A transmembrane-anchored chimeric focal adhesion kinase is constitutively activated and phosphorylated at tyrosine residues identical to pp125FAK.
Focal adhesion kinase, pp125FAK, is a nonmyristylated cytosolic tyrosine kinase unrelated to protein-tyrosine kinase families categorized to date. The kinase activity and tyrosine phosphorylation of pp125FAK are induced by beta 1 and beta 3 integrin-mediated cell adherence or aggregation. pp125FAK is also a tyrosine phosphorylation substrate in v-src-transformed cells and is localized to focal adhesion contracts of adherent fibroblasts and carcinoma cells. In this report, we have transiently expressed in COS cells a transmembrane-anchored chimeric receptor kinase, CD2FAK, consisting of CD2 and pp125FAK. We analyzed its kinase activity and tyrosine phosphorylation and compared to those of pp125FAK. We found that CD2FAK exhibited constitutive kinase activity and a high basal tyrosine phosphorylation level when COS transfectants were suspended in serum-free media. The kinase activity of CD2FAK was similarly up-regulated upon beta 1 integrin-mediated cell adherence as the endogenous pp125FAK. Both CD2FAK and pp125FAK appeared to be active as autophosphorylating kinases as shown by mutation of the ATP binding site. We determined the major tyrosine phosphorylation site, Tyr397, identical for both the constitutively activated CD2FAK and pp125FAK in response to beta 1 integrin-mediated cell adherence by site-directed mutagenesis. Deletions of the NH2- or the COOH-terminal noncatalytic domain of FAK, including Tyr397 did not lead to abolition of the kinase activity of pp125FAK or CD2FAK. Taken together, CD2FAK exhibits properties of an activated pp125FAK and the kinase activity does not appear to require tyrosine phosphorylation in vitro or in vivo.
Chan
P Y
PY
Department of Inflammation, Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, Washington 98121.
Kanner
S B
SB
Whitney
G
G
Aruffo
A
A
D000818:Animals; D002384:Catalysis; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D004789:Enzyme Activation; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017384:Sequence Deletion; D014443:Tyrosine / Q000378:metabolism*; D015854:Up-Regulation
D015815:Cell Adhesion Molecules; D011993:Recombinant Fusion Proteins; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases