Src transduces erythropoietin-induced differentiation signals through phosphatidylinositol 3-kinase

In this study, we examined the molecular mechanism of erythropoietin-initia ted signal transduction of erythroid differentiation through Src and phosph atidylinositol 3-kinase (Pi3-kinase). Antisense oligonucleotides against sr c but not lyn inhibited the formation of erythropoietin-dependent colonies derived from human bone marrow cells and erythropoietin-induced differentia tion of K562 human erythroleukaemia cells. Antisense p85 alpha oligonucleot ide or LY294002, a selective inhibitor of PI3-kinase, independently inhibit ed the formation of erythropoietin-dependent colonies. In K562 cells, Src a ssociated with PI3-kinase in response to erythropoietin. Antisense src RNA expression in K562 cells inhibited the erythropoietin-induced activation of PI3-kinase and its association with erythropoietin receptor. PPI, a select ive inhibitor of the Src family, reduced erythropoietin-induced tyrosine ph osphorylation. of erythropoietin receptor and its association with PI3-kina se in F-36P human erythroleukaemia cells. The coexpression experiments and in vitro kinase assay further demonstrated that Src directly tyrosine-phosp horylated erythropoietin receptor, and associated with PI3-kinase. In vitro binding experiments proved that glutathione S-transferase-p85 alpha N- or C-terminal SH2 domains independently bound to erythropoietin receptor, whic h was tyrosine-phosphorylated by Src. Taken together, Src transduces the er ythropoietin-induced erythroid differentiation signals by regulating PI3-ki nase activity.