Elucidation of signal transduction induced by thrombopoietin.

我们最近报道血小板生成素诱导正常血小板中Crkl的酪氨酸磷酸化。在这项研究中，我们证明，血小板生成素诱导协会的Crkl与酪氨酸磷酸化的95- 100-kD蛋白在血小板和UT 7/TPO细胞，血小板生成素依赖性巨核细胞系。利用针对STAT 5的特异性抗体，我们证明Crkl免疫沉淀物中的95至100 kD蛋白是STAT 5。以β-酪蛋白启动子STATS结合位点为探针，我们还证明了Crkl抗血清能使STATS-DNA复合物发生超移位，提示Crkl是细胞核中STATS-DNA复合物的组成部分，而且白细胞介素-3（IL-3）、粒细胞-巨噬细胞集落刺激因子（GM-CSF）和促红细胞生成素也能以刺激依赖的方式诱导应答细胞中Crkl-STATS复合物的形成。在体外，谷胱甘肽5-转移酶（GST）-Crkl通过其5 H2结构域与STATS诱导结合.我们已经报道了胶原蛋白和血小板生成素诱导人血小板中Wiskott-Aldrich综合征蛋白的酪氨酸磷酸化。我们在体外由CD 34（+）细胞产生的高度纯化的人集落形成单位红细胞中检测了促红细胞生成素的信号传导。我们已经报道了凝血酶产生截短形式的TPO可能是血小板相关的TPO代谢的一个值得注意的事件。我们已经表明，血小板生成素和其他激动剂可以通过不同的机制诱导Vav的酪氨酸磷酸化，并且Vav也可以通过其重新分布到细胞骨架而参与血小板聚集期间的信号传导。

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.