Regulation of PKCα Activity by C1-C2 Domain Interactions

Abstract

In this study, the role of interdomain interactions involving the C1 and C2 domains in the mechanism of activation of PKC was investigated. Using an in vitro assay containing only purified recombinant proteins and the phorbol ester, 4␤-12-O-tetradecanoylphorbol-13-acetate (TPA), but lacking lipids, it was found that PKC␣ bound specifically, and with high affinity, to a ␣C1A-C1B fusion protein of the same isozyme. The ␣C1A-C1B domain also potently activated the isozyme in a phorbol ester-and diacylglycerol-dependent manner. The level of this activity was comparable with that resulting from membrane association induced under maximally activating conditions. Furthermore, it was found that ␣C1A-C1B bound to a peptide containing the C2 domain of PKC␣. The ␣C1A-C1B domain also activated conventional PKC␤I, -␤II, and -␥ isoforms, but not novel PKC␦ or -⑀. PKC␦ and -⑀ were each activated by their own C1 domains, whereas PKC␣, -␤I, -␤II, or -␥ activities were unaffected by the C1 domain of PKC␦ and only slightly activated by that of PKC⑀. PKC activity was unaffected by its own C1 domain and those of the other PKC isozymes. Based on these findings, it is proposed that the activating conformational change in PKC␣ results from the dissociation of intra-molecular interactions between the ␣C1A-C1B domain and the C2 domain. Furthermore, it is shown that PKC␣ forms dimers via intermolecular interactions between the C1 and C2 domains of two neighboring molecules. These mechanisms may also apply for the activation of the other conventional and novel PKC isozymes.The 10 closely related isozymes that constitute the protein kinase C (PKC) 1 family of serine/threonine kinases each occupy critical nodes in the complex cellular signal transduction networks that regulate diverse cellular processes, including: secretion, proliferation, differentiation, apoptosis, permeability, migration, and hypertrophy (1-7). In common with many signaling proteins, the structure of PKC is modular, consisting of a C-terminal catalytic region containing the active site, and a regulatory region with conserved domains that mediate membrane association and activation. PKC isozymes are classified according to the structural and functional differences in these conserved domains (8, 9). In the case of the "conventional" PKC␣, -␤I/␤II, and -␥ isozymes, these include the activatorbinding C1 domains, and the Ca 2ϩ -binding C2 domain. The C1 domains consist of a tandem C1A and C1B arrangement, each of which can potentially bind the endogenous activator, diacylglycerol and exogenous activators including phorbol esters. The "novel" PKC␦, -⑀, -, -, and -isozymes, contain C2 domains that lack Ca 2ϩ binding ability, while retaining functional C1A and C1B domains. The "atypical" PKC, -, and -regulatory domains also lack a functional C2 domain and contain a single C1 domain that lacks the ability to bind activators, the function of which remains obscure. Each isozyme becomes catalytically competent by undergoing multiple serine/threonine and tyrosine phosphorylations that...

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A Critical Intramolecular Interaction for Protein Kinase Cϵ Translocation

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