Roles of ionic residues of the C1 domain in protein kinase C-alpha activation and the origin of phosphatidylserine specificity

On the basis of extensive structure-function studies of protein kinase C-Cy (PRC-OL), We have proposed an activation mechanism for conventional PKCs i n which the C2 domain and the C1 domain interact sequentially with membrane s (Medkova, M., and Cho, W. (1999) J. Biol Chem. 274, 19852-19881), To furt her elucidate the interactions between the C1 and C2 domains during PKC act ivation and the origin of phosphatidylserine specificity, we mutated severa l charged residues in two CI domains (Cla and Gib) of PHC-alpha. We then me asured the membrane binding affinities, activities, and monolayer penetrati on of these mutants. Results indicate that cationic residues of the C1a dom ain, most notably Arg(77), interact nonspecifically with anionic phospholip ids prior to the membrane penetration of hydrophobic residues. The mutation of a single aspartate (Asp(55)) in the C1a domain to Ala or Lys resulted i n dramatically reduced phosphatidylserine specificity in vesicle binding, a ctivity, and monolayer penetration. In particular, D55A showed much higher vesicle affinity, activity, and monolayer penetration power than wild type under nonactivating conditions, i.e. with phosphatidylglycerol and in the a bsence of Ca2+, indicating that Asp(55) in involved in the tethering of the Cia domain to another part of PKC-alpha, which keeps it in an inactive con formation at the resting state, Based on these results, we propose a refine d model for the activation of conventional PKC, in which phosphatidylserine specifically disrupts the C1a domain tethering by competing with Asp(55), which then leads to membrane penetration and diacylglycerol binding of the Cia domain and PKC activation.