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)

We have investigated the signalling pathways involved in the stimulation of glycogen and fatty acid synthesis by insulin in rat fat cells using wortmannin, an inhibitor of phosphatidylinositol 3-kinase, and rapamycin, which blocks activation of p70 ribosomal S6 protein kinase (p70S6K). Insulin produced a decrease in the activity of glycogen synthase kinase-3 which is likely to be important in the observed stimulation of glycogen synthase. Both of these actions were found to be sensitive to inhibition by wortmannin. Activation of three processes is involved in the stimulation of fatty acid synthesis from glucose by insulin, namely glucose uptake, acetyl-CoA carboxylase and pyruvate dehydrogenase. Whereas wortmannin largely abolished the effects of insulin on glucose utilization and acetyl-CoA carboxylase activity, it was without effect on the stimulation of pyruvate dehydrogenase. Although epidermal growth factor stimulated mitogen-activated protein kinase to a greater extent than insulin, it was unable to mimic the effect of insulin on glycogen synthase, glycogen synthase kinase-3, glucose utilization, acetyl-CoA carboxylase or pyruvate dehydrogenase. Rapamycin also failed to have any appreciable effect on stimulation of these parameters by insulin, although it did block the effect of insulin on p70S6K. We conclude that the activity of phosphatidylinositol 3-kinase is required for the effects of insulin on glycogen synthesis, glucose uptake and acetyl-Co-AN carboxylase, but is not involved in signalling to pyruvate dehydrogenase. Activation of mitogen-activated protein kinase or p70S6K, however, does not appear to be sufficient to bring about the stimulation of fatty acid or glycogen synthesis. Altogether is seems likely that at least four distinct signalling pathways are involved in the effects of insulin on rat fat cells.
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PMID:Multiple signalling pathways involved in the stimulation of fatty acid and glycogen synthesis by insulin in rat epididymal fat cells. 748 1

Lactogens [prolactin (Prl) and growth hormone] stimulate phosphorylation of the 40S ribosomal protein, S6, in Nb2 cells by mechanisms that do not involve participation of cAMP or protein kinase A, protein kinase C, or cGMP-dependent protein kinase. However, inhibition of tyrosine kinase (TK) abrogates Prl-mediated macromolecular biosynthesis. Inasmuch as lactogen signaling may involve sequential activation of protein kinases, the effect of Prl on the well-characterized mitogen-activated protein kinase (MAPK) and S6 kinase (S6K), the enzyme responsible for S6 phosphorylation in vivo, and their relationship to Nb2 macromolecular biosynthesis and mitogenesis were investigated. The results show that MAPK stimulation is transient (peak activity, 30 min) and precedes that of S6K, which reaches a maximum at 1.5-2 h, and slowly returns towards control levels at 6 h. Both staurosporine which inhibits GH receptor-associated kinase (JAK2) and genistein (GEN), an inhibitor of membrane-associated and cytoplasmic TKs, abrogate Prl-stimulated TK, MAPK, and S6K. Rapamycin (RAP), a specific inhibitor of p70S6K, completely blocks S6K but does not affect TK and MAPK. TK and MAPK activity correlates with Prl-stimulated anabolism, i.e., protein and DNA synthesis and mitogenesis. Thus, concentrations of STR and GEN which abrogate TK and MAPK inhibit anabolism virtually 100%. However, RAP, which inhibits S6K (ca. 100%) but not TK or MAPK, only delays Prl-mediated anabolism. These results indicate that Prl signaling in Nb2 cells involves a protein kinase cascade and that regulation of receptor-associated kinase, TK, and MAPK correlates with anabolism. The role of S6K (and S6 phosphorylation) appears to be ancillary.
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PMID:Stimulation of receptor-associated kinase, tyrosine kinase, and MAP kinase is required for prolactin-mediated macromolecular biosynthesis and mitogenesis in Nb2 lymphoma. 784 Jun 14

We have previously shown that insulin causes inactivation of glycogen synthase kinase-3 (GSK-3) in Chinese hamster ovary cells over-expressing the human insulin receptor (CHO.T cells). We now show that serum and phorbol ester also cause rapid inactivation of GSK-3, both in CHO.T cells and in the nontransfected parental cell line, CHO.K1 cells. Rapamycin was without effect on the inactivation of GSK-3 by insulin, serum or phorbol ester, indicating that the p70 S6 kinase pathway is not involved. In contrast, wortmannin, a potent inhibitor of phosphatidylinositol 3-kinase, blocked the effects of both insulin and serum on GSK-3 activity, and also substantially reduced the activation of both p90 S6 kinase (by insulin) and mitogen-activated protein (MAP) kinase (by insulin and serum). These findings imply (i) that GSK-3 activity is regulated by a cascade involving MAP kinase and p90 S6 kinase and (ii) that wortmannin affects an early step in the MAP kinase pathway. One can infer from this that GSK-3 may be an important regulatory enzyme for the control of several biosynthetic pathways, key enzymes in which are regulated by GSK-3-mediated phosphorylation. Wortmannin had a smaller effect on the activation of MAP kinase by phorbol ester, indicating that phorbol esters may stimulate MAP kinase by a different or additional mechanism to that employed by insulin or serum. Wortmannin had very little effect on the inactivation of GSK-3 by phorbol ester: possible reasons for this are discussed.
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PMID:Wortmannin inhibits the effects of insulin and serum on the activities of glycogen synthase kinase-3 and mitogen-activated protein kinase. 794 34

Glycogen synthase kinase-3 (GSK3) is inactivated in vitro by p70 S6 kinase or MAP kinase-activated protein kinase-1 beta (MAPKAP kinase-1 beta; also known as Rsk-2). Here we show that GSK3 isoforms are inhibited by 40% within minutes after stimulation of the rat skeletal-muscle cell line L6 with insulin-like growth factor-1 (IGF-1) or insulin. GSK3 was similarly inhibited in rabbit skeletal muscle after an intravenous injection of insulin. Inhibition resulted from increased phosphorylation of GSK3, probably at a serine/threonine residue(s), because it was reversed by incubation with protein phosphatase-2A. Rapamycin blocked the activation of p70 S6 kinase by IGF-1 in L6 cells, but had no effect on the inhibition of GSK3 or the activation of MAPKAP kinase-1 beta. In contrast, wortmannin, a potent inhibitor of PtdIns 3-kinase, prevented the inactivation of GSK3 and the activation of MAPKAP kinase-1 beta and p70 S6 kinase by IGF-1 or insulin. Wortmannin also blocked the activation of p74raf-1. MAP kinase kinase and p42 MAP kinase, but not the formation of GTP-Ras by IGF-1. The results suggest that the stimulation of glycogen synthase by insulin/IGF-1 in skeletal muscle involves the MAP-KAP kinase-1-catalysed inhibition of GSK3, as well as the previously described activation of the glycogen-associated form of protein phosphatase-1.
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PMID:The inhibition of glycogen synthase kinase-3 by insulin or insulin-like growth factor 1 in the rat skeletal muscle cell line L6 is blocked by wortmannin, but not by rapamycin: evidence that wortmannin blocks activation of the mitogen-activated protein kinase pathway in L6 cells between Ras and Raf. 794 42

The cyclin-dependent kinase (Cdk) enzymes, when associated with the G1 cyclins D and E, are rate-limiting for entry into the S phase of the cell cycle. During T-cell mitogenesis, antigen-receptor signalling promotes synthesis of cyclin E and its catalytic partner, Cdk2, and interleukin-2 (IL-2) signalling activates cyclin E/Cdk2 complexes. Rapamycin is a potent immunosuppressant which specifically inhibits G1-to-S-phase progression, leading to cell-cycle arrest in yeast and mammals. Here we report that IL-2 allows Cdk activation by causing the elimination of the Cdk inhibitor protein p27Kip1, and that this is prevented by rapamycin. By contrast, the Cdk inhibitor p21 is induced by IL-2 and this induction is blocked by rapamycin. Our results show that p27Kip1 governs Cdk activity during the transition from quiescence to S phase in T lymphocytes and that p21 function may be restricted to cycling cells.
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PMID:Interleukin-2-mediated elimination of the p27Kip1 cyclin-dependent kinase inhibitor prevented by rapamycin. 799 Sep 32

IL-2 stimulates the proliferative response of various lymphoid cells. Previous studies showed an increase in intracellular levels of cAMP concomitant with an increase in phosphorylation of discrete proteins by protein kinase A at late G1 phase in mitogen-stimulated lymphocytes. Thus, experiments were undertaken to study nuclear proteins that bind to the cAMP-responsive enhancer (CRE) in cloned T lymphocytes stimulated with IL-2. With the use of a 32P-labeled CRE consensus sequence in a DNA binding gel mobility shift assay, we showed that IL-2 stimulation resulted in the induction of two major DNA-protein complexes at late G1/S during the cell cycle. This binding was competed in a dose-dependent manner by a nonlabeled CRE oligonucleotide but was not competed by a nonlabeled AP-1 oligonucleotide. Rapamycin, a potent immunosuppressant, which arrests IL-2-stimulated T lymphocytes at G1/S, inhibited the IL-2-induced CRE binding activities concomitantly with inhibition of DNA synthesis. By using specific Abs in a gel mobility shift assay, we identified two known CREB/ATF transcription factors in the IL-2-induced CRE complexes: the CRE binding factor (CREB), and ATF1. The induction of CREB binding by IL-2 was not associated with an increase in its abundance but was associated with a major increase in CREB phosphorylation that was particularly prominent at late G1/S. However, we found that G1/S progression induced by IL-2 was not associated with an increase in the intracellular levels of cAMP. These results suggest that 1) the transcription factors CREB and ATF1 and possibly other CRE binding proteins may have an important role in the modulation of specific gene expression at G1/S during cell cycle progression induced by IL-2. 2) The involvement of these CRE binding transcription factors in IL-2-stimulated cells is regulated via a mechanism that is not cAMP dependent.
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PMID:Regulation of cAMP-responsive enhancer binding proteins during cell cycle progression in T lymphocytes stimulated by IL-2. 799 60

Rapamycin (RAP) inhibits several biologic responses in the YAC-1 T cell lymphoma, including the serum-driven proliferation and cyclin A mRNA expression, the induction of Ly-6E Ag expression by IFN, and the induction of IFN-gamma production by IL-1. RAP also suppresses the enzymatic activity of the 70 kDa S6 protein kinase (pp70s6k). To define the mechanistic relationship between these multiple effects of RAP, we have generated stable somatic mutants with altered sensitivities to this drug. A first series of mutants, represented by the R19, 4R16, and 10R13 clones, showed markedly reduced sensitivity to the inhibitory effect of RAP on all biologic responses tested and on pp70s6k activity. Two other mutant types, R103 and R125, were both highly sensitive to RAP-mediated suppression of proliferation, of IL-1-induced IFN-gamma production, and of pp70s6k activity but differed in their Ly-6E response. This response was not affected by RAP in the R125 clone and was enhanced in the R103 clone. Therefore, the inhibitory effects of RAP on proliferation and IL-1-mediated IFN-gamma induction both appear associated with the inhibition of pp70s6k activity, whereas the modulation of Ly-6E induction is independent from the latter. Moreover, the cellular binding of [3H]dihydro-FK-506 was found to be blocked by RAP in all mutant types to the same extent as in wild-type YAC-1 cells, suggesting that the altered sensitivity to the effects of RAP in these mutants is not due to an inability of the drug to enter the cells or to interact with FKBP. Further biochemical characterization of the mutant cells described here is expected to help clarify the mechanisms of RAP action.
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PMID:Relationship between multiple biologic effects of rapamycin and the inhibition of pp70S6 protein kinase activity. Analysis in mutant clones of a T cell lymphoma. 830 Nov 50

Rapamycin has potent immunosuppressive properties reflecting its ability to disrupt cytokine signaling that promotes lymphocyte growth and differentiation. In IL-2-stimulated T cells, rapamycin impedes progression through the G1/S transition of the proliferation cycle, resulting in a mid-to-late G1 arrest. Two major biochemical alterations underlie this mode of action. The first one affects the phosphorylation/activation of the p70 S6 kinase (p70s6k), an early event of cytokine-induced mitogenic response. By inhibiting this enzyme, whose major substrate is the 40S ribosomal subunit S6 protein, rapamycin reduces the translation of certain mRNA encoding for ribosomal proteins and elongation factors, thereby decreasing protein synthesis. A second, later effect of rapamycin in IL-2-stimulated T cells is an inhibition of the enzymatic activity of the cyclin-dependent kinase cdk2-cyclin E complex, which functions as a crucial regulator of G1/S transition. This inhibition results from a prevention of the decline of the p27 cdk inhibitor, that normally follows IL-2 stimulation. To mediate these biochemical alterations, rapamycin needs to bind to intracellular proteins, termed FKBP, thereby forming a unique effector molecular complex. However, neither(p70s6k) inhibition, nor p27-induced cdk2-cyclin E inhibition are directly caused by the FKBP-rapamycin complex. Instead, this complex physically interacts with a novel protein, designated "mammalian target of rapamycin" (mTOR), which has sequence homology with the catalytic domain of phosphatidylinositol kinases and may therefore be itself a kinase. mTOR may act upstream of (p70s6K) and cdk2-cyclin E in a linear or bifurcated pathway of growth regulation. Molecular dissection of this pathway should further unravel cytokine-mediated signaling processes and help devise new immunosuppressants.
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PMID:Mechanism of action of the immunosuppressant rapamycin. 859 3

It is well established that insulin and serum stimulate gene expression at the level of mRNA translation in animal cells, and previous studies have mainly focused on the initiation process. Here we show that, in Chinese hamster ovary cells expressing the human insulin receptor, insulin causes decreased phosphorylation of elongation factor eEF-2 and that this is associated with stimulation of the rate of peptide-chain elongation. eEF-2 is phosphorylated by a very specific Ca 2+/calmodulin-dependent protein kinase (eEF-2 kinase) causing its complete inactivation. The decrease in eEF-2 phosphorylation induced by insulin reflects a fall in eEF-2 kinase activity. Rapamycin, a macrolide immunosuppressant which blocks the signalling pathway leading to the stimulation of the 70/85 kDa ribosomal protein S6 kinases, substantially blocks the activation of elongation, the fall in eEF-2 phosphorylation and the decrease in eEF-2 kinase activity, suggesting that p7O S6 kinase (p70s6k) and eEF-2 kinase may tie on a common signalling pathway. Wortmannin, an inhibitor of phosphatidylinositide-3-OH kinase, had similar effects. eEF-2 kinase was phosphorylated in vitro by purified p70s6k but this had no significant effect on the in vitro activity of eEF-2 kinase.
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PMID:Regulation of translation elongation factor-2 by insulin via a rapamycin-sensitive signalling pathway. 864 Dec 94

There is mounting evidence that in fat and other insulin-sensitive cells activation of protein synthesis may involve the dissociation of a protein (4E-BP1) from eukaryotic initiation factor (eIF)-4E thus allowing formation of the eIF-4F complex. This study compares the effects of insulin and epidermal growth factor (EGF) on the phosphorylation of 4E-BP1 in fat-cells (followed by gel-shift assays and incorporation of 32P) and on its association with eIF-4E. Several lines of evidence suggest that mitogenactivated protein kinase (MAP kinase) is not involved in these effects of insulin. Insulin causes much more extensive phosphorylation and dissociation of 4E-BP1 from eIF-4E than EGF, although EGF activates MAP kinase to a much greater extent than insulin. Moreover, MAP kinase does not phosphorylate 4E-BP1 when it is complexed with eIF-4E. In contrast, insulin activates the 40S ribosomal protein S6 kinase (p70S6K) 18-fold compared with a 2-fold activation by EGF, and the time course of this activation is similar to the phosphorylation and dissociation of 4E-BP1. Rapamycin, a specific inhibitor of the activation of this latter kinase, inhibits dissociation of 4E-BP1 from eIF-4E in cells incubated with insulin but reveals a phosphorylated from of 4E-BP1 which remains bound to eIF-4E. It is concluded that in rat epididymal fat-cells, the effects of insulin on 4E-BP1 involves multiple phosphorylation events. One phosphorylation event is rapamycin-insensitive, occurs only on bound 4E-BP1 and does not initiate dissociation. The second event does result in dissociation and is blocked by rapamycin, suggesting that the p70S6K signalling pathway is involved: p70S6K itself is probably not involved directly as this kinase does not phosphorylate 4E-BP1 in vitro.
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PMID:Both rapamycin-sensitive and -insensitive pathways are involved in the phosphorylation of the initiation factor-4E-binding protein (4E-BP1) in response to insulin in rat epididymal fat-cells. 868 86


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