Bryostatin 1 protects protein kinase C-δ from down-regulation in mouse keratinocytes in parallel with its inhibition of phorbol ester-induced differentiation

Bryostatin 1 and phorbol-12-myristate-13-acetate (PMA) are both potent activators of protein kinase C (PKC), although in primary mouse keratinocytes bryostatin 1 does not induce differentiation and blocks PMA-induced differentiation. We report here that in primary mouse keratinocytes PMA caused translocation of PKC-ε to the Triton X-100-soluble fraction with an approximately 2-order of magnitude higher potency, compared with translocation of PKC-α and PKC-δ. The kinetics of translocation were fastest for PKC-ε, slower for PKC-α, and slowest for PKC-δ. At 5-20 min bryostatin 1 showed potency similar to that of PMA for translocating PKC-α, higher potency for translocating PKC-δ, and lower potency for translocating PKC-ε. At a later time (6 hr), bryostatin 1 was 1-2 orders magnitude more potent than PMA for causing loss of PKC-α, -δ, and -ε from the soluble fraction. Bryostatin 1 was 40-fold more potent than PMA for down-regulating PKC-α and showed a biphasic dose-response curve for down-regulating PKC-δ. Bryostatin 1 at 0.1-1 nM down-regulated PKC-δ to a similar extent as did PMA. Bryostatin 1 at 100 nM to 1 μM, on the other hand, failed to induce down-regulation, and these high (100 nM to 1 μM) doses of bryostatin 1 showed noncompetitive inhibition of PKC-δ down-regulation by 1 μM PMA after coapplication. This protected portion of PKC-δ retained kinase activity. The dose-response curve for bryostatin 1 protection of PKC-δ from down- regulation by PMA correlated with bryostatin 1 inhibition of the effects of PMA on cornified envelope formation (a marker of differentiation) and epidermal growth factor binding. Although PKC-ε was readily translocated by both PMA and bryostatin 1, the PKC-ε originally associated with the particulate fraction showed no down-regulation by either of these agents. We hypothesize that differential regulation of PKC isozymes by PMA and bryostatin 1 may contribute to the different patterns of biological responses that they induce.