Paxillin phosphorylation, actin polymerization, noise temperature, and the sustained phase of swine carotid artery contraction

Histamine stimulation of swine carotid artery induces both contraction and actin polymerization. The importance of stimulus-induced actin polymerization is not known. Tyrosine phosphorylation of the scaffolding protein paxillin is thought to be an important regulator of actin polymerization. Noise temperature, hysteresivity, and phase angle are rheological measures of the fluidity of a tissue, i.e., whether the muscle is more a "Hookean solid" or a "Newtonian liquid." Y118 paxillin phosphorylation, crossbridge phosphorylation, actin polymerization, noise temperature, hysteresivity, phase angle, real stiffness, and stress were measured in intact swine carotid arteries that were depolarized with high K⁺ or stimulated with histamine. The initial rapid force development phase of high-K⁺ or histamine-induced contraction was associated with increased crossbridge phosphorylation but no significant change in Y118 paxillin phosphorylation, actin polymerization, noise temperature, hysteresivity, or phase angle. This suggests that the initial contraction was caused by the increase in crossbridge phosphorylation and did not alter the tissue's rheology. Only after full force development was there a significant increase in Y118 paxillin phosphorylation and actin polymerization associated with a significant decrease in noise temperature and hysteresivity. These data suggest that some part of the sustained contraction may depend on stimulated actin polymerization and/or a transition to a more "solid" rheology. Supporting this contention was the finding that an inhibitor of actin polymerization, latrunculin-A, reduced force while increasing noise temperature/hysteresivity. Further research is needed to determine whether Y118 paxillin phosphorylation, actin polymerization, and changes in rheology could have a role in arterial smooth muscle contraction. cytoskeleton; hysteresivity; latch hypothesis; vascular smooth muscle     

Heat shock protein 20-mediated force suppression in forskolin-relaxed swine carotid artery

Increases in cyclic nucleotide levels induce smooth muscle relaxation by deactivation [reductions in myosin regulatory light chain (MRLC) phosphorylation (e.g., by reduced [Ca²⁺])] or force suppression (reduction in force without reduction in MRLC phosphorylation). Ser¹⁶-heat shock protein 20 (HSP20) phosphorylation is the proposed mediator of force suppression. We evaluated three potential hypotheses whereby Ser¹⁶-HSP20 phosphorylation could regulate smooth muscle force: 1) a threshold level of HSP20 phosphorylation could inactivate a thin filament as a whole, 2) phosphorylation of a single HSP20 could fully inactivate a small region of a thin filament, or 3) HSP20 phosphorylation could weakly inhibit myosin binding at either the thin- or thick-filament level. We tested these hypotheses by analyzing the dependence of force on Ser¹⁶-HSP20 phosphorylation in swine carotid media. First, we determined that swine HSP20 has a second phosphorylation site at Ser¹⁵⁷. Ser¹⁵⁷-HSP20 phosphorylation values were high and did not change during contractile activation or forskolin-induced relaxation. Forskolin significantly increased Ser¹⁶-HSP20 phosphorylation. The relationship between Ser¹⁶-HSP20 phosphorylation and force remained linear and was shifted downward in partially activated muscles relaxed with forskolin. Neither forskolin nor nitroglycerin induced actin depolymerization as detected using the F/G-actin ratio method in smooth muscle homogenates. These results suggest that force suppression does not occur in accordance with the first hypothesis (inactivation of a thin filament as a whole). Our data are more consistent with the second and third hypotheses that force suppression is mediated by full or partial inhibition of local myosin binding at the thin- or thick-filament level. cAMP; cGMP; nitric oxide; vascular smooth muscle     

Light chain phosphorylation regulates the movement of smooth muscle myosin on actin filaments

In smooth muscles there is no organized sarcomere structure wherein the relative movement of myosin filaments and actin filaments has been documented during contraction. Using the recently developed in vitro assay for myosin-coated bead movement (Sheetz, M.P., and J.A. Spudich, 1983, Nature (Lond.)., 303:31-35), we were able to quantitate the rate of movement of both phosphorylated and unphosphorylated smooth muscle myosin on ordered actin filaments derived from the giant alga, Nitella. We found that movement of turkey gizzard smooth muscle myosin on actin filaments depended upon the phosphorylation of the 20-kD myosin light chains. About 95% of the beads coated with phosphorylated myosin moved at velocities between 0.15 and 0.4 micron/s, depending upon the preparation. With unphosphorylated myosin, only 3% of the beads moved and then at a velocity of only approximately 0.01-0.04 micron/s. The effects of phosphorylation were fully reversible after dephosphorylation with a phosphatase prepared from smooth muscle. Analysis of the velocity of movement as a function of phosphorylation level indicated that phosphorylation of both heads of a myosin molecule was required for movement and that unphosphorylated myosin appears to decrease the rate of movement of phosphorylated myosin. Mixing of phosphorylated smooth muscle myosin with skeletal muscle myosin which moves at 2 microns/s resulted in a decreased rate of bead movement, suggesting that the more slowly cycling smooth muscle myosin is primarily determining the velocity of movement in such mixtures.     

Effects of microtubule disruption on force, velocity, stiffness and [Ca(2+)](i) in porcine coronary arteries

Force generated by smooth muscle cells is believed to result from the interaction of actin and myosin filaments and is regulated through phosphorylation of the myosin regulatory light chain (LC(20)). The role of other cytoskeleton filaments, such as microtubules and intermediate filaments, in determining the mechanical output of smooth muscle is unclear. In cultured fibroblasts, microtubule disruption results in large increases in force similar to contractions associated with LC(20) phosphorylation (15). One hypothesis, the "tensegrity" or "push-pull" model, attributes this increase in force to the disruption of microtubules functioning as rigid struts to resist force generated by actin-myosin interaction (9). In porcine coronary arteries, the disruption of microtubules by nocodazole (11 microM) also elicited moderate but significant increases in isometric force (10-40% of a KCl contracture), which could be blocked or reversed by taxol (a microtubule stabilizer). We tested whether this nocodazole-induced force was accompanied by changes in coronary artery stiffness or unloaded shortening velocity, parameters likely to be highly sensitive to microtubule resistance elements. Few changes were seen, ruling out push-pull mechanisms for the increase in force by nocodazole. In contrast, the intracellular calcium concentration, measured by fura 2 in the intact artery, was increased by nocodazole in parallel with force, and this was inhibited and/or reversed by taxol. Our results indicate that microtubules do not significantly contribute to vascular smooth muscle mechanical characteristics but, importantly, may play a role in modulation of Ca(2+) signal transduction.     

Latrunculin B increases force fluctuation-induced relengthening of ACh-contracted, isotonically shortened canine tracheal smooth muscle.

We hypothesized that differences in actin filament length could influence force fluctuation-induced relengthening (FFIR) of contracted airway smooth muscle and tested this hypothesis as follows. One-hundred micromolar ACh-stimulated canine tracheal smooth muscle (TSM) strips set at optimal reference length (Lref) were allowed to shorten against 32% maximal isometric force (Fmax) steady preload, after which force oscillations of +/-16% Fmax were superimposed. Strips relengthened during force oscillations. We measured hysteresivity and calculated FFIR as the difference between muscle length before and after 20-min imposed force oscillations. Strips were relaxed by ACh removal and treated for 1 h with 30 nM latrunculin B (sequesters G-actin and promotes depolymerization) or 500 nM jasplakinolide (stabilizes actin filaments and opposes depolymerization). A second isotonic contraction protocol was then performed; FFIR and hysteresivity were again measured. Latrunculin B increased FFIR by 92.2 +/- 27.6% Lref and hysteresivity by 31.8 +/- 13.5% vs. pretreatment values. In contrast, jasplakinolide had little influence on relengthening by itself; neither FFIR nor hysteresivity was significantly affected. However, when jasplakinolide-treated tissues were then incubated with latrunculin B in the continued presence of jasplakinolide for 1 more h and a third contraction protocol performed, latrunculin B no longer substantially enhanced TSM relengthening. In TSM treated with latrunculin B + jasplakinolide, FFIR increased by only 3.03 +/- 5.2% Lref and hysteresivity by 4.14 +/- 4.9% compared with its first (pre-jasplakinolide or latrunculin B) value. These results suggest that actin filament length, in part, determines the relengthening of contracted airway smooth muscle.     

Smooth muscle myosin cross-bridge interactions modulate actin filament sliding velocity in vitro

Although it is generally believed that phosphorylation of the regulatory light chain of myosin is required before smooth muscle can develop force, it is not known if the overall degree of phosphorylation can also modulate the rate at which cross-bridges cycle. To address this question, an in vitro motility assay was used to observe the motion of single actin filaments interacting with smooth muscle myosin copolymers composed of varying ratios of phosphorylated and unphosphorylated myosin. The results suggest that unphosphorylated myosin acts as a load to slow down the rate at which actin is moved by the faster cycling phosphorylated cross-bridges. Myosin that was chemically modified to generate a noncycling analogue of the "weakly" bound conformation was similarly able to slow down phosphorylated myosin. The observed modulation of actin velocity as a function of copolymer composition can be accounted for by a model based on mechanical interactions between cross-bridges.     

Phosphorylation of tropomyosin extends cooperative binding of myosin beyond a single regulatory unit

Tropomyosin (Tm) is one of the major phosphoproteins comprising the thin filament of muscle. However, the specific role of Tm phosphorylation in modulating the mechanics of actomyosin interaction has not been determined. Here we show that Tm phosphorylation is necessary for long-range cooperative activation of myosin binding. We used a novel optical trapping assay to measure the isometric stall force of an ensemble of myosin molecules moving actin filaments reconstituted with either natively phosphorylated or dephosphorylated Tm. The data show that the thin filament is cooperatively activated by myosin across regulatory units when Tm is phosphorylated. When Tm is dephosphorylated, this "long-range" cooperative activation is lost and the filament behaves identically to bare actin filaments. However, these effects are not due to dissociation of dephosphorylated Tm from the reconstituted thin filament. The data suggest that end-to-end interactions of adjacent Tm molecules are strengthened when Tm is phosphorylated, and that phosphorylation is thus essential for long range cooperative activation along the thin filament.     

Inhibition of the relative movement of actin and myosin by caldesmon and calponin.

Contractile activity of myosin II in smooth muscle and non-muscle cells requires phosphorylation of myosin by myosin light chain kinase. In addition, these cells have the potential for regulation at the thin filament level by caldesmon and calponin, both of which bind calmodulin. We have investigated this regulation using in vitro motility assays. Caldesmon completely inhibited the movement of actin filaments by either phosphorylated smooth muscle myosin or rabbit skeletal muscle heavy meromyosin. The amount of caldesmon required for inhibition was decreased when tropomyosin is present. Similarly, calponin binding to actin resulted in inhibition of actin filament movement by both smooth muscle myosin and skeletal muscle heavy meromyosin. Tropomyosin had no effect on the amount of calponin needed for inhibition. High concentrations of calmodulin (10 microM) in the presence of calcium completely reversed the inhibition. The nature of the inhibition by the two proteins was markedly different. Increasing caldesmon concentrations resulted in graded inhibition of the movement of actin filaments until complete inhibition of movement was obtained. Calponin inhibited actin sliding in a more "all or none" fashion. As the calponin concentration was increased the number of actin filaments moving was markedly decreased, but the velocity of movement remained near control values.     

Phosphorylation of vascular smooth muscle caldesmon by endogenous kinase.

Caldesmon was phosphorylated up to 1.2 molPi/mol using a partially purified endogenous kinase fraction. The phosphorylation site was within the C-terminal 99 amino acids. We were also able to phosphorylate caldesmon incorporated into native and synthetic smooth muscle thin filaments. Phosphorylation did not alter caldesmon binding to actin or inhibition of actomyosin ATPase. It also did not change Ca2+ sensitivity in native thin filaments. Phosphorylated caldesmon bound to myosin less than unphosphorylated caldesmon, especially when the myosin was also not phosphorylated. This work did not support the hypothesis that caldesmon function is modulated by phosphorylation.     

Myosin phosphorylation triggers actin polymerization in vascular smooth muscle

A variety of contractile stimuli increases actin polymerization, which is essential for smooth muscle contraction. However, the mechanism(s) of actin polymerization associated with smooth muscle contraction is not fully understood. We tested the hypothesis that phosphorylated myosin triggers actin polymerization. The present study was conducted in isolated intact or beta-escin-permeabilized rat small mesenteric arteries. Reductions in the 20-kDa myosin regulatory light chain (MLC20) phosphorylation were achieved by inhibiting MLC kinase with ML-7. Increases in MLC20 phosphorylation were achieved by inhibiting myosin light chain phosphatase with microcystin. Isometric force, the degree of actin polymerization as indicated by the F-actin-to-G-actin ratio, and MLC20 phosphorylation were determined. Reductions in MLC20 phosphorylation were associated with a decreased force development and actin polymerization. Increased MLC20 phosphorylation was associated with an increased force generation and actin polymerization. We also found that a heptapeptide that mimics the actin-binding motif of myosin II enhanced microcystin-induced force generation and actin polymerization without affecting MLC20 phosphorylation in beta-escin-permeabilized vessels. Collectively, our data demonstrate that MLC20 phosphorylation is capable of triggering actin polymerization. We further suggest that the binding of myosin to actin triggers actin polymerization and enhances the force development in arterial smooth muscle.     

THE ULTRASTRUCTURE OF STRIATED MUSCLE AT VARIOUS SARCOMERE LENGTHS

1. Rest and equilibrium length muscle sarcomeres are composed of thin filaments (actin) which traverse the sarcomeres from the Z membranes up to the H band; at this level the filaments are considerably thicker and less numerous. 2. Shortening of muscle is associated with a transformation of thin into thick filaments in the A band. 3. These observations are discussed in terms of interaction of actin and myosin to form a supercoiled structure as the basis of contraction.     

Tropomyosin interacts with phosphorylated HSP27 in agonist-induced contraction of smooth muscle

Displacement of the contractile protein tropomyosin from actin filament exposes the myosin-binding sites on actin, resulting in actin-myosin interaction and muscle contraction. The objective of the present study was to better understand the interaction of tropomyosin with heat shock protein (HSP)27 in contraction of smooth muscle cells of the colon. We investigated the possibility of a direct protein-protein interaction of tropomyosin with HSP27 and the role of phosphorylated HSP27 in this interaction. Immunoprecipitation studies on rabbit smooth muscle cells indicate that upon acetylcholine-induced contraction tropomyosin shows increased association with HSP27 phosphorylated at Ser82 and Ser78. Transfection of smooth muscle cells with HSP27 phosphorylation mutants indicated that the association of tropomyosin with HSP27 could be affected by HSP27 phosphorylation. In vitro binding studies with glutathione S-transferase (GST)-tagged HSP27 mutant proteins show that tropomyosin has greater direct interaction to phosphomimic HSP27 mutant compared with wild-type and nonphosphomimic HSP27. Our data suggest that, in response to a contractile agonist, HSP27 undergoes a rapid phosphorylation that may strengthen its interaction with tropomyosin. acetylcholine; fusion proteins; serine     

Smooth muscle and skeletal muscle myosins produce similar unitary forces and displacements in the laser trap.

Purified smooth muscle myosin in the in vitro motility assay propels actin filaments at 1/10 the velocity, yet produces 3-4 times more force than skeletal muscle myosin. At the level of a single myosin molecule, these differences in force and actin filament velocity may be reflected in the size and duration of single motion and force-generating events, or in the kinetics of the cross-bridge cycle. Specifically, an increase in either unitary force or duty cycle may explain the enhanced force-generating capacity of smooth muscle myosin. Similarly, an increase in attached time or decrease in unitary displacement may explain the reduced actin filament velocity of smooth muscle myosin. To discriminate between these possibilities, we used a laser trap to measure unitary forces and displacements from single smooth and skeletal muscle myosin molecules. We analyzed our data using mean-variance analysis, which does not rely on scoring individual events by eye, and emphasizes periods in the data with constant properties. Both myosins demonstrated multiple but similar event populations with discrete peaks at approximately +11 and -11 nm in displacement, and 1.5 and 3.5 pN in force. Mean attached times for smooth muscle myosin were longer than for skeletal-muscle myosin. These results explain much of the difference in actin filament velocity between these myosins, and suggest that an increased duty cycle is responsible for the enhanced force-generating capacity of smooth over skeletal-muscle myosin.     

Forces measured with micro-fabricated cantilevers during actomyosin interactions produced by filaments containing different myosin isoforms and loop 1 structures

Background

There is evidence that the actin-activated ATP kinetics and the mechanical work produced by muscle myosin molecules are regulated by two surface loops, located near the ATP binding pocket (loop 1), and in a region that interfaces with actin (loop 2). These loops regulate force and velocity of contraction, and have been investigated mostly in single molecules. There is a lack of information of the work produced by myosin molecules ordered in filaments and working cooperatively, which is the actual muscle environment.

Methods

We use micro-fabricated cantilevers to measure forces produced by myosin filaments isolated from mollusk muscles, skeletal muscles, and smooth muscles containing variations in the structure of loop 1 (tonic and phasic myosins). We complemented the experiments with in-vitro assays to measure the velocity of actin motility.

Results

Smooth muscle myosin filaments produced more force than skeletal and mollusk myosin filaments when normalized per filament overlap. Skeletal muscle myosin propelled actin filaments in a higher sliding velocity than smooth muscle myosin. The values for force and velocity were consistent with previous studies using myosin molecules, and suggest a close correlation with the myosin isoform and structure of surface loop 1.

General significance

The technique using micro-fabricated cantilevers to measure force of filaments allows for the investigation of the relation between myosin structure and contractility, allowing experiments to be conducted with an array of different myosin isoforms. Using the technique we observed that the work produced by myosin molecules is regulated by amino-acid sequences aligned in specific loops.     

Force spectroscopy reveals multiple "closed states" of the muscle thin filament

Tropomyosin (Tm) plays a critical role in regulating the contraction of striated muscle. The three-state model of activation posits that Tm exists in three positions on the thin filament: "blocked" in the absence of calcium when myosin cannot bind, "closed" when calcium binds troponin and Tm partially covers the myosin binding site, and "open" after myosin binding forces Tm completely off neighboring sites. However, we recently showed that actin filaments decorated with phosphorylated Tm are driven by myosin with greater force than bare actin filaments. This result cannot be explained by simple steric hindrance and suggests that Tm may have additional effects on actin-myosin interactions. We therefore tested the hypothesis that Tm and its phosphorylation state affect the rate at which single actin-myosin bonds form and rupture. Using a laser trap, we measured the time necessary for the first bond to form between actin and rigor heavy meromyosin and the load-dependent durations of those bonds. Measurements were repeated in the presence of subsaturating myosin-S1 to force Tm from the closed to the open state. Maximum bond lifetimes increased in the open state, but only when Tm was phosphorylated. While the frequency with which bonds formed was extremely low in the closed state, when a bond did form it took significantly less time to do so than with bare actin. These data suggest there are at least two closed states of the thin filament, and that Tm provides additional points of contact for myosin.     

Inhibition of the ATP-dependent interaction of actin and myosin by the catalytic domain of the myosin light chain kinase of smooth muscle: possible involvement in smooth muscle relaxation

Myosin light chain kinase (MLCK) phosphorylates the light chain of smooth muscle myosin enabling its interaction with actin. This interaction initiates smooth muscle contraction. MLCK has another role that is not attributable to its phosphorylating activity, i.e., it inhibits the ATP-dependent movement of actin filaments on a glass surface coated with phosphorylated myosin. To analyze the inhibitory effect of MLCK, the catalytic domain of MLCK was obtained with or without the regulatory sequence adjacent to the C-terminal of the domain, and the inhibitory effect of the domain was examined by the movement of actin filaments. All the domains work so as to inhibit actin filament movement whether or not the regulatory sequence is included. When the domain includes the regulatory sequence, calmodulin in the presence of calcium abolishes the inhibition. Since the phosphorylation reaction is not involved in regulating the movement by MLCK, and a catalytic fragment that shows no kinase activity also inhibits movement, the kinase activity is not related to inhibition. Higher concentrations of MLCK inhibit the binding of actin filaments to myosin-coated surfaces as well as their movement. We discuss the dual roles of the domain, the phosphorylation of myosin that allows myosin to cross-bridge with actin and a novel function that breaks cross-bridging.     

The N-terminal region of twitchin binds thick and thin contractile filaments: redundant mechanisms of catch force maintenance

Catch force maintenance in invertebrate smooth muscles is probably mediated by a force-bearing tether other than myosin cross-bridges between thick and thin filaments. The phosphorylation state of the mini-titin twitchin controls catch. The C-terminal phosphorylation site (D2) of twitchin with its flanking Ig domains forms a phosphorylation-sensitive complex with actin and myosin, suggesting that twitchin is the tether (Funabara, D., Osawa, R., Ueda, M., Kanoh, S., Hartshorne, D. J., and Watabe, S. (2009) J. Biol. Chem. 284, 18015-18020). Here we show that a region near the N terminus of twitchin also interacts with thick and thin filaments from Mytilus anterior byssus retractor muscles. Both a recombinant protein, including the D1 and DX phosphorylation sites with flanking 7th and 8th Ig domains, and a protein containing just the linker region bind to thin filaments with about a 1:1 mol ratio to actin and K(d) values of 1 and 15 μM, respectively. Both proteins show a decrease in binding when phosphorylated. The unphosphorylated proteins increase force in partially activated permeabilized muscles, suggesting that they are sufficient to tether thick and thin filaments. There are two sites of thin filament interaction in this region because both a 52-residue peptide surrounding the DX site and a 47-residue peptide surrounding the D1 site show phosphorylation-dependent binding to thin filaments. The peptides relax catch force, confirming the region's central role in the mechanism of catch. The multiple sites of thin filament interaction in the N terminus of twitchin in addition to those in the C terminus provide an especially secure and redundant mechanical link between thick and thin filaments in catch.     

Role of vimentin in smooth muscle force development

Vimentin intermediate filaments undergo spatial reorganization in cultured smooth muscle cells in response to contractile activation; however, the role of vimentin in the physiological properties of smooth muscle has not been well elucidated. Tracheal smooth muscle strips were loaded with antisense oligonucleotides (ODNs) against vimentin and then cultured for 2 days to allow for protein degradation. Treatment with vimentin antisense, but not sense, ODNs suppressed vimentin protein expression; neither vimentin antisense nor sense ODNs affected protein levels of desmin and actin. Force development in response to ACh stimulation or KCl depolarization was lower in vimentin-deficient tissues than in vimentin sense ODN- or non-ODN-treated muscle strips. Passive tension was also depressed in vimentin-depleted muscle tissues. Vimentin downregulation did not attenuate increases in myosin light chain (MLC) phosphorylation in response to contractile stimulation or basal MLC phosphorylation. In vimentin sense ODN-treated or non-ODN-treated smooth muscle strips, the desmosomal protein plakoglobin was primarily localized in the cell periphery. The membrane-associated localization of plakoglobin was reduced in vimentin-depleted muscle tissues. These studies suggest that vimentin filaments play an important role in mediating active force development and passive tension, which are not regulated by MLC phosphorylation. Vimentin downregulation impairs the structural organization of desmosomes, which may be associated with the decrease in force development. intermediate filaments; cytoskeleton; contraction; desmin     

In vitro movement of actin filaments on gizzard smooth muscle myosin: requirement of phosphorylation of myosin light chain and effects of tropomyosin and caldesmon

ATP-dependent movement of actin filaments on smooth muscle myosin was investigated by using the in vitro motility assay method in which myosin was fixed on the surface of a coverslip in a phosphorylated or an unphosphorylated state. Actin filaments slid on gizzard myosin phosphorylated with myosin light chain kinase (MLCK) at a rate of 0.35 micron/s, but did not slide at all on unphosphorylated myosin. The movement of actin filaments on phosphorylated myosin was stopped by perfusion of phosphatase. Subsequent perfusion with a solution containing MLCK, calmodulin, and Ca2+ enabled actin filaments to move again. The sliding velocities on monophosphorylated and diphosphorylated myosin by MLCK were not different. Actin filaments did not move on myosin phosphorylated with protein kinase C (PKC). The sliding velocity on myosin phosphorylated with both MLCK and PKC was identical to that on myosin phosphorylated only with MLCK. Gizzard tropomyosin enhanced the sliding velocity to 0.76 micron/s. Gizzard caldesmon decreased the sliding velocity with increase in its concentration. At a 5-fold molar ratio of caldesmon to actin, the movement stopped completely. This inhibitory effect of caldesmon was relieved upon addition of excess calmodulin and Ca2+.     

The Association of Cortactin with Profilin-1 Is Critical for Smooth Muscle Contraction

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.