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.
Wierenga
Albertus T J
AT
University Hospital Groningen, Department of Hematology, Groningen, The Netherlands.
Vogelzang
Irma
I
Eggen
Bart J L
BJ
Vellenga
Edo
E
D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004789:Enzyme Activation; D015672:Erythroid Precursor Cells / Q000378:metabolism*; D004921:Erythropoietin / Q000494:pharmacology*; D006801:Humans; D048728:MAP Kinase Kinase Kinase 1; D008894:Milk Proteins; D020928:Mitogen-Activated Protein Kinases / Q000502:physiology*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D038744:Ribosomal Protein S6 Kinases, 90-kDa / Q000502:physiology*; D050796:STAT3 Transcription Factor; D050799:STAT5 Transcription Factor; D012694:Serine / Q000378:metabolism*; D015534:Trans-Activators / Q000378:metabolism*; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D008894:Milk Proteins; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D050799:STAT5 Transcription Factor; D015534:Trans-Activators; D004921:Erythropoietin; D012694:Serine; D017346:Protein Serine-Threonine Kinases; D038744:Ribosomal Protein S6 Kinases, 90-kDa; C116919:mitogen and stress-activated protein kinase 1; D020928:Mitogen-Activated Protein Kinases; D048728:MAP Kinase Kinase Kinase 1; C482576:MAP3K1 protein, human