Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Protein kinase C-theta (PKC-eta) is a member of the protein kinase C family that is characterized by Ca2+ independence and restricted histone kinase activity (Dekker, L. V., Parker, P. J., and McIntyre, P. (1992) FEBS Lett. 312, 195-199). Here we have investigated the molecular basis of this low histone kinase activity by limited proteolysis and site-directed mutagenesis. It is shown that a 46-kDa C-terminal tryptic fragment, representing the catalytic domain of PKC-eta, can phosphorylate histone. The Km value for histone of this catalytic fragment is 25-fold lower than that of intact PKC-eta. Thus, sites in the N-terminal regulatory domain upstream of the trypsin cleavage site (near residue 320) restrict histone kinase activity of intact PKC-eta. Deletion of the "Vo domain" (residues 2-137) generates a PKC-eta mutant that shows the same cofactor dependence and substrate phosphorylation as wild-type PKC-eta, indicating that the relevant sites do not appear to lie in the Vo domain but between amino acid 137 and the start of the catalytic domain. Deletion of the pseudosubstrate region (residue 155-171) generates a cofactor-independent kinase that has high histone kinase activity. A pseudosubstrate site point mutation in which the alanine residue at position 161 is replaced with a glutamic acid residue shows the same properties as the pseudosubstrate site deletion mutant. Km values for histone for both mutants are similar to that observed for the catalytic fragment. Therefore, in addition to its role in conferring cofactor dependence, the pseudosubstrate site also mediates the low histone kinase activity of wild-type PKC-eta. The data are discussed in the light of current models for PKC activation.
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PMID:Mutagenesis of the regulatory domain of rat protein kinase C-eta. A molecular basis for restricted histone kinase activity. 839 39

Sequence analysis of a nearly full-length murine c-akt cDNA clone and comparison with v-akt revealed the following: (a) The entire coding region of c-akt is identical to that of v-akt with the exception of five G to A transitions that do not alter the reading frame. The 3' untranslated regions of v-akt and c-akt are also identical with the exception of three single-base differences. (b) The recombination event that gave rise to v-akt occurred between the virus at nucleotide 785 from the Gag ATG codon and the 5' untranslated region of c-akt to 60 bp 5' from the c-akt ATG codon. (c) Three nucleotides absent from both Gag and c-akt were inserted at the junction between the two genes. The outcome of these events was to place, in frame, a 63-bp fragment between Gag and Akt. The resulting v-akt oncogene is predicted to encode a tripartite Gag (p12, p15, delta p30)-X-c-akt protein product. The c-akt protein contains, starting from its amino terminus, a src homology 2-like (SH2-like) domain, a domain rich in glutamic acid residues, part of which is predicted to form an amphipathic helix, and a kinase domain encoding a serine-threonine kinase with high degree of homology to members of the protein kinase C (PKC) family. The mouse c-akt is 90% homologous to human AKT1/RAC at the nucleic acid level and 98% homologous at the amino acid level. c-akt in the mouse is composed of 13 exons. The first exon contains a 5' untranslated GC-rich region. Since the recombination that gave rise to v-akt occurred with the 5' untranslated region, we hypothesize that the transduction of c-akt was preceded by provirus insertion upstream from or within the 5' untranslated region and in the same transcriptional orientation as the gene. c-akt was mapped by fluorescence in situ hybridization (FISH) to mouse chromosome 12 and rat chromosome 6 in close proximity to the Igh locus.
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PMID:Structure, expression and chromosomal mapping of c-akt: relationship to v-akt and its implications. 843 58

Adducin is a 200-kDa heterodimeric protein of the cortical cytoskeleton of mammalian erythrocytes. Analogs are also abundant in brain and several other tissues. In vitro, adducin bundles F-actin and enhances the binding of spectrin to actin. Previous studies have established that the beta subunit of adducin binds calmodulin (CaM) in a Ca(2+)-dependent fashion with intermediate affinity (approximately 200 nM) and that this activity is destroyed by proteolysis. We have confirmed the trypsin sensitivity of CaM binding by beta-adducin and the existence of a 38- to 39-kDa protease-resistant core. Calpain I digestion generates a larger core fragment (49 kDa) that is also devoid of CaM-binding activity. Use of recombinant beta-adducin peptides generated from partial cDNA clones identified strong CaM-binding activity within the protease-sensitive domain in residues 425-461: KQQKEKTRWLNTPNTYLRVNVADEVQRNMGSPRPKTT in single-letter amino acid codes. This region of the molecule is highly conserved between mouse, rat, and human and shares structural features with CaM-binding sequences in other proteins. Multiple flanking PEST sequences (sequences rich in proline, glutamic acid, serine, and threonine residues that enhance proteolytic sensitivity) may contribute to the protease sensitivity of this region. Consensus sequences for phosphorylation by cAMP-dependent kinases and by protein kinase C (or CaM-dependent kinase) are also found within or near this CaM-binding domain. Collectively, these data suggest a structural basis for the regulation of adducin by Ca(2+)-dependent CaM binding and possibly by covalent phosphorylation and calpain I-mediated proteolysis as well.
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PMID:Calmodulin-binding domain of recombinant erythrocyte beta-adducin. 847 88

Pleckstrin is the major substrate phosphorylated on serine and threonine in response to stimulation of human platelets by thrombin (Abrams, C. S., Zhao, W., Belmonte, E., and Brass, L. F. (1995) J. Biol. Chem. 270, 23317-23321). We now show that pleckstrin in platelets is in a complex with inositol polyphosphate 5-phosphatase I (5-phosphatase I). This enzyme hydrolyzes the 5-phosphate from inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate and thus serves as a calcium signal-terminating enzyme, since the substrates but not the products mobilize intracellular calcium. Pleckstrin co-immunoprecipitates with 5-phosphatase I in homogenates of platelets. Platelet homogenates fractionated by anion exchange chromatography show co-elution of pleckstrin and 5-phosphatase I. Fractions containing phosphorylated pleckstrin have 7-fold greater 5-phosphatase activity than those containing unphosphorylated pleckstrin. Mixing experiments with recombinant 5-phosphatase I and pleckstrin in vitro show that they form a stoichiometric complex. A mutant form of pleckstrin, in which the serine and threonine residues that are phosphorylated by protein kinase C are substituted with glutamic acid (pseudophosphorylated pleckstrin), activates recombinant 5-phosphatase I 2-3-fold while native unphosphorylated pleckstrin does not stimulate the enzyme. Thus pleckstrin functions to terminate calcium signaling in platelets when it is phosphorylated by binding to and activating 5-phosphatase I.
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PMID:Phosphorylation of platelet pleckstrin activates inositol polyphosphate 5-phosphatase I. 899 61

The 78-kDa protein kinase Mekk1 plays an important role in the stress response pathway that involves the activation of downstream kinases Sek1 and stress-activated protein kinase/c-Jun NH2-terminal kinase. Conserved serine and threonine residues located between the kinase subdomains VII and VIII of many protein kinases are phosphorylated for maximal kinase activation. Two threonine residues within this region in Mekk1 at positions 560 and 572, but not the serine at 557, were shown to be essential for catalytic activity in this study. When these threonine residues were replaced with alanine, there was a significant loss in phosphotransferase activity toward the primary substrate, Sek1, and a large decrease in autophosphorylation activity. Site-directed mutagenesis demonstrated that these threonine residues cannot be replaced with either serine or glutamic acid for preservation of phosphotransferase activity. Further examination of the Mekk1 mutants isolated from 32P-labeled transfected COS cells showed that Thr-560 and Thr-572 were indeed phosphorylated after two-dimensional tryptic-chymotryptic phosphopeptide analysis. Additional determinants in the NH2-terminal domain of Mekk1 also play a role in the regulation of Mekk1 activity. Although Pak3 and PKC can activate Mekk1 in vivo, this interaction is indirect and independent, since there was no direct phosphorylation of Mekk1 by Pak3 or PKC or of Pak3 by PKC, respectively.
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PMID:Identification of two essential phosphorylated threonine residues in the catalytic domain of Mekk1. Indirect activation by Pak3 and protein kinase C. 906 12

As one of the first steps to elucidate the relationship between the structure and function of CTP:phosphocholine cytidylytransferase (EC 2.7.7.15) in plants, the cytidylyltransferase cDNA of Arabidopsis thaliana was cloned and characterized. The A. thaliana cytidylyltransferase cDNA is 1447 bp long and contains an open reading frame of 993 bp coding for a protein of 331 amino acids. The deduced structure of the enzyme was composed of three main regions; the catalytic domain in the N-terminal half, the hydrophilic C-terminal region and the amphipathic domain in the middle. The catalytic domain region was relatively well conserved among different organisms, showing 76 and 72% homology with the rat and yeast protein sequences, respectively. The hydropathy profile revealed that the C-terminal non-catalytic portion of the protein was very hydrophilic, highly enriched in negatively charged aspartic acid and glutamic acid residues. In the region between the catalytic domain and the C-terminal region, there was an amphipathic alpha-helical domain, which was believed to bind the membrane surface in the active formation. Unlike animal counterparts, there was only one potential site of phosphorylation by protein kinase C and none by Ca2+/calmodulin protein kinase II in the C-terminal region. The identity of cytidylyltransferase cDNA was verified by successful transformation of a yeast mutant defective in the enzyme activity, using an expression vector inserted with the A. thaliana cytidylyltransferase cDNA. This was further confirmed by in vivo analysis of the enzyme reaction product after labeling the yeast transformants with radioactive phosphocholine. Southern analysis indicated the presence of a single copy of the citidylyltransferase gene in A. thaliana.
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PMID:Cloning of CTP:phosphocholine cytidylyltransferase cDNA from Arabidopsis thaliana. 908 66

The proteolytic processing of the beta-amyloid precursor protein (APP) is regulated by neurotransmitters. Stimulation of metabotropic glutamate receptors (mGluRs) has been shown to increase the release of soluble amyloid precursor protein derivatives (APPs) from cultured cells. We examined the effects of mGluR agonists on APP processing in cortical and hippocampal slices from rat brain. Incubation of the slices in the presence of L-glutamic acid (500 microM), trans-(1S,3R)-1-amino-1,3-cyclopentane dicarboxylic acid (1-100 microM) or quisqualic acid (1-100 microM) increased APP release into the medium, relative to the amount of APPs released during incubation in normal Krebs-Ringer buffer under basal conditions. N-Methyl-D-aspartate (1-320 microM), (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (1-100 microM) or kainic acid (5-500 microM) did not alter APP release. The increases in APP release induced by L-glutamic acid (500 microM), trans-(1S,3R)-1-amino-1,3-cyclopentane dicarboxylic acid (10 microM) or quisqualic acid (10 microM) were blocked by 100 microM (+/-)-alpha-methyl-4-carboxyphenylglycine, a selective antagonist of mGluRs. Incubation of the slices in the presence of 1 microM phorbol-12-myrisate-13-acetate, an activator of protein kinase C (PKC), also increased APP release, and an inhibitor of PKC, GF-109203X (1 microM), blocked this response as well as the release evoked by mGluR agonists. These data show that activation of mGluR increases APP release from brain slices via PKC-dependent mechanisms.
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PMID:Metabotropic glutamate receptor agonists increase release of soluble amyloid precursor protein derivatives from rat brain cortical and hippocampal slices. 910 92

The activation of p70s6k is associated with multiple phosphorylations at two sets of sites. The first set, S411, S418, T421, and S424, reside within the autoinhibitory domain, and each contains a hydrophobic residue at -2 and a proline at +1. The second set of sites, T229 (in the catalytic domain) and T389 and S404 (in the linker region), are rapamycin sensitive and flanked by bulky aromatic residues. Here we describe the identification and mutational analysis of three new phosphorylation sites, T367, S371, and T447, all of which have a recognition motif similar to that of the first set of sites. A mutation of T367 or T447 to either alanine or glutamic acid had no apparent effect on p70s6k activity, whereas similar mutations of S371 abolished kinase activity. Of these three sites and their surrounding motifs, only S371 is conserved in p70s6k homologs from Drosophila melanogaster, Arabidopsis thaliana, and Saccharomyces cerevisiae, as well as many members of the protein kinase C family. Serum stimulation increased S371 phosphorylation; unlike the situation for specific members of the protein kinase C family, where the homologous site is regulated by autophosphorylation, S371 phosphorylation is regulated by an external mechanism. Phosphopeptide analysis of S371 mutants further revealed that the loss of activity in these variants was paralleled by a block in serum-induced T389 phosphorylation, a phosphorylation site previously shown to be essential for kinase activity. Nevertheless, the substitution of an acidic residue at T389, which mimics phosphorylation at this site, did not rescue mutant p70s6k activity, indicating that S371 phosphorylation plays an independent role in regulating intrinsic kinase activity.
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PMID:Dual requirement for a newly identified phosphorylation site in p70s6k. 927 40

As previously described, confluent AKR-2B fibroblasts rapidly disintegrate upon removal of serum. Platelet-derived growth factor isoforms AB or BB (PDGF-AB, -BB) added immediately after serum deprivation caused complete survival of the cells without initiating proliferation (Simm et al., 1994, J. Cell. Physiol. 160, 295). Here the role of cAMP as a protective agent was investigated by using forskolin or 8-Br-cAMP. Both reagents afforded high cellular protection. The phorbolester TPA, an activator of protein kinase C isoforms, also exerted a high protection against cell death (ED50 = 7 nM). Unexpectedly colchicine (ED50 = 1.5 microM) an inhibitor of tubulin polymerization also protected cells from death. The protective effects of PDGF-BB and TPA were dependent on protein synthesis as indicated by their complete suppression by cycloheximide (CHx). Surprisingly, forskolin and 8-Br-cAMP remained effective even in the presence of CHx. Detailed studies of several signalling pathways were performed. These investigations showed no interference between PDGF-BB and cAMP-dependent pathways at the early stage of signal transduction. As previously described, the ICE-like protease inhibitor tyr-val-ala-asp-chloromethylketone (YVAD-cmk) protected cells from death (Simm et al., 1997, J. Cell Sci. 110, 819-828). As shown here, a substantial protection was also achieved by the addition of two other caspase inhibitors: asp-glu-val-asp-aldehyde (DEVD-cho; ED50 = 100 microM) and benzoylcarbonyl-asp-glu-val-asp-chloromethylketone (Z-DEVD-cmk; ED50 = 100 microM). The activity of caspases was studied using either tyr-val-ala-asp-aminomethylcoumarine (YVAD-amc) or asp-glu-val-asp-aminomethylcoumarine (DEVD-amc) as substrates. There was no activation of a YVADase, whereas as pronounced increase in DEVDase activity was found with a maximum 3 h after serum removal. Cross competition experiments in vitro showed that the latter activity is inhibited also by low concentrations of YVAD-cmk (300-600 nM), suggesting that both inhibitors inactivated the same target protease. Remarkably all tested protective reagents lead to an inhibition of the DEVDase activity in intact cells. Since these reagents act via distinct intracellular pathways, the existence of a regulatory element upstream of the DEVDase is proposed which integrates signals from a variety of pathways.
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PMID:Multiple intracellular pathways interfere with the activation of a CPP32-like protease induced by serum deprivation of AKR-2B cells. 957 Sep 18

The cellular localization of protein kinase C (PKC) is intimately associated with the regulation of its biological activity. Previously we have demonstrated that the redistribution of PKC to the plasma membrane in response to physiological stimuli is followed by a rapid returning of PKC back to the cytoplasm (Feng, X., Zhang, J., Barak, L. S., Meyer, T., Caron, M. G., and Hannun, Y. A. (1998) J. Biol. Chem. 273, 10755-10762). Although the process of PKC membrane targeting has been extensively studied, the molecular mechanism underlying the dissociation of membrane-bound PKC remains unclear. In the present study, by examining the dynamic distribution of wild-type PKC betaII and its kinase-deficient mutant (K371R), we demonstrate that kinase activity is required for PKC membrane dissociation. Moreover, the inability of PKC betaII(K371R) to dissociate from the plasma membrane in cells overexpressing wild-type PKC betaII suggests that autophosphorylation activity of the kinase might be essential for its membrane dissociation. This was further supported by mutational analysis of two in vivo autophosphorylation sites on PKC betaII. The replacement of Ser660 or Thr641 by alanine (S660A or T641A) was found to synergistically reduce the reversal of PKC betaII membrane translocation, whereas the replacement of the same amino acids by glutamic acid (S660E or T641E), an amino acid commonly used to mimic phosphate, results in mutants behaving similar to wild-type PKC betaII. These findings point to an essential role for autophosphorylation in the dissociation of activated PKC from the plasma membrane and suggest that, like PKC membrane translocation, the returning of PKC to the cytoplasm after its activation is also delicately regulated.
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PMID:An essential role for autophosphorylation in the dissociation of activated protein kinase C from the plasma membrane. 975 33


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