Focal adhesion kinase (FAK) controls adhesion-dependent cell motility, survival, and proliferation. FAK has kinase-dependent and kinase-independent functions, both of which play major roles in embryogenesis and tumor invasiveness. The precise mechanisms of FAK activation are not known. Using x-ray crystallography, small angle x-ray scattering, and biochemical and functional analyses, we show that the key step for activation of FAK's kinase-dependent functions-autophosphorylation of tyrosine-397-requires site-specific dimerization of FAK. The dimers form via the association of the N-terminal FERM domain of FAK and are stabilized by an interaction between FERM and the C-terminal FAT domain. FAT binds to a basic motif on FERM that regulates co-activation and nuclear localization. FAK dimerization requires local enrichment, which occurs specifically at focal adhesions. Paxillin plays a dual role, by recruiting FAK to focal adhesions and by reinforcing the FAT:FERM interaction. Our results provide a structural and mechanistic framework to explain how FAK combines multiple stimuli into a site-specific function. The dimer interfaces we describe are promising targets for blocking FAK activation.

Phosphoglucose isomerase (EC 5.3.1.9) catalyzes the second step in glycolysis, the reversible isomerization of D-glucose 6-phosphate to D-fructose 6-phosphate. The reaction mechanism involves acid-base catalysis with proton transfer and proceeds through a cis-enediol(ate) intermediate. 5-Phospho-D-arabinonohydroxamic acid (5PAH) is a synthetic small molecule that resembles the reaction intermediate, differing only in that it has a nitrogen atom in place of C1. Hence, 5PAH is the best inhibitor of the isomerization reaction reported to date with a Ki of 2 ؋ 10 ؊7 M. Here we report the crystal structure of rabbit phosphoglucose isomerase complexed with 5PAH at 1.9 Å resolution. The interaction of 5PAH with amino acid residues in the enzyme active site supports a model of the catalytic mechanism in which Glu-357 transfers a proton between C1 and C2 and Arg-272 helps stabilize the intermediate. It also suggests a mechanism for proton transfer between O1 and O2. P hosphoglucose isomerase (PGI; EC 5.3.1.9) is a cytosolic glycolytic enzyme that catalyzes the reversible isomerization of D-glucose 6-phosphate (G6P) to D-fructose 6-phosphate (F6P). In addition to isomerase activity, PGI has been shown to display anomerase activity between pyranose anomers of G6P (1), between furanose anomers of F6P (2), and between those of D-mannose 6-phosphate (3), as well as C-2-epimerase activity on G6P (4). It also has roles in protein glycosylation, gluconeogenesis, and the pentose phosphate pathway (5). This central role in the metabolism of phosphorylated sugars explains the strong impact of PGI deficiency in humans (6, 7), as well as the interest as a therapeutic target in parasite metabolism (8). The PGI polypeptide chain, or perhaps a variation thereof, also has extracellular roles as neuroleukin, autocrine motility factor, and differentiation and maturation mediator (9-12). These proteins promote antibody secretion by mononuclear cells, tumor cell motility, and differentiation of leukemia cells and a promyelocytic cell line (HL-60 cells), respectively. More recently, PGI has also been identified as the antigen involved in rheumatoid arthritis of a mouse line (13), as a specific inhibitor toward myofibril-bound serine proteinase (14), and as the surface antigen involved in sperm agglutination (15). Because very little is known about how PGI is precisely involved in these various biological processes, this moonlighting protein (16) has been the subject of intense investigations.Biochemical characterization of PGI began over 50 years ago and includes inhibitor studies (1,(17)(18)(19)(20)(21)(22)(23)(24)(25), labeling studies (26-32), solvent exchange (33, 34), mutagenesis (35,36), and pH profile studies (20,37). A proposed multistep catalytic mechanism includes a ring-opening step followed by an isomerization step. The isomerization step proceeds via general acid-base catalysis with proton transfer. In the G6P to F6P direction, abstraction of the proton from C2 by an active site amino acid residue yields a 1,2-cis-enediol(ate) in...