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Abstract
Emerging evidence suggests important differences among protein kinase C (PKC) isozymes in terms of their regulation and biological functions. PKC is regulated by multiple interdependent mechanisms, including enzymatic activation, translocation of the enzyme in response to activation, phosphorylation, and proteolysis. As part of our ongoing studies to define the factors contributing to the specificity of PKC isozymes, we prepared chimeras between the catalytic and regulatory domains of PKCalpha, -delta, and -epsilon. These chimeras, which preserve the overall structure of the native PKC enzymes, were stably expressed in NIH 3T3 fibroblasts. Their intracellular distribution was similar to that of the endogenous enzymes, and they responded with translocation upon treatment with phorbol 12-myristate 13-acetate (PMA). We found that the potency of PMA for translocation of the PKCalpha/x chimeras from the soluble fraction was influenced by the catalytic domain. The ED50 for translocation of PKCalpha/alpha was 26 nM, in marked contrast to the ED50 of 0.9 nM in the case of the PKCalpha/epsilon chimera. In addition to this increase in potency, the site of translocation was also changed; the PKCalpha/epsilon chimera translocated mainly into the cytoskeletal fraction. PKCx/epsilon chimeras displayed twin isoforms with different mobilities on Western blots. PMA treatment increased the proportion of the higher mobility isoform. The two PKCx/epsilon isoforms differed in their localization; moreover, their localization pattern depended on the regulatory domain. Our results emphasize the complex contributions of the regulatory and catalytic domains to the overall behavior of PKC.
Highlights
protein kinase C (PKC)␣ and -⑀ isoforms preferentially inhibited phospholipase C activity [3], whereas the PKC and -⑀ isoforms linked the mast cell high affinity receptor for IgE to the expression of c-fos and c-jun [4]
We have determined that these PKC chimeras can be stably expressed in NIH 3T3 cells, bind [3H]PDBu, and exhibit cofactordependent kinase activity as do the wild type PKC isozymes (Table I, Fig. 1)
A major objective is to dissect the factors regulating the flow of information through the families of PKC isoforms present in specific cell types
Summary
Vol 272, No 35, Issue of August 29, pp. 22148 –22153, 1997 Printed in U.S.A. The Catalytic Domain of Protein Kinase C Chimeras Modulates the Affinity and Targeting of Phorbol Ester-induced Translocation*. As part of our ongoing studies to define the factors contributing to the specificity of PKC isozymes, we prepared chimeras between the catalytic and regulatory domains of PKC␣, -␦, and -⑀. These chimeras, which preserve the overall structure of the native PKC enzymes, were stably expressed in NIH 3T3 fibroblasts. As part of our ongoing studies to explore the basis of specificity of PKC isozymes, we have prepared chimeras between the regulatory and the catalytic domains of PKC␣, -␦, and -⑀ and investigated their behavior in intact cells. We report here that the catalytic domain of PKC influences both the potency of phorbol ester for PKC translocation and the compartment to which PKC is translocated in response to phorbol ester
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