Engagement of integrin receptors with extracellular ligands gives rise to the formation of complex multiprotein structures that link the ECM to the cytoplasmic actin cytoskeleton. These adhesive complexes are dynamic, often heterogeneous structures, varying in size and organization. In motile cells, sites of adhesion within ®lopodia and lamellipodia are relatively small and transient and are referred to as`focal complexes,' whereas adhesions underlying the body of the cell and localized to the ends of actin stress ®bers are referred to as`focal adhesions'. Signal transduction through focal complexes and focal adhesions has been implicated in the regulation of a number of key cellular processes, including growth factor induced mitogenic signals, cell survival and cell locomotion. The formation and remodeling of focal contacts is a dynamic process under the regulation of protein tyrosine kinases and small GTPases of the Rho family. In this review, we consider the role of the focal complex associated protein tyrosine kinase, Focal Adhesion Kinase (FAK), in the regulation of cell movement with the emphasis on how FAK regulates the¯ow of signals from the ECM to the actin cytoskeleton. Oncogene (2000) 19, 5606 ± 5613.

Focal adhesion kinase (FAK) is a member of a family of nonreceptor protein-tyrosine kinases that regulates integrin and growth factor signaling pathways involved in cell migrationThe ability of cells to respond appropriately to environmental cues is critical to maintaining cellular, tissue, and organism homeostasis. One such environmental cue is derived from cellular adhesion to the extracellular matrix. The loss of adhesiondependent cellular regulation can lead to increased cellular proliferation, decreased cell death, changes in cellular differentiation status, and altered cellular migratory capacity, all of which are critical components of cell carcinogenesis and metastatic progression.The FAK 4 family kinases (which include FAK and Pyk2) regulate cell adhesion, migration, and proliferation in a variety of cell types (for review see Refs. 1-3). Adhesion of cells to the extracellular matrix is mediated by heterodimeric transmembrane integrin receptors located within sites of close opposition to the underlying matrix called focal adhesions. Integrin engagement and clustering stimulates FAK phosphorylation on Tyr 397 , creating a high affinity binding site for Src and Src family kinases. The FAK⅐Src complex phosphorylates many components of the focal adhesion, resulting in changes in adhesion dynamics and the initiation of signaling cascades. In addition to FAK catalytic activity, FAK also functions as a scaffold to organize structural and signaling proteins within focal adhesions.The importance of FAK as a regulator of normal cellular function is underscored by the number of cancers reported to have alterations in FAK expression and/or activity, including colon, breast, thyroid, prostate, cervical, ovarian, head and neck, oral, liver, stomach, sarcoma, glioblastoma, and melanoma (4, 5). Additionally, alterations in FAK expression and/or activity have been associated with tumorigenesis and increased metastatic potential (4, 5). Currently, it is unclear how the catalytic and/or scaffolding function of FAK contributes to tumor progression. To date studies of FAK function have relied on the expression of dominant interfering mutants or elimination of FAK expression by genetic knock-out, antisense oligonucleotide expression, or small interfering RNA.Herein, we report the biochemical and cellular characterization of a novel small molecule inhibitor, PF-573,228 (here after referred to as PF-228), that targets FAK catalytic activity. The inhibitor interacts with FAK in the ATP-binding pocket and effectively blocks the catalytic activity of recombinant FAK protein or endogenous FAK expressed in a variety of normal and cancer cell lines. Treatment of cells with PF-228 blocked FAK phosphorylation on Tyr 397 and concomitantly reduced the tyrosine phosphorylation of paxillin, a recognized downstream effector of FAK signaling. Drug treatment of normal and cancer cells resulted in decreased cell migration and inhibited adhesion turnover, biological activities previously ascribed to FAK. Interestingly, inhibition of FAK activi...

ASAP1 (ADP ribosylation factor [ARF]-GTPase-activating protein [GAP] containing SH3, ANK repeats, and PH domain) is a phospholipid-dependent ARF-GAP that binds to and is phosphorylated by pp60Src . Using affinity chromatography and yeast two-hybrid interaction screens, we identified ASAP1 as a major binding partner of protein tyrosine kinase focal adhesion kinase (FAK). Glutathione S-transferase pull-down and coimmunoprecipitation assays showed the binding of ASAP1 to FAK is mediated by an interaction between the C-terminal SH3 domain of ASAP1 with the second proline-rich motif in the C-terminal region of FAK. Transient overexpression of wild-type ASAP1 significantly retarded the spreading of REF52 cells plated on fibronectin. In contrast, overexpression of a truncated variant of ASAP1 that failed to bind FAK or a catalytically inactive variant of ASAP1 lacking GAP activity resulted in a less pronounced inhibition of cell spreading. Transient overexpression of wild-type ASAP1 prevented the efficient organization of paxillin and FAK in focal adhesions during cell spreading, while failing to significantly alter vinculin localization and organization. We conclude from these studies that modulation of ARF activity by ASAP1 is important for the regulation of focal adhesion assembly and/or organization by influencing the mechanisms responsible for the recruitment and organization of selected focal adhesion proteins such as paxillin and FAK. INTRODUCTIONAttachment of cells to the extracellular matrix (ECM) is primarily mediated by the integrin family receptors (Hynes, 1992). Engagement of heterodimeric integrin receptors leads to the clustering of integrins and recruitment of numerous proteins to form multi-protein complexes on the cytoplasmic face of the plasma membrane termed focal adhesions (Burridge et al., 1988). Focal adhesions serve to anchor actin cytoskeleton to the plasma membrane and to provide a linkage between the extracellular environment and the cytoplasm (Burridge and Chrzanowska-Wodnicka, 1996). The recruitment of cytoskeletal proteins and the assembly of focal adhesions are functionally important for a number of cellular processes, including cell migration, survival, and proliferation (Lauffenburger and Horwitz, 1996). In the case of migrating cells (or cells spreading on ECM proteins), there is a requirement for the coordinated reorganization of the actin cytoskeleton and the formation of new attachments with the substratum (Huttenlocher et al., 1995;Bretscher, 1996). This process is temporally and spatially controlled, consistent with integrins functioning as both cell adhesion receptors and as initiators of signaling cascades that convey signals from ECM to actin cytoskeleton (Bretscher, 1996).Because integrins are catalytically inactive, their signaling ability is dependent upon the recruitment and activation of Article published online ahead of print. Mol. Biol. Cell 10.1091/ mbc.E02-01-0018. Article and publication date are at www.molbiolcell.org/cgi/doi/10.1091/mbc.E02-01-0018.* Corresponding a...

Integrins are transmembrane proteins that serve as primary sensors of the extracellular matrix (ECM) environment. In response to interactions with the ECM, integrins initiate signaling pathways that regulate cell migration, growth, and survival. Advances in imaging have contributed to the understanding of the dynamic nature of these cell-ECM interactions and the complexes that form at these sites and have provided insights into their regulation and signal organizing functions.

The process of cell migration is initiated by protrusion at the leading edge of the cell, the formation of peripheral adhesions, the exertion of force on these adhesions, and finally the release of the adhesions at the rear of the cell. Focal adhesion kinase (FAK) is intimately involved in the regulation of this process, although the precise mechanism(s) whereby FAK regulates cell migration is unclear. We have used two approaches to reduce FAK expression in fibroblasts. Treatment of cells with FAK-specific siRNAs substantially reduced FAK expression and inhibited the spreading of fibroblasts in serum-free conditions, but did not affect the rate of spreading in the presence of serum. In contrast with the wild-type cells, the FAK siRNA-treated cells exhibited multiple extensions during cell spreading. The extensions appeared to be inappropriately formed lamellipodia as evidenced by the localization of cortactin to lamellipodial structures and the inhibition of such structures by expression of dominant-negative Rac. The wild-type phenotype was restored by reexpressing wild-type FAK in the knockdown cells, but not by expression of FAK containing a point mutation at the autophosphorylation site (FAK Y397F). In wound-healing assays, FAK knockdown cells failed to form broad lamellipodia, instead forming multiple leading edges. Similar results were obtained using primary mouse embryo fibroblasts from FAK-flox mice in which Cre-mediated excision was used to ablate the expression of FAK. These data are consistent with a role for FAK in regulating the formation of a leading edge during cell migration by coordinating integrin signaling to direct the correct spatial activation of membrane protrusion.