Regulation Of Smooth Muscle Myosin Research Articles

Graded effects of unregulated smooth muscle myosin on intestinal architecture, intestinal motility and vascular function in zebrafish

ABSTRACTSmooth muscle contraction is controlled by the regulated activity of the myosin heavy chain ATPase (Myh11). Myh11 mutations have diverse effects in the cardiovascular, digestive and genitourinary systems in humans and animal models. We previously reported a recessive missense mutation, meltdown (mlt), which converts a highly conserved tryptophan to arginine (W512R) in the rigid relay loop of zebrafish Myh11. The mlt mutation disrupts myosin regulation and non-autonomously induces invasive expansion of the intestinal epithelium. Here, we report two newly identified missense mutations in the switch-1 (S237Y) and coil-coiled (L1287M) domains of Myh11 that fail to complement mlt. Cell invasion was not detected in either homozygous mutant but could be induced by oxidative stress and activation of oncogenic signaling pathways. The smooth muscle defect imparted by the mlt and S237Y mutations also delayed intestinal transit, and altered vascular function, as measured by blood flow in the dorsal aorta. The cell-invasion phenotype induced by the three myh11 mutants correlated with the degree of myosin deregulation. These findings suggest that the vertebrate intestinal epithelium is tuned to the physical state of the surrounding stroma, which, in turn, governs its response to physiologic and pathologic stimuli. Genetic variants that alter the regulation of smooth muscle myosin might be risk factors for diseases affecting the intestine, vasculature, and other tissues that contain smooth muscle or contractile cells that express smooth muscle proteins, particularly in the setting of redox stress.

Mutant Analysis and Computational Analysis of the Essential Light Chain and Regulatory Light Chain Interactions with Respect to Regulation of Smooth Muscle Myosin

To understand how smooth muscle myosin's (SMM) light chains are involved in phosphorylation dependent regulation of ATP hydrolysis, we expressed single, double and triple amino acid mutants of the heavy chain, regulatory (RLC) or essential light chain (ELC) in the area of the Ca2+-binding loop of the ELC. We mutated this area because of similarities to scallop smooth myosin's Ca2+ regulatory mechanism. Since the ELC is not required to maintain the off state, we reasoned that mutations in this region would specifically alter the activity of the on (phosphorylated) state of SMM. Mutations were made to disrupt hydrogen bonding between the ELC and RLC and the heavy chain (HC) based on scallop crystal structures. All mutant ATPase activities and actin sliding velocities (ASVs) were essentially identical to wild type in the unphosphorylated. For the phosphorylated states, one class of mutant showed normal ATPase activity and ASVs; a second class showed similarly depressed ATPase activity and ASVs; and a third class with differentially depressed ATPase activity and ASVs. We also created recovery mutants that restored (and recoupled) ATPase rates with ASVs. We then used course grain discrete molecular dynamics and force constant profiling to reconcile changes in the interactions of ELC, RLC & HC in that region, with changes at the ATPase site. We created a prepowerstroke smooth muscle myosin model with an ELC Ca2+ loop from scallop that interacted weakly with the RLC and used FlexServe (http://mmb.pcb.ub.es/FlexServ/) to compute changes in flexibility between the native (1QVI) and modified model. We see that changes in the ELC RLC interaction have direct effects on lever arm flexibility and active site protein dynamics.

Kinetic and motor functions mediated by distinct regions of the regulatory light chain of smooth muscle myosin

To understand the importance of selected regions of the regulatory light chain (RLC) for phosphorylation-dependent regulation of smooth muscle myosin (SMM), we expressed three heavy meromyosins (HMMs) containing the following RLC mutants; K12E in a critical region of the phosphorylation domain, GTDP 95–98/AAAA in the central hinge, and R160C a putative binding residue for phosphorylated S19. Single-turnover actin-activated Mg 2+-ATPase ( V max and K ATPase) and in vitro actin-sliding velocities were examined for both unphosphorylated (up-) and phosphorylated (p-) states. Turnover rates for the up-state (0.007–0.030 s − 1 ) and velocities (no motion) for all constructs were not significantly different from the up-wild type (WT) indicating that they were completely turned off. The apparent binding constants for actin in the presence of ATP ( K ATPase) were too weak to measure as expected for fully regulated constructs. For p-HMM containing GTDP/AAAA, we found that both ATPase and motility were normal. The data suggest that the native sequence in the central hinge between the two lobes of the RLC is not required for turning the HMM off and on both kinetically and mechanically. For p-HMM containing R160C, all parameters were normal, suggesting that R160C is not involved in coordination of the phosphorylated S19. For p-HMM containing K12E, the V max was 64% and the actin-sliding velocity was ∼ 50% of WT, suggesting that K12 is an important residue for the ability to sense or to promote the conformational changes required for kinetic and mechanical activation.

Folding and regulation in myosins II and V

The enzymatic activity of many myosins is regulated by various means including calcium binding, phosphorylation or binding of receptor molecules. In this review we compare and contrast the regulation of smooth muscle myosin II and myosin Va with particular emphasis on the structural basis for the regulation. Both myosins adopt folded compact conformations in their off states, but the details of the conformations are markedly different. In the regulated smooth muscle myosin II, the key feature is an asymmetric interaction between the two heads of the molecule with contributions of specific tail-head interactions. In myosin V the key feature is an interaction between the heads and the globular tail domain.

Essential light chain modulates phosphorylation-dependent regulation of smooth muscle myosin.

To examine the functional role of the essential light chain (ELC) in the phosphorylation-dependent regulation of smooth muscle myosin, we replace the native light chain in smooth muscle myosin with bacterially expressed chimeric ELCs in which one or two of the four helix-loop-helix domains of chicken gizzard ELC were substituted by the corresponding domains of scallop (Aquipecten irradians) ELC. All of these myosins, regardless of the ELC mutations or regulatory light chain (RLC) phosphorylation, showed normal subunit constitutions and NH(4)(+)/EDTA-ATPase activities, both of which were similar to those of native myosin. None of the ELC mutations changed the actin-activated ATPase activity of myosin in the absence of RLC phosphorylation. However, in the presence of RLC phosphorylation, the substitution of domain 1 or 2 in the ELC significantly decreased the actin-activated ATPase activity, whereas the substitution of both of these domains did not change the activity. In contrast to myosin, the domain 2 substitution in the ELC did not affect the actin-activated ATPase activity of single-headed myosin subfragment 1. These results suggest an interhead interaction between domains 1 and 2 of ELCs which is required to attain the full actin-activated ATPase activity of smooth muscle myosin in the presence of RLC phosphorylation.

The interaction between the regulatory light chain domains on two heads is critical for regulation of smooth muscle myosin.

Recent findings have suggested that the interaction between the two heads is critical for phosphorylation-dependent regulation of smooth muscle myosin. We hypothesized that the interaction between the two regulatory light chains on two heads of myosin dictates the regulation of myosin motor function. To evaluate this notion, we engineered and characterized smooth muscle heavy meromyosin (HMM), which is composed of one entire HMM heavy chain and one motor domain truncated heavy chain containing the S2 rod and regulatory light chain (RLC) binding site, as well as the bound RLC (SMDHMM). SMDHMM was inactive for both actin-translocating activity and actin-activated ATPase activity in the dephosphorylated state, demonstrating that the interaction between the two RLC domains on the two heads and/or a motor domain and a RLC domain in a distinct head is sufficient for the inhibition of smooth muscle myosin motor activity. When phosphorylated, SMDHMM was activated for both actin-translocating activity and actin-activated ATPase activity; however, these activities were lower than those of double-headed HMM, implying partial release of inhibition by phosphorylation in SMDHMM and/or cooperativity between the two heads of smooth muscle myosin. The present results indicate that the RLC domain is critical for phosphorylation-dependent regulation of smooth muscle myosin motor activity. On the other hand, similar to double-headed HMM, SMDHMM showed both "folded" and "extended" conformations, and the ratio of those conformations is dependent on ionic strength, suggesting that the RLC domain is sufficient to regulate the conformational transition in myosin.

Registration of the rod is not critical for the phosphorylation-dependent regulation of smooth muscle myosin.

A recent report has suggested that the interaction between the head and the rod region of smooth muscle myosin at S2 is important for the phosphorylation-mediated regulation of myosin motor activity [Trybus, K. M., Freyzon, Y., Faust, L. Z., and Sweeney, H. L. (1997) Proc. Natl. Acad. Sci. U.S.A. 74, 48-52]. To investigate whether specific amino acid residues at S2 or whether the registration of the 7-residue/28-residue repeat appearing in the alpha-helical coiled-coil structure of the rod are critical for such an interaction, two smooth muscle myosin mutants were constructed in which the N-terminal sequences of S2 were deleted to various extents. One mutant contained a deletion of 71 residues at the position immediately C-terminal to the invariant proline (Pro849) linking the S1 domain directly to the downstream sequence of the rod, while in another mutant, 53 residues were deleted at a position 56 residues downstream of Pro849. Despite these alterations which change the registration of both the 28-residue repeat and the 7-residue repeat found in myosin rod sequence, both myosin mutants showed a stable double-headed structure by electron microscopic observation. Both the actin-activated ATPase activity and the actin translocating activity of the mutants were completely regulated by the phosphorylation of the regulatory light chain. The actin sliding velocity of the two mutant myosins was the same as the wild-type recombinant myosin. Furthermore, the head configuration critical for myosin filament formation (extended or folded) was unchanged in either mutant. These results indicate that neither the specific amino acid residues nor the registration of the amino acid repeat in S2 is critical for the head configuration. These results indicate that neither a specific amino acid sequence at the head-rod junction nor the rod sequence registration is critical for the regulation of smooth muscle myosin.

Thin-filament linked regulation of smooth muscle myosin.

Phosphorylation of the regulatory light chain subunit of smooth muscle myosin is sufficient, but not necessary for muscle contraction. It has been suggested that thin-filament regulation may also contribute to the regulation of contraction. A hallmark feature of regulated thin filaments, previously described for vertebrate skeletal muscle, is the capacity of strong-binding or rigor-like cross bridges to "turn-on" the actin filament. Turned-on thin filaments stimulate cross-bridge attachment even in the absence of calcium. The present study utilized an in vitro sliding-filament motility assay to test for thin-filament regulation of both unphosphorylated and phosphorylated smooth muscle myosins. Regulated thin-filaments were reconstituted from skeletal muscle actin and chicken gizzard smooth muscle tropomyosin (TmCG), and then turned-on either (1) by rigor cross bridges at low concentrations of MgATP, or (2) by adding N-ethyl-maleimide-modified skeletal subfragment S1(NEM-S1), which forms rigor-like bonds in the presence of MgATP. For control actin.TmCG filaments, force production by unphosphorylated myosin was 0.5% of that produced by thiophosphorylated myosin. The force exerted on actin.Tm filaments by both unphosphorylated and phosphorylated myosins was increased by reducing the [MgATP] to 10-100 microM MgATP (rigor-dependent activation). Force was also increased by actin.TmCG filaments that had been turned-on by NEM-S1 binding, with force production by unphosphorylated myosin increased 80-fold vs. 2.3-fold for thiophosphorylated myosin. TmCG was required for increased force production with both low MgATP and NEM-S1. Unloaded filament velocity for NEM-S1-activated thin filaments was 0.72 micron/sec with unphosphorylated myosin compared to 1.24 microns/sec with thiophosphorylated myosin. Taken together, these results suggest that thin-filament regulation may play a role in the activation of both unphosphorylated and phosphorylated smooth muscle myosins and suggest a possible mechanism for activation of slowly cycling unphosphorylated cross bridges (i.e. latch-state) during tonic contractions of smooth muscle.

A Long, Weakly Charged Actin-binding Loop Is Required for Phosphorylation-dependent Regulation of Smooth Muscle Myosin

Chimeric substitution of the weak actin-binding loop (ABL) from chicken skeletal muscle myosin for that of gizzard smooth muscle heavy meromyosin (HMM) causes activation of the dephosphorylated mutant (SABL HMM; Rovner, A. S., Freyzon, Y., and Trybus, K. M. (1995) J. Biol. Chem. 270, 30260-30263). The present study determined whether this loss of regulation is due to the greater positive charge density (5 versus 3 clustered lysine residues) or lesser length (14 versus 26 residues) of the mutant ABL. Charge augmentation had little effect on regulation of expressed mutants, but elimination of the 12 N-terminal amino acids from the wild-type ABL significantly increased actin-activated ATPase activity of the dephosphorylated relative to the phosphorylated molecule while conferring the ability to move actin filaments in vitro on the former. Addition of the same 12 residues to the SABL mutant increased the ratio of phosphorylated to dephosphorylated ATPase activity while imparting wild type-like regulation to motility. However, full actin activation of dephosphorylated ATPase activity required both the shorter length and greater positive charge density found in the SABL loop. These results demonstrate that, compared with skeletal, both the greater length and lesser positive charge density of the smooth muscle myosin ABL are required for proper phosphorylation-mediated regulation of the molecule.

A specific amino acid sequence at the head-rod junction is not critical for the phosphorylation-dependent regulation of smooth muscle myosin.

It has been suggested that the structure at the head-rod junction of smooth muscle myosin is important for the phosphorylation-mediated regulation of myosin motor activity. To investigate whether a specific amino acid sequence at the head-rod junction is critical for the regulation, three smooth muscle myosin mutants in which the sequence at the N-terminal end of S2 is deleted to various extents were expressed in Sf9 cells; 28, 56, and 84 amino acid residues, respectively, at the position immediately C-terminal to the invariant proline (Pro849) were deleted, and the S1 domain was directly linked to the downstream sequence of the rod. The mutant myosins were expressed, purified, and biochemically characterized. All three myosin mutants showed a stable double-headed structure based upon electron microscopic observation. Both the actin-activated ATPase activity and the actin translocating activity of the mutants were completely regulated by the phosphorylation of the regulatory light chain. The actin sliding velocity of the three mutant myosins was the same as the wild-type recombinant myosin. These results indicate that a specific amino acid sequence at the head-rod junction is not required for the regulation of smooth muscle myosin. The results also suggest that there is no functionally important interaction between the regulatory light chain and the heavy chain at the head-rod junction.

A Hinge at the Central Helix of the Regulatory Light Chain of Myosin Is Critical for Phosphorylation-dependent Regulation of Smooth Muscle Myosin Motor Activity

The motor function of smooth muscle myosin is activated by phosphorylation of the regulatory light chain (RLC) at Ser19. However, the molecular mechanism by which the phosphorylation activates the motor function is not yet understood. In the present study, we focused our attention on the role of the central helix of RLC for regulation. The flexible region at the middle of the central helix (Gly95-Pro98) was substituted or deleted to various extents, and the effects of the deletion or substitution on the regulation of the motor activity of myosin were examined. Deletion of Gly95-Asp97, Gly95-Thr96, or Thr96-Asp97 decreased the actin-translocating activity of myosin a little, but the phosphorylation-dependent regulation of the motor activity was not disrupted. In contrast, the deletion of Gly95-Pro98 of RLC completely abolished the actin translocating activity of phosphorylated myosin. However, the unregulated myosin long subfragment 1 containing this RLC mutant showed motor activity the same as that containing the wild type RLC. Since long subfragment 1 motor activity is unregulated by phosphorylation, i.e. constitutively active, these results suggest that the deletion of these residues at the central helix of RLC disrupts the phosphorylation-mediated activation mechanism but not the motor function of myosin itself. On the other hand, the elimination of Pro98 or substitution of Gly95-Pro98 by Ala resulted in the activation of actin translocating activity of dephosphorylated myosin, whereas it did not affect the motor activity of phosphorylated myosin. Together, these results clearly indicate the importance of the hinge at the central helix of RLC on the phosphorylation-mediated regulation of smooth muscle myosin.

Mapping phosphorylation site of the regulatory light chain in smooth muscle myosin by immuno-electron microscopy.

Phosphorylation site responsible for the regulation of smooth muscle myosin was mapped using a polyclonal antibody against a phosphorylated hendecapeptide corresponding to the amino acid sequence around Ser-19 in the regulatory light chain. Phosphorylated myosin mixed with the antibody was rotary-shadowed and was examined by electron microscopy. The antibody binding site was located in the head portion of myosin and the average distance from the head-rod junction was about 3nm toward the tip of myosin head. The results indicate that the phosphorylated Ser-19 in regulatory light chain is a little more extended toward the adjacent essential light chain in reference to the resolved N-terminal residues of the regulatory light chain in the three dimensional structure of myosin heads from other sources, in which the structure of the N-terminal portions homologous to the phosphorylated Ser-19 was not resolved (Rayment, I. et al. (1993) Science 261, 50-58; Xie, X. et al. (1994) Nature 368, 306-312). Intramolecular interaction through the introduced phosphoryl group may be the primary results in the regulatory light chain which releases the motor domain from its suppressed state.

Restoration of Phosphorylation-dependent Regulation to the Skeletal Muscle Myosin Regulatory Light Chain

Regulation of the ATPase activity of smooth and nonmuscle myosin II involves reversible phosphorylation of the regulatory light chain (RLC). The RLC from skeletal muscle myosin (skRLC) is unable to confer regulation (myosin is locked in an inactive state) to smooth muscle myosin when substituted for the endogenous smooth RLC (smRLC). Studies of chimeric light chains comprised of the N- or C-terminal half of each skRLC and smRLC suggest that the structural basis for the loss of this regulation is within the C-terminal half of the RLC (Trybus, K.M., and Chatman, T.A. (1993) J. Biol. Chem. 268, 4412-4419). The purpose of this study is to delineate the structural elements within the C-terminal half of the smRLC that are absent in the skRLC and are necessary for regulation. By sequence comparison, six residues, Arg-103, Arg-123, Met-129, Gly-130, Arg-143, and Arg-160, which are conserved in regulated myosin RLCs but missing in nonregulated myosin RLCs, were identified in smRLC. To test whether these amino acids provide the missing structural elements necessary for phosphorylation-mediated regulation, a skRLC was engineered that replaced the corresponding skRLC amino acids (positions 100, 120, 126, 127, 140, and 157, respectively) with their smRLC counterparts. Using a newly developed RLC exchange procedure, the purified mutant protein was evaluated for its ability to regulate chicken gizzard smooth muscle myosin. Substitution of the six conserved amino acids into the skRLC completely restored phosphorylation-mediated regulation. Thus, a subset of these amino acids, including four basic arginine residues located in the E, F, G, and H helices which are missing in skRLC, may be the structural coordinates for the phosphorylserine in the N terminus. Based on this result, the regulation of glycogen phosphorylase is discussed as a model for the regulation of smooth muscle myosin.

Smooth Muscle Myosin II and Alpha Smooth Muscle Actin Expression in the Baboon (Papio Anubis) Uterus is Associated with Glandular Secretory Activity and Stromal Cell Transformation1

The objective of this study was to investigate the localization and hormonal regulation of smooth muscle myosin II (SMM II) and alpha smooth muscle actin (alpha SMA) in the baboon uterus, since cytoskeletal proteins are involved in secretory function and morphological transformation. Uterine tissue was obtained from baboons 1) during the menstrual cycle, 2) following steroid treatment of ovariectomized baboons, 3) during pregnancy (Days 14-60 postovulation [PO]), and 4) during simulated pregnancy (Days 18-32 PO). Tissues were processed for immunocytochemical localization of SMM II or alpha SMA with specific polyclonal or monoclonal antibodies, respectively. SMM II stained all smooth muscle cells of blood vessels and myometrium regardless of treatment. Glandular epithelial staining was present only in endometrium obtained during the luteal phase or following estrogen and progesterone treatment. Staining intensity was greater in the basalis than in the functionalis. The number of glands staining positive for SMM II on Days 18-32 of pregnancy and simulated pregnancy was variable. Glandular stain was absent after Day 32 PO. These immunocytochemical data were confirmed by immunoblot analysis of glandular cytosolic extracts. Stromal staining for SMM II was present under the luminal epithelium during simulated pregnancy (Days 18-32), on Day 25 of steroid treatment in the simulated-pregnant controls, and in nonimplantation sites during pregnancy. In contrast, alpha SMA staining was low or absent in all uterine cell types in ovariectomized baboons. Under estrogen-dominated conditions (follicular phase and estrogen treatment), alpha SMA staining was present in smooth muscle cells, and this staining persisted throughout the remaining treatment periods. Glandular epithelial staining for alpha SMA was absent in all treatment groups. However, alpha SMA staining in stromal fibroblasts underneath the luminal epithelium was evident as early as Day 14 of pregnancy and Day 18 of simulated pregnancy. The number of stromal fibroblasts that stained positive increased in the surface region of the functionalis between Days 18 and 32 PO, and the staining extended throughout the upper functionalis region. There was a decrease in the number of positively stained stromal fibroblasts, particularly at the implantation site, between Days 32 and 40 of pregnancy. By Days 50-60 of pregnancy, this staining was almost absent. The induction of alpha SMA in stromal fibroblasts in the functionalis region in pregnant baboons was confirmed by immunoblot analysis of stromal cell cytosol extracts. We conclude that the progesterone-induced glandular expression of SMM II may be involved in uterine secretory function and that alpha SMA expression in stromal fibroblasts during pregnancy and after long-term steroid treatment is associated with the decidualization process.

Requirement of the two-headed structure for the phosphorylation dependent regulation of smooth muscle myosin

It is known for smooth muscle myosin that while acto-HMM ATPase activity is regulated by phosphorylation, acto-S-1 ATPase activity is not regulated. To clarify the heavy chain structure required for the regulation, smooth muscle myosin containing 7 different lengths of the S-2 portion were expressed in Sf9 insect cells using Baculovirus expression system. Myosin containing longer than 991 residues of heavy chain formed a stable two-headed structure while myosin with shorter than 944 residues of heavy chain formed a single-headed structure, indicating that the residues Gln 945-Asp 991 are critical for the fomation of the two-headed structure. The actin activated ATPase activity of myosin mutants having a two-headed structure was activated by phosphorylation while that of myosin mutants that failed to form the two-headed structure was completely independent of phosphorylation. These results suggest that the two-headed structure is critical for the phosphorylation-dependent regulation.

Two Heads Are Required for Phosphorylation-dependent Regulation of Smooth Muscle Myosin

Recent structural evidence (Rayment, I., Holden, H. M., Whittaker, M., Yohn, C. B., Lorenz, M., Holmes, K. C., and Milligan, R. A. (1993) Science 261, 58-65) suggests that the two heads of skeletal muscle myosin interact when the protein is bound to filamentous actin. Direct chemical cross-linking experiments show that the two heads of smooth muscle myosin interact in the presence of filamentous actin and the absence of ATP (Onishi, H., Maita, T., Matsuda, G., and Fujiwara, K. (1992) Biochemistry 31, 1201-1210). Head-head interactions may be important in the mechanism of phosphorylation-dependent regulation of smooth muscle myosin. To explore the structural elements essential for phosphorylation-dependent regulation, we purified a proteolytic fragment of chicken gizzard myosin containing only one head attached to an intact tail. This molecule contained a partially digested regulatory light chain, which was replaced with exogenously added intact light chain in either the thiophosphorylated or the unphosphorylated state. Control experiments showed that this replacement was nearly quantitative and did not alter the actin-activated ATPase of this myosin. Electron micrographs confirmed that the single-headed preparation contained an intact form of single-headed myosin. The unphosphorylated single-headed myosin hydrolyzed ATP rapidly and moved actin filaments in an in vitro motility assay. Phosphorylation had minimal effects upon these properties. Therefore, we conclude that phosphorylation-dependent regulation in this myosin requires two heads. These findings may have important implications in studies of other regulated motor proteins that contain two motor domains.

Function of the NH2-terminal domain of the regulatory light chain on the regulation of smooth muscle myosin.

The role of the NH2-terminal domain of the 20,000-dalton light chain on the regulatory function of smooth muscle myosin was studied by exchanging it in myosin with various mutant forms. The 10 S to 6 S conformational transition as well as the thick filament formation were significantly influenced by the deletion or substitution of the amino acid residues at the NH2-terminal side of the phosphorylation site (Ser19). Whereas the deletion of Ser1-Thr10 did not significantly affect these functions, further deletion of Lys11-Arg16 completely abolished the formation of 10 S conformation and induced thick filament formation. Among the residues in this region, Arg13 and Arg16 were most important for these functions since substitution of these residues by Glu or Ala significantly altered these functions. Similar substitutions of Lys11 and Lys12 also stabilized the 6 S conformation and thick filament formation but less effectively. While the 6 S conformation was stabilized, the deletion of NH2-terminal residues did not activate the actin-activated ATPase activity. This suggests that stabilization of the 6 S conformation is not directly coupled with activation of actomyosin ATPase activity but rather a more defined conformational change around the phosphorylation site is necessary for activation. Such a change also influences the 6 S to 10 S conformation and, therefore, the filament formation. To support this notion, substitution of Lys11 and Lys12 by Glu-Glu inhibited the phosphorylation-induced activation of actomyosin ATPase activity.

Regulation of smooth muscle myosin

It is well established that light chain phosphorylation is required before a smooth muscle can generate force. The apparent modulation of shortening velocity by phosphorylation during sustained contractions may be accounted for by a mechanical interaction between rapidly cycling phosphorylated crossbridges and slowly or non-cycling dephosphorylated crossbridges. Latchbridges, force-producing dephosphorylated crossbridges, have been proposed to explain why force levels remain high at low levels of phosphorylation. The role of the thin-filament-associated proteins caldesmon and calponin in regulation remains enigmatic, but their inhibitory properties in solution would be consistent with a possible involvement in maintenance of a relaxed state.