CYSTEINE-RICH REGIONS OF PROTEIN-KINASE C-DELTA ARE FUNCTIONALLY NONEQUIVALENT - DIFFERENCES BETWEEN CYSTEINE-RICH REGIONS OF NON-CALCIUM-DEPENDENT PROTEIN-KINASE C-DELTA AND CALCIUM-DEPENDENT PROTEIN-KINASE C-GAMMA

Regulatory domain elements of the non-calcium-dependent protein kinase C delta (nPKC delta), including either or both of the cysteine-rich r egions Cys1(delta) and Cys2(delta), were expressed as fusion proteins with glutathione-S-transferase and characterized using Liposomal or mi xed micellar phorbol ester binding assays, Fusion proteins containing Cys2(delta) bound phorbol-12,13-dibutyrate (PDBu) efficiently in the a ssay employing phosphatidylserine (PS) vesicles, while no significant binding was seen for proteins containing only Cys1(delta). Likewise, i n mixed micellar assays, fusion proteins with Cys2(delta) bound PDBu w ith high affinity (K-d: 14-37 nM) and to significant stoichiometric le vels (0.23-0.66 mol/mol), but no binding could be detected for protein s with Cys1(delta) only. The PS dependence of PDBu binding to Cys2(del ta) was highly cooperative with Hill numbers lying in the range of 2.5 -5.2. These results demonstrate the presence of striking functional di fferences between the cysteine-rich regions of nPKC delta and the calc ium-dependent isoform, cPKC gamma, where both cysteine-rich regions re present functional PDBu binding elements.