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)

In adipose and muscle, insulin stimulates glucose uptake and glycogen synthase activity. Phosphatidylinositol 3-kinase (PI3K) activation is necessary but not sufficient for these metabolic actions of insulin. The insulin-stimulated translocation of phospho-c-Cbl to lipid rafts, via its association with CAP, comprises a second pathway regulating GLUT4 translocation. In 3T3-L1 adipocytes, overexpression of a dominant negative CAP mutant (CAP Delta SH3) completely blocked the insulin-stimulated glucose transport and glycogen synthesis but only partially inhibited glycogen synthase activation. In contrast, CAP Delta SH3 expression did not affect glycogen synthase activation by insulin in the absence of extracellular glucose. Moreover, CAP Delta SH3 has no effect on the PI3K-dependent activation of protein phosphatase-1 or phosphorylation of glycogen synthase kinase-3. These results indicate blockade of the c-Cbl/CAP pathway directly inhibits insulin-stimulated glucose uptake, which results in secondary inhibition of glycogen synthase activation and glycogen synthesis.
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PMID:Activation of glycogen synthase by insulin in 3T3-L1 adipocytes involves c-Cbl-associating protein (CAP)-dependent and CAP-independent signaling pathways. 1122 22

Phosphatidylinositol 4,5 bisphosphate (PIP(2)) is widely implicated in cytoskeleton regulation, but the mechanisms by which PIP(2) effect cytoskeletal changes are not defined. We used recombinant adenovirus to infect CV1 cells with the mouse type I phosphatidylinositol phosphate 5-kinase alpha (PIP5KI), and identified the players that modulate the cytoskeleton in response to PIP(2) signaling. PIP5KI overexpression increased PIP(2) and reduced phosphatidylinositol 4 phosphate (PI4P) levels. It promoted robust stress-fiber formation in CV1 cells and blocked PDGF-induced membrane ruffling and nucleated actin assembly. Y-27632, a Rho-dependent serine/threonine protein kinase (ROCK) inhibitor, blocked stress-fiber formation and inhibited PIP(2) and PI4P synthesis in cells. However, Y-27632 had no effect on PIP(2) synthesis in lysates, although it inhibited PI4P synthesis. Thus, ROCK may regulate PIP(2) synthesis by controlling PI4P availability. PIP5KI overexpression decreased gelsolin, profilin, and capping protein binding to actin and increased that of ezrin. These changes can potentially account for the increased stress fiber and nonruffling phenotype. Our results establish the physiological role of PIP(2) in cytoskeletal regulation, clarify the relation between Rho, ROCK, and PIP(2) in the activation of stress-fiber formation, and identify the key players that modulate the actin cytoskeleton in response to PIP(2).
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PMID:Phosphatidylinositol 4,5-bisphosphate induces actin stress-fiber formation and inhibits membrane ruffling in CV1 cells. 1123 45

Previous clinical studies showed an apparent correlation between hypertension and insulin resistance, and patients with diabetes are known to have increased blood pressure responsiveness to salt loading. To investigate the effect of high salt intake on insulin sensitivity and the insulin signaling pathway, a high-salt diet (8% NaCl) or a normal diet was given to 7-week-old SD rats for 2 weeks. High salt-fed rats developed slightly but significantly higher systolic blood pressure than controls (133 +/- 2 vs. 117 +/- 2 mmHg, P < 0.001), with no change in food intake or body weight. High salt-fed rats were slightly hyperglycemic (108.5 +/- 2.8 vs. 97.8 +/- 2.5 mg/dl, P = 0.01) and slightly hyperinsulinemic (0.86 +/- 0.07 vs. 0.61 +/- 0.06 ng/ml, P = 0.026) in the fasting condition, as compared with controls. Hyperinsulinemic-euglycemic clamp study revealed a 52.7% decrease in the glucose infusion rate and a 196% increase in hepatic glucose production in high salt-fed rats, which also showed a 66.4% decrease in 2-deoxyglucose uptake into isolated skeletal muscle and a 44.5% decrease in insulin-induced glycogen synthase activation in liver, as compared with controls. Interestingly, despite the presence of insulin resistance, high salt-fed rats showed enhanced insulin-induced tyrosine phosphorylation of insulin receptor substrate (IRS)-1, IRS-2 (liver and muscle), and IRS-3 (liver only). Phosphatidylinositol (PI) 3-kinase activities associated with IRS and phosphotyrosine in the insulin-stimulated condition increased 2.1- to 4.1-fold, as compared with controls. Insulin-induced phosphorylation of Ser-473 of Akt and Ser-21 of glycogen synthase kinase-3 also increased 2.9- and 2-fold, respectively, in the liver of the high salt-fed rats. Therefore, in both the liver and muscle of high salt-fed rats, intracellular insulin signaling leading to PI 3-kinase activation is enhanced and insulin action is attenuated. The hyperinsulinemic-euglycemic clamp study showed that decreased insulin sensitivity induced with a high-salt diet was not reversed by administration of pioglitazone. The following can be concluded: 1) a high-salt diet may be a factor promoting insulin resistance, 2) the insulin-signaling step impaired by high salt intake is likely to be downstream from PI 3-kinase or Akt activation, and 3) this unique insulin resistance mechanism may contribute to the development of diabetes in patients with hypertension.
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PMID:Insulin resistance with enhanced insulin signaling in high-salt diet-fed rats. 1124 77

Phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/Akt) is thought to serve as an oncogenic signaling pathway which can be activated by Ras. The role of PI3K/Akt in Ras-mediated transformation of intestinal epithelial cells is currently not clear. Here we demonstrate that inducible expression of oncogenic Ha-Ras results in activation of PKB/Akt in rat intestinal epithelial cells (RIE-iHa-Ras), which was blocked by treatment with inhibitors of PI3K activity. The PI3K inhibitor, LY-294002, partially reversed the morphological transformation induced by Ha-Ras and resulted in a modest stimulation of apoptosis. The most pronounced phenotypic alteration following inhibition of PI3K was induction of G(1) phase cell cycle arrest. LY-294002 blocked the Ha-Ras-induced expression of cyclin D1, cyclin-dependent kinase (CDK) 2, and increased the levels of p27(kip). Both LY-294002 and wortmannin significantly reduced anchorage-independent growth of RIE-iHa-Ras cells. Forced expression of both the constitutively active forms of Raf (DeltaRaf-22W or Raf BXB) and Akt (Akt-myr) resulted in transformation of RIE cells that was not achieved by transfection with either the Raf mutant construct or Akt-myr alone. These findings delineate an important role for PI3K/Akt in Ras-mediated transformation of intestinal epithelial cells.
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PMID:Akt/PKB activity is required for Ha-Ras-mediated transformation of intestinal epithelial cells. 1127 13

A20 IIA1.6 B cells cotransfected with FcalphaR and wild-type gamma-chain (wt-ITAM (immunoreceptor tyrosine-based activation motif)) or FcalphaR and gamma-chain, in which the wt-ITAM was substituted with the FcgammaRIIA ITAM (IIA-ITAM), were used to investigate cell signaling events influencing presentation of FcalphaR-targeted exogenous Ag in the context of MHC class II. wt-ITAM cells presented FcalphaR-targeted OVA more efficiently than IIA-ITAM transfectants to OVA-specific T cell hybridomas. Phosphatidylinositol 3-kinase (PI 3-kinase) inhibition abrogated Ag presentation, suggesting that FcalphaR may trigger a PI 3-kinase-dependent signal transduction pathway, and thus phosphatidylinositol-dependent protein kinase (PDK1) and protein kinase B alpha (PKBalpha) activation. Cross-linking FcalphaR on wt-ITAM or IIA-ITAM cells triggered equivalent PI 3-kinase-dependent activation of PKBalpha. Furthermore, FcalphaR cross-linking triggered recruitment of PDK1 and serine-phosphorylated PKBalpha to capped cell surface FcalphaR irrespective of the gamma-chain ITAM. Although FcalphaR endocytosis was accompanied by translocation of PDK1 and phospho-PKBalpha to FcalphaR-containing vesicles in both transfectants, this was decreased in IIA-ITAM cells, and a significant proportion of PDK1 and PKBalpha remained at the plasma membrane. In wt-ITAM cells, PDK1 and serine-phosphorylated PKBalpha translocated to lysosomal-associated membrane glycoprotein 1- and cathepsin B-containing vesicles, consistent with MHC class II peptide-loading compartments (MIIC) described by other groups. Our data indicate that translocation of signal transduction mediators to MIIC-like compartments accompanies efficient presentation of receptor-targeted Ag, and suggest a mechanism connecting signaling to the Ag-processing pathway.
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PMID:Fc alpha receptor cross-linking causes translocation of phosphatidylinositol-dependent protein kinase 1 and protein kinase B alpha to MHC class II peptide-loading-like compartments. 1131 98

Phosphatidylinositol 3-kinase (PI 3-kinase) is a cytoplasmic signaling molecule that is recruited to activated growth factor receptors and has been shown to be involved in regulation of stimulated exocytosis and endocytosis. One of the downstream signaling molecules activated by PI 3-kinase is the protein kinase Akt. Previous studies have indicated that PI 3-kinase is necessary for basal Na(+)/H(+) exchanger 3 (NHE3) transport and for fibroblast growth factor-stimulated NHE3 activity in PS120 fibroblasts. However, it is not known whether activation of PI 3-kinase is sufficient to stimulate NHE3 activity or whether Akt is involved in this PI 3-kinase effect. We used an adenoviral infection system to test the possibility that activation of PI 3-kinase or Akt alone is sufficient to stimulate NHE3 activity. This hypothesis was investigated in PS120 fibroblasts stably expressing NHE3 after somatic gene transfer using a replication-deficient recombinant adenovirus containing constitutively active catalytic subunit of PI 3-kinase or constitutively active Akt. The adenovirus construct used was engineered with an upstream ecdysone promoter to allow time-regulated expression. Adenoviral infection was nearly 100% at 48 h after infection. Forty-eight hours after infection (24 h after activation of the ecdysone promoter), PI 3-kinase and Akt amount and activity were increased. Increases in both PI 3-kinase activity and Akt activity stimulated NHE3 transport. In addition, a membrane-permeant synthetic 10-mer peptide that binds polyphosphoinositides and increases PI 3-kinase activity similarly enhanced NHE3 transport activity and also increased the percentage of NHE3 on the plasma membrane. The magnitudes of stimulation of NHE3 by constitutively active PI 3-kinase, PI 3-kinase peptide, and constitutively active Akt were similar to each other. These results demonstrate that activation of PI 3-kinase or Akt is sufficient to stimulate NHE3 transport activity in PS120/NHE3 cells.
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PMID:Constitutively active phosphatidylinositol 3-kinase and AKT are sufficient to stimulate the epithelial Na+/H+ exchanger 3. 1137 99

We determined the involvement of Tyr-1158 within the regulatory loop of the insulin receptor (IR) in the generation of insulin-specific responses in situ. For this purpose chimeric receptors with an epidermal growth factor (EGF) receptor extracellular domain and an IR cytoplasmic domain (EIR) were constructed, which allow activation of the cytoplasmic IR domain without activation of endogenous wt-IRs. Tyr-1158 of the chimera EIR was exchanged for Phe, creating a mutant chimeric receptor (EIR-Y1158F). Chimeric receptors were expressed in 3T3-L1 pre-adipocytes, which do not show insulin-specific responses upon EGF stimulation. We found that pre-adipocytes expressing EIR-Y1158F were impaired in their ability to stimulate glycogen synthesis and DNA synthesis upon maximal stimulation with EGF. EIR-Y1158F was impaired in its ability to phosphorylate insulin receptor substrate (IRS)-1 and induce downstream signals of IRS-1 phosphorylation, such as the association of IRS-1 with phosphatidyl-inositol-3'-kinase and the activation of protein kinase B (Akt). In contrast with the phosphorylation of IRS-1, the phosphorylation of IRS-2 and extracellular regulated protein kinase-1/-2 was normal in EIR-Y1158F expressing cells. These observations suggest that the level of IRS-1 phosphorylation rather than the level of IRS-2 phosphorylation mediates insulin-induced glycogen synthesis and DNA synthesis in 3T3-L1 pre-adipocytes.
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PMID:IRS-1 tyrosine phosphorylation reflects insulin-induced metabolic and mitogenic responses in 3T3-L1 pre-adipocytes. 1147 Oct 71

Phosphatidylinositol 3-kinase (PI3-kinase) activates protein kinase B (also known as Akt), which phosphorylates and activates a cyclic nucleotide phosphodiesterase 3B. Increases in cyclic nucleotide concentrations inhibit agonist-induced contraction of vascular smooth muscle. Thus we hypothesized that the PI3-kinase/Akt pathway may regulate vascular smooth muscle tone. In unstimulated, intact bovine carotid artery smooth muscle, the basal phosphorylation of Akt was higher than that in cultured smooth muscle cells. The phosphorylation of Akt decreases in a time-dependent manner when incubated with the PI3-kinase inhibitor, LY-294002. Agonist (serotonin)-, phorbol ester (phorbol 12,13-dibutyrate; PDBu)-, and depolarization (KCl)-induced contractions of vascular smooth muscles were all inhibited in a dose-dependent fashion by LY-294002. However, LY-294002 did not inhibit serotonin- or PDBu-induced increases in myosin light chain phosphorylation or total O(2) consumption, suggesting that inhibition of contraction was not mediated by reversal or inhibition of the pathways that lead to smooth muscle activation and contraction. Treatment of vascular smooth muscle with LY-294002 increased the activity of cAMP-dependent protein kinase and increased the phosphorylation of the cAMP-dependent protein kinase substrate heat shock protein 20 (HSP20). These data suggest that activation of the PI3-kinase/Akt pathway in unstimulated smooth muscle may modulate vascular smooth muscle tone (allow agonist-induced contraction) through inhibition of the cyclic nucleotide/HSP20 pathway and suggest that cyclic nucleotide-dependent inhibition of contraction is dissociated from the myosin light chain contractile regulatory pathways.
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PMID:PI3-kinase/Akt modulates vascular smooth muscle tone via cAMP signaling pathways. 1156 68

Cell migration requires precise coordination of many signaling pathways to achieve directed motility. We report here that NIH3T3 fibroblasts expressing a dominant negative PKA subunit (dnPKA) show diminished migration in response to serum or growth factors. This effect is not a general effect on cell motility, but rather a decreased capacity to enhance migration in response to stimuli. Control (neo) and dnPKA cells show very similar haptotactic migration toward fibronectin, but dnPKA cells show reduced stimulation of migration in response to EGF/PDGF or serum. These effects were not due to alterations in cell growth or adhesion to fibronectin. Forskolin, which elevates cyclic adenosine monophosphate (cAMP) levels, dramatically inhibited neo cell motility in a scrape migration assay, although dnPKA cell migration was unaffected. The MEK selective inhibitor U0126 and the phosphatidyl-inositol-3 kinase (PI3K) inhibitor LY294002 inhibited migrating neo cells and were able to further inhibit residual dnPKA cell migration. Our data show that intermediate or well-controlled levels of PKA activity are required for optimal growth factor-stimulated migration in fibroblasts. PKA may play an important role in the signaling processes that lead to motility.
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PMID:Inhibition of PKA blocks fibroblast migration in response to growth factors. 1164 Aug 85

The proto-oncogene ErbB2 is known to be amplified and to play an important role in the development of about one-third of human breast cancers. Phosphatidylinositol 3-kinase (PI3K), which is often activated in ErbB2-overexpressing breast cancer cells, is known to regulate cell proliferation and cell survival. Selective inhibitors of the PI3K pathway were used to assess the relevance of PI3K signaling in the anchorage-independent growth of a series of human mammary carcinoma cell lines. Wortmannin, LY294002, and rapamycin at concentrations that did not affect MAPK phosphorylation but substantially inhibited PI3K, Akt, and p70(S6K) significantly suppressed the soft agar growth of tumor cell lines that overexpress ErbB2 but not the growth of tumor lines with low ErbB2 expression. A similar growth inhibition of ErbB2-overexpressing carcinoma lines was observed when a dominant negative p85(PI3K) mutant was introduced into these cells. Forced expression of ErbB2 in breast cancer lines originally expressing low ErbB2 levels augmented receptor expression and sensitized those lines to LY294002- and rapamycin-mediated inhibition of colony formation. Furthermore, treatment with LY294002 resulted in the selective increase of cyclin-dependent kinase inhibitors p21(Cip1) or p27(Kip1) and suppression of cyclin E-associated Cdk2 kinase activity in ErbB2-overexpressing lines, which may account for their hypersensitivity toward inhibitors of the PI3K pathway in anchorage-independent growth. Our results indicate that the PI3K/Akt/p70(S6K) pathway plays an enhanced role in the anchorage-independent growth of ErbB2-overexpressing breast cancer cells, therefore providing a molecular basis for the selective targeting of this signaling pathway in the treatment of ErbB2-related human breast malignancies.
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PMID:ErbB2-overexpressing human mammary carcinoma cells display an increased requirement for the phosphatidylinositol 3-kinase signaling pathway in anchorage-independent growth. 1170 27


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