HIGHLIGHTED TOPICS
Signal Transduction in Smooth Muscle
Invited Review: Regulation of myosin phosphorylation in smooth muscle

Department of Physiology, University of Cologne, D-50931 Koeln, Germany

	ABSTRACT

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Phosphorylation of the regulatory light chains of myosin II (rMLC) by the Ca²⁺/calmodulin-dependent myosin light-chain kinase (MLCK) and dephosphorylation by a type 1 phosphatase (MLCP), which is targeted to myosin by a regulatory subunit (MYPT1), are the predominant mechanisms of regulation of smooth muscle tone. The activities of both enzymes are modulated by several protein kinases. MLCK is inhibited by the Ca²⁺/calmodulin-dependent protein kinase II, whereas the activity of MLCP is increased by cGMP and perhaps also cAMP-dependent protein kinases. In either case, this results in a decrease in the Ca²⁺ sensitivity of rMLC phosphorylation and force production. The activity of MLCP is inhibited by Rho-associated kinase, one of the effectors of the monomeric GTPase Rho, and protein kinase C, leading to an increase in Ca²⁺ sensitivity. Hence, smooth muscle tone appears to be regulated by a network of activating and inactivating intracellular signaling cascades.

calcium sensitivity of smooth muscle contraction; myosin light-chain kinase; myosin light-chain phosphatase

	INTRODUCTION

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A KEY EVENT IN THE REGULATION of smooth muscle contraction is the phosphorylation/dephosphorylation of the regulatory light chains of myosin II (rMLC) catalyzed, respectively, by the Ca²⁺- and calmodulin-activated myosin light-chain kinase (MLCK) and a type 1 myosin phosphatase (MLCP). Several excellent recent reviews addressed the biochemical properties of these enzymes (22, 31, 82). The extent of rMLC phosphorylation and, hence, of the amplitude of force production depends on the balance of the activities of MLCK and MLCP. However, the relation between force, rMLC phosphorylation, and intracellular Ca²⁺ concentration ([Ca²⁺]_i) is not unique because both MLCK and MLCP are targets for intracellular signaling cascades, which modulate their respective activities independent of changes in [Ca²⁺]_i. Under certain conditions, force is also regulated independent of changes in rMLC phosphorylation levels perhaps by thin filament-associated proteins (4). In any case, smooth muscle tone appears to depend on a network of activating and inhibiting intracellular signals integrating incoming extracellular signals where [Ca²⁺]_i is just one, albeit important, player. The focus of this review is on the regulation of force and Ca²⁺ sensitivity by rMLC phosphorylation.

	REGULATION OF CONTRACTION BY RMLC PHOSPHORYLATION

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Activation of smooth muscle by different agonists or by electrical depolarization typically results in a rapid increase in [Ca²⁺]_i due to influx of extracellular Ca²⁺ through voltage-gated Ca²⁺ channels and release from sarcoplasmic reticulum, which reaches maximal values within a few seconds (for review, see Ref. 78). Ca²⁺ binds to calmodulin and activates MLCK, leading to phosphorylation of rMLC predominantly at Ser-19 (Fig. 1), which can reach maximal levels within 4 s (13, 30). Phosphorylation of rMLC allows the myosin ATPase to be activated by actin and the muscle to contract (see Ref. 29 for review). The onset of significant phosphorylation and tension development in electrically stimulated smooth muscle occurs after a lag period of about 200-500 ms, in which the major delay following Ca²⁺ release is due to prephosphorylating reactions (for review, see Ref. 77). rMLC may also be phosphorylated in a Ca²⁺-independent manner by Rho-associated kinase (ROK) (2) and one or more unidentified staurosporine-sensitive kinases (47, 95). There is no evidence that direct phosphorylation of rMLC by ROK plays a significant role in regulation of smooth muscle contraction (26, 85). The physiological role of the (other) kinase(s) is also not clear at present. Ca²⁺-independent phosphorylation of rMLC of myosin II may be important for regulation of contraction in nonmuscle cells (43).

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Activation scheme of smooth muscle contraction. G, heterotrimeric GTP-binding protein; R, receptor; L, ligand; IP3, inositol 1,4,5-trisphosphate; SR, sarcoplasmic reticulum; CaM, calmodulin; MLCK, myosin light-chain kinase; MLCP, myosin light-chain phosphatase; A, actin; M, unphosphorylated myosin; Mp, phosphorylated myosin; AM and AMp, force-generating cross bridges.

The precise coupling between force and rMLC phosphorylation is quite variable and often nonlinear. Under steady-state conditions, maximal force can be attained at ~0.2-0.3 mol P_i/mol rMLC, leading to a rather steep dependence of force on phosphorylation (9, 35, 74). The relation between force and rMLC phosphorylation may also depend on the agonist used for stimulation (83). In addition, phosphorylation often declines during tension maintenance (see Ref. 29 for review; also see Refs. 13, 85). With the assumption that maximal force reflects the activity of a large portion of cross bridges, nonphosphorylated myosin cross bridges must then contribute to force generation (Fig. 1). In principle, force-generating, dephosphorylated cross bridges could be generated by dephosphorylating attached cross bridges, which are thought to have a slow detachment rate compared with phosphorylated cross bridges (61) or by the cooperative attachment of dephosphorylated cross bridges (3, 37, 80, 93; reviewed in Ref. 4). Cooperative attachment is possibly regulated by calponin and caldesmon (1, 55).

Under most conditions, dephosphorylation of rMLC precedes relaxation of force (10, 21). However, under certain conditions, smooth muscle relaxed at high levels of rMLC phosphorylation (5, 34, 56, 87); however, the mechanism is not clear at present. An interesting hypothesis suggests that actomyosin interaction of phosphorylated myosin may be inhibited by the small heat shock protein, HSP20 (69).

	REGULATION OF CA2+ SENSITIVITY OF RMLC PHOSPHORYLATION AND CONTRACTION

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In addition to their effects on [Ca²⁺]_i, contractile agonists increase Ca²⁺ sensitivity of contraction. On the other hand, relaxation mediated by an increase in intracellular cAMP or cGMP is often associated with a decrease in Ca²⁺ sensitivity. There are in principle two ways to modulate Ca²⁺ sensitivity of contraction (Fig. 2): 1) by altering the balance between the activities of rMLC kinase(s) and MLCP at a constant [Ca²⁺]_i, which would not affect the relation between force and phosphorylation, and 2) by rMLC phosphorylation-independent regulatory mechanisms such as caldesmon (91) or calponin (55) and perhaps also heat shock proteins (Ref. 69; Fig. 2). In this case, the coupling between force and phosphorylation will be altered. Much more is known about modulation of Ca²⁺ sensitivity of rMLC phosphorylation, and this review will focus on this mechanism.

View larger version (29K): [in this window] [in a new window]   Fig. 2.   A: signaling cascades that modulate Ca2+ sensitivity of phosphorylation of regulatory light chains of myosin (rMLC) and contraction. B: increasing the ratio of active MLCK to active MLCP will lead to an increase in Ca2+ sensitivity of rMLC phosphorylation (a) and contraction (b). A decrease in this ratio will have the opposite effect. Modulating the MLCK-to-MLCP ratio will not affect the dependence of force on rMLC phosphorylation (c, solid line). Ca2+ sensitivity can be decreased independent of rMLC phosphorylation, which will affect the dependence of force on rMLC phosphorylation (c; dashed line). MLCP consists of a regulatory subunit (MYPT), a catalytic subunit (PP1c), and a 20-kDa subunit (M20). MLCK is phosphorylated by the Ca2+- and calmodulin-dependent protein kinase II (CaMKII); MLCP can be inhibited by phosphorylated CPI-17 and phosphorylation of MYPT by Rho kinase and arachidonic acid. Arachidonic acid inhibits MLCP activity by dissociation of the holoenzmye, as well as activation of protein kinase C (PKC) and Rho kinase. Rho kinase is the downstream effector of RhoA, which is activated by guanosine exchange factors (GEF) and inactivated by GTPase-activating proteins (GAP). RhoA is also inactivated by protein kinase G (PKG). Relaxation induced by cAMP and cGMP may in addition be mediated by PKG- and protein kinase A (PKA)-dependent phosphorylation of heat shock protein 20 (HSP20). This possibly leads to inhibition of actomyosin interaction and contraction.

Inhibition of MLCP as a Major Mechanism of Ca²+ Sensitization

A key event in agonist-induced Ca²⁺ sensitization is the G protein-dependent inhibition of MLCP (41, 46). MLCP consists of three subunits: a 110- to 130-kDa regulatory subunit (MYPT1), an ~37-kDa catalytic subunit (PP-1C), and a 20-kDa subunit of unknown function (22). Several mechanisms have been identified that inhibit MLCP (reviewed in Ref. 22): 1) phosphorylation of the MYPT1; 2) inhibition by an endogenous, smooth muscle-specific phosphopeptide, CPI-17; and 3) dissociation of the holoenzyme by arachidonic acid (Fig. 2).

Rho/Rho kinase pathway (Fig. 2). Phosphorylation of MYPT1 by ROK leads to inhibition of MLCP (12, 39). Other substrates of ROK include CPI-17 and calponin (32, 44). ROK is activated by the Ras-related monomeric GTPase, Rho (55), which, in permeabilized smooth muscle, increased force at constant Ca²⁺ concentration (19, 23) and enhanced carbachol-induced sensitization (64). Agonist-induced Ca²⁺ sensitization was inhibited by bacterial exoenzymes, which inactivate Rho by ADP ribosylation (exoenzyme C3, EDIN) or monoglucosylation (toxin B; reviewed in Refs. 65, 79). In the intact smooth muscle, the tonic phase of the contraction but not the initial phasic response was inhibited by membrane-permeant forms of these compounds (15, 64) as well as by a synthetic inhibitor of ROK, Y27632 (14, 85, 90, 99). The effects of these agents on contraction were associated with the respective changes in rMLC phosphorylation (19, 51, 85), possibly due to inhibition of MLCP (85). The precise mechanism of G-protein-coupled receptor activation that leads to activation of RhoA in smooth muscle is not yet known. There is some evidence that heterotrimeric G proteins belonging to G_q, G_12/13, and G_1-2 may be located upstream of RhoA (reviewed in Ref. 71). The activity of Rho itself is regulated by proteins that regulate the exchange of GDP for GTP and the GTPase activity of Rho (Fig. 2; see Ref. 71 for review).

There are, however, concerns as to the access of ROK to its substrate MYPT1 because both Rho and ROK have to be recruited from a cytosolic pool to the cell membrane for activation (15, 86). Perhaps, a kinase that copurifies with the holoenzyme of MLCP (25), and related to Zipper-interacting protein (ZIP) kinase, could be a link between activated ROK and MYPT1 (52). This kinase phosphorylates MYPT1 (25, 52) and may be responsible for thiophosphorylation-induced Ca²⁺ sensitization (67, 89).

Protein kinase C. Phorbolester-induced contractions (reviewed in Refs. 24, 59, 94) were associated with inhibition of MLCP (27, 54). This may be due to protein kinase C (PKC)-dependent phosphorylation of CPI-17, a smooth muscle-specific protein inhibitor of MLCP, by PKC (11, 42). The importance of conventional or novel PKCs for regulation of contraction was, however, questioned (16, 26). Interestingly, agonist-induced sensitization was inhibited by peptide inhibitors of atypical PKCs (16). Atypical PKCs are activated by arachidonic acid, which increases Ca²⁺ sensitivity of force production in permeabilized smooth muscle (18) and is released on stimulation of smooth muscle (20). The arachidonic acid-induced increase in Ca²⁺ sensitivity could be effected not only by activation of PKC and perhaps phosphorylation of CPI-17 but also by dissociation of the MLCP holoenzyme (18) and activation of ROK (12). In intact smooth muscle, agonist-induced phosphorylation of CPI-17 is inhibited by PKC inhibitors but interestingly also by the relatively specific ROK inhibitor Y27632 (40). Thus PKC and Rho/ROK pathways may converge on CPI-17 (40, 50).

Signals That Lead to a Decrease in Ca²+ Sensitivity

Cyclic nucleotide-dependent protein kinases (protein kinase A, protein kinase G). It is well established that cAMP and cGMP decrease Ca²⁺ sensitivity of contraction in both the intact and permeabilized smooth muscle (33, 36, 58, 66, 75, 92). In vitro, protein kinase A phosphorylates MLCK at two sites, site A and B, located COOH terminal to the calmodulin binding domain (8, 96). Phosphorylation of site A but not site B decreased the affinity of MLCK for the Ca²⁺/calmodulin complex (8), and it was thought that this is responsible for desensitization. However, agents that elevate cAMP in vivo have negligible effects both on the phosphorylation of site A and on Ca²⁺ activation of MLCK (57, 81). This suggests that cAMP desensitizes smooth muscle by an alternative, not yet defined mechanism. Phosphorylation of MLCK by protein kinase G, which occurred only when calmodulin was not bound to MLCK, had no effect on the activity of MLCK (63). Therefore, it was proposed (66) and later experimentally confirmed that MLCP was activated by cGMP (49) either directly (84) or perhaps indirectly by phosphorylation of telokin (98) or inhibition of RhoA (73). Thus Rho could integrate activating and relaxing pathways. It will be interesting to see whether protein kinase A also inactivates Rho in smooth muscle as it does in nonmuscle cells (48). Relaxation induced by nitrovasodilators also occurs independent of rMLC dephosphorylation and could be mediated by phosphorylation of HSP20 (6, 69).

Ca²+/calmodulin-dependent protein kinase II. Site A of MLCK is, however, significantly phosphorylated in the presence of agents that increase [Ca²⁺]_i (81), which was abolished by inhibitors of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) (88). Phosphorylation of MLCK by CaMKII was associated with a decrease in Ca²⁺ sensitivity of rMLC phosphorylation (88). It was suggested that this acts as a negative feedback mechanism to inhibit high levels of rMLC phosphorylation (97). More recently, a stimulatory role of CaMKII activation on contraction has been proposed (38, 70).

	FUTURE RESEARCH

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In addition to changes in [Ca²⁺]_i, several signal transduction pathways have been identified that regulate phosphorylation of rMLC and contraction. Most likely, more pathways will be identified in the near future; for example, for nonmuscle cells, it is known that MLCK activity is upregulated by mitogen-activated protein kinase (60, 62) and downregulated by p21-activated kinase (17, 72). Little is known in a quantitative way about the relative importance of the individual signaling cascades (26, 45). It is also obvious that these signaling cascades interact, creating a signaling network that may have emergent properties such as memory (7, 68). It will be important to know how these signaling cascades are altered in different disease states such as coronary artery spasms (75) and hypertension (28, 90).

	ACKNOWLEDGEMENTS

The art work of D. Metzler is gratefully acknowledged.

	FOOTNOTES

This work was supported by the Deutsche Forschungsgemeinschaft.

Address for reprint requests and other correspondence: G. Pfitzer, Dept. of Physiology, Univ. of Cologne, Robert-Koch-Str. 39, D-50931 Koeln, Germany (E-mail: gabriele.pfitzer{at}uni-koeln.de).

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