EXPRESSION AND ACTIVATION OF PROTEIN-KINASE-C ISOFORMS IN A HUMAN MEGAKARYOCYTIC CELL-LINE

Megakaryocytes undergo a unique differentiation program, becoming poly ploid through repeated cycles of DNA synthesis without concomitant cel l division. We have shown previously that phorbol 12-myristate 13-acet ate (PMA) induces the Dami human megakaryocytic cell line to become po lyploid and to express platelet-specific proteins, including von Wille brand factor (VWF) and glycoprotein Ib (GpIb). Phorbol esters are thou ght to regulate gene expression principally through the activation of protein kinase C (PKC), a family of structurally related kinases with potentially unique activation requirements and substrate specificities . A survey of PKC isoforms in Dami cells revealed that, by both Wester n and Northern analyses, PKC isoforms alpha, beta, delta, epsilon, eta , theta, and zeta were reproducibly detected. PKC-gamma was not detect ed. In order to define the role of individual PKC isoforms in megakary ocytic maturation, PMA and 2-deoxyphorbol 13-phenylacetate 20-acetate (dPPA), a putative selective activator of the PKC-beta 1 isotype, were compared for their effects on Dami cell maturation. Treatment with ei ther dPPA or PMA caused Dami cells to cease proliferating, to become p olyploid, and to express vWF. We also examined dPPA and PMA for their ability to activate and to downregulate expression of different PKC is oforms. Fifteen-minute treatment with PMA resulted in the translocatio n of PKC isoforms alpha, epsilon, and theta from the cytosolic to the membrane fraction; twenty-four hour treatment resulted in the downregu lation of these isoforms. In contrast, dPPA was found to be a potent a ctivator of PKC-epsilon alone and exhibited weaker effects on alpha an d theta. These data suggest that PKC isoforms beta, delta, eta, and ze ta, which appear not to be activated by either phorbol ester, are unli kely to be primarily involved in megakaryocytic maturation in response to these agents. The isoforms that are translocated by both phorbol e sters-PKC isoforms alpha and theta, and particularly epsilon-are more likely to transduce the signals that stimulate Dami cell differentiati on.