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

Robert A Ivey,Mini P Sajan,Robert V Farese

doi:10.1074/jbc.m114.565671

Abstract

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.

Highlights

The mechanisms whereby atypical PKCs are silenced and activated by phosphatidylinositol 3,4,5(PO4)3 are uncertain
Activation of atypical PKCs (aPKCs) by PIP3 versus PIP2—In keeping with previous findings of increased overall 32PO4 labeling of aPKC during incubation of immunoprecipitated aPKC with PIP3, but not PIP2 (11), we found that PIP3, but not PIP2, increased both enzyme activity and autophosphorylation of aPKC immunoprecipitated from mouse liver (Fig. 2)
These findings suggested that both arginine 126 and arginine 131 were required for binding of PIP3 to the pseudosubstrate. This dovetails with the fact that replacement of either arginine 126 or arginine 131 with alanine in the pseudosubstrate led to constitutive activation of aPKC and loss of the ability of PIP3 to cause further activation, as shown above. These findings show that arginine residues in the pseudosubstrate sequence play a key role in maintaining aPKC in an inactive state under basal conditions

Summary

Background

The mechanisms whereby atypical PKCs (aPKCs) are silenced and activated by phosphatidylinositol 3,4,5(PO4) are uncertain. 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. Given the fact that the aPKC pseudosubstrate sequence, like the aPKC consensus substrate recognition sequence, contains basic arginine residues that flank a short sequence that, in substrates, contains phosphorylatable threonine or serine residues, we examined the importance of the five basic arginine residues that are common to pseudosubstrate sequences of all aPKCs for their ability to maintain aPKC in an inactive state and serve as a target used by PIP3 to provoke a dissociation of the pseudosubstrate from the substrate-binding site and, thereby, promote kinase activation

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION