Hecker Timothy P, Ding Qiang, Rege Tanya A, Hanks Steven K, Gladson Candece L
Department of Pathology, Division of Neuropathology, The University of Alabama at Birmingham, Birmingham, AL 35294, USA.
Oncogene. 2004 May 13;23(22):3962-71. doi: 10.1038/sj.onc.1207541.
Focal adhesion kinase (FAK) signaling may be mediated through the modulation of Ras activity. We have shown previously that grade III malignant astrocytoma biopsy samples exhibit elevated levels of FAK, and that overexpression of FAK in U-251MG malignant astrocytoma cells promotes the phosphorylation of Shc, a potential upstream mediator of Ras activity. Here, we report that overexpression of FAK promotes Ras activity in U-251MG malignant astrocytoma cells cultured in aggregate suspension or as monolayers adherent to vitronectin. The overexpression of FAK also promoted the association of FAK with p120RasGAP, which is a negative regulator of Ras activity, in the U-251MG cells cultured in aggregate suspension, with this association being abrogated upon plating of the cells onto vitronectin. An association of FAK with p120RasGAP also was observed in malignant astrocytoma biopsy samples, but not in normal brain samples. As overexpression of FAK in U-251MG cells in aggregate suspension culture reduced the amount of p120RasGAP complexed with active Ras, we hypothesize that the association of FAK with p120 RasGAP may facilitate Ras activity. The overexpression of a mutated FAK in which the Y397 had been mutated to F did not result in the formation of the FAK/p120RasGAP complex and did not promote Ras activity, indicating that the Y397 residue of FAK plays a role in the formation of this complex and in the activation of Ras. Moreover, the overexpression of mutated FAK (397F) was found to inhibit anchorage-independent growth. These data provide the basis for a previously undescribed mechanism in which the elevated expression of FAK can promote Ras activity through its competitive recruitment of p120RasGAP, thereby diminishing the association of p120RasGAP with active Ras.
粘着斑激酶（FAK）信号传导可能通过调节Ras活性来介导。我们之前已经表明，III级恶性星形细胞瘤活检样本中FAK水平升高，并且在U-251MG恶性星形细胞瘤细胞中FAK的过表达促进了Shc的磷酸化，Shc是Ras活性的潜在上游介质。在此，我们报告FAK的过表达促进了在聚集体悬浮培养或粘附于玻连蛋白的单层培养的U-251MG恶性星形细胞瘤细胞中的Ras活性。在聚集体悬浮培养的U-251MG细胞中，FAK的过表达还促进了FAK与p120RasGAP（Ras活性的负调节因子）的结合，当将细胞接种到玻连蛋白上时这种结合被消除。在恶性星形细胞瘤活检样本中也观察到FAK与p120RasGAP的结合，但在正常脑样本中未观察到。由于在聚集体悬浮培养的U-251MG细胞中FAK的过表达减少了与活性Ras复合的p120RasGAP的量，我们推测FAK与p120RasGAP的结合可能促进Ras活性。Y397已突变为F的突变型FAK的过表达不会导致FAK/p120RasGAP复合物的形成，也不会促进Ras活性，表明FAK的Y397残基在该复合物的形成和Ras的激活中起作用。此外，发现突变型FAK（397F）的过表达抑制非锚定依赖性生长。这些数据为一种先前未描述的机制提供了基础，即FAK的表达升高可通过其对p120RasGAP的竞争性募集来促进Ras活性，从而减少p120RasGAP与活性Ras的结合。
