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.

Abstract

The regulatory domain of protein kinase C gamma (PKC gamma) contains the following functional elements which can interact with lipids: the pseudosubstrate motif within the first variable region (V1), cysteine-rich domains, Cys1 and Cys2 which contain zinc and bind phorbol dibutyrate (PDBu)/diacylglycerol, and the calcium-dependent lipid binding domain (CaLB). The function of individual or combined segments of the regulatory 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 (Kd = 14-17 nM). GST-Cys1Cys2 yielded a protein with a PDBu binding affinity of 3.4 nM, in the presence of calcium, similar to that of intact PKC gamma (Kd = 2.6 nM). 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 gamma. While Hill numbers were similar under all conditions, the PS concentration necessary for half-maximal PDBu binding was dependent upon the nature and presence of divalent cations. The PS requirement was lowest in the presence of calcium for GST-Cys1, GST-Cys2, and GST-Cys1Cys2 (Km for PS = 11, 14, and 12 mol %, respectively) but still significantly above the value for intact PKC gamma (5.4 mol %). The data establish Cys1 and Cys2 as independent PDBu binding domains that are modulated by divalent cations. While PDBu binding affinity to a GST-V1Cys1 fusion protein (Kd = 36 nM) was comparable to that of GST-Cys1, the CaLB domain dramatically reduced PDBu binding affinity of GST-Cys2CaLB (Kd = 912 nM). This effect of the CaLB domain on PDBu binding to Cys2 suggests that PDBu/diacylglycerol binding to native PKC gamma may occur at Cys1 and that the Cys2 domain may serve another regulatory function.