Thrombopoietin induces association of Crk1 with STAT5 but not STAT3 in human platelets

Crkl, a 39-kD SH2, SH3 domain-containing adapter protein, is constitutively tyrosine phosphorylated in hematopoietic cells from chronic myelogenous le ukemia (CML) patients. We recently reported that thrombopoietin induces tyr osine phosphorylation of Crkl in normal platelets. In this study, we demons trate that thrombopoietin induces association of Crkl with a tyrosine phosp horylated 95- to 100-kD protein in platelets and in UT7/TPO cells, a thromb opoietin-dependent megakaryocytic cell line. With specific antibodies again st STATE, we demonstrate that the 95- to 100-kD protein in Crkl immunopreci pitates is STAT5. This coimmunoprecipitation was specific in that Crkl immu noprecipitates do not contain STATE, although STATE becomes tyrosine phosph orylated in thrombopoietin-stimulated platelets. The coimmunoprecipitaion o f Crkl with STATE was inhibited by the immunizing peptide for Crkl antisera or phenyl phosphate (20 mmol/L). After denaturing of Crkl immunoprecipitat es, Crkl was still immunoprecipitated by Crkl antisera. However, coimmunopr ecipitation of STATE was not observed. Coincident with STATE tyrosine phosp horylation, thrombopoietin induces activation of STATE DNA-binding activity as demonstrated by electrophoretic mobility shift assays (EMSA). Using a p -casein promoter STAT5 binding site as a probe, we have also demonstrated t hat Crkl antisera supershift the STAT5-DNA complex, suggesting that Crkl is a component of the complex in the nucleus. Furthermore, interleukin-3 (IL- 3), granulocyte-macrophage colony-stimulating factor (GMCSF), and erythropo ietin also induce Crkl-STAT5 complex formation in responding cells in a sti mulation-dependent manner. In vitro, glutathione S-transferase (GST)-Crkl b ound to STATE inducibly through its SH2 domain. These results indicate that thrombopoietin, IL-3, GM-CSF, and erythropoietin commonly induce associati on of STAT5 and Crkl and that the complex translocates to the nucleus and b inds to DNA. Interestingly, such association between STAT5 and Crkl was not observed in cytokine-stimulated murine cells, suggesting an intriguing pos sibility that components of the human STAT5-DNA complex may be different fr om those of the murine counterpart. (C) 1998 by The American Society of Hem atology.