Elucidation of signal transduction induced by thrombopoietin.

我们最近报道了促血小板生成素诱导正常血小板CRK1酪氨酸磷酸化。在这项研究中，我们证明了在血小板和UT7/TPO细胞中，血小板生成素诱导CRK1与酪氨酸磷酸化的95-LOO-KD蛋白相关联，UT7/TPO是一种依赖于血小板生成素的巨核细胞系。利用针对STAT5的特异性抗体，我们证明了CRK1免疫沉淀物中的95-to LOO-KD蛋白是STAT5。以β-酪蛋白启动子STATS结合位点为探针，我们还证明了Crk1抗血清使STATS-DNA复合体发生超位，提示Crk1是核内该复合体的一个组成部分。此外，白介素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以刺激依赖的方式在反应细胞中诱导Crk1-STATS复合体的形成。在体外，谷胱甘肽5-转移酶(GST)-CRK1通过其5H2结构域与STATS诱导结合。我们已报道胶原蛋白和血小板生成素诱导人血小板Wiskott-Aldrich综合征蛋白酪氨酸磷酸化。我们在高纯度的人类集落形成单位--红系细胞中检测了促红细胞生成素的信号，红系细胞是从CD34(+)细胞体外产生的。我们已经报道了凝血酶产生截短形式的TPO可能是一个值得注意的事件，因为TPO与血小板相关的代谢。我们已经证明，促血小板生成素和其他激动剂可能通过不同的机制诱导Vav的酪氨酸磷酸化，Vav也可能通过其重新分布到细胞骨架而参与血小板聚集过程中的信号传递。

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.