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. 2009 Sep 30;85(3):571–581. doi: 10.1093/cvr/cvp322

Role of FRNK tyrosine phosphorylation in vascular smooth muscle spreading and migration

Yevgeniya E Koshman 1,†, Steven J Engman 1,†, Taehoon Kim 1, Rekha Iyengar 1, Kyle K Henderson 1, Allen M Samarel 1,*
PMCID: PMC2802202  PMID: 19793767

Abstract

Aims

Focal adhesion kinase (FAK) and its autonomously expressed, C-terminal inhibitor FAK-related non-kinase (FRNK), are important regulators of vascular smooth muscle cell (VSMC) spreading and migration. However, the mechanisms of FRNK-mediated inhibition of FAK-dependent signalling are not fully defined. The aim of this study was to determine the potential role of FRNK tyrosine phosphorylation in regulating these processes.

Methods and results

Rat carotid arteries were balloon-injured and FAK and FRNK expression and phosphorylation were examined by immunocytochemistry, immunoprecipitation, and western blotting with total and phosphospecific antibodies. FAK and FRNK expression increased four- and nine-fold, respectively, in α-smooth muscle actin-positive VSMCs of injured arteries when compared with contralateral control arteries, and the upregulated FRNK was phosphorylated at residues Y168 and Y232. In A7r5 cells (an embryonic rat VSMC line), endogenously expressed FRNK was also phosphorylated at Y168 and Y232 under basal conditions, and Y168/Y232 phosphorylation increased in response to angiotensin II treatment. When overexpressed in A7r5 cells and adult rat aortic smooth muscle cells (RASM), wild-type (wt) GFP-tagged FRNK was also phosphorylated at residues Y168 and Y232, and GFP-wtFRNK inhibited cell spreading and migration. Mutation of GFP-FRNK at Y168 (GFP-Y168F-FRNK) abrogated FRNK-mediated inhibition of cell spreading and migration, but did not affect its localization in VSMC focal adhesions or its ability to inhibit FAK tyrosine phosphorylation.

Conclusion

Phosphorylation of Y168 on FRNK may represent a novel mechanism by which FRNK inhibits cell spreading and migration in VSMCs.

Keywords: Focal adhesion kinase, FAK-related non-kinase, A7r5, Signal transduction

1. Introduction

Vascular smooth muscle cell (VSMC) spreading and migration are critical functions during vasculogenesis and pathologic vascular remodelling. Focal adhesion kinase (FAK) is a ubiquitously expressed non-receptor protein tyrosine kinase that is crucial to both processes in numerous cell types, including VSMCs.1–3 Fibroblasts isolated from FAK−/− mice demonstrate defective cell migration, which is restored by FAK re-expression.1–4 In contrast, elevated FAK expression in many tumour cell lines is associated with increased cellular migration and invasion.5–8

FAK possesses autocatalytic tyrosine kinase activity, but also functions as a scaffolding protein by providing phosphotyrosine binding sites for additional signalling proteins.9 The phosphorylation status of specific residues appears to regulate specific cellular processes. FAK phosphorylation at Y861 regulates cell migration in prostate carcinoma and endothelial cells,10–12 whereas FAK phosphorylation at Y925 may regulate cellular proliferation rate.13–16 Additionally, FAK Y925 may play a role in focal adhesion turnover thus potentially linking this phosphorylation site to both cellular proliferation and migration.17

FAK-related non-kinase (FRNK) is the autonomously expressed C-terminal region of FAK, which lacks the N-terminal and central catalytic domains but retains the focal adhesion targeting (FAT) sequence.18–22 Endogenous FRNK expression is primarily limited to arterial smooth muscle cells, with increased expression levels observed in embryonic vascular tissue with low-level expression in the adult. However, FRNK expression markedly increases in adult arteries in response to vascular injury.3,20–22 FRNK is thought to function as a dominant-negative inhibitor of FAK by competitively displacing FAK from focal adhesions23,24 and thus has been frequently used as an experimental tool to investigate FAK-dependent signalling. Since FRNK inhibits cell spreading and migration in cultured VSMCs, it may also negatively regulate these processes in vivo.3,20,21

FAK-deficient cells display a reduced rate of cell migration and spreading, and have larger and more numerous focal adhesions.1 In contrast, FRNK-overexpressing cells display reduced cell spreading and migration, as well as the loss of focal adhesion complexes and cell detachment.24–26 These phenotypic differences raise the possibility that FRNK may have independent functions in cell signalling in addition to its role as a competitive inhibitor of FAK.

FRNK retains the Y861 and Y925 phosphorylation sites present on FAK. Since the Y861 and Y925 residues function in FAK-dependent cell migration and proliferation, phosphorylation of the corresponding residues on FRNK (Y168 and Y232) may be necessary for the phenotype seen with FRNK overexpression. To date, reports on FRNK phosphorylation have been conflicting.10,27,28 For instance, both serine27 and tyrosine10,28 phosphorylation of FRNK have been described, but serine phosphorylation at residues S148 and S151 does not regulate the inhibitory effect of FRNK. However, the function of FRNK tyrosine phosphorylation is not known. Therefore the objective of the present study was to determine whether FRNK undergoes tyrosine phosphorylation in vivo and in cultured VSMCs, and to analyse the functional significance of these potential phosphorylation sites.

2. Methods

2.1. Materials and reagents

A detailed description of the materials used in this study is provided in the online supplement (see Supplementary material online).

2.2. Carotid artery balloon injury

Loyola University Medical Center's Institutional Animal Care and Use Committee approved all procedures involving animals, which were handled in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Balloon injury of the right common carotid artery was accomplished using a 2.5F double-lumen balloon catheter (NuMED, Inc., Hopkinton, NY), as previously described.29 A detailed description of the procedure is provided in the online supplement (see Supplementary material online).

2.3. Cell culture

Rat aortic smooth muscle cells (RASM) were isolated as previously described30 and maintained in DMEM containing 10% FBS. Cells up to the ninth passage were used. A7r5 cells were a gift from Dr Kenneth Byron, Loyola University Medical Center. Cells up to the 15th passage were used 2–7 days after plating.

2.4. Immunoprecipitation, SDS–PAGE, and western blotting

A detailed description of these methods is provided in the online supplement (see Supplementary material online).

2.5. Expression plasmids and site-directed mutagenesis

Wild-type chick FRNK was kindly provided by Dr Tom Parsons, University of Virginia, and cloned in-frame into pEGFP-C2 (Clontech, Palo Alto, CA) as previously described.24 Mutagenesis of the GFP-FRNK expression plasmid was performed using the Stratagene QuikChange Kit (Stratagene, La Jolla, CA). Two sets of 35mer oligo primers were used to generate the desired mutations (Y168F, Y232F, Y168,232F, and L341S mutations, respectively) which were confirmed by DNA sequencing. Plasmids were then amplified and purified using Qiagen Maxiprep kits (Valencia, CA).

2.6. Transfection

A7r5 cells grown on 100 mm dishes were transfected with expression plasmids (20 µg) using SuperFect transfection reagent (Qiagen) in serum- and antibiotic-free medium. After 2–3 h, cells were rinsed once with phosphate-buffered saline (PBS), fresh growth medium containing 10% FBS was then added, and the cells were maintained in culture until sufficient transgene expression occurred as assessed by GFP-fluorescence.

2.7. Cell fixation and confocal microscopy

A7r5 cells grown on Permanox® chamberslides were transfected with plasmids expressing GFP-wtFRNK, GFP-Y168F-FRNK, GFP-Y232F-FRNK, GFP-Y168,232F-FRNK, and GFP-L341S-FRNK (4 µg DNA, 72 h). Cells were fixed in 2% paraformaldehyde in PBS, permeabilized with 1% Triton X-100 in PBS, and counterstained with rhodamine-conjugated phalloidin. Fluorescently labelled cells were viewed with a Zeiss LSM 510 laser scanning confocal microscope.

2.8. Adenoviral constructs

Replication-defective adenoviruses (Adv) expressing GFP, wtFRNK, GFP-wtFRNK, and GFP-Y168F-FRNK were generated as previously described.24 The multiplicity of viral infection (MOI) was determined by dilution assay in HEK293 cells grown in 96 well clusters. RASM were growth-arrested in serum-free culture medium for at least 1 h prior to infection. Cells were incubated (24 h, 37°C) with Adv in serum-free medium, and the medium was replaced with serum-free DMEM for an additional 24 h.

2.9. FAK and FRNK localization in VSMCs

RASM and A7r5 cells grown on Permonox® chamberslides were infected with Adv-GFP, Adv-GFP-wtFRNK, and Adv-GFP-Y168F-FRNK (300moi, 48 h). Cells were then fixed, permeabilized, and immunostained with N-terminal FAK antibody (which recognizes FAK but not FRNK) followed by rhodamine-conjugated goat anti-mouse IgG, and then viewed on a Zeiss HBO-100 microscope (Carl Zeiss, Inc., Oberkochen, Germany) fitted with an AxioCam HRM digital camera running AxioVision AC Ver. 4.5 software.

2.10. Cell spreading assays

A detailed description of the cell spreading assays performed in RASM and A7r5 cells is provided in the online supplement (see Supplementary material online).

2.11. Cell migration assays

A detailed description of the cell migration assays performed in RASM and A7r5 cells is provided in the online supplement (see Supplementary material online).

2.12. Data analysis

Results were expressed as either mean ± SD or mean ± SEM. Normality was assessed using the Kolmogorov–Smirnov test, and homogeneity of variance was assessed using Levene's test. Data from multiple groups were compared by two-way analysis of variance (ANOVA), or one-way ANOVA followed by Tukey test, Student–Newman–Keuls test, or the Holm–Sidak test. Data from two groups were compared by unpaired t-test, paired t-test, Mann–Whitney Rank Sum test, or Wilcoxin Signed Rank test, where appropriate. Differences among means were considered significant at P < 0.05. Data were analysed using SigmaStat, Ver. 3.1 (Jandel Scientific, San Rafael, CA).

3. Results

3.1. Endogenous FRNK is tyrosine-phosphorylated at Y168 and Y232

In initial experiments, we made use of the carotid artery balloon injury model to examine whether endogenously expressed FRNK undergoes tyrosine phosphorylation in vivo. As seen in Figure 1, balloon-injured arteries demonstrated a marked accumulation of α-SMA-positive VSMCs (red, top panel) near the site of rupture of the internal elastic lamina, which were visualized in the same section by tissue autofluorescence (green). An adjacent section was also stained with total FAK/FRNK antibody (red, bottom panel), which revealed that FAK and FRNK were highly expressed in the α-SMA-positive cells, and their number and intensity greatly increased after balloon injury. Co-localization of α-SMA, FAK/FRNK, FAK, and pY397FAK within the neointima of a similarly injured artery is depicted in the online supplement (see Supplementary material online, Figure S1).

Figure 1.

Figure 1

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α-SMA and FAK/FRNK localization in balloon-injured carotid artery. Frozen sections of balloon-injured rat carotid artery were immunostained with α-SMA and rhodamine-conjugated goat anti-mouse IgG (upper panel, red). In the same section, the internal elastic lamina were visualized by tissue autofluorescence (green). Cell nuclei were stained with DAPI (blue). An adjacent section was immunostained with FAK/FRNK antibody and rhodamine-conjugated goat anti-rabbit IgG (bottom panel, red).

A quantitative analysis of FAK and FRNK expression was then performed using SDS–PAGE and western blotting. As seen in Figure 2A, a polyclonal antibody specific for the C-terminal portion of FAK cross-reacted with several proteins in control and injured arteries. The largest protein band (125 kDa) also cross-reacted with an anti-FAK kinase-domain monoclonal antibody, as well as a polyclonal antibody specific for FAK phosphorylated at Y397 (pFAK-Y397; see Supplementary material online, Figure S2) indicating that this band was endogenous FAK. Of particular interest, a 41/43 kDa doublet was also detected, which is consistent with the apparent molecular weight of endogenous FRNK observed in other cell types.3,23,27,31 This doublet co-migrated with proteins expressed in HEK293 cells infected with Adv-FRNK. However, the doublet did not cross-react with either the kinase-domain or pFAK-Y397 antibodies (see Supplementary material online, Figure S2), indicating that the 41/43 kDa bands were endogenous FRNK, or less likely, C-terminal degradation products of endogenous FAK. Quantitative western blotting demonstrated an approximately nine-fold increase in the level of FRNK, and a ∼four-fold increase in the level of FAK (relative to GAPDH) 2 weeks after balloon injury (Figure 2B). Immunoprecipitation of FAK and FRNK (using a polyclonal antibody directed towards the C-terminus of human FAK) demonstrated the substantial increase in FAK and FRNK expression (Figure 2C), and also revealed that FRNK was tyrosine-phosphorylated at Y168 (Figure 2D) and Y232 (Figure 2E), corresponding to the Y861 and Y925 phosphorylation sites on FAK.

Figure 2.

Figure 2

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FRNK is tyrosine-phosphorylated after balloon injury. (A) Injured and contralateral, control carotid arteries were harvested 2 weeks after balloon injury. Tissue lysates (100 µg total protein) were subjected to SDS–PAGE and western blotting. The migration of molecular weight standards is indicated to the left of the blot, and the position of endogenous FAK and FRNK are indicated to the right. Blots were also probed with GAPDH antibody to ensure equal loading. (B) Data (mean ± SD) from n = 4 pairs of arteries were compared by paired t-test. *P < 0.05. Similar tissue extracts (1000 µg total protein) were immunoprecipitated using anti-FAK/FRNK antibody. Blots were probed with an antibody to total FAK (C), FAK phosphorylated at Y861 (D), and FAK phosphorylated at Y925 (E). Non-specific IgG bands are depicted to ensure equal loading.

FRNK was also expressed in A7r5 cells, and tyrosine-phosphorylated under basal conditions. AngII stimulation markedly increased the tyrosine phosphorylation of both proteins at both sites (see Supplementary material online, Figure S3).

3.2. GFP-FRNK is tyrosine-phosphorylated when overexpressed in VSMCs

To determine whether exogenous FRNK also undergoes tyrosine phosphorylation, growth-arrested RASM, and A7r5 cells were maintained in control medium, or infected with either Adv-GFP or Adv-GFP-wtFRNK (300MOI; 48 h). Cell lysates were either directly analysed by western blotting, or immunoprecipitated with FAK/FRNK or GFP antibody. As seen in Figure 3A, western blots identified both endogenous FAK (125 kDa) and overexpressed GFP-FRNK (67 kDa). Immunoprecipitates of the same samples probed with an anti-pTyr antibody revealed that GFP-FRNK was tyrosine-phosphorylated (Figure 3B). Furthermore, as was demonstrated for endogenous FRNK, overexpressed GFP-wtFRNK in A7r5 cells was also tyrosine-phosphorylated at Y168 and Y232 under basal conditions. Ang II stimulation further increased GFP-FRNK phosphorylation at both sites (Figure 3D and E). Similar results were obtained in RASM (data not shown).

Figure 3.

Figure 3

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GFP-FRNK is tyrosine-phosphorylated in VSMCs. Growth-arrested RASM were maintained in control medium (UI), or infected with Adv-GFP or Adv-GFP-FRNK (300MOI, 48 h). Cell lysates were then subjected to SDS–PAGE and western blotting before or after immunoprecipitation with C-terminal FAK/FRNK antibody. (A) Cell lysates (100 µg total protein) were probed with FAK antibody. (B) Immunoprecipitates (500 µg total protein) were probed with pTyr antibody. (C–E) A7r5 cells were infected with Adv-GFP or Adv-GFP-FRNK (300MOI, 48 h) and maintained in control medium, or stimulated with AngII (50 nmol/L, 30 min). Cell lysates (500 µg) were immunoprepitated with GFP antibody, and probed with GFP (C), pY861 (D) or pY925 (E) phosphospecific antibodies. Non-specific IgG bands are depicted to ensure equal loading.

3.3. GFP-wtFRNK inhibits VSMC cell spreading

Growth-arrested RASM were then infected (300MOI, 48 h) with Adv-GFP or Adv-GFP-wtFRNK, and cells were re-plated onto fibronectin-coated dishes. To avoid effects from delayed cell attachment, non-adherent cells were first removed by washing with fresh culture medium 15 min after re-plating. Adherent RASM were then allowed to spread for a total of 15–480 min prior to fixation and Coumassie blue staining. As seen in Figure 4A and B, GFP-wtFRNK inhibited VSMC spreading at every time point examined. To ensure that the differences in cell surface area at each timepoint were not due to an overall decrease in cell volume prior to attachment, similarly infected RASM were analysed by flow cytometry. Cells were gated for GFP fluorescence and the average cell size compared. As seen in Figure 4C, there was no difference in the size of suspended cells expressing GFP vs. GFP-wtFRNK.

Figure 4.

Figure 4

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GFP-FRNK expression inhibits RASM spreading. RASM were infected with either Adv-GFP or Adv-GFP-FRNK (300MOI, 48 h), trypsinized, and resuspended in 10% serum-containing medium. In (A) and (B), cells were replated on fibronectin (FN)-coated dishes and allowed to attach and spread for varying time periods (15–480 min). (A) Depicts a representative low power image of RASM fixed and stained after 120 min of spreading. Quantitative analysis of two separate experiments with 179–429 cells per timepoint for each experiment is depicted in (B). Data are means ± SEM; *P < 0.001 comparing GFP vs. GFP-FRNK infected cells at each timepoint. In (C), cells were gated for appropriate side-scatter and GFP-fluorescence and the average size of Adv-GFP and Adv-GFP-FRNK infected RASM in suspension was compared by analysis of forward-scatter data. The composite displays the overlay of Adv-GFP and Adv-GFP-FRNK forward-scatter data.

3.4. Mutation of potential tyrosine phosphorylation sites does not alter GFP-FRNK focal adhesion localization

To analyse whether mutations at Y168 and/or Y232 resulted in the loss of FAT, plasmids expressing wt and mutant GFP-FRNK were transfected into A7r5 cells, and examined by confocal microscopy. Previous studies have demonstrated that mutation of leucine at position 1034 to serine on FAK (analogous to L341S on FRNK) resulted in the loss of focal adhesion localization of both FAK and FRNK.4,32,33 Therefore, as a control for the loss of focal adhesion localization, we also generated an expression plasmid expressing GFP-L341S-FRNK. As seen in Figure 5A, only the L341S mutation resulted in the loss of focal adhesion localization. Mutations in neither Y168F nor Y232F (nor their combination) affected GFP-FRNK localization.

Figure 5.

Figure 5

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Localization and cell spreading of GFP-FRNK mutants in A7r5 cells. (A) A7r5 cells were transfected with plasmids expressing GFP-wtFRNK, GFP-Y168F-FRNK, GFP-Y232F-FRNK, GFP-Y168,232F-FRNK, or GFP-L341S-FRNK. Cells were maintained in serum-containing medium for 48 h prior to fixation, permeabilization, and counterstaining with rhodamine-phalloidin. Optical sections (∼1 µm) were obtained at the cell-substratum interface. Green, GFP fluorescence; Red, rhodamine fluorescence; Yellow, co-localization. (B and C) Transfected A7r5 cells (20 µg, 72 h) were sorted by FACS for GFP-fluorescence, and re-plated onto fibronectin-coated dishes. Attached cells were fixed, stained, and cell surface area was determined by image analysis 4 h after re-plating. The quantitative analysis of two individual experiments representing 244–394 cells in each group are depicted. (B) Depicts mean ± SEM of GFP vs. GFP-wtFRNK expressing cells; *P < 0.05 vs. GFP. (C) Depicts mean ± SEM of GFP-wtFRNK vs. mutant GFP-FRNK expressing cells; *P < 0.05 vs. wtGFP-FRNK.

3.5. GFP-FRNK phosphorylation on Y168 is necessary for GFP-FRNK-mediated inhibition of VSMC cell spreading and migration

To examine whether mutation of potential tyrosine phosphorylation sites affected the ability of GFP-FRNK to inhibit FAK-dependent cell spreading, A7r5 cells were transfected with plasmids expressing GFP, GFP-wtFRNK, GFP-Y168F-FRNK, GFP-Y232F-FRNK, and GFP-Y168,232F-FRNK and sorted by flow cytometry. Brightly fluorescent cells were re-plated on fibronectin-coated dishes. Non-adherent cells were removed by rinsing with fresh 10% serum-containing medium 15 min after plating. Cells were then maintained for a total of 4 h prior to fixation, Coumassie blue staining, and image analysis. As seen in Figure 5B, GFP-FRNK expressing A7r5 cells had a significantly smaller surface area than control, GFP expressing cells, which is similar to the results observed with RASM (Figure 4). Mutation of the Y168 residue (Y168F) resulted in the abrogation of GFP-wtFRNK-mediated inhibition of cell spreading (Figure 5C). Data were analysed by two-way ANOVA, and mutation of the Y168 site was highly significant (P < 0.00008), whereas mutation of the Y232 site had no significant effect (P = 0.924). Also, there was no statistically significant interaction between mutation of the Y168 and Y232 sites (P = 0.160). That is, the effect of the Y168 mutation was unaffected by the mutational status of Y232.

To further examine the role of FRNK-Y168 tyrosine phosphorylation in RASM, we generated a replication-defective Adv encoding GFP-Y168F-FRNK, and analysed its ability to inhibit cell spreading when compared with cells expressing GFP or GFP-wtFRNK. Here, however, only cells demonstrating GFP fluorescence were analysed, and cells were examined at multiple time points (15–480 min) without cell sorting. As seen in Figure 6A, cells expressing GFP-wtFRNK were considerably less spread than either GFP or GFP-Y168F-FRNK expressing cells. Quantitative analysis of three experiments comprising ∼100 cells in each group indicated that both time and transgene were significant factors in the degree of RASM spreading on the fibronectin substratum, and that mutation of the Y168 site markedly abrogated the inhibitory effect of GFP-FRNK on cell spreading at each timepoint (Figure 6B). However, at some timepoints, there was still a modest inhibition of cell spreading observed with the Y168 mutant when compared with GFP.

Figure 6.

Figure 6

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Mutation of the Y168 site inhibits GFP-FRNK-mediated inhibition of cell spreading and migration. RASM were infected with Adv-GFP, Adv-GFP-wtFRNK, or Adv-GFP-Y168F-FRNK (300MOI, 48 h). RASM were then re-plated onto fibronectin-coated cell culture dishes and allowed to attach and spread for various lengths of time (15–480 min) prior to cell surface area analysis (A and B). *P < 0.001 for GFP-wtFRNK vs. both GFP and GFP-Y168F-FRNK expressing cells; #P < 0.02 for GFP vs. GFP-Y168F-FRNK by one-way and two-way ANOVA. In (C) and (D), adenovirally infected cells were stimulated with PDGF (10 ng/mL) and were allowed to migrate through a Matrigel-coated membrane for 4 h. Fluorescently labelled cells were visualized and counted. Results from each experiment were normalized to GFP-infected cells. Cell migration was calculated by counting the number of cells that migrated to the lower side of the membrane. *P < 0.001 for GFP-wtFRNK vs. both GFP and GFP-Y168F-FRNK expressing cells.

To examine the functional significance of FRNK-Y168 phosphorylation on cell migration, RASM were infected (300MOI, 48 h) with Adv-GFP, Adv-GFP-wtFRNK, and Adv-GFP-Y168F-FRNK. Adenovirally infected cells were suspended in serum free medium and placed in the upper chamber of Matrigel-coated Boyden chambers. As seen in Figure 6C and D, mutation of the Y168 residue (Y168F) resulted in the abrogation of GFP-wtFRNK-mediated inhibition of cell migration. Similar results were observed in A7r5 cells (data not shown).

3.6. Effects of GFP-wtFRNK and GFP-Y168F-FRNK overexpression on endogenous FAK localization and phosphorylation

Imaging and western blotting experiments were performed to determine whether overexpression of GFP-wtFRNK or GFP-Y168F-FRNK affected the localization and phosphorylation of endogenous FAK in VSMCs. A7r5 cells were infected (100moi, 48 h) with Adv-GFP, Adv-GFP-wtFRNK, or Adv-GFP-Y168F-FRNK, and the cells were fixed, permeabilized, and counterstained with anti-FAK kinase-domain antibody (which recognizes only endogenous FAK). As seen in Figure 7A, both wild-type as well as the mutant FRNK displaced endogenous FAK from focal adhesions, when compared with cells overexpressing GFP, or to uninfected cells. The displaced FAK was detected in a cytoplasmic and perinuclear staining pattern with little or no co-localization with the exogenously expressed GFP-wtFRNK or GFP-Y168F-FRNK mutant. Similar results were observed in RASM (data not shown). Overall, these results are similar to those observed in A7r5 cells transfected with GFP-FRNK expression plasmids (Figure 5A), and indicate that mutation of the FRNK-Y168 phosphorylation site did not affect FRNK's ability to target to focal adhesions, and to displace endogenous FAK.

Figure 7.

Figure 7

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Effects of GFP-wtFRNK and GFP-Y168F-FRNK on FAK localization and phosphorylation. (A) A7r5 cells infected (100moi, 48 h) with Adv-GFP, Adv-GFP-wtFRNK, or Adv-GFP-Y168F-FRNK were fixed, permeabilized, and GFP was detected by fluorescence microscopy (green). The same cells were counterstained with kinase-domain FAK antibody and rhodamine-conjugated goat anti-mouse IgG (red). Cell nuclei were stained with DAPI (blue), and co-localization of GFP and FAK were represented in the merged image (yellow). (B) Tyrosine phosphorylation of endogenous FAK was analysed by western blotting in A7r5 cells infected (300moi, 48) with GFP, GFP-wtFRNK, and GFP-Y168F-FRNK. Paired cultures were stimulated (10 min) with AngII (50 nmol/L). (C) Quantitative analysis of FAK phosphorylation (relative to GAPDH) at Y397, Y861, and Y925; data are means ± SD, n = 3 expts.

To determine whether GFP-wtFRNK or GFP-Y168F-FRNK inhibited endogenous FAK tyrosine phosphorylation, A7r5 cells were infected (300moi, 48 h) with Adv-GFP, Adv-GFP-wtFRNK, or Adv-GFP-Y168F-FRNK, and then stimulated with AngII (100 nmol/L, 10 min). A representative western blot is shown in Figure 7B, and the results of three experiments are summarized in Figure 7C. A7r5 cells infected with Adv-GFP contained substantial amounts of FAK phosphorylated at Y397, Y861, and Y925. AngII stimulation resulted in a further ∼two-fold increase in FAK tyrosine phosphorylation. In contrast, both GFP-wtFRNK and GFP-Y168F-FRNK markedly reduced basal and AngII-induced FAK phosphorylation at all three sites.

We also examined the effects of GFP-wtFRNK and GFP-Y168-FRNK overexpression on the expression and phosphorylation of endogenous FRNK. As seen in Supplementary material online, Figure S4, both constructs reduced endogenous FRNK expression and phosphorylation at both sites.

4. Discussion

In this report, we have identified a novel role for FRNK tyrosine phosphorylation in the inhibition of VSMC spreading and migration. When overexpressed, wtFRNK was sufficient to inhibit VSMC spreading and migration. These functions were associated with the tyrosine phosphorylation of FRNK. Mutation of Y168 (but not Y232) resulted in the abrogation of FRNK-mediated inhibition of cell spreading and migration, indicating an important role for FRNK tyrosine phosphorylation in this cellular phenotype.

Although FRNK overexpression is commonly used as a laboratory tool to dissect FAK-dependent signalling pathways, FRNK is also an endogenously expressed protein in VSMCs. Despite low basal FRNK expression levels in adult tissues, FRNK expression is high in smooth muscle cell-containing tissues in the neonatal rat,3 and its expression is increased in response to arterial injury in the adult animal. Hayasaka et al.20 further demonstrated that FRNK promoter activity is specifically increased in medial and neointimal VSMCs following vascular injury. As seen in Figures 1 and 2, we now show that both FRNK and FAK are upregulated in balloon-injured arteries, and that the upregulated FAK and FRNK were tyrosine-phosphorylated at Y168 and Y232. Similarly, Zhou et al.28 showed that stimulation of mouse neuroblastoma N1E-115 cells with the cannabinoid agonist HU-210 resulted in a time-dependent increase in endogenously expressed FRNK phosphotyrosine content. HU-210-stimulated FRNK phosphorylation correlated with the induction of neurite retraction34 without an alteration in FAK phosphotyrosine content. Furthermore, Owens et al.35 have shown that FAK is upregulated in diseased human arteries, although its tyrosine phosphorylation, and the expression and phosphorylation of FRNK were not addressed. Nevertheless, these data suggest that the signalling pathways responsible for FAK phosphorylation may differ from those involving FRNK, and that FRNK phosphorylation may regulate smooth muscle cell signalling independently of its effects on FAK.

Overexpression of wild-type GFP-FRNK inhibited A7r5 and RASM cell spreading. Although adenovirally mediated GFP-FRNK expression resulted in a ∼30% decrease in RASM surface area, only an 8% decrease in cell surface area was seen in transfected and FACS-sorted A7r5 cells. This difference might be due to the lower level of expression achieved by transient transfection (when compared with adenoviral-mediated transgene expression), or could be due to inherent differences in the cell types. Although the Y232F mutation had no effect on GFP-FRNK-mediated inhibition of cell spreading, the Y168F mutation abrogated GFP-FRNK-mediated inhibition independently of the Y232F mutation.

Since both FRNK-Y168 and FRNK-Y232 residues are located near the FAT domain, it is conceivable that GFP-Y168F-FRNK failed to inhibit A7r5 cell spreading and migration because it lost its ability to target to focal adhesions. Our current studies demonstrate however, that although mutation of L1034 in the FAT domain eliminates FAT, all of the other mutants, including the Y168F mutant, target to focal adhesions in a similar pattern to GFP-wtFRNK. Thus, mutation of Y168F in GFP-FRNK does not inhibit FRNK-mediated inhibition of cell spreading and migration through mislocalization of GFP-FRNK.

The mechanisms responsible for FRNK inhibition of FAK signalling, and the role of FRNK Y168 phosphorylation are presently uncertain. FAK localization to focal adhesions is required for its activation, and the loss of FAK from these sites results in decreased FAK activation.36 Since FRNK contains the FAT domain of FAK, we and others have proposed that FRNK inhibits FAK-dependent signalling by competitively displacing FAK from focal adhesions.23,24 However, another possibility is that FRNK inhibits FAK signalling by acting as a sink for FAK binding proteins.37 Our evidence that FRNK can undergo tyrosine phosphorylation independently of FAK suggests that other factors besides FRNK localization are important for its inhibitory function. These factors may also be responsible for the phenotypic differences observed between FAK-null and FRNK-overexpressing cells.1,3,24–26

Increased and dysregulated phosphorylation of FAK at Y861 correlates with an increase in migratory capacity of prostate cancer cells10 and vascular endothelial cells.11 Conversely, inhibition of FAK-Y861 phosphorylation with Src-kinase inhibitors resulted in an inhibition of migration, suggesting an important role for Src-mediated FAK-Y861 phosphorylation in this process. As VSMC migration and proliferation are integral to neointimal formation in response to balloon angioplasty-induced injury, our current studies indicate that the phosphorylation status of individual FRNK tyrosine residues may also play a role in vascular remodelling. However, additional studies will be necessary to define the tyrosine kinase(s) responsible for FRNK phosphorylation, and to evaluate the role of FRNK in limiting the processes of vasculogenesis and neointimal development.

Supplementary material

Supplementary material is available at Cardiovascular Research online.

Funding

These studies were supported in part by NIH RO1 HL34328 and a grant from the Dr Ralph and Marian Falk Medical Research Trust. S.J.E. was a recipient of an American Heart Association Predoctoral Fellowship during the time these studies were performed.

Supplementary Material

[Supplementary Data]
cvp322_index.html (707B, html)

Acknowledgements

The authors thank Drs Areck Ucuzian and Howard Greisler for help with the Boyden chamber assays, and Mss Maxie Turim and Kuldeep Patel with immunofluorescent microscopy.

Conflict of interest: none declared.

References

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