Constitutive Phosphorylation of GATA-1 at Serine.sup.26 Attenuates the Colony-Forming Activity of Erythrocyte-Committed Progenitors

We previously reported that IL-3 signaling induces phosphorylation of GATA-1 at the serine.sup.26 position, which contributes to IL-3-mediated anti-apoptotic response. Here, we demonstrate that phosphorylation of GATA-1 at serine.sup.26 is also transiently induced in cells of the erythroid lineage (primary erythroblasts and erythrocyte-committed progenitors [EPs]) by erythropoietin (EPO), the principal cytokine regulating erythropoiesis. To examine whether phosphorylation of GATA-1 at serine.sup.26 would have any impact on erythropoiesis, mutant mice carrying either a glutamic acid (GATA-1.sup.S26E) or alanine (GATA-1.sup.S26A) substitution at serine.sup.26 were generated. Neither GATA-1.sup.S26E nor GATA-1.sup.S26A mice showed any significant difference from control mice in peripheral blood cell composition under either steady state or stress conditions. The erythroblast differentiation in both mutant mice also appeared to be normal. However, a moderate reduction in the CFU-E progenitor population was consistently observed in the bone marrow of GATA-1.sup.S26E, but not GATA-1.sup.S26A mice, suggesting that such defect was compensated for within the bone marrow. Surprisingly, reduced CFU-E progenitor population in GATA-1.sup.S26E mice was mainly due to EPO-induced growth suppression of GATA-1.sup.S26E EPs, albeit in the absence of EPO these cells manifested a survival advantage. Further analyses revealed that EPO-induced growth suppression of GATA-1.sup.S26E EPs was largely due to the proliferation block resulted from GATA-1.sup.S26E -mediated transcriptional activation of the gene encoding the cell cycle inhibitor p21.sup.Waf1/Cip1 . Taken together, these results suggest that EPO-induced transient phosphorylation of GATA-1 at serine.sup.26 is dispensable for erythropoiesis. However, failure to dephosphorylate this residue following its transient phosphorylation significantly attenuates the colony-forming activity of EPs.