Elucidation of signal transduction induced by thrombopoietin.

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I.We recently reported that thrombopoietin induces tyrosine phosphorylation of Crkl in normal platelets. In this study, we demonstrate that thrombopoietin induces association of Crkl with a tyrosine phosphorylated 95- to lOO-kD protein in platelets and in UT7/TPO cells, a thrombopoietin-dependent megakaryocytic cell line. With specific antibodies against STAT5, we demonstrate that the 95- to lOO-kD protein in Crkl immunoprecipitates is STAT5. Using a beta-casein promoter STATS binding site as a probe, we have also demonstrated that Crkl antisera supershift the STATS-DNA complex, suggesting that Crkl is a component of the complex in the nucleus.Furthermore, interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and erythropoietin also induce Crkl-STATS complex formation in responding cells in a stimulation-dependent manner. In vitro, glutathione 5-transferase (GST)-Crkl bound to STATS inducibly through its 5H2 domain.2. We have reported that collagen and thrombopoietin induce tyrosine phosphorylation of Wiskott-Aldrich syndrome protein in human platelets.3. We examined signaling by erythropoietin in highly purified human colony forming unit-erythroid cells, generated in vitro from CD34(+) cells.4. We have reported the generation of truncated forms of TPO by thrombin may be a notable event in view of the platelet-related metabolism of TPO.5. We have shown that thrombopoietin and other agonists may induce tyrosine phosphorylation of Vav by different mechanisms and Vav may also be involved in signaling during platelet aggregation by its redistribution to the cytoskeleton.