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)

The effects of protein phosphorylation and dephosphorylation on glucose transport activity reconstituted from adipocyte membrane fractions and its relationship to the phosphorylation state of the adipose/muscle-type glucose transporter (GLUT4) were studied. In vitro phosphorylation of membranes in the presence of ATP and protein kinase A produced a stimulation of the reconstituted glucose transport activity in plasma membranes and low-density microsomes (51% and 65% stimulation respectively), provided that the cells had been treated with insulin prior to isolation of the membranes. Conversely, treatment of membrane fractions with alkaline phosphatase produced an inhibition of reconstituted transport activity. However, in vitro phosphorylation catalysed by protein kinase C failed to alter reconstituted glucose transport activity in membrane fractions from both basal and insulin-treated cells. In experiments run under identical conditions, the phosphorylation state of GLUT4 was investigated by immunoprecipitation of glucose transporters from membrane fractions incubated with [32P]ATP and protein kinases A and C. Protein kinase C stimulated a marked phosphate incorporation into GLUT4 in both plasma membranes and low-density microsomes. Protein kinase A, in contrast to its effect on reconstituted glucose transport activity, produced a much smaller phosphorylation of the GLUT4 in plasma membranes than in low-density microsomes. The present data suggest that glucose transport activity can be modified by protein phosphorylation via an insulin-dependent mechanism. However, the phosphorylation of the GLUT4 itself was not correlated with changes in its reconstituted transport activity.
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PMID:Phosphorylation of the adipose/muscle-type glucose transporter (GLUT4) and its relationship to glucose transport activity. 163 3

The acute effects of insulin, adenosine, and isoproterenol on the activity, subcellular distribution, and phosphorylation state of the GLUT4 glucose transporter isoform were investigated in rat adipocytes under conditions carefully controlled to monitor changes in cAMP-dependent protein kinase (A-kinase) activity. In contrast to GLUT1, which has not been shown to be phosphorylated even when cells are exposed to any of the above agents, GLUT4 was partially phosphorylated (0.1-0.2 mol/mol) when the activity of the A-kinase was suppressed, and remained unchanged in response to insulin. Isoproterenol elicited a 64% inhibition of insulin-stimulated glucose transport activity in the absence, but not the presence, of adenosine receptor agonists. However, in either the presence or the absence of agonists, A-kinase was activated as assessed by examining the phosphorylation of the major adipocyte A-kinase substrate, perilipin. Similarly, under either condition, phosphorylation of GLUT4 was enhanced 1.4-fold in the intracellular membranes, but no significant change was observed in the plasma membrane. In the absence of adenosine receptor agonists, isoproterenol exerted a small (14%) but significant inhibition of the insulin-induced translocation of GLUT4 but had no effect on the translocation of GLUT1. Thus, changes in the phosphorylation state and/or subcellular distribution of GLUT4 cannot account for the inhibition of insulin-stimulated glucose activity induced by isoproterenol.
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PMID:Phosphorylation state of the GLUT4 isoform of the glucose transporter in subfractions of the rat adipose cell: effects of insulin, adenosine, and isoproterenol. 176 64

The long-term hypoglycemic activity of sulphonylurea drugs has been attributed, in part at least, to the stimulation of glucose utilization in extra-pancreatic tissues. The novel sulphonylurea, glimepiride, gives rise to a longer lasting reduction in the blood sugar level in dogs and rabbits compared to glibenclamide (Geisen K, Drug Res 38: 1120-1130, 1988). This cannot be explained adequately by elevated plasma insulin levels. This study investigated whether this prolonged hypoglycemic phase was based on the drug's abilities to stimulate glucose utilization and affect the underlying regulatory mechanisms in insulin-sensitive cells in vitro. It was found that in the absence of added insulin, glimepiride and glibenclamide (1-50 microM) stimulated lipogenesis (3T3 adipocytes) and glycogenesis (isolated rat diaphragm) approximately 4.5- and 2.5-fold, respectively, and reduced the isoproterenol-stimulated lipolysis (rat adipocytes) up to 40-60%. The increased glucose utilization was correlated with a 3-4-fold higher 2-deoxyglucose transport rate and amount of GLUT4 at the plasma membrane, as well as with increased activities of key metabolic enzymes (glycerol-3-phosphate acyltransferase, glycogen synthase) within the same concentration range. Furthermore, the low Km cAMP-specific phosphodiesterase was activated 1.8-fold, whereas the cytosolic cAMP level and protein kinase A activity ratios were significantly lowered after incubation of isoproterenol-stimulated rat adipocytes with the sulphonylureas. In many of the aspects studied the novel sulphonylurea, glimepride, exhibited slightly lower ED50-values than glibenclamide. This study demonstrates correlations existing between drug-induced stimulation of glucose transport/metabolism and cAMP degradation/protein kinase A inhibition as well as between the relative efficiencies of glimepiride and glibenclamide in inducing these extra-pancreatic processes. Therefore, it is suggested that the stimulation of glucose utilization by sulphonylureas is mediated by a decrease of cAMP-dependent phosphorylation of GLUT4 and glucose metabolizing enzymes. The therapeutic relevance of extra-pancreatic effects of sulphonylureas, in general, and of the differences between glimepiride and glibenclamide as observed in vitro in this work, in particular, remain to be elucidated.
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PMID:Stimulation of glucose utilization in 3T3 adipocytes and rat diaphragm in vitro by the sulphonylureas, glimepiride and glibenclamide, is correlated with modulations of the cAMP regulatory cascade. 809 11

Downstream mediators of insulin signaling are thought to include multiple cytoplasmic serine/threonine kinases such as the product of the cellular proto-oncogene c-raf-1. To investigate a role for the Raf-1 protein kinase in insulin-stimulated glucose transport, a gene encoding an oncogenically activated Raf-1 mutant was introduced into 3T3-L1 fibroblasts by retroviral gene transfer. Expression of activated Raf-1 in differentiated 3T3-L1 adipocytes markedly increases hexose uptake compared to control adipocytes or those infected with the retroviral vector. Basal 2-deoxyglucose uptake in adipocytes expressing activated Raf-1 is approximately 40-fold higher than in parental adipocytes, and insulin further increases uptake about 1.2-fold. As determined by the plasma membrane "sheet" assay, Raf-1-expressing adipocytes contain greatly elevated levels of the ubiquitous glucose transporter (GLUT1) on the cell surface in the absence or presence of insulin. Total cellular GLUT1 protein is increased about 5-fold. In contrast, activated Raf-1 affects neither the expression of the "insulin-responsive" glucose transporter (GLUT4) nor its cellular distribution; GLUT4 is virtually undetectable on the plasma membrane in the absence of insulin and translocates normally following the addition of hormone. These data suggest that activation of Raf-1 mediates the chronic effect of insulin on hexose uptake but is not sufficient for the rapid translocation of GLUT4. Moreover, the differential effects of activated Raf-1 expression on the two transporter isoforms define divergent signaling pathways by which insulin regulates glucose transport in cultured adipocytes.
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PMID:A role for Raf-1 in the divergent signaling pathways mediating insulin-stimulated glucose transport. 814 13

To investigate the mechanism responsible for the inhibition of glucose transport by dibutyryl cAMP (Bt2cAMP), two different transporter isoforms (GLUT1 and GLUT4) and several GLUT1/4 chimeric transporters were expressed in Chinese hamster ovary (CHO) cells by using a Sindbis virus expression system. Bt2cAMP inhibited GLUT4-mediated 2-deoxy[3H]glucose (2DOG) uptake by 50% but was without effect on GLUT1-mediated uptake. When the subcellular distribution of GLUT4 was assessed by quantitative immunocytochemistry, neither the overall concentration of GLUT4 nor the regional distribution of GLUT-4 within the plasma membrane was found to be altered by Bt2cAMP. Thus, inhibition of 2DOG uptake by Bt2cAMP appears to be due to a decrease in transporter activity rather than a decrease in the number of transporters exposed at the plasma membrane. By using chimeric transporters, a region of GLUT4 necessary for the inhibitory effect of Bt2cAMP was localized to the last 29 amino acids in the COOH terminus. This intracellular region contains the site (Ser488) phosphorylated in vitro by cAMP-dependent protein kinase (cAdPK). Changing Ser488 to an Ala abolished phosphorylation of GLUT4; however, the inhibitory effect of Bt2cAMP on glucose transport was not diminished by this mutation. Therefore, phosphorylation of GLUT4 was not required for the inhibition. The effects of other nucleotides on GLUT4 transport activity were assessed to investigate the role of cAdPK. Uptake of 2DOG by GLUT4 was inhibited by 8-bromo-AMP, but not by 8-bromo-cAMP, suggesting that the inhibitory effect did not involve activation of cAdPK. Results consistent with this interpretation were obtained with CHO cells (line 10248), which express a cAdPK that is resistant to activation by cAMP. No difference in the concentrations of Bt2cAMP required to inhibit GLUT4-mediated transport was observed in normal CHO cells and 10248 cells. The results presented suggest that the inhibitory effects of Bt2cAMP could be mediated by direct binding of a nucleotide to GLUT4 at a site involving the intracellular COOH terminus of the transporter.
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PMID:GLUT4 phosphorylation and inhibition of glucose transport by dibutyryl cAMP. 839 69

We examined the question of whether insulin activates protein kinase C (PKC)-zeta in L6 myotubes, and the dependence of this activation on phosphatidylinositol (PI) 3-kinase. We also evaluated a number of issues that are relevant to the question of whether diacylglycerol (DAG)-dependent PKCs or DAG-insensitive PKCs, such as PKC-zeta, are more likely to play a role in insulin-stimulated glucose transport in L6 myotubes and other insulin-sensitive cell types. We found that insulin increased the enzyme activity of immunoprecipitable PKC-zeta in L6 myotubes, and this effect was blocked by PI 3-kinase inhibitors, wortmannin and LY294002; this suggested that PKC-zeta operates downstream of PI 3-kinase during insulin action. We also found that treatment of L6 myotubes with 5 microM tetradecanoyl phorbol-13-acetate (TPA) for 24 h led to 80-100% losses of all DAG-dependent PKCs (alpha, beta1, beta2, delta, epsilon) and TPA-stimulated glucose transport (2-deoxyglucose uptake); in contrast, there was full retention of PKC-zeta, as well as insulin-stimulated glucose transport and translocation of GLUT4 and GLUT1 to the plasma membrane. Unlike what has been reported in BC3H-1 myocytes, TPA treatment did not elicit increases in PKCbeta2 messenger RNA or protein in L6 myotubes, and selective retention of this PKC isoform could not explain the retention of insulin effects on glucose transport after prolonged TPA treatment. Of further interest, TPA acutely activated membrane-associated PI 3-kinase in L6 myotubes, and acute effects of TPA on glucose transport were inhibited, not only by the PKC inhibitor, LY379196, but also by both wortmannin and LY294002; this suggested that DAG-sensitive PKCs activate glucose transport through cross-talk with phosphatidylinositol (PI) 3-kinase, rather than directly through PKC. Also, the cell-permeable, myristoylated PKC-zeta pseudosubstrate inhibited insulin-stimulated glucose transport both in non-down-regulated and PKC-depleted (TPA-treated) L6 myotubes; thus, the PKC-zeta pseudosubstrate appeared to inhibit a protein kinase that is required for insulin-stimulated glucose transport but is distinct from DAG-sensitive PKCs. In keeping with the latter dissociation of DAG-sensitive PKCs and insulin-stimulated glucose transport, LY379196, which inhibits PKC-beta (preferentially) and other DAG-sensitive PKCs at relatively low concentrations, inhibited insulin-stimulated glucose transport only at much higher concentrations, not only in L6 myotubes, but also in rat adipocytes, BC3H-1 myocytes, 3T3/L1 adipocytes and rat soleus muscles. Finally, stable and transient expression of a kinase-inactive PKC-zeta inhibited basal and insulin-stimulated glucose transport in L6 myotubes. Collectively, our findings suggest that, whereas PKC-zeta is a reasonable candidate to participate in insulin stimulation of glucose transport, DAG-sensitive PKCs are unlikely participants.
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PMID:Evidence for involvement of protein kinase C (PKC)-zeta and noninvolvement of diacylglycerol-sensitive PKCs in insulin-stimulated glucose transport in L6 myotubes. 934 99

Electroporation of rat adipocytes with guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) elicited sizable insulin-like increases in glucose transport and GLUT4 translocation. Like insulin, GTPgammaS activated membrane phosphatidylinositol (PI) 3-kinase in rat adipocytes, but, unlike insulin, this activation was blocked by Clostridium botulinum C3 transferase, suggesting a requirement for the small G-protein, RhoA. Also suggesting that Rho may operate upstream of PI 3-kinase during GTPgammaS action, the stable overexpression of Rho in 3T3/L1 adipocytes provoked increases in membrane PI 3-kinase activity. As with insulin treatment, GTPgammaS stimulation of glucose transport in rat adipocytes was blocked by C3 transferase, wortmannin, LY294002, and RO 31-8220; accordingly, the activation of glucose transport by GTPgammaS, as well as insulin, appeared to require Rho, PI 3-kinase, and another downstream kinase, e.g. protein kinase C-zeta (PKC-zeta) and/or protein kinase N (PKN). Whereas insulin activated both PKN and PKC-zeta, GTPgammaS activated PKN but not PKC-zeta. In transfection studies in 3T3/L1 cells, stable expression of wild-type Rho and PKN activated glucose transport, and dominant-negative forms of Rho and PKN inhibited insulin-stimulated glucose transport. In transfection studies in rat adipocytes, transient expression of wild-type and constitutive Rho and wild-type PKN provoked increases in the translocation of hemagglutinin (HA)-tagged GLUT4 to the plasma membrane; in contrast, transient expression of dominant-negative forms of Rho and PKN inhibited the effects of both insulin and GTPgammaS on HA-GLUT4 translocation. Our findings suggest that (a) GTPgammaS and insulin activate Rho, PI 3-kinase, and PKN, albeit by different mechanisms; (b) each of these signaling substances appears to be required for, and may contribute to, increases in glucose transport; and (c) PKC-zeta may contribute to increases in glucose transport during insulin, but not GTPgammaS, action.
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PMID:Comparative effects of GTPgammaS and insulin on the activation of Rho, phosphatidylinositol 3-kinase, and protein kinase N in rat adipocytes. Relationship to glucose transport. 951 46

Previous studies have indicated a role for calmodulin in hypoxia-and insulin-stimulated glucose transport. However, since calmodulin interacts with multiple protein targets, it is unknown which of these targets is involved in the regulation of glucose transport. In the present study, we have used the calcium-dependent calmodulin protein kinase II (CAMKII) inhibitor 1-[N, O-bis-(5-isoquinolinesulphonyl) -N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62) to investigate the possible role of this enzyme in the regulation of glucose transport in isolated rat soleus and epitrochlearis muscles. KN-62 did not affect basal 2-deoxyglucose transport, but it did inhibit both insulin- and hypoxia-stimulated glucose transport activity by 46 and 40% respectively. 1-[N,O-Bis-(1, 5-isoquinolinesulphonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine (KN-04), a structural analogue of KN-62 that does not inhibit CAMKII, had no effect on hypoxia-or insulin-stimulated glucose transport. Accordingly, KN-62 decreased the stimulated cell-surface GLUT4 labelling by a similar extent as the inhibition of glucose transport (insulin, 49% and hypoxia, 54%). Additional experiments showed that KN-62 also inhibited insulin- and hypoxia-stimulated transport by 37 and 40% respectively in isolated rat epitrochlearis (a fast-twitch muscle), indicating that the effect of KN-62 was not limited to the slow-twitch fibres of the soleus. The inhibitory effect of KN-62 on hypoxia-stimulated glucose transport appears to be specific to CAMKII, since KN-62 did not inhibit hypoxia-stimulated 45Ca efflux from muscles pre-loaded with 45Ca, or hypoxia-stimulated glycogen breakdown. Additionally, KN-62 affected neither insulin-stimulated phosphoinositide 3-kinase nor Akt activity, suggesting that the effects of KN-62 are not due to non-specific effects of this inhibitor on these regions of the insulin-signalling cascade. The results of the present study suggest that CAMKII might have a distinct role in insulin- and hypoxia-stimulated glucose transport, possibly in the vesicular trafficking of GLUT4.
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PMID:1-[N, O-bis-(5-isoquinolinesulphonyl)-N-methyl-L-tyrosyl]-4- phenylpiperazine (KN-62), an inhibitor of calcium-dependent camodulin protein kinase II, inhibits both insulin- and hypoxia-stimulated glucose transport in skeletal muscle. 1021 90

To characterize the contribution of glycogen synthase kinase 3beta (GSK3beta) inactivation to insulin-stimulated glucose metabolism, wild-type (WT-GSK), catalytically inactive (KM-GSK), and uninhibitable (S9A-GSK) forms of GSK3beta were expressed in insulin-responsive 3T3-L1 adipocytes using adenovirus technology. WT-GSK, but not KM-GSK, reduced basal and insulin-stimulated glycogen synthase activity without affecting the -fold stimulation of the enzyme by insulin. S9A-GSK similarly decreased cellular glycogen synthase activity, but also partially blocked insulin stimulation of the enzyme. S9A-GSK expression also markedly inhibited insulin stimulation of IRS-1-associated phosphatidylinositol 3-kinase activity, but only weakly inhibited insulin-stimulated Akt/PKB phosphorylation and glucose uptake, with no effect on GLUT4 translocation. To further evaluate the role of GSK3beta in insulin signaling, the GSK3beta inhibitor lithium was used to mimic the consequences of insulin-stimulated GSK3beta inactivation. Although lithium stimulated the incorporation of glucose into glycogen and glycogen synthase enzyme activity, the inhibitor was without effect on GLUT4 translocation and pp70 S6 kinase. Lithium stimulation of glycogen synthesis was insensitive to wortmannin, which is consistent with its acting directly on GSK3beta downstream of phosphatidylinositol 3-kinase. These data support the hypothesis that GSK3beta contributes to insulin regulation of glycogen synthesis, but is not responsible for the increase in glucose transport.
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PMID:The role of glycogen synthase kinase 3beta in insulin-stimulated glucose metabolism. 1036 40

Chronic hyperglycemia causes insulin resistance, termed glucose toxicity. Herein we studied chronic glucose-dependent regulation of the glucose transport system in adipocytes. 3T3-L1 adipocytes were incubated for up to 24 h with low (1 mM) or high (25 mM) glucose, and glucose transport was subsequently analyzed. 100 nM insulin was present throughout the experiments. 24 h incubation with 1 mM glucose caused a 2.3+/-0.4 fold increase in glucose transport activity, compared to the values obtained with 25 mM glucose. This difference was not observed when 24 h incubation was carried out without insulin. Glucose transport activity was not increased at 3 or 6 h incubation with 1 mM glucose, but was increased at 12 h, which closely paralleled increased expression of GLUT1. In addition to increased GLUT1 expression, more efficient translocation of GLUT1 to the plasma membrane was observed when incubated with 1 mM glucose compared to 25 mM glucose. The addition of azaserin or deprivation of glutamine at 25 mM glucose did not increase the glucose transport activity to the level obtained with 1 mM glucose. PD98059 did not affect glucose transport activity when incubated with 1 mM or 25 mM glucose. In conclusion, the present study is the first to show that, in 3T3-L1 adipocytes, chronic exposure to low (1 mM) and high (25 mM) glucose leads to different insulin-stimulated glucose transport activities. These differences result from the difference in the expression and plasma membrane distribution of GLUT1, but not of GLUT4, and the hexosamine biosynthesis pathway or extracellular signal-regulated protein kinase is not involved.
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PMID:Regulation of insulin-stimulated glucose transport by chronic glucose exposure in 3T3-L1 adipocytes. 1050 86


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