Dusko Ilic

FAK integrates growth-factor and integrin signals to promote cell migration

Here we show that cells lacking focal adhesion kinase (FAK) are refractory to motility signals from platelet-derived and epidermal growth factors (PDGF and EGF respectively), and that stable re-expression of FAK rescues these defects. FAK associates with activated PDGF- and EGF-receptor (PDGFR and EGFR) signalling complexes, and expression of the band-4.1-like domain at the FAK amino terminus is sufficient to mediate an interaction with activated EGFR. However, efficient EGF-stimulated cell migration also requires FAK to be targeted, by its carboxy-terminal domain, to sites of integrin-receptor clustering. Although the kinase activity of FAK is not needed to promote PDGF- or EGF-stimulated cell motility, kinase-inactive FAK is transphosphorylated at the indispensable Src-kinase-binding site, FAK Y397, after EGF stimulation of cells. Our results establish that FAK is an important receptor-proximal link between growth-factor-receptor and integrin signalling pathways.

Differential regulation of cell motility and invasion by FAK

Cell migration and invasion are fundamental components of tumor cell metastasis. Increased focal adhesion kinase (FAK) expression and tyrosine phosphorylation are connected with elevated tumorigenesis. Null mutation of FAK results in embryonic lethality, and FAK−/− fibroblasts exhibit cell migration defects in culture. Here we show that viral Src (v-Src) transformation of FAK−/− cells promotes integrin-stimulated motility equal to stable FAK reexpression. However, FAK−/− v-Src cells were not invasive, and FAK reexpression, Tyr-397 phosphorylation, and FAK kinase activity were required for the generation of an invasive cell phenotype. Cell invasion was linked to transient FAK accumulation at lamellipodia, formation of a FAK–Src-p130Cas–Dock180 signaling complex, elevated Rac and c-Jun NH2-terminal kinase activation, and increased matrix metalloproteinase expression and activity. Our studies support a dual role for FAK in promoting cell motility and invasion through the activation of distinct signaling pathways.

Pyk2 and Src-family protein-tyrosine kinases compensate for the loss of FAK in fibronectin-stimulated signaling events but Pyk2 does not fully function to enhance FAK- cell migration.

The focal adhesion kinase (FAK) protein‐tyrosine kinase (PTK) links transmembrane integrin receptors to intracellular signaling pathways. We show that expression of the FAK‐related PTK, Pyk2, is elevated in fibroblasts isolated from murine fak−/− embryos (FAK−) compared with cells from fak+/+ embryos (FAK+). Pyk2 was localized to perinuclear regions in both FAK+ and FAK− cells. Pyk2 tyrosine phosphorylation was enhanced by fibronectin (FN) stimulation of FAK− but not FAK+ cells. Increased Pyk2 tyrosine phosphorylation paralleled the time‐course of Grb2 binding to Shc and activation of ERK2 in FAK− cells. Pyk2 in vitro autophosphorylation activity was not enhanced by FN plating of FAK− cells. However, Pyk2 associated with active Src‐family PTKs after FN but not poly‐L‐lysine replating of the FAK− cells. Overexpression of both wild‐type (WT) and kinase‐inactive (Ala457), but not the autophosphorylation site mutant (Phe402) Pyk2, enhanced endogenous FN‐stimulated c‐Src in vitro kinase activity in FAK− cells, but only WT Pyk2 overexpression enhanced FN‐stimulated activation of co‐transfected ERK2. Interestingly, Pyk2 overexpression only weakly augmented FAK− cell migration to FN whereas transient FAK expression promoted FAK− cell migration to FN efficiently compared with FAK+ cells. Significantly, repression of endogenous Src‐family PTK activity by p50csk overexpression inhibited FN‐stimulated cell spreading, Pyk2 tyrosine phosphorylation, Grb2 binding to Shc, and ERK2 activation in the FAK− but not in FAK+ cells. These studies show that Pyk2 and Src‐family PTKs combine to promote FN‐stimulated signaling events to ERK2 in the absence of FAK, but that these signaling events are not sufficient to overcome the FAK− cell migration defects.

Nuclear FAK Promotes Cell Proliferation and Survival through FERM-Enhanced p53 Degradation

FAK is known as an integrin- and growth factor-associated tyrosine kinase promoting cell motility. Here we show that, during mouse development, FAK inactivation results in p53- and p21-dependent mesodermal cell growth arrest. Reconstitution of primary FAK-/-p21-/- fibroblasts revealed that FAK, in a kinase-independent manner, facilitates p53 turnover via enhanced Mdm2-dependent p53 ubiquitination. p53 inactivation by FAK required FAK FERM F1 lobe binding to p53, FERM F2 lobe-mediated nuclear localization, and FERM F3 lobe for connections to Mdm2 and proteasomal degradation. Staurosporine or loss of cell adhesion enhanced FERM-dependent FAK nuclear accumulation. In primary human cells, FAK knockdown raised p53-p21 levels and slowed cell proliferation but did not cause apoptosis. Notably, FAK knockdown plus cisplatin triggered p53-dependent cell apoptosis, which was rescued by either full-length FAK or FAK FERM re-expression. These studies define a scaffolding role for nuclear FAK in facilitating cell survival through enhanced p53 degradation under conditions of cellular stress.

Integrin signaling: it's where the action is

Recent advances highlight a critical role for integrin receptors for extracellular matrix in determining where in cells critical signals are transduced. Integrins are shown to activate signaling intermediates at specific surface membrane locations, to promote nuclear translocation of factors that activate gene transcription, and to recruit and augment the signaling power of receptors for growth factors.

PyK2 and FAK connections to p190Rho guanine nucleotide exchange factor regulate RhoA activity, focal adhesion formation, and cell motility

Integrin binding to matrix proteins such as fibronectin (FN) leads to formation of focal adhesion (FA) cellular contact sites that regulate migration. RhoA GTPases facilitate FA formation, yet FA-associated RhoA-specific guanine nucleotide exchange factors (GEFs) remain unknown. Here, we show that proline-rich kinase-2 (Pyk2) levels increase upon loss of focal adhesion kinase (FAK) in mouse embryonic fibroblasts (MEFs). Additionally, we demonstrate that Pyk2 facilitates deregulated RhoA activation, elevated FA formation, and enhanced cell proliferation by promoting p190RhoGEF expression. In normal MEFs, p190RhoGEF knockdown inhibits FN-associated RhoA activation, FA formation, and cell migration. Knockdown of p190RhoGEF-related GEFH1 does not affect FA formation in FAK−/− or normal MEFs. p190RhoGEF overexpression enhances RhoA activation and FA formation in MEFs dependent on FAK binding and associated with p190RhoGEF FA recruitment and tyrosine phosphorylation. These studies elucidate a compensatory function for Pyk2 upon FAK loss and identify the FAK–p190RhoGEF complex as an important integrin-proximal regulator of FA formation during FN-stimulated cell motility.

Intrinsic FAK activity and Y925 phosphorylation facilitate an angiogenic switch in tumors

Elevated focal adhesion kinase (FAK) expression occurs in advanced cancers, yet a signaling role for FAK in tumor progression remains undefined. Here, we suppressed FAK activity in 4T1 breast carcinoma cells resulting in reduced FAK Y925 phosphorylation, Grb2 adaptor protein binding to FAK, and signaling to mitogen-activated protein (MAP) kinase (MAPK). Loss of a FAK-Grb2-MAPK linkage did not affect 4T1 cell proliferation or survival in culture, yet FAK inhibition reduced vascular endothelial growth factor (VEGF) expression and resulted in small avascular tumors in mice. This FAK-Grb2-MAPK linkage was essential in promoting angiogenesis as reconstitution experiments using Src-transformed FAK-null fibroblasts revealed that point mutations affecting FAK catalytic activity (R454) or Y925 phosphorylation (F925) disrupted the ability of FAK to promote MAPK- and VEGF-associated tumor growth. Notably, in both FAK-inhibited 4T1 and Src-transformed FAK-null cells, constitutively activated (CA) mitogen-activated protein kinase kinase 1 (MEK1) restored VEGF production and CA-MEK1 or added VEGF rescued tumor growth and angiogenesis. These studies provide the first biological support for Y925 FAK phosphorylation and define a novel role for FAK activity in promoting a MAPK-associated angiogenic switch during tumor progression.

Focal Adhesion Kinase Is Required for Blood Vessel Morphogenesis

Abstract— The nonreceptor tyrosine kinase focal adhesion kinase (FAK) is a point of convergence for signals from extracellular matrix, soluble factors, and mechanical stimuli. Targeted disruption of the fak gene in mice leads to death at embryonic day 8.5 (E8.5). FAK−/− embryos have severely impaired blood vessel development. Gene expression and in vitro differentiation studies revealed that endothelial cell differentiation was comparable in FAK−/− and wild-type E8.5 embryos. We examined the role of FAK in blood vessel morphogenesis using an in vitro tubulogenesis assay and three different culture systems: FAK+/+ and FAK−/− embryoid bodies, FAK+/+ and FAK−/− endothelial cells, and human umbilical vein endothelial cells expressing antisense FAK, a dominant-negative fragment of FAK, or wild-type FAK. In all of these systems, endothelial cells deficient in FAK expression or function displayed a severely reduced ability to form tubules in Matrigel. These studies demonstrate clearly that the vascular defects in FAK−/− mice result from the inability of FAK-deficient endothelial cells to organize themselves into vascular networks, rather than from defects in tissue-specific differentiation.

FAK promotes organization of fibronectin matrix and fibrillar adhesions

Targeted disruption of the focal adhesion kinase (FAK) gene in mice is lethal at embryonic day 8.5 (E8.5). Vascular defects in FAK-/- mice result from the inability of FAK-deficient endothelial cells to organize themselves into vascular network. We found that, although fibronectin (FN) levels were similar, its organization was less fibrillar in both FAK-/- endothelial cells and mesoderm of E8.5 FAK-/- embryos, as well as in mouse embryonic fibroblasts isolated from mutant embryos. FAK catalytic activity, proline-rich domains, and location in focal contacts were all required for proper allocation and patterning of FN matrix. Cells lacking FAK in focal adhesions fail to translocate supramolecular complexes of integrin-bound FN and focal adhesion proteins along actin filaments to form mature fibrillar adhesions. Taken together, our data suggest that proper FN allocation and organization are dependent on FAK-mediated remodeling of focal adhesions.

v-Src SH3-enhanced Interaction with Focal Adhesion Kinase at β1 Integrin-containing Invadopodia Promotes Cell Invasion

In viral Src (v-Src)-transformed cells, focal adhesion kinase (FAK) associates with v-Src by combined v-Src SH2 and gain-of-function v-Src SH3 domain binding to FAK. Here we assess the significance of the Arg-95 to Trp gain-of-function mutation in the v-Src SH3 domain through comparisons of Src−/− fibroblasts transformed with either Prague C v-Src or a point mutant (v-Src-RT) containing a normal (Arg-95) SH3 domain. Both v-Src isoforms exhibited equivalent kinase activity, enhanced Src−/− cell motility, and stimulated cell growth in both low serum and soft agar. The stability of a v-Src·FAK signaling complex and FAK phosphorylation at Tyr-861 and Tyr-925 were reduced in v-Src-RT- compared with v-Src-transformed cells. v-Src but not v-Src-RT promoted Src−/− cell invasion through a reconstituted Matrigel basement membrane barrier and v-Src co-localized with FAK and β1 integrin at invadopodia. In contrast, v-Src-RT exhibited a partial perinuclear and focal contact distribution in Src−/− cells. Adenovirus-mediated FAK overexpression promoted v-Src-RT recruitment to invadopodia, the formation of a v-Src-RT·FAK signaling complex, and reversed the v-Src-RT invasion deficit. Adenovirus-mediated inhibition of FAK blocked v-Src-stimulated cell invasion. These studies establish that gain-of-function v-Src SH3 targeting interactions with FAK at β1 integrin-containing invadopodia act to stabilize a v-Src·FAK signaling complex promoting cell invasion.