Previously we have demonstrated that focal adhesion kinase (FAK)-promoted migration on fibronectin (FN) by its overexpression in CHO cells is dependent on FAK autophosphorylation at Y397 and subsequent binding of Src to this site. In this report, we have examined the role of FAK association with Grb2 and p130Cas, two downstream events of the FAK/Src complex that could mediate integrin-stimulated activation of extracellular signal-regulated kinases (Erks). We show that a Y925F FAK mutant was able to promote cell migration as efficiently as FAK and that the transfected FAK demonstrated no detectable association with Grb2 in CHO cells. In contrast, cells expressing a FAK P712/715A mutant demonstrated a level of migration comparable to that of control cells. This mutation did not affect FAK kinase activity, autophosphorylation, or Src association but did significantly reduce p130Cas association with FAK. Furthermore, FAK expression in CHO cells increased tyrosine phosphorylation of p130Cas and its subsequent binding to several SH2 domains, which depended on both the p130Cas binding site and the Src binding site. However, we did not detect increased activation of Erks in cells expressing FAK, and the MEK inhibitor PD98059 did not decrease FAK-promoted cell migration. Finally, we show that coexpression of p130Cas further increased cell migration on FN and coexpression of the p130Cas SH3 domain alone functioned as a dominant negative mutant and decreased cell migration. Together, these results demonstrate that p130Cas, but not Grb2, is a mediator of FAK-promoted cell migration and suggest that FAK/ p130Cas complex targets downstream pathways other than Erks in mediating FAK-promoted cell migration.

Because signal transducer and activator of transcription 3 (STAT3) is constitutively activated in most human solid tumors and is involved in the proliferation, angiogenesis, immune evasion, and antiapoptosis of cancer cells, researchers have focused on STAT3 as a target for cancer therapy. We screened for natural compounds that inhibit the activity of STAT3 using a dual-luciferase assay. Cryptotanshinone was identified as a potent STAT3 inhibitor. Cryptotanshinone rapidly inhibited STAT3 Tyr705 phosphorylation in DU145 prostate cancer cells and the growth of the cells through 96 hours of the treatment. Inhibition of STAT3 Tyr705 phosphorylation in DU145 cells decreased the expression of STAT3 downstream target proteins such as cyclin D1, survivin, and Bcl-xL. To investigate the cryptotanshinone inhibitory mechanism in DU145 cells, we analyzed proteins upstream of STAT3. Although phosphorylation of Janus-activated kinase (JAK) 2 was inhibited by 7 Mmol/L cryptotanshinone at 24 hours, inhibition of STAT3 Tyr705 phosphorylation occurred within 30 minutes and the activity of the other proteins was not affected. These results suggest that inhibition of STAT3 phosphorylation is caused by a JAK2-independent mechanism, with suppression of JAK2 phosphorylation as a secondary effect of cryptotanshinone treatment. Continuing experiments revealed the possibility that cryptotanshinone might directly bind to STAT3 molecules. Cryptotanshinone was colocalized with STAT3 molecules in the cytoplasm and inhibited the formation of STAT3 dimers. Computational modeling showed that cryptotanshinone could bind to the SH2 domain of STAT3. These results suggest that cryptotanshinone is a potent anticancer agent targeting the activation STAT3 protein. It is the first report that cryptotanshinone has antitumor activity through the inhibition of STAT3.

T he migration of vascular endothelial cells (ECs) plays an important role in angiogenesis and postangioplasty wound healing. Cell migration is a coordinated process consisting of adhesion at the leading edge and detachment at the rear (1, 2). The focal adhesions (FAs), cytoskeleton, and signaling pathways that mediate cell migration need to respond to diverse extracellular signals and translate them into precisely regulated intracellular responses. There have been many studies on EC migration in response to gradients of soluble chemicals (chemotaxis) and immobilized extracellular matrix (haptotaxis; refs. 3-6). However, the effect of mechanical environment on EC migration is not well understood.ECs are constantly subjected to shear stress, the tangential component of hemodynamic force caused by blood flow. It has been shown that shear stress induces EC monolayer remodeling, e.g., increase of stress fibers and alterations in gene expression (7,8). Shear stress can modulate EC migration in wounding area and vascular stent surface (9-12), but the kinetics and molecular mechanism of EC migration in response to shear stress remain to be determined.Integrins are transmembrane adhesion receptors that link the extracellular matrix to cytoskeletal proteins and signaling molecules at FAs (13-15). Integrin-matrix binding activates the signaling cascade at FAs to modulate cell migration (13,14). Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that colocalizes with integrins at FAs. FAK mediates the FA dynamics and signaling in response to growth factors and integrin-ligand binding (16,17). Phosphorylation of FAK at Tyr-397 [p-FAK(Y397)] upon cell adhesion allows FAK to associate with Src, which triggers downstream signaling events such as phosphorylation of mitogen-activated kinases, p130 cas , and paxillin to mediate cell adhesion and migration (18)(19)(20)(21)(22)(23)(24). Recent studies show that FAK is required for mechanosensing and persistent migration of fibroblasts (25, 26). We and others have shown that shear stress induces a transient activation of FAK in EC monolayer (27)(28)(29). These previous studies focused on the analysis of the global activity of FAK by using traditional biochemical assays; the subcellular distribution and dynamics of FAK at FAs and the role of this spatial dynamics in cell migration in response to mechanical and chemical stimuli remain to be determined.Here, we defined the kinetics of shear stress-induced directional migration of ECs. By expressing green fluorescence protein (GFP)-tagged FAK, we demonstrated the molecular dynamics of FAK at FAs in migrating ECs in response to shear stress and serum. The results showed that p-FAK(Y397) was correlated with FAK dynamics at FAs. Our findings indicate that the spatial dynamics of signaling at FAs is critical in directional migration, and that mechanotaxis is an important mechanism controlling EC migration. Materials and MethodsCell Culture. Cell culture reagents were obtained from GIBCO͞ BRL. Bovine aortic ECs (BAECs) before passa...

Focal adhesion kinase (FAK) has been implicated to play a key role in integrin-mediated signal transduction in cell migration. Grb7 is an Src homology (SH) 2-containing and pleckstrin homology domain-containing molecule, which shares significant homology with the Caenorhabditis elegans gene for Mig-10 involved in cell migration during embryogenesis. Here, we report that the SH2 domain of Grb7 can directly interact with FAK through Tyr-397, a major autophosphorylation site in vitro and in vivo. This interaction is cell adhesion-dependent, suggesting that the FAK-Grb7 complex is involved in integrin signaling. Using tetracycline-regulated expression system, we showed that overexpression of Grb7 enhanced cell migration toward fibronectin, whereas overexpression of its SH2 domain alone inhibited cell migration. In addition, we found that phosphorylation of FAK or p130 cas was not affected by the expression of either Grb7 or its SH2 domain alone, suggesting that Grb7 is downstream of FAK and does not compete with Src for binding to FAK in vivo. Taken together, these results suggest that the FAK-Grb7 complex plays a role in cell migration stimulated by integrin signaling through FAK. Focal adhesion kinase (FAK)1 is a cytoplasmic tyrosine kinase that has been shown to play a critical role in integrinmediated signal transduction (1-3). FAK is localized to focal contacts in many adherent cells, and it rapidly becomes activated and autophosphorylated following cell adhesion to extracellular matrix proteins or integrin clustering by antibodies (1, 4 -6). The major tyrosine phosphorylation site of FAK has been mapped to Tyr-397, the phosphorylation of which creates binding sites for the Src homology (SH) 2 domains of other intracellular signaling molecules. Indeed, both Src family kinases (7-10) and phosphatidylinositol 3-kinase (PI3K) (11, 12) have been shown to form complexes with FAK at this site through their SH2 domains in cell adhesion-dependent manner. The formation of FAK-Src family kinase complexes have been proposed to allow phosphorylation of additional sites on FAK, which include Tyr-925 for binding the SH2 domain of Grb2 (13). Finally, FAK has been shown to interact with p130 cas through its proline-rich sequences at the COOH-terminal domain (14, 15) and cytoskeletal proteins paxillin and talin by sequences also located in the COOH terminus (16 -18). FAK interactions with these and potentially other proteins are believed to mediate the functions of FAK in integrin-dependent signal transduction.Recent studies have suggested several roles for integrin signaling through FAK in regulation of cell survival (19,20), proliferation (21, 22), spreading (23), and migration (21,24,25). Using Chinese hamster ovary (CHO) cells as a model system, we have previously shown that overexpression of FAK stimulated cell migration and mutation of Tyr-397 to Phe abolished its ability to promote cell migration (25). Furthermore, we have identified p130cas as a downstream mediator of FAKSrc family kinases in the regulation of cell migr...