Erythropoietin induces tyrosine phosphorylation of Jak2, STAT5A, and STAT5B in primary cultured human erythroid precursors.
We examined signaling by erythropoietin in highly purified human colony forming unit-erythroid cells, generated in vitro from CD34(+) cells. We found that erythropoietin induces tyrosine phosphorylation of Jak2, STAT5A, and STAT5B. Tyrosine phosphorylation of Jak2 reaches a peak around 10 minutes after stimulation and is maximum at 5 U/mL of erythropoietin. Tyrosine phosphorylation of STAT5 is accompanied by the translocation of activated STAT5 to the nucleus as shown by electrophoretic mobility shift assay (EMSA) using 32Pi-labeled STAT5 binding site in the beta-casein promoter. Tyrosine phosphorylation STAT1 or STAT3 was not detected in human erythroid precursors after stimulation with erythropoietin. Crkl, an SH2/SH3 adapter protein, becomes coimmunoprecipitated specifically with STAT5 from erythropoietin-stimulated erythroid cells; although it was shown to become associated with c-Cbl in the studies using cell lines. Thus, human erythroid precursors can be expanded in vitro in sufficient numbers and purity to allow its usage in signal transduction studies. This report sets a basis for further studies on signaling in primary cultured human erythroid precursors, which in turn contribute to our better understanding in the differentiation processes of erythrocytes and their precursors.
Oda
A
A
Division of Hematology, Department of Internal Medicine, Keio University, Tokyo, Japan.
Sawada
K
K
Druker
B J
BJ
Ozaki
K
K
Takano
H
H
Koizumi
K
K
Fukada
Y
Y
Handa
M
M
Koike
T
T
Ikeda
Y
Y
D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004900:Erythroblasts / Q000378:metabolism*; D004921:Erythropoietin / Q000494:pharmacology*; D006801:Humans; D053614:Janus Kinase 2; D008894:Milk Proteins; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins; D050799:STAT5 Transcription Factor; D015398:Signal Transduction / Q000187:drug effects*; D015534:Trans-Activators / Q000378:metabolism*; D025521:Tumor Suppressor Proteins; D014443:Tyrosine / Q000378:metabolism
D004268:DNA-Binding Proteins; D008894:Milk Proteins; D011518:Proto-Oncogene Proteins; D050799:STAT5 Transcription Factor; C494092:STAT5A protein, human; C494095:STAT5B protein, human; D015534:Trans-Activators; D025521:Tumor Suppressor Proteins; D004921:Erythropoietin; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; C507924:JAK2 protein, human; D053614:Janus Kinase 2