title: Requirements for pseudosubstrate arginine residues during autoinhibition and phosphatidylinositol 3,4,5-(PO₄)₃-dependent activation of atypical PKC.
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.
authors.affiliation: From the Medical and Research Services, James A. Haley Veterans Medical Center, Tampa, Florida 33612 and.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Ivey
authors.affiliation: From the Medical and Research Services, James A. Haley Veterans Medical Center, Tampa, Florida 33612 and the Division of Endocrinology and Metabolism, Department of Internal Medicine, University of South Florida College of Medicine, Tampa, Florida 33612.
authors.forename: Mini P
authors.initials: MP
authors.lastname: Sajan
authors.affiliation: From the Medical and Research Services, James A. Haley Veterans Medical Center, Tampa, Florida 33612 and the Division of Endocrinology and Metabolism, Department of Internal Medicine, University of South Florida College of Medicine, Tampa, Florida 33612 rfarese@health.usf.edu.
authors.forename: Robert V
authors.initials: RV
authors.lastname: Farese
mesh_terms: D041721:3T3-L1 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D057507:Mice, 129 Strain; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000378:metabolism; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000494:pharmacology; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism* / Q000494:pharmacology; D010718:Phosphatidylserines / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D000099293:Protein Kinase C-lambda; D000099221:Protein Kinase C zeta
chemical_list: D001120:Arginine; D007527:Isoenzymes; D010455:Peptides; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D010718:Phosphatidylserines; D011493:Protein Kinase C; D000099293:Protein Kinase C-lambda; D000099221:Protein Kinase C zeta; C060974:phosphatidylinositol 3,4,5-triphosphate; C055525:phosphatidylinositol 3-phosphate
keywords: Diabetes; Insulin Resistance; Phosphatidylinositol Kinase (PI Kinase); Protein Kinase C (PKC); Signal Transduction