Critical role for PI 3-kinase in the control of erythropoietin- induced erythroid progenitor proliferation. Short title: PI 3-kinase controls erythroid progenitor proliferation

The production of red blood cells is tightly regulated by erythropoietin (Epo). The phosphoinositide 3-kinase (PI 3-kinase) pathway was previously shown to be activated in response to erythropoietin (Epo). We studied the role of this pathway in the control of Epoinduced survival and proliferation of primary human erythroid progenitors. We show that PI 3-kinase associates with four tyrosine phosphorylated proteins in primary human erythroid progenitors, namely IRS2, SHIP, Gab1 and the Epo receptor (EpoR). Using different in vitro systems, we demonstrate that three alternative pathways independently lead to Epo-induced activation of PI 3-kinase and its downstream effectors, Akt, FKHRL1 and P70S6 kinase: through direct association of PI 3-kinase to the last tyrosine residue (Tyr 479) of the Epo receptor (EpoR), through recruitment and phosphorylation of Gab proteins via either Tyr 343 or Tyr 401 of the EpoR or through phosphorylation of IRS2 adaptor protein. The MAP kinase pathway was also activated by Epo in erythroid progenitors but we found that this process is independent of PI 3-kinase activation. In erythroid progenitors, the functional role of PI 3kinase was both to prevent apoptosis and to stimulate cell proliferation in response to Epo stimulation. Finally, our results show that PI 3-kinase-mediated proliferation of erythroid progenitors in response to Epo mainly occurs through modulation of the E3 ligase SCF which, in turn downregulates p27 CDK inhibitor via proteasome degradation. E-Mail: [email protected] only. For personal use at PENN STATE UNIVERSITY on February 23, 2013. bloodjournal.hematologylibrary.org From