NONEQUIVALENT ROLES FOR THE FIRST AND 2ND ZINC FINGERS OF PROTEIN-KINASE-C-DELTA - EFFECT OF THEIR MUTATION ON PHORBOL ESTER-INDUCED TRANSLOCATION IN NIH-3T3-CELLS

Classical and novel protein kinase C (PKC) isozymes contain two, so-ca lled cysteine-rich zinc finger domains that represent the binding site s for phorbol esters and the diacylglycerols, X-ray crystallographic, mutational, and modeling studies are providing detailed understanding of the interactions between the phorbol esters and individual PKC zinc fingers, In the present study, we explore the roles of the individual zinc fingers in the context of the intact enzyme, Our approach was to mu tate either the first, the second, or both zinc fingers of PKC del ta, to express the mutated enzyme in NIH 3T3 cells, and to monitor the effect of the mutations on the dose-response curve for translocation induced by phorbol 12-myristate 13-acetate. The introduced mutations c hange into glycine the consensus proline in the phorbol ester binding loop of the zinc finger; in the isolated zinc finger, this mutation ca uses a 125-fold decrease in phorbol ester binding affinity, We observe d that mutation in the first zinc finger caused almost no shift in the dose-response curve for translocation; mutation in the second zinc fi nger caused a al-fold shift, whereas mutation in both zinc fingers cau sed a 138-fold shift, We conclude that the zinc fingers in the intact PRC are not equivalent and that the second zinc finger plays the predo minant role in translocation of protein kinase C gamma in response to phorbol 12-myristate 13-acetate, Our findings have important implicati ons for the understanding and design of PKC inhibitors targeted to the zinc finger domains.