title: The origin of C1A-C2 interdomain interactions in protein kinase Calpha.
abstract: The regulatory domain of protein kinase Calpha (PKCalpha) contains three membrane-targeting modules, two C1 domains (C1A and C1B) that bind diacylglycerol and phorbol ester, and the C2 domain that is responsible for the Ca2+-dependent membrane binding. Accumulating evidence suggests that C1A and C2 domains of PKCalpha are tethered in the resting state and that the tethering is released upon binding to the membrane containing phosphatidylserine. The homology modeling and the docking analysis of C1A and C2 domains of PKCalpha revealed a highly complementary interface that comprises Asp55-Arg252 and Arg42-Glu282 ion pairs and a Phe72-Phe255 aromatic pair. Mutations of these residues in the predicted C1A-C2 interface showed large effects on in vitro membrane binding, enzyme activity, phosphatidylserine selectivity, and cellular membrane translocation of PKCalpha, supporting their involvement in interdomain interactions. In particular, D55A (or D55K) and R252A (or R252E) mutants showed much higher basal membrane affinity and enzyme activity and faster subcellular translocation than wild type, whereas a double charge-reversal mutant (D55K/R252E) behaved analogously to wild type, indicating that a direct electrostatic interaction between the two residues is essential for the C1A-C2 tethering. Collectively, these studies provide new structural insight into PKCalpha C1A-C2 interdomain interactions and the mechanism of lipid-mediated PKCalpha activation.
authors.affiliation: Department of Chemistry, University of Illinois, Chicago, Illinois 60607-7061, USA.
authors.forename: Robert V
authors.initials: RV
authors.lastname: Stahelin
authors.forename: Jiyao
authors.initials: J
authors.lastname: Wang
authors.forename: Nichole R
authors.initials: NR
authors.lastname: Blatner
authors.forename: John D
authors.initials: JD
authors.lastname: Rafter
authors.forename: Diana
authors.initials: D
authors.lastname: Murray
authors.forename: Wonhwa
authors.initials: W
authors.lastname: Cho
mesh_terms: D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D018076:DNA, Complementary / Q000378:metabolism; D004075:Diglycerides / Q000737:chemistry; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007313:Insecta; D007477:Ions; D007700:Kinetics; D008055:Lipids / Q000737:chemistry; D051379:Mice; D018613:Microscopy, Confocal; D008954:Models, Biological; D008956:Models, Chemical; D008958:Models, Molecular; D009154:Mutation; D015240:Phorbol 12,13-Dibutyrate / Q000737:chemistry; D010703:Phorbol Esters / Q000737:chemistry; D010718:Phosphatidylserines / Q000737:chemistry; D011312:Pressure; D011485:Protein Binding; D051571:Protein Kinase C-alpha / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D055672:Static Electricity; D020349:Surface Plasmon Resonance
chemical_list: C011439:1,2-diacylglycerol; D018076:DNA, Complementary; D004075:Diglycerides; D007477:Ions; D008055:Lipids; D010703:Phorbol Esters; D010718:Phosphatidylserines; D049452:Green Fluorescent Proteins; D015240:Phorbol 12,13-Dibutyrate; D051571:Protein Kinase C-alpha