Document Detail

Functional involvement of protein kinase C-betaII and its substrate, myristoylated alanine-rich C-kinase substrate (MARCKS), in insulin-stimulated glucose transport in L6 rat skeletal muscle cells.
MedLine Citation:
PMID:  19252893     Owner:  NLM     Status:  MEDLINE    
AIMS/HYPOTHESIS: Insulin stimulates phosphorylation cascades, including phosphatidylinositol-3-kinase (PI3K), phosphatidylinositol-dependent kinase (PDK1), Akt, and protein kinase C (PKC). Myristoylated alanine-rich C-kinase substrate (MARCKS), a PKCbetaII substrate, could link the effects of insulin to insulin-stimulated glucose transport (ISGT) via phosphorylation of its effector domain since MARCKS has a role in cytoskeletal rearrangements.
METHODS: We examined phosphoPKCbetaII after insulin treatment of L6 myocytes, and cytosolic and membrane phosphoMARCKS, MARCKS and phospholipase D1 in cells pretreated with LY294002 (PI3K inhibitor), CG53353 (PKCbetaII inhibitor) or W13 (calmodulin inhibitor), PI3K, PKCbetaII and calmodulin inhibitors, respectively, before insulin treatment, using western blots. ISGT was examined after cells had been treated with inhibitors, small inhibitory RNA (siRNA) for MARCKS, or transfection with MARCKS mutated at a PKC site. MARCKS, PKCbetaII, GLUT4 and insulin receptor were immunoblotted in subcellular fractions with F-actin antibody immunoprecipitates to demonstrate changes following insulin treatment. GLUT4 membrane insertion was followed after insulin with or without CG53353.
RESULTS: Insulin increased phosphoPKCbetaII(Ser660 and Thr641); LY294002 blocked this, indicating its activation by PI3K. Insulin treatment increased cytosolic phosphoMARCKS, decreased membrane MARCKS and increased membrane phospholipase D1 (PLD1), a protein regulating glucose transporter vesicle fusion resulted. PhosphoMARCKS was attenuated by CG53353 or MARCKS siRNA. MARCKS siRNA blocked ISGT. Association of PKCbetaII and GLUT4 with membrane F-actin was enhanced by insulin, as was that of cytosolic and membrane MARCKS. ISGT was attenuated in myocytes transfected with mutated MARCKS (Ser152Ala), whereas overproduction of wild-type MARCKS enhanced ISGT. CG53353 blocked insertion of GLUT4 into membranes of insulin treated cells.
CONCLUSIONS/INTERPRETATION: The results suggest that PKCbetaII is involved in mediating downstream steps of ISGT through MARCKS phosphorylation and cytoskeletal remodelling.
D S Chappell; N A Patel; K Jiang; P Li; J E Watson; D M Byers; D R Cooper
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.     Date:  2009-02-28
Journal Detail:
Title:  Diabetologia     Volume:  52     ISSN:  1432-0428     ISO Abbreviation:  Diabetologia     Publication Date:  2009 May 
Date Detail:
Created Date:  2009-04-06     Completed Date:  2009-07-29     Revised Date:  2014-09-14    
Medline Journal Info:
Nlm Unique ID:  0006777     Medline TA:  Diabetologia     Country:  Germany    
Other Details:
Languages:  eng     Pagination:  901-11     Citation Subset:  IM    
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MeSH Terms
Cell Differentiation
Chromones / pharmacology
DNA, Complementary / genetics
Deoxyglucose / metabolism
Glucose / metabolism*
Insulin / pharmacology*
Intracellular Signaling Peptides and Proteins / genetics,  metabolism*
Membrane Proteins / genetics,  metabolism*
Morpholines / pharmacology
Muscle, Skeletal / drug effects,  enzymology,  metabolism*
Myoblasts / cytology,  drug effects,  enzymology,  metabolism
Phosphoserine / metabolism
Phosphothreonine / metabolism
Protein Kinase C / genetics,  metabolism*
Protein Kinase C beta
RNA, Small Interfering / genetics
Grant Support
Reg. No./Substance:
0/Chromones; 0/DNA, Complementary; 0/Insulin; 0/Intracellular Signaling Peptides and Proteins; 0/Membrane Proteins; 0/Morpholines; 0/RNA, Small Interfering; 1114-81-4/Phosphothreonine; 125267-21-2/myristoylated alanine-rich C kinase substrate; 154447-36-6/2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; 17885-08-4/Phosphoserine; 9G2MP84A8W/Deoxyglucose; EC Kinase C; EC Kinase C beta; IY9XDZ35W2/Glucose

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