| Erythropoietin (EPO), the major cytokine regulator of red blood cell production, activates many different signal transduction pathways upon engagement of its receptor (EPO-R). Gene targeting studies have revealed that deletion of EPO, EPO-R, or the tyrosine kinase JAK2 results in embryonic lethality around day 12.5 due to hematopoietic defects. Hence, tyrosine phosphorylation of EPO-R and/or downstream substrates of the EPO-R are critical for normal erythroid development.; Early structure-function studies that examined regions of the EPO-R required for growth, differentiation and cell survival focused on tyrosine 343. Mutation of EPO-R Y343 impaired EPO-dependent growth and blocked EPO-dependent CFU-E development. STAT5 is an important JAK2 substrate and is able to bind to Y343, yet STAT5a/b-/- mice have a mild erythroid phenotype. Therefore, we initiated a Cloning of Ligand Target (COLT) screen to identify proteins that interact with EPO-R Y343 in an SH2 domain-dependent manner. One of the clones isolated encodes PLCgamma1. Our studies have demonstrated that PLCgamma1 and the related protein PLCgamma2 bind multiple EPO-R tyrosines in response to EPO stimulation, but have a critical requirement for EPO-R Y479. Activation of ERK1/2 by EPO is also linked to EPO-R Y479. We have evidence that PLCgamma1 constitutively associates with the Grb2/SOS2 complex, providing a novel mechanism for EPO-dependent ERK1/2 activation.; EPO has been reported to activate STAT1, STAT3, and STAT5a/b transcription factors in erythroid cell lines. To date, studies have focused on STAT5 as the primary target of EPO-activated JAK2. It was recently reported that EPO-R cytoplasmic tyrosines are dispensable for viability in vivo. Interestingly, we have shown that activation of STAT1 by EPO does not require EPO-R cytoplasmic tyrosines. Therefore, we decided to examine erythropoiesis in STAT1-deficient mice. Clonogenic assays demonstrated that STAT1-/- mice had increased numbers of erythroid progenitors in the spleen, and that these progenitors were hyperresponsive to EPO. Signalling experiments also revealed increased EPO-dependent phosphorylation of STAT5a/b, ERK1/2, and PKB in STAT1-/- mice. Although hematological parameters were normal, there was delayed differentiation and increased apoptosis of erythroblasts in STAT1-deficient bone marrow and spleen. These results reveal a novel function of STAT1 as an important regulator of erythropoiesis. |
