Tumor cell invasion into surrounding tissue contributes significantly to poor clinical prognosis of glioblastoma. The increased migratory commitment of these cells precludes effective tumor resection, reduces the efficacy of radiation treatment, and increases their resistance to chemotherapeutic agents. The related focal adhesion kinases FAK and Pyk2 have been implicated as proximal integrin effectors coupling integrin-matrix interactions with intracellular signaling events in a variety of cell types. We have previously reported that Pyk2 contributes substantially to glioma cell migration while FAK stimulated cell proliferation and inhibited migration. Notably, the N-terminal Pyk2 FERM domain is necessary for stimulation of migration as a N-terminally deleted variant of Pyk2 failed to stimulate migration and expression of an autonomous Pyk2 FERM domain significantly inhibited glioma cell migration. To investigate the role of the Pyk2 FERM domain in regulating Pyk2 activity, a 3D model of the FERM domain was developed and refined using established protocols of threading and multiple molecular dynamic simulations. Based on this model, several residues were selected for mutagenesis. W104 lies at the base a hydrophobic groove between lobe 1 (F1) and lobe 3 (F3) that has been previously implicated in protein-protein interactions in other FERM domain proteins. Y135 and D350 may be involved in an interaction between F2 and F3 that stabilizes the overall domain structure. These residues were substituted by site directed mutagenesis and their effect on Pyk2 function was analyzed following expression in SF767 glioma cells. All of the mutants were expressed at a level comparable to wild type Pyk2. Substitution at W104 led to a loss of Pyk2 phosphorylation comparable to that observed for the catalytically inactive variant K457R. Substitution of Y135 also reduced Pyk2 phosphorylation while substitution of D350 did not alter Pyk2 phosphorylation. Loss of Pyk2 phosphorylation correlated directly with inhibition of migration as the Pyk2 W104A and Y135C mutants failed to stimulate SF767 glioma cell in vitro migration. When expressed as autonomous FERM domains, the W104A and Y135C mutants failed to inhibit glioma cell migration while the wild type Pyk2 FERM domain significantly inhibited migration. Finally, the immunohistochemical staining patterns of wild type Pyk2 and the W104A and Y135C Pyk2 variants suggest that the loss of function may be due to aberrant subcellular localization and inability of engage in protein-protein interactions required for glioma cell migration. We conclude that the N-terminal FERM domain plays a critical role in Pyk2 function by directing its specific subcellular localization necessary for stimulating glioma cell migration.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]