ROLE OF PROTEIN-PHOSPHORYLATION IN EPO-MEDIATED EARLY SIGNAL-TRANSDUCTION - ANALYSIS IN THE EPO-REACTIVE CELL-LINE ELM-I-1 TRANSFECTED WITHA C-FOS-ENHANCER PROMOTER-LUCIFERASE REPORTER GENE/

To investigate the role of protein phosphorylation in the early phase of EPO-mediated signal transduction, we EPO-stimulated a murine erythr oid cell line ELM-I-1 transformed by plasmids comprised of the c-fos e nhancer/promoter linked to the luciferase gene. Using this reporter ge ne system, we previously showed that EPO-induced activation of the c-f os promoter can be detected rapidly and sensitively as an elevation of cellular luciferase activity. In this study, we first examined the ro le of protein tyrosine phosphorylation. The tyrosine phosphatase inhib itor orthovanadate not only induced luciferase activity by itself but enhanced the action of EPO. On the other hand, the tyrosine kinase inh ibitors erbstatin and herbimycin suppressed the effect of EPO. Next, t he role of protein kinase C (PKC) in the EPO response was assessed. Th e PKC activator phorbol myristate acetate (PMA) not only induced lucif erase activity by itself but enhanced the action of Epo. On the other hand, the PKC inhibitor 1-(5-isoquinolynyl-sulfonyl)-2-methylpiperazin e (H-7) suppressed the effect of Epo and PMA, whereas a nonspecific pr otein kinase inhibitor, N-(2-Guanidinoethyl)-5-Isoquinolinesulfornamin e (HA(1004)) inhibited the action of neither Epo nor PMA. Another know n PKC inhibitor staurosporine (STSP) did not inhibit but rather enhanc ed the effect of Epo. This action of STSP was blocked by H-7 but not b y HA(1004). These results suggest that the EPO-mediated early signal t ransduction pathway leading to c-fos expression involves protein-tyros ine phosphorylation, is modulated by tyrosine phosphatase activity and is positively regulated by PKC.