Insulin Activates Protein Kinases C-ζ and C-λ by an Autophosphorylation-dependent Mechanism and Stimulates Their Translocation to GLUT4 Vesicles and Other Membrane Fractions in Rat Adipocytes

• Published in: The Journal of biological chemistry 1999-09, Vol.274 (36), p.25308
• Main Authors: Mary L. Standaert, Gautam Bandyopadhyay, Liliam Perez, Debbie Price, Lamar Galloway, Andrew Poklepovic, Minni P. Sajan, Vitorria Cenni, Alessandra Sirri, Jorge Moscat, Alex Toker, Robert V. Farese
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.274.36.25308

In rat adipocytes, insulin provoked rapid increases in ( a ) endogenous immunoprecipitable combined protein kinase C (PKC)-ζ/λ activity in plasma membranes and microsomes and ( b ) immunoreactive PKC-ζ and PKC-λ in GLUT4 vesicles. Activity and autophosphorylation of immunoprecipitable epitope-tagged PKC-ζ and PKC-λ 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-ζ and protein kinase B, we compared their activation. Both RO 31-8220 and myristoylated PKC-ζ pseudosubstrate blocked insulin-induced activation and autophosphorylation of PKC-ζ/λ but did not inhibit PDK-1-dependent ( a ) protein kinase B phosphorylation/activation or ( b ) threonine 410 phosphorylation in the activation loop of PKC-ζ. Also, insulin in situ and PIP 3 in vitro activated and stimulated autophosphorylation of a PKC-ζ 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-ζ 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-ζ. Our findings suggest that insulin activates both PKC-ζ and PKC-λ 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-ζ and λ, and subsequent autophosphorylation and/or transphosphorylation.