Insulin and PIP3 Activate PKC-ζ by Mechanisms That Are Both Dependent and Independent of Phosphorylation of Activation Loop (T410) and Autophosphorylation (T560) Sites

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Abstract
Activation of protein kinase C-ζ (PKC-ζ) by insulin requires phosphatidylinositol (PI) 3-kinase-dependent increases in phosphatidylinositol-3,4,5-(PO4)3 (PIP3) 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-ζ. 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-ζ and T410E-PKC-ζ, insulin and PIP3 did not stimulate autophosphorylation of T560A, T560E, T410A/T560E, T410E/T560A, or T410E/T560E mutant forms of PKC-ζ; thus, T560 appeared to be the sole autophosphorylation site. Activating effects of insulin and/or PIP3 on enzyme activity were completely abolished in T410A-PKC-ζ, partially compromised in T560A-PKC-ζ, T410E/T560A-PKC-ζ, and T410A/T560E-PKC-ζ, and largely intact in T410E-PKC-ζ, T560E-PKC-ζ, and T410E/T560E-PKC-ζ. 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-ζ, T560A-PKC-ζ, T410E/T560A, and T410A/T560E-PKC-ζ but not T410E-PKC-ζ, T560E-PKC-ζ, or T410E/T560E-PKC-ζ. Insulin, but not PIP3, activated truncated, pseudosubstrate-lacking forms of PKC-ζ and PKC-λ by a wortmannin-sensitive mechanism, apparently involving PI 3-kinase/PDK-1-dependent phosphorylations but independent of PIP3-dependent conformational activation. Our findings suggest that insulin, via PIP3, provokes increases in PKC-ζ enzyme activity through (a) PDK-1-dependent T410 loop phosphorylation, (b) T560 autophosphorylation, and (c) phosphorylation-independent/conformational-dependent relief of pseudosubstrate autoinhibition.