A PHORBOL ESTER BINDING DOMAIN OF PROTEIN KINASE-C-GAMMA - HIGH-AFFINITY BINDING TO A GLUTATHIONE-S-TRANSFERASE CYS2 FUSION PROTEIN/

Cysteine-rich regions of protein kinase C (PKC) are implicated in diac ylglycerol-dependent regulation of kinase activity. The second cystein e-rich region (residues 92-173) of PKCgamma was expressed as a fusion protein with glutathione-S-transferase in Escherichia coli and purifie d to homogeneity by affinity chromatography. This fusion protein displ ayed high affinity phorbol dibutyrate (PDBu) binding (K(d) 23 nM). The phosphatidylserine dependence of PDBu binding was highly cooperative with Hill numbers (near 4.5) similar to those previously reported for PKCgamma (Burns, D. J., and Bell, R. M. (1991) J. Biol. Chem. 266,1833 0-18338). The fusion protein specifically bound 4beta-hydroxy-PDBu but not the 4alpha-stereoisomer. Furthermore, sn-1,2-dioctanoylglycerol ( diC8) stereoselectively competed for PDBu binding. The cysteine-rich r egion was sufficient for association of the fusion protein to liposome preparations containing phosphatidylserine and phosphatidylcholine. A ssociation was significantly enhanced in a stereospecific manner by th e presence of PDBu as well as diC8. These results establish that a sin gle cysteine-rich domain (residues 92-173) of PKCgamma contains region s necessary and sufficient for lipid-dependent stereospecific interact ions with PDBu and diC8. Furthermore, the region is sufficient to conf er translocation of a fusion protein to liposomes in a PDBu- and diC8- dependent fashion. Thus, a single cysteine-rich region of PKCgamma dis plays many of the properties characteristic of PKC.