The catalytic domain of protein kinase Cδ confers protection from down- regulation induced by Bryostatin 1

Abstract

Bryostatin 1 (Bryo) has been shown to induce biphasic dose-response curves for down-regulating protein kinase Cδ (PKCδ) as well as for protecting PKCδ from down-regulation induced by phorbol 12-myristate 13- acetate (PMA). To identify regions within PKCδ that confer these responses to Bryo, we utilized reciprocal PKCα and PKCδ chimeras (PKCα/δ and PKCδ/α) constructed by exchanging the regulatory and catalytic domains of these PKCs. These chimeras and wild-type PKCα/α and PKCδ/δ constructed in the same way were stably expressed in NIH 3T3 fibroblasts. Twenty-four h of treatment with Bryo induced a biphasic dose-response curve for down- regulating both wild-type PKCδ/δ and the PKCα/δ chimera. In contrast, Bryo led to a nearly complete down-regulation of both PKCα/α and PKCδ/α and also produced a faster mobility form of these species on SDS- polyacrylamide gel electrophoresis. The nature of both the regulatory and, to a lesser extent, the catalytic domains affected the potency of Bryo to down- regulate the chimeric PKC proteins as well as to protect PKCα/δ and PKCδ/δ from down-regulation. Bryo at high concentrations also inhibited the down-regulation of PKCδ/δ and PKCα/δ induced by 1 μM PMA when co- applied. The portion of PKC protected by Bryo from down-regulation by either Bryo or PMA was localized in the particulate fraction of the cells. We conclude that the catalytic domain of PKCδ confers protection from down- regulation induced by Bryo or Bryo plus PMA, suggesting that this domain contains the isotype-specific determinants involved in the unique effect of Bryo on PKCδ.