Induced focal adhesion kinase (FAK) expression in FAK-null cells enhances cell spreading and migration requiring both auto- and activation loop phosphorylation sites and inhibits adhesion-dependent tyrosine phosphorylation of Pyk2

Abstract

Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase involved in integrin-mediated control of cell behavior. Following cell adhesion to components of the extracellular matrix, FAK becomes phosphorylated at multiple sites, including tyrosines 397, 576, and 577. Tyr-397 is an autophosphorylation site that promotes interaction with c-Src or Fyn. Tyr-576 and Tyr-577 lie in the putative activation loop of the kinase domain, and FAK catalytic activity may be elevated through phosphorylation of these residues by associated Src family kinase. Recent studies have implicated FAK as a positive regulator of cell spreading and migration. To further study the mechanism of adhesion-induced FAK activation and the possible role and signaling requirements for FAK in cell spreading and migration, we utilized the tetracycline repression system to achieve inducible expression of either wild-type FAK or phosphorylation site mutants in fibroblasts derived from FAK-null mouse embryos. Using these Tet-FAK cells, we demonstrated that both the FAK autophosphorylation and activation loop sites are critical for maximum adhesion-induced FAK activation and FAK-enhanced cell spreading and migration responses. Negative effects on cell spreading and migration, as well as decreased phosphorylation of the substrate p130(Cas), were observed upon induced expression of the FAK autophosphorylation site mutant. These negative effects appear to result from an inhibition of integrin-mediated signaling by the FAK-related kinase Pyk2/CAKbeta/RAFTK/CadTK.

FIG. 1

Inducible FAK expression in Tet-FAK cells. (A) Induction time course. Parallel cultures of exponentially growing Tet-FAK(WT)-46 cells were induced by tetracycline withdrawal (0 h), and cell lysates were prepared at the times indicated for analysis of FAK expression. (B) Expression levels. The indicated Tet-FAK clones were either maintained in the presence of tetracycline (+ tet.) or induced by tetracycline withdrawal for 2 days (− tet.), and then total cell lysates were prepared and analyzed for relative FAK expression levels. For both panels A and B, cells were lysed in RIPA buffer, and 30 μg of total protein was loaded per lane for assessment of FAK levels by immunoblotting (IB) with C-20 antibody and detection with ¹²⁵I-labeled protein A. Control samples (MEF +/+ lanes) contained 30 μg of total protein prepared from normal mouse embryo fibroblasts. Relative expression levels were quantitated by phosphorimage volume integration.

FIG. 2

Immunolocalization of FAK in induced Tet-FAK cells. Tet-FAK(WT)-46, Tet-FAK(F397)-21, and Tet-FAK(F576/F577)-16 cells were induced for 2 days and then plated overnight on fibronectin-coated coverslips. Double-label indirect immunofluorescence was carried out by using monoclonal antibody 8d4 against talin (left panels) and polyclonal antibody C-20 against FAK (right panels). Bar = 30 μm.

FIG. 3

Relative tyrosine phosphorylation of WT-, F397-, and F576/F577-FAK in Tet-FAK cells growing under normal culture conditions. Tet-FAK cells were induced for either 2 days [Tet-FAK(WT)-46 and Tet-FAK(F576/F577)-16] or 12 h [Tet-FAK(F397)-21] to obtain near-equal levels of expression of the FAK variants, and then FAK was immunoprecipitated (IP) from RIPA buffer lysates containing 650 μg of total protein. The immunoprecipitates were then divided equally for immunoblot (IB) analysis by using either antiphosphotyrosine antibody 4G10 (top panel) or anti-FAK antibody C-20 (bottom panel). Immunoblots were developed by enhanced chemiluminescence, and signals were quantitated from digitized images.

FIG. 4

In vitro kinase assays. (A) Inhibition of c-Src but not FAK by PD161430. Increasing concentrations of PD161430 (0 to 220 μM) were added to mixtures for kinase reactions carried out on either coimmunoprecipitates of c-Src and p120^ctn (top panel) or baculovirus-expressed FAK (bottom panel). PD161430 inhibited c-Src phosphorylation of p120^ctn with a 50% inhibitory concentration of ∼0.2 μM but had little or no effect on FAK autophosphorylation at concentrations up to 220 μM. (B and C) F576/F577-FAK shows reduced adhesion-dependent tyrosine phosphorylation and in vitro autophosphorylation activity. WT-FAK or F576/F577-FAK was immunoprecipitated (IP) from NP-40 buffer lysates of induced Tet-FAK(WT)-46 or Tet-FAK(F576/F577)-16 cells, respectively, under either attached (Att) (serum-starved 14 h), suspended (Sus), or fibronectin-replated (Fn) conditions. The immunoprecipitates were then divided equally for assessment of in vivo FAK tyrosine phosphorylation by immunoblotting (IB) with antiphosphotyrosine antibody 4G10 (top panel), FAK recovery by immunoblotting with anti-FAK antibody C-20 (middle panel), and in vitro FAK phosphorylation from kinase assays carried out either in the absence or presence of 22 μM PD161430 (bottom panel). In panel C, in vitro FAK phosphorylation from three independent kinase assays is plotted as mean activity (+SEM) relative to that of WT-FAK assayed from attached cells in the absence of PD161430. (D) F397-FAK and F576/F577-FAK show reduced phosphorylation of poly(GluTyr). WT, -F397, and F576/F577-FAK were immunoprecipitated from NP-40 buffer lysates prepared from induced Tet-FAK(WT)-46, Tet-FAK(F397)-21, or Tet-FAK(F576/F577)-16 cells, respectively, and used to assess vitro kinase phosphorylation of poly(GluTyr) in kinase assays carried out either in the absence or presence of 22 μM PD161430. Data from three independent assays are plotted as mean activities (+SEM) relative to that of WT-FAK assayed from attached (Att) cells in the absence of PD161430.

FIG. 5

Early spreading analysis of Tet-FAK cells. Induced or noninduced Tet-FAK(WT)-46, Tet-FAK(F397)-21, or Tet-FAK(F576/F577)-16 cells were plated onto fibronectin-coated tissue culture dishes and, after 30 min, phase-contrast light microscopic images were captured for spreading analysis. Note full lamellipod extension of induced WT-FAK cells and more apparent filopod extension of induced F397-FAK cells. Bar = 37.5 μm.

FIG. 6

Late spreading analysis of Tet-FAK cells. Induced and noninduced Tet-FAK(WT)-46, Tet-FAK(F397)-21, and Tet-FAK(F576/F577)-16 cells were plated onto fibronectin-coated glass coverslips and, after 20 h, phase-contrast light microscopic images were captured for spreading analysis. Note the highly spread morphology of induced F397-FAK cells. Bar = 75 μm.

FIG. 7

Analysis of cell migration changes following induced expression of wild-type FAK versus phosphorylation site FAK mutants. Cell migration was determined by using the modified Boyden chamber assay for noninduced versus induced cell clones as follows: Tet-tTA (clones 13 and 3), Tet-FAK(WT) (clones 70 and 46), Tet-FAK(F397) (clones 18 and 21), and Tet-FAK(F576/F577) (clones 16 and 29). The assay was carried out for 5 h, with 10% FBS as the attractant, and migrating cells were counted and averaged from four or more representative wells. The percentage of change in migration was determined by dividing the number of cells migrating under induced (without tetracycline) conditions by the number of cells migrating under noninduced (with tetracycline) conditions. Three independent experiments (inductions) were carried out for each cell clone, and the average percentages of change in migration (±SEM) are shown.

FIG. 8

Pyk2 adhesion-dependent tyrosine phosphorylation is impaired by FAK expression. (A) Relative Pyk2 levels were determined by immunoblot (IB) analysis of total cell lysates (30 μg of total protein) from FAK-null cells (parental population to Tet-FAK clones) versus normal mouse embryo fibroblasts. (B) Induced or noninduced Tet-FAK(F397)-21 cells were serum starved for 2 h and then lysed when either attached (Att.), trypsinized and held in suspension for 30 min (Susp.), or replated onto fibronectin for either 30 min (Fn-30′) or 60 min (Fn-60′). Either FAK or Pyk2 was immunoprecipitated (IP) from 300 μg of total protein lysates and then divided equally for immunoblot (IB) detection of either FAK, Pyk2, or phosphotyrosine (pTyr).

FIG. 9

Induced FAK expression alters Cas phosphotyrosine levels. Tet-FAK(WT)-46, Tet-FAK(F397)-21, and Tet-FAK(F576/F577)-29 cells were either maintained in the presence of tetracycline or induced to express near-equal amounts of WT-, F397, and F576/F577-FAK protein, respectively, and then cells from either attached (Att) (serum starved for 14 h), suspended (Sus), or fibronectin-replated (Fn) (30 min) conditions were lysed in RIPA buffer. FAK (top panel) and Cas (bottom panel) levels were assessed by immunoblot (IB) analysis of 20 μg of total protein from the lysates. Two hundred eighty micrograms of total protein from the same lysates was subjected to immunoprecipitation (IP) by using PY20 antiphosphotyrosine (pTyr) antibody, followed by Cas immunoblot analysis, to determine relative levels of tyrosine phosphorylation of Cas (middle panel).

FIG. 10

Model for reciprocal catalytic activation by FAK and c-Src (or Fyn) with potential signal amplification loop and possible downstream signaling events promoting cell spreading and motility. See text for details.