Although netrins are an important family of neuronal guidance proteins, intracellular mechanisms that mediate netrin function are not well understood. Here we show that netrin-1 induces tyrosine phosphorylation of proteins including focal adhesion kinase (FAK) and the Src family kinase Fyn. Blockers of Src family kinases inhibited FAK phosphorylation and axon outgrowth and attraction by netrin. Dominant-negative FAK and Fyn mutants inhibited the attractive turning response to netrin. Axon outgrowth and attraction induced by netrin-1 were significantly reduced in neurons lacking the FAK gene. Our results show the biochemical and functional links between netrin, a prototypical neuronal guidance cue, and FAK, a central player in intracellular signaling that is crucial for cell migration.Axon outgrowth and pathfinding are crucial for the formation of a normal nervous system. Extracellular cues that control these processes have been discovered in the past two decades. The netrin family of proteins can both promote axon outgrowth and guide the direction of axon pathfinding in many regions of the nervous system including the neocortex and the spinal cord 1-4 .In Caenorhabditis elegans, the gene unc-6 encodes the prototypical member of the netrin family 1,5 . In mammals, studies of commissural axons in the spinal cord showed a diffusible guidance activity in the floorplate 6 . The floorplate was able to promote the outgrowth of commissural axons and to attract them 7-10 . Biochemical purification of the axon-promoting activity from the chick brain led to the isolation of netrin-1, which was then shown to act as both a factor promoting axon outgrowth and a guidance cue to attract axons 2,3 . SequenceCorrespondence should be addressed to Y.R. (raoyi@pcg.wustl.edu). 6 These authors contributed equally to this work. COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests. NIH Public Access Author ManuscriptNat Neurosci. Author manuscript; available in PMC 2008 March 10. Published in final edited form as:Nat Neurosci. 2004 November ; 7(11): 1222-1232. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscriptsimilarities between UNC-6 and netrin-1 proteins established a remarkably conserved family of molecular cues in invertebrates and vertebrates 2,3,5,11-15 .The receptors for the netrins are 16,17 ) and in C. elegans, and their corresponding mammalian homologs are DCC (deleted in colorectal cancer), neogenin 19,20 and UNC5A-D (also known as . DCC/ UNC-40 seems to be required for both the attractive and repulsive responses, whereas UNC-5 is required only for repulsion [17][18][19][20]25,26 . Heterodimerization of UNC-40 with UNC-5 may convert UNC-40 from mediating attraction to repulsion 27 . There is also evidence, however, that DCC/UNC-40 can mediate repulsion in the absence of UNC-5 in specific cells in C. elegans 28 . In contrast to our knowledge of the multiple roles of netrins and their receptors, much less is known about intracellular signal transductio...

Cancer cells require sustained oncogenic signaling in order to maintain their malignant properties. It is, however, unclear whether they possess other dependencies that can be exploited therapeutically. We report here that in a large fraction of human breast cancers, the gene encoding focal adhesion kinase (FAK), a core component of integrin signaling, was amplified and FAK mRNA was overexpressed. A mammary gland-specific deletion of Fak in mice did not seem to affect normal mammary epithelial cells, and these mice were protected from tumors initiated by the polyoma middle T oncoprotein (PyMT), which activates Ras and PI3K. FAK-deficient PyMT-transformed cells displayed both growth arrest and apoptosis, as well as diminished invasive and metastatic capacity. Upon silencing of Fak, mouse mammary tumor cells transformed by activated Ras became senescent and lost their invasive ability. Further, Neu-transformed cells also underwent growth arrest and apoptosis if integrin β4-dependent signaling was simultaneously inactivated. Human breast cancer cells carrying oncogenic mutations that activate Ras or PI3K signaling displayed similar responses upon silencing of FAK. Mechanistic studies indicated that FAK sustains tumorigenesis by promoting Src-mediated phosphorylation of p130 Cas . These results suggest that FAK supports Ras-and PI3K-dependent mammary tumor initiation, maintenance, and progression to metastasis by orchestrating multiple core cellular functions, including proliferation, survival, and avoidance of senescence.

Morphogenesis of a vascular network requires dynamic vessel growth and regression. To investigate the cellular mechanism underlying this process, we deleted focal adhesion kinase (FAK), a key signaling mediator, in endothelial cells (ECs) using Tie2-Cre mice. Targeted FAK depletion occurred efficiently early in development, where mutants exhibited a distinctive and irregular vasculature, resulting in hemorrhage and lethality between embryonic day (e) 10.5 and 11.5. Capillaries and intercapillary spaces in yolk sacs were dilated before any other detectable abnormalities at e9.5, and explants demonstrate that the defects resulted from the loss of FAK and not from organ failure. Time-lapse microscopy monitoring EC behavior during vascular formation in explants revealed no apparent decrease in proliferation or migration but revealed increases in cell retraction and death leading to reduced vessel growth and increased vessel regression. Consistent with this phenotype, ECs derived from mutant embryos exhibited aberrant lamellipodial extensions, altered actin cytoskeleton, and nonpolarized cell movement. This study reveals that FAK is crucial for vascular morphogenesis and the regulation of EC survival and morphology.

Axonal growth cones respond to adhesion molecules and extracellular matrix components by rapid morphological changes and growth rate modification. Neurite outgrowth mediated by the neural cell adhesion molecule (NCAM) requires the src family tyrosine kinase p59 fyn in nerve growth cones, but the molecular basis for this interaction has not been defined. The NCAM140 isoform, which is found in migrating growth cones, selectively co-immunoprecipitated with p59fyn from nonionic detergent (Brij 96) extracts of early postnatal mouse cerebellum and transfected rat B35 neuroblastoma and COS-7 cells. p59 fyn did not associate significantly with the NCAM180 isoform, which is found at sites of stable neural cell contacts, or with the glycophosphatidylinositol-linked NCAM120 isoform. fak , and p59 fyn and activate the catalytic function of these tyrosine kinases, initiating a signaling cascade that may modulate growth cone migration.

In response to αβ1 integrin signaling, transducers such as focal adhesion kinase (FAK) become activated, relaying to specific machineries and triggering distinct cellular responses. By conditionally ablating Fak in skin epidermis and culturing Fak-null keratinocytes, we show that FAK is dispensable for epidermal adhesion and basement membrane assembly, both of which require αβ1 integrins. FAK is also dispensible for proliferation/survival in enriched medium. In contrast, FAK functions downstream of αβ1 integrin in regulating cytoskeletal dynamics and orchestrating polarized keratinocyte migration out of epidermal explants. Fak-null keratinocytes display an aberrant actin cytoskeleton, which is tightly associated with robust, peripheral focal adhesions and microtubules. We find that without FAK, Src, p190RhoGAP, and PKL–PIX–PAK, localization and/or activation at focal adhesions are impaired, leading to elevated Rho activity, phosphorylation of myosin light chain kinase, and enhanced tensile stress fibers. We show that, together, these FAK-dependent activities are critical to control the turnover of focal adhesions, which is perturbed in the absence of FAK.

Macrophages are a key component of the innate immune system. In this study, we investigate how focal adhesion kinase (FAK) and the related kinase Pyk2 integrate adhesion signaling and growth factor receptor signaling to regulate diverse macrophage functions. Primary bone marrow macrophages isolated from mice in which FAK is conditionally deleted from cells of the myeloid lineage exhibited elevated protrusive activity, altered adhesion dynamics, impaired chemotaxis, elevated basal Rac1 activity, and a marked inability to form stable lamellipodia necessary for directional locomotion. The contribution of FAK to macrophage function in vitro was substantiated in vivo by the finding that recruitment of monocytes to sites of inflammation was impaired in the absence of FAK. Decreased Pyk2 expression in primary macrophages also resulted in a diminution of invasive capacity. However, the combined loss of FAK and Pyk2 had no greater effect than the loss of either molecule alone, indicating that both kinases function within the same pathway to promote invasion.

Abstract. Src-related nonreceptor protein tyrosine kinases in nerve growth cones (p59 fyn, pp60"srL and pp62c'Y~0 are potential intracellular signaling molecules for cell adhesion molecule-directed axonal growth. To determine whether src-related tyrosine kinases mediate NCAM-dependent neurite outgrowth, cultures of cerebellar and sensory neurons from fyn-, src-, and yesminus mice were analyzed for neurite outgrowth on monolayers of NCAM140-transfected L fibroblasts. NCAM-dependent neurite outgrowth was selectively inhibited in cultures of cerebellar and dorsal root ganglion neurons from fyn-, but not src-or yes-mice. Neurite outgrowth by fyn-, src-, or yes-neurons on untransfected fibroblast monolayers was unaffected, indicating that these kinases do not contribute significantly to axon growth on at least some integrins or other adhesive substrates present on fibroblasts. This study demonstrates that p59" is an essential component of the NCAM signaling pathway leading to axonal growth.

The formation of neuronal networks in the central nervous system (CNS) requires precise control of axonal branch development and stabilization. Here we show that cell-specific ablation of the murine gene Ptk2 (more commonly known as fak), encoding focal adhesion kinase (FAK), increases the number of axonal terminals and synapses formed by neurons in vivo. Consistent with this, fak mutant neurons also form greater numbers of axonal branches in culture because they have increased branch formation and reduced branch retraction. Expression of wild-type FAK, but not that of several FAK variants that prevent interactions with regulators of Rho family GTPases including the p190 Rho guanine nuclear exchange factor (p190RhoGEF), rescues the axonal arborization phenotype observed in fak mutant neurons. In addition, expression of a mutant p190RhoGEF that cannot associate with FAK results in a phenotype very similar to that of neurons lacking FAK. Thus, FAK functions as a negative regulator of axonal branching and synapse formation, and it seems to exert its actions, in part, through Rho family GTPases.