Insulin and PIP3 activate PKC-zeta by mechanisms that are both dependent and independent of phosphorylation of activation loop (T410) and autophosphorylation (T560) sites.
Activation of protein kinase C-zeta (PKC-zeta) by insulin requires phosphatidylinositol (PI) 3-kinase-dependent increases in phosphatidylinositol-3,4,5-(PO(4))(3) (PIP(3)) and phosphorylation of activation loop and autophosphorylation sites, but actual mechanisms are uncertain. Presently, we examined: (a) acute effects of insulin on threonine (T)-410 loop phosphorylation and (b) effects of (i) alanine (A) and glutamate (E) mutations at T410 loop and T560 autophosphorylation sites and (ii) N-terminal truncation on insulin-induced activation of PKC-zeta. Insulin acutely increased T410 loop phosphorylation, suggesting enhanced action of 3-phosphoinositide-dependent protein kinase-1 (PDK-1). Despite increasing in vitro autophosphorylation of wild-type PKC-zeta and T410E-PKC-zeta, insulin and PIP(3) did not stimulate autophosphorylation of T560A, T560E, T410A/T560E, T410E/T560A, or T410E/T560E mutant forms of PKC-zeta; thus, T560 appeared to be the sole autophosphorylation site. Activating effects of insulin and/or PIP(3) on enzyme activity were completely abolished in T410A-PKC-zeta, partially compromised in T560A-PKC-zeta, T410E/T560A-PKC-zeta, and T410A/T560E-PKC-zeta, and largely intact in T410E-PKC-zeta, T560E-PKC-zeta, and T410E/T560E-PKC-zeta. Activation of the T410E/T560E mutant suggested a phosphorylation-independent mechanism. As functional correlates, insulin effects on epitope-tagged GLUT4 translocation were compromised by expression of T410A-PKC-zeta, T560A-PKC-zeta, T410E/T560A, and T410A/T560E-PKC-zeta but not T410E-PKC-zeta, T560E-PKC-zeta, or T410E/T560E-PKC-zeta. Insulin, but not PIP(3), activated truncated, pseudosubstrate-lacking forms of PKC-zeta and PKC-lambda by a wortmannin-sensitive mechanism, apparently involving PI 3-kinase/PDK-1-dependent phosphorylations but independent of PIP(3)-dependent conformational activation. Our findings suggest that insulin, via PIP(3), provokes increases in PKC-zeta enzyme activity through (a) PDK-1-dependent T410 loop phosphorylation, (b) T560 autophosphorylation, and (c) phosphorylation-independent/conformational-dependent relief of pseudosubstrate autoinhibition.
Standaert
M L
ML
J. A. Haley Veterans' Hospital Research Service, and Department of Internal Medicine, University of South Florida College of Medicine, Tampa, Florida 33612, USA.
Bandyopadhyay
G
G
Kanoh
Y
Y
Sajan
M P
MP
Farese
R V
RV
D064413:3-Phosphoinositide-Dependent Protein Kinases; D017667:Adipocytes / Q000201:enzymology; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D004789:Enzyme Activation / Q000235:genetics; D018698:Glutamic Acid / Q000235:genetics; D007328:Insulin / Q000737:chemistry / Q000494:pharmacology*; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000737:chemistry / Q000502:physiology*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000502:physiology; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000494:pharmacology; D017384:Sequence Deletion; D013379:Substrate Specificity / Q000235:genetics; D013912:Threonine / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D000099293:Protein Kinase C-lambda; D000099221:Protein Kinase C zeta
D064413:3-Phosphoinositide-Dependent Protein Kinases; D018698:Glutamic Acid; D007328:Insulin; D007527:Isoenzymes; D019869:Phosphatidylinositol 3-Kinases; D018129:Phosphatidylinositol Phosphates; D011493:Protein Kinase C; D017346:Protein Serine-Threonine Kinases; D011994:Recombinant Proteins; D013912:Threonine; D000099293:Protein Kinase C-lambda; D000099221:Protein Kinase C zeta; C060974:phosphatidylinositol 3,4,5-triphosphate