Myosin Regulatory Light Chain Diphosphorylation Slows Relaxation of Arterial Smooth Muscle

Background: The regulatory light chains of smooth muscle myosin are phosphorylated at Ser¹⁹ and Thr¹⁸.

Results: Phosphorylation at Thr¹⁸ does not increase force elicited by Ser¹⁹ phosphorylation, but reduces the rate of relaxation.

Conclusion: Diphosphorylation slows relaxation compared with monophosphorylation at Ser¹⁹.

Significance: Knowledge of the functional effects of myosin diphosphorylation is important for understanding the underlying causes of hypercontractility.

Abstract

The principal signal to activate smooth muscle contraction is phosphorylation of the regulatory light chains of myosin (LC₂₀) at Ser¹⁹ by Ca²⁺/calmodulin-dependent myosin light chain kinase. Inhibition of myosin light chain phosphatase leads to Ca²⁺-independent phosphorylation at both Ser¹⁹ and Thr¹⁸ by integrin-linked kinase and/or zipper-interacting protein kinase. The functional effects of phosphorylation at Thr¹⁸ on steady-state isometric force and relaxation rate were investigated in Triton-skinned rat caudal arterial smooth muscle strips. Sequential phosphorylation at Ser¹⁹ and Thr¹⁸ was achieved by treatment with adenosine 5′-O-(3-thiotriphosphate) in the presence of Ca²⁺, which induced stoichiometric thiophosphorylation at Ser¹⁹, followed by microcystin (phosphatase inhibitor) in the absence of Ca²⁺, which induced phosphorylation at Thr¹⁸. Phosphorylation at Thr¹⁸ had no effect on steady-state force induced by Ser¹⁹ thiophosphorylation. However, phosphorylation of Ser¹⁹ or both Ser¹⁹ and Thr¹⁸ to comparable stoichiometries (0.5 mol of P_i/mol of LC₂₀) and similar levels of isometric force revealed differences in the rates of dephosphorylation and relaxation following removal of the stimulus: t_½ values for dephosphorylation were 83.3 and 560 s, and for relaxation were 560 and 1293 s, for monophosphorylated (Ser¹⁹) and diphosphorylated LC₂₀, respectively. We conclude that phosphorylation at Thr¹⁸ decreases the rates of LC₂₀ dephosphorylation and smooth muscle relaxation compared with LC₂₀ phosphorylated exclusively at Ser¹⁹. These effects of LC₂₀ diphosphorylation, combined with increased Ser¹⁹ phosphorylation (Ca²⁺-independent), may underlie the hypercontractility that is observed in response to certain physiological contractile stimuli, and under pathological conditions such as cerebral and coronary arterial vasospasm, intimal hyperplasia, and hypertension.

Introduction

Smooth muscle contraction is activated by an increase in cytosolic free Ca²⁺ concentration ([Ca²⁺]_i), whereupon Ca²⁺ saturates the four Ca²⁺-binding sites of calmodulin (1). (Ca²⁺)₄-calmodulin activates myosin light chain kinase (MLCK),² which catalyzes phosphorylation of the motor protein myosin II at Ser¹⁹ of its two 20-kDa regulatory light chain subunits (LC₂₀) (2). This simple phosphorylation reaction markedly increases the actin-activated MgATPase activity of myosin, which provides the energy for cross-bridge cycling and the development of force or shortening of the muscle (3). MLCK is also capable of phosphorylating LC₂₀ at Thr¹⁸ in vitro, but this requires very high (unphysiological) concentrations of the kinase (4, 5). Relaxation follows the removal of Ca²⁺ from the cytosol, which inactivates MLCK, and myosin is dephosphorylated by myosin light chain phosphatase (MLCP), a type 1 Ser/Thr phosphatase (6).

We and others have demonstrated that smooth muscle contraction can be elicited in the absence of Ca²⁺ by treatment with inhibitors of type 1 protein phosphatases (7–19). For example, treatment of Triton-skinned rat caudal arterial smooth muscle strips with the membrane-impermeant phosphatase inhibitor microcystin in the absence of Ca²⁺ (presence of EGTA) elicited a slow, sustained contractile response that correlated with LC₂₀ phosphorylation (16). Further investigation revealed that this Ca²⁺-independent phosphorylation occurred at both Ser¹⁹ and Thr¹⁸, referred to as diphosphorylation (16). The kinase responsible was shown not to be MLCK on the basis of the following observations: (i) purified MLCK is inactive in the absence of Ca²⁺ (20–22); (ii) LC₂₀ diphosphorylation requires unphysiologically high MLCK concentrations (5); (iii) MLCK inhibitors have no effect on Ca²⁺-independent, microcystin-induced LC₂₀ diphosphorylation and contraction of Triton-skinned tissue (16, 19); (iv) removal of endogenous calmodulin by treatment of Triton-skinned smooth muscle strips with the calmodulin antagonist trifluoperazine in the presence of Ca²⁺ does not affect Ca²⁺-independent, microcystin-induced LC₂₀ diphosphorylation and contraction (23); (v) endogenous LC₂₀ in smooth muscle myofilaments is phosphorylated in the absence of Ca²⁺ at Ser¹⁹ or Thr¹⁸ alone, as well as at both sites (16), whereas purified MLCK (at high concentration) only phosphorylates Thr¹⁸ after Ser¹⁹ has been phosphorylated (4); (vi) stimuli that induce maximal activation of MLCK in smooth muscle tissues (e.g. membrane depolarization of intact vascular smooth muscle strips with an optimal KCl concentration, or addition of a maximal concentration of Ca²⁺ to permeabilized strips) induce LC₂₀ phosphorylation exclusively at Ser¹⁹ (23, 24); (vii) Ca²⁺-independent LC₂₀ kinase activity can be separated from MLCK chromatographically (16); and (viii) the Ca²⁺-independent LC₂₀ kinase, unlike MLCK, does not use ATPγS as a substrate (this study). We purified this Ca²⁺-independent LC₂₀ kinase activity from chicken gizzard myofilaments and identified it as integrin-linked kinase (ILK) (17). Bacterially expressed ILK phosphorylated LC₂₀ in intact myosin in a Ca²⁺-independent manner (17). Approximately 50% of cellular ILK was retained in Triton-skinned smooth muscle and may be associated with MLCP because purified phosphatase preparations contain co-purifying ILK (19). It should be noted that ILK has often been described as a pseudokinase (25), but the evidence for its bona fide kinase activity is substantial (26, 27). Zipper-interacting protein kinase (ZIPK) has also been implicated in the diphosphorylation of LC₂₀ (18, 28), although inhibition of ZIPK activity in Triton-skinned rat caudal arterial smooth muscle did not affect microcystin-induced LC₂₀ diphosphorylation or contraction (19), suggesting that ILK is likely the responsible kinase in these conditions.

The diphosphorylation site in LC₂₀ is highly evolutionarily conserved: the sequence around Thr¹⁸–Ser¹⁹ (Arg-Ala-Thr-Ser-Asn-Val-Phe-Ala-Met-Phe; residues 16–25), is identical throughout the animal kingdom and is also found in a homolog of LC₂₀ (29) in the genome of the unicellular choanoflagellate Monosiga brevicollis (30); choanoflagellates appear to be the closest living relatives of metazoans (30, 31). LC₂₀ isoforms are also found in non-muscle myosin II, and contain phosphorylation sites corresponding to Thr¹⁸ and Ser¹⁹ of smooth muscle LC₂₀ that play an important role in regulation of motility (32).

The functional effects of phosphorylation of LC₂₀ at Ser¹⁹ and Thr¹⁸ have been investigated in vitro using purified LC₂₀ or intact myosin as substrates at high concentrations of MLCK. Ikebe and Hartshorne (4) showed that the actin-activated MgATPase activity of diphosphorylated myosin was 2–3-fold greater than that of myosin phosphorylated exclusively at Ser¹⁹. This increase in actomyosin MgATPase activity can be attributed to a doubling of the V_max when both sites are phosphorylated (33–35). In the in vitro motility assay, however, myosin phosphorylated at both Ser¹⁹ and Thr¹⁸ moved actin filaments at a rate similar to myosin phosphorylated at Ser¹⁹ alone (35, 36).

LC₂₀ diphosphorylation has been observed in various smooth muscle tissues treated with a variety of contractile stimuli (37–41), and several instances of diphosphorylation of LC₂₀ have been reported in pathological conditions associated with hypercontractility (42–46). This prompted us to further investigate the functional effects of LC₂₀ diphosphorylation in vascular smooth muscle.

EXPERIMENTAL PROCEDURES

Materials

All chemicals were analytical grade unless otherwise indicated and purchased from EMD Chemicals (Gibbstown, NJ). Triton X-100 and ATPγS were purchased from Sigma, microcystin-LR from Alexis Biochemicals (San Diego, CA), calyculin-A and okadaic acid from Calbiochem, and dithiothreitol (DTT) from ICN Biochemicals (Aurora, OH). Calmodulin (47) and MLCK (48) were purified from chicken gizzard as previously described. Antibodies to LC₂₀ (polyclonal anti-pan LC₂₀) were from Santa Cruz Biotechnology (Santa Cruz, CA) and used at 1:500 dilution; phosphospecific antibodies to LC₂₀ phosphorylated at Ser¹⁹ (monoclonal anti-pS19-LC₂₀) were from Cell Signaling (Danvers, MA) and used at 1:1,000 dilution; phosphospecific antibodies to LC₂₀ phosphorylated at Thr¹⁸ (polyclonal anti-pT18-LC₂₀) were from 21^st Century Biochemicals (Marlboro, MA) and used at 1:2,000 dilution; phosphospecific antibodies to LC₂₀ phosphorylated at both Thr¹⁸ and Ser¹⁹ (polyclonal anti-pT18,pS19-LC₂₀) were from Cell Signaling and used at 1:500 dilution. Polyclonal phosphospecific antibodies to MYPT1 phosphorylated at Thr⁶⁹⁷ or Thr⁸⁵⁵ were purchased from Upstate USA (Charlottesville, VA) and used at 1:1,000 dilution. Polyclonal anti-actin was from Cytoskeleton Inc. (Denver, CO) and used at 1:1,000 dilution. Secondary antibodies coupled to horseradish peroxidase were purchased from Chemicon (Temecula, CA).

Buffer Compositions

HEPES-Tyrode (H-T) buffer contained 137 mm NaCl, 2.7 mm KCl, 1 mm MgCl₂, 1.8 mm CaCl₂, 5.6 mm glucose, 10 mm HEPES, pH 7.4. Ca²⁺-free H-T buffer contained 140.6 mm NaCl, 2.7 mm KCl, 1 mm MgCl₂, 5.6 mm glucose, 10 mm HEPES, pH 7.4. Buffer A contained 30 mm TES, 0.5 mm DTT, 50 mm KCl, 5 mm K₂EGTA, 150 mm sucrose, pH 7.4. pCa 9 solution contained 4 mm K₂EGTA, 5.83 mm MgCl₂, 0.5 mm dithioerythritol, 20 mm TES, pH 6.9, and an ATP regenerating system composed of 3.9 mm Na₂ATP, 7.56 mm potassium propionate, 16.2 mm phosphocreatine, and 30 units/ml of creatine kinase. The free [Ca²⁺] of this pCa 9 solution was determined to be 6 nm using fura-2. pCa 4.5 solution contained 4 mm CaEGTA, 5.66 mm MgCl₂, 0.5 mm dithioerythritol, 20 mm TES, pH 6.9, and the ATP regenerating system.

Tissue Preparation and Force Measurements

Caudal arteries were removed from male Sprague-Dawley rats (300–350 g) that had been anesthetized with halothane and euthanized according to protocols consistent with the standards of the Canadian Council on Animal Care and approved by the University of Calgary Animal Care and Use Committee. The arteries were cleaned of excess adventitia and adipose tissue in Ca²⁺-free H-T buffer. Segments were placed over a 0.31-mm needle and moved back and forth 40 times to remove the endothelium, cut into helical strips (1.5 × 6 mm), mounted on a Grass isometric force transducer (model FT03C) connected to a PowerLab (ADInstruments) 8-channel recording device with a resting tension of 0.45 g and incubated for 20 min in H-T buffer (bath volume = 0.8 ml). Tissues were stimulated at least twice with H-T buffer containing 87 mm KCl (the increase in [KCl] was balanced by a decrease in [NaCl]) with a 20-min interval of relaxation in Ca²⁺-free H-T buffer. Muscle strips were then incubated in Ca²⁺-free H-T buffer and either used for experiments with intact tissue or were skinned (demembranated) as follows. Tissues for skinning were incubated for 5 min in Buffer A and subsequently demembranated by incubation for 2 h in Buffer A containing 1% (v/v) Triton X-100. Skinned tissues were then washed 3 times (5 min each) in pCa 9 solution prior to treatments described in the figure legends.

Quantification of LC₂₀ Phosphorylation Levels

At selected times during experimental protocols, tissues were immersed in cold 10% trichloroacetic acid, acetone, 10 mm DTT, washed three times (1 min each) with acetone/DTT, and lyophilized for 36 h. Dried tissues were immersed in 1 ml of SDS gel sample buffer (2% (w/v) SDS, 100 mm DTT, 10% (v/v) glycerol, 0.01% bromphenol blue, 60 mm Tris-HCl, pH 6.8), heated to 95 °C for 2 min, cooled to room temperature, and rotated overnight at 4 °C. Samples (40 μl) were subjected to phosphate affinity SDS-PAGE using an acrylamide-pendant phosphate-binding tag (Phos-tag SDS-PAGE with 12.5% acrylamide) at 30 mA/gel for 70 min in mini-gels in which 0.05 mm Phos-tag acrylamide (NARD Institute, Japan) and 0.1 mm MnCl₂ were incorporated into the running gel (49). Separated proteins were transferred to PVDF membranes (Roche Applied Science) overnight at 27 volts and 4 °C in 25 mm Tris-HCl, pH 7.5, 192 mm glycine, 10% (v/v) methanol. Proteins were fixed on the membrane by treatment with 0.5% glutaraldehyde in phosphate-buffered saline (137 mm NaCl, 2.68 mm KCl, 10 mm Na₂HPO₄, 1.76 mm KH₂PO₄) for 45 min. Membranes were then incubated with 5% nonfat dried milk in Tris-buffered saline containing Tween (TBST: 20 mm Tris-HCl, pH 7.5, 137 mm NaCl, 3 mm KCl, 0.05% Tween 20) for 1–2 h, followed by primary antibody in TBST overnight at 4 °C. Following washout of the primary antibody, membranes were incubated with secondary antibody (anti-rabbit or anti-mouse IgG-horseradish peroxidase conjugate in TBST at 1:10,000 dilution) for 2 h at room temperature, washed with TBST (4 × 5 min), and then with TBS (1 × 5 min) before chemiluminescence signal detection using the Super-Signal West Femto reagent (Thermo Scientific, Rockford, IL). The emitted light was detected and quantified with a chemiluminescence imaging analyzer (LAS3000mini; Fujifilm) and images were analyzed with MultiGauge version 3.0 software.

Data Analysis

Values are presented as the mean ± S.E., with n indicating the number of animals used; several muscle strips were used from each animal. Statistical analyses were performed with SigmaPlot and data were analyzed by Student's t test, with p < 0.05 considered to indicate statistically significant differences.

RESULTS

Ca²⁺-independent, Microcystin-induced LC₂₀ Diphosphorylation and Contraction

Fig. 1A show the time course of Ca²⁺-independent contraction of Triton-skinned rat caudal arterial smooth muscle strips in response to the phosphatase inhibitor microcystin (t_½ = 451.1 ± 13.4 s (n = 8)). Tissues were immersed in TCA/acetone/DTT at the indicated times during the contractile response, washed with acetone, lyophilized, and tissue proteins were extracted in SDS gel sample buffer. Phosphorylated and unphosphorylated forms of LC₂₀ were separated by Phos-tag SDS-PAGE (49) and detected by Western blotting with anti-pan LC₂₀, which recognizes all forms of the protein (Fig. 1B, panel a). The three separated bands were identified by Western blotting with phosphospecific antibodies to LC₂₀ (Fig. 1B, panels b-d). In resting tissue in the absence of Ca²⁺ (lane 1), only unphosphorylated LC₂₀ was detected. Treatment with microcystin in the absence of Ca²⁺ induced a time-dependent increase in mono- and diphosphorylated LC₂₀. The monophosphorylated band contained a mixture of LC₂₀ phosphorylated exclusively at Ser¹⁹ (Fig. 1B, panel b) and LC₂₀ phosphorylated exclusively at Thr¹⁸ (Fig. 1B, panel c). The antibody to Thr(p)¹⁸-LC₂₀ also recognized diphosphorylated LC₂₀ (Fig. 1B, panel c), identified as containing both Thr(P)¹⁸ and Ser(P)¹⁹ in Fig. 1B, panel d. The cumulative quantitative data in Fig. 1C show the time-dependent increase in mono- and diphosphorylation, and the corresponding decrease in unphosphorylated LC₂₀ in response to microcystin in the absence of Ca²⁺.

FIGURE 1.

Ca²⁺-independent contraction and LC₂₀ diphosphorylation in Triton-skinned rat caudal arterial smooth muscle in response to microcystin at pCa 9. A, Triton-skinned rat caudal arterial smooth muscle strips mounted on a force transducer in pCa 9 solution were treated with microcystin (1 μm). A typical contractile response is shown. Separate tissues were harvested at the indicated times during the contraction for analysis of LC₂₀ phosphorylation by Phos-tag SDS-PAGE and Western blotting (B) with antibodies to LC₂₀ (panel a), Ser(P)¹⁹-LC₂₀ (panel b), Thr(P)¹⁸-LC₂₀ (panel c), and Thr(P)¹⁸,Ser(P)¹⁹-LC₂₀ (panel d). Numbers below the gel lanes correspond to the time points in A. C, cumulative quantitative data showing the proportions of unphosphorylated (0P, closed circles), mono- (1P, open circles), and diphosphorylated LC₂₀ (2P, closed inverted triangles) as a function of time. Values represent the mean ± S.E. (n = 3).

Ca²⁺-independent, Calyculin-A-induced LC₂₀ Diphosphorylation and Contraction

Treatment of intact rat caudal arterial smooth muscle with the membrane-permeant phosphatase inhibitor calyculin-A in Ca²⁺-free solution also induced LC₂₀ mono- and diphosphorylation, which correlated with force development with a t_½ of 1326 ± 96 s (n = 6) (Fig. 2). In this case, the amount of monophosphorylated LC₂₀ detected was significantly less (Fig. 2C) than was observed in the Triton-skinned tissue in response to microcystin (Fig. 1C). It is also noteworthy that the steady-state force achieved in response to calyculin-A in the absence of Ca²⁺ appeared to be significantly higher than the force induced by a strong depolarizing stimulus (87 mm KCl) (Fig. 2A). This prompted us to address the question: does LC₂₀ diphosphorylation elicit more steady-state isometric force than monophosphorylation?

FIGURE 2.

Ca²⁺-independent contraction and LC₂₀ diphosphorylation in intact rat caudal arterial smooth muscle in response to calyculin-A in Ca²⁺-free solution. A, intact rat caudal arterial smooth muscle strips mounted on a force transducer in Ca²⁺-containing H-T buffer were induced to contract 3 times with 87 mm KCl. The tissue was then washed extensively with Ca²⁺-free H-T buffer (which emptied intracellular Ca²⁺ stores because the contractile response to caffeine was abolished; data not shown) prior to treatment with calyculin-A (0.5 μm) in Ca²⁺-free solution. Separate tissues were harvested at the indicated times during the contraction for analysis of LC₂₀ phosphorylation by Phos-tag SDS-PAGE and Western blotting (B) with antibodies to LC₂₀ (panel a), Ser(P)¹⁹-LC₂₀ (panel b), Thr(P)¹⁸-LC₂₀ (panel c), and Thr(P)¹⁸,Ser(P)¹⁹-LC₂₀ (panel d). Numbers below the gel lanes correspond to the time points in A. C, cumulative quantitative data showing the proportions of unphosphorylated (0P, closed circles), mono- (1P, open circles), and diphosphorylated LC₂₀ (2P, closed inverted triangles) as a function of time. Values represent the mean ± S.E. (n = 3).

KCl-induced LC₂₀ Monophosphorylation and Contraction

We first demonstrated that an increase in cytosolic free Ca²⁺ concentration induced exclusively monophosphorylation of LC₂₀ at Ser¹⁹. Ca²⁺ entry via voltage-gated Ca²⁺ channels was activated by KCl-induced membrane depolarization of intact rat caudal arterial smooth muscle strips, which induced a rapid contractile response (t_½ = 10.2 ± 0.2 s (n = 29)) (Fig. 3A). Analysis of the LC₂₀ phosphorylation time course revealed phosphorylation at Ser¹⁹ (Fig. 3B, panel b) with no phosphorylation at Thr¹⁸ (Fig. 3B, panel c) or diphosphorylation at Thr¹⁸ and Ser¹⁹ (Fig. 3B, panels a and d). LC₂₀ phosphorylation stoichiometry peaked at ∼0.6 mol of P_i/mol of LC₂₀ (Fig. 3C).

FIGURE 3.

Contraction and LC₂₀ phosphorylation in intact rat caudal arterial smooth muscle in response to KCl-induced depolarization in the presence of Ca²⁺. A, intact rat caudal arterial smooth muscle strips were treated with 87 mm KCl in Ca²⁺-containing H-T buffer and the contractile response was recorded. Separate tissues were harvested at the indicated times during the contraction for analysis of LC₂₀ phosphorylation by Phos-tag SDS-PAGE and Western blotting with antibodies to LC₂₀ (panel a), Ser(P)¹⁹-LC₂₀ (panel b), Thr(P)¹⁸-LC₂₀ (panel c), and Thr(P)¹⁸,Ser(P)¹⁹-LC₂₀ (panel d). Numbers below the gel lanes correspond to the time points in A. MC denotes control tissue (Triton-skinned rat caudal arterial smooth muscle treated with microcystin at pCa 9 for 60 min) to identify unphosphorylated, mono-, and diphosphorylated LC₂₀ bands. C, cumulative quantitative data showing the time course of LC₂₀ phosphorylation stoichiometry; as shown in panel b, phosphorylation occurred exclusively at Ser¹⁹ under these conditions. Values represent the mean ± S.E. (n = 3).

Effects on Force and LC₂₀ Phosphorylation of Sequential Treatment with Ca²⁺ and Microcystin

Similarly, addition of Ca²⁺ to Triton-skinned rat caudal arterial smooth muscle induced phosphorylation of LC₂₀ exclusively at Ser¹⁹ (Fig. 4G, lanes A in panels a-d) with a t_½ of 151.7 ± 4.8 s (n = 23) and an LC₂₀ phosphorylation level of ∼0.5 mol of P_i/mol of LC₂₀ (Table 1). Addition of microcystin at the plateau of a Ca²⁺-induced contraction resulted in a further increase in force of ∼25% (Fig. 4B and Table 2), which correlated with LC₂₀ diphosphorylation (Fig. 4G, lanes B in panels a–d, and Table 1). If microcystin and Ca²⁺ were added together, a rapid contraction occurred (t_½ of 65.3 ± 2.3 s (n = 15) compared with 151.7 ± 4.8 s (n = 23) for Ca²⁺ alone and 451.1 ± 13.4 s (n = 8) for microcystin at pCa 9), which was again accompanied by LC₂₀ diphosphorylation (Fig. 4G, lanes C in panels a–d, and Table 1). No force development or LC₂₀ phosphorylation was observed in the absence of Ca²⁺ and phosphatase inhibitor (Fig. 4, D and G, lanes D in panels a–d, and Table 1). If contraction was evoked by addition of microcystin in the absence of Ca²⁺, subsequent addition of Ca²⁺ elicited further force development (∼20%; Fig. 4F and Table 2) and LC₂₀ diphosphorylation (Fig. 4G, lanes F in panels a–d, and Table 1) compared with control (Fig. 4, E and G, lanes E, and Tables 1 and 2). A more detailed analysis of the (Ca²⁺ + microcystin)-induced contraction revealed rapid phosphorylation of LC₂₀ at Ser¹⁹ that can be attributed to MLCK activation by Ca²⁺, and a slower rate of phosphorylation at Thr¹⁸, due to ILK activity that is unmasked by the phosphatase inhibitor (Fig. 5).

FIGURE 4.

Effect of microcystin on Ca²⁺-induced contraction and Ca²⁺ on microcystin-induced contraction of Triton-skinned rat caudal arterial smooth muscle. A–F, Triton-skinned rat caudal arterial smooth muscle strips mounted on a force transducer in pCa 9 solution were treated as indicated. G, LC₂₀ phosphorylation at the end of the protocols shown in A–F was analyzed by Phos-tag SDS-PAGE and Western blotting with antibodies to LC₂₀ (panel a), Ser(P)¹⁹-LC₂₀ (panel b), Thr(P)¹⁸-LC₂₀ (panel c), and Thr(P)¹⁸,Ser(P)¹⁹-LC₂₀ (panel d). Letters below the gel lanes correspond to panels A–F. Results are representative of at least 3 independent experiments.

TABLE 1.

Quantification of LC₂₀ mono- and diphosphorylation in Triton-skinned rat caudal arterial smooth muscle

LC₂₀ phosphorylation levels were quantified by Phos-tag SDS-PAGE (see Fig. 4G, panel a) in tissues treated as described in the legend to Fig. 4, A–F. Values indicate the levels of unphosphorylated (0P), monophosphorylated (1P), and diphosphorylated LC₂₀ (2P) under the conditions indicated.

Conditions	% Total LC20			n
	0P	1P	2P
pCa4.5/pCa 4.5	52.1 ± 3.2	47.9 ± 3.2	0	4
pCa 4.5/MC, pCa 4.5	0	40.3 ± 2.6	59.6 ± 2.7	5
MC, pCa 4.5/MC, pCa 4.5	0	27.8 ± 3.8	71.0 ± 4.3	4
pCa 9/pCa 9	100	0	0	3
MC, pCa 9/MC, pCa 9	41.7 ± 3.1	30.6 ± 2.5	27.7 ± 2.5	5
MC, pCa 9/MC, pCa 4.5	0	34.3 ± 4.3	64.4 ± 4.6	5

TABLE 2.

The effects on steady-state isometric force of sequential treatment of Triton-skinned rat caudal arterial smooth muscle with Ca²⁺ and microcystin

Steady-state force measurements were made under the conditions indicated in the legend to Fig. 4, A–F. Values of Force (%) indicate the levels of steady-state force at the end of the protocol compared to that before transfer to the final bathing solution. For example, in the case of Fig. 4B, where the tissue was contracted with pCa 4.5 and then transferred to pCa 4.5 solution containing microcystin, steady-state force in the presence of Ca²⁺ and microcystin was 124.5 ± 2.2% of that in the presence of Ca²⁺ alone.

Conditions	Force (%)	n
pCa 4.5/pCa 4.5	105.1 ± 1.4	4
pCa 4.5/MC, pCa 4.5	124.5 ± 2.2	5
MC, pCa 4.5/MC, pCa 4.5	100.8 ± 0.3	4
MC, pCa 9/MC, pCa9	106.1 ± 1.8	4
MC, pCa 9/MC, pCa 4.5	121.4 ± 3.0	5

FIGURE 5.

Contraction and LC₂₀ diphosphorylation in Triton-skinned rat caudal arterial smooth muscle in response to microcystin at pCa 4.5. A, Triton-skinned rat caudal arterial smooth muscle strips mounted on a force transducer in pCa 9 solution were treated with microcystin (1 μm) at pCa 4.5. Separate tissues were harvested at the indicated times during the contraction for analysis of LC₂₀ phosphorylation by Phos-tag SDS-PAGE and Western blotting (B) with antibodies to LC₂₀ (panel a), Ser(P)¹⁹-LC₂₀ (panel b), Thr(P)¹⁸-LC₂₀ (panel c), and Thr(P)¹⁸,Ser(P)¹⁹-LC₂₀ (panel d). Numbers below the gel lanes correspond to the time points in A. C, cumulative quantitative data showing the proportions of unphosphorylated (0P, closed circles), mono- (1P, open circles), and diphosphorylated LC₂₀ (2P, closed inverted triangles) as a function of time. Values represent the mean ± S.E. (n = 3).

Effects on Force and LC₂₀ Phosphorylation of Combined Treatment with KCl and Calyculin-A

Calyculin-A treatment of intact rat caudal arterial smooth muscle in the presence of extracellular Ca²⁺ elicited a slow, sustained contraction (Fig. 6, green trace) with a t_½ of 1206 ± 102 s (n = 6), which was indistinguishable from the calyculin-A-induced contraction in Ca²⁺-free solution (t_½ = 1326 ± 96 s (n = 6)) (Fig. 2A). Membrane depolarization in the presence of extracellular Ca²⁺ elicited a rapid increase in force (t_½ = 10.2 ± 0.2 s (n = 29)), which subsequently declined to a steady-state level (Figs. 3A and 6, red trace). The simultaneous application of KCl and calyculin-A in the presence of extracellular Ca²⁺ elicited a contractile response (Fig. 6, black trace) that matched the superimposed contractions due to membrane depolarization (Fig. 6, red trace) and phosphatase inhibition (Fig. 6, green trace): the initial rapid contractile response in the presence of KCl and calyculin-A occurred with a t_½ of 11.2 ± 0.6 s (n = 6), i.e. similar to the contraction induced by KCl treatment alone (t_½ = 10.2 ± 0.2 s (n = 29)), whereas the slow, sustained contractile response occurred with a t_½ of 1110 ± 84 s (n = 3), i.e. similar to the contraction induced by calyculin-A in Ca²⁺-free solution (t_½ = 1326 ± 96 s (n = 6)). We hypothesize that the biphasic contractile response to KCl and calyculin-A involves two distinct mechanisms: the rapid response is attributable to membrane depolarization-mediated Ca²⁺ entry and MLCK activation, and the slow response to calyculin-A-mediated inhibition of MLCP with unmasking of Ca²⁺-independent LC₂₀ kinase activity. These mechanisms are supported by measurements of site-specific LC₂₀ phosphorylation during the time course of contraction in the presence of extracellular Ca²⁺ and following addition of both KCl and calyculin-A (Fig. 7). Thus, there was a rapid initial increase in LC₂₀ monophosphorylation (Fig. 7B, panel a), which occurred exclusively at Ser¹⁹ (Fig. 7B, panels b and c), followed by a slight dephosphorylation (Fig. 7C) leading to partial relaxation (Fig. 7A). It was only at prolonged incubation times that diphosphorylation of LC₂₀ was observed (Fig. 7B, panels a and d), which correlated with the slow, sustained phase of contraction (Fig. 7A).

FIGURE 6.

Comparison of the time courses of contraction of intact rat caudal arterial smooth muscle in response to: (i) KCl in the presence of extracellular Ca²⁺, (ii) calyculin-A in the presence of extracellular Ca²⁺, and (iii) a combination of KCl and calyculin-A in the presence of extracellular Ca²⁺. Membrane-intact rat caudal arterial smooth muscle strips, mounted on a force transducer in Ca²⁺-containing H-T buffer, were treated with KCl (87 mm) (red trace), calyculin-A (0.5 μm) (green trace), or both KCl and calyculin-A (black trace). The arrow indicates the time of application of the contractile stimulus.

FIGURE 7.

Contraction and LC₂₀ diphosphorylation in intact rat caudal arterial smooth muscle in response to KCl and calyculin-A in the presence of extracellular Ca²⁺. A, membrane-intact rat caudal arterial smooth muscle strips, mounted on a force transducer in Ca²⁺-containing H-T buffer, were treated with KCl (87 mm) and calyculin-A (0.5 μm). Separate tissues were harvested at the indicated times during the contraction for analysis of LC₂₀ phosphorylation by Phos-tag SDS-PAGE and Western blotting (B) with antibodies to LC₂₀ (panel a), Ser(P)¹⁹-LC₂₀ (panel b), Thr(P)¹⁸-LC₂₀ (panel c), and Thr(P)¹⁸,Ser(P)¹⁹-LC₂₀ (panel d). Numbers below the gel lanes correspond to the time points in A. C, cumulative quantitative data showing the proportions of unphosphorylated (0P, closed circles), mono- (1P, open circles), and diphosphorylated LC₂₀ (2P, closed inverted triangles) as a function of time. Values represent the mean ± S.E. (n = 3).

Stoichiometric Phosphorylation of LC₂₀ at Ser¹⁹ in Triton-skinned Tissue

The results described above suggest that phosphorylation of LC₂₀ at Thr¹⁸ may increase the level of force that is achieved in intact or Triton-skinned rat caudal arterial smooth muscle as a result of Ser¹⁹ phosphorylation. Alternatively, the observed increases in force could be due to an increase in the total level of Ser¹⁹ phosphorylation, rather than phosphorylation at Thr¹⁸. To distinguish between these possibilities, it would be necessary to achieve stoichiometric phosphorylation exclusively at Ser¹⁹ and then observe whether or not phosphorylation at Thr¹⁸ has an additional effect on steady-state force. The next step, therefore, was to achieve stoichiometric phosphorylation exclusively at Ser¹⁹. Unfortunately, treatment of intact tissue with an optimal KCl concentration to elicit a maximal increase in [Ca²⁺]_i, leading to maximal activation of MLCK, does not lead to stoichiometric phosphorylation of LC₂₀ at Ser¹⁹ (Fig. 3). This is due to competing dephosphorylation of LC₂₀ by MLCP, which is constitutively active. Likewise, in Triton-skinned tissue, addition of a maximal [Ca²⁺] fails to elicit stoichiometric LC₂₀ phosphorylation at Ser¹⁹ for the same reason (Fig. 4G, lane A in panel a, and Table 1). We tested the possibility that the stoichiometry of LC₂₀ phosphorylation could be increased by addition of exogenous calmodulin and MLCK to Triton-skinned tissue in the presence of Ca²⁺, recognizing the caveat that, if the MLCK concentration was too high, it would phosphorylate Thr¹⁸ as well. Whereas the addition of calmodulin in the absence or presence of MLCK did increase LC₂₀ phosphorylation slightly, there remained a significant amount of unphosphorylated LC₂₀, and a low level of LC₂₀ diphosphorylation was observed (supplemental Fig. S1 and Table S1). This approach was, therefore, unsuitable for achieving stoichiometric phosphorylation at Ser¹⁹ in the absence of Thr¹⁸ phosphorylation.

An alternative approach to achieve stoichiometric LC₂₀ phosphorylation was to use ATPγS to thiophosphorylate LC₂₀: MLCK uses ATPγS as a substrate (50), but the thiophosphorylated protein is not a substrate for MLCP (51). This approach was used successfully with Triton-skinned rat caudal arterial smooth muscle (Fig. 8). Triton-skinned tissues were shown to be viable by contraction at pCa 4.5 in the presence of ATP and an ATP regenerating system, and relaxation following removal of Ca²⁺ (Fig. 8A). Following removal of ATP, incubation with ATPγS in the presence of Ca²⁺, but absence of ATP or an ATP regenerating system, resulted in stoichiometric thiophosphorylation of LC₂₀ at Ser¹⁹ (Fig. 8B, lanes 2 and 3). It is noteworthy that thiophosphorylated LC₂₀ migrates more rapidly upon Phos-tag SDS-PAGE than does phosphorylated LC₂₀, which enables clear discrimination between phosphorylated and thiophosphorylated forms of the protein.

FIGURE 8.

Stoichiometric thiophosphorylation of LC₂₀ at Ser¹⁹ in Triton-skinned rat caudal arterial smooth muscle. A, the viability of Triton-skinned rat caudal arterial smooth muscle strips was initially verified by transfer from relaxing solution (pCa 9) to pCa 4.5 solution containing ATP and an ATP regenerating system (RS), which induced a contractile response. Tissues were then relaxed by 3 washes in pCa 9 solution containing ATP and RS. ATP was then removed by 6 washes in pCa 9 solution without ATP or RS. Tissues were then incubated in pCa 4.5 solution containing ATPγS (4 mm) in the absence of ATP and RS. Excess ATPγS was then removed by washing twice with pCa 9 solution without ATP or RS. Contraction was evoked by transfer to pCa 9 solution containing ATP and RS. Once steady-state force was established, microcystin (1 μm) was added in pCa 9 solution containing ATP and RS. Tissues were harvested at the indicated times during this protocol for Phos-tag SDS-PAGE and Western blotting with anti-pan LC₂₀ (B), as shown by the arrows in A (the numbers correspond to the lanes in B): (i) lanes 1 and 8, tissue incubated at pCa 9 showing exclusively unphosphorylated LC₂₀; (ii) lanes 2 and 3, pCa 4.5 + ATPγS in the absence of ATP and RS; (iii) lane 4, pCa 9 in the absence of ATP and RS following thiophosphorylation; (iv) lane 5, at the plateau of force development following transfer to pCa 9 solution containing ATP and RS; (v) lanes 6 and 7, following treatment with microcystin at pCa 9 in the presence of ATP and RS. An additional control is included in lane 9: Triton-skinned tissue treated with microcystin at pCa 9 for 60 min to identify unphosphorylated (0P), monophosphorylated (1P), and diphosphorylated (2P) LC₂₀ bands. Thiophosphorylated forms of LC₂₀ are indicated as follows: 1SP, monothiophosphorylated LC₂₀; 2SP, dithiophosphorylated LC₂₀; 1SP1P, LC₂₀ thiophosphorylated at one site and monophosphorylated at the other. Data are representative of 8 independent experiments.

ATPγS is not hydrolyzed by activated myosin and therefore does not support cross-bridge cycling and contraction (20, 52, 53). Stoichiometric thiophosphorylation at Ser¹⁹ (Fig. 8B, lanes 2 and 3) was, therefore, not accompanied by contraction (Fig. 8A). Transfer to pCa 9 solution containing ATP and an ATP regenerating system following washout of ATPγS resulted in a rapid contractile response (t_½ = 21.2 ± 0.2 s (n = 8)) and steady-state force corresponding to 85.4 ± 1.9% (n = 8) of the pCa 4.5-induced contraction (Fig. 8A). Once the steady-state force was achieved, microcystin was added at pCa 9 in the presence of ATP and an ATP regenerating system. No additional force development was observed (77.3 ± 4.2% (n = 5) of pCa 4.5-induced contraction), although significant di(thio)phosphorylation of LC₂₀ did occur (Fig. 8B, lanes 6 and 7, and Table 3).

TABLE 3.

Thiophosphorylation of LC₂₀ in Triton-skinned rat caudal arterial smooth muscle

Values represent percentage of total LC₂₀ ± S.E. (n = 3). *, #, and ^ indicate values are not statistically significantly different from each other; 0P, unphosphorylated LC₂₀; 1SP, monothiophosphorylated LC₂₀; 1P, monophosphorylated LC₂₀; 1SP1P, LC₂₀ thiophosphorylated at one site and phosphorylated at the other; 2P, diphosphorylated LC₂₀; RS, ATP regenerating system; MC, microcystin.

Conditions	0P	1SP	1P	1SP1P	2P
ATPγS, pCa 4.5, no RS	6.0 ± 2.5*	88.4 ± 4.8#	0	0	5.6 ± 4.3^
Then pCa 9, no RS	11.1 ± 3.4*	81.6 ± 3.9#	0	0	7.2 ± 2.7^
Then pCa 9, RS	16.6 ± 4.7*	80.0 ± 4.4#	0	0	3.4 ± 1.9^
Then MC, pCa 9, RS	13.9 ± 3.2	54.7 ± 4.0	3.4 ± 2.1	14.0 ± 9.2	13.9 ± 8.6

The identities of the thiophosphorylated LC₂₀ species as depicted in Fig. 8B were verified by the use of phosphospecific antibodies (supplemental Fig. S2). Incubation of Triton-skinned rat caudal arterial smooth muscle strips with ATPγS and microcystin at pCa 9, in the absence of ATP and an ATP regenerating system, failed to elicit thiophosphorylation of LC₂₀ (supplemental Fig. S3, lanes 3 and 4). This is in contrast to incubation with ATPγS at pCa 4.5, in the absence of ATP and an ATP regenerating system, which led to LC₂₀ monothiophosphorylation (supplemental Fig. S3, lane 2) at Ser¹⁹ (supplemental Fig. S2, lanes 3–5).

Effects of Diphosphorylation of LC₂₀ on the Rates of Dephosphorylation and Relaxation

Finally, we investigated the possibility that LC₂₀ diphosphorylation may affect relaxation, rather than contraction, by comparing the rates of dephosphorylation and relaxation of Triton-skinned rat caudal arterial smooth muscle following monophosphorylation of LC₂₀ at pCa 4.5 or diphosphorylation of LC₂₀ at pCa 9 in the presence of okadaic acid. Okadaic acid was chosen as the phosphatase inhibitor for these experiments, rather than microcystin, because its effects are readily reversible (54), whereas microcystin can covalently modify the catalytic subunit of type 1 protein phosphatase, resulting in irreversible inhibition of the phosphatase (55). Indeed, we have observed that microcystin-induced contractions cannot be reversed by washout of the inhibitor (data not shown).

Comparable levels of phosphorylation of LC₂₀ were achieved with pCa 4.5 (0.48 ± 0.02 mol of P_i/mol of LC₂₀ (n = 4)) or okadaic acid treatment at pCa 9 (0.49 ± 0.09 mol of P_i/mol of LC₂₀ (n = 5)), with monophosphorylation occurring exclusively in response to Ca²⁺ and both mono- and diphosphorylation being detected in the presence of okadaic acid, as expected (Fig. 9C). The steady-state force generated by okadaic acid at pCa 9 was 83.3 ± 1.4% (n = 9) of that at pCa 4.5 (supplemental Fig. S4). Relaxation was initiated by transfer to pCa 9 solution and the time courses of LC₂₀ dephosphorylation and relaxation were quantified (Fig. 9, A and B, respectively). The rate of dephosphorylation of LC₂₀ was markedly reduced in the tissues in which LC₂₀ had been diphosphorylated compared with tissues containing exclusively monophosphorylated LC₂₀ (Fig. 9A): t_½ values were 83.3 s for Ca²⁺-treated tissue and 560 s for okadaic acid-treated tissue. This correlated with a reduction in the rate of relaxation (Fig. 9B): t_½ values were 560 s for Ca²⁺-treated tissue and 1293 s for okadaic acid-treated tissue. The slower rate of dephosphorylation following okadaic acid treatment cannot be explained by slow washout of the inhibitor because MYPT1-Thr⁶⁹⁷ and -Thr⁸⁵⁵ (the inhibitory phosphorylation sites in the myosin targeting subunit of MLCP) (56) were maximally dephosphorylated at the first time point analyzed during the relaxation, i.e. when force was at 90% (supplemental Fig. S5).

FIGURE 9.

Comparison of the time courses of relaxation and LC₂₀ dephosphorylation in Triton-skinned rat caudal arterial smooth muscle following contraction with Ca²⁺ or okadaic acid in the absence of Ca²⁺. Triton-skinned tissues that had been contracted with Ca²⁺ (open circles) or okadaic acid (20 μm) at pCa 9 (closed circles) were transferred to pCa 9 solution and the time courses of dephosphorylation (A) and relaxation (B) were followed. Tissues were harvested at 10, 20, 30, 40, 50, 75, and 100% relaxation and LC₂₀ phosphorylation levels were quantified by Phos-tag SDS-PAGE and Western blotting with anti-pan LC₂₀. Values represent the mean ± S.E. (n = 5). Representative Western blots are shown in C.

DISCUSSION

LC₂₀ diphosphorylation has been observed in several smooth muscle tissues treated with various contractile stimuli, including carbachol- (37) and neurally stimulated bovine tracheal smooth muscle (38), prostaglandin-F_2α-stimulated rabbit thoracic aorta (39, 40), and angiotensin II-stimulated rat renal efferent arterioles (41). LC₂₀ diphosphorylation has also been observed in pathological cases of smooth muscle hypercontractility, for example, coronary artery spasm (44, 45), cerebral vasospasm after subarachnoid hemorrhage (43, 46), and intimal hyperplasia (42). More recently, Cho et al. (57) provided evidence for enhanced Ca²⁺-independent LC₂₀ diphosphorylation and force generation in β-escin-permeabilized mesenteric arterial smooth muscle rings of spontaneously hypertensive rats compared with normotensive Wistar Kyoto controls. Furthermore, phenylephrine induced significant LC₂₀ diphosphorylation in the spontaneously hypertensive rat arteries. Evidence was also presented that ZIPK contributes to the Ca²⁺-independent LC₂₀ diphosphorylation through phosphorylation of MYPT1 at Thr⁶⁹⁷ and possibly direct phosphorylation of LC₂₀, and the expression level of ZIPK, but not ILK, was greater in spontaneously hypertensive rats than Wistar Kyoto tissues (57). Collectively, these data suggest that LC₂₀ diphosphorylation may account for the hypercontractility observed in smooth muscle tissues in response to certain contractile stimuli and in pathological situations. It was, therefore, important to determine the functional effect of LC₂₀ phosphorylation on smooth muscle contractility. The results of these studies led to the following conclusions.

(i) Treatment of Triton-skinned rat caudal arterial smooth muscle with the phosphatase inhibitor microcystin in the absence of Ca²⁺ induced a slow, sustained contraction, as previously observed (16), which correlated with LC₂₀ phosphorylation at Ser¹⁹ and Thr¹⁸ (Fig. 1).

(ii) Similar results were obtained when intact tissues were treated with the membrane-permeant phosphatase inhibitor calyculin-A in the absence of extracellular and stored Ca²⁺ (Fig. 2). However, an interesting difference between the Triton-skinned and intact tissues was observed: microcystin treatment of skinned tissue induced monophosphorylation at Ser¹⁹ and Thr¹⁸ at similar rates (Fig. 1B, panels b and c), in addition to diphosphorylation (Fig. 1B, panel d), whereas no monophosphorylation was observed at Thr¹⁸ following calyculin-A treatment of intact tissue in the absence of extracellular Ca²⁺ (Fig. 2B, panel c), but instead Ser¹⁹ monophosphorylation was followed by Thr¹⁸ phosphorylation to form the diphosphorylated species (Fig. 2B). This suggests that LC₂₀ phosphorylation at the two sites was random in the Triton-skinned tissue experiments but sequential in the intact tissue experiments. A possible explanation would be that distinct kinases are involved in the two situations, the most likely candidates being ILK and ZIPK, and we have provided evidence that ILK is responsible for microcystin-induced Ca²⁺-independent contraction of Triton-skinned rat caudal arterial smooth muscle (19).

(iii) The level of steady-state force induced by calyculin-A in the absence of Ca²⁺ is significantly greater than that induced by a maximally effective concentration of KCl, i.e. an optimal Ca²⁺ signal (Fig. 2A). This would be consistent with diphosphorylation of LC₂₀ increasing steady-state force compared with Ser¹⁹ monophosphorylation. Indeed, addition of microcystin to Triton-skinned tissue pre-contracted at pCa 4.5 (Fig. 4B), or of Ca²⁺ to tissue pre-contracted with microcystin in the absence of Ca²⁺ (Fig. 4F), evoked a significant increase in steady-state force (Table 2), which correlated with increases in LC₂₀ diphosphorylation (Fig. 4G and Table 1). However, Ser¹⁹ phosphorylation stoichiometry also increased under these conditions (from ∼0.5 mol of P_i/mol of LC₂₀ to ∼1 mol of P_i/mol of LC₂₀) (Table 1), suggesting that the enhanced force responses could be due to increased phosphorylation at Ser¹⁹ (whether in the form of monophosphorylated or diphosphorylated LC₂₀).

(iv) In intact (Fig. 3) and Triton-skinned tissue (Fig. 4A and G), Ca²⁺ elicited exclusively monophosphorylation of LC₂₀ at Ser¹⁹, as expected.

(v) The fact that the rate of contraction of Triton-skinned rat caudal arterial smooth muscle in response to Ca²⁺ was significantly faster (t_½ ∼ 150 s) than that in response to microcystin at pCa 9 (t_½ ∼ 450 s) suggested that it may be possible to induce maximal phosphorylation at Ser¹⁹ before achieving diphosphorylation, and thereby determine more convincingly if diphosphorylation causes additional force development. Furthermore, treatment with microcystin at pCa 4.5 caused a significant increase in the rate of contraction (t_½ ∼ 65 s) compared with Ca²⁺ alone (t_½ ∼ 150 s) or microcystin alone (t_½ ∼ 450 s). Detailed analysis of the (Ca²⁺ + microcystin)-induced contraction of Triton-skinned rat caudal arterial smooth muscle revealed rapid phosphorylation of LC₂₀ at Ser¹⁹ (which can be attributed to MLCK activation by Ca²⁺) and a slower rate of phosphorylation at Thr¹⁸ (due to ILK activity that is unmasked by the phosphatase inhibitor) (Fig. 5). The observation that no additional force was evoked as diphosphorylated LC₂₀ appeared argues that Thr¹⁸ phosphorylation likely does not increase steady-state force beyond that achieved by phosphorylation at Ser¹⁹.

(vi) The combination of KCl and calyculin-A in the presence of Ca²⁺ induced a biphasic contractile response of intact tissue (Fig. 6), which corresponds to the combined contractile responses to KCl in the presence of Ca²⁺ and calyculin-A in the absence or presence of Ca²⁺. In this case, the initial rapid phasic contraction correlated with Ser¹⁹ phosphorylation, and the slow sustained contractile response with the diphosphorylation of LC₂₀ (Fig. 7). The contractile effects of KCl and calyculin-A, however, could be explained entirely by Ser¹⁹ phosphorylation.

It was necessary, therefore, to devise a way to achieve stoichiometric phosphorylation at Ser¹⁹ without Thr¹⁸ phosphorylation, and then observe whether subsequent phosphorylation at Thr¹⁸ has an effect on steady-state force development. This was achieved by using ATPγS to evoke close-to-stoichiometric thiophosphorylation at Ser¹⁹ with very little dithiophosphorylation (Fig. 8B and Table 3). Subsequent phosphorylation of LC₂₀ at Thr¹⁸ (Fig. 8B) failed to elicit an increase in force (Fig. 8A). We conclude, therefore, that phosphorylation at Ser¹⁹ of LC₂₀ accounts for maximal force development, and no further force results from additional phosphorylation at Thr¹⁸.

We then turned our attention to the possibility that diphosphorylation may affect relaxation rather than contraction by comparing the time courses of dephosphorylation of LC₂₀ and relaxation of Triton-skinned muscle strips that had been pre-contracted under conditions that evoked phosphorylation exclusively at Ser¹⁹ or at both Ser¹⁹ and Thr¹⁸ to the same overall phosphorylation stoichiometry. The rates of dephosphorylation and relaxation were significantly slower in the case of diphosphorylated LC₂₀ (Fig. 9). We conclude, therefore, that diphosphorylation of LC₂₀ at Thr¹⁸ and Ser¹⁹ has a marked effect on relaxation compared with monophosphorylation at Ser¹⁹.

The mechanism underlying the reduction in the rate of dephosphorylation of diphosphorylated LC₂₀ compared with Ser¹⁹-monophosphorylated LC₂₀ remains to be determined. A possibility is that the K_m of MLCP for diphosphorylated LC₂₀ may be significantly higher than that for LC₂₀ phosphorylated exclusively at Ser¹⁹. Although such kinetic comparisons have not been performed to date, in vitro assays indicated that dephosphorylation of diphosphorylated LC₂₀ (whether free or in intact myosin) occurred by a random mechanism, with dephosphorylation at Ser¹⁹ and Thr¹⁸ occurring at similar rates (5).

The principal conclusions from this study are: (i) the level of steady-state force is dictated by the level of Ser¹⁹ phosphorylation and is unaffected by Thr¹⁸ phosphorylation; and (ii) Thr¹⁸ phosphorylation reduces the rate of LC₂₀ dephosphorylation and relaxation, supporting a sustained contractile response. There is abundant literature indicating that most contractile stimuli elicit phosphorylation exclusively at Ser¹⁹ and this can be explained by Ca²⁺-induced activation of MLCK, with or without a modest degree of Ca²⁺ sensitization due to MLCP inhibition (58). Specific stimuli and pathophysiological situations associated with hypercontractility induce LC₂₀ diphosphorylation at Thr¹⁸ and Ser¹⁹. This can be explained by increased MLCP inhibition, unmasking constitutive Ca²⁺-independent LC₂₀ kinase activity (ILK and/or ZIPK), and potentially an increase in activity of Ca²⁺-independent LC₂₀ kinases, leading to an increase in Ser¹⁹ phosphorylation (force) and Thr¹⁸ phosphorylation (sustained contraction). ILK and ZIPK are therefore potential therapeutic targets for the treatment of cerebral and coronary vasospasm, intimal hyperplasia, hypertension, and other conditions associated with hypercontractility.