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)

In the fission yeast Schizosaccharomyces pombe, the execution of Start requires the activity of the Cdc2 protein kinase and the Cdc10/Sct1 transcription complex. The loss of any of these genes leads to G1 arrest and activation of the mating pathway under appropriate conditions. We have undertaken a genetic and biochemical analysis of these genes and their protein products to elucidate the molecular mechanism that governs the regulation of Start. We demonstrate that serine-196 of Cdc10 is phosphorylated in vivo and provide evidence that suggests that phosphorylation of this residue is required for Cdc10 function. Substitution of serine-196 of Cdc10 with alanine (Cdc10 S196A) leads to inactivation of Cdc10. We show that Cdc10 S196A is incapable of associating with Sct1 to form a heteromeric complex, whereas substitution of this serine with aspartic acid (S196D) restores DNA-binding activity by allowing Cdc10 to associate with Sct1. Furthermore, we demonstrate that Cdc2 activity is required for the formation of the heteromeric Sct1/Cdc10 transcription complex and that the Cdc10 S196D mutation alleviates this requirement. We thus provide biochemical evidence to demonstrate one mechanism by which the Cdc2 protein kinase may regulate Start in the fission yeast cell cycle.
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PMID:The Cdc2 protein kinase controls Cdc10/Sct1 complex formation. 920 19

Protein kinase CK2 (casein kinase 2) is a ubiquitous Ser/Thr protein kinase involved in cell proliferation. Mutation of the alpha subunit of the Xenopus laevis CK2 to change aspartic acid 156 to alanine (CK2alphaA156) resulted in an inactive enzyme. The CK2alphaA156 mutant, however, binds the regulatory subunit as measured by retention of beta on a nickel chelating column mediated by (His)6-tagged CK2alphaA156. Addition of CK2alphaA156 also caused beta to shift sedimentation in a sucrose gradient from a beta2 dimer (52 kDa) to an alpha2beta2 tetramer (130,000 kDa). CK2alphaA156 can trap the beta subunit in an inactive complex reducing the stimulation of casein phosphorylation caused by addition of beta to wild-type alpha. This competitive effect depends on the ratio of alpha/alphaA156 and on the amount of beta available. Since beta inhibits the phosphorylation of calmodulin by CK2alpha, the addition of CK2alphaA156, in this case, increases calmodulin phosphorylation by the alpha and beta combination. These results suggest that CK2alphaA156 may be a useful dominant-negative mutant that can serve to explore the multiple functions of CK2beta.
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PMID:An inactive mutant of the alpha subunit of protein kinase CK2 that traps the regulatory CK2beta subunit. 923 69

Srp1p, the protein encoded by SRP1 of the yeast Saccharomyces cerevisiae, is a yeast nuclear localization signal (NLS) receptor protein. We have previously reported isolation of a protein kinase from yeast extracts that phosphorylates Srp1p complexed with NLS peptides/proteins. From partial amino acid sequences of the four subunits of the purified kinase, we have now identified this protein kinase to be identical to yeast casein kinase II (CKII). It was previously thought that autophosphorylation of the 36 kDa subunit of the yeast enzyme was stimulated by the substrate, GST-Srp1p. However, with the use of a more refined system, no stimulation of autophosphorylation of the 36 kDa subunit of yeast CKII was observed. Biochemical and mutational analyses localized the in vitro phosphorylation site of Srp1p by CKII to serine 67. It was shown that, in the absence of NLS peptides/proteins, phosphorylation of the intact Srp1p protein is very weak, but deletion of the C-terminal end causes great stimulation of phosphorylation without NLS peptides/proteins. Thus, the CKII phosphorylation site is apparently masked in the intact protein structure by the presence of a C-terminal region, probably between amino acids 403 and 516. Binding of NLS peptides/proteins most likely causes a change in protein conformation, exposing the CKII phosphorylation site. Mutational alterations of serine 67, the CKII phosphorylation site, to valine (S67V) and aspartic acid (S67D) were not found to cause any significant deleterious effects on cell growth. Analysis of in vivo phosphorylation showed that at least 30% of the wild type Srp1p molecules are phosphorylated in growing cells, and that the phosphorylation is mostly at the serine 67 CKII site. The ability of Srp1p purified from E coli and treated with calf intestinal phosphatase to bind a SV40 T-antigen NLS peptide was compared with that of Srp1p which was almost fully phosphorylated by CKII. No significant difference was observed. It appears that NLS binding does not require any phosphorylation of Srp1p, either by CKII or by some other protein kinase.
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PMID:Phosphorylation of Srp1p, the yeast nuclear localization signal receptor, in vitro and in vivo. 925 33

Nuclear localization sequence (NLS)-dependent nuclear import of SV40 large tumor antigen (T-Ag) fusion proteins is regulated by phosphorylation sites for casein kinase II (CKII) and the cyclin-dependent kinase Cdc2 amino-terminal to the NLS (amino acids 126-132). Between the T-Ag CKII and Cdc2 sites is a site (Ser120) for the double-stranded DNA-dependent protein kinase (dsDNA-PK), which we show here for the first time to play a role in regulating T-Ag nuclear import. We replaced Ser120 by aspartic acid or alanine using site-directed mutagenesis and assessed the effects on nuclear transport kinetics both in vivo (microinjected cells) and in vitro (mechanically perforated cells) in HTC rat hepatoma cells. Maximal nuclear accumulation of the Asp120 and Ala120 protein derivatives was approximately 40% and 70% reduced in vivo, respectively, compared with that of the wild type protein, and similarly reduced in vitro, although to a lesser extent. This implies that the dsDNA-PK site regulates the maximal level of nuclear accumulation, normally functioning to enhance T-Ag nuclear transport; the higher accumulation of the Asp120 protein compared with the Ala120 protein indicates that negative charge at the dsDNA-PK site is mechanistically important in regulating nuclear import. The Asp120 protein accumulated in the nucleus at a faster rate than the wild type protein, implying that phosphorylation at Ser120 may also regulate the nuclear import rate. CKII phosphorylation of the Asp120 protein in cytosol or by purified CKII was approximately 30% higher than that of the Ser120 and Ala120 proteins, while negative charge at the CKII site increased dsDNA-PK phosphorylation of Ser120 by approximately 80% compared with wild type, implying physical and functional interactions between the two phosphorylation sites. Quantitation of NLS recognition by the importin 58/97 subunits using an enzyme-linked immunosorbent assay indicated that while the Ala120 protein derivative had a binding affinity very similar to that of wild type, the Asp120 derivative showed 40% higher affinity. In vitro CKII phosphorylation increased importin binding by about 30% in all cases. These results imply that negative charge at the dsDNA-PK site may enhance nuclear import through increasing both NLS recognition by importin subunits, and phosphorylation at the CKII site, which itself also facilitates NLS recognition by importin 58/97.
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PMID:SV40 large tumor antigen nuclear import is regulated by the double-stranded DNA-dependent protein kinase site (serine 120) flanking the nuclear localization sequence. 926 64

The catalytic domains of the Raf family of protein kinases (deltaRaf) differ in their ability to activate MEK in vitro and in vivo and in their ability to oncogenically transform mammalian cells. The kinase domain of B-Raf is more active than the equivalent portion of Raf-1 which in turn is more active than A-Raf. In Raf-1 the phosphorylation or mutation to aspartic acid of two key tyrosine residues upstream of the ATP binding site has been demonstrated to significantly potentiate catalytic activity. In A-Raf the analogous amino acids are also tyrosine whereas in B-Raf they are aspartic acid. To determine if these differences in amino acid sequence influence the relative catalytic activity of the Raf kinase domains we constructed forms of deltaA-Raf, deltaB-Raf and deltaRaf-1 that encode either aspartic acid [DD], phenylalanine [FF] or tyrosine [YY] at these positions. These proteins were expressed both in mammalian cells as fusions with the hormone binding domain of the estrogen receptor and as epitope-tagged proteins in Sf9 insect cells to test their oncogenic and catalytic potentials. When expressed in Rat1 or 3T3 cells in the presence of hormone all of the deltaRaf-1:ER and deltaA-Raf:ER proteins were transforming with the exception of the [FF] form of deltaA-Raf. In general the [DD] forms of the deltaRaf-1:ER and deltaA-Raf:ER proteins were the most potently oncogenic which correlated with their ability to elicit activation of the MAP kinase pathway. Consistent with the transformation data, the catalytic activity of the [DD] forms of deltaA-Raf:ER and deltaRaf-1:ER was about ten times greater than the cognate [FF] and [YY] forms of the proteins. By contrast all of the deltaB-Raf:ER proteins were highly transforming and deltaB-Raf catalytic activity was largely unaffected by mutation of the aforementioned aspartic acids to either tyrosine or phenylalanine. Similar results were obtained with epitope-tagged forms of deltaA-Raf, deltaB-Raf and deltaRaf-1 expressed in Sf9 cells. These data provide support for the model that key tyrosine residues in the protein kinase domains of A-Raf and Raf-1 are important in the regulation of catalytic activity. In addition they demonstrate that the higher intrinsic activity of B-Raf cannot be explained simply by the presence of aspartic acids at the analogous positions.
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PMID:Mutations of critical amino acids affect the biological and biochemical properties of oncogenic A-Raf and Raf-1. 928 56

Neurotrophin modulation of NMDA receptors in cultured murine and isolated rat neurons. J. Neurophysiol. 78: 2363-2371, 1997. Patch-clamp and calcium imaging techniques were used to assess the acute effects of the neurotrophins, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and nerve growth factor (NGF), on the responses of cultured and acutely isolated hippocampal and cultured striatal neurons to the glutamate receptor agonist N-methyl--aspartic acid (NMDA). The effects of BDNF on NMDA-activated currents were examined in greater detail. Currents evoked by NMDA, and the accompanying changes in intracellular calcium, were enhanced by low concentrations of the neurotrophins (1-20 ng/ml). The potentiation by the neurotrophins was rapid in onset and offset (<1 s). The neurotrophins also reduced desensitization of these currents in most cells. The enhancement of NMDA-activated currents by BDNF was observed using both perforated and whole cell patch recording techniques and could be demonstrated in outside-out patches. Furthermore, its effects were not attenuated by pretreatment with the protein kinase inhibitors genistein or 1-(5-isoquinolynesulfony)2-methylpiperazine (H7). Therefore, the actions of BDNF do not appear to be mediated by phosphorylation. Similar enhancements were observed with NT-3 and NT-4 and with NGF despite the fact that hippocampal neurons lack TrkA receptors. All together this evidence suggests that the enhancement of NMDA-evoked currents is unlikely to be mediated through the activation of growth factor receptors. Modulation of NMDA responses by BDNF was dependent on the concentration of extracellular glycine. The most pronounced potentiation by BDNF was observed at low concentrations, whereas no potentiation was observed in saturating concentrations of glycine, suggesting that BDNF may have increased the affinity of the NMDA receptor for glycine. However, the competitive glycine-site antagonist 7-chloro-kynurenic acid blocked the enhancement by BDNF without shifting the dose-inhibition relationship for this antagonist, and Mg2+ consistently depressed the potentiation of NMDA-evoked currents by BDNF, indicating that BDNF does not alter glycine affinity. BDNF also reversibly increased the probability of opening of NMDA channels recorded from outside-out patches taken from cultured hippocampal neurons. Other unrelated peptides including dynorphin and somatostatin also caused a glycine-dependent enhancement of NMDA currents and depressed the currents in saturating concentrations of glycine. In contrast, a shortened analogue dynorphin (6-17), which lacks N-terminus glycine residues, and another peptide met-enkephalin were without effects on NMDA currents recorded in low concentrations of glycine. Our results suggest that neurotrophins and other peptides can serve as glycine-like ligands for the NMDA receptor.
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PMID:Neurotrophin modulation of NMDA receptors in cultured murine and isolated rat neurons. 935 88

Steroidogenic acute regulatory protein (StAR) plays a critical role in steroid hormone synthesis. StAR is thought to increase the delivery of cholesterol to the inner mitochondrial membrane where P450scc resides. Tropic hormones acting through the intermediacy of cAMP rapidly increase pregnenolone synthesis, and this rapid steroidogenic response is believed to be due to StAR's action. The StAR protein contains two consensus sequences for phosphorylation catalyzed by protein kinase A that are conserved across all species in which the amino acid sequence of the StAR protein has been determined. We demonstrated that human StAR expressed in COS-1 cells exists in at least four species detectable by two-dimensional gel electrophoresis followed by Western blotting. The two more acidic species disappeared after treatment of the cell extracts with alkaline phosphatase. 32P was incorporated into StAR protein immunoprecipitated from COS-1 cell extracts, and a 10-min treatment with 8-bromo-cAMP increased 32P incorporation into the StAR preprotein. StAR protein generated by in vitro transcription/translation was phosphorylated by the protein kinase A catalytic subunit in the presence of [gamma-32P]ATP. Mutation of potential sites for protein kinase A-mediated phosphorylation at serine 57 and serine 195 to alanines, individually, reduced 32P incorporation from labeled ATP into StAR preprotein produced by in vitro transcription/translation when incubated with protein kinase A catalytic subunit. 32P labeling of StAR protein expressed in COS-1 cells was also reduced when serine 57 or serine 195 were mutated to alanines. A double mutant in which both serine 57 and serine 195 were changed to alanines displayed markedly reduced 32P incorporation. To determine the functional significance of StAR phosphorylation, we tested the steroidogenic activity of the wild-type StAR and mutated StAR proteins in COS-1 cells expressing the human cholesterol side chain cleavage enzyme system. Mutation of the conserved protein kinase A phosphorylation site at serine 57 had no effect on pregnenolone synthesis. However, mutation of the serine residue at 195 resulted in an approximately 50% reduction in pregnenolone production. The S195A mutant construct did not yield the more acidic species of StAR detected in two-dimensional Western blots, indicating that the mutation affected the ability of the protein to be post-translationally modified. Mutation of the corresponding serine residues in murine StAR (Ser56 and Ser194) to alanines yielded results that were similar to those obtained with human StAR; the S56A mutant displayed a modest reduction in steroidogenic activity, whereas the S194A mutant had approximately 40% of the activity of murine wild-type StAR. In contrast to the human S195A mutation, conversion of serine 195 to an aspartic acid residue had no effect on steroidogenic activity, consistent with the idea that a negative charge at this site modulates StAR function. Our observations suggest that phosphorylation of serine 194/195 increases the biological activity of StAR and that this post- or co-translational event accounts, in part, for the immediate effects of cAMP on steroid production.
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PMID:Phosphorylation of steroidogenic acute regulatory protein (StAR) modulates its steroidogenic activity. 940 83

In vivo p53 is multiply phosphorylated by different protein kinases suggesting a central role for phosphorylation in modulating p53 function. In addition, p53 was found to be associated with two protein kinases, p34cdc2 and protein kinase CK2. Here we report the precise mapping of the interaction sites of p53-p34cdc2 complexes. The p34cdc2 binding site on human p53 maps to one distinct C-terminal site LQIRGRERFE (aa 330-339) close to the corresponding phosphorylation site at serine 315. In order to test whether phosphorylation of p53 might influence the binding of p53 to p34cdc2 phosphorylation mutants of the C-terminus of p53, which mimick permanent phosphorylation, were tested on their ability to bind to p34cdc2 in vitro. Substitution of serine 315 (the p34cdc2 phosphorylation site) with aspartic acid had only little effect on complex formation whereas an exchange of serine 392 (the protein kinase CK2 phosphorylation site) to aspartic acid resulted in a significant reduced relative binding affinity of p53 to p34cdc2. The same result was obtained when the C-terminus of p53 was phosphorylated by purified protein kinase CK2 prior to examination of complex formation. In addition, the specificity of the complex formation has been checked by competition experiments with full length p53 proteins and the influence of cyclin B on complex formation was examined.
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PMID:Fine mapping and regulation of the association of p53 with p34cdc2. 946 49

In Streptomyces coelicolor A3(2), bldA mutants that lack the tRNA for the rare leucine codon UUA fail to make the red undecylprodigiosin antibiotic complex. To find out why, red-pigmented while bald (Pwb) derivatives of a bldA mutant were isolated. Using a cloning strategy that allowed for (and demonstrated) dominance of the mutations, they were localized to the red gene cluster. By using insert-mediated integration of a phi C31 phage-based vector, one of the Pwb mutations was more precisely located between red structural genes to a segment of approximately 1 kb about 4 kb from the known pathway-specific regulatory gene redD. The segment contained most of an ORF (redZ) encoding a protein (RedZ) with end-to-end similarity to response regulators of diverse function from a variety of bacteria. Remarkably, in RedZ hydrophobic residues replace nearly all of the charged residues that usually make up the phosphorylation pocket present in typical response regulators, including the aspartic acid residue that is normally phosphorylated by a cognate sensory protein kinase. A single TTA codon in redZ provided a potential explanation for the bldA-dependence of undecylprodigiosin synthesis. This codon was unchanged in three Pwb mutants, but further analysis of one of the mutants revealed a potential up-promoter mutation. It seems possible that a combination of low-level natural translation of the UUA codon by a charged non-cognate tRNA, coupled with increased transcription of redZ in the Pwb mutant allows the accumulation of a threshold level of the RedD protein.
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PMID:A response-regulator-like activator of antibiotic synthesis from Streptomyces coelicolor A3(2) with an amino-terminal domain that lacks a phosphorylation pocket. 953 42

Protein P0, an essential component of the eukaryotic ribosomal stalk, is found phosphorylated in the ribosome. Substitution of serine 302 in the amino acid sequence of the Saccharomyces cerevisiae P0 by either aspartic acid or cysteine abolishes in vitro and in vivo phosphorylation of the protein. On the contrary, the replacement of this serine by a threonine results in an increase in the protein phosphorylation under both sets of conditions. Therefore, this serine residue, which is part of a consensus casein kinase II modification site, SDDD, seems to be the phosphorylation site in protein P0. The effect of the mutations on the protein activity has been tested in S. cerevisiae W303dGP0 and D67dGP0, both of which carry a genomic P0 gene under the control of the GAL1 promoter. Transformation of the mutated genes in S. cerevisiae W303dGP0 allows cell growth at 30 degreesC in glucose-to repress the wild-type P0 expression-at the same rate as controls, and the ribosomes contain a normal amount of the other stalk components. A similar absence of effect of the mutations on growth was found in strain D67dGP0, which has ribosomes deprived of the P1 and P2 proteins. Therefore, P0 phosphorylation is not a requirement for ribosome activity in standard growth conditions either in the presence or in the absence of the other stalk proteins. However, a phenotypic effect is detected in the case of strain D67 transformed with the overphosphorylated threonine containing P0, which contrary to the wild-type and the other mutated proteins is unable to support cell growth at 37 degreesC in the presence of either 0.3 M NaCl or 0.8 M sorbitol. In vitro polymerizing tests indicate that this effect is not due to the thermosensitivity of the mutated protein. The results indicate that although P0 phosphorylation is not required for the overall ribosome activity, it may affect the expression of specific proteins involved in metabolic processes such as osmoregulation.
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PMID:Phosphorylation of ribosomal protein P0 is not essential for ribosome function but can affect translation. 984 29


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