Odai H, Sasaki K, Iwamatsu A, Hanazono Y, Tanaka T, Mitani K, Yazaki Y, Hirai H
Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Japan.
J Biol Chem. 1995 May 5;270(18):10800-5. doi: 10.1074/jbc.270.18.10800.
Granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (Epo) are hematopoietic growth factors that regulate proliferation and differentiation of hematopoietic cells. They elicit and control a cascade of biochemical events, the earliest of which is tyrosine phosphorylation of several cellular proteins. Grb2/Ash is composed of SH2 and SH3 domains. The SH2 domain binds to tyrosine-phosphorylated proteins, and the SH3 domains bind to proteins containing proline-rich regions. It is considered that Grb2/Ash functions as an adapter protein linking tyrosine kinases and Ras in downstream of receptors for growth factors in fibroblasts. However, the mechanisms of signal transduction through Grb2/Ash and the roles of proteins associated with Grb2/Ash remain to be determined in hematopoietic cells. By means of the binding experiments using the glutathione S-transferase fusion protein including the full-length Grb2/Ash, we have found that Shc and unidentified 130- and 135-kDa proteins are associated with Grb2/Ash and that they are tyrosine phosphorylated by treatment with GM-CSF or Epo in a human leukemia cell line, UT-7. We have purified the 130-kDa protein (pp130) using the glutathione S-transferase-Grb2/Ash affinity column. The amino acid sequence analysis of the three peptides derived from the in situ protease digestion of the purified pp130 showed that the pp130 was identical to the human c-cbl proto-oncogene product (c-Cbl). c-Cbl constitutively binds to the SH3 domain of Grb2/Ash both in vitro and in vivo but not to the SH2 domain of Grb2/Ash, and the binding of Grb2/Ash to c-Cbl or Sos was not altered by GM-CSF stimulation. Moreover, c-Cbl (pp130) becomes tyrosine phosphorylated rapidly and transiently depending on GM-CSF or Epo stimulation. These findings strongly suggest that c-Cbl is implicated in the signal transduction of GM-CSF or Epo in hematopoietic cells and that c-Cbl is involved in another signaling pathway different from the Ras signaling pathway.
粒细胞-巨噬细胞集落刺激因子（GM-CSF）和促红细胞生成素（Epo）是调节造血细胞增殖和分化的造血生长因子。它们引发并控制一系列生化事件，其中最早的是几种细胞蛋白的酪氨酸磷酸化。Grb2/Ash由SH2和SH3结构域组成。SH2结构域与酪氨酸磷酸化蛋白结合，SH3结构域与富含脯氨酸区域的蛋白结合。据认为，Grb2/Ash在成纤维细胞生长因子受体下游作为连接酪氨酸激酶和Ras的衔接蛋白发挥作用。然而，在造血细胞中，通过Grb2/Ash的信号转导机制以及与Grb2/Ash相关蛋白的作用仍有待确定。通过使用包含全长Grb2/Ash的谷胱甘肽S-转移酶融合蛋白进行结合实验，我们发现在人白血病细胞系UT-7中，Shc以及未鉴定的130 kDa和135 kDa蛋白与Grb2/Ash相关，并且它们在GM-CSF或Epo处理后会发生酪氨酸磷酸化。我们使用谷胱甘肽S-转移酶-Grb2/Ash亲和柱纯化了130 kDa蛋白（pp130）。对纯化的pp130原位蛋白酶消化产生的三个肽段进行氨基酸序列分析表明，pp130与人c-cbl原癌基因产物（c-Cbl）相同。c-Cbl在体外和体内均组成性地与Grb2/Ash的SH3结构域结合，但不与Grb2/Ash的SH2结构域结合，并且GM-CSF刺激不会改变Grb2/Ash与c-Cbl或Sos的结合。此外，c-Cbl（pp130）会根据GM-CSF或Epo刺激迅速且短暂地发生酪氨酸磷酸化。这些发现强烈表明，c-Cbl参与造血细胞中GM-CSF或Epo的信号转导，并且c-Cbl参与了与Ras信号通路不同的另一条信号通路。
