Skip to main content
Key Specialties
Cardiology Diabetology Endocrinology Gastroenterology Geriatrics and Gerontology Gynecology and Obstetrics Hematology Infectious Disease Internal Medicine Nephrology Neurology Oncology Pediatrics Respiratory Medicine Rheumatology Surgery
Congress Coverage
2024 ACC Congress 2023 ESMO Congress 2023 EASD Congress 2023 ERS Congress 2023 ESC Congress
Clinical Collections
Advances in Alzheimer’s

Keynote webinar | Spotlight on medication adherence

LIVE: Thursday 27th June 2024, 18:00-19:30 (CEST)
Join our expert panel to discover why you need to understand the drivers of non-adherence in your patients, and how you can optimize medication adherence in your clinics to drastically improve patient outcomes.
ADVANCED SEARCH
Log in
Top
Diabetologia
Published in: Diabetologia 5/2009

01-05-2009 | Article

Functional involvement of protein kinase C-βII and its substrate, myristoylated alanine-rich C-kinase substrate (MARCKS), in insulin-stimulated glucose transport in L6 rat skeletal muscle cells

Authors: D. S. Chappell, N. A. Patel, K. Jiang, P. Li, J. E. Watson, D. M. Byers, D. R. Cooper

Published in: Diabetologia | Issue 5/2009

Login to get access

Abstract

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 PKCβII 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 phosphoPKCβII after insulin treatment of L6 myocytes, and cytosolic and membrane phosphoMARCKS, MARCKS and phospholipase D1 in cells pretreated with LY294002 (PI3K inhibitor), CG53353 (PKCβII inhibitor) or W13 (calmodulin inhibitor), PI3K, PKCβII 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, PKCβII, 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 phosphoPKCβII(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 PKCβII 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 PKCβII is involved in mediating downstream steps of ISGT through MARCKS phosphorylation and cytoskeletal remodelling.
Literature
1.
Osterhoff MA, Heuer S, Pfeiffer M et al (2008) Identification of a functional protein kinase C beta promoter polymorphism in humans related to insulin-resistance. Mol Genet Metab 93:210–215PubMedCrossRef
2.
Ikeda Y, Suehiro T, Osaki F, Tsuzura S, Kumon Y, Hashimoto K (2004) Polymorphisms in the 5′-upstream region of the PKCbeta gene in Japanese patients with Type 2 diabetes. Diabet Med 21:1113–1120PubMedCrossRef
3.
Araki S, Ng DP, Krolewski B et al (2003) Identification of a common risk haplotype for diabetic nephropathy at the protein kinase C-beta1 (PRKCB1) gene locus. J Am Soc Nephrol 14:2015–2024PubMedCrossRef
4.
Cortright RN, Azevedo JL Jr, Zhou Q et al (2000) Protein kinase C modulates insulin action in human skeletal muscle. Am J Physiol Endocrinol Metab 278:E553–562PubMed
5.
Sampson SR, Cooper DR (2006) Specific protein kinase C isoforms as transducers and modulators of insulin signaling. Mol Genet Metab 89:32–47PubMedCrossRef
6.
Pillay TS, Xiao S, Keranen L, Olefsky JM (2004) Regulation of the insulin receptor by protein kinase C isoenzymes: preferential interaction with beta isoenzymes and interaction with the catalytic domain of betaII. Cell Signal 16:97–104PubMedCrossRef
7.
Formisano P, Beguinot F (2001) The role of protein kinase C isoforms in insulin action. J Endocrinol Invest 24:460–467PubMed
8.
Newton AC (1995) Protein kinase C: structure, function, and regulation. J Biol Chem 270:28495–28498PubMedCrossRef
9.
10.
Chalfant CE, Mischak H, Watson JE et al (1995) Regulation of alternative splicing of protein kinase Cbeta by insulin. J Biol Chem 270:13326–13332PubMedCrossRef
11.
Chalfant CE, Watson JE, Bisnauth LD et al (1998) Insulin regulates protein kinase CbetaII expression through enhanced exon inclusion in L6 skeletal muscle cells. A novel mechanism of insulin- and insulin-like growth factor-i-induced 5′ splice site selection. J Biol Chem 273:910–916PubMedCrossRef
12.
Patel NA, Apostolatos HS, Mebert K et al (2004) Insulin regulates protein kinase CbetaII alternative splicing in multiple target tissues: development of a hormonally responsive heterologous minigene. Mol Endocrinol 18:899–911PubMedCrossRef
13.
Chalfant CE, Ohno S, Konno Y et al (1996) A carboxy-terminal deletion mutant of protein kinase C beta II inhibits insulin-stimulated 2-deoxyglucose uptake in L6 rat skeletal muscle cells. Mol Endocrinol 10:1273–1281PubMedCrossRef
14.
Arnold TP, Standaert ML, Hernandez H et al (1993) Effects of insulin and phorbol esters on MARCKS (myristoylated alanine-rich C-kinase substrate) phosphorylation (and other parameters of protein kinase C activation) in rat adipocytes, rat soleus muscle and BC3H-1 myocytes. Biochem J 295(Pt 1):155–164PubMed
15.
Arbuzova A, Schmitz AA, Vergeres G (2002) Cross-talk unfolded: MARCKS proteins. Biochem J 362:1–12PubMedCrossRef
16.
Yarmola EG, Edison AS, Lenox RH, Bubb MR (2001) Actin filament cross-linking by MARCKS: characterization of two actin-binding sites within the phosphorylation site domain. J Biol Chem 276:22351–22358PubMedCrossRef
17.
McLaughlin S, Wang J, Gambhir A, Murray D (2002) PIP(2) and proteins: interactions, organization, and information flow. Annu Rev Biophys Biomol Struct 31:151–175PubMedCrossRef
18.
Sundaram M, Cook HW, Byers DM (2004) The MARCKS family of phospholipid binding proteins: regulation of phospholipase D and other cellular components. Biochem Cell Biol 82:191–200PubMedCrossRef
19.
Yang H, Wang X, Sumners C, Raizada MK (2002) Obligatory role of protein kinase Cbeta and MARCKS in vesicular trafficking in living neurons. Hypertension 39:567–572PubMedCrossRef
20.
Frohman MA, Morris AJ (1999) Phospholipase D structure and regulation. Chem Phys Lipids 98:127–140PubMedCrossRef
21.
22.
Huang P, Altshuller YM, Hou JC, Pessin JE, Frohman MA (2005) Insulin-stimulated plasma membrane fusion of Glut4 glucose transporter-containing vesicles is regulated by phospholipase D1. Mol Biol Cell 16:2614–2623PubMedCrossRef
23.
Vlahos CJ, Matter WF, Hui KY, Brown RF (1994) A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J Biol Chem 269:5241–5248PubMed
24.
Wick MJ, Dong LQ, Riojas RA, Ramos FJ, Liu F (2000) Mechanism of phosphorylation of protein kinase B/Akt by a constitutively active 3-phosphoinositide-dependent protein kinase-1. J Biol Chem 275:40400–40406PubMedCrossRef
25.
Dong LQ, Liu F (2005) PDK2: the missing piece in the receptor tyrosine kinase signaling pathway puzzle. Am J Physiol Endocrinol Metab 289:E187–196PubMedCrossRef
26.
Buchdunger E, Mett H, Trinks U et al (1995) 4,5-bis(4-fluoroanilino)phthalimide: a selective inhibitor of the epidermal growth factor receptor signal transduction pathway with potent in vivo antitumor activity. Clin Cancer Res 1:813–821PubMed
27.
Morash SC, Douglas D, McMaster CR, Cook HW, Byers DM (2005) Expression of MARCKS effector domain mutants alters phospholipase D activity and cytoskeletal morphology of SK-N-MC neuroblastoma cells. Neurochem Res 30:1353–1364PubMedCrossRef
28.
Czech MP, Buxton JM (1993) Insulin action on the internalization of the GLUT4 glucose transporter in isolated rat adipocytes. J Biol Chem 268:9187–9190PubMed
29.
Toker A, Meyer M, Reddy KK et al (1994) Activation of protein kinase C family members by the novel polyphosphoinositides PtdIns-3,4-P2 and PtdIns-3,4,5-P3. J Biol Chem 269:32358–32367PubMed
30.
Yang C, Watson RT, Elmendorf JS, Sacks DB, Pessin JE (2000) Calmodulin antagonists inhibit insulin-stimulated GLUT4 (glucose transporter 4) translocation by preventing the formation of phosphatidylinositol 3,4,5-trisphosphate in 3T3L1 adipocytes. Mol Endocrinol 14:317–326PubMedCrossRef
31.
Arbuzova A, Murray D, McLaughlin S (1998) MARCKS, membranes, and calmodulin: kinetics of their interaction. Biochim Biophys Acta 1376:369–379PubMed
32.
Hartwig JH, Thelen M, Rosen A, Janmey PA, Nairn AC, Aderem A (1992) MARCKS is an actin filament crosslinking protein regulated by protein kinase C and calcium-calmodulin. Nature 356:618–622PubMedCrossRef
33.
Blackshear PJ (1993) The MARCKS family of cellular protein kinase C substrates. J Biol Chem 268:1501–1504PubMed
34.
Braiman L, Sheffi-Friedman L, Bak A, Tennenbaum T, Sampson SR (1999) Tyrosine phosphorylation of specific protein kinase C isoenzymes participates in insulin stimulation of glucose transport in primary cultures of rat skeletal muscle. Diabetes 48:1922–1929PubMedCrossRef
35.
Brozinick JT Jr., Berkemeier BA, Elmendorf JS (2007) “Actin”g on GLUT4: membrane & cytoskeletal components of insulin action. Curr Diabetes Rev 3:111–122PubMedCrossRef
36.
37.
Wright DC, Geiger PC, Rheinheimer MJ, Han DH, Holloszy JO (2004) Phorbol esters affect skeletal muscle glucose transport in a fiber type-specific manner. Am J Physiol Endocrinol Metab 287:E305–309PubMedCrossRef
38.
Song XM, Ryder JW, Kawano Y, Chibalin AV, Krook A, Zierath JR (1999) Muscle fiber type specificity in insulin signal transduction. Am J Physiol 277:R1690–1696PubMed
39.
Blobe GC, Stribling DS, Fabbro D, Stabel S, Hannun YA (1996) Protein kinase C beta II specifically binds to and is activated by F-actin. J Biol Chem 271:15823–15830PubMedCrossRef
40.
Uberall F, Giselbrecht S, Hellbert K et al (1997) Conventional PKC-alpha, novel PKC-epsilon and PKC-theta, but not atypical PKC-lambda are MARCKS kinases in intact NIH 3T3 fibroblasts. J Biol Chem 272:4072–4078PubMedCrossRef
41.
Simpson F, Whitehead JP, James DE (2001) GLUT4-at the cross roads between membrane trafficking and signal transduction. Traffic 2:2–11PubMedCrossRef
42.
Iannazzo L (2001) Involvement of B-50 (GAP-43) phosphorylation in the modulation of transmitter release by protein kinase C. Clin Exp Pharmacol Physiol 28:901–904PubMedCrossRef
43.
Baron W, de Vries EJ, de Vries H, Hoekstra D (1999) Protein kinase C prevents oligodendrocyte differentiation: modulation of actin cytoskeleton and cognate polarized membrane traffic. J Neurobiol 41:385–398PubMedCrossRef
44.
Wang J, Arbuzova A, Hangyas-Mihalyne G, McLaughlin S (2001) The effector domain of myristoylated alanine-rich C kinase substrate binds strongly to phosphatidylinositol 4,5-bisphosphate. J Biol Chem 276:5012–5019PubMedCrossRef
45.
Pershadsingh HA, Shade DL, Delfert DM, McDonald JM (1987) Chelation of intracellular calcium blocks insulin action in the adipocyte. Proc Natl Acad Sci USA 84:1025–1029PubMedCrossRef
46.
Whitehead JP, Molero JC, Clark S, Martin S, Meneilly G, James DE (2001) The role of Ca2+ in insulin-stimulated glucose transport in 3T3-L1 cells. J Biol Chem 276:27816–27824PubMedCrossRef
47.
Bruton JD, Katz A, Westerblad H (1999) Insulin increases near-membrane but not global Ca2+ in isolated skeletal muscle. Proc Natl Acad Sci USA 96:3281–3286PubMedCrossRef
Metadata
Title
Functional involvement of protein kinase C-βII and its substrate, myristoylated alanine-rich C-kinase substrate (MARCKS), in insulin-stimulated glucose transport in L6 rat skeletal muscle cells
Authors
D. S. Chappell
N. A. Patel
K. Jiang
P. Li
J. E. Watson
D. M. Byers
D. R. Cooper
Publication date
01-05-2009
Publisher
Springer-Verlag
Published in
Diabetologia / Issue 5/2009
Print ISSN: 0012-186X
Electronic ISSN: 1432-0428
DOI
https://doi.org/10.1007/s00125-009-1298-7

Other articles of this Issue 5/2009

Diabetologia 5/2009 Go to the issue

Commentary

A role for islet neogenesis in curing diabetes

Research Letter

Mutations in G6PC2 do not contribute to monogenic forms of early infancy diabetes and beta cell dysfunction

Letter

Left ventricular diastolic function and exercise capacity in diabetes. Reply to Brassard P, Poirier P [letter]

Letter

Which diet is best for diabetes?

For Debate

Glucose allostasis: Emperor’s new clothes?

Review

The vascular actions of insulin control its delivery to muscle and regulate the rate-limiting step in skeletal muscle insulin action
Live Webinar | 27-06-2024 | 18:00 (CEST)

Keynote webinar | Spotlight on medication adherence

Reserve your place

Live: Thursday 27th June 2024, 18:00-19:30 (CEST)

WHO estimates that half of all patients worldwide are non-adherent to their prescribed medication. The consequences of poor adherence can be catastrophic, on both the individual and population level.

Join our expert panel to discover why you need to understand the drivers of non-adherence in your patients, and how you can optimize medication adherence in your clinics to drastically improve patient outcomes.

Prof. Kevin Dolgin
Prof. Florian Limbourg
Prof. Anoop Chauhan
Developed by: Springer Medicine
Obesity Clinical Trial Summary

At a glance: The STEP trials

Read more

A round-up of the STEP phase 3 clinical trials evaluating semaglutide for weight loss in people with overweight or obesity.

Developed by: Springer Medicine

Highlights from the ACC 2024 Congress

Year in Review: Pediatric cardiology

Pediatric Cardiology Video

Watch Dr. Anne Marie Valente present the last year's highlights in pediatric and congenital heart disease in the official ACC.24 Year in Review session.

Year in Review: Pulmonary vascular disease

Cardiopulmonary Comorbidity Video

The last year's highlights in pulmonary vascular disease are presented by Dr. Jane Leopold in this official video from ACC.24.

Year in Review: Valvular heart disease

Valvular Heart Disease Video

Watch Prof. William Zoghbi present the last year's highlights in valvular heart disease from the official ACC.24 Year in Review session.

Year in Review: Heart failure and cardiomyopathies

Heart Failure Video

Watch this official video from ACC.24. Dr. Biykem Bozkurt discusses last year's major advances in heart failure and cardiomyopathies.

ACC 2024 Congress Coverage

Year in Review: Heart failure and cardiomyopathies

Heart Failure Video

Watch this official video from ACC.24. Dr. Biykem Bozkurt discusses last year's major advances in heart failure and cardiomyopathies.

Watch now

Semaglutide benefits people with type 2 diabetes and obesity-related heart failure

16-04-2024 Type 2 Diabetes News

Incentivised to exercise

11-04-2024 Lifestyle Modifications News

New intervention for STEMI-related cardiogenic shock

10-04-2024 Myocardial Infarction News

» View more highlights on the ACC 2024 congress hub page

Image Credits