Journal of Cell Science 101, 671-679 (1992) Printed in Great Britain © The Company of Biologists Limited 1992 671 Protein kinase C isotypes in human erythroleukemia cell proliferation and differentiation BARBARA A. HOCEVAR 1 , DWIGHT M. MORROW 2 , MARK L. TYKOCINSKI 2 and ALAN P. FIELDS 1 * 'Department of Pharmacology and 2 Pathology, Case Western Reserve University School of Medicine, 2119 Abington Road, Cleveland, Ohio 44106, USA •Author for correspondence Summary The human erythroleukemia (K562) cell line is induced to differentiate into megakaryocytic cells by treatment with the tumor promoter phorbol myristate acetate (PMA). PMA-induced differentiation is characterized by (1) almost complete cessation of cellular proliferation, (2) expression of the megakaryocytic cell surface marker glycoprotein Ilb/nia (gpHIa), (3) increased secretion of granulocyte/macrophage-colony stimulating factor (GM-CSF) and (4) increased secretion of interleukin-6 (TL-6). PMA-induced differentiation is dose-dependent with maximal activity seen at 10 nM PMA. In contrast, bryostatin (bryo), a structurally distinct protein kinase C (PKC) activator, fails to induce megakaryocytic differentiation or growth arrest at the concentrations tested (0.01-100 nM). Rather, bryo inhibits PMA- induced growth arrest and megakaryocytic differen- tiation in a dose-dependent fashion (full inhibition at 100 nM). The divergent biological effects of PMA and bryo correspond to the differential activation and translo- cation of PKC isotypes in K562 cells. PKC isotype analysis demonstrates that undifferentiated cells express both a and fa PKC but no detectable ft, yorf PKC. Treatment of cells with either PMA or bryo leads to rapid translocation of both a and fa PKC from the cytosol to the non-nuclear particulate fraction. How- ever, bryo also induces selective translocation of fa PKC to the nuclear membrane. Nuclear fa PKC is function- ally active as evidenced by the time-dependent phos- phorylation of lamin B, a previously identified nuclear PKC substrate. These data indicate that the divergent effects of PMA and bryo on erythroleukemia cell proliferation and differentiation correspond to differen- tial activation of fa PKC at the nuclear membrane. Nuclear activation of fa PKC by bryo appears to generate a dominant, proliferative signal that overrides the PMA-induced differentiation signal. Therefore, the a and fa PKC isotypes exhibit distinct translocation and activation profiles during megakaryocytic differen- tiation and proliferation, indicating that they play distinct roles in these cellular processes. Key words: lamin B phosphorylation, bryostatin 1, nuclear protein kinase C. Introduction The Ca 2+ - and phospholipid-dependent protein kinase, protein kinase C (PKC), functions in cell surface signal transduction for a variety of external stimuli related to cellular proliferation and differentiation (reviewed by Nishizuka, 1986; Coussens et al., 1986). Molecular cloning studies demonstrate that PKC belongs to a multigene family containing at least nine distinct isotypes (Nishizuka, 1986; Coussens et al., 1986; Parker et al., 1986; Knopf et al., 1986; Ohno et al., 1987; Ono et al., 1988; Nishizuka, 1988). Though the PKC isotypes have distinct kinetic and immunological properties (Huang et al., 1986) little is known about the specific functions of the individual isotypes in whole cells. PKC isotypes exhibit tissue-specific (Nishizuka, 1988; Brandt et al., 1987) and regio-specific expression patterns (Nishizuka, 1988), suggesting that different isotypes function to regulate the wide variety of PKC-mediated cellular processes observed in different cell types (Brandt et al., 1987). Activation of PKC has been implicated in the proliferation and differentiation of both normal and leukemic hematopoietic cells. In the human promyelo- cytic leukemia (HL60) cell line, PKC isotype levels and activities change during dimethylsulfoxide (DMSO)- and l<*,25-dihydroxyvitamin D 3 -induced differentiation (Makowske et al., 1988; Solomon et al., 1991). Likewise, both hexamethylene bisacetamide (HMBA)- and DMSO-induced erythroid differentiation of murine erythroleukemia cells are accompanied by changes in PKC isotype levels and activity (Melloni et al., 1987;