Direct phosphorylation of focal adhesion kinase by c-Src: evidence using a modified nucleotide pocket kinase and ATP analog.
A single mutation in the nucleotide binding pocket of select protein kinases allows for use of a bulky, substituted-ATP analog not used by the wild-type kinase [1]. Using this approach with the protein tyrosine kinase c-Src, we have generated a mutant T338G and expressed it in Src/Yes/Fyn null fibroblasts (SYF1) at near endogenous levels. T338G Src exhibits high specificity for a substituted ATP analog N(6)-2-phenyl ethyl ATP (peATP), which is not used by wild-type c-Src in autophosphorylation nor substrate phosphorylation assays. By employing the T338G Src mutant and [gamma-(32)P]peATP analog, we demonstrate that c-Src can directly phosphorylate focal adhesion kinase (Fak) in vitro. We also show that incubation of permeabilized, T338 Src-expressing cells with peATP causes an increase in Fak tyrosine phosphorylation not observed in wild-type Src cells. Taken together, these data provide evidence that Src directly phosphorylates Fak and demonstrates the limitations of using this modified ATP strategy for analysis of direct substrates of protein kinases in permeabilized cells.
Chaudhary
Anu
A
Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N., A2-025, Seattle, WA 98109, USA.
Brugge
Joan S
JS
Cooper
Jonathan A
JA
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D016466:CHO Cells; D000081247:CSK Tyrosine-Protein Kinase; D002463:Cell Membrane Permeability / Q000187:drug effects; D006224:Cricetinae; D004072:Digitonin / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D051379:Mice; D010751:Phosphopyruvate Hydratase / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D014162:Transfection; D019061:src-Family Kinases
C459592:N(6)-2-phenylethyladenosine triphosphate; D000255:Adenosine Triphosphate; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496051:Ptk2 protein, mouse; D019061:src-Family Kinases; D010751:Phosphopyruvate Hydratase; D004072:Digitonin