Fer-mediated cortactin phosphorylation is associated with efficient fibroblast migration and is dependent on reactive oxygen species generation during integrin-mediated cell adhesion

Abstract

The molecular details linking integrin engagement to downstream cortactin (Ctn) tyrosine phosphorylation are largely unknown. In this report, we show for the first time that Fer and Ctn are potently tyrosine phosphorylated in response to hydrogen peroxide (H2O2) in a variety of cell types. Working with catalytically inactive fer and src/yes/fyn-deficient murine embryonic fibroblasts (ferDR/DR and syf MEF, respectively), we observed that H2O2-induced Ctn tyrosine phosphorylation is primarily dependent on Fer but not Src family kinase (SFK) activity. We also demonstrated for the first time that Fer is activated by fibronectin engagement and, in concert with SFKs, mediates Ctn tyrosine phosphorylation in integrin signaling pathways. Reactive oxygen species (ROS) scavengers or the NADPH oxidase inhibitor, diphenylene iodonium, attenuated integrin-induced Fer and Ctn tyrosine phosphorylation. Taken together, these findings provide novel genetic evidence that a ROS-Fer signaling arm contributes to SFK-mediated Ctn tyrosine phosphorylation in integrin signaling. Lastly, a migration defect in ferDR/DR MEF suggests that integrin signaling through the ROS-Fer-Ctn signaling arm may be linked to mechanisms governing cell motility. These data demonstrate for the first time an oxidative link between integrin adhesion and an actin-binding protein involved in actin polymerization.

FIG. 1.

Fer and Ctn are robustly phosphorylated on tyrosine in response to H₂O₂ in wt but not fer^DR/DR MEF. (A) Subconfluent monolayers of MEF were treated with 0 to 10 mM H₂O₂ for 0 to 90 min. Cells were then harvested and anti-Fer antibodies were used for IP analysis, followed by IB with anti-pY and anti-Fer antibodies, as indicated. (B) Fer phosphorylation was assessed as above in wt and fer^DR/DR MEF after treatment with increasing concentrations of H₂O₂ for 10 min. (C and D) Total Ctn, pY421-Ctn, or pY466-Ctn were assessed by IB in lysates from subconfluent monolayers of wt and fer^DR/DR MEF treated with 0 to 9 mM H₂O₂ for 10 min (C) or 2 mM H₂O₂ for 0 to 90 min (D). (E) Densitometric analyses of the phosphotyrosine profiles of Fer in panel A and Ctn at Y421 and Y466 in panel D. (F) Fer and Ctn phosphorylation were assessed by IP/IB or IB as described above after 10-min challenges with the indicated H₂O₂ concentrations in wt, fer^DR/DR, or fer^DR/DR MEF infected with lentivirus encoding a Fer-DsRed protein (fer^DR/DR-Rescue).

FIG. 2.

SFKs do not play a major role in cortactin tyrosine phosphorylation downstream of H₂O₂. The effect of PP2 on H₂O₂-stimulated Fer and Ctn phosphorylation in wt or fer^DR/DR MEF was tested by IP/IB or IB analyses. Both normal and high exposures of pY421-Ctn and pY466-Ctn are shown in order to accentuate the relative differences in H₂O₂-stimulated Ctn phosphorylation in wt and fer^DR/DR MEF, as well as to demonstrate that low levels of H₂O₂-stimulated Ctn phosphorylation in fer^DR/DR MEF are dose-dependently inhibited by PP2.

FIG. 3.

H₂O₂-induced cortactin tyrosine phosphorylation in syf triple-deficient MEF correlates with Fer activation. (A) Tyrosine phosphorylation of Fer and Ctn was assessed by IP/IB (Fer) or IB (Ctn) in H₂O₂-treated wt and syf MEF. (B) A plot of Fer pY to Fer ratios versus Ctn pY to Ctn ratios from four independent experiments reveals a strong biphasic correlation in the level of activation of these proteins in response to H₂O₂.

FIG. 4.

Increasing Fer expression in syf MEF directly increased the levels of H₂O₂-induced Ctn tyrosine phosphorylation. (A) syf MEF were infected with increasing amount of lentiviruses encoding Fer-DsRed or kinase-dead FerK592R-DsRed. The cells were then challenged with H₂O₂, and assessed by IP/IB or IB for Fer and Ctn expression and tyrosine phosphorylation. (B and C) The blots in panel A were analyzed by densitometry, and pY421-Ctn to Ctn and pY466-Ctn to Ctn ratios were expressed as functions of Fer-DsRed and FerK592R-DsRed expression levels. The data show a linear correlation between pY421-Ctn phosphorylation and both Fer-DsRed and FerK592R-DsRed expression (B; R² = 0.9786 and 0.8565, respectively). A linear correlation between pY466-Ctn phosphorylation and Fer-DsRed and FerK592R-DsRed expression was also observed (C; R² = 0.9913 and 0.7935, respectively). The responses of pY421-Ctn and pY466-Ctn tyrosine phosphorylation to expression of Fer-DsRed in syf MEF were calculated to be 10.5- and 5.8-fold greater, respectively, than the responses of phosphorylation at these residues to expression of FerK592R-DsRed.

FIG. 5.

Cortactin tyrosine phosphorylation is dependent on cell substratum-induced Fer tyrosine kinase activity. (A) wt MEF were plated on Fn or PLL for the indicated times and harvested. IP/IB analyses showed that Fer is tyrosine phosphorylated in response to Fn and PLL plating. Statistical analyses of each time point showed that Fn-induced Fer tyrosine phosphorylation was significantly different from the level of Fer phosphorylation in suspended cells (P = 9.3 × 10⁻⁹ − 0.046). (B) wt and fer^DR/DR MEF were plated on Fn for 0 to 60 min. IP/IB analyses showed that Fer was inducibly phosphorylated in response to cell adhesion in wt but not fer-deficient MEF. Two-way ANOVA analyses showed a statistically significant difference in Fer activation in wt and fer^DR/DR MEF (P = 0.0058). (C to F) Soluble cell lysates were prepared from cells harvested after adhesion to Fn for the indicated times. IB analysis using phosphospecific antibodies against pY421-Ctn (C) and pY466-Ctn (D) showed that both sites were strongly phosphorylated in wt but not fer^DR/DR MEF in response to substratum adhesion (two-way ANOVA; P = 10⁻⁴ and 10⁻⁵, respectively). IB analyses using phosphospecific antibodies against pY418 of Src (E) and pY397 of Fak (F) showed that the kinetic phosphorylation profiles of these key adhesion proteins were normal in adhering fer^DR/DR MEF (two-way ANOVA; P = 0.08 and 0.96, respectively).

FIG. 6.

H₂O₂ scavengers and the NADPH oxidase inhibitor, DPI, inhibit Fer and cortactin phosphorylation induced by cell substratum engagement. (A) Fn-induced tyrosine phosphorylation of Fer at 25 min (expressed as a ratio of Fer pY to Fer in IP/IB analysis) was attenuated by the H₂O₂ scavengers NAC and Tiron and the NADPH oxidase inhibitor, DPI (*, P = 0.04, 0.01, and 0.002, respectively). The mitochondrial superoxide production inhibitor rotenone caused a partial inhibitory effect on Fer pY, although this change was not statistically significant. (B) Fn-induced phosphorylation of Ctn at 25 min (expressed as a ratio of pY421-Ctn to total Ctn) was also attenuated by NAC, Tiron, and DPI (*, P = 0.0044, 0.0007, and 0.0046, respectively). Rotenone had no effect on adhesion-induced Ctn phosphorylation.

FIG. 7.

SFKs promote Fer and Ctn tyrosine phosphorylation downstream of integrins. wt and syf MEF were plated on Fn and subsequently analyzed by IP/IB. Reduced Fer activation and Ctn phosphorylation at residues Y421 and Y466 are observed in syf MEF in response to adhesion on Fn at 25 min. This reduction is most pronounced at 40 min.

FIG. 8.

Ctn tyrosine phosphorylation is compromised in fer^DR/DR fibroblasts. MEF were allowed to adhere to Fn-coated surfaces and fixed after 30 min. Cells were stained with antibodies against total Ctn (red) and Fer (green) and with phosphospecific antibodies against pY421 or pY466 of Ctn (green) or against pY418 of Src (green), as indicated. (A to D) Ctn staining was observed at the cell periphery and in the perinuclear region in both wt (A and C) and fer^DR/DR (B and D) MEF. Consistent with the biochemical adhesion data in Fig. 5, phosphorylation at residues Y421 and Y466 of Ctn was detected in wt fibroblasts (A and C) but was undetectable or only faintly visible in fer^DR/DR fibroblasts (B and D). (E and F) Phosphorylation of residue Y418 of Src, Yes, and Fyn was observed in peripheral regions of both wt and fer^DR/DR MEF. (G and H) Cytoplasmic staining of Fer was observed in both wt and fer^DR/DR MEF. Localization of Fer in the periphery and its colocalization with Ctn in these regions were more predominant in wt MEF.

FIG. 9.

fer^DR/DR MEF have defects in migration in vitro. (A and B) Migration assays of wt, fer^DR/DR, and fer^DR/DR MEF rescued by lentiviral-mediated expression of Fer-DsRed (A; Rescue) or retroviral-mediated expression of Fer-Myc (B; Rescue). (A) Detailed kinetic profiles of scrape-wound areas (expressed as percentages of the initial wound area ± SD) for wt, fer^DR/DR, and fer-Rescue MEF over 18 h. Initial wound areas, expressed as means ± SD, were 161,447 ± 20,659 μm² for wt MEF, 176,044 ± 16,763 μm² for fer^DR/DR MEF, and 154,958 ± 9,317 μm² for fer-Rescue MEF. Kinetic migration profiles of fer^DR/DR MEF show a severe delay in wound closure which is partially rescued by expression of Fer-DsRed (*, P = 0.0001). (B) Relative migration in transwell assays of wt MEF, fer^DR/DR MEF, and fer^DR/DR MEF rescued by retroviral-mediated expression of epitope-tagged Myc-Fer. fer^DR/DR MEF exhibited impaired migration in transwell assays which was rescued by retroviral transduction of a Fer-Myc fusion protein (*, P = 0.0019; **, P = 0.0036). The y axis represents the average number of migrated cells per insert acquired from four ×20 fields of view. Three transwell inserts were used per cell type, per experiment.

FIG. 10.

Model of Fer involvement in integrin-induced Ctn phosphorylation. Ligand engagement of integrins leads to activation of NADPH oxidase and production of ROS, which in turn leads to inhibition of PTP. PTP inhibition is involved in the inactivation of SFKs at the plasma membrane and plays a complex role in SYF regulation, including both stimulatory and inhibitory dephosphorylation at Y519 and Y418, respectively (51). LMW-PTP inhibits Fak (9), and we propose that PTP may also be involved in the inhibition of Fer. Collectively, integrin-induced ROS production correlates with Fer, Fak, and SYF activation, and subsequent phosphorylation of Ctn, which in turn contributes to the regulation of cell migration. This model predicts that Fer can act both independently of SFK and Fak and downstream from them during oxidative signaling from integrins to Ctn. It emphasizes a novel ROS-Fer-Ctn signaling arm involved in the regulation of cell migration downstream of integrin signaling pathways.