Abstract
Mounting evidence suggests that the FAK N-terminal (FERM) domain controls FAK phosphorylation and function; however, little is known regarding the role of the C terminal (FAT) domain in FAK regulation. We identified a patient-derived FAK mutant, in which a 27-amino acid segment was deleted from the C-terminal FAT domain (named FAK-Del33). When FAK-Del33 was overexpressed in specific tumor cell lines, Y397 phosphorylation increased compared with that observed in cells expressing FAK-WT. Here, we attempt to unveil the mechanism of this increased phosphorylation. Using cell biology experiments, we show that FAK-Del33 is incapable of co-localizing with paxillin, and has constitutively high Y397 phosphorylation. With a kinase-dead mutation, it showed phosphorylation of FAK-Del33 has enhanced through auto-phosphorylation. It was also demonstrated that phosphorylation of FAK-Del33 is not Src dependent or enhanced intermolecular interactions, and that the hyperphosphorylation can be lowered using increasing amounts of transfected FERM domain. This result suggests that Del33 mutation disrupting of FAT's structural integrity and paxillin binding capacity leads to incapable of targeting Focal adhesions, but has gained the capacity for auto-phosphorylation in cis.
Highlights
Focal adhesion kinase (FAK) plays a key role in promoting adhesion, proliferation, and cell migration and in regulating anchorage-dependent anti-apoptotic signals [1]
The results shows that Y397 phosphorylation increased when FAK-Del33 was expressed in Hep3B, MDA-MB-468 and MDA-MB-435s cells, and similar tyrosine phosphorylation levels were maintained when FAK-Del33 was expressed in the other cell lines (Fig 2B)
We provide interesting insight into the possible mechanism of Y397 phosphorylation in a focal adhesion targeting (FAT) domain-defective mutant (FAK-Del33)
Summary
Focal adhesion kinase (FAK) plays a key role in promoting adhesion, proliferation, and cell migration and in regulating anchorage-dependent anti-apoptotic signals [1]. FAK is associated with tissue functions, such as vascular development, axon outgrowth, and dendrite formation, and diseases, such as cardiomyocyte-induced hypertrophy, fibrosis, and epithelial cancer [2,3]. Auto-phosphorylation at Y397 is a key event in FAK activation [4]. Y397 phosphorylation creates a high-affinity binding site for the SH2 domain of Src family kinase, which leads to Src recruitment and activation [5]. Src recruitment facilitates FAK phosphorylation at tyrosine residues 576/7, 861, and 925, resulting in the activation of other focal adhesion proteins, such as paxillin [6]. Y397 auto-phosphorylation allows FAK to function as an adapter protein that communicates with various signal transduction pathways
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