ERYTHROPOIETIN INDUCES TYROSINE PHOSPHORYLATION OF JAK2, STAT5A, AND STAT5B IN PRIMARY CULTURED HUMAN ERYTHROID PRECURSORS

We examined signaling by erythropoietin in highly purified human colon y forming unit-erythroid cells, generated in vitro from CD34(+) cells. We found that erythropoietin induces tyrosine phosphorylation of Jak2 , STAT5A, and STATES. Tyrosine phosphorylation of Jak2 reaches a peak around 10 minutes after stimulation and is maximum at 5 U/mL of erythr opoietin. Tyrosine phosphorylation of STATE is accompanied by the tran slocation of activated STATE to the nucleus as shown by electrophoreti c mobility shift assay (EMSA) using (32)Pi-labeled STATE binding site in the p-casein promoter. Tyrosine phosphorylation STAT1 or STAT3 was not detected in human erythroid precursors after stimulation with eryt hropoietin. Crkl, an SH2/SH3 adapter protein, becomes coimmunoprecipit ated specifically with STATE from erythropoietin-stimulated erythroid cells; although it was shown to become associated with c-Cbl in the st udies using cell lines. Thus, human erythroid precursors can be expand ed in vitro in sufficient numbers and purity to allow its usage in sig nal 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 pr ocesses of erythrocytes and their precursors. (C) 1998 by The American Society of Hematology.