Insulin activates protein kinases C-zeta and C-lambda by an autophosphorylation-dependent mechanism and stimulates their translocation to GLUT4 vesicles and other membrane fractions in rat adipocytes.
In rat adipocytes, insulin provoked rapid increases in (a) endogenous immunoprecipitable combined protein kinase C (PKC)-zeta/lambda activity in plasma membranes and microsomes and (b) immunoreactive PKC-zeta and PKC-lambda in GLUT4 vesicles. Activity and autophosphorylation of immunoprecipitable epitope-tagged PKC-zeta and PKC-lambda were also increased by insulin in situ and phosphatidylinositol 3,4,5-(PO(4))(3) (PIP(3)) in vitro. Because phosphoinositide-dependent kinase-1 (PDK-1) is required for phosphorylation of activation loops of PKC-zeta and protein kinase B, we compared their activation. Both RO 31-8220 and myristoylated PKC-zeta pseudosubstrate blocked insulin-induced activation and autophosphorylation of PKC-zeta/lambda but did not inhibit PDK-1-dependent (a) protein kinase B phosphorylation/activation or (b) threonine 410 phosphorylation in the activation loop of PKC-zeta. Also, insulin in situ and PIP(3) in vitro activated and stimulated autophosphorylation of a PKC-zeta mutant, in which threonine 410 is replaced by glutamate (but not by an inactivating alanine) and cannot be activated by PDK-1. Surprisingly, insulin activated a truncated PKC-zeta that lacks the regulatory (presumably PIP(3)-binding) domain; this may reflect PIP(3) effects on PDK-1 or transphosphorylation by endogenous full-length PKC-zeta. Our findings suggest that insulin activates both PKC-zeta and PKC-lambda in plasma membranes, microsomes, and GLUT4 vesicles by a mechanism requiring increases in PIP(3), PDK-1-dependent phosphorylation of activation loop sites in PKC-zeta and lambda, and subsequent autophosphorylation and/or transphosphorylation.
Standaert
M L
ML
J. A. Haley Veterans' Hospital Research Service and the Department of Internal Medicine, University of South Florida College of Medicine, Tampa, Florida 33612, USA.
Bandyopadhyay
G
G
Perez
L
L
Price
D
D
Galloway
L
L
Poklepovic
A
A
Sajan
M P
MP
Cenni
V
V
Sirri
A
A
Moscat
J
J
Toker
A
A
Farese
R V
RV
D017667:Adipocytes / Q000378:metabolism* / Q000648:ultrastructure; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003594:Cytoplasmic Granules / Q000378:metabolism; D051275:Glucose Transporter Type 4; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D007328:Insulin / Q000494:pharmacology*; D007527:Isoenzymes; D051379:Mice; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D009124:Muscle Proteins; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects*; D000099293:Protein Kinase C-lambda; D000099221:Protein Kinase C zeta
D051275:Glucose Transporter Type 4; D007004:Hypoglycemic Agents; D007328:Insulin; D007527:Isoenzymes; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D011493:Protein Kinase C; D000099293:Protein Kinase C-lambda; D000099221:Protein Kinase C zeta; C495693:Slc2a4 protein, mouse; C495694:Slc2a4 protein, rat