A PHORBOL ESTER BINDING DOMAIN OF PROTEIN KINASE-C-GAMMA - DELETION ANALYSIS OF THE CYS2 DOMAIN DEFINES A MINIMAL 43-AMINO ACID PEPTIDE

Cysteine-rich regions of protein kinase C (PKC) are critical for the l ipid-dependent regulation of activity and are implicated in the coordi nation of zinc. A glutathione S-transferase fusion protein containing the second cysteine-rich region, Cys2, of PKCgamma with bound zinc wit h a stoichiometry of 1.8 +/- 0.1 mol of zinc/mol of protein. Deletion analysis within this cysteine-rich region defined amino acids essentia l for zinc coordination. An NH2-terminal histidine (His102) and a COOH -terminal cysteine (Cys151) were both critical for the coordination of distinct zinc atoms. Both represent the ultimate residues of a 50-ami no acid consensus motif with six conserved cysteines and two conserved histidines present in the cysteine-rich regions of all PKC isoforms. Removal of histidine His102 abolished phorbol ester binding, while del etion of cysteine Cys151 did not. Deletion of valine (Val147) greatly diminished phorbol ester binding, which was completely lost only when valine (Val144) was also deleted. No significant further reduction in zinc stoichiometry below one resulted even when three COOH-terminal co nserved cysteines (Cys151, Cys143, and Cys135) and a conserved histidi ne (His140) were deleted. These results are consistent with a model in which two zinc atoms are tetracoordinated per cysteine-rich region in two independent coordination spheres that are not functionally equiva lent. These analyses determine a minimal peptide (residues 102-144) of 43 amino acids capable of [H-3]PDBu binding.