Cell Motility and the Cytoskeleton 18:81-85 (1991)
Views and Reviews Regulation of Smooth Muscle Myosin Kathleen M. Trybus Rosenstiel Research Center, Brandeis University, Waltham, Massachusetts
INTRODUCTION
Phosphorylation of the myosin regulatory light chain is the key event necessary to initiate force development in smooth muscle. The native thin-filament also has the potential to regulate and/or modulate actomyosin interactions, because of the presence of the actin-binding proteins tropomyosin, caldesmon, and calponin. The interplay between the thick and thin-filament-based regulatory systems might explain some of the properties unique to smooth muscle. ACTIVATION OF MYOSIN LIGHT CHAIN KINASE
The pathway leading to myosin phosphorylation begins with an increase in cytoplasmic calcium concentration. Unlike skeletal muscle, calcium release in smooth muscles can be triggered by voltage-independent pathways, such as via the second messenger inositol trisphosphate (IP,), a process known as “pharmomechanical coupling. IP, causes calcium release from the sarcoplasmic reticulum, mediated by an IP, receptor which is believed to be a member of a new class of intracellular ligand-gated ion channels [Somlyo et al., 1988bl. Calcium-calmodulin binds to myosin light chain kinase (MLCK) and displaces the “pseudosubstrate” from the active site, thus activating MLCK to phosphorylate the 20 kd regulatory light chain of myosin. The inhibitory pseudosubstrate sequence found in MLCK is similar to that around Ser 19 of the myosin regulatory light chain, the natural substrate for MLCK [Pearson et al., 19881. ”
ing physiological (pH 7-7.5, 0.1-0.15 M KCl), synthetic dephosphorylated myosin filaments are unstable in the presence of MgATP. The filament depolymerizes to an assembly-incompetent monomer in which the a-helical coiled-coil tail is folded into thirds and the heads are bent toward the rod, as illustrated in Fig. 1 . Dephosphorylated folded monomers are enzymatically inactive and “trap” the products of ATP hydrolysis, which are released at 0.0002-0.0005 sec-’, the lowest turnover rate observed for any myosin to date [Cross et al., 19861. Phosphorylation destabilizes this conformation, possibly by altering a binding site for the tail, and causes the rod to extend and assemble into filaments (Fig. 1). Phosphorylated myosin filaments have an actin activated ATPase activity of 0.3/sec (24”C), approximately 500-fold higher than dephosphorylated folded myosin. From these observations it could not be deduced if myosin conformation or the state of phosphorylation were the primary determinant of activity. Since dephosphorylated myosin filaments are known to exist in relaxed smooth muscle cells [Somlyo et al., 19811, it is important to determine if the activity of this species is inhibited by dephosphorylation alone, or whether an inhibitory thin-filament based regulatory system needs to be invoked. The activity of dephosphorylated myosin in filamentous form could be determined under standard solvent conditions only by the addition of an anti-rod monoclonal antibody which blocked depolymerization in the presence of MgATP. Dephosphorylated myosin filaments had an actin-activated activity of O.O02/sec, only 5-fold higher than the dephosphorylated folded monomer, and at least 100-fold lower than phosphorylated myosin filaments. This result establishes that phosphor-
LIGHT CHAIN PHOSPHORYLATION REGULATES ACTIN-ACTIVATED ATPase ACTIVITY
Light chain phosphorylation has profound effects on two aspects of smooth muscle myosin function in vitro: its ability to assemble into a filament, and its enzymatic activity. Under solvent conditions approximat0 1991 Wiley-Liss, Inc.
Accepted October 23, 1990. Address reprint requests to Kathleen M . Trybus, Rosenstiel Research Center, Brandeis University, Waltham, MA 02254.
82
Trybus O.B/sec ACTIVE
O.OOZ/sec
INACTIVE
DEPHOSPHORYLATED
FILAMENTS
PHOSPHORYLATED FILAMENTS
t
t
TRANSIENT UNFOLDED M0N0ME R
FOLDED MONOMER
INACTIVE 0.0005/sec
Fig 1
ylation is the primary regulator of activity independent of large changes in myosin conformation [Trybus, 19891. Dephosphorylated heavy meromyosin, which does not fold or form filaments, had an actin-activated ATPase activity remarkably similar to that of dephosphorylated myosin filaments [Sellers, 19851. Phosphorylation must therefore directly affect product release from the active site. A “direct” effect, however, refers to changes occurring over a distance equal to half the length of the myosin head. By analogy with other myosins, the regulatory light chain of smooth muscle myosin is localized at the headhod junction, which is at least 100 A distant from the active site. Photoaffinity labeling showed that the 17 kd class of light chains is located near the active site of smooth muscle myosin [Okamoto et al., 19861, and it is possible that this subunit is the link between distant regions on the head. The three levels of enzymatic activity that can be distinguished are summarized in Figure 1: active phosphorylated myosin filaments (0.3/sec), inactive dephosphorylated myosin filaments (O.OOYsec), and enzymatically incompetent soluble folded dephosphorylated monomers (~0.0005/sec).Intramolecular interaction of the myosin tail with the headheck region in the folded monomer inhibits product release to a somewhat greater extent than can be achieved by possible intermolecular interactions within the filament. DOES FOLDED MYOSIN EXIST IN A SMOOTH MUSCLE CELL?
Filaments containing dephosphorylated myosin exist in relaxed smooth muscle cells [Somlyo et al., 19811, but it is less clear if the dephosphorylated folded monomer is formed in vivo. If folded monomers are not present, an unanswered question is why synthetic fila-
ments so readily disassemble in vitro, but native filaments remain assembled in the cellular milieu. One possibility is that a critical concentration of folded monomers does exist, but the myosin concentration in the cell exceeds this value such that dephosphorylated filaments are in equilibrium with this pool [KendrickJones et al., 19871. Alternatively, a yet unidentified protein exists which stabilizes the native filament. Results obtained from optical and electron microscopy experiments suggest that assembly-disassembly does occur to some extent during a contraction-relaxation cycle. Electron micrographs of cross-sections of the smooth anococcygeus muscle fixed during rest or contraction directly showed that myosin filament density increased upon contraction [Gillis et al., 19881. This observation is consistent with the finding that the birefringence of this muscle also increased during contraction, and was reversed during relaxation [GodfraindDeBecker and Gillis, 19881. What would be the advantage of this disassembly? Both dephosphorylated filaments and dephosphorylated monomers are enzymatically “off” and utilize only low amounts of MgATP. The folded monomer is soluble, however, and could diffuse throughout the cell without binding to actin and reassemble in a different location upon phosphorylation. Although this mechanism would appear to be better suited to a non-muscle cell, the architecture of a smooth muscle cell is not well established and similar processes might occur although to a lesser extent. MODULATION OF CYCLING RATES BY PHOSPHORYLATED-DEPHOSPHORYLATED CROSSBRIDGE INTERACTIONS
Results from solution actin-activated ATPases suggested that phosphorylation/dephosphorylation by itself
Regulation of Smooth Muscle Myosin
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Fig. 2.
efficiently regulates the activity of filamentous myosin; an in vitro motility assay further demonstrated that light chain phosphorylation actually controls actin movement. The rate at which single actin filaments move over myosin bound to a nitrocellulose substrate can be quantitated in the motility assay. Dephosphorylated myosin filaments did not support motility, but phosphorylated myosin moved actin at approximately 0.2-0.4 p d s e c (20°C, 25 mM KCI) [Umemoto and Sellers, 1990; Warshaw et al., 19901, approximately 10-fold slower than the rate observed with skeletal muscle myosin. Surprisingly, dephosphorylated filaments held the actin in a rigid rigor-like conformation even in the presence of MgATP. Since phosphate release is the kinetic step regulated by light chain phosphorylation, dephosphorylated heads are predominantly in a “weak-binding’’ conformation which rapidly attaches and detaches from actin, with an equilibrium constant of approximately lo4 M-’. The strength of this interaction is, however, enough to prevent actin from diffusing into solution. There are few times when myosin in the cell is either completely phosphorylated or completely dephosphorylated. Most often, the filament is submaximally phosphorylated and consists of mixtures of myosin molecules that have 0, l , or 2 heads phosphorylated. Figure 2 illustrates the response of a smooth muscle to calcium. The initial rise in calcium and light chain phosphorylation is accompanied by rapid shortening (dashed line) and tension development (solid line). As calcium levels decrease, phosphatase activity predominates over kinase
activity, and phosphorylation levels drop. Correlated with the decrease in phosphorylation is a decrease in shortening velocity, while force levels remain high, a situation unique to smooth muscle [Dillon et al., 1981; Hai and Murphy, 19881. The apparent correlation of phosphorylation and shortening velocity during sustained contractions suggested that phosphorylation may be more than a simple “on-off’’ switch. Shortening velocity is independent of crossbridge number, so the declining velocity cannot be accounted for by a decrease in the number of active heads. Solution studies showed that the cycling rate of phosphorylated myosin did not change when it was copolymerized with dephosphorylated myosin, arguing against a model where the intrinsic crossbridge cycling rate is modulated by cooperative interactions within the filament. In the motility assay, however, which is considered to be the in vitro analog of unloaded shortening velocity in a muscle, the presence of dephosphorylated myosin in a copolymer with phosphorylated myosin significantly decreased the rate of actin movement [Sellers et al., 1985; Warshaw et al., 19901. This modulation of velocity was interpreted as the result of mechanical interactions between the two populations of crossbridges: faster cycling phosphorylated crossbridges move actin more slowly because they are working against a load imposed by the slowly or non-cycling dephosphorylated heads. The decrease in shortening velocity in a muscle might also be due to dephosphorylated bridges acting as an internal load.
84
Trybus
it could promote the formation of bridges that detach more slowly from actin, a property associated with the To explain why force levels remain high when postulated latchbridge. There have also been suggestions phosphorylation levels are low, the existence of “latchthat caldesmon could be involved in the latch state bebridges” was proposed [Hai and Murphy, 19891. Latchcause caldesmon has a binding site both for actin and bridges can only be formed by dephosphorylation of at- myosin which could physically prevent crossbridge distached phosphorylated crossbridges. They sustain force sociation. There is, however, no evidence suggesting and dissociate very slowly from actin, thus contributing that caldesmon crosslinks native thin filaments to myosin to the high economy of force maintenance in smooth [Moody et al., 19901, or that crosslinked dephosphorymuscles. Myosin that is dephosphorylated when not atlated bridges can support tension. tached to actin simply results in the relaxed state. The Despite the solution data which suggest that caldesobserved physiological data can be modeled by assuming mon inhibits actin-activated ATPase activity, fiber studthat the latchbridge state exists; another regulatory sysies do not support the idea that both calcium and phostem is not required to fit the experimental data. There has phorylation are required to initiate contraction. If the been no direct demonstration, however, of the existence calcium dependency of phosphorylation is removed eiof a long-lived, force producing, attached dephosphoryther through the use of a calcium-independent MLCK lated crossbridge. An alternative explanation of why fragment [Itoh et al., 19891, or by thiophosphorylating force remains high at low phosphorylation levels is that the light chains [Somlyo et al., 1988a1, calcium is no the presence of a few attached phosphorylated crosslonger required to initiate contraction. If caldesmon is bridges permits attachment and force development by present in saturating amounts on the thin filament in the dephosphorylated bridges [Somlyo et al., 1988al. cell, this result cannot be accounted for since caldesmon should prevent phosphorylated myosin from binding to actin. The two types of experiments might be reconciled, THIN-FILAMENT-BASED however, if some caldesmon-free thin filaments are REGULATORY MECHANISMS present, as suggested from quantitation of the actin: Does a second regulatory system, in addition to caldesmon ratio in various smooth muscles [Haeberle light chain phosphorylation, modulate and/or regulate and Hathaway, 19901. smooth muscle contractility? Obvious candidates include the thin-filament-associated proteins caldesmon and calponin. Calponin is a calcium, calmodulin-binding SUMMARY protein antigenically related to troponin-T from skeletal It is well established that light chain phosphorylamuscle [Takahashi et al., 19881, which inhibits actomyosin activity in a phosphorylation-dependent manner tion is required before a smooth muscle can generate [Winder and Walsh, 19901. Caldesmon is a 740 A-long, force. The apparent modulation of shortening velocity by 87 kd protein that binds actin, tropomyosin, calmodulin, phosphorylation during sustained contractions may be and myosin. Caldesmon inhibits actin-activated ATPase accounted for by a mechanical interaction between rapactivity, but the mechanism by which it does is still idly cycling phosphorylated crossbridges and slowly controversial. Marston [ 19881 has suggested that caldes- or non-cycling dephosphorylated crossbridges. Latchmon inhibits product release, while Chalovich and col- bridges, force-producing dephosphorylated crossbridges , leagues believe that caldesmon prevents binding of have been proposed to explain why force levels remain M.ADP.Pi to actin [Hemric and Chalovich, 19881. Cal- high at low levels of phosphorylation. The role of the cium-calmodulin reverses the inhibition of activity, al- thin-filament-associated proteins caldesmon and calpothough a different calcium-binding protein may perform nin in regulation remains enigmatic, but their inhibitory properties in solution would be consistent with a possible this role in vivo. Based on the observations that light chain phos- involvement in maintenance of a relaxed state. phorylation has little effect on the binding of heavy meromyosin.ADP.Pi to actin [Greene and Sellers, 19871, and that dephosphorylated myosin binds to actin in the ACKNOWLEDGMENTS presence of MgATP in the motility assay, a possible role I thank Susan Lowey and Dave Warshaw for helpfor caldesmon could be to prevent weak actomyosin interactions in the relaxed state. Phosphorylation/de- ful comments regarding this manuscript, and Dave Warphosphorylation would, however, still control the rate of shaw for providing Figure 2. KT is supported by grants cycling between weak and strong binding states. Alter- from NIH (HL38113) and the American Heart Associanatively, if caldesmon slows the rate of product release, tion (89061 I). LATCHBRIDGES
Regulation of Smooth Muscle Myosin
REFERENCES Cross, R.A., Cross, K.E., and Sobieszek, A. (1986): ATP-linked monomer-polymer equilibrium of smooth muscle myosin: the free folded monomer traps ADP.P,. EMBO J. 5:2637-2641. Dillon, P.F., Aksoy, M.O., Driska, S.P., and Murphy, R.A. (198 1): Myosin phosphorylation and the cross-bridge cycle in arterial smooth muscle. Science 21 1:495-497. Gillis, J.M., Cao, M.L., and Godfraind-DeBecker, A . (1988): Density of myosin filaments in the rat anococcygeus muscle, at rest and in contraction. 11. J. Muscle Res. Cell Motil. 9:18-28. Godfraind-DeBecker, A., and Gillis, J.M. (1988): Analysis of the birefringence of the smooth muscle anococcygeus of the rat, at rest and in contraction. I. J. Muscle Res. Cell Motil. 9:9-17. Greene, L.E., and Sellers, J.R. (1987): Effect of phosphorylation on the binding of smooth muscle heavy meromyosin. ADP to actin. J. Biol. Chem. 262:4177-4181. Haeberle, J.R., and Hathaway, D.R. (1990): Heterogeneous distribution of caldesmon among thin filaments in smooth muscle. Biophys. J. 57:166a. Hai, C., and Murphy, R.A. (1988): Regulation of shortening velocity by cross-bridge phosphorylation in smooth muscle. Am. J. Physiol. 255:C86-C94. Hai, C., and Murphy, R.A. (1989): Ca2+, cross-bridge phosphorylation, and contraction. Annu. Rev. Physiol. 5 1:285-298. Hemric, M.E., and Chalovich, J.M. (1988): Effect of caldesmon on the ATPase activity and the binding of smooth and skeletal myosin subfragments to actin. J. Biol. Chem. 263:1878-1885. Itoh, T., Ikebe, M., Kargacin, G.J., Hartshorne, D.J., Kemp, B.E., and Fay, F.S. (1989): Effects of modulators of myosin light chain kinase activity in single smooth muscle cells. Nature 338:164-167. Kendrick-Jones, J., Smith, R.C., Craig, K., and Citi, S. (1987): Polymerization of vertebrate non-muscle and smooth muscle myosins. J . Mol. Biol. 198:244-252. Marston, S. (1988): Aorta caldesmon inhibits actin-activation of thiophosphorylated heavy meromyosin Mg2' -ATPase activity by slowing the rate of product release. FEBS Lett. 238:147-150. Moody, C., Lehman, W., and Craig R. (1990): Caldesmon and the structure of smooth muscle thin filaments: electron microscopy of isolated thin filaments. J. Muscle Res. Cell Motil. 11:176185.
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Okamoto, Y., Sekine, T., Grammer, J., and Yount, R.G. (1986): The essential light chains constitute part of the active site of smooth muscle myosin. Nature 324:78-80. Pearson, R.B., Wettenhal, R.E.H., Means, A.R., Hartshorne, D.J., and Kemp, B.E. (1988): Autoregulation of enzymes by pseudosubstrate prototypes: myosin light chain kinase. Science 241 : 970-973. Sellers, J.R. (1985): Mechanism of the phosphorylation dependent regulation of smooth muscle heavy meromyosin. J. Biol. Chem. 260: 15815-1 58 19. Sellers, J.R., Spudich, J.A., and Sheetz, M.P. (1985): Light chain phosphorylation regulates the movement of smooth muscle myosin on actin filaments. J. Cell Biol. 101:1897-1902. Somlyo, A.V., Butler, T.M., Bond, M., and Somlyo, A.P. (1981): Myosin filaments have non-phosphorylated light chains in relaxed smooth muscle. Nature 294567-569. Somlyo, A.V., Goldman, Y .E., Fujimori, T., Bond, M., Trentham, D.R., and Somlyo, A.P. (1988a): Crossbridge kinetics, cooperativity, and negatively strained crossbridges in vertebrate smooth muscle. J. Gen. Physiol. 91:165-192. Somlyo, A.P., Walker, J.W., Goldman, Y.E., Trentham, D.R., Kobayashi, S . , Kitazawa, T., and Somlyo, A.V. (1988b): Inositol trisphosphate, calcium, and muscle contraction. Phi10s. Trans. R. SOC.Lond. [Biol.] 320:399-414. Takahashi, K., Hiwada, K., and Kokubu, T. (1988): Vascular smooth muscle calponin. A novel troponin T-like protein. Hypertension 11:620-626. Trybus, K.M. (1989): Filamentous smooth muscle myosin is regulated by phosphorylation. J. Cell Biol. 109:2887-2894. Umemoto, S . , and Sellers, J. (1990): Characterization of in v i m motility assays using smooth muscle and cytoplasmic myosins. J . Biol. Chem. 265:14864-14869. Washaw, D.M., Desrosiers, J.M., Work, S . S . , and Trybus, K.M. (1990): Smooth muscle myosin cross-bridge interactions modulate actin filament sliding velocity in vitro. J. Cell Biol. 111: 453-463. Winder, S.J., and Walsh, M.P. (1990): Smooth muscle calponin. Inhibition of actomyosin MgATPase and regulation by phosphorylation. J. Biol. Chem. 265: 10148-10155.
Views and Reviews Regulation of Smooth Muscle Myosin Kathleen M. Trybus Rosenstiel Research Center, Brandeis University, Waltham, Massachusetts
INTRODUCTION
Phosphorylation of the myosin regulatory light chain is the key event necessary to initiate force development in smooth muscle. The native thin-filament also has the potential to regulate and/or modulate actomyosin interactions, because of the presence of the actin-binding proteins tropomyosin, caldesmon, and calponin. The interplay between the thick and thin-filament-based regulatory systems might explain some of the properties unique to smooth muscle. ACTIVATION OF MYOSIN LIGHT CHAIN KINASE
The pathway leading to myosin phosphorylation begins with an increase in cytoplasmic calcium concentration. Unlike skeletal muscle, calcium release in smooth muscles can be triggered by voltage-independent pathways, such as via the second messenger inositol trisphosphate (IP,), a process known as “pharmomechanical coupling. IP, causes calcium release from the sarcoplasmic reticulum, mediated by an IP, receptor which is believed to be a member of a new class of intracellular ligand-gated ion channels [Somlyo et al., 1988bl. Calcium-calmodulin binds to myosin light chain kinase (MLCK) and displaces the “pseudosubstrate” from the active site, thus activating MLCK to phosphorylate the 20 kd regulatory light chain of myosin. The inhibitory pseudosubstrate sequence found in MLCK is similar to that around Ser 19 of the myosin regulatory light chain, the natural substrate for MLCK [Pearson et al., 19881. ”
ing physiological (pH 7-7.5, 0.1-0.15 M KCl), synthetic dephosphorylated myosin filaments are unstable in the presence of MgATP. The filament depolymerizes to an assembly-incompetent monomer in which the a-helical coiled-coil tail is folded into thirds and the heads are bent toward the rod, as illustrated in Fig. 1 . Dephosphorylated folded monomers are enzymatically inactive and “trap” the products of ATP hydrolysis, which are released at 0.0002-0.0005 sec-’, the lowest turnover rate observed for any myosin to date [Cross et al., 19861. Phosphorylation destabilizes this conformation, possibly by altering a binding site for the tail, and causes the rod to extend and assemble into filaments (Fig. 1). Phosphorylated myosin filaments have an actin activated ATPase activity of 0.3/sec (24”C), approximately 500-fold higher than dephosphorylated folded myosin. From these observations it could not be deduced if myosin conformation or the state of phosphorylation were the primary determinant of activity. Since dephosphorylated myosin filaments are known to exist in relaxed smooth muscle cells [Somlyo et al., 19811, it is important to determine if the activity of this species is inhibited by dephosphorylation alone, or whether an inhibitory thin-filament based regulatory system needs to be invoked. The activity of dephosphorylated myosin in filamentous form could be determined under standard solvent conditions only by the addition of an anti-rod monoclonal antibody which blocked depolymerization in the presence of MgATP. Dephosphorylated myosin filaments had an actin-activated activity of O.O02/sec, only 5-fold higher than the dephosphorylated folded monomer, and at least 100-fold lower than phosphorylated myosin filaments. This result establishes that phosphor-
LIGHT CHAIN PHOSPHORYLATION REGULATES ACTIN-ACTIVATED ATPase ACTIVITY
Light chain phosphorylation has profound effects on two aspects of smooth muscle myosin function in vitro: its ability to assemble into a filament, and its enzymatic activity. Under solvent conditions approximat0 1991 Wiley-Liss, Inc.
Accepted October 23, 1990. Address reprint requests to Kathleen M . Trybus, Rosenstiel Research Center, Brandeis University, Waltham, MA 02254.
82
Trybus O.B/sec ACTIVE
O.OOZ/sec
INACTIVE
DEPHOSPHORYLATED
FILAMENTS
PHOSPHORYLATED FILAMENTS
t
t
TRANSIENT UNFOLDED M0N0ME R
FOLDED MONOMER
INACTIVE 0.0005/sec
Fig 1
ylation is the primary regulator of activity independent of large changes in myosin conformation [Trybus, 19891. Dephosphorylated heavy meromyosin, which does not fold or form filaments, had an actin-activated ATPase activity remarkably similar to that of dephosphorylated myosin filaments [Sellers, 19851. Phosphorylation must therefore directly affect product release from the active site. A “direct” effect, however, refers to changes occurring over a distance equal to half the length of the myosin head. By analogy with other myosins, the regulatory light chain of smooth muscle myosin is localized at the headhod junction, which is at least 100 A distant from the active site. Photoaffinity labeling showed that the 17 kd class of light chains is located near the active site of smooth muscle myosin [Okamoto et al., 19861, and it is possible that this subunit is the link between distant regions on the head. The three levels of enzymatic activity that can be distinguished are summarized in Figure 1: active phosphorylated myosin filaments (0.3/sec), inactive dephosphorylated myosin filaments (O.OOYsec), and enzymatically incompetent soluble folded dephosphorylated monomers (~0.0005/sec).Intramolecular interaction of the myosin tail with the headheck region in the folded monomer inhibits product release to a somewhat greater extent than can be achieved by possible intermolecular interactions within the filament. DOES FOLDED MYOSIN EXIST IN A SMOOTH MUSCLE CELL?
Filaments containing dephosphorylated myosin exist in relaxed smooth muscle cells [Somlyo et al., 19811, but it is less clear if the dephosphorylated folded monomer is formed in vivo. If folded monomers are not present, an unanswered question is why synthetic fila-
ments so readily disassemble in vitro, but native filaments remain assembled in the cellular milieu. One possibility is that a critical concentration of folded monomers does exist, but the myosin concentration in the cell exceeds this value such that dephosphorylated filaments are in equilibrium with this pool [KendrickJones et al., 19871. Alternatively, a yet unidentified protein exists which stabilizes the native filament. Results obtained from optical and electron microscopy experiments suggest that assembly-disassembly does occur to some extent during a contraction-relaxation cycle. Electron micrographs of cross-sections of the smooth anococcygeus muscle fixed during rest or contraction directly showed that myosin filament density increased upon contraction [Gillis et al., 19881. This observation is consistent with the finding that the birefringence of this muscle also increased during contraction, and was reversed during relaxation [GodfraindDeBecker and Gillis, 19881. What would be the advantage of this disassembly? Both dephosphorylated filaments and dephosphorylated monomers are enzymatically “off” and utilize only low amounts of MgATP. The folded monomer is soluble, however, and could diffuse throughout the cell without binding to actin and reassemble in a different location upon phosphorylation. Although this mechanism would appear to be better suited to a non-muscle cell, the architecture of a smooth muscle cell is not well established and similar processes might occur although to a lesser extent. MODULATION OF CYCLING RATES BY PHOSPHORYLATED-DEPHOSPHORYLATED CROSSBRIDGE INTERACTIONS
Results from solution actin-activated ATPases suggested that phosphorylation/dephosphorylation by itself
Regulation of Smooth Muscle Myosin
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I Latchbridge IInternal Load1 Ldsdl
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Fig. 2.
efficiently regulates the activity of filamentous myosin; an in vitro motility assay further demonstrated that light chain phosphorylation actually controls actin movement. The rate at which single actin filaments move over myosin bound to a nitrocellulose substrate can be quantitated in the motility assay. Dephosphorylated myosin filaments did not support motility, but phosphorylated myosin moved actin at approximately 0.2-0.4 p d s e c (20°C, 25 mM KCI) [Umemoto and Sellers, 1990; Warshaw et al., 19901, approximately 10-fold slower than the rate observed with skeletal muscle myosin. Surprisingly, dephosphorylated filaments held the actin in a rigid rigor-like conformation even in the presence of MgATP. Since phosphate release is the kinetic step regulated by light chain phosphorylation, dephosphorylated heads are predominantly in a “weak-binding’’ conformation which rapidly attaches and detaches from actin, with an equilibrium constant of approximately lo4 M-’. The strength of this interaction is, however, enough to prevent actin from diffusing into solution. There are few times when myosin in the cell is either completely phosphorylated or completely dephosphorylated. Most often, the filament is submaximally phosphorylated and consists of mixtures of myosin molecules that have 0, l , or 2 heads phosphorylated. Figure 2 illustrates the response of a smooth muscle to calcium. The initial rise in calcium and light chain phosphorylation is accompanied by rapid shortening (dashed line) and tension development (solid line). As calcium levels decrease, phosphatase activity predominates over kinase
activity, and phosphorylation levels drop. Correlated with the decrease in phosphorylation is a decrease in shortening velocity, while force levels remain high, a situation unique to smooth muscle [Dillon et al., 1981; Hai and Murphy, 19881. The apparent correlation of phosphorylation and shortening velocity during sustained contractions suggested that phosphorylation may be more than a simple “on-off’’ switch. Shortening velocity is independent of crossbridge number, so the declining velocity cannot be accounted for by a decrease in the number of active heads. Solution studies showed that the cycling rate of phosphorylated myosin did not change when it was copolymerized with dephosphorylated myosin, arguing against a model where the intrinsic crossbridge cycling rate is modulated by cooperative interactions within the filament. In the motility assay, however, which is considered to be the in vitro analog of unloaded shortening velocity in a muscle, the presence of dephosphorylated myosin in a copolymer with phosphorylated myosin significantly decreased the rate of actin movement [Sellers et al., 1985; Warshaw et al., 19901. This modulation of velocity was interpreted as the result of mechanical interactions between the two populations of crossbridges: faster cycling phosphorylated crossbridges move actin more slowly because they are working against a load imposed by the slowly or non-cycling dephosphorylated heads. The decrease in shortening velocity in a muscle might also be due to dephosphorylated bridges acting as an internal load.
84
Trybus
it could promote the formation of bridges that detach more slowly from actin, a property associated with the To explain why force levels remain high when postulated latchbridge. There have also been suggestions phosphorylation levels are low, the existence of “latchthat caldesmon could be involved in the latch state bebridges” was proposed [Hai and Murphy, 19891. Latchcause caldesmon has a binding site both for actin and bridges can only be formed by dephosphorylation of at- myosin which could physically prevent crossbridge distached phosphorylated crossbridges. They sustain force sociation. There is, however, no evidence suggesting and dissociate very slowly from actin, thus contributing that caldesmon crosslinks native thin filaments to myosin to the high economy of force maintenance in smooth [Moody et al., 19901, or that crosslinked dephosphorymuscles. Myosin that is dephosphorylated when not atlated bridges can support tension. tached to actin simply results in the relaxed state. The Despite the solution data which suggest that caldesobserved physiological data can be modeled by assuming mon inhibits actin-activated ATPase activity, fiber studthat the latchbridge state exists; another regulatory sysies do not support the idea that both calcium and phostem is not required to fit the experimental data. There has phorylation are required to initiate contraction. If the been no direct demonstration, however, of the existence calcium dependency of phosphorylation is removed eiof a long-lived, force producing, attached dephosphoryther through the use of a calcium-independent MLCK lated crossbridge. An alternative explanation of why fragment [Itoh et al., 19891, or by thiophosphorylating force remains high at low phosphorylation levels is that the light chains [Somlyo et al., 1988a1, calcium is no the presence of a few attached phosphorylated crosslonger required to initiate contraction. If caldesmon is bridges permits attachment and force development by present in saturating amounts on the thin filament in the dephosphorylated bridges [Somlyo et al., 1988al. cell, this result cannot be accounted for since caldesmon should prevent phosphorylated myosin from binding to actin. The two types of experiments might be reconciled, THIN-FILAMENT-BASED however, if some caldesmon-free thin filaments are REGULATORY MECHANISMS present, as suggested from quantitation of the actin: Does a second regulatory system, in addition to caldesmon ratio in various smooth muscles [Haeberle light chain phosphorylation, modulate and/or regulate and Hathaway, 19901. smooth muscle contractility? Obvious candidates include the thin-filament-associated proteins caldesmon and calponin. Calponin is a calcium, calmodulin-binding SUMMARY protein antigenically related to troponin-T from skeletal It is well established that light chain phosphorylamuscle [Takahashi et al., 19881, which inhibits actomyosin activity in a phosphorylation-dependent manner tion is required before a smooth muscle can generate [Winder and Walsh, 19901. Caldesmon is a 740 A-long, force. The apparent modulation of shortening velocity by 87 kd protein that binds actin, tropomyosin, calmodulin, phosphorylation during sustained contractions may be and myosin. Caldesmon inhibits actin-activated ATPase accounted for by a mechanical interaction between rapactivity, but the mechanism by which it does is still idly cycling phosphorylated crossbridges and slowly controversial. Marston [ 19881 has suggested that caldes- or non-cycling dephosphorylated crossbridges. Latchmon inhibits product release, while Chalovich and col- bridges, force-producing dephosphorylated crossbridges , leagues believe that caldesmon prevents binding of have been proposed to explain why force levels remain M.ADP.Pi to actin [Hemric and Chalovich, 19881. Cal- high at low levels of phosphorylation. The role of the cium-calmodulin reverses the inhibition of activity, al- thin-filament-associated proteins caldesmon and calpothough a different calcium-binding protein may perform nin in regulation remains enigmatic, but their inhibitory properties in solution would be consistent with a possible this role in vivo. Based on the observations that light chain phos- involvement in maintenance of a relaxed state. phorylation has little effect on the binding of heavy meromyosin.ADP.Pi to actin [Greene and Sellers, 19871, and that dephosphorylated myosin binds to actin in the ACKNOWLEDGMENTS presence of MgATP in the motility assay, a possible role I thank Susan Lowey and Dave Warshaw for helpfor caldesmon could be to prevent weak actomyosin interactions in the relaxed state. Phosphorylation/de- ful comments regarding this manuscript, and Dave Warphosphorylation would, however, still control the rate of shaw for providing Figure 2. KT is supported by grants cycling between weak and strong binding states. Alter- from NIH (HL38113) and the American Heart Associanatively, if caldesmon slows the rate of product release, tion (89061 I). LATCHBRIDGES
Regulation of Smooth Muscle Myosin
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