Differential roles of the tandem C1 domains of protein kinase C delta in the biphasic down-regulation induced by bryostatin 1

Bryostatin 1 (Bryo), currently in clinical trials, has been shown to induce a biphasic concentration-response curve for down-regulating protein kinase C (PKC) delta, with protection of the enzyme from down-regulation at high Bryo doses. In our ongoing studies to identify the basis for this unique be havior of PKC delta, we examined the participation of the two ligand bindin g sites (C1a and C1b) in the regulatory domain of the enzyme. Three mutants of PKC delta prepared by introducing a point mutation in either C1a or C1b or both C1a and C1b were overexpressed in NM 3T3 cells. All of the constru cts retained a biphasic response to down-regulation assessed after 24-h tre atment with Bryo. However, the roles of the individual C1 domains were diff erent 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 affi nities for the ligand. For protection from down-regulation, a reduced prote ction was observed for the C1a mutant, which showed a broader biphasic curv e compared with those for wild-type PKC delta and the C1b mutant. Like wild -type PKC delta, all of the mutants showed the same subcellular partitionin g 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 delta constructs. We conclude that the C1 dom ains of PKC delta do not have equivalent roles in inducing protection again st Bryo-induced down-regulation. The Cia domain plays a critical role in co nferring the degree of protection at high concentrations of Bryo. Elucidati on of the differential effect of Bryo on PKC delta may suggest strategies f or the design of novel ligands with Bryo-like activities.