Erythropoietin-induced serine 727 phosphorylation of STAT3 in erythroid cells is mediated by a MEK-, ERK-, and MSK1-dependent pathway

Objective. Erythropoietin (EPO) is a key regulator of erythropoiesis, playing a role in both the proliferation and differentiation of erythroid cells. One of the signal transduction molecules activated upon EPO stimulation is signal transducer and activator of transcription (STAT) 3. Besides tyrosine 705 phosphorylation of STAT3, serine 727 phosphorylation has been described upon EPO stimulation. In the present study, we investigated which molecular pathways mediate the STAT3 serine 727 phosphorylation and the functional implications of this phosphorylation. Methods. The EPO-dependent erythroid cell line ASE2 was used to investigate which signaling routes were involved in the STAT3 serine 727 phosphorylation. Western blotting using phosphospecific antibodies was used to assess the phosphorylation status of STAT3 molecules. Transfection analysis was performed to investigate the transactivational potential of STAT3, and quantitative RT-PCR was used to study the in vivo gene expression of STAT3-responsive genes. Results. Western blotting of extracts of cells exposed to various chemical inhibitors revealed that the MEK inhibitors PD98059 and U0126 abrogated the EPO-mediated STAT3 serine 727 phosphorylation without an effect on tyrosine phosphorylation. Further analysis showed that MSK1 is activated downstream of ERK, and retroviral transductions with kinase-inactive MSK1 revealed that MSK1 is necessary for STAT3 serine phosphorylation. Furthermore, the STAT3-mediated transactivation was reduced by blocking the STAT3 serine phosphorylation with the MEK inhibitor U0126 or by expression of kinase-inactive MSK1. Conclusions. The EPO-induced STAT3 serine 727 phosphorylation is mediated by a pathway involving MEK, ERK, and MSK1. Furthermore, serine phosphorylation of STAT3 augments the transactivational potential of STAT3. (C) 2003 International Society for Experimental Hematology. Published by Elsevier Science Inc.