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Differential Roles of the Tandem C1 Domains of Protein Kinase C in the Biphasic Down-Regulation Induced by Bryostatin 1

Laboratory of Cellular Carcinogenesis and Tumor Promotion [P. S. L., K. B., K. M. H., M. B., D. B., P. M. B.], and Laboratory of Experimental Carcinogenesis [S. H. G.], National Cancer Institute, NIH, Bethesda, Maryland 20892, and Cancer Research Institute, Arizona State University, Tempe, Arizona 85287 [G. R. P.]

Bryostatin 1 (Bryo), currently in clinical trials, has been shown to induce a biphasic concentration-response curve for down-regulating protein kinase C (PKC) , with protection of the enzyme from down-regulation at high Bryo doses. In our ongoing studies to identify the basis for this unique behavior of PKC, we examined the participation of the two ligand binding sites (C1a and C1b) in the regulatory domain of the enzyme. Three mutants of PKC prepared by introducing a point mutation in either C1a or C1b or both C1a and C1b were overexpressed in NIH 3T3 cells. All of the constructs retained a biphasic response to down-regulation assessed after 24-h treatment with Bryo. However, the roles of the individual C1 domains were different for the two phases of the response. For down-regulation, both the C1a and the C1b mutants displayed equivalent 3–4-fold reductions in their affinities for the ligand. For protection from down-regulation, a reduced protection was observed for the C1a mutant, which showed a broader biphasic curve compared with those for wild-type PKC and the C1b mutant. Like wild-type PKC, all of the mutants showed the same subcellular partitioning of the protected enzyme to the particulate fraction of the cells, arguing against changes in sensitivity to Bryo due to differences in localization. Likewise, relatively similar patterns of localization were observed using green fluorescent protein-PKC constructs. We conclude that the C1 domains of PKC do not have equivalent roles in inducing protection against Bryo-induced down-regulation. The C1a domain plays a critical role in conferring the degree of protection at high concentrations of Bryo. Elucidation of the differential effect of Bryo on PKC may suggest strategies for the design of novel ligands with Bryo-like activities.

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