Characterization of an activated mutant of focal adhesion kinase: 'SuperFAK'.
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.
Gabarra-Niecko
Veronica
V
Department of Cell and Developmental Biology, 534 Taylor Hall CB 7090, University of North Carolina, Chapel Hill, NC 27599, U.S.A.
Keely
Patricia J
PJ
Schaller
Michael D
MD
D000595:Amino Acid Sequence; D000818:Animals; D002448:Cell Adhesion / Q000502:physiology; D002465:Cell Movement / Q000502:physiology; D002478:Cells, Cultured; D002642:Chick Embryo; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004789:Enzyme Activation; D005347:Fibroblasts / Q000201:enzymology; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D022001:Focal Adhesions / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D051419:Paxillin; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D014443:Tyrosine / Q000378:metabolism; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
D003598:Cytoskeletal Proteins; C496061:PXN protein, human; D051419:Paxillin; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; D019061:src-Family Kinases