The role of focal adhesion kinase binding in the regulation of tyrosine phosphorylation of paxillin.
Focal adhesion kinase (FAK) and paxillin are focal adhesion-associated, phosphotyrosine-containing proteins that physically interact. A previous study has demonstrated that paxillin contains two binding sites for FAK. We have further characterized these two binding sites and have demonstrated that the binding affinity of the carboxyl-terminal domain of FAK is the same for each of the two binding sites. The presence of both binding sites increases the affinity for FAK by 5-10-fold. A conserved paxillin sequence called the LD motif has been implicated in FAK binding. We show that mutations in the LD motifs in both FAK-binding sites are required to dramatically impair FAK binding in vitro. A paxillin mutant containing point mutations in both FAK-binding sites was characterized. The mutant exhibited reduced levels of phosphotyrosine relative to wild type paxillin in subconfluent cells growing in culture, following cell adhesion to fibronectin and in src-transformed fibroblasts. These results suggest that paxillin must bind FAK for maximal phosphorylation in response to cell adhesion and that FAK may function to direct tyrosine phosphorylation of paxillin in the process of transformation by the src oncogene.
Thomas
J W
JW
Department of Cell Biology and Anatomy, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
Cooley
M A
MA
Broome
J M
JM
Salgia
R
R
Griffin
J D
JD
Lombardo
C R
CR
Schaller
M D
MD
D000818:Animals; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism*; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D009154:Mutation; D051419:Paxillin; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction; D014443:Tyrosine
D015815:Cell Adhesion Molecules; D003598:Cytoskeletal Proteins; D051419:Paxillin; D010750:Phosphoproteins; C496063:Pxn protein, rat; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496052:Ptk2 protein, rat