THE REGULATORY REGION OF PROTEIN-KINASE C-GAMMA - STUDIES OF PHORBOL ESTER BINDING TO INDIVIDUAL, AND COMBINED FUNCTIONAL SEGMENTS EXPRESSED AS GLUTATHIONE-S-TRANSFERASE FUSION PROTEINS INDICATE A COMPLEX MECHANISM OF REGULATION BY PHOSPHOLIPIDS, PHORBOL ESTERS, AND DIVALENT-CATIONS

The regulatory domain of protein kinase C gamma (PKC gamma) contains t he following functional elements which can interact with lipids: the p seudosubstrate motif within the first variable region (V1), cysteine r ich domains, Cys1 and Cys2 which contain zinc and bind phorbol dibutyr ate (PDBu)/diacylglycero1, and the calcium dependent lipid binding dom ain (CaLB). The function of individual or combined segments of the reg ulatory domain was investigated, using glutathione S-transferase (GST) fusion proteins and mixed micellar or liposomal assays. GST-Cys1 and GST-Cys2 bound PDBu with comparable affinity (K-d = 14-17 nM). GST-Cys 1Cys2 yielded a protein with a PDBu binding affinity of 3.4 nM, in the presence of calcium, similar to that of intact PKC gamma (K-d = 2.6 n M). The phosphatidylserine (PS) dependence of PDBu binding was highly cooperative for all fusion proteins tested with Hill numbers (n) lying in the range of 3.5-4.8, similar to values obtained for intact PKC ga mma. While Hill numbers were similar under all conditions, the PS conc entration necessary for half-maximal PDBu binding was dependent upon t he nature and presence of divalent cations. The PS requirement was low est in the presence of calcium for GST-Cys1, GST-Cys2, and GST-Cys1Cys 2 (K-m for PS = 11, 14, and 12 mol %, respectively) but still signific antly above the value for intact PKC gamma (5.4 mol %). The data estab lish Cys1 and Cys2 as independent PDBu binding domains that are modula ted by divalent cations. While PDBu binding affinity to a GST-V1Cys1 f usion protein (K-d = 36 nM) was comparable to that of GST-Cys1, the Ca LB domain dramatically reduced PDBu binding affinity of GST-Cys2CaLB ( K-d = 912 nM). This effect of the CaLB domain on PDBu binding to Cys2 suggests that PDBu/diacylglycerol binding to native PKC gamma may occu r at Cys1 and that the Cys2 domain may serve another regulatory functi on.