The carboxyl terminus of protein kinase c provides a switch to regulate its interaction with the phosphoinositide-dependent kinase, PDK-1.
The function of protein kinase C family members depends on two tightly coupled phosphorylation mechanisms: phosphorylation of the activation loop by the phosphoinositide-dependent kinase, PDK-1, followed by autophosphorylation at two positions in the COOH terminus, the turn motif, and the hydrophobic motif. Here we address the molecular mechanisms underlying the regulation of protein kinase C betaII by PDK-1. Co-immunoprecipitation studies reveal that PDK-1 associates preferentially with its substrate, unphosphorylated protein kinase C, by a direct mechanism. The exposed COOH terminus of protein kinase C provides the primary interaction site for PDK-1, with co-expression of constructs of the carboxyl terminus effectively disrupting the interaction in vivo. Disruption of this interaction promotes the autophosphorylation of protein kinase C, suggesting that the binding of PDK-1 to the carboxyl terminus protects it from autophosphorylation. Studies with constructs of the COOH terminus reveal that the intrinsic affinity of PDK-1 for phosphorylated COOH terminus is over an order of magnitude greater than that for unphosphorylated COOH terminus, contrasting with the finding that PDK-1 does not bind phosphorylated protein kinase C effectively. However, effective binding of the phosphorylated species can be induced by the activated conformation of protein kinase C. This suggests that the carboxyl terminus becomes masked following autophosphorylation, a process that can be reversed by the conformational changes accompanying activation. Our data suggest a model in which PDK-1 provides two points of regulation of protein kinase C: 1) phosphorylation of the activation loop, which is regulated by the intrinsic activity of PDK-1, and 2) phosphorylation of the carboxyl terminus, which is regulated by the release of PDK-1 to allow autophosphorylation.
Gao
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Department of Pharmacology, University of California at San Diego, La Jolla, California 92093-0640, USA.
Toker
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Newton
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D064413:3-Phosphoinositide-Dependent Protein Kinases; D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry* / Q000378:metabolism; D008954:Models, Biological; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D011487:Protein Conformation; D011493:Protein Kinase C / Q000737:chemistry* / Q000378:metabolism*; D064546:Protein Kinase C beta; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection
D018076:DNA, Complementary; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D064413:3-Phosphoinositide-Dependent Protein Kinases; D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C; D064546:Protein Kinase C beta