Role and regulation of Abelson tyrosine kinase in Crk-associated substrate/profilin-1 interaction and airway smooth muscle contraction

Abstract

Background: Airway smooth muscle contraction is critical for maintenance of appropriate airway tone, and has been implicated in asthma pathogenesis. Smooth muscle contraction requires an "engine" (myosin activation) and a "transmission system" (actin cytoskeletal remodeling). However, the mechanisms that control actin remodeling in smooth muscle are not fully elucidated. The adapter protein Crk-associated substrate (CAS) regulates actin dynamics and the contraction in smooth muscle. In addition, profilin-1 (Pfn-1) and Abelson tyrosine kinase (c-Abl) are also involved in smooth muscle contraction. The interplays among CAS, Pfn-1 and c-Abl in smooth muscle have not been previously investigated.

Methods: The association of CAS with Pfn-1 in mouse tracheal rings was evaluated by co-immunoprecipitation. Tracheal rings from c-Abl conditional knockout mice were used to assess the roles of c-Abl in the protein-protein interaction and smooth muscle contraction. Decoy peptides were utilized to evaluate the importance of CAS/Pfn-1 coupling in smooth muscle contraction.

Results: Stimulation with acetylcholine (ACh) increased the interaction of CAS with Pfn-1 in smooth muscle, which was regulated by CAS tyrosine phosphorylation and c-Abl. The CAS/Pfn-1 coupling was also modified by the phosphorylation of cortactin (a protein implicated in Pfn-1 activation). In addition, ACh activation promoted the spatial redistribution of CAS and Pfn-1 in smooth muscle cells, which was reduced by c-Abl knockdown. Inhibition of CAS/Pfn-1 interaction by a decoy peptide attenuated the ACh-induced actin polymerization and contraction without affecting myosin light chain phosphorylation. Furthermore, treatment with the Src inhibitor PP2 and the actin polymerization inhibitor latrunculin A attenuated the ACh-induced c-Abl tyrosine phosphorylation (an indication of c-Abl activation).

Conclusions: Our results suggest a novel activation loop in airway smooth muscle: c-Abl promotes the CAS/Pfn-1 coupling and actin polymerization, which conversely facilitates c-Abl activation. The positive feedback may render c-Abl in active state after contractile stimulation.

Keywords: Actin cytoskeleton; Crk-associated substrate; Excitation-contraction coupling; Profilin-1; Smooth muscle; c-Abl kinase.

Fig. 1

Increases in the association and translocation of CAS with Pfn-1 upon acetylcholine stimulation are regulated by c-Abl. a Mouse tracheal rings from c-Abl^-lox and c-Abl^smko mice were treated with acetylcholine (ACh) (100 μM, 5 min) or left untreated (UT) followed by CAS immunoprecipitation. CAS immunoprecipitates were separated by SDS-PAGE and blotted with antibodies against CAS and Pfn-1. Data are mean ± SE (n = 4). b Human airway smooth muscle (HASM) cells expressing control shRNA or c-Abl shRNA were stimulated with ACh (100 μM, 5 min) or left untreated. The cellular localization of CAS and Pfn-1 was evaluated by immunofluorescent microscopy. Arrows indicate a single line scan to analyze the fluorescent intensity for each cell. The inset plots represent fluorescent intensity of the line scan indicated by the arrows. c Fluorescent ratios of cell periphery over interior for CAS or Pfn-1 in cells were calculated. Data are mean ± SE (n = 26–27 cells from 4 independent experiments). UT, untreated. ** P < 0.01

Fig. 2

c-Abl knockout inhibits actin filament polymerization in response to ACh stimulation. a Mouse tracheal rings from c-Abl^-lox and c-Abl^smko mice were treated with acetylcholine (ACh) (100 μM, 5 min) or left untreated (UT). F/G-actin ratios were evaluated using the fractionation assay. Data are mean ± SE (n = 5). b Representative images illustrating the effects of c-Abl knockout on F/G-actin ratios. Sections of trachealis from c-Abl^-lox and c-Abl^smko mice were stained with DNase I (for G-actin) or phalloidin (for F-actin). c ACh-induced increases in F/G-actin ratios evaluated by fluorescent microscopy are reduced in c-Abl^smko mice (n = 4). *P < 0.05, **P < 0.01

Fig. 3

Smooth muscle contraction and CAS phosphorylation, but not myosin light chain phosphorylation, are regulated by c-Abl. a & b Contractile force was reduced in tissues from c-Abl^smko than in c-Abl^-lox mice, which is time- and dose-dependent (n = 5–7). Contractile force is normalized to the force induced by 10⁻⁴ M ACh. c Immunoblot analysis was used to assess myosin light chain phosphorylation at Ser-19 of extracts from tracheal rings of c-Abl^-lox and c-Abl^smko mice. Data are mean ± SE (n = 4). d ACh-induced CAS phosphorylation at Tyr-410 was reduced in tracheal rings from c-Abl^smko mice as compared to c-Abl^-lox mice. Data are mean ± SE (n = 6). **P < 0.01. NS, not significant

Fig. 4

Treatment with CAS peptide attenuates CAS/Pfn-1 coupling and spatial redistribution of CAS and Pfn-1 upon contractile stimulation. a Mouse tracheal rings were pretreated with control (Con) or CAS peptide (2.5 μg/ml) for 30 min. They were then stimulated with ACh (100 μM, 5 min), or left untreated (UT). The protein-protein interaction was evaluated by co-immunoprecipitation. Data are mean ± SE (n = 4). b HASM cells pretreated with control or CAS peptide were stimulated with ACh (100 μM, 5 min), or were untreated. The spatial localization of CAS and Pfn-1 in the cells was assessed by immunostaining. Arrows indicate a single line scan to analyze the fluorescent intensity for each cell. The inset plots represent fluorescent intensity of the line scan indicated by the arrows. c Fluorescent ratios of cell periphery over interior for CAS or Pfn-1 in cells were calculated. Data are mean ± SE (n = 26 cells from 4 independent experiments). **P < 0.01. UT, untreated

Fig. 5

Effects of CAS-peptide on CAS phosphorylation, F/G-actin ratios, contraction and myosin phosphorylation. a Mouse tracheal rings pretreated with control or CAS peptide were stimulated with ACh (100 μM, 5 min), or left unstimulated. CAS phosphorylation at Tyr-410 was determined by immunoblot analysis. Data are mean ± SE (n = 4). b Mouse tracheal tissues that had been pretreated with control or CAS peptide were stimulated with ACh or left untreated. F/G-actin ratios in tissues were evaluated using the fractionation assay. Values represent mean ± SE (n = 5). c Contractile response of mouse tracheal rings to ACh (10⁻⁵ M) was determined, after which they were treated with peptides (2.5 μg/ml) for 30 min. They were then stimulated with different concentration of ACh. Contractile force is normalized to contraction induced by 10⁻⁵ M ACh before treatment with the peptides (n = 7–8). d Myosin light chain phosphorylation in mouse tracheal segments pretreated with peptides was assessed by immunoblot analysis. Values represent mean ± SE (n = 4). **P < 0.01; NS, not significant

Fig. 6

CAS/Pfn-1 coupling and cortactin/Pfn-1 interaction are regulated by phosphorylation. a HASM cells expressing wild type (WT) CAS and CAS-SD mutant were stimulated with ACh (100 μM, 5 min), or left unstimulated. The protein-protein interaction was evaluated by co-immunoprecipitation. Data are mean ± SE (n = 4). b ACh-induced interaction of cortactin with Pfn-1 is enhanced in cells expressing non-phosphorylated CAS mutant. Cortactin/Pfn-1 interaction was evaluated for HASM cells expressing CAS-WT and CAS-SD. Data are mean ± SE (n = 4). c Association of CAS with Pfn-1 upon ACh stimulation is increased in cells expressing non-phosphorylated cortactin mutant. CAS/Pfn-1 coupling was assessed for HASM cells expressing cortactin-NP. Values are mean ± SE (n = 4). NP, non-phosphorylated; cort, cortactin. **P < 0.01

Fig. 7

c-Abl phosphorylation is regulated by Src and actin polymerization. a Mouse tracheal rings pretreated with the Src inhibitor PP2 (10 μM, 30 min) were stimulated with ACh or unstimulated. c-Abl phosphorylation was evaluated by immunoblotting. Data are mean ± SE (n = 4). b Treatment with PP2 attenuates contractile response of tracheal segments to ACh (n = 4–6). Contractile force is normalized to the force induced by 10⁻⁵ M ACh before addition of PP2. c Immunoblotting was used to evaluate c-Abl phosphorylation of unstimulated and stimulated mouse tracheal rings pretreated with the actin polymerization inhibitor latrunculin A (1 μM, 30 min). (n = 4). d Contraction of mouse tracheal rings were reduced by treatment with latrunculin A (Lat-A). Contractile force is normalized to the force induced by 10⁻⁵ M ACh before addition of Lat-A. **P < 0.01; NS, not significant

Fig. 8

Novel mechanisms for regulation of actin dynamics. Upon agonist activation, c-Abl mediates CAS phosphorylation, which promotes the association of CAS with Pfn-1 to activate Pfn-1. In addition, c-Abl also catalyzes cortactin phosphorylation and increases the coupling of cortactin with Pfn-1. Activated Pfn-1 facilitates actin polymerization. Src mediates c-Abl activation in response to contractile stimulation. Furthermore, actin polymerization provides a positive feedback for c-Abl activation