Requirements for Pseudosubstrate Arginine Residues during Autoinhibition and Phosphatidylinositol 3,4,5-(PO4)3-dependent Activation of Atypical PKC

Atypical PKC (aPKC) isoforms are activated by the phosphatidylinositol 3-kinase product phosphatidylinositol 3,4,5-(PO₄)₃ (PIP₃). How PIP₃ activates aPKC is unknown. Although Akt activation involves PIP₃ binding to basic residues in the Akt pleckstrin homology domain, aPKCs lack this domain. Here we examined the role of basic arginine residues common to aPKC pseudosubstrate sequences. Replacement of all five (or certain) arginine residues in the pseudosubstrate sequence of PKC-ι by site-directed mutagenesis led to constitutive activation and unresponsiveness to PIP₃ in vitro or insulin in vivo. However, with the addition of the exogenous arginine-containing pseudosubstrate tridecapeptide to inhibit this constitutively active PKC-ι, PIP₃-activating effects were restored. A similar restoration of responsiveness to PIP₃ was seen when exogenous pseudosubstrate was used to inhibit mouse liver PKC-λ/ζ maximally activated by insulin or ceramide and a truncated, constitutively active PKC-ζ mutant lacking all regulatory domain elements and containing “activating” glutamate residues at loop and autophosphorylation sites (Δ1–247/T410E/T560E-PKC-ζ). NMR studies suggest that PIP₃ binds directly to the pseudosubstrate. The ability of PIP₃ to counteract the inhibitory effects of the exogenous pseudosubstrate suggests that basic residues in the pseudosubstrate sequence are required for maintaining aPKCs in an inactive state and are targeted by PIP₃ for displacement from the substrate-binding site during kinase activation.