Protein kinase C (PKC) is an integral part of a major signal transduction pathway for external stimuli. PKC has been implicated in the proliferation and differentiation of both normal and leukemic hematopoietic cells. Phorbol esters (i.e., PMA), which bind to and activate PKC directly, can induce terminal differentiation of both promyelocytic leukemia HL-60 and erythroleukemia K562 cells. In contrast, bryostatin 1 (bryo), a non- phorbol ester PKC activator, stimulates continued cell division and blocks the cytostatic effects of PMA in these cell lines. The divergent effects of PMA and bryo correlate with the differential translocation and activation of alpha and beta11 PKC in response to PMA and bryo. PMA and bryo activate both alpha and beta11 PKC, however, bryo but not PMA causes selective translocation and activation of beta11 PKC at the nucleus. In the proposed studies the expression, cellular distribution and activation state of the major PKC isotypes (alpha, beta1, beta11, gamma and epsilon) will be examined in K562 cells during proliferation and PMA-induced megakaryocytic differentiation. The role of individual PKC isotypes in proliferation and differentiation will be probed by assessing the effect of overexpressing and inhibiting the expression of individual PKC isotypes on the proliferative and differentiation capacity of K562 cells. The molecular basis for the selective translocation and activation of beta11 PKC at the nucleus in response to bryo will be assessed by constructing and expressing alpha/beta11 PKC chimeras in K562 cells. PKC chimeras will be assayed for translocation and activation phenotype in response to PMA and bryo in intact cells. Likewise, PKC chimeras will be expressed in the baculovirus expression system, purified from Sf9 insect cells and characterized for co-factor requirements, activator responsiveness and substrate specificity in-vitro. The aims of this proposal will be achieved using a combination of biochemical, immunological, pharmacological and molecular biological approaches.