Characterization of an activated mutant of focal adhesion kinase: 'SuperFAK'
Characterization of an activated mutant of focal adhesion kinase: 'SuperFAK'
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DOI：
10.1042/bj20020065
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发表时间：
2002-08-01
影响因子：
4.1
通讯作者：
Schaller, MD
中科院分区：
文献类型：
--
作者：
Gabarra-Niecko, V;Keely, PJ;Schaller, MD
粘着斑激酶(FAK)是一种非受体酪氨酸激酶，在细胞的黏附、扩散、迁移、增殖和存活等正常过程中发挥重要作用。此外，FAK在多种癌细胞和肿瘤中过表达，并可能在人类癌症的发展中发挥作用。作为模拟异常的FAK信号在癌症发生中的作用的前奏，本研究的目标是设计FAK的点突变，以增强酶的活性。在FAK中引入了一些被报道为激活其他酪氨酸激酶突变的替换。以成纤维细胞生长因子受体3的K650E(Lys(650->；)Glu)突变体为基础，用谷氨酸取代FAK激活环中的两个赖氨酸残基，从而产生SuperFAK。两个大脑特异的外显子被改造成禽类FAK，产生了FAK6.7。与野生型FAK相比，SuperFAK和FAK6.7在体外表现出更高的催化活性。SuperFAK和FAK6.7在成纤维细胞中的表达导致FAK底物的过度磷酸化。虽然SuperFAK和FAK6.7的催化活性在很大程度上与细胞黏附无关，但下游底物的酪氨酸磷酸化依赖于黏附。此外，由于SuperFAK表现出与野生型FAK相同的招募Src家族激酶的能力，底物的酪氨酸磷酸化可能是由于FAK的直接磷酸化。除了增强生化信号外，SuperFAK还增加了上皮细胞的运动性。SuperFAK和FAK6.7可能是研究异常FAK信号在人类疾病中的潜在作用的有价值的分子工具。
Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that plays an important role in normal cellular processes such as adhesion, spreading, migration, proliferation and survival. In addition, FAK is overexpressed in a variety of cancer cells and tumours and may play a role in the development of human cancer. As a prelude to modelling the role of aberrant FAK signalling in the initiation of cancer, the goal of the present study was to engineer point mutations in FAK that would enhance enzymic activity. A number of substitutions that were reported as activating mutations in other tyrosine kinases were introduced into FAK. Glutamic acid substitutions for two lysine residues in the activation loop of FAK, based upon the K650E (Lys(650-->)Glu) mutant of fibroblast-growth-factor receptor 3, were made to create 'SuperFAK'. Two brain-specific exons were engineered into avian FAK to create FAK6.7. SuperFAK and, to a lesser extent, FAK6.7, exhibited increased catalytic activity in vitro compared with wild-type FAK. The expression of SuperFAK and FAK6.7 in fibroblasts led to hyperphosphorylation of FAK substrates. Although the catalytic activity of SuperFAK and FAK6.7 was largely independent of cell adhesion, tyrosine phosphorylation of downstream substrates was adhesion-dependent. Further, since SuperFAK exhibited the same ability as wild-type FAK to recruit Src family kinases, tyrosine phosphorylation of substrates was likely due to direct phosphorylation by FAK. In addition to enhanced biochemical signalling, SuperFAK also increased the motility of epithelial cells. SuperFAK and FAK6.7 may be valuable molecular tools to investigate the potential role of aberrant FAK signalling in human disease.