Ship1 (SH2 inositol 5-phosphatase 1) has been shown to be a target of tyrosine phosphorylation downstream of cytokine and immunoregulatory receptors. In addition to its catalytic activity on phosphatidylinositol substrates, it can serve as an adaptor protein in binding Shc and Grb2. Erythropoietin (EPO), the primary regulator of erythropoiesis, has been shown to activate the tyrosine phosphorylation of Shc, resulting in recruitment of Grb2. However, the mechanism by which the erythropoietin receptor (EPO-R) recruits Shc remains unknown. EPO activates the tyrosine phosphorylation of Ship1, resulting in the interdependent recruitment of Shc and Grb2. Ship1 is recruited to the EPO-R in an SH2-dependent manner. Utilizing a panel of EPO-R deletion and tyrosine mutants, we have discovered remarkable redundancy in Ship1 recruitment. EPO-R Tyr401 appears to be a major site of Ship1 binding; however, Tyr429 and Tyr431 can also serve to recruit Ship1. In addition, we have shown that EPO stimulates the formation of a ternary complex consisting of Ship1, Shc, and Grb2. Ship1 may modulate several discrete signal transduction pathways. EPO-dependent activation of ERK1/2 and protein kinase B (PKB)/Akt was examined utilizing a panel of EPO-R deletion mutants. Activation of ERK1/2 was observed in EPO-RΔ99, which retains only the most proximal tyrosine, Tyr343. In contrast, EPO-dependent PKB activation was observed in EPO-RΔ43, but not in EPO-RΔ99. It appears that EPO-dependent PKB activation is downstream of a region that indirectly couples to phosphatidylinositol 3-kinase.
This work was supported in part by Medical Research Council of Canada Grant MT 13612 and by the National Cancer Institute of Canada and the University of Toronto Faculty of Medicine Dean's Research Funds (all to D. L. B.). This work was performed in partial fulfillment of the requirements for a Ph.D. degree from the University of Toronto (J. M. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
