ERYTHROPOIETIN INDUCES THE TYROSINE PHOSPHORYLATION OF INSULIN-RECEPTOR SUBSTRATE-2 - AN ALTERNATE PATHWAY FOR ERYTHROPOIETIN-INDUCED PHOSPHATIDYLINOSITOL 3-KINASE ACTIVATION

In this report, we demonstrate that insulin receptor substrate-a (IRS- P) is phosphorylated on tyrosine following treatment of UT-7 cells wit h erythropoietin. We have investigated the expression of IRS-1 and IRS -S in several cell lines with erythroid and/or megakaryocytic features , and we observed that IRS-P was expressed in all cell lines tested. I n contrast, we did not detect the expression of IRS-1 in these cells. In response to erythropoietin, IRS-2 was immediately phosphorylated on tyrosine, with maximal phosphorylation between 1 and 5 min. Tyrosine- phosphorylated IRS-P was associated with phosphatidylinositol 3-kinase and with a 140-kDa protein that comigrated with the phosphatidylinosi tol-3,4,5-trisphosphate B-phosphatase, SHIP. Moreover, IRS-2 was const itutively associated with the erythropoietin receptor. We did not obse rve the association of IRS-2 with JAK2, Grb2, or PTP1D. Using BaF3 cel ls transfected with mutated erythropoietin receptors, we demonstrate t hat neither the tyrosine residues of the intracellular domain nor the last 109 amino acids of the erythropoietin receptor are required for e rythropoietin-induced IRS-P tyrosine phosphorylation. Altogether, our results indicate that erythropoietin-induced IRS-S tyrosine phosphoryl ation could account for the previously reported activation of phosphat idylinositol 3-kinase mediated by erythropoietin receptors mutated in the phosphatidylinositol 3-kinase-binding site.