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.
Project description:Actin filament polymerization plays a critical role in the regulation of smooth muscle contraction. However, our knowledge regarding modulation of the actin cytoskeleton in smooth muscle just begins to accumulate. In this study, stimulation with acetylcholine (ACh) induced an increase in the association of the adapter protein c-Abl interactor 1 (Abi1) with neuronal Wiskott-Aldrich syndrome protein (N-WASP) (an actin-regulatory protein) in smooth muscle cells/tissues. Furthermore, contractile stimulation activated N-WASP in live smooth muscle cells as evidenced by changes in fluorescence resonance energy transfer efficiency of an N-WASP sensor. Abi1 knockdown by lentivirus-mediated RNAi inhibited N-WASP activation, actin polymerization, and contraction in smooth muscle. However, Abi1 silencing did not affect myosin regulatory light chain phosphorylation at Ser-19 in smooth muscle. In addition, c-Abl tyrosine kinase and Crk-associated substrate (CAS) have been shown to regulate smooth muscle contraction. The interaction of Abi1 with c-Abl and CAS has not been investigated. Here, contractile activation induced formation of a multiprotein complex including c-Abl, CAS, and Abi1. Knockdown of c-Abl and CAS attenuated the activation of Abi1 during contractile activation. More importantly, Abi1 knockdown inhibited c-Abl phosphorylation at Tyr-412 and the interaction of c-Abl with CAS. These results suggest that Abi1 is an important component of the cellular process that regulates N-WASP activation, actin dynamics, and contraction in smooth muscle. Abi1 is activated by the c-Abl-CAS pathway, and Abi1 reciprocally controls the activation of its upstream regulator c-Abl.
Project description:Profilin-1 (Pfn-1) is an actin-regulatory protein that has a role in modulating smooth muscle contraction. However, the mechanisms that regulate Pfn-1 in smooth muscle are not fully understood. Here, stimulation with acetylcholine induced an increase in the association of the adapter protein cortactin with Pfn-1 in smooth muscle cells/tissues. Furthermore, disruption of the protein/protein interaction by a cell-permeable peptide (CTTN-I peptide) attenuated actin polymerization and smooth muscle contraction without affecting myosin light chain phosphorylation at Ser-19. Knockdown of cortactin by lentivirus-mediated RNAi also diminished actin polymerization and smooth muscle force development. However, cortactin knockdown did not affect myosin activation. In addition, cortactin phosphorylation has been implicated in nonmuscle cell migration. In this study, acetylcholine stimulation induced cortactin phosphorylation at Tyr-421 in smooth muscle cells. Phenylalanine substitution at this position impaired cortactin/Pfn-1 interaction in response to contractile activation. c-Abl is a tyrosine kinase that is necessary for actin dynamics and contraction in smooth muscle. Here, c-Abl silencing inhibited the agonist-induced cortactin phosphorylation and the association of cortactin with Pfn-1. Finally, treatment with CTTN-I peptide reduced airway resistance and smooth muscle hyperreactivity in a murine model of asthma. These results suggest that the interaction of cortactin with Pfn-1 plays a pivotal role in regulating actin dynamics, smooth muscle contraction, and airway hyperresponsiveness in asthma. The association of cortactin with Pfn-1 is regulated by c-Abl-mediated cortactin phosphorylation.
Project description:Abl is a nonreceptor tyrosine kinase that is required for smooth muscle contraction. However, the mechanism by which Abl regulates smooth muscle contraction is not completely understood. In the present study, Abl underwent phosphorylation at Tyr412 (an index of Abl activation) in smooth muscle in response to contractile activation. Treatment with a cell-permeable decoy peptide, but not the control peptide, attenuated Abl phosphorylation during contractile stimulation. Treatment with the decoy peptide did not affect the association of Abl with the cytoskeletal protein vinculin and the spatial location of vinculin in smooth muscle. Inhibition of Abl phosphorylation by the decoy peptide attenuated the agonist-induced phosphorylation of Crk-associated substrate (CAS), an adapter protein participating in the signaling processes that regulate force development in smooth muscle. Additionally, previous studies have shown that contractile stimulation triggers the dissociation of CAS from the vimentin network, which is important for cytoskeletal signaling and contraction in smooth muscle. In this report, the decrease in the amount of CAS in cytoskeletal vimentin in response to contractile activation was reversed by the Abl inhibition with the decoy peptide. Moreover, force development and the enhancement of F-actin-to-G-actin ratios (an indication of actin polymerization) upon contractile activation were also attenuated by the Abl inhibition. However, myosin phosphorylation induced by contractile activation was not affected by the inhibition of Abl. These results suggest that Abl regulates the dissociation of CAS from the vimentin network, actin polymerization, and contraction by modulating CAS phosphorylation in smooth muscle.
Project description: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.
Project description:Abl is a nonreceptor tyrosine kinase that has a role in regulating migration and adhesion of nonmuscle cells as well as smooth muscle contraction. The role of Abl in smooth muscle cell proliferation has not been investigated. In this study, treatment with endothelin-1 (ET-1) and platelet-derived growth factor (PDGF) increased Abl phosphorylation at Tyr(412) (an indication of Abl activation) in vascular smooth muscle cells. To assess the role of Abl in smooth muscle cell proliferation, we generated stable Abl knockdown cells by using lentivirus-mediated RNA interference. ET-1- and PDGF-induced cell proliferation was attenuated in Abl knockdown cells compared with cells expressing control shRNA and uninfected cells. Abl silencing also arrested cell cycle progression from G(0)/G(1) to S phase. Furthermore, activation of smooth muscle cells with ET-1 and PDGF induced phosphorylation of ERK1/2 and Akt. Abl knockdown attenuated ERK1/2 phosphorylation in smooth muscle cells stimulated with ET-1 and PDGF. However, Akt phosphorylation upon stimulation with ET-1 and PDGF was not reduced. Because Abl is known to regulate actin polymerization in smooth muscle, we also evaluated the effects of inhibition of actin polymerization on phosphorylation of ERK1/2. Pretreatment with the actin polymerization inhibitor latrunculin-A also blocked ERK1/2 phosphorylation during activation with ET-1 and PDGF. The results suggest that Abl may regulate smooth muscle cell proliferation by modulating actin dynamics and ERK1/2 phosphorylation during mitogenic activation.
Project description:Actin dynamics plays an essential role in regulating airway smooth muscle contraction. The mechanisms that regulate actin dynamics in smooth muscle are not completely understood. Glia maturation factor (GMF) is a protein that has been reported to inhibit actin nucleation and to induce actin network debranching in vitro. The role of GMF in human smooth muscle cells and tissues has not been investigated. In this study, knockdown of GMF-? by RNA interference enhanced actin polymerization and contraction in human airway smooth muscle (HASM) cells and tissues without affecting myosin phosphorylation (another important biochemical change during contractile activation). Activation of HASM cells and tissues with acetylcholine induced dissociation of GMF-? from Arp2 of the Arp2/3 complex. Acetylcholine stimulation also increased GMF-? phosphorylation at Tyr-104. GMF-? phosphorylation at this residue was mediated by c-Abl tyrosine kinase. The GMF-? mutant Y104F (phenylalanine substitution at Tyr-104) had higher association with Arp2 in HASM cells upon contractile activation. Furthermore, expression of mutant Y104F GMF-? attenuated actin polymerization and contraction in smooth muscle. Thus, we propose a novel mechanism for the regulation of actin dynamics and smooth muscle contraction. In unstimulated smooth muscle, GMF-? binds to the Arp2/3 complex, which induces actin disassembly and retains lower levels of F-actin. Upon contractile stimulation, phosphorylation at Tyr-104 mediated by c-Abl tyrosine kinase leads to the dissociation of GMF-? from Arp2/3, by which GMF-? no longer induces actin disassembly. Reduced actin disassembly renders F-actin in higher level, which facilitates smooth muscle contraction.
Project description:Vinculin localizes to membrane adhesion junctions where it links actin filaments to the extracellular matrix by binding to the integrin-binding protein talin at its head domain (Vh) and to actin filaments at its tail domain (Vt). Vinculin can assume an inactive (closed) conformation in which Vh and Vt bind to each other, masking the binding sites for actin and talin, and an active (open) conformation in which the binding sites for talin and actin are exposed. We hypothesized that the contractile activation of smooth muscle tissues might regulate the activation of vinculin and thereby contribute to the regulation of contractile tension. Stimulation of tracheal smooth muscle tissues with acetylcholine (ACh) induced the recruitment of vinculin to cell membrane and its interaction with talin and increased the phosphorylation of membrane-localized vinculin at the C-terminal Tyr-1065. Expression of recombinant vinculin head domain peptide (Vh) in smooth muscle tissues, but not the talin-binding deficient mutant head domain, VhA50I, inhibited the ACh-induced recruitment of endogenous vinculin to the membrane and the interaction of vinculin with talin and also inhibited vinculin phosphorylation. Expression of Vh peptide also inhibited ACh-induced smooth muscle contraction and inhibited ACh-induced actin polymerization; however, it did not affect myosin light chain phosphorylation, which is necessary for cross-bridge cycling. Inactivation of RhoA inhibited vinculin activation in response to ACh. We conclude that ACh stimulation regulates vinculin activation in tracheal smooth muscle via RhoA and that vinculin activation contributes to the regulation of active tension by facilitating connections between actin filaments and talin-integrin adhesion complexes and by mediating the initiation of actin polymerization.
Project description:The tyrosine phosphorylated protein Crk-associated substrate (CAS) has previously been shown to participate in the cellular processes regulating dynamic changes in the actin architecture and arterial constriction. In the present study, treatment of rat mesenteric arteries with phenylephrine (PE) led to the increase in CAS tyrosine phosphorylation and the association of CAS with the adapter protein CrkII. CAS phosphorylation was catalyzed by Abl in an in vitro study. To determine the role of Abl tyrosine kinase in arterial vessels, plasmids encoding Abl short hairpin RNA (shRNA) were transduced into mesenteric arteries by chemical loading plus liposomes. Abl silencing diminished increases in CAS phosphorylation on PE stimulation. Previous studies have shown that assembly of the multiprotein compound containing CrkII, neuronal Wiskott-Aldrich Syndrome Protein (N-WASP) and the Arp2/3 (Actin Related Protein) complex triggers actin polymerization in smooth muscle as well as in nonmuscle cells. In this study, Abl silencing attenuated the assembly of the multiprotein compound in resistance arteries on contractile stimulation. Furthermore, the increase in F/G-actin ratios (an index of actin assembly) and constriction on contractile stimulation were reduced in Abl-deficient arterial segments compared with control arteries. However, myosin regulatory light chain phosphorylation (MRLCP) elicited by contractile activation was not inhibited in Abl-deficient arteries. These results suggest that Abl may play a pivotal role in mediating CAS phosphorylation, the assembly of the multiprotein complex, actin assembly, and constriction in resistance arteries. Abl does not participate in the regulation of myosin activation in arterial vessels during contractile stimulation.
Project description:Smooth muscle cell migration is essential for many diverse biological processes such as pulmonary/cardiovascular development and homeostasis. Abi1 (Abelson interactor 1) is an adapter protein that has been implicated in nonmuscle cell migration. However, the role and mechanism of Abi1 in smooth muscle migration are largely unknown. Here, Abi1 knockdown by shRNA reduced human airway smooth muscle cell migration, which was restored by Abi1 rescue. Abi1 localized at the tip of lamellipodia and its protrusion coordinated with F-actin at the leading cell edge of live cells. In addition, we identified profilin-1 (Pfn-1), a G-actin transporter, as a new partner for Abi1. Abi1 knockdown reduced the recruitment of Pfn-1 to the leading cell edge. Moreover, Abi1 knockdown reduced the localization of the actin-regulatory proteins c-Abl (Abelson tyrosine kinase) and N-WASP (neuronal Wiskott-Aldrich Syndrome Protein) at the cell edge without affecting other migration-related proteins including pVASP (phosphorylated vasodilator stimulated phosphoprotein), cortactin and vinculin. Furthermore, we found that c-Abl and integrin ?1 regulated the positioning of Abi1 at the leading edge. Taken together, the results suggest that Abi1 regulates cell migration by affecting Pfn-1 and N-WASP, but not pVASP, cortactin and focal adhesions. Integrin ?1 and c-Abl are important for the recruitment of Abi1 to the leading edge.
Project description:Raf-1 is a serine/threonine protein kinase that has an essential role in cell proliferation. The mechanisms that regulate Raf-1 in airway smooth muscle are not well understood. In this study, treatment with platelet-derived growth factor (PDGF) induced spatial redistribution of Raf-1 from the cytoplasm to the periphery of human airway smooth muscle cells. Moreover, a pool of Raf-1 was found in F-actin of human airway smooth muscle cells. Activation with PDGF led to an increase in the association of Raf-1 with cytoskeletal actin. Treatment of cells with the actin polymerization inhibitor latrunculin A (LAT-A), but not the microtubule depolymerizer nocodazole, inhibited the interaction of Raf-1 with actin in response to PDGF activation. Because abelson tyrosine kinase (Abl) is known to specifically regulate actin dynamics in smooth muscle, the role of Abl in modulating the coupling of Raf-1 with actin was also evaluated. Abl knockdown by RNA interference attenuated the association of Raf-1 with actin, which is recovered by Abl rescue. Treatment with LAT-A, but not nocodazole, inhibited the spatial redistribution of Raf-1 during PDGF activation. However, treatment with both LAT-A and nocodazole attenuated smooth muscle cell proliferation. Finally, Abl knockdown attenuated the redistribution of Raf-1 and cell proliferation, which were restored by Abl reexpression. The results suggest a novel mechanism that the interaction of Raf-1 with cytoskeletal actin is critical for Raf-1 redistribution and airway smooth muscle cell proliferation during activation with the growth factor.