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 doseresponse 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 phorbol ester binding affinity. We observed that mutation in the first zinc finger caused almost no shift in the doseresponse 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 cysteine-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 ...

Mechanism of Diacylglycerol-induced Membrane Targeting and Activation of Protein Kinase Cδ

“…Our monolayer penetration data, in conjunction with the SPR vesicle binding data showing that the C1A domain hydrophobic site mutations reduce the vesicle affinity of PKC␦ mainly by k d , indicate that membrane penetration of the C1A domain is an essential step in its function in membrane binding and activation of PKC␦. Our measurements also showed that the C1B domain of PKC␦ has higher affinity for phorbol esters than the C1A domain, which is consistent with previous reports showing the importance of the C1B domain in phorbol ester-induced membrane targeting and activation of PKC␦ (67). Therefore, these data represent another example demonstrating disparate mechanisms for DAG-and phorbol ester-induced PKC activation (38).…”

β2-Chimaerin Is a High Affinity Receptor for the Phorbol Ester Tumor Promoters

“…Structural differences among the different receptors conferring unique patterns of interactions with phorbol esters and phospholipids may regulate the association with membranes and/or the cytoskeleton. Recent studies (14) showed that the first and second cysteine-rich domains in PKC␦ have nonequivalent roles in translocation. A proline to glycine mutation in position 11 of the second cysteine-rich domain reduces the EC 50 for translocation by about 20-fold.…”

“…We named this vector pCR3⑀. The ⑀-tag proved to work well for immunodetection with a commercial anti-PKC⑀ antibody (Life Technologies, Inc.) and, importantly, it does not affect subcellular localization in tagged phorbol ester receptors (14,31). A 1.4-kilobase XhoI-MluI fragment comprising the full-length ␤2-chimaerin was ligated in the pCR3⑀ vector to generate pCR3⑀-␤2.…”

“…The cysteine-rich domains have been implicated in the association of PKC with membranes upon phorbol ester binding, a process referred to as translocation in intact cells. The molecular basis of this subcellular compartmentalization is currently the subject of intense research; recent studies on the structural aspects of translocation revealed that in PKC␦ the second cysteine-rich domain plays the predominant role in the translocation to membranes (14).…”

Taxonomy and function of C1 protein kinase C homology domains

“…Some of these single domains bind phorbol esters and diacylglycerol, while others clearly do not (On0 et al, 1989;Ahmed et al, 1991;Bums & Bell, 1991;Kazanietz et al, 1994). Individual domains may have non-equivalent roles within the context of a tandem repeat (Szallasi et al, 1996), but the properties of isolated recombinant first and second domains are not substantially different (Kazanietz et al, 1994). It has become clear over the past five years that the fundamental structural unit is the single, rather than the double, cysteine-rich region.…”