Department of Biochemistry, McGill University, Montréal Québec H3A 1Y6, Canada.
J Cell Sci. 2012 Jun 15;125(Pt 12):2940-53. doi: 10.1242/jcs.100834. Epub 2012 Feb 24.
Invasive carcinoma cells form actin-rich matrix-degrading protrusions called invadopodia. These structures resemble podosomes produced by some normal cells and play a crucial role in extracellular matrix remodeling. In cancer, formation of invadopodia is strongly associated with invasive potential. Although deregulated signals from the receptor tyrosine kinase Met (also known as hepatocyte growth factor are linked to cancer metastasis and poor prognosis, its role in invadopodia formation is not known. Here we show that stimulation of breast cancer cells with the ligand for Met, hepatocyte growth factor, promotes invadopodia formation, and in aggressive gastric tumor cells where Met is amplified, invadopodia formation is dependent on Met activity. Using both GRB2-associated-binding protein 1 (Gab1)-null fibroblasts and specific knockdown of Gab1 in tumor cells we show that Met-mediated invadopodia formation and cell invasion requires the scaffold protein Gab1. By a structure-function approach, we demonstrate that two proline-rich motifs (P4/5) within Gab1 are essential for invadopodia formation. We identify the actin regulatory protein, cortactin, as a direct interaction partner for Gab1 and show that a Gab1-cortactin interaction is dependent on the SH3 domain of cortactin and the integrity of the P4/5 region of Gab1. Both cortactin and Gab1 localize to invadopodia rosettes in Met-transformed cells and the specific uncoupling of cortactin from Gab1 abrogates invadopodia biogenesis and cell invasion downstream from the Met receptor tyrosine kinase. Met localizes to invadopodia along with cortactin and promotes phosphorylation of cortactin. These findings provide insights into the molecular mechanisms of invadopodia formation and identify Gab1 as a scaffold protein involved in this process.
侵袭性癌细胞形成富含肌动蛋白的基质降解突起，称为侵袭伪足。这些结构类似于一些正常细胞产生的破骨细胞，在细胞外基质重塑中发挥着关键作用。在癌症中，侵袭伪足的形成与侵袭潜能密切相关。尽管受体酪氨酸激酶 Met（也称为肝细胞生长因子）的信号失调与癌症转移和预后不良有关，但它在侵袭伪足形成中的作用尚不清楚。在这里，我们发现配体与 Met 刺激乳腺癌细胞可促进侵袭伪足的形成，并且在 Met 扩增的侵袭性胃肿瘤细胞中，侵袭伪足的形成依赖于 Met 的活性。我们使用 GRB2 相关结合蛋白 1（Gab1）缺失的成纤维细胞和肿瘤细胞中 Gab1 的特异性敲低，表明 Met 介导致侵袭伪足的形成和细胞侵袭需要支架蛋白 Gab1。通过结构功能方法，我们证明 Gab1 内的两个富含脯氨酸的基序（P4/5）对于侵袭伪足的形成是必不可少的。我们确定肌动蛋白调节蛋白 cortactin 是 Gab1 的直接相互作用伙伴，并表明 Gab1-cortactin 相互作用依赖于 cortactin 的 SH3 结构域和 Gab1 的 P4/5 区域的完整性。在 Met 转化的细胞中，cortactin 和 Gab1 都定位于侵袭伪足的玫瑰花结中，并且 cortactin 与 Gab1 的特异性解偶联会破坏 Met 受体酪氨酸激酶下游的侵袭伪足生物发生和细胞侵袭。Met 与 cortactin 一起定位于侵袭伪足，并促进 cortactin 的磷酸化。这些发现为侵袭伪足形成的分子机制提供了深入的了解，并确定 Gab1 作为参与该过程的支架蛋白。
