Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.1 (protein kinase)
 81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A second messenger-independent serine/threonine protein kinase from lactating goat mammary gland is purified and characterized. The purification steps include: homogenization, ultracentrifugation, ammonium sulphate precipitation, DEAE-Sepharose, phosphocellulose, hydrophobic and Mono Q columns. On the final step of purification the enzyme is revealed as a single band of mol wt 45,000 on silver-stained SDS-PAGE. Mg2+ and K+ are necessary for its optimum activity. Phosvitin and casein are substrates for the enzyme but kemptide, RRREEETEEE, protamine and histone mixture are all poorly phosphorylated. The kinase is inhibited by quercetin, heparin, random tyrosine- and glutamic acid-containing polymers, Ca2+, NaF, 2,3-bis-phosphoglycerate. 1 mM Mn2+ affects positively the basal level of the kinase activity but 5 mM Mn2+ completely suppress the effect of 10 mM Mg2+. Km of this enzyme for ATP is 1.57 microM and pH optimum is from 6 to 7. Isolation of this kinase is facilitated by its unusually high affinity for phosphocellulose.
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PMID:Identification and purification of a novel serine/threonine messenger-independent growth-related protein kinase from lactating goat mammary gland. 162 98

The insulin-stimulated protein kinase (ISPK) was purified over 50,000-fold from extracts of rabbit skeletal muscle by a procedure involving chromatography on phosphocellulose, fractionation with ammonium sulphate, and further chromatography on DEAE-cellulose, phenyl-Superose, Mono S and Mono Q. About 10 micrograms enzyme was isolated from 800 g muscle (one rabbit) in four days with an overall recovery of 5%. The purified enzyme showed a single protein-staining band of apparent molecular mass 91 kDa when analysed by SDS/polyacrylamide gel electrophoresis. The ISPK comigrated during SDS/polyacrylamide gel electrophoresis with the enzyme S6 kinase II from Xenopus eggs, and was recognised in immunoblotting and immunoprecipitation experiments by antibodies raised against S6 kinase II. The substrate specificities of ISPK and S6 kinase II were also very similar and like S6 kinase II, ISPK that had been inactivated by protein phosphatase 2A could be reactivated by incubation with mitogen-activated protein kinase and MgATP. ISPK was distinct from an insulin-stimulated 70-kDa S6 kinase from rat liver in both substrate specificity and immunological cross reactivity. It is concluded that ISPK is closely related in structure to S6 kinase II and may be a mammalian equivalent of this enzyme. The possibility that ISPK is involved in mediating a number of the actions of insulin is discussed.
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PMID:Purification and characterisation of the insulin-stimulated protein kinase from rabbit skeletal muscle; close similarity to S6 kinase II. 165 Dec 43

DEAE-cellulose column chromatography of Neurospora crassa soluble mycelial extracts leads to the resolution of three major protein kinase activity peaks designated PKI, PKII, and PKIII. PKII activity is stimulated by Ca2+ and Neurospora or brain calmodulin. Maximal stimulation was observed at 2 microM-free Ca2+ and 1 microgram/ml of the modulator. The stimulatory effect of the Ca(2+)-calmodulin complex was blocked by EGTA and by some calmodulin antagonists such as phenothiazine drugs or compound 48/80. PKII phosphorylates different proteins, among which histone II-A at a low concentration and CDPKS, the synthetic peptide specific for Ca(2+-)-calmodulin dependent protein kinase, are the best substrates. Some phosphorylation can be detected in the absence of any exogenous acceptor. PKII activity assayed in the presence of histone II-A or in the absence of exogenous phosphate acceptor (autophosphorylation) co-elute in a DEAE-cellulose column at 0.28 NaCl. As result of the autophosphorylation reaction of the purified enzyme a main phosphorylated component of 70 kDa was resolved by SDS-polyacrylamide gel electrophoresis. It is possible that this component is an active part of this enzyme.
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PMID:Ca2+ calmodulin-dependent protein kinase activity in the ascomycetes Neurospora crassa. 165 80

Limited proteolysis of catalytic and regulatory subunits of cyclic AMP-dependent protein kinase (A-pk), cyclic AMP phosphodiesterase, glycogen synthase, and histones by fungal protease (type XIX) was analyzed by the digested peptide bands in SDS polyacrylamide gel electrophoresis. The modulatory effects on proteolysis by nucleotides, polypeptides, and phospholipids may greatly depend on the intrinsic nature of substrates. The proteolysis of the regulatory subunit of A-pk and glycogen synthase was not regulated by nucleotides and nucleic acids. In comparison, phosphatidyl serine, cardiolipin, and pepstatin A stimulated the proteolysis of the catalytic subunit of A-pk. Whereas, lambda DNA (Hind III digest), t-RNA, GTP-, phosphatidyl serine, sphingosine inhibited the proteolysis of cyclic AMP phosphodiesterase. Moreover, MS2 RNA, lambda DNA, t-RNA, dGTP, Phosphatidyl serine, phosphatidyl inositol, antipain, and chymostatin exerted inhibitory proteolytic effect on histone VIII-S. Some of these agents also had similar inhibitory effect on other types of histones (types III-S and VII-S). The inhibitory effect of phosphatidyl serine on proteolysis of histone may be due to their interaction which was monitored by the drastic increase of uv absorbance.
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PMID:Regulation of fungal proteolysis on cyclic AMP-dependent protein kinase, cyclic AMP phosphodiesterase, glycogen synthase and histones. 165 82

The regulatory subunit of cAMP-dependent protein kinase in yeast, encoded by the BCY1 gene, is known to be required under certain conditions such as growth on nonfermentable carbon sources and entry into stationary phase. We have identified novel isoforms of Bcy1 in cells under these conditions. The isoforms are distinguishable by their migration on one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 2-dimensional nonequilibrium pH gradient gel electrophoresis. The isoforms observed by one-dimensional SDS-PAGE bind cAMP, as determined by [32P]8-azido-cAMP labeling (diagnostic of Bcy1 protein). Proteins isolated from cells grown to stationary phase in rich medium exhibit five antibody-reactive bands, by one-dimensional SDS-PAGE immunoblot analysis, with apparent molecular masses of 50, 52, 55, 59 and 61 kDa. Total Bcy1 protein increases at least 8-fold between exponential and stationary phase. Analysis of proteins from a variety of yeast mutants indicated that 1) many of the observed modifications of Bcy1 are dependent upon the presence of the Ser-145 phosphorylation site; 2) the appearance of the 59- and 61-kDa bands is dependent upon the presence of Yak1 kinase; and 3) Bcy1 protein is modified even in the absence of cAMP-dependent protein kinase catalytic subunits. Cells carrying the bcy1(ala145) allele exhibit non-wild type growth, indicating that these modifications may be functionally significant.
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PMID:Bcy1, the regulatory subunit of cAMP-dependent protein kinase in yeast, is differentially modified in response to the physiological status of the cell. 165 93

Endogenous phosphorylation of proteins in cell suspensions of collecting tubes was studied. Using SDS disc electrophoresis in polyacrylamide gel with subsequent autoradiography, it was shown that vasopressin increases the 32P incorporation into two proteins with molecular masses of 15 kDa and 33 kDa, which serve as endogenous substrates for cAMP-dependent protein kinase. The hormone-dependent phosphorylation of these proteins was typical of the membrane fraction of collecting tube cells but was absent in the cytosolic fraction. The results obtained are suggestive of the direct involvement of vasopressin in the regulation of membrane protein phosphorylation by cAMP-dependent protein kinase which may increase the permeability of cells for H2O.
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PMID:[Phosphorylation of proteins in collecting tube cells under the effect of vasopressin]. 165 15

The lysosomal enzyme binding receptor protein isolated from monkey brain by phosphomannan-Sepharose affinity chromatography was phosphorylated by [gamma-32P] ATP by protein kinases tightly associated with the receptor protein. A greater than 200 kDa protein was phosphorylated on both serine and tyrosine residues and a approximately 45 kDa protein was phosphorylated on only serine residues as evidenced by SDS-gel electrophoresis, autoradiography and phosphoamino acid analysis [(Panneerselvam, Ramamoorthy & Balasubramanian (1987) Biochem Biophys Res Commun, 147, 927-935)]. 125I-labelled lysosomal enzymes could be cross-linked to the receptor protein in the presence of disuccinimidyl suberate. Phosphorylation of the receptor on both serine and tyrosine residues was inhibited by quercetin, polylysine and polymyxin B. Catalytic subunit of cyclic AMP-dependent protein kinase preferentially phosphorylated the approximately 45 kDa protein. In the presence of Triton X-100, phosphorylation of a few additional protein bands on non-tyrosine residues was observed. There was a marked reduction in the efficiency of binding lysosomal enzymes by the phosphorylated receptor protein in comparison to the unphosphorylated receptor protein.
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PMID:Lysosomal enzyme binding receptor protein from monkey brain and its phosphorylation. 166 94

Epstein-Barr virus encodes two small RNAs, EBER-1 and -2, that are abundantly expressed in latently infected cells. Recent evidence suggests a role for EBER-1 in regulation of translation since this RNA is able to prevent the inhibition of protein synthesis by double-stranded RNA in rabbit reticulocyte lysates. We show here that EBER-1 that has been synthesized in vitro forms a complex with the dsRNA-activated inhibitor of protein synthesis DAI, a protein kinase that specifically phosphorylates polypeptide chain initiation factor eIF-2. Gel retardation assays and UV crosslinking experiments indicate that complex formation is specific for EBER-1 and requires the presence of some secondary structure in the molecule. RNA competition studies show that EBER-1-DAI complex formation is not inhibited in the presence of other small RNA species, heparin or the synthetic double-stranded RNA, poly(I).poly(C). SDS gel analysis reveals the existence of two forms of the crosslinked complex, of 64-68kDa and 46-53kDa, both of which are recognized by anti-DAI antibodies in immunoprecipitation experiments. These data suggest that EBER-1 regulates protein synthesis through its ability to interact with DAI.
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PMID:Binding of Epstein-Barr virus small RNA EBER-1 to the double-stranded RNA-activated protein kinase DAI. 167 26

Limited proteolysis of tyrosine hydroxylase (TH) by calpain, Ca(2+)-activated neutral protease, was studied. Cleavage of TH with calpain in vitro produced molecules having Mrs of approximately 57,000 and 56,000 in SDS-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence, Ser-Pro-Arg-Phe-Val, of the 56-kDa species indicated that calpain cleaved off the N-terminal region (residues 1-30) encoded by the first exon including Ser-8 and Ser-19, the phosphorylation sites by proline-directed protein kinase (PDPK) and by Ca2+/calmodulin-dependent protein kinase II (kinase II), respectively, from the native TH. The removal of the N-terminal region from the native molecule induced a slight but significant activation of TH at pH 7.0. The native TH behaved as the tetramer with an Mr of 240,000. In contrast, calpain-cleaved TH showed the monomeric Mr by gel permeation chromatography and increased Ki for catecholamine which inhibits the native TH in competition to the coenzyme, DL-6-methyl-5,6,7,8-tetrahydropterin (6MPH4). These results imply that calpain cleavage would effectively release TH from the feedback inhibition by removal of the N-terminal region resulting in disruption of the quaternary structure.
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PMID:Limited proteolysis of tyrosine hydroxylase by Ca(2+)-activated neutral protease (calpain). 168 1

Recently we reported the purification of a mitogen-activated S6 kinase from Swiss mouse 3T3 fibroblasts and rat liver. The rat liver protein was cleaved with cyanogen bromide or trypsin and 17 of the resulting peptides were sequenced. DNA primers were generated from 3 peptides that had homology to sequences of the conserved catalytic domain of protein kinases. These primers were used in the polymerase chain reaction to obtain a 0.4-kilobase DNA fragment. This fragment was either radioactively labeled and hybridized to Northern blots of poly(A)+ mRNA or used to screen a rat liver cDNA library. Northern blot analysis revealed four transcripts of 2.5, 3.2, 4.0, and 6.0 kilobases, and five S6 kinase clones were obtained by screening the library. Only two of the clones, which were identical, encoded a full-length protein. This protein had a molecular weight of 56,160, which correlated closely to that of the dephosphorylated kinase determined by SDS/PAGE. The catalytic domain of the kinase resembles that of other serine/threonine kinases belonging to the second messenger subfamily of protein kinases.
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PMID:Cloning of the mitogen-activated S6 kinase from rat liver reveals an enzyme of the second messenger subfamily. 169 26


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