Role of c-Abl tyrosine kinase in smooth muscle cell migration

Abstract

c-Abl is a nonreceptor protein tyrosine kinase that has a role in regulating smooth muscle cell proliferation and contraction. The role of c-Abl in smooth muscle cell migration has not been investigated. In the present study, c-Abl was found in the leading edge of smooth muscle cells. Knockdown of c-Abl by RNA interference attenuated smooth muscle cell motility as evidenced by time-lapse microscopy. Furthermore, the actin-associated proteins cortactin and profilin-1 (Pfn-1) have been implicated in cell migration. In this study, cell adhesion induced cortactin phosphorylation at Tyr-421, an indication of cortactin activation. Phospho-cortactin and Pfn-1 were also found in the cell edge. Pfn-1 directly interacted with cortactin in vitro. Silencing of c-Abl attenuated adhesion-induced cortactin phosphorylation and Pfn-1 localization in the cell edge. To assess the role of cortactin/Pfn-1 coupling, we developed a cell-permeable peptide. Treatment with the peptide inhibited the interaction of cortactin with Pfn-1 without affecting cortactin phosphorylation. Moreover, treatment with the peptide impaired the recruitment of Pfn-1 to the leading edge and cell migration. Finally, β1-integrin was required for the recruitment of c-Abl to the cell edge. Inhibition of actin dynamics impaired the spatial distribution of c-Abl. These results suggest that β1-integrin may recruit c-Abl to the leading cell edge, which may regulate cortactin phosphorylation in response to cell adhesion. Phosphorylated cortactin may facilitate the recruitment of Pfn-1 to the cell edge, which promotes localized actin polymerization, leading edge formation, and cell movement. Conversely, actin dynamics may strengthen the recruitment of c-Abl to the leading edge.

Keywords: actin cytoskeleton; adapter protein; cell migration; smooth muscle; tyrosine kinase.

Fig. 1.

Abelson tyrosine kinase (c-Abl) is localized in the leading cell edge and is required for smooth muscle cell motility. A: c-Abl is localized in the leading edge. Human airway smooth muscle (HASM) cells were plated on collagen-coated coverslips for 30 min. Immunofluorescent microscopy was used to evaluate the spatial localization of c-Abl. Arrows indicate leading edge. Scale bar, 10 μm. B: knockdown (KD) of c-Abl in cells by lentivirus-mediated gene transduction. Blots of extracts from uninfected (UI) cells, cells transduced with lentivirus encoding control (Con) short hairpin RNA (shRNA) or c-Abl (KD) shRNA were probed using antibodies against c-Abl and GAPDH. Ratios of c-Abl to GAPDH protein in cells transduced with virus are normalized to those in uninfected cells. *P < 0.05 compared with UI cells and cells expressing Con shRNA (n = 4). C: illustrative diagram of cell migration analysis. A cell moves from position 1 to position 2 along the path of the solid line. The path the of solid line represents total distance. The path of the dashed line represents net distance. Directionality is net distance divided by total distance (26). D–G: migration of UI cells, cells expressing control shRNA, c-Abl KD cells, and cells treated with GNF-5 (1 μM) or imatinib (IM; 1 μM) was evaluated by time-lapse microscopy. The net path, total path, velocity, and directionality of cells were assessed using the NIH ImageJ software (n = 29–31). *P < 0.05 compared with UI cells and Con shRNA-treated cells.

Fig. 2.

Cortactin undergoes phosphorylation upon cell adhesion and interacts with profilin-1 (Pfn-1) in vitro and in cells. A: adhesion to extracellular matrix promotes cortactin phosphorylation at Tyr-421 in cells. Cells were plated on collagen-uncoated or coated dishes for 30 min. Cortactin phosphorylation in these cells was evaluated by immunoblotting using phospho-cortactin (p-Cortactin; Tyr-421) antibody and total cortactin antibody. Cortactin phosphorylation in cells treated with coated surfaces is normalized to that in uncoated surfaces. Values represent means ± SE (n = 6). *P < 0.05. B: far Western analysis of cortactin interaction with Pfn-1 in vitro. Cortactin immunoprecipitates were separated by SDS-PAGE and transferred to membranes. Lane a, immobilization of cortactin (Cort) on the membrane probed using cortactin antibody. Lane b, detection of Pfn-1 on immobilized cortactin suggests the association of cortactin with Pfn-1 in vitro. Lane c, no detection of Pfn-1 in the control membrane. Lane d, minor detection of Pfn-1 on the immobilized Y421F cortactin. Immunoblots (IB) are representative of 4 identical experiments. C: colocalization of c-Abl, cortactin, and Pfn-1 in the leading edge. The spatial distribution of these molecules in adherent cells was evaluated by immunofluorescent microscopy. Arrows indicate leading edge. Scale bar, 10 μm. p-Cort, phosphorylated cortactin. D: cells were plated on collagen-uncoated (a) or coated (b) coverslips for 30 min. Antibodies against phosphorylated cortactin was used to assess morphology of the cells. Images are representatives of 35 cells. Arrows indicate leading edge. Scale bar, 10 μm.

Fig. 3.

c-Abl regulates cortactin phosphorylation and Pfn-1 localization. A: adhesion-induced cortactin phosphorylation is mediated by c-Abl. c-Abl KD cells and cells expressing control shRNA were plated on collagen-coated culture dishes for 30 min. Immunoblot analysis was used to determine cortactin phosphorylation. The level of cortactin phosphorylation in KD cells is normalized to that in cells expressing control shRNA (n = 6). *P < 0.05. B: cortactin phosphorylation and Pfn-1 recruitment in the cell edge are reduced in c-Abl KD cells. c-Abl KD cells and cells expressing control shRNA were plated for 30 min. The spatial localization of phospho-cortactin and Pfn-1 of the cells was evaluated by immunofluorescent microscopy. Green fluorescent protein (GFP) signals indicate successful infection of cells. Scale bar, 10 μm. C: quantification analysis of cortactin phosphorylation and Pfn-1 in the cell edge. Fluorescence intensity of corresponding proteins in c-Abl KD cells is normalized to that in cells expressing control shRNA (n = 28). *P < 0.05. D: c-Abl catalyzes cortactin phosphorylation in vitro. c-Abl mediated cortactin phosphorylation 30 min after the initiation of reaction was determined as described in materials and methods. The phosphorylation level after c-Abl treatment is normalized to the level of cortactin phosphorylation in the absence of c-Abl. *P < 0.05, significantly higher phosphorylation levels in the presence of c-Abl compared with the level without c-Abl treatment (n = 6). E: c-Abl promotes F-actin formation in the cell edge. Representative images show the effects of c-Abl shRNA on F-actin staining in the cell edge. Stable c-Abl KD cells and cells expressing control shRNA were plated on collagen-coated coverslips for 30 min. F-actin was visualized by rhodamine-phalloidin staining. Scale bar, 10 μm. Cells expressing control shRNA (a and b) showed higher staining of F-actin in the leading edge, whereas c-Abl KD cells (c and d) exhibited lower levels of F-actin in the edge. Red, phalloidin; green, GFP. Arrowheads indicate cell edge. F: quantification analysis of F-actin in the cell edge. Fluorescent intensity of F-actin in c-Abl KD cells is normalized to that in cells expressing control shRNA (n = 26). *P < 0.05. G: illustrative diagrams of image analysis. Six to 10 line scans are traced across the leading edge. Each line provides a fluorescence intensity profile. An average of peak intensity for all lines is calculated for each cell. The intensity of c-Abl KD cells is normalized to that of control cells.

Fig. 4.

Characterization of the cell-permeable peptide CTTN-I. A: blots of cortactin immunoprecipitates (IP) from adherent cells treated with peptides were probed with the use of antibodies against Pfn-1 and cortactin. Ratios of Pfn-1 to cortactin in cells treated with CTTN-I peptide are normalized to the values in cells treated with control peptide. Values represent means ± SE (n = 4). B: blots of adherent cells treated with CTTN-I or control peptides for 30 min were detected with antibodies against phospho-cortactin (Tyr-421) and total cortactin. Phosphorylation levels in cells treated with CTTN-I peptide are normalized to the levels in cells treated with control peptide. Values represent means ± SE (n = 6). C: representative images showing the effects of peptides on phosphorylated cortactin, Pfn-1, and F-actin in the cell edge. Cells were plated on collagen-coated coverslips in the present of CTTN-I or control peptides for 30 min. Phospho-cortactin and Pfn-1 were evaluated by immunofluorescent microscopy. F-actin was visualized by phalloidin staining. Scale bar, 10 μm. D: quantification analysis of cortactin phosphorylation, Pfn-1, and F-actin in the cell edge. Fluorescence intensity of corresponding proteins in cells treated with CTTN-I peptide is normalized to that in cells treated with control peptide (n = 28–32). *P < 0.05.

Fig. 5.

Treatment with CTTN-I peptide inhibits cell motility. Cells were treated with 1 μg/ml control peptide or CTTN-I peptide, and their migration was evaluated as described in materials and methods. The net path (A), total path (B), velocity (C), and directionality (D) of cells were assessed using the NIH ImageJ software (n = 28). *P < 0.05 compared with cells treated with control peptide.

Fig. 6.

β₁-Integrin promotes the recruitment of c-Abl in the cell edge. A: representative image illustrating the cellular localization of β₁-integrin in normal HASM cells 30 min after adhesion. Scale bar, 10 μm. B: representative immunoblots showing the effects of β₁-integrin antisense or sense on protein expression. Blots of cells transfected with β₁-integrin sense or antisense for 2 days were probed with the use of antibodies against β₁-integrin and GAPDH. C: ratios of β₁-integrin to GAPDH in antisense-treated cells are normalized to those in sense-treated cells (n = 6). *P < 0.05. D: representative micrographs illustrating the role of β₁-integrin in c-Abl localization. c-Abl is localized in the leading edge of cells treated with β₁-integrin sense (a) but is barely detected in cells treated with β₁-integrin antisense (b). Arrows indicate the cell edge. E: fluorescence intensity of c-Abl in the periphery of cells treated with antisense is normalized to that in cells treated with sense (n = 26–30). *P < 0.05.

Fig. 7.

Actin polymerization modulates spatial distribution of c-Abl. A: representative micrographs illustrating the roles of actin polymerization in c-Abl localization. Cells were treated with or without 1 μM latrunculin-A (LAT-A) for 15 min and then plated on collagen-coated coverslips for 30 min. The spatial localization of c-Abl was assessed by immunofluorescent microscopy. Arrows indicate cell edge. Scale bar, 10 μm. B: fluorescence intensity of c-Abl in the edge of cells treated with latrunculin A is normalized to that in control cells. *P < 0.01, significantly lower intensity in cells treated with latrunculin A than in control cells (n = 25–29).

Fig. 8.

Proposed mechanism. During adhesion and spreading, c-Abl is recruited to the leading cell edge via β₁-integrin, catalyzing cortactin phosphorylation. Phosphorylated cortactin may recruit Pfn-1 to the cell periphery, which promotes local actin polymerization, leading edge formation, and smooth muscle cell migration. Additionally, actin polymerization may facilitate the recruitment of c-Abl to the cell edge.