kinase 1 (AtSERK1) gene encodes a receptor-like protein kinase that is transiently expressed during embryogenesis. To determine the intrinsic biochemical properties of the AtSERK1 protein, we have expressed the intracellular catalytic domain as a glutathione S-transferase fusion protein in Escherichia coli. The AtSERK1-glutathione S-transferase fusion protein mainly autophosphorylates on threonine residues (K(m) for ATP, 4 x 10(-6) m), and the reaction is Mg(2+) dependent and inhibited by Mn(2+). A K330E substitution in the kinase domain of AtSERK1 abolishes all kinase activity. The active AtSERK1(kin) can phosphorylate inactive AtSERK1(K330E) protein, suggesting an intermolecular mechanism of autophosphorylation. The AtSERK1 kinase protein was modeled using the insulin receptor kinase as a template. On the basis of this model, threonine residues in the AtSERK1 activation loop of catalytic subdomain VIII are potential targets for phosphorylation. AtSERK1 phosphorylation on myelin basic protein and casein showed tyrosine, serine, and threonine as targets, demonstrating that AtSERK1 is a dual specificity kinase. Replacing Thr-468 with either alanine or glutamic acid not only obliterated the ability of the AtSERK1 protein to be phosphorylated but also inhibited phosphorylation on myelin basic protein and casein, suggesting that Thr-468 is essential for AtSERK-mediated signaling.
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PMID:Role of threonines in the Arabidopsis thaliana somatic embryogenesis receptor kinase 1 activation loop in phosphorylation. 1150 54

Capacitation represents the final maturational steps that render mammalian sperm competent to fertilize, either in vivo or in vitro. Capacitation is defined as a series of events that enables sperm to bind the oocyte and undergo the acrosome reaction in response to the zona pellucida. Although the molecular mechanisms involved are not fully understood, sperm protein phosphorylation is associated with capacitation. The hypothesis of this study is that protein tyrosine phosphorylation and kinase activity mediate capacitation of porcine sperm. Fresh sperm were incubated in noncapacitating or capacitating media for various times. Proteins were extracted with SDS, subjected to SDS-PAGE, and immunoblotted with an antiphosphotyrosine antibody. An M(r) 32 000 tyrosine-phosphorylated protein (designated as p32) appeared only when the sperm were incubated in capacitating medium and concomitant with capacitation as assessed by the ionophore-induced acrosome reaction. The p32 was soluble in Triton X-100. Fractionation of sperm proteins with Triton X-114 demonstrated that after capacitation, this tyrosine phosphoprotein is located in both the cytosol and the membrane. Enzyme renaturation of sperm proteins was conducted in gels with or without either poly glu:tyr (a tyrosine kinase substrate) or kemptide (a protein kinase A substrate). An M(r) 32 000 enzyme with kinase behavior was observed in all gels but was preferentially phosphorylated on tyrosine, as assessed by phosphorimagery and by thin layer chromotography to identify the phosphoamino acids. Indirect immunolocalization showed that the phosphotyrosine residues redistribute to the acrosome during capacitation, which is an appropriate location for a protein involved in the acquisition of fertility.
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PMID:Capacitation is associated with tyrosine phosphorylation and tyrosine kinase-like activity of pig sperm proteins. 1151 42


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