A PUTATIVE PHOSPHATIDYLSERINE BINDING MOTIF IS NOT INVOLVED IN THE LIPID REGULATION OF PROTEIN-KINASE-C

Protein kinase C is specifically regulated by diacylglycerol and the a mino phospholipid, phosphatidylserine. The molecular basis for the pho sphatidylserine specificity was recently proposed to arise hom the pre sence of a putative phosphatidylserine binding motif, FXFXLKXXXKXR, lo calized in the C2 domain of protein kinase C (Igarashi, K., Kaneda, M. , Yamaji, A. Saido, T. C., Kikkawa, Il,, One, U,, Inoue, a, and Umeda, M, (1995) J. Biol. Chem. 270, 29075-29078). To determine whether this moth mediates the interaction of protein kinase C with phosphatidylse rine, the carboxyl terminal basic residues were mutated to Ala in prot ein kinase C beta II (K236A and R238A), and the phosphatidylserine reg ulation of the mutant enzyme was examined. Membrane binding and activi ty measurements revealed that the phosphatidylserine regulation for th e mutant protein was indistinguishable from that of wild-type protein kinase C. Specifically, neither the apparent membrane affinity for pho sphatidylserine-containing membranes in the presence or absence of dia cylglycerol nor the phosphatidyl-serine-dependence for activation was affected by removal of the conserved basic residues at the carboxyl te rminus of the consensus sequence. In addition, a synthetic peptide cor responding to the amino terminus of the consensus sequence (FTFNVK) ha d no effect on the concentration of phosphatidylserine resulting in ha lf-maximal activation of protein kinase C. These results reveal that t he carboxyl-terminal basic residues in the consensus moth FXFXLKXXXKXR are not responsible for the phosphatidylserine selectivity of protein kinase C and that, furthermore, the region of the C2 domain containin g this moth is not involved in the membrane binding of protein kinase C.