Non-equivalent Roles for the First and Second Zinc Fingers of Protein Kinase Cδ

Abstract

Classical and novel protein kinase C (PKC) isozymes contain two, so-called cysteine-rich zinc finger domains that represent the binding sites 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 mutate either the first, the second, or both zinc fingers of PKC, 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 change into glycine the consensus proline in the phorbol ester binding loop of the zinc finger; in the isolated zinc finger, this mutation causes a 125-fold decrease in phor-bol ester binding affinity. We observed that mutation in the first zinc finger caused almost no shift in the dose-response curve for translocation; mutation in the second zinc finger caused a 21-fold shift, whereas mutation in both zinc fingers caused a 138-fold shift. We conclude that the zinc fingers in the intact PKC are not equivalent and that the second zinc finger plays the predominant role in translocation of protein kinase C in response to phorbol 12-myristate 13-acetate. Our findings have important implications for the understanding and design of PKC inhibitors targeted to the zinc finger domains. Protein kinase C (PKC) 1 comprises a family of isozymes that mediate signal transduction for the lipophilic second messenger diacylglycerol, regulating a wide array of cellular processes (see Refs. 1 and 2 for review). PKC possesses two functional domains, an N-terminal regulatory domain and a C-terminal catalytic domain. Within the regulatory domain lie two cys-teine-rich zinc fingers, responsible for recognition by PKC of diacylglycerol or their ultrapotent analogs, the phorbol esters. In isolation, the individual first and second zinc fingers bind phorbol ester with similar affinities (3, 4), and the molecular details of the interaction between the phorbol esters and the zinc finger domain are beginning to emerge. NMR spectroscopy has yielded the solution structure of the second zinc finger of PKC (5, 6); the structure of the complex between the second zinc finger of PKC and phorbol 13-acetate has been solved by x-ray crystallography (7). Site-directed mutagenesis has further highlighted key residues within the zinc finger structure required for ligand binding (8), and computer modeling is providing insight into how other high affinity ligands, e.g. the indole alkaloids, interact with the zinc finger. An issue that is only beginning to be addressed is the role of the individual zinc fingers within the context of the intact PKC molecule. In pioneering studies, Bell and co-workers (9) had reported that the binding affinity of the second zinc finger of PKC was reduced in constructs containing sequences C-terminal to the zinc finger. Riedel and co-workers (10) analyzed the effect of the deletion of the first or second zinc finger of PKC in a phenotypic yeast assay; they reported that PMA showed a comparable loss in potency for activation of the deletion mutants at either site; mezerein activated predominantly through the first zinc finger, whereas ()-indolactam V required both intact zinc fingers for activity. Unfortunately, interpretation of the results of deletion analysis is clouded by the extent of potential disruption of the structure of the holoen-zyme; for example, the deletion of the first zinc finger also included the pseudosubstrate domain immediately N-terminal to it. As an alternative approach, we have introduced a proline to glycine mutation into the first or second or both zinc fingers of PKC. We had previously reported that this mutation, in the isolated zinc finger, preserves binding activity but causes a substantial loss (125-fold) in binding affinity (8). We evaluated the quantitative influence of these mutations on recognition of phorbol 12-myristate 13-acetate in the intact cell, using as an end point translocation of the mutant PKC from the soluble to the detergent-soluble particulate fraction. We report here that the zinc fingers in the intact PKC are not equivalent for inducing translocation and that the second zinc finger in the intact PKC has approximately 25-fold higher affinity for PMA than does the first zinc finger under these cellular conditions.