Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.26 (GSK)
 6,788 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The L-myc protein migrates as three distinct differentially phosphorylated bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This phosphorylation can be rapidly increased either by treatment with the protein kinase C (PKC) activator phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or by inhibition of serine/threonine protein phosphatases with okadaic acid. In vitro mutagenesis and phosphoamino acid analyses define the N-terminal serine residues 38 and 42 of L-myc as critical targets for the PKC-dependent phosphorylation. These are the exclusive sites of phosphorylation in the N-terminal third of the L-myc protein, and can be phosphorylated in vitro by glycogen synthase kinase 3 beta (GSK-3 beta). A mutant L-myc protein in which these serines have been replaced by alanine residues does not show heterogeneous electrophoretic migration or hyperphosphorylation in response to PKC activation, and is not a substrate for GSK-3 beta in vitro. Similar potential phosphorylation sites are present in c-myc and N-myc in a highly conserved region thought to represent a transcriptional activation domain. We suggest that N-terminal phosphorylation of the L-myc protein is a means of rapid regulation of this oncoprotein, possibly mediated in vivo by the action of GSK-3.
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PMID:Activation of protein kinase C increases phosphorylation of the L-myc trans-activator domain at a GSK-3 target site. 131 97

Type 1 protein phosphatases (PP-1) comprise a group of widely distributed enzymes that specifically dephosphorylate serine and threonine residues of certain phosphoproteins. They all contain an isoform of the same catalytic subunit, which has an extremely conserved primary structure. One of the properties of PP-1 that allows one to distinguish them from other serine/threonine protein phosphatases is their sensitivity to inhibition by two proteins, termed inhibitor 1 and inhibitor 2, or modulator. The latter protein can also form a 1:1 complex with the catalytic subunit that slowly inactivates upon incubation. This complex is reactivated in vitro by incubation with MgATP and protein kinase FA/GSK-3. In the cell the type 1 catalytic subunit is associated with noncatalytic subunits that determine the activity, the substrate specificity, and the subcellular location of the phosphatase. PP-1 plays an essential role in glycogen metabolism, calcium transport, muscle contraction, intracellular transport, protein synthesis, and cell division. The activity of PP-1 is regulated by hormones like insulin, glucagon, alpha- and beta-adrenergic agonists, glucocorticoids, and thyroid hormones.
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PMID:The structure, role, and regulation of type 1 protein phosphatases. 135 Feb 40

We have isolated the full-length sequence for a unique human kinase, designated TTK. TTK was initially identified by screening of a T cell expression library with anti-phosphotyrosine antibodies. The kinases most closely related to TTK are the SPK1 serine, threonine and tyrosine kinase, the Pim1, PBS2, and CDC2 serine/threonine kinases, and the TIK kinase which was also identified through screening of an expression library with anti-phosphotyrosine antibodies. However, the relationships are distant with less than 25% identity. Nevertheless, TTK is highly conserved throughout phylogeny with hybridizing sequences being detected in mammals, fish, and yeast. TTK mRNA is present at relatively high levels in testis and thymus, tissues which contain a large number of proliferating cells, but is not detected in most other benign tissues. Freshly isolated cells from most malignant tumors assessed expressed TTK mRNA. As well, all rapidly proliferating cell lines tested expressed TTK mRNA. Escherichia coli expressing the complete kinase domain of TTK contain markedly elevated levels of phosphoserine and phosphothreonine as well as slightly increased levels of phosphotyrosine. Taken together, these findings suggest that expression of TTK, a previously unidentified member of the family of kinases which can phosphorylate serine, threonine, and tyrosine hydroxyamino acids, is associated with cell proliferation.
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PMID:Expression of TTK, a novel human protein kinase, is associated with cell proliferation. 163 25

In resting human epithelial and fibroblastic cells, c-Jun is phosphorylated on serine and threonine at five sites, three of which are phosphorylated in vitro by glycogen synthase kinase 3 (GSK-3). These three sites are nested within a single tryptic peptide located just upstream of the basic region of the c-Jun DNA-binding domain (residues 227-252). Activation of protein kinase C results in rapid, site-specific dephosphorylation of c-Jun at one or more of these three sites and is coincident with increased AP-1-binding activity. Phosphorylation of recombinant human c-Jun proteins in vitro by GSK-3 decreases their DNA-binding activity. Mutation of serine 243 to phenylalanine blocks phosphorylation of all three sites in vivo and increases the inherent trans-activation ability of c-Jun at least 10-fold. We propose that c-Jun is present in resting cells in a latent, phosphorylated form that can be activated by site-specific dephosphorylation in response to protein kinase C activation.
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PMID:Activation of protein kinase C decreases phosphorylation of c-Jun at sites that negatively regulate its DNA-binding activity. 184 81

The mechanism for synergistic phosphorylation by glycogen synthase kinase 3 (GSK-3) and casein kinase II was studied using a synthetic peptide which contains the sequence of a potentially important proline/serine-rich regulatory region of rabbit muscle glycogen synthase. The peptide, Ac-PRPAS(3a)VPPS(3b)PSLS(3c)RHSS(4)PHQS(5) EDEEEP-amide, has five known phosphorylation sites of the native enzyme designated sites 3a, 3b, 3c, 4, and 5, which are spaced every fourth residue. The peptide was phosphorylated specifically at site 5 by casein kinase II with an apparent Km of 23 microM, but it was not phosphorylated by GSK-3. However, after initial phosphorylation of site 5 by casein kinase II, the peptide became an effective substrate for GSK-3 with an apparent Km of 2 microM. GSK-3 introduced up to four phosphates and appeared to catalyze the sequential modification of sites 4, 3c, 3b, and 3a, respectively. The results can be explained if GSK-3 recognizes the sequence -SXXXS(P). Phosphorylation of site 5 by casein kinase II creates this recognition site. Thereafter, each successive phosphorylation introduced by GSK-3 generates a new recognition site. The results provide a molecular basis to explain the synergistic action of casein kinase II and GSK-3 that is also observed with native glycogen synthase. In addition, this investigation emphasizes how protein recognition sites in some cellular targets may have to be formed post-translationally.
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PMID:Formation of protein kinase recognition sites by covalent modification of the substrate. Molecular mechanism for the synergistic action of casein kinase II and glycogen synthase kinase 3. 282 Sep 93

A form of glycogen synthase kinase designated GSK-M3 was purified 4000-fold from rat skeletal muscle by phosphocellulose, Affi-Gel blue, Sephacryl S-300 and carboxymethyl-Sephadex column chromatography. Separation of GSK-M from the catalytic subunit of the cAMP-dependent protein kinase was facilitated by converting the catalytic subunit to the holoenzyme form by addition of the regulatory subunit prior to the gel filtration step. GSK-M had an apparent Mr 62,000 (based on gel filtration), an apparent Km of 11 microM for ATP, and an apparent Km of 4 microM for rat skeletal muscle glycogen synthase. The kinase had very little activity with 0.2 mM GTP as the phosphate donor. Kinase activity was not affected by the addition of cyclic nucleotides, EGTA, heparin, glucose 6-P, glycogen, or the heat-stable inhibitor of cAMP-dependent protein kinase. Phosphorylation of glycogen synthase from rat skeletal muscle by GSK-M reduced the activity ratio (activity in the absence of Glc-6-P/activity in the presence of Glc-6-P X 100) from 90 to 25% when approximately 1.2 mol of phosphate was incorporated per mole of glycogen synthase subunit. Phosphopeptide maps of glycogen synthase obtained after digestion with CNBr or trypsin showed that this kinase phosphorylated glycogen synthase in serine residues found in the peptides containing the sites known as site 2, which is located in the N-terminal CNBr peptide, and site 3, which is located in the C-terminal CNBr peptide of glycogen synthase. In addition to phosphorylating glycogen synthase, GSK-M phosphorylated inhibitor 2 and activated ATP-Mg-dependent protein phosphatase. Activation of the protein phosphatase by GSK-M was dependent on ATP and was virtually absent when ATP was replaced with GTP. GSK-M had minimal activity toward phosphorylase b, casein, phosvitin, and mixed histones. These data indicate that GSK-M, a major form of glycogen synthase kinase from rat skeletal muscle, differs from the known glycogen synthase kinases isolated from rabbit skeletal muscle.
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PMID:Characterization of GSK-M, a glycogen synthase kinase from rat skeletal muscle. 282 16

Prior phosphorylation of its substrate has been shown to be important for substrate recognition by the protein kinase glycogen synthase kinase-3 (GSK-3). Phosphorylation of glycogen synthase by GSK-3 is known to be enhanced by the previous action of casein kinase II and the sequence -SXXXS(P)- was proposed as the minimal recognition determinant for GSK-3. The glycogen binding subunit of type 1 phosphoprotein phosphatase has been shown to be phosphorylated by cyclic AMP-dependent protein kinase at serine-13 in the sequence KPGFS(5)PQPS(9)RRGS(13)ESSEEVYV (F.B. Caudwell, A. Hiraga, and P. Cohen (1986) FEBS Lett. 194, 85-89). Inspection of the sequence revealed potential GSK-3 sites at residues 5 and 9. Using a synthetic peptide with the above sequence, we found that phosphorylation of serine-13 by cyclic AMP-dependent protein kinase permitted the recognition of serine-9 and serine-5 by GSK-3. The work provides another example of a substrate for GSK-3 and demonstrates that the action of GSK-3 is linked to the presence of phosphate in the substrate and not the action of any particular protein kinase. In the course of the analyses, a novel feature of trypsin cleavage of phosphopeptides was noted. In the sequence -SRRGS(P)- trypsin acted uniquely after the first arginine whereas in the sequence -S(P)RRGS(P)- it cleaved randomly at either arginine residue. The fact that GSK-3 could phosphorylate a peptide derived from a phosphatase subunit also raises the possibility that GSK-3 might be involved in controlling glycogen-associated type 1 phosphatase and, more generally, in mediating cyclic AMP control of protein phosphorylation in cells.
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PMID:Phosphoserine as a recognition determinant for glycogen synthase kinase-3: phosphorylation of a synthetic peptide based on the G-component of protein phosphatase-1. 285 Jul 71

We have examined phosphorylation of nerve growth factor (NGF) receptor in cultured sympathetic neurons and PC12 cells. Dissociated rat superior cervical ganglion neurons or PC12 cells were incubated with 32Pi to label cellular phosphoproteins. Membrane proteins were solubilized, and NGF receptor proteins were immunoprecipitated with the monoclonal antibody 192-IgG. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography showed that NGF receptor components of Mr = 80,000 and Mr = 210,000 were phosphorylated. Phosphorylation of neither species was affected by treating the cells with NGF or phorbol 12-myristate 13-acetate. When the 80,000-Da protein was subjected to complete trypsin proteolysis and then analyzed by reverse phase liquid chromatography, two 32P-labeled peptides were resolved. The more hydrophobic peptide accounted for most of the 32P and contained only phosphoserine; the other peptide contained phosphoserine and phosphothreonine. No phosphotyrosine was detected in the receptor proteins. When receptor molecules from nonlabeled PC12 cells were immunoprecipitated and then incubated in vitro with [gamma-32P]ATP and the cAMP-independent protein kinase FA/GSK-3, phosphorylation occurred predominantly on serine and to a lesser extent on threonine. However, the immunoprecipitated receptor proteins neither autophosphorylated nor were they detectably phosphorylated by cAMP-dependent protein kinase, casein kinase II, or protein kinase C (the Ca2+/phospholipid-dependent enzyme). We conclude that binding units of the NGF receptor are phosphorylated constitutively in at least two sites in intact cells and that they can be phosphorylated by FA/GSK-3 in vitro.
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PMID:Phosphorylation of nerve growth factor receptor proteins in sympathetic neurons and PC12 cells. In vitro phosphorylation by the cAMP-independent protein kinase FA/GSK-3. 302 30

Rat liver glycogen synthase was purified to homogeneity by an improved procedure that yielded enzyme almost exclusively as a polypeptide of Mr 85,000. The phosphorylation of this enzyme by eight protein kinases was analyzed by cleavage of the enzyme subunit followed by mapping of the phosphopeptides using polyacrylamide gel electrophoresis in the presence of SDS, reverse-phase high-performance liquid chromatography and thin-layer electrophoresis. Cyclic AMP-dependent protein kinase, phosphorylase kinase, protein kinase C and the calmodulin-dependent protein kinase all phosphorylated the same small peptide (approx. 20 amino acids) located in a 14 kDa CNBr-fragment (CB-1). Calmodulin-dependent protein kinase and protein kinase C also modified second sites in CB-1. A larger CNBr-fragment (CB-2) of approx. 28 kDa was the dominant site of action for casein kinases I and II, FA/GSK-3 and the heparin-activated protein kinase. The sites modified were all localized in a 14 kDa species generated by trypsin digestion. Further proteolysis with V8 proteinase indicated that FA/GSK-3 and the heparin-activated enzyme recognized the same smaller peptide within CB-2, which may also be phosphorylated by casein kinase 1. Casein kinase 1 also modified a distinct peptide, as did casein kinase II. The results lead us to suggest homology to the muscle enzyme with regard to CB-1 phosphorylation and the region recognized by FA/GSK-3, which in rabbit muscle is characterized by a high density of proline and serine residues. A striking difference with the muscle isozyme is the apparent lack of phosphorylations corresponding to the muscle sites 1a and 1b. These results provide further evidence for the presence of liver- and muscle-specific glycogen synthase isozymes in the rat. That the isozymes differ subtly as to phosphorylation sites may provide a clue to the functional differences between the isozymes.
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PMID:Multiple phosphorylation sites of rat liver glycogen synthase. 309 Oct 84

The inhibitory potencies of bioflavonoids on various tyrosine protein kinases and serine/threonine protein kinases were investigated. The phosphotransferase activity of an oncogene product, pp130fps, and a growth factor receptor, insulin receptor, were inhibited by myricetin, a derivative of quercetin. However, tyrosine kinase activity in the particulate fraction from human platelets (PM-TPK) was resistant to myricetin. Apparent Ki values of myricetin for tyrosine protein kinases of pp130fps and insulin receptor were 1.8 and 2.6 microM, respectively. The Ki values for serine/threonine kinase activities of myosin light chain kinase (MLC-kinase), casein kinase I, casein kinase II, cAMP-dependent protein kinase, and protein kinase C were 1.7 microM, 9.0 microM, 0.6 microM, 27.5 microM, and 12.1 microM, respectively. Lineweaver-Burk plots revealed that myricetin competitively inhibits pp130fps tyrosine kinase, myosin light chain kinase, casein kinase I and II with ATP, but does not inhibit other protein kinases. Since myricetin is a hydroxylated derivative of quercetin, the inhibitory effects of a series of seven flavonoids with various numbers of hydroxy residues were examined. Structure activity studies exhibited that the inhibitory potencies of the flavonoids for tyrosine kinases of pp130fps and insulin receptor correlated with the number of hydroxy residues on the flavone rings (gamma = 0.974 and 0.926, respectively), whereas the hydroxylation influenced to a lesser extent the inhibitory potencies for serine/threonine protein kinase. The hydroxy residues at position 3' and 5' did not affect the activities of cAMP-dependent protein kinase, and protein kinase C, and the hydroxylation at position 5' is detrimental for the inhibition of MLC-kinase, and casein kinase I and II. Thus, flavonoids may be useful tools to elucidate the active site of tyrosine and serine/threonine protein kinases.
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PMID:Differential effects of flavonoids as inhibitors of tyrosine protein kinases and serine/threonine protein kinases. 316 98


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