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 inhibition or constitutive activation of glycogen synthase kinase-3 (GSK3) on glycogen synthase (GS) activity, abundance, and glycogen deposition in L6 rat skeletal muscle cells were investigated. GS protein expression increased approximately 5-fold during differentiation of L6 cells (comparing cells at the end of day 5 with those at the beginning of day 3). However, exposure of undifferentiated myoblasts (day 3) to 50 microM SB-415286, a GSK3 inhibitor, led to a significant elevation in GS protein that was not accompanied by changes in the abundance of GLUT4, another late differentiation marker. In contrast, stable expression of a constitutively active form of GSK3beta (GSK3S9A) led to a significant reduction (approximately 80%) in GS protein that was antagonized by SB-415286. Inhibition of GSK3 or expression of the constitutively active GSK3S9A did not result in any detectable changes in GS mRNA abundance. However, the increase in GS protein in undifferentiated myoblasts or that seen following incubation of cells expressing GSK3S9A with GSK3 inhibitors was blocked by cycloheximide suggesting that GSK3 influences GS abundance possibly via control of mRNA translation. Consistent with the reduction in GS protein, cells expressing GSK3S9A were severely glycogen depleted as judged using a specific glycogen-staining antibody. Inhibiting GSK3 in wild-type or GSK3S9A-expressing cells using SB-415286 resulted in an attendant activation of GS, but not that of glucose transport. However, GS activation alone was insufficient for stimulating glycogen deposition. Only when muscle cells were incubated simultaneously with insulin and SB-415286 or with lithium (which stimulates GS and glucose transport) was an increase in glycogen accretion observed. Our findings suggest that GSK3 activity is an important determinant of GS protein expression and that while glycogen deposition in muscle cells is inherently dependent upon the activity/expression of GS, glucose transport is a key rate-determining step in this process.
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PMID:Constitutive activation of GSK3 down-regulates glycogen synthase abundance and glycogen deposition in rat skeletal muscle cells. 1563 69

PKCzeta (protein kinase Czeta) is a serine/threonine protein kinase controlled by insulin, various growth factors and phosphoinositide 3-kinase. It has been implicated in controlling glucose transport in response to insulin by the translocation of GLUT4-(glucose transporter 4) containing vesicles to the plasma membrane in stimulated cells. How PKCzeta modulates GLUT4 vesicle trafficking remains unknown. A yeast two-hybrid screen using full-length human PKCzeta identified 80K-H protein as an interactor with PKCzeta. GST (glutathione S-transferase) pull-down assays with GST-tagged 80K-H constructs confirmed the interaction and showed that the N-terminal portion of 80K-H was not required for the interaction. Immunoprecipitates of endogenous PKCzeta from Cho cells, 3T3-L1 adipocytes or L6 myotubes contained endogenous 80K-H, demonstrating a physiological interaction. Insulin stimulation enhanced the association 3-5-fold. Immunoprecipitates of endogenous 80K-H contained endogenous munc18c and immunoprecipitates of endogenous munc18c contained endogenous PKCzeta, with insulin markedly increasing the amount of co-immunoprecipitated protein in each case. These results show that insulin triggers interactions in vivo between PKCzeta, 80K-H and munc18c. Overexpression of 80K-H constructs mimicked the action of insulin in stimulating both glucose uptake and translocation of Myc-tagged GLUT4 in Cho cells, with the level of effect proportional to the ability of the constructs to associate with munc18c. These results identify 80K-H as a new player involved in GLUT4 vesicle transport and identify a link between a kinase involved in the insulin signalling cascade, PKCzeta, and a known component of the GLUT4 vesicle trafficking pathway, munc18c. The results suggest a model whereby insulin triggers the formation of a PKCzeta-80K-H-munc18c complex that enhances GLUT4 translocation to the plasma membrane.
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PMID:Identification of 80K-H as a protein involved in GLUT4 vesicle trafficking. 1570 89

Exercise induces a rapid increase in expression of the GLUT4 isoform of the glucose transporter in skeletal muscle. One of the signals responsible for this adaptation appears to be an increase in cytosolic Ca(2+). Myocyte enhancer factor 2A (MEF2A) is a transcription factor that is involved in the regulation of GLUT4 expression. It has been reported that the Ca(2+)-regulated phosphatase calcineurin mediates the activation of MEF2 by exercise. It has also been shown that the expression of activated calcineurin in mouse skeletal muscle results in an increase in GLUT4. These findings suggest that increases in cytosolic Ca(2+) induce increased GLUT4 expression by activating calcineurin. However, we have obtained evidence that this response is mediated by a Ca(2+)-calmodulin-dependent protein kinase. The purpose of this study was to test the hypothesis that calcineurin is involved in mediating exercise-induced increases in GLUT4. Rats were exercised on 5 successive days using a swimming protocol. One group of swimmers was given 20 mg/kg body weight of cyclosporin, a calcineurin inhibitor, 2 h before exercise. A second group was given vehicle. GLUT4 protein was increased approximately 80%, GLUT4 mRNA was increased approximately 2.5-fold, MEF2A protein was increased twofold, and hexokinase II protein was increased approximately 2.5-fold 18 h after the last exercise bout. The cyclosporin treatment completely inhibited calcineurin activity but did not affect the adaptive increases in GLUT4, MEF2A, or hexokinase expression. We conclude that calcineurin activation does not mediate the adaptive increase in GLUT4 expression induced in skeletal muscle by exercise.
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PMID:Calcineurin does not mediate exercise-induced increase in muscle GLUT4. 1573 36

We investigated the role of the kallikrein-kinin system in cardiac function and glucose utilization in the streptozotocin (STZ)-induced diabetic rat model using a gene transfer approach. Adenovirus harboring the human tissue kallikrein gene was administered to rats by intravenous injection at 1 week after STZ treatment. Human kallikrein transgene expression was detected in the serum and urine of STZ-induced diabetic rats after gene transfer. Kallikrein gene delivery significantly reduced blood glucose levels and cardiac glycogen accumulation in STZ-induced diabetic rats. Kallikrein gene transfer also significantly attenuated elevated plasma triglyceride and cholesterol levels, food and water intake, and loss of body weight gain, epididymal fat pad, and gastrocnemius muscle weight in STZ-induced diabetic rats. However, these effects were blocked by icatibant, a kinin B2 receptor antagonist. Cardiac function was significantly improved after kallikrein gene transfer as evidenced by increased cardiac output and +/-delta P/delta t (maximum speed of contraction/relaxation), along with elevated cardiac sarco(endo)plasmic reticulum (Ca2+ + Mg2+)-ATPase (SERCA)-2a, phosphorylated phospholamban, NOx and cAMP levels, and GLUT4 translocation into plasma membranes of cardiac and skeletal muscle. Kallikrein gene delivery also increased Akt and glycogen synthase kinase (GSK)-3beta phosphorylation, resulting in decreased GSK-3beta activity in the heart. These results indicate that kallikrein through kinin formation protects against diabetic cardiomyopathy by improving cardiac function and promoting glucose utilization and lipid metabolism.
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PMID:Kallikrein gene delivery improves serum glucose and lipid profiles and cardiac function in streptozotocin-induced diabetic rats. 1585 48

Insulin causes the rapid translocation of the glucose transporter GLUT4 from intracellular sites to the plasma membrane in fat and muscle cells. There is considerable evidence that the signaling to this trafficking process is downstream of the insulin-activated protein kinase Akt. One Akt substrate that connects signaling to trafficking is a 160 kDa GTPase activating protein for Rabs. Another potential connecting substrate is the protein Synip, which associates with the SNARE syntaxin4. A recent study presents evidence that Akt phosphorylates Synip on serine 99, at least in vitro, and proposes that this phosphorylation enables GLUT4 translocation by causing the dissociation of Synip from syntaxin4. In the present study we show that marked overexpression of Synip mutant S99A, which lacks this phosphorylation site, has no effect on insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes. This finding is strong evidence that phosphorylation of Synip on serine 99 is not required for GLUT4 translocation.
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PMID:Synip phosphorylation does not regulate insulin-stimulated GLUT4 translocation. 1591 52

AS160 is a newly described substrate for the protein kinase Akt that links insulin signaling and GLUT4 trafficking. In this study, we determined the expression of and in vivo insulin action on AS160 in human skeletal muscle. In addition, we compared the effect of physiological hyperinsulinemia on AS160 phosphorylation in 10 lean-to-moderately obese type 2 diabetic and 9 healthy subjects. Insulin infusion increased the phosphorylation of several proteins reacting with a phospho-Akt substrate antibody. We focused on AS160, as this Akt substrate has been linked to glucose transport. A 160-kDa phosphorylated protein was identified as AS160 by immunoblot analysis with an AS160-specific antibody. Physiological hyperinsulinemia increased AS160 phosphorylation 2.9-fold in skeletal muscle of control subjects (P < 0.001). Insulin-stimulated AS160 phosphorylation was reduced 39% (P < 0.05) in type 2 diabetic patients. AS160 protein expression was similar in type 2 diabetic and control subjects. Impaired AS160 phosphorylation was related to aberrant Akt signaling; insulin action on Akt Ser(473) phosphorylation was not significantly reduced in type 2 diabetic compared with control subjects, whereas Thr(308) phosphorylation was impaired 51% (P < 0.05). In conclusion, physiological hyperinsulinemia increases AS160 phosphorylation in human skeletal muscle. Moreover, defects in insulin action on AS160 may impair GLUT4 trafficking in type 2 diabetes.
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PMID:Insulin-stimulated phosphorylation of the Akt substrate AS160 is impaired in skeletal muscle of type 2 diabetic subjects. 1591 90

Recently, we described a 160 kDa protein (designated AS160, for Akt substrate of 160 kDa) with a predicted Rab GAP (GTPase-activating protein) domain that is phosphorylated on multiple sites by the protein kinase Akt. Phosphorylation of AS160 in adipocytes is required for insulin-stimulated translocation of the glucose transporter GLUT4 to the plasma membrane. The aim of the present study was to determine whether AS160 is in fact a GAP for Rabs, and, if so, what its specificity is. We first identified a group of 16 Rabs in a preparation of intracellular vesicles containing GLUT4 by MS. We then prepared the recombinant GAP domain of AS160 and examined its activity against many of these Rabs, as well as several others. The GAP domain was active against Rabs 2A, 8A, 10 and 14. There was no significant activity against 14 other Rabs. GAP activity was further validated by the finding that the recombinant GAP domain with the predicted catalytic arginine residue replaced by lysine was inactive. Finally, it was found by immunoblotting that Rabs 2A, 8A and 14 are present in GLUT4 vesicles. These results indicate that AS160 is a Rab GAP, and suggest novel Rabs that may participate in GLUT4 translocation.
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PMID:AS160, the Akt substrate regulating GLUT4 translocation, has a functional Rab GTPase-activating protein domain. 1597 98

The increase in skeletal muscle glucose uptake during exercise results from a coordinated increase in rates of glucose delivery (higher capillary perfusion), surface membrane glucose transport, and intracellular substrate flux through glycolysis. The mechanism behind the movement of GLUT4 to surface membranes and the subsequent increase in transport by muscle contractions is largely unresolved, but it is likely to occur through intracellular signaling involving Ca(2+)-calmodulin-dependent protein kinase, 5'-AMP-activated protein kinase, and possibly protein kinase C.
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PMID:Skeletal muscle glucose uptake during exercise: how is it regulated? 1602 14

Recently, we described a 160 kDa protein with a Rab GTPase activating protein domain that is phosphorylated on multiple sites by the protein kinase Akt (designated AS160). Phosphorylation of AS160 in adipocytes is required for insulin-stimulated translocation of the glucose transporter GLUT4 to the plasma membrane. In the present study, we searched for proteins that interact with the GTPase activating protein (GAP) domain region of AS160 by the yeast two-hybrid screen. This search indicated that calmodulin bound to a small domain just amino terminal to the GAP domain of AS160, and this association has been confirmed by three other methods, including co-immunoprecipitation from lysates of adipocytes. The association was Ca ion dependent. The role of calmodulin binding to AS160 in insulin-stimulated GLUT4 translocation was examined through the generation of a point mutant of AS160 that did not bind calmodulin. This mutation did not interfere with the capacity of AS160 lacking Akt phosphorylation sites to inhibit GLUT4 translocation. Consequently, calmodulin binding is probably not required for the participation of AS160 in insulin-stimulated GLUT4 translocation.
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PMID:Calmodulin binds to the Rab GTPase activating protein required for insulin-stimulated GLUT4 translocation. 1605 84

AMP-activated protein kinase (AMPK) promotes glucose transport, maintains ATP stores, and prevents injury and apoptosis during ischemia. AMPK has several direct molecular targets in the heart but also may interact with other stress-signaling pathways. This study examined the role of AMPK in the activation of the p38 mitogen-activated protein kinase (MAPK). In isolated heart muscles, the AMPK activator 5-aminoimidazole-4-carboxy-amide-1-beta-D-ribofuranoside (AICAR) increased p38 MAPK activation. In AMPK-deficient mouse hearts, expressing a kinase-dead (KD) alpha2 catalytic subunit, p38 MAPK activation was markedly reduced during low-flow ischemia (2.3- versus 7-fold in wild-type hearts, P<0.01) and was similarly reduced during severe no-flow ischemia in KD hearts (P<0.01 versus ischemic wild type). Knockout of the p38 MAPK upstream kinase, MAPK kinase 3 (MKK3), did not affect ischemic activation of either AMPK or p38 MAPK in transgenic mkk3(-/-) mouse hearts. Ischemia increased p38 MAPK recruitment to transforming growth factor-beta-activated protein kinase 1-binding protein 1 (TAB1), a scaffold protein that promotes p38 MAPK autophosphorylation. Moreover, TAB1 was associated with the alpha2 catalytic subunit of AMPK. p38 MAPK recruitment to TAB1/AMPK complexes required AMPK activation and was reduced in ischemic AMPK-deficient transgenic mouse hearts. The potential role of p38 MAPK in mediating the downstream action of AMPK to promote glucose transport was also assessed. The p38 MAPK inhibitor SB203580 partially inhibited both AICAR- and hypoxia-stimulated glucose uptake and GLUT4 translocation. Activation of p38 MAPK by anisomycin also increased glucose transport in heart muscles. Thus, AMPK has an important role in promoting p38 MAPK activation in the ischemic heart by inducing p38 MAPK autophosphorylation through interaction with the scaffold protein TAB1.
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PMID:AMP-activated protein kinase activates p38 mitogen-activated protein kinase by increasing recruitment of p38 MAPK to TAB1 in the ischemic heart. 1617 88


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