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)

Protein kinase B lies "downstream" of phosphatidylinositide (PtdIns) 3-kinase and is thought to mediate many of the intracellular actions of insulin and other growth factors. Here we show that FKHR, a human homologue of the DAF16 transcription factor in Caenorhabditis elegans, is rapidly phosphorylated by human protein kinase Balpha (PKBalpha) at Thr-24, Ser-256, and Ser-319 in vitro and at a much faster rate than BAD, which is thought to be a physiological substrate for PKB. The same three sites, which all lie in the canonical PKB consensus sequences (Arg-Xaa-Arg-Xaa-Xaa-(Ser/Thr)), became phosphorylated when FKHR was cotransfected with either PKB or PDK1 (an upstream activator of PKB). All three residues became phosphorylated when 293 cells were stimulated with insulin-like growth factor 1 (IGF-1). The IGF-1-induced phosphorylation was abolished by the PtdIns 3-kinase inhibitor wortmannin but not by PD 98059 (an inhibitor of the mitogen-activated protein kinase cascade) or by rapamycin. These results indicate that FKHR is a physiological substrate of PKB and that it may mediate some of the physiological effects of PKB on gene expression. DAF16 is known to be a component of a signaling pathway that has been partially dissected genetically and includes homologues of the insulin/IGF-1 receptor, PtdIns 3-kinase and PKB. The conservation of Thr-24, Ser-256, and Ser-319 and the sequences surrounding them in DAF16 therefore suggests that DAF16 is also a direct substrate for PKB in C. elegans.
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PMID:Phosphorylation of the transcription factor forkhead family member FKHR by protein kinase B. 1035 75

Protein kinase B and p70 S6 kinase are members of the cyclic AMP-dependent/cyclic GMP-dependent/protein kinase C subfamily of protein kinases and are activated by a phosphatidylinositol 3-kinase-dependent pathway when cells are stimulated with insulin or growth factors. Both of these kinases are activated in cells by phosphorylation of a conserved residue in the kinase domain (Thr-308 of protein kinase B (PKB) and Thr-252 of p70 S6 kinase) and another conserved residue located C-terminal to the kinase domain (Ser-473 of PKB and Thr-412 of p70 S6 kinase). Thr-308 of PKBalpha and Thr-252 of p70 S6 kinase are phosphorylated by 3-phosphoinositide-dependent protein kinase-1 (PDK1) in vitro. Recent work has shown that PDK1 interacts with a region of protein kinase C-related kinase-2, termed the PDK1 interacting fragment (PIF). Interaction with PIF converts PDK1 from a form that phosphorylates PKB at Thr-308 alone to a species capable of phosphorylating Ser-473 as well as Thr-308. This suggests that PDK1 may be the enzyme that phosphorylates both residues in vivo. Here we demonstrate that PDK1 is capable of phosphorylating p70 S6 kinase at Thr-412 in vitro. We study the effect of PIF on the ability of PDK1 to phosphorylate p70 S6 kinase. Surprisingly, we find that PDK1 bound to PIF is no longer able to interact with or phosphorylate p70 S6 kinase in vitro at either Thr-252 or Thr-412. The expression of PIF in cells prevents insulin-like growth factor 1 from inducing the activation of the p70 S6 kinase and its phosphorylation at Thr-412. Overexpression of PDK1 in cells induces the phosphorylation of p70 S6 kinase at Thr-412 in unstimulated cells, and a catalytically inactive mutant of PDK1 prevents the phosphorylation of p70 S6K at Thr-412 in insulin-like growth factor 1-stimulated cells. These observations indicate that PDK1 regulates the activation of p70 S6 kinase and provides evidence that PDK1 mediates the phosphorylation of p70 S6 kinase at Thr-412.
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PMID:Evidence that 3-phosphoinositide-dependent protein kinase-1 mediates phosphorylation of p70 S6 kinase in vivo at Thr-412 as well as Thr-252. 1060 11

The interaction of BAD (Bcl-2/Bcl-X(L)-antagonist, causing cell death) with Bcl-2/Bcl-X(L) is thought to neutralize the anti-apoptotic effects of the latter proteins, and may represent one of the mechanisms by which BAD promotes apoptosis. A variety of survival signals are reported to induce the phosphorylation of BAD at Ser(112) or Ser(136), triggering its dissociation from Bcl-2/Bcl-X(L). Ser(136) is thought to be phosphorylated by protein kinase B (PKB, also called Akt), which is activated when cells are exposed to agonists that stimulate phosphatidylinositol 3-kinase (PI3K). In contrast, Ser(112) is reported to be phosphorylated by mitogen-activated protein (MAP) kinase-activated protein kinase-1 (MAPKAP-K1, also called RSK) and by cAMP-dependent protein kinase (PKA). Here we identify Ser(155) as a third phosphorylation site on BAD. We find that Ser(155) is phosphorylated preferentially by PKA in vitro and is the only residue in BAD that becomes phosphorylated when cells are exposed to cAMP-elevating agents. The phosphorylation of BAD at Ser(155) prevents it from binding to Bcl-X(L) and promotes its interaction with 14-3-3 proteins. We also provide further evidence that MAPKAP-K1 mediates the phosphorylation of Ser(112) in response to agonists that activate the classical MAP kinase pathway. However insulin-like growth factor 1, a potent activator of PI3K and PKB does not increase the phosphorylation of Ser(136) in BAD-transfected HEK-293 cells, and nor is the basal level of Ser(136) phosphorylation suppressed by inhibitors of PI3K.
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PMID:Regulation of BAD by cAMP-dependent protein kinase is mediated via phosphorylation of a novel site, Ser155. 1088 Mar 54

The brain has enormous anabolic needs during early postnatal development. This study presents multiple lines of evidence showing that endogenous brain insulin-like growth factor 1 (Igf1) serves an essential, insulin-like role in promoting neuronal glucose utilization and growth during this period. Brain 2-deoxy-d- [1-(14)C]glucose uptake parallels Igf1 expression in wild-type mice and is profoundly reduced in Igf1-/- mice, particularly in those structures where Igf1 is normally most highly expressed. 2-Deoxy-d- [1-(14)C]glucose is significantly reduced in synaptosomes prepared from Igf1-/- brains, and the deficit is corrected by inclusion of Igf1 in the incubation medium. The serine/threonine kinase Akt/PKB is a major target of insulin-signaling in the regulation of glucose transport via the facilitative glucose transporter (GLUT4) and glycogen synthesis in peripheral tissues. Phosphorylation of Akt and GLUT4 expression are reduced in Igf1-/- neurons. Phosphorylation of glycogen synthase kinase 3beta and glycogen accumulation also are reduced in Igf1-/- neurons. These data support the hypothesis that endogenous brain Igf1 serves an anabolic, insulin-like role in developing brain metabolism.
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PMID:Insulin-like growth factor 1 regulates developing brain glucose metabolism. 1095 33

Insulin-like growth factor binding protein (IGFBP)-1 has been shown to alter cellular responses to insulin-like growth factor 1 (IGF-1). Human IGFBP-1 undergoes serine phosphorylation, and this enhances both its affinity for IGF-1 by six- to eightfold and its capacity to inhibit IGF-1 actions. To investigate the physiological role of IGFBP-1 in vivo, transgenic mice have been generated using either the human IGFBP-1 or rat IGFBP-1 transgene. Both lines of mice expressed high concentrations of IGFBP-1 in serum and tissues; however, human IGFBP-1 transgenic mice did not show glucose intolerance and exhibited no significant intrauterine growth retardation, whereas rat IGFBP-1 transgenic mice showed fasting hyperglycemia and intrauterine growth restriction. The aim of this study was to investigate the physiological differences in the phosphorylation state of human IGFBP-1 and rat IGFBP-1 in these transgenic mice. The phosphorylation status of IGFBP-1 in transgenic mouse serum was analyzed by nondenaturing PAGE. Almost all of the IGFBP-1 in serum from the human IGFBP-1 transgenic mice was present as a nonphosphorylated form. Most of the rat IGFBP-1 in the serum of the mice expressing the rat IGFBP-1 was phosphorylated. Immunoprecipitation showed that mouse hepatoma (Hepa 1-6) cells (exposed to [32P]H3PO4) secrete 32P-labeled IGFBP-1. When the human IGFBP-1 transgene was transfected into Hepa 1-6 cells, all of the IGFBP-1 was secreted in the nonphosphorylated form. However, when the rat IGFBP-1 transgene was transfected into these cells, phosphorylated forms of IGFBP-1 were secreted. To confirm this result, the mouse hepatoma cell protein kinase was partially purified. This kinase activity phosphorylated mouse and rat IGFBP-1 in vitro, but it did not phosphorylate human IGFBP-1. Scatchard analysis showed that the affinity of phosphorylated rat IGFBP-1 for IGF-1 was 3.9-fold higher than that of nonphosphorylated human IGFBP-1. We conclude that the mouse IGFBP-1 kinase activity cannot phosphorylate human IGFBP-1, whereas it can phosphorylate rat IGFBP-1. The phosphorylation state of human IGFBP-1 may account for part of the phenotypic differences noted in the two studies of transgenic mice, and it is an important determinant of the capacity of human IGFBP-1 to inhibit IGF-1 actions in vivo.
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PMID:Physiological differences in insulin-like growth factor binding protein-1 (IGFBP-1) phosphorylation in IGFBP-1 transgenic mice. 1114 91

AMP-activated protein kinase (AMPK) is tightly regulated by the cellular AMP:ATP ratio and plays a central role in the regulation of energy homeostasis. Previously, AMPK was reported to phosphorylate serine 621 of Raf-1 in vitro. In the present study, we investigated a possible role of AMPK in extracellular signal-regulated kinase (Erk) cascades, using 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR), a cell-permeable activator of AMPK and antisense RNA experiments. Activation of AMPK by AICAR in NIH-3T3 cells resulted in drastic inhibitions of Ras, Raf-1, and Erk activation induced by insulin-like growth factor 1 (IGF-1). Expression of an antisense RNA for the AMPK catalytic subunit decreased the AMPK activity and significantly diminished the AICAR effect on IGF-1-induced Ras activation and the subsequent Erk activation, indicating that its effect is indeed mediated by AMPK. Phosphorylation of Raf-1 serine 621, however, was not involved in AMPK-mediated inhibition of Erk cascades. In contrast to IGF-1, AICAR did not block epidermal growth factor (EGF)-dependent Raf-1 and Erk activation, but our results demonstrated that multiple Raf-1 upstream pathways induced by EGF were differentially affected by AICAR: inhibition of Ras activation and simultaneous induction of Ras-independent Raf activation. The activities of IGF-1 and EGF receptor were not affected by AICAR. Taken together, our results suggest that AMPK differentially regulate Erk cascades by inhibiting Ras activation or stimulating the Ras-independent pathway in response to the varying energy status of the cell.
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PMID:Effects of stimulation of AMP-activated protein kinase on insulin-like growth factor 1- and epidermal growth factor-dependent extracellular signal-regulated kinase pathway. 1126 1

Thyrotropin (TSH)-initiated cell cycle progression from G1 to S phase in FRTL-5 thyroid cells requires serum, insulin, or insulin-like growth factor 1 (IGF-1) and involves activation of 3-hydroxy-3-methylglutaryl-CoA reductase, geranylgeranylation of RhoA, p27Kip1 degradation, and activation of cyclin-dependent kinase (cdk) 2. In the present report, we show that the serine-threonine kinase Akt is an important mediator of insulin/IGF-1/serum effects on cell cycle progression in FRTL-5 thyroid cells. The phosphoinositol (OH) 3 kinase inhibitors, Wortmannin (WM) and Ly294002 (LY), block the ability of insulin/IGF-1 to reduce p27 expression, to induce expression of cyclins E, D1, and A as well as cdk 2 and 4, and to phosphorylate retinoblastoma protein. They also inhibit insulin/IGF-1-increased DNA synthesis and cell cycle entrance (S+G2/M). Insulin/IGF-1 rapidly induced activation of Aktl in a PI3 kinase-dependent manner, and increased Aktl RNA levels. Most importantly, FRTL-5 cells transfected with a constitutively active form of Aktl have higher basal rates of DNA synthesis and no longer require exogenous insulin/IGF-1 or serum for TSH-induced growth. In sum, Aktl appears to have an important role in insulin/IGF-1 regulation of FRTL-5 thyroid cell growth and cell cycle progression.
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PMID:Regulation of FRTL-5 thyroid cell growth by phosphatidylinositol (OH) 3 kinase-dependent Akt-mediated signaling. 1134 32

The physiological performance of an organ depends on an interplay between changes in cellular function and organ size, determined by cell growth, proliferation and death. Nowhere is this more evident than in the endocrine pancreas, where disturbances in function or mass result in severe disease. Recently, the insulin signal-transduction pathway has been implicated in both the regulation of hormone secretion from beta cells in mammals as well as the determination of cell and organ size in Drosophila melanogaster. A prominent mediator of the actions of insulin and insulin-like growth factor 1 (IGF-1) is the 3'-phosphoinositide-dependent protein kinase Akt, also known as protein kinase B (PKB). Here we report that overexpression of active Akt1 in the mouse beta cell substantially affects compartment size and function. There was a significant increase in both beta-cell size and total islet mass, accompanied by improved glucose tolerance and complete resistance to experimental diabetes.
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PMID:Regulation of pancreatic beta-cell growth and survival by the serine/threonine protein kinase Akt1/PKBalpha. 1159 Apr 37

The involvement of mitogen-activated protein (MAP) kinases in the mitogenic effect of thyrotropin (TSH) is not fully elucidated. In FRTL-5 cells, we found that the MAP kinase kinase (MEK) inhibitors UO126 and PD98059 substantially decreased TSH-induced DNA synthesis, indicating that MAP kinases are involved in the TSH-stimulated proliferative response. Accordingly, TSH, forskolin (FSK) and 8-bromo-cAMP induced a rapid (3 min) and transient activation of ERK1/2, as assessed by phosphorylation of myelin basic protein and ERK1/2. This effect was cAMP-dependent and protein kinase A (PKA)-independent. The activation of Rap1 and B-Raf was involved in the mechanism of MAP kinase stimulation by TSH. TSH induced rapid (3 min) GDP/GTP exchange and activation of Rap1. After a 3-min exposure to FSK, B-Raf was recruited to a vesicular compartment, where it colocalized with Rap1. Both activation of Rap1 and translocation of B-Raf were PKA-independent. The Rap1 dominant negative Rap1N17 significantly reduced TSH-stimulated but not insulin-like growth factor 1-stimulated ERK1/2 phosphorylation, whereas the Ras dominant negative RasN17 inhibited the effect of both agonists. In conclusion, our results document that TSH increases intracellular cAMP, which rapidly stimulates MAP kinase cascade independent of PKA. This novel mechanism could integrate other pathways involved in TSH-stimulated proliferative response.
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PMID:Thyrotropin activates mitogen-activated protein kinase pathway in FRTL-5 by a cAMP-dependent protein kinase A-independent mechanism. 1164 20

Protein phosphatase 2A (PP2A) is a multimeric serine/threonine phosphatase that carries out multiple functions. Although numerous observations suggest that PP2A plays a major role in downregulation of the mitogen-activated protein (MAP) kinase pathway, the precise mechanisms are unknown. To clarify the role of PP2A in growth factor (insulin, epidermal growth factor [EGF], and insulin-like growth factor 1 [IGF-1]) stimulation of the Ras/MAP kinase pathway, simian virus 40 small t antigen was expressed in Rat-1 fibroblasts which overexpress insulin receptors. Small t antigen is known to specifically inhibit PP2A by binding to the A PP2A regulatory subunit, interfering with the ability of PP2A to bind to its cellular substrates. Overexpressed small t protein was coimmunoprecipitated with PP2A and inhibited cellular PP2A activity but did not inhibit protein phosphatase 1 (PP1) activity. Insulin, IGF-1, and EGF stimulation also inhibited PP2A activity. Growth factor-stimulated Ras, Raf-1, MAP kinase, and mitogen-activated extracellular-signal-regulated kinase kinase (MEK) activities were elevated in small-t-antigen-expressing cells. Furthermore, Shc tyrosine phosphorylation and its association with Grb2 were also elevated in small-t-antigen-expressing cells. Expression levels of Shc, Ras, MEK, or MAP kinase and phosphorylation of insulin, EGF, and IGF-1 receptors were not altered. Interestingly, we found that PP2A associated with Shc in the basal state and dissociated in response to insulin and EGF and that this dissociation was inhibited by 65% in small-t-antigen-expressing cells. In addition, we found that PP2A associates with the phosphotyrosine-binding domain (PTB domain) of Shc and that phosphorylation of tyrosine 317 of Shc was required for PP2A-Shc dissociation. We conclude (i) that PP2A negatively regulates the Ras/MAP kinase pathway by binding to Shc, inhibiting tyrosine phosphorylation; (ii) that the Shc-PP2A association is mediated by the Shc PTB domain but the interaction is independent of phosphotyrosine binding, indicating a new molecular function for the PTB domain; (iii) that growth factor stimulation, or small-t-antigen expression, causes dissociation of the PP2A-Shc complex, facilitating Shc phosphorylation and downstream activations of the Ras/MAP kinase pathway; and (iv) that this defines a new mechanism of small-t-antigen action to promote mitogenesis.
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PMID:Protein phosphatase 2A forms a molecular complex with Shc and regulates Shc tyrosine phosphorylation and downstream mitogenic signaling. 1188 20


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