The hematopoietic growth factor erythropoietin upregulates the proto- oncogene c-myc via a protein kinase C (PKC) dependent pathway. Recently, it has been shown that this is due exclusively to activation of the PKC epsilon isoform. I hypothesize that PKC epsilon activation is mediated by activated (tyrosine-phosphorylated) phospholipase Cgamma (PLCgamma), and that PLC gamma is phosphorylated by the upstream transducer element JAK2, thus identifying a dual role for jAK2 in Epo signaling. PLCgamma activation by Epo will be shown by immunoprecipitation of PLC gamma followed by probing with anti-phosphotyrosine antibodies. The physical association of PLC gamma and JAK2 in Epo-treated cells will be documented by co-immunoprecipitation and western blotting. The functions of each member of the proposed JAK2 yields PLC gamma yields PKC epsilon yields to c-myc pathway will be elucidated with loss-of function experiments using antisense oligodeoxynucleotides. Finally, the role of this pathway in regulating Epo's mitogenic and anti-apoptotic actions will be studied. The results will provide a functional linkage between upstream signaling elements, downstream changes in c-myc expression, and erythroid cell growth.
