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)

In the yeast Saccharomyces cerevisiae, three genes TPK1, TPK2, and TPK3 encode catalytic subunits of cAMP-dependent protein kinase. We have purified and characterized the catalytic subunit, C1, encoded by the TPK1 gene. In order to purify C1 completely free of C2 and C3, a strain was constructed that contained only the TPK1 gene and genetic disruptions of the other two TPK genes. The cellular level of C1 was increased by expressing the genes for C1 (TPK1) and yeast regulatory subunit (BCY1) on multiple copy plasmids within this strain. Purification was accomplished by a two-column procedure in which holoenzyme was chromatographed on Sephacryl-200, then bound to an anti-regulatory subunit immunoaffinity column. Pure C1 was released from the antibody column by addition of cAMP. The protein migrated on a sodium dodecyl sulfate-polyacrylamide gel with an Mr of 52,000. Kinetic analysis showed that the apparent Km for ATP and Leu-Arg-Arg-Ala-Ser-Leu-Gly was 33 and 101 microM, respectively. The kcat was determined to be 640 min-1. The protein weakly autophosphorylated, incorporating less than 0.1 mol of phosphate/mol of catalytic subunit. NH2-terminal sequencing revealed that the protein was blocked.
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PMID:Purification and characterization of C1, the catalytic subunit of Saccharomyces cerevisiae cAMP-dependent protein kinase encoded by TPK1. 328 29

A protein kinase, able to phosphorylate casein, phosvitin, and glycogen synthase, was purified approximately 9000-fold from rabbit liver, and appeared analogous to an enzyme studied by Itarte and Huang (Itarte, E., and Huang, K.-P. (1979) J. Biol. Chem. 254, 4052-4057). This enzyme, designated here casein kinase-1, was shown to be a distinct glycogen synthase kinase and in particular to be different from the protein kinase GSK-3 (Hemmings, B.A., Yellowlees, D., Kernohan, J.C., and Cohen, P. (1981) Eur. J. Biochem. 119, 443-451). Casein kinase-1 had native molecular weight of 30,000 as judged by gel filtration. The enzyme phosphorylated beta-casein A or B better than kappa-casein or alpha s1-casein, and modified only serine residues in beta-casein B and phosvitin. The apparent Km for ATP was 11 microM, and GTP was ineffective as a phosphoryl donor. The phosphorylation of glycogen synthase by casein kinase-1 was inhibited by glycogen, half-maximally at 2 mg/ml, and by heparin, half-maximally at 0.5-1.0 microgram/ml, but was unaffected by Ca2+ and/or calmodulin, or by cyclic AMP. Phosphorylation of muscle glycogen synthase proceeded to a stoichiometry of at least 6 phosphates/subunit with reduction in the +/- glucose-6-P activity ratio to less than 0.4. Phosphate was introduced into both a COOH-terminal CNBr fragment (CB-2) as well as a NH2-terminal fragment (CB-1). At a phosphorylation stoichiometry of 6 phosphates/subunit, 84% of the phosphate was associated with CB-2 and 6.5% with CB-1. The remainder of the phosphate was introduced into another CNBr fragment of apparent molecular weight 16,500. Phosphorylation by casein kinase-1 correlated with reduced electrophoretic mobilities, as analyzed on polyacrylamide gels in the presence of sodium dodecyl sulfate, of the intact glycogen synthase subunit, as well as the CNBr fragments CB-1 and CB-2.
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PMID:Glycogen synthase kinases. Classification of a rabbit liver casein and glycogen synthase kinase (casein kinase-1) as a distinct enzyme. 632 24

Inhibitor-2 (I-2) inhibits the free catalytic subunit of type 1 phosphatase (CS1) and controls the cyclic inactivation/activation of CS1 in the ATP-Mg-dependent protein phosphatase complex. We report here the effect of mutations on these two properties of I-2. Substitution of Thr-72 with Ala, Asp, or Glu generated complexes with CS1 that could not be activated. Mutation of Ser-86 did not affect activation by glycogen synthase kinase-3 (GSK-3) alone but impaired synergistic activation by casein kinase II and GSK-3. Mutations in the region between Thr-72 and Ser-86 did not alter the inhibitory potency of I-2 but prevented complete inactivation of CS1. A mutant without the 35 NH2-terminal residues exhibited an IC50 for CS1 200-fold higher than that of wild-type I-2. However, it formed an inactive phosphatase complex with CS1, which was activated by GSK-3. A mutant with the 59 COOH-terminal residues deleted retained full inhibitory activity and formed an inactive complex that could not be activated by GSK-3. We conclude that the NH2-terminal region of I-2 is involved in inhibition, that the sequence between Thr-72 and Ser-86 is necessary for the conversion of CS1 from an active to an inactive conformation, and that the COOH terminus is required for activation by GSK-3. Thus, different functional domains of I-2 may interact with distinct regions of CS1.
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PMID:Domains of phosphatase inhibitor-2 involved in the control of the ATP-Mg-dependent protein phosphatase. 796 54

Addition of a nitrogen-source to glucose-repressed, nitrogen-starved G0 cells of the yeast Saccharomyces cerevisiae in the presence of a fermentable carbon source induces growth and causes within a few minutes a five-fold, protein-synthesis-independent increase in the activity of trehalase. Nitrogen-activated trehalase could be deactivated in vitro by alkaline phosphatase treatment, supporting the idea that the activation is triggered by phosphorylation. Yeast strains containing only one of the three TPK genes (which encode the catalytic subunit of cAMP-dependent protein kinase) showed different degrees of nitrogen-induced trehalase activation. The order of effectiveness was different from that previously reported for glucose-induced activation of trehalase in glucose-depressed yeast cells. Further reduction of TPK-encoded catalytic subunit activity by partially inactivating point mutations in the remaining TPK gene further diminished nitrogen-induced trehalase activation, while deletion of the BCY1 gene (which encodes the regulatory subunit) in the same strains resulted in an increase in the extent of activation. Deletion of the RAS genes in such a tpkw1 bcy1 strain had no effect. These results are consistent with mediation of nitrogen-induced trehalase activation by the free catalytic subunits alone. They support our previous conclusion that cAMP does not act as second messenger in this nitrogen-induced activation process and our suggestion that a novel nitrogen-induced signaling pathway integrates with the cAMP pathway at the level of the free catalytic subunits of protein kinase A. Western blot experiments showed that the differences in the extent of trehalase activation were not due to differences in trehalase expression. On the other hand, we cannot completely exclude that protein kinase A influences the nitrogen-induced activation mechanism itself rather than acting directly on trehalase. However, any such alternative explanation requires the existence of an additional, yet unknown, mechanism for activation of trehalase besides the well-established regulation by protein kinase A.
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PMID:Activation of trehalase during growth induction by nitrogen sources in the yeast Saccharomyces cerevisiae depends on the free catalytic subunits of cAMP-dependent protein kinase, but not on functional Ras proteins. 799 5

Rabbit skeletal muscle glycogen synthase, a rate-limiting enzyme for glycogen biosynthesis, is regulated by multisite phosphorylation. The protein kinase glycogen synthase kinase 3 (GSK-3) phosphorylates 4 Ser residues (Ser-640, Ser-644, Ser-648, and Ser-652; also known as sites 3a, 3b, 3c, and 4, respectively) at the COOH terminus of the subunit. Phosphorylation of these sites by GSK-3 is sequential, from COOH- to NH2-terminal, and is wholly dependent on prior phosphorylation by casein kinase II at Ser-656 (site 5). Expression in Escherichia coli was used to generate mutant forms of glycogen synthase, S640A, S644A, and S648A, in which site 3a, site 3b, or site 3c was changed to Ala, respectively. The purified enzymes had -/+ glucose-6-P activity ratios in the range of 0.8-0.9. Phosphorylation by casein kinase II and GSK-3 gave results consistent with the model of obligate sequential action of GSK-3. Phosphorylation at site 5, sites 4 + 5, or sites 3c + 4 + 5 had no measurable effect on activity. When sites 3b + 3c + 4 + 5 were phosphorylated, modest inactivation resulted. Additional phosphorylation at site 3a, however, was potently inactivating, reducing the -/+ glucose-6-P activity ratio to 0.1 and increasing the glucose-6-P concentration needed for half-maximal activation by an order of magnitude. Introduction of each additional phosphate, in the order site 4, 3c, 3b, and 3a, caused an incremental reduction in the mobility of the subunit when analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The results of this study demonstrate that GSK-3 phosphorylation of site 3a (Ser-640), and to a lesser extent, site 3b, correlates with inactivation of glycogen synthase by GSK-3. Evidence is also presented for an allosteric mechanism of inactivation whereby modification of one subunit influences the activity state of adjacent subunits.
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PMID:Inactivation of rabbit muscle glycogen synthase by glycogen synthase kinase-3. Dominant role of the phosphorylation of Ser-640 (site-3a). 822 27

We report the identification of the PPS1 gene of Saccharomyces cerevisiae. The deduced amino acid sequence of PPS1p shows similarity with protein-tyrosine phosphatases (PTPases) and is most closely related to a subfamily of PTPases that are capable of dephosphorylating phosphoseryl and phosphothreonyl residues as well as phosphotyrosyl residues. Analysis of the predicted amino acid sequence suggests that the protein consists of an active phosphatase domain, an inactive phosphatase-like domain, and an NH2-terminal extension. Mutation of the catalytic cysteinyl residue in the active phosphatase domain reduced the in vitro activity of the mutant protein to less than 0.5% of wild type activity, while mutation of the corresponding cysteinyl residue of the inactive phosphatase-like domain had no effect on in vitro activity. The PPS1 protein was expressed in Escherichia coli, and the protein was shown to catalyze the hydrolysis of p-nitrophenyl phosphate, dephosphorylate phosphotyrosyl, and phosphothreonyl residues in synthetic diphosphorylated peptides and to inactivate the human ERK1 protein. PPS1 transcript abundance is coregulated with that of the divergently transcribed DPB3 gene, which codes for a subunit of DNA polymerase II, with both transcripts showing peak abundance in S phase. pps1Delta mutant strains did not differ from PPS1 strains under any of the conditions tested, but overexpression of the PPS1 protein in S. cerevisiae led to synchronous growth arrest and to aberrant DNA synthesis. A screen for suppressors of this growth arrest identified the RAS2 gene as a multicopy suppressor of the PPS1p overexpression arrest. The arrest was not suppressed by the presence of multicopy RAS1, TPK2, or TPK3 genes or by the presence of 5 mM cAMP in the growth medium, suggesting that PPS1 functions in a pathway involving RAS2, but not TPK kinases or adenylate cyclase.
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PMID:The PPS1 gene of Saccharomyces cerevisiae codes for a dual specificity protein phosphatase with a role in the DNA synthesis phase of the cell cycle. 908 70

Mutation of the adenomatous polyposis coli (APC) tumor suppressor gene initiates the majority of colorectal (CR) cancers. One consequence of this inactivation is constitutive activation of beta-catenin/Tcf-mediated transcription. To further explore the role of the APC/beta-catenin/Tcf pathway in CR tumorigenesis, we searched for mutations in genes implicated in this pathway in CR tumors lacking APC mutations. No mutations of the gamma-catenin (CTNNG1), GSK-3alpha (GSK3A), or GSK-3beta (GSK3B) genes were detected. In contrast, mutations in the NH2-terminal regulatory domain of beta-catenin (CTNNB1) were found in 13 of 27 (48%) CR tumors lacking APC mutations. Mutations in the beta-catenin regulatory domain and APC were observed to be mutually exclusive, consistent with their equivalent effects on beta-catenin stability and Tcf transactivation. In addition, we found that CTNNB1 mutations can occur in the early, adenomatous stage of CR neoplasia, as has been observed previously with APC mutations. These results suggest that CTNNB1 mutations can uniquely substitute for APC mutations in CR tumors and that beta-catenin signaling plays a critical role in CR tumorigenesis.
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PMID:Mutational analysis of the APC/beta-catenin/Tcf pathway in colorectal cancer. 951 95

Occupational exposure to asphalt fumes may pose a health risk. Experimental studies using animal and in vitro models indicate that condensates from asphalt fumes are genotoxic and can promote skin tumorigenesis. Enhanced activity of activator protein-1 (AP-1) is frequently associated with the promotion of skin tumorigenesis. The current study investigated the effect of exposure to asphalt fumes on AP-1 activation in mouse JB6 P+ epidermal cells and the skin of transgenic mice expressing the AP-1 luciferase reporter gene. Asphalt fumes were generated from a dynamic generation system that simulated road-paving conditions. Exposure to asphalt fumes significantly increased AP-1 activity in JB6 P+ cells as well as in cultured keratinocytes isolated from transgenic mice expressing AP-1 reporter. In addition, topical application of asphalt fumes by painting the tail skin of mice increased AP-1 activity by 14-fold. Exposure to asphalt fumes promoted basal as well as epidermal growth factor-stimulated anchorage-independent growth of JB6 P+ cells in soft agar. It activated phosphatidylinositol 3-kinase and induced phosphorylation of Akt at Ser-473/Thr-308, and concurrently activated downstream p70 S6 kinase as well as glycogen synthase kinase-3beta. Asphalt fumes transiently activated c-Jun NH2-terminal kinases without affecting extracellular signal-regulated kinases and p38 mitogen-activated protein kinases. Further study indicated that blockage of phosphatidylinositol 3-kinase activation eliminated asphalt fume-stimulated AP-1 activation and formation of anchorage-independent colonies in soft agar. This is the first report showing that exposure to asphalt fumes can activate AP-1 and intracellular signaling that may promote skin tumorigenesis, thus providing important evidence on the potential involvement of exposure to asphalt fumes in skin carcinogenesis.
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PMID:Exposure to asphalt fumes activates activator protein-1 through the phosphatidylinositol 3-kinase/Akt signaling pathway in mouse epidermal cells. 1294

Various cellular signaling pathways, such as phosphatidylinositol 3-kinase, calcineurin, Janus kinase 2/signal transducer and activator of transcription 3 (STAT3), and mitogen-activated protein kinase (MAPK) have been suggested to play an important role in skeletal muscle growth. Old muscle, compared with young muscle, lacks the ability to completely regrow its muscle mass after an atrophy-induced stimulus. it is hypothesized that defects and/or delays in the activation of specific cell signaling pathways of aged soleus muscle limit the potential for growth. To test this, 42 male Fischer 344 x Brown Norway rats, 30 mo old, were hindlimb immobilized for 10 days, and their muscle samples were compared with muscle samples analyzed from 3- to 4-mo-old rats in a previous report (Childs TE, Spangenburg EE, Vyas DR, and Booth FW. Am J Physiol Cell Physiol: 285: C391-C398, 2003). After 10 days, the immobilization was removed and rats were allowed to ambulate for a series of days. Alterations in the activation or deactivation status of specific signaling pathways were determined by comparing the phosphorylation (phos) and total concentration of specific signaling proteins (pan) through Western blotting with the 10-day immobilization group. Various cell signals and their respective time groups of the old rats were shown to be significantly different compared with the 10-day immobilization group. For example, peak increases during recovery from the immobilization were observed at 1) the third recovery day for calcineurin B-pan and 2) the sixth recovery day for glycogen synthase kinase-3beta-phos, p70 S6 kinase (p70S6k) -phos and -pan, calcineurin A-pan, STAT3-phos and -pan, p44 MAPK-pan, and p42 MAPK-pan. In contrast, Akt-pan, c-Jun NH2-terminal kinase-phos, and p38 MAPK-phos were observed to decrease from 10-day immobilization values to control levels. Also, Aktphos was unchanged among all groups. In a follow-up experiment in which muscle samples from both the present study and a previous study (Childs TE, Spangenburg EE, Vyas DR, and Booth FW. Am J Physiol Cell Physiol: 285: C391-C398, 2003) were reanalyzed together, the recovery-induced increase in p70S6k-phos from immobilization-atrophy was significantly attenuated in soleus muscles of the old group.
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PMID:Responsiveness of cell signaling pathways during the failed 15-day regrowth of aged skeletal muscle. 1451 1

Integrin-linked kinase (ILK), bound to the cytoplasmic tails of integrin beta1, beta2, and beta3, is thought to signal through AKT and glycogen synthase kinase-3beta (GSK-3beta) for survival and proliferation regulation. To determine the role of ILK in the cellular radiation response, stably transfected A549 lung cancer cells overexpressing either wild-type (ILK-wk) or hyperactive ILK (ILK-hk) were studied for survival, signaling, proliferation, and examined in immunofluorescence and adhesion assays. Strong radiosensitization was observed in ILK-hk in contrast to ILK-wk mutants and empty vector controls. ILK small interfering RNA transfections showed radioresistance similar to irradiation on fibronectin. AKT, GSK-3beta-cyclin D1, mitogen-activated protein kinase kinase 1/2-mitogen-activated protein kinase, and c-Jun NH2-terminal kinase signaling was dysregulated in irradiated ILK-hk mutants. Immunofluorescence stainings of ILK-hk cells indicated disturbed ILK and paxillin membrane localization with concomitant decrease in focal adhesions. Profound ILK-hk-dependent changes in morphology were characterized by spindle-like cell shape, cell size reduction, increased cell protrusions, strong formation of membranous f-actin rings, and significantly reduced adhesion to matrix proteins. Additionally, ILK-wk and ILK-hk overexpression impaired beta1-integrin clustering and protein Tyr-phosphorylation. Taken together, the data provide evidence that ILK signaling modulates the cellular radiation response involving diverse signaling pathways and through changes in f-actin-based processes such as focal adhesion formation, cell adhesion, and spreading. Identification of ILK and its signaling partners as potential targets for tumor radiosensitization might promote innovative anticancer strategies by providing insight into the mechanism of cell adhesion-mediated radioresistance, oncogenic transformation, and tumor growth and spread.
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PMID:Overexpression of hyperactive integrin-linked kinase leads to increased cellular radiosensitivity. 1531 8


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