Autophosphorylation-independent and -dependent Functions of Focal Adhesion Kinase during Development
Autophosphorylation-independent and -dependent Functions of Focal Adhesion Kinase during Development
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DOI：
10.1074/jbc.m109.067280
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发表时间：
2009-12-11
影响因子：
4.8
通讯作者：
Enslen, Herve
中科院分区：
文献类型：
--
作者：
Corsi, Jean-Marc;Houbron, Christophe;Enslen, Herve
粘着斑激酶（FAK）调节许多细胞功能，对从胚胎发育到癌症进展的过程至关重要。虽然Tyr-397的自磷酸化似乎是体外FAK功能所必需的，但其在体内的作用尚未确定。我们使用缺失编码Tyr-397的外显子15的突变小鼠（fak Delta）解决了这个问题。所得到的突变蛋白FAK（Delta/Delta）是以正常水平表达的活性激酶。我们的研究结果表明，FAK自磷酸化的要求在发展过程中有所不同。FAK（Delta/Delta）胚胎正常发育至胚胎日（E）12.5，与FAK-无效胚胎在E8.5的致死率形成对比。因此，Tyr-397上的自磷酸化对于FAK实现其功能是不需要的，直到妊娠中晚期。然而，FAK（Delta/Delta）胚胎在E13.5-14.5时显示出萎缩、水肿、动脉形成延迟、血管重塑缺陷、多器官异常和总体发育迟缓，并且此后死亡，这证明FAK自身磷酸化对于正常发育也是必要的。来自突变胚胎的成纤维细胞具有正常的星状形态和黏着斑蛋白、Src家族成员、p53和Pyk 2的表达。相比之下，在FAK（Delta/Delta）成纤维细胞和内皮细胞中，扩展和板状伪足的形成随着粘着斑的大小和数量的增加而改变，富含FAK（Delta/Delta）。FAK突变也降低成纤维细胞增殖。这些结果表明FAK在体内的生理功能是通过自身磷酸化非依赖性和自身磷酸化依赖性机制实现的。
Focal adhesion kinase (FAK) regulates numerous cellular functions and is critical for processes ranging from embryo development to cancer progression. Although autophosphorylation on Tyr-397 appears required for FAK functions in vitro, its role in vivo has not been established. We addressed this question using a mutant mouse (fak Delta) deleted of exon 15, which encodes Tyr-397. The resulting mutant protein FAK(Delta/Delta) is an active kinase expressed at normal levels. Our results demonstrate that the requirement for FAK autophosphorylation varies during development. FAK(Delta/Delta) embryos developed normally up to embryonic day (E) 12.5, contrasting with the lethality at E8.5 of FAK-null embryos. Thus, autophosphorylation on Tyr-397 is not required for FAK to achieve its functions until late mid-gestation. However, FAK(Delta/Delta) embryos displayed hemorrhages, edema, delayed artery formation, vascular remodeling defects, multiple organ abnormalities, and overall developmental retardation at E13.5-14.5, and died thereafter demonstrating that FAK autophosphorylation is also necessary for normal development. Fibroblasts derived from mutant embryos had a normal stellate morphology and expression of focal adhesion proteins, Src family members, p53, and Pyk2. In contrast, in FAK(Delta/Delta) fibroblasts and endothelial cells, spreading and lamellipodia formation were altered with an increased size and number of focal adhesions, enriched in FAK(Delta/Delta). FAK mutation also decreased fibroblast proliferation. These results show that the physiological functions of FAK in vivo are achieved through both autophosphorylation-independent and autophosphorylation-dependent mechanisms.