Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2001 Nov;21(22):7852-61.
doi: 10.1128/MCB.21.22.7852-7861.2001.

Activation of protein kinase C zeta induces serine phosphorylation of VAMP2 in the GLUT4 compartment and increases glucose transport in skeletal muscle

L Braiman  1 , A Alt, T Kuroki, M Ohba, A Bak, T Tennenbaum, S R Sampson
Affiliations

Activation of protein kinase C zeta induces serine phosphorylation of VAMP2 in the GLUT4 compartment and increases glucose transport in skeletal muscle

L Braiman et al. Mol Cell Biol. 2001 Nov.

Abstract

Insulin stimulates glucose uptake into skeletal muscle tissue mainly through the translocation of glucose transporter 4 (GLUT4) to the plasma membrane. The precise mechanism involved in this process is presently unknown. In the cascade of events leading to insulin-induced glucose transport, insulin activates specific protein kinase C (PKC) isoforms. In this study we investigated the roles of PKC zeta in insulin-stimulated glucose uptake and GLUT4 translocation in primary cultures of rat skeletal muscle. We found that insulin initially caused PKC zeta to associate specifically with the GLUT4 compartments and that PKC zeta together with the GLUT4 compartments were then translocated to the plasma membrane as a complex. PKC zeta and GLUT4 recycled independently of one another. To further establish the importance of PKC zeta in glucose transport, we used adenovirus constructs containing wild-type or kinase-inactive, dominant-negative PKC zeta (DNPKC zeta) cDNA to overexpress this isoform in skeletal muscle myotube cultures. We found that overexpression of PKC zeta was associated with a marked increase in the activity of this isoform. The overexpressed, active PKC zeta coprecipitated with the GLUT4 compartments. Moreover, overexpression of PKC zeta caused GLUT4 translocation to the plasma membrane and increased glucose uptake in the absence of insulin. Finally, either insulin or overexpression of PKC zeta induced serine phosphorylation of the GLUT4-compartment-associated vesicle-associated membrane protein 2. Furthermore, DNPKC zeta disrupted the GLUT4 compartment integrity and abrogated insulin-induced GLUT4 translocation and glucose uptake. These results demonstrate that PKC zeta regulates insulin-stimulated GLUT4 translocation and glucose transport through the unique colocalization of this isoform with the GLUT4 compartments.

PubMed Disclaimer

Figures

FIG. 1
FIG. 1
Effects of insulin on activity of PKCζ in internal membrane fraction. Myotubes after 6 days in culture were transferred to a serum-free, low-glucose medium for 24 h. PKCζ was immunoprecipitated from different cell fractions of control cells and cells stimulated with 100 nM insulin for the times indicated. The immunoprecipitates were then assayed for PKC activity as described in Materials and Methods. Each bar represents the mean ± standard error of duplicate measurements in each of three experiments (P < 0.005).
FIG. 2
FIG. 2
Overexpression of PKC isoforms α and ζ in skeletal muscle cells by recombinant adenovirus constructs. (A) Western blots of PKCs α and ζ in control cells (C) and in cells overexpressing (OE) either one of these isoforms. Myotubes after 6 days in culture were infected with PKCα or PKCζ recombinant adenovirus (as described in Materials and Methods). Whole-cell lysates were prepared from control and infected cells 16 h postinfection, and the samples were subjected to SDS-PAGE and immunoblotted with anti-PKC antibodies. The blot is representative of five separate experiments. (B) Comparison of PKC activity in control and PKC-overexpressing myotubes. Myotubes were treated as for Fig. 2A, after which PKC isoforms α and ζ were immunoprecipitated from control cells and cells overexpressing PKCα or -ζ with specific anti-PKC antibodies. The specific PKC immunoprecipitates were then assayed for PKC activity as described in Materials and Methods. Each bar represents the mean ± standard error of duplicate measurements in each of three experiments (P < 0.005).
FIG. 3
FIG. 3
Effects of PKCζ or PKCα overexpression on basal and insulin-induced glucose uptake. PKC isoforms were overexpressed as for Fig. 2A. Sixteen hours postinfection, control cells and cells overexpressing PKCζ or PKCα were either untreated (empty bars) or treated with insulin for 30 min (filled bars) and were assayed for 2-DG uptake as described in Materials and Methods. Data are expressed as fold increase over the basal level determined in control, non-insulin-stimulated cultures. Each bar represents the mean ± standard error of duplicate measurements in each of three experiments (P < 0.005).
FIG. 4
FIG. 4
Effects of overexpression of PKCζ on distribution of GLUTs. (A) GLUT4 distribution. PKC isoforms were overexpressed as for Fig. 2A. Sixteen hours postinfection, control (CON) cells and cells overexpressing PKCζ or PKCα were either untreated or treated with insulin (INS) for 30 min and were fractionated to plasma membrane (P.M.), internal membrane (I.M.), and very-light-density microsome (V.L.D.M.) fractions. The samples were subjected to SDS-PAGE and were immunoblotted (IB) with anti-GLUT4 antibody. The blot shown is representative of five separate experiments. The graph represents the densitometry measurements (mean ± standard error) of five Western blot analyses. Empty bars, plasma membrane; black-bars, internal membrane (I.M.); gray bars, very-light-density micosomes. (B) Western blots of the distribution of GLUT1 (upper set) and GLUT3 (lower set). PKC isoforms were overexpressed and the cells were treated as for panel A. Following treatment, cells were fractionated to plasma membrane (P.M.) or internal membrane (I.M.) fractions.
FIG. 5
FIG. 5
PKCζ distribution in cytosolic (Cyto) and membrane fractions of skeletal myotubes. Cells were infected as for Fig. 2A. Control (C) cells and cells overexpressing (O.E.) PKCζ were fractionated into vesicular (V.M.) and plasma (P.M.) fractions, and the samples were subjected to SDS-PAGE and immunoblotted with anti-PKCζ antibody. The blot shown is representative of three separate experiments.
FIG. 6
FIG. 6
Western blots showing effects of insulin (IN) on distribution of PKC isoforms and GLUT4 in cytosolic (CYTO), vesicular (V.M.), and plasma (P.M.) membrane fractions of skeletal muscle. Cells were infected with PKC viral constructs and 16 to 20 h postinfection were either untreated or stimulated with insulin for the designated times (in minutes) and fractionated on sucrose gradient (as described in Materials and Methods). The cytosolic, vesicular, and plasma membrane fractions were subjected to SDS-PAGE and immunoblotted (IB) with specific anti-PKC (A) or anti-GLUT4 (B) antibodies. The blots shown are representative of three separate experiments. Graphs of densitometry measurements (mean ± standard error) of the appropriate Western blot of PKC isoform distribution are shown to the right of each blot. OD, optical density. White bars, plasma membranes; black bars, vesicular membranes; gray bars, cytosol.
FIG. 7
FIG. 7
Induction of VAMP2 serine phosphorylation by insulin and PKCζ. Cells were infected as for Fig. 2A, and 16 h postinfection, control (CON) and infected cells were treated with insulin for the times indicated. (A) Western blots of serine phosphorylation of VAMP2 by insulin (INS) and overexpression of PKCζ. Whole-cell lysates were immunoprecipitated (I.P.) with anti-VAMP2 antibody and immunoblotted (I.B.) with antiphosphoserine (p-ser) and anti-VAMP3. The blots shown are representative of three separate experiments; time is given in minutes below blots. (B) PKC activity assay showing serine phosphorylation of synaptobrevin-2 fragments by immunoprecipitated PKCζ. Cells were infected as for Fig. 2A, and, 16 h postinfection, control and infected cells were treated with insulin for 5 min. Whole-cell lysates were immuno-precipitated with anti-PKCζ antibody and were added to an activity assay in which the synaptobrevin-2 fragments served as the substrate. (C) Western blots of serine phosphorylation of VAMP3 by insulin (INS) and PKCζ. Whole-cell lysates were immunoprecipitated with anti-VAMP3 antibody and immunoblotted with antiphosphoserine and anti-VAMP3. The blots shown are representative of three separate experiments. Time is given in minutes below blots.
FIG. 8
FIG. 8
(A) Insulin-(INS)-induced translocation of VAMP2 in noninfected (CON) and WTPKCζ- and DNPKCζ-overexpressing cells. PKC isoforms were overexpressed as for Fig. 2A. Sixteen hours postinfection, noninfected cells and cells overexpressing WTPKCζ or DNPKCζ were either untreated or treated with insulin for the designated times. Whole-cell lysates were fractionated on sucrose gradient (as described in Materials and Methods). The cytosolic, vesicular, and plasma membrane (P.M.) fractions were then immunoprecipitated (IP) with anti-GLUT4 antibody, subjected to SDS-PAGE and immunoblotted (IB) with specific anti-VAMP2 antibody. V.L.D.N., very-light-density membrane. Time is given in minutes below blots. (B) Recycling of GLUT4 following insulin stimulation of WTPKCζ-overexpressing cells. WTPKCζ was overexpressed as for Fig. 2A. Sixteen hours postinfection, cells were either untreated or treated with insulin for the designated times. Whole-cell lysates were fractionated on sucrose gradient (as described in Materials and Methods). The internal membrane (I.M.) and plasma membrane fractions were then subjected to SDS-PAGE and immunoblotted with specific anti-GLUT4 antibody.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bandyopadhyay G, Kanoh Y, Sajan M P, Standaert M L, Farese R V. Effects of adenoviral gene transfer of wild-type, constitutively active, and kinase-defective protein kinase C-lambda on insulin-stimulated glucose transport in L6 myotubes. Endocrinology. 2000;141:4120–4127. - PubMed
    1. Bandyopadhyay G, Standaert M L, Galloway L, Moscat J, Farese R V. Evidence for involvement of protein kinase C (PKC)-zeta and noninvolvement of diacylglycerol-sensitive PKCs in insulin-stimulated glucose transport in L6 myotubes. Endocrinology. 1997;138:4721–4731. - PubMed
    1. Bandyopadhyay G, Standaert M L, Kikkawa U, Ono Y, Moscat J, Farese R V. Effects of transiently expressed atypical (zeta, lambda), conventional (alpha, beta) and novel (delta, epsilon) protein kinase C isoforms on insulin-stimulated translocation of epitope-tagged GLUT4 glucose transporters in rat adipocytes: specific interchangeable effects of protein kinases C-zeta and C-lambda. Biochem J. 1999;337:461–470. - PMC - PubMed
    1. Braiman L, Alt A, Kuroki T, Ohba M, Bak A, Tennenbaum T, Sampson S R. Protein kinase Cdelta mediates insulin-induced glucose transport in primary cultures of rat skeletal muscle. Mol Endocrinol. 1999;13:2002–2012. - PubMed
    1. Braiman L, Alt A, Kuroki T, Ohba M, Bak A, Tennenbaum T, Sampson S R. Insulin induces specific interaction between insulin receptor and PKC in primary cultured skeletal muscle. Mol Endocrinol. 2001;15:565–574. - PubMed

Publication types

MeSH terms

LinkOut - more resources

-