Holgado-Madruga Marina, Wong Albert J
Department of Microbiology and Immunology, The Kimmel Cancer Institute, Thomas Jefferson University, 233 South 10th Street, 1002 BLSB, Philadelphia, PA 19107, USA.
Cancer Res. 2004 Mar 15;64(6):2007-15. doi: 10.1158/0008-5472.can-03-2886.
Grb2-associated binder 1 (Gab1) is a docking protein that is tyrosine phosphorylated following the activation of multiple cytokine receptors and receptor tyrosine kinases. Its function then is to recruit and activate multiple signaling molecules. In our previous work, we showed that Gab1 enhances cell growth and induces the transformed phenotype in NIH3T3 cells downstream of the epidermal growth factor (EGF) receptor. In this report, we analyze how it produces these effects. Because SHP-2 is the major binding partner of Gab1, we mutated its binding site in the Gab1 cDNA (Gab1/DeltaSHP-2). This construct was stably overexpressed in NIH3T3 cells (3T3-Gab1/DeltaSHP-2) and in the wild-type Gab1 cDNA (3T3-Gab1) or an empty expression vector (3T3-CTR). Our findings show that after EGF stimulation, Gab1/DeltaSHP-2 has a higher level of tyrosine phosphorylation at early time points than Gab1. Gab1/DeltaSHP-2 recruits more phosphatidylinositol 3'-kinase than Gab1 after EGF triggering, which accounts for a higher and more sustained AKT activation in 3T3-Gab1/DeltaSHP-2 cells relative to 3T3-Gab1 fibroblasts. Moreover, 3T3-Gab1/DeltaSHP-2 cells demonstrate a higher level of extracellular-regulated kinase 1 activation at early time points of EGF stimulation. However, there was an unexpected decrease in c-fos promoter induction in 3T3-Gab1/DeltaSHP-2 cells when compared with 3T3-Gab1 cells. Additionally, the 3T3-Gab1/DeltaSHP-2 cells show a reversion of the transformed phenotype, including fewer morphologic changes, an increase in stress fiber cytoskeletal organization, and a decrease in cell proliferation and anchorage independent growth. These results reveal that the Gab1/SHP-2 interaction is essential for cell growth and transformation but that this must occur through a novel pathway that is independent of extracellular-regulated kinase or AKT. On the basis of its role in growth and transformation, the Gab1/SHP-2 interaction may become an attractive target for the pharmacologic intervention of malignant cell growth.
Grb2相关结合蛋白1（Gab1）是一种对接蛋白，在多种细胞因子受体和受体酪氨酸激酶激活后会发生酪氨酸磷酸化。其功能是招募并激活多种信号分子。在我们之前的研究中，我们发现Gab1在表皮生长因子（EGF）受体下游可增强NIH3T3细胞的生长并诱导其转化表型。在本报告中，我们分析了它是如何产生这些效应的。由于SHP-2是Gab1的主要结合伙伴，我们在Gab1 cDNA中突变了其结合位点（Gab1/DeltaSHP-2）。该构建体在NIH3T3细胞（3T3-Gab1/DeltaSHP-2）、野生型Gab1 cDNA（3T3-Gab1）或空表达载体（3T3-CTR）中稳定过表达。我们的研究结果表明，在EGF刺激后，Gab1/DeltaSHP-2在早期时间点的酪氨酸磷酸化水平高于Gab1。在EGF触发后，Gab1/DeltaSHP-2比Gab1招募更多的磷脂酰肌醇3'-激酶，这解释了相对于3T3-Gab1成纤维细胞，3T3-Gab1/DeltaSHP-2细胞中AKT激活水平更高且更持久。此外，在EGF刺激的早期时间点，3T3-Gab1/DeltaSHP-2细胞表现出更高水平的细胞外调节激酶1激活。然而，与3T3-Gab1细胞相比，3T3-Gab1/DeltaSHP-2细胞中c-fos启动子诱导出现意外下降。此外，3T3-Gab1/DeltaSHP-2细胞显示出转化表型的逆转，包括形态变化减少、应力纤维细胞骨架组织增加以及细胞增殖和锚定非依赖性生长减少。这些结果表明，Gab1/SHP-2相互作用对于细胞生长和转化至关重要，但这必须通过一条独立于细胞外调节激酶或AKT的新途径发生。基于其在生长和转化中的作用，Gab1/SHP-2相互作用可能成为恶性细胞生长药物干预的一个有吸引力的靶点。
