Phosphorylation of myosin light chain kinase: a cellular mechanism for Ca2+ desensitization

Phosphorylation of the regulatory light chain of myosin by the Ca²⁺/calmodulin-dependent myosin light chain kinase plays an important role in smooth muscle contraction, nonmuscle cell shape changes, platelet contraction, secretion, and other cellular processes. Smooth muscle myosin light chain kinase is also phosphorylated, and recent results from experiments designed to satisfy the criteria of Krebs and Beavo for establishing the physiological significance of enzyme phosphorylation have provided insights into the cellular regulation and function of this phosphorylation in smooth muscle. The multifunctional Ca²⁺/calmodulin-dependent protein kinase II phosphorylates myosin light chain kinase at a regulatory site near the calmodulin-binding domain. This phosphorylation increases the concentration of Ca²⁺/calmodulin required for activation and hence increases the Ca²⁺ concentrations required for myosin light chain kinase activity in cells. However, the concentration of cytosolic Ca²⁺ required to effect myosin light chain kinase phosphorylation is greater than that required for myosin light chain phosphorylation. Phosphorylation of myosin light chain kinase is only one of a number of mechanisms used by the cell to down regulate the Ca²⁺ signal in smooth muscle. Since both smooth and nonmuscle cells express the same form of myosin light chain kinase, this phosphorylation may play a regulatory role in cellular processes that are dependent on myosin light chain phosphorylation.     

α1-Adrenergic signaling mechanisms in contraction of resistance arteries

Our goal in this review is to provide a comprehensive, integrated view of the numerous signaling pathways that are activated by ₁-adrenoceptors and control actin-myosin interactions (i.e., crossbridge cycling and force generation) in mammalian arterial smooth muscle. These signaling pathways may be categorized broadly as leading either to thick (myosin) filament regulation or to thin (actin) filament regulation. Thick filament regulation encompasses both "Ca²⁺ activation" and "Ca²⁺-sensitization" as it involves both activation of myosin light chain kinase (MLCK) by Ca²⁺-calmodulin and regulation of myosin light chain phosphatase (MLCP) activity. With respect to Ca²⁺ activation, adrenergically induced Ca²⁺ transients in individual smooth muscle cells of intact arteries are now being shown by high resolution imaging to be sarcoplasmic reticulum-dependent asynchronous propagating Ca²⁺ waves. These waves differ from the spatially uniform increases in [Ca²⁺] previously assumed. Similarly, imaging during adrenergic activation has revealed the dynamic translocation, to membranes and other subcellular sites, of protein kinases (e.g., Ca²⁺-activated protein kinases, PKCs) that are involved in regulation of MLCP and thus in "Ca²⁺ sensitization" of contraction. Thin filament regulation includes the possible disinhibition of actin-myosin interactions by phosphorylation of CaD, possibly by mitogen-activated protein (MAP) kinases that are also translocated during adrenergic activation. An hypothesis for the mechanisms of adrenergic activation of small arteries is advanced. This involves asynchronous Ca²⁺ waves in individual SMC, synchronous Ca²⁺ oscillations (at high levels of adrenergic activation), Ca²⁺ sparks, "Ca²⁺-sensitization" by PKC and Rho-associated kinase (ROK), and thin filament mechanisms.Electronic Supplementary Material Supplementary material is available for this article if you access the article at . A link in the frame on the left on that page takes you directly to the supplementary material.Abbreviations 2-APB 2-Aminoethoxydiphenylborate - ABS-1 Actin binding sequence no. 1 - BK Large conductance potassium channel - CaD Caldesmon - CaM Calmodulin - CaMKinase II Calmodulin kinase II - CaP Calponin - CICR Ca²⁺-induced Ca²⁺ release - CPA Cyclopiazonic acid - CPI-17 Protein kinase C-potentiated 17 kDa inhibitor protein - 2,4-DCB 2,4-Dichlorobenzamil - DAG Diacylglycerol - DHP Dihydropyridine - DOG 1,2-Dioctanoyl-sn-glycerol - ERK Extracellular-regulated kinase - FDS Frequent discharge sites - FRAP Fluorescence recovery after photobleaching - FRET Fluorescence resonance energy transfer - GEF Guanine nucleotide exchange factor - GS17C Fluorophore peptide antagonist of caldesmon - HA-1077 1-(5-Isoquinolinesulfonyl)homopiperazine, Di-HCl Salt - IICR InsP_3-induced Ca²⁺ release - ILK Integrin-linked kinase - InsP₃R 1,4,5-Trisphosphate receptor - IVC Inferior vena cava - jCaTs Junctional calcium transients - LC20 20,000 Da light chain of smooth muscle myosin - M20 Small noncatalytic subunit of myosin phosphatase - M130 Large noncatalytic subunit of myosin phosphatase - MAP kinase Mitogen-activated protein kinase - MEK MAPK kinase - ML-9 1-(5-Chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride - MLCK Myosin light chain kinase - MLCP Myosin light chain phosphatase - MLC₂₀ Myosin light chain 20 - MP Myosin phosphatase - MYPT1 Targeting subunit of myosin phosphatase - NCX Na/Ca exchanger - NE Norepinephrine - p160ROCK A rho kinase - PAK P21-activated kinase - PE Phenylephrine - PGF2 Prostaglandin factor 2 - PKC Protein kinase C - PKC- Protein kinase C- - PKN Rho effector, protein kinase C-related kinase - PL Plasmalemma - PLC Phospholipase C - PL-jSR Plasmalemma-junctional sarcoplasmic reticulum - PMA Phorbol 12-myristate 13-acetate - PP1c Catalytic subunit of myosin phosphatase - PSF Point spread function - PMCA Plasmalemma Ca²⁺ pumping ATPase - PM-SR Plasma membrane-sarcoplasmic reticulum - ROK Rho-associated kinase - RYR Ryanodine receptor - SBB Superficial buffer barrier - SERCA Sarcoplasmic reticulum Ca²⁺ ATPase - Ser/Thr Serine/threonine - SMC Smooth muscle cell - SMPP-1M Smooth muscle phosphatase-1M - SOC Store-operated channels - SR Sarcoplasmic reticulum - STOCs Spontaneous transient outward currents - TnI Inhibitory subunit troponin I - TPEN N,N,NN-tetrakis (2-pyridylmethyl) ethylenediamine - Tyr Tyrosine - UTP Uridine 5-triphosphate - VSMC Vascular smooth muscle cells - ZIP kinase Zipper interacting protein kinase The French version of this article is available in the form of electronic supplementary material and can be obtained by using the Springer Link server located at     

A multifunctional cyclic nucleotide- and Ca2+-independent protein kinase from rabbit skeletal muscle

A cyclic nucleotide- and Ca²⁺-independent protein kinase, initially identified as a glycogen synthase kinase (Itarte, E. and Huang, K.-P. (1979) J. Biol. Chem. $\text{254}$, 4052–4057), was also found to phosphorylate phosphorylase kinase and troponin from skeletal muscle as well as myosin light chain and myosin light chain kinase from both smooth and skeletal muscles. With the exception of myosin light chain from skeletal muscle, all the above-mentioned proteins are also substrates for the multifunctional cAMP-dependent protein kinase. The results suggest that this cyclic nucleotide- and Ca²⁺-independent protein kinase, like cAMP-dependent protein kinase, may have multiple cellular functions.     

Phosphorylation of smooth muscle myosin by type II Ca2+/calmodulin-dependent protein kinase

Brain type II Ca²⁺/calmodulin-dependent protein kinase was found to phoshorylate smooth muscle myosin, incorporating maximally 2 mol of phosphoryl per mol of myosin, exclusively on the 20,000 dalton light chain subunit. After maximal phosphorylation of myosin or the isolated 20,000 dalton light chain subunit by myosin light chain kinase, the addition of type II Ca²⁺/calmodulin-dependent protein kinase led to no further incorporation indicating the two kinases phosphorylated a common site. This conclusion was supported by two dimensional mapping of tryptic digests of myosin phosphorylated by the two kinases. By phosphoamino acid analysis the phosphorylated residue was identified as a serine. The phosphorylation by type II Ca ²⁺/calmodulin-dependent protein kinase of myosin resulted in enhancement of its actin-activated Mg²⁺-ATPase activity. Taken together, these data strongly support the conclusion that type II Ca²⁺/calmodulin-dependent protein kinase phosphorylates the same amino acid residue on the 20,000 dalton light chain subunit of smooth muscle myosin as is phosphorylated by myosin light chain kinase and suggest an alternative mechanism for the regulation of actin-myosin interaction.Abbreviations SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis - EGTA Ethylene Glycol Bis (-amino-ethyl ether)-N,N,N,N-Tetraacetic Acid - DTT Dithiothreitol - LC₂₀ Gizzard Smooth Muscle Phosphorylatable 20 kDa Myosin Light Chain - LC₁₇ Gizzard Smooth Muscle, 17 kDa Myosin Light Chain - H Chain Gizzard Smooth Muscle 200 kDa Myosin Heavy Chain - TPCK L-1-Tosylamido-2-Phenylethyl Chloromethyl Ketone - MOPS 3-(N-morpholino) Propanesulfonic Acid     

Ca2+-phospholipid dependent phosphorylation of smooth muscle myosin

Isolated myosin light chain from chicken gizzard has been shown to serve as a substrate for Ca²⁺-activated phospholipid-dependent protein kinase. Autoradiography showed that Ca²⁺-activated phospholipid-dependent protein kinase phosphorylated mainly the 20,000-dalton light chain of chicken gizzard myosin. Exogenously added calmodulin had no effect on myosin light chain phosphorylation catalyzed by the enzyme. The 20,000-dalton myosin light chain, both in the isolated form and in the whole myosin form, served as the substrate for this enzyme. In contrast to the isolated myosin light chain, the light chain of whole myosin was phosphorylated to a lesser extent by the Ca²⁺-activated phospholipid dependent kinase. Our results suggest the involvement of phospholipid in regulating Ca²⁺-dependent phosphorylation of the 20,000-dalton light chain of smooth muscle myosin.     

Phosphorylation of myosin light chain by protease activated kinase I

Protease activated kinase I from rabbit reticulocytes has been shown to phosphorylate the P-light chain of myosin light chains isolated from rabbit skeletal muscle. The enzyme is not activated by Ca²⁺ and calmodulin or phospholipids. Protease activated kinase I is not inhibited by trifluoperazine at concentrations up to 200 μM or by the antibody to the Ca²⁺, calmodulin-dependent myosin light chain kinase from rabbit skeletal muscle. Two-dimensional peptide mapping of chymotryptic digests of myosin P-light chain show the site phosphorylated by the protease activated kinase is different from that phosphorylated by the Ca²⁺, calmodulin-dependent myosin light chain kinase.     

Effects of purified myosin light chain kinase on myosin light chain phosphorylation and catecholamine secretion in digitonin-permeabilized chromaffin cells

Many non-muscle cells including chromaffin cells contain actin and myosin. The 20,000 dalton light chain subunits of myosin can be phosphorylated by a Ca²⁺/calmodulin-dependent enzyme, myosin light chain kinase. In tissues other than striated muscle, light chain phosphorylation is required for actin-induced myosin ATPase activity. The possibility that actin and myosin are involved in catecholamine secretion was investigated by determining whether increased phosphorylation in the presence of [-³²P]ATP of myosin light chain by myosin light chain kinase enhances secretion from digitonin-treated chromaffin cells. In the absence of exogenous myosin light chain kinase, 1 M Ca²⁺ caused a 30–40% enhancement of the phosphorylation of a 20 kDa protein. This protein was identified on 2-dimensional gels as myosin light chain by its comigration with purified myosin light chain. Purified myosin light chain kinase (400 g/ml) in the presence of calmodulin (10 M) caused little or no enhancement of myosin light chain phosphorylation in the absence of Ca²⁺ in digitonin-treated cells. In the presence of 1 M Ca²⁺, myosin light chain kinase (400 g/ml) caused an approximately two-fold increase in myosin light chain phosphorylation in digitonin-treated cells in 5 min. The phosphorylation required permeabilization of the cells by digitonin and occurred within the cells rather than in the medium. Myosin light chain kinase-induced phosphorylation of myosin light chain was maximal at 1 M. Ca²⁺. Under identical conditions to those of the phosphorylation experiments, secretion was unaltered by myosin light chain kinase. The experiments indicate that the phosphorylation of myosin light chain by myosin light chain kinase is not a limiting factor in secretion in digitonin-treated chromaffin cells and suggest that the activation of myosin is not directly involved in secretion from the cells. The experiments also demonstrate the feasibility of investigation of effects of exogenously added proteins on secretion in digitonin-treated cells.Abbreviations EGTA ethyleneglycol-bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - KGEPM solution containing potassium glutamate, EGTA, PIPES and MgCl₂ - NE norepinephrine - PIPES piperazine-N,-N-bis-(2-ethanesulfonic acid) - PSS physiological salt solution     

Evidence that myosin light chain phosphorylation regulates contraction in the body wall muscles of the sea cucumber

The Ca²⁺ activation mechanism of the longitudinal body wall muscles of Parastichopus californicus (sea cucumber) was studied using skinned muscle fiber bundles. Reversible phosphorylation of the myosin light chains correlated with Ca²⁺-activated tension and relaxation. Pretreatment of the skinned fibers with ATPγS and high Ca²⁺ (10^-5M) resulted in irreversible thiophosphorylation of the myosin light chains and activation of a Ca²⁺ insensitive tension. In contrast, pretreatment with low Ca²⁺ (10^-8M) and ATPγS results in no thiophosphorylation of the myosin light chains or irreversible activation of tension. These results are consistent with a Ca²⁺-sensitive myosin light chain kinase/phosphatase system being responsible for the activation of the muscle. Other agents known to have an effect upon the Ca²⁺-activated tension in skinned vertebrate smooth muscle fibers (trifluoperazine, catalytic subunit of the cyclic AMP-dependent protein kinase, and calmodulin) did not have an effect on myosin light chain phosphorylation or Ca²⁺-activated tension. These results suggest a different type of myosin light chain kinase than is found in vertebrate smooth muscle is responsible for the activation of parastichopus longitudinal body wall muscle.     

Myosin Loop 2 Is Involved in the Formation of a Trimeric Complex of Twitchin, Actin, and Myosin

Molluscan smooth muscles exhibit a low energy cost contraction called catch. Catch is regulated by twitchin phosphorylation and dephosphorylation. Recently, we found that the D2 fragment of twitchin containing the D2 site (Ser-4316) and flanking immunoglobulin motifs (TWD2-S) formed a heterotrimeric complex with myosin and with actin in the region that interacts with myosin loop 2 (Funabara, D., Hamamoto, C., Yamamoto, K., Inoue, A., Ueda, M., Osawa, R., Kanoh, S., Hartshorne, D. J., Suzuki, S., and Watabe, S. (2007) J. Exp. Biol. 210, 4399–4410). Here, we show that TWD2-S interacts directly with myosin loop 2 in a phosphorylation-sensitive manner. A synthesized peptide, CAQNKEAETTGTHKKRKSSA, based on the myosin loop 2 sequence (loop 2 peptide), competitively inhibited the formation of the trimeric complex. Isothermal titration calorimetry showed that TWD2-S binds to the loop 2 peptide with a K_a of (2.44 ± 0.09) × 10⁵ m⁻¹ with two binding sites. The twitchin-binding peptide of actin, AGFAGDDAP, which also inhibited formation of the trimeric complex, bound to TWD2-S with a K_a of (5.83 ± 0.05) × 10⁴ m⁻¹ with two binding sites. The affinity of TWD2-S to actin and myosin was slightly decreased with an increase of pH, but this effect could not account for the marked pH dependence of catch in permeabilized fibers. The complex formation also showed a moderate Ca²⁺ sensitivity in that in the presence of Ca²⁺ complex formation was reduced.Molluscan smooth muscles, such as mussel anterior byssus retractor muscle (ABRM)² and adductor muscle, exhibit a low energy cost phase of tension maintenance termed catch. Catch muscle develops active tension following an increase of the intracellular [Ca²⁺] induced by secretion of acetylcholine. Myosin is activated by direct binding of Ca²⁺ to the regulatory myosin light chain and initiates a relative sliding between thick and thin filaments (1). After a decrease of intracellular [Ca²⁺] to resting levels, the catch state is formed where tension is maintained over long periods of time with little energy consumption (2, 3). Catch tension is abolished by secretion of serotonin and an increase of intracellular [cAMP] with the resulting activation of cAMP-dependent protein kinase and phosphorylation of twitchin (4, 5). Twitchin phosphorylation is required for relaxation of the muscle from catch. For this cycle to repeat, dephosphorylation of twitchin is necessary (6). Thus, in this scheme, twitchin is a major regulator of the catch state.Molluscan twitchin is known as a myosin-binding protein belonging to the titin/connectin superfamily. It is a single polypeptide of 530 kDa containing multiple repeats of immunoglobulin (Ig) and fibronectin type 3-like motifs in addition to a single kinase domain homologous to the catalytic domain of myosin light chain kinase of vertebrate smooth muscle (7). There are several possible phosphorylation sites in molluscan twitchin recognized by cAMP-dependent protein kinase, and two, D1 and D2, have been identified. The D1 phosphorylation site (Ser-1075) is in the linker region between the 7th and 8th Ig motifs (numbering from the N terminus). The D2 site (Ser-4316) is in the linker region between the 21st and 22nd Ig motifs. Additional sites are found close to D1, but are thought not to be vital for catch regulation.The molecular mechanisms underlying development and maintenance of the catch state have been controversial for several years. One theory proposes that catch reflected attached frozen or slowly cycling cross-bridges (8, 9). What distinguished the attached cross-bridge from the detached relaxed state is not clear. Also it was suggested that interactions between thick filaments, other than cross-bridges, or between thin and thick filaments are responsible for the catch contraction (10). In either of the latter cases, the cross-bridge (myosin head) was not involved.Recently we found that a twitchin fragment including the D2 phosphorylation site and its flanking Ig motifs (TWD2-S) interacted with myosin and actin in a phosphorylation-sensitive manner, and it was suggested that this trimeric complex contributed to tension maintenance in catch (11). TWD2-S bound to a region of the actin molecule known also to interact with loop 2 of myosin that is involved in the ATP-driven movement of myosin with actin (12). In the present study, we show that the myosin loop 2 binds to TWD2-S using competitive cosedimentation assays and isothermal titration calorimetry (ITC). These techniques were applied to also study in more detail the interactions of the twitchin-binding peptide of actin (identified in the previous study (11)). In addition, the effects of pH and Ca²⁺ on the binding of TWD2-S to myosin and actin were investigated.     

Myosin Light Chain Kinase Is Necessary for Tonic Airway Smooth Muscle Contraction

Different interacting signaling modules involving Ca²⁺/calmodulin-dependent myosin light chain kinase, Ca²⁺-independent regulatory light chain phosphorylation, myosin phosphatase inhibition, and actin filament-based proteins are proposed as specific cellular mechanisms involved in the regulation of smooth muscle contraction. However, the relative importance of specific modules is not well defined. By using tamoxifen-activated and smooth muscle-specific knock-out of myosin light chain kinase in mice, we analyzed its role in tonic airway smooth muscle contraction. Knock-out of the kinase in both tracheal and bronchial smooth muscle significantly reduced contraction and myosin phosphorylation responses to K⁺-depolarization and acetylcholine. Kinase-deficient mice lacked bronchial constrictions in normal and asthmatic airways, whereas the asthmatic inflammation response was not affected. These results indicate that myosin light chain kinase acts as a central participant in the contractile signaling module of tonic smooth muscle. Importantly, contractile airway smooth muscles are necessary for physiological and asthmatic airway resistance.     

Myosin light chain kinase and the role of myosin light chain phosphorylation in skeletal muscle

Skeletal muscle myosin light chain kinase (skMLCK) is a dedicated Ca²⁺/calmodulin-dependent serine–threonine protein kinase that phosphorylates the regulatory light chain (RLC) of sarcomeric myosin. It is expressed from the MYLK2 gene specifically in skeletal muscle fibers with most abundance in fast contracting muscles. Biochemically, activation occurs with Ca²⁺ binding to calmodulin forming a (Ca²⁺)₄•calmodulin complex sufficient for activation with a diffusion limited, stoichiometric binding and displacement of a regulatory segment from skMLCK catalytic core. The N-terminal sequence of RLC then extends through the exposed catalytic cleft for Ser15 phosphorylation. Removal of Ca²⁺ results in the slow dissociation of calmodulin and inactivation of skMLCK. Combined biochemical properties provide unique features for the physiological responsiveness of RLC phosphorylation, including (1) rapid activation of MLCK by Ca²⁺/calmodulin, (2) limiting kinase activity so phosphorylation is slower than contraction, (3) slow MLCK inactivation after relaxation and (4) much greater kinase activity relative to myosin light chain phosphatase (MLCP). SkMLCK phosphorylation of myosin RLC modulates mechanical aspects of vertebrate skeletal muscle function. In permeabilized skeletal muscle fibers, phosphorylation-mediated alterations in myosin structure increase the rate of force-generation by myosin cross bridges to increase Ca²⁺-sensitivity of the contractile apparatus. Stimulation-induced increases in RLC phosphorylation in intact muscle produces isometric and concentric force potentiation to enhance dynamic aspects of muscle work and power in unfatigued or fatigued muscle. Moreover, RLC phosphorylation-mediated enhancements may interact with neural strategies for human skeletal muscle activation to ameliorate either central or peripheral aspects of fatigue.     

Signaling Processes for Initiating Smooth Muscle Contraction upon Neural Stimulation

Relationships among biochemical signaling processes involved in Ca²⁺/calmodulin (CaM)-dependent phosphorylation of smooth muscle myosin regulatory light chain (RLC) by myosin light chain kinase (MLCK) were determined. A genetically-encoded biosensor MLCK for measuring Ca²⁺-dependent CaM binding and activation was expressed in smooth muscles of transgenic mice. We performed real-time evaluations of the relationships among [Ca²⁺]_i, MLCK activation, and contraction in urinary bladder smooth muscle strips neurally stimulated for 3 s. Latencies for the onset of [Ca²⁺]_i and kinase activation were 55 ± 8 and 65 ± 6 ms, respectively. Both increased with RLC phosphorylation at 100 ms, whereas force latency was 109 ± 3 ms. [Ca²⁺]_i, kinase activation, and RLC phosphorylation responses were maximal by 1.2 s, whereas force increased more slowly to a maximal value at 3 s. A delayed temporal response between RLC phosphorylation and force is probably due to mechanical effects associated with elastic elements in the tissue. MLCK activation partially declined at 3 s of stimulation with no change in [Ca²⁺]_i and also declined more rapidly than [Ca²⁺]_i during relaxation. The apparent desensitization of MLCK to Ca²⁺ activation appears to be due to phosphorylation in its calmodulin binding segment. Phosphorylation of two myosin light chain phosphatase regulatory proteins (MYPT1 and CPI-17) or a protein implicated in strengthening membrane adhesion complexes for force transmission (paxillin) did not change during force development. Thus, neural stimulation leads to rapid increases in [Ca²⁺]_i, MLCK activation, and RLC phosphorylation in phasic smooth muscle, showing a tightly coupled Ca²⁺ signaling complex as an elementary mechanism initiating contraction.Increases in [Ca²⁺]_i3 in smooth muscle cells lead to Ca²⁺/CaM-dependent MLCK activation and RLC phosphorylation. Phosphorylation of RLC increases actin-activated myosin MgATPase activity leading to myosin cross-bridge cycling with force development (1–3).The activation of smooth muscle contraction may be affected by multiple cellular processes. Previous investigations show that free Ca²⁺/CaM is limiting for kinase activation despite the abundance of total CaM (4–6). The extent of RLC phosphorylation is balanced by the actions of MLCK and myosin light chain phosphatase, which is composed of three distinct protein subunits (7). The myosin phosphatase targeting subunit, MYPT1, in smooth muscle binds to myosin filaments, thus targeting the 37-kDa catalytic subunit (type 1 serine/threonine phosphatase, PP1c) to phosphorylated RLC. RLC phosphorylation and muscle force may be regulated by additional signaling pathways involving phosphorylation of RLC by Ca²⁺-independent kinase(s) and inhibition of myosin light chain phosphatase, processes that increase the contraction response at fixed [Ca²⁺]_i (Ca²⁺-sensitization) (8–14). Many studies indicate that agonist-mediated Ca²⁺-sensitization most often reflects decreased myosin light chain phosphatase activity involving two major pathways including MYPT1 phosphorylation by a Rho kinase pathway and phosphorylation of CPI-17 by PKC (8, 14–16). Additionally, phosphorylation of MLCK in its calmodulin-binding sequence by a Ca²⁺/calmodulin-dependent kinase pathway has been implicated in Ca²⁺ desensitization of RLC phosphorylation (17–19). How these signaling pathways intersect the responses of the primary Ca²⁺/CaM pathway during physiological neural stimulation is not known.There is also evidence that smooth muscle contraction requires the polymerization of submembranous cytoskeletal actin filaments to strengthen membrane adhesion complexes involved in transmitting force between actin-myosin filaments and external force-transmitting structures (20–23). In tracheal smooth muscle, paxillin at membrane adhesions undergoes tyrosine phosphorylation in response to contractile stimulation by an agonist, and this phosphorylation increases concurrently with force development in response to agonist. Expression of nonphosphorylatable paxillin mutants in tracheal muscle suppresses acetylcholine-induced tyrosine phosphorylation of paxillin, tension development, and actin polymerization without affecting RLC phosphorylation (24, 25). Thus, paxillin phosphorylation may play an important role in tension development in smooth muscle independently of RLC phosphorylation and cross-bridge cycling.Specific models relating signaling mechanisms in the smooth muscle cell to contraction dynamics are limited when cells in tissues are stimulated slowly and asynchronously by agonist diffusing into the preparation. Field stimulation leading to the rapid release of neurotransmitters from nerves embedded in the tissue avoids these problems associated with agonist diffusion (26, 27). In urinary bladder smooth muscle, phasic contractions are brought about by the parasympathetic nervous system. Upon activation, parasympathetic nerve varicosities release the two neurotransmitters, acetylcholine and ATP, that bind to muscarinic and purinergic receptors, respectively. They cause smooth muscle contraction by inducing Ca²⁺ transients as elementary signals in the process of nerve-smooth muscle communication (28–30). We recently reported the development of a genetically encoded, CaM-sensor for activation of MLCK. The CaM-sensor MLCK contains short smooth muscle MLCK fused to two fluorophores, enhanced cyan fluorescent protein and enhanced yellow fluorescent protein, linked by the MLCK calmodulin binding sequence (6, 14, 31). Upon dimerization, there is significant FRET from the donor enhanced cyan fluorescent protein to the acceptor enhanced yellow fluorescent protein. Ca²⁺/CaM binding dissociates the dimer resulting in a decrease in FRET intensity coincident with activation of kinase activity (31). Thus, CaM-sensor MLCK is capable of directly monitoring Ca²⁺/CaM binding and activation of the kinase in smooth muscle tissues where it is expressed specifically in smooth muscle cells of transgenic mice. We therefore combined neural stimulation with real-time measurements of [Ca²⁺]_i, MLCK activation, and force development in smooth muscle tissue from these mice. Additionally, RLC phosphorylation was measured precisely at specific times following neural stimulation in tissues frozen by a rapid-release electronic freezing device (26, 27). Results from these studies reveal that physiological stimulation of smooth muscle cells by neurotransmitter release leads to rapid increases in [Ca²⁺]_i, MLCK activation, and RLC phosphorylation at similar rates without the apparent activities of Ca²⁺-independent kinases, inhibition of myosin light chain phosphatase, or paxillin phosphorylation. Thus, the elemental processes for phasic smooth muscle contraction are represented by this tightly coupled Ca²⁺ signaling complex.     

Biochemical markers of contraction in human myometrial smooth muscle cells in culture

Summary Phosphorylation of a light chain subunit of myosin by Ca²⁺ and calmodulin-dependent myosin light chain kinase is believed to be essential for smooth muscle contraction. The biochemical properties of the myosin phosphorylation system in human myometrial smooth muscle cells in monolayer culture were compared with those of human myometrial tissue and nonmuscle cells in culture. Native myosin was isolated from other cellular proteins of crude homogenates by polyacrylamide gel electrophoresis (in the presence of pyrophosphate) and quantified by densitometry. The myosin content of myometrial smooth muscle cells in culture and that of myometrial tissue were similar and four- to five-fold greater than that of human endometrial stromal cells or skin fibroblasts in culture. The specific activities of myosin light chain kinase in homogenates of myometrial smooth muscle cells that were maintained in culture and in myometrial tissue were similar (2.05±0.18 and 1.60±0.37 nmol phosphate incorporated per min per mg protein, respectively). On the other hand, enzyme activity in skin fibroblasts was only 5% of that in myometrial smooth muscle cells. Myosin light chain kinase activity in myometrial smooth muscle cells was dependent upon Ca²⁺ and was inhibited reversibly by the calmodulin antagonist, calmidazolium. The intracellular Ca²⁺ concentration measured by quin2 fluorescence was 0.12 μM in resting cells and increased in a concentration-dependent manner with KC1 to a maximal value of 0.47 μM. These results indicate that biochemical processes important for smooth muscle contraction are retained in human myometrial smooth muscle cells in culture. This research was supported by grants HL26043, HD11149, and GM07062 from the National Institutes of Health, Bethesda, MD.     

Further characterization and thiophosphorylation of smooth muscle myosin

(i) Myosin from chicken gizzards was purified by a modification of an earlier procedure (M. N. Malik, 1978,Biochemistry17, 27–32). When this myosin, as well as that prepared by the method of A. Sobieszek and R. D. Bremel (1975,Eur. J. Biochem.55, 49–60), was analyzed by gradient slab gel using the discontinuous buffer system of Neville (1971,J. Biol. Chem.246, 6328–6334), a closely spaced doublet in the heavy chain and four light chains were observed as opposed to one heavy chain and two light chains with the method of Weber and Osborn (1969, J. Biol. Chem.244, 4406–4412). These findings raise the possibility of the existence of myosin isoenzymes in smooth muscle. (ii) The purified gizzard myosin was found to be free of kinase and phosphatase. Phosphorylation or thiophosphorylation of myosin was observed only by exogenously adding kinase. A maximum of 1.2 mol of ³²P/mol of myosin and 2.3 mol of ³⁵S/mol of myosin were obtained. The actin-activated ATPase activity depended upon the extent of thiophosphorylation of myosin; a four- to fivefold increase in the activity was observed when myosin was fully thiophosphorylated. Thiophosphorylated myosin was found to be more stable than phosphorylated myosin.     

Protein kinase network in the regulation of phosphorylation and dephosphorylation of smooth muscle myosin light chain

The contraction of smooth muscle is regulated primarily by intracellular Ca²⁺ signal. It is well established that the elevation of the cytosolic Ca²⁺ level activates myosin light chain kinase, which phosphorylates 20 kDa regulatory myosin light chain and activates myosin ATPase. The simultaneous measurement of cytosolic Ca²⁺ concentration and force development revealed that the alteration of the Ca²⁺-sensitivity of the contractile apparatus as well as the Ca²⁺ signal plays a critical role in the regulation of smooth muscle contraction. The fluctuation of an extent of myosin phosphorylation for a given change in Ca²⁺ concentration is considered to contribute to the major mechanisms regulating the Ca²⁺-sensitivity. The level of myosin phosphorylation is determined by the balance between phosphorylation and dephosphorylation. The phosphorylation level for a given Ca²⁺ elevation is increased either by Ca²⁺-independent activation of phosphorylation process or inhibition of dephosphorylation. In the last decade, the isolation and cloning of myosin phosphatase facilitated the understanding of regulatory mechanism of dephosphorylation process at the molecular level. The inhibition of myosin phosphatase can be achieved by (1) alteration of hetrotrimeric structure, (2) phosphorylation of 110 kDa regulatory subunit MYPT1 at the specific site and (3) inhibitory protein CPI-17 upon its phosphorylation. Rho-kinase was first identified to phosphorylate MYPT1, and later many kinases were found to phosphorylate MYPT1 and inhibit dephosphorylation of myosin. Similarly, the phosphorylation of CPI-17 can be catalysed by multiple kinases. Moreover, the myosin light chain can be phosphorylated by not only authentic myosin light chain kinase in a Ca²⁺-dependent manner but also by multiple kinases in a Ca²⁺-independent manner, thus adding a novel mechanism to the regulation of the Ca²⁺-sensitivity by regulating the phosphorylation process. It is now clarified that the protein kinase network is involved in the regulation of myosin phosphorylation and dephosphorylation. However, the physiological role of each component remains to be determined. One approach to accomplish this purpose is to investigate the effects of the dominant negative mutants of the signalling molecule on the smooth muscle contraction. In this regards, a protein transduction technique utilizing the cell-penetrating peptides would provide a useful tool. In the preliminary study, we succeeded in introducing a fragment of MYPT1 into the arterial strips, and found enhancement of contraction.     

Phosphorylation of a putative myosin light chain inChara by calcium-dependent protein kinase

Summary Ca²⁺-dependent protein kinase (CDPK) has been proposed to mediate inhibition by Ca²⁺ of cytoplasmic streaming in the green algaChara. We have identified the in vivo substrate(s) of CDPK inChara by using vacuolar perfusion of individual internodal cells with [-³²P]ATP. Phosphorylation of several polypeptides is enhanced when perfusions are performed at 10^–4M free Ca²⁺ compared to <10^–9M free Ca²⁺. The Ca²⁺-stimulated phosphorylation of these proteins is inhibited by the presence of a monoclonal antibody to soybean CDPK. One of these proteins is 16 to 18kDa and is recognized by an antibody against gizzard myosin light chains. These results demonstrate that inChara, several polypeptides are phophorylated by CDPK and one of these proteins has been tentatively identified as a myosin light chain. These observations support the hypothesis that Ca²⁺-regulated phosphorylation of myosin is involved in the regulation of cytoplasmic streaming.Abbreviations CDPK calcium-dependent protein kinase - mAb monoclonal antibody     

Signal transduction and motility ofDictyostelium

This review is concerned with the roles of cyclic GMP and Ca²⁺ ions in signal transduction for chemotaxis ofDictyostelium. These molecules are involved in signalling between the cell surface cyclic AMP receptors and cytoskeletal myosin II involved in chemotactic cell movement. Evidence is presented for uptake and/or eflux of Ca²⁺ being regulated by cyclic GMP. The link between Ca²⁺, cyclic GMP and chemotactic cell movement has been explored using streamer F mutants whose primary defect is in the structural gene for the cyclic GMP-specific phosphodiesterase. This mutation causes the mutants to produce an abnormally prolonged peak of cyclic GMP accumulation in response to stimulation with the chemoattractant cyclic AMP. The production and relay of cyclic AMP signals is normal in these mutants, but certain events associated with movement are (like the cyclic GMP response) abnormally prolonged in the mutants. These events include Ca²⁺ uptake, myosin II association with the cytoskeleton and regulation of both myosin heavy and light chain phosphorylation. These changes can be correlated with changes in the shape of the amoebae after chemotactic stimulation. Other mutants in which the accumulation of cyclic GMP in response to cyclic AMP stimulation was absent produced no myosin II responses.A model is described in which cyclic GMP (directly or indirectly via Ca²⁺) regulates accumulation of myosin II on the cytoskeleton by regulating phosphorylation of the myosin heavy and light chain kinases.     

Influence of myosin heavy chains on the Ca2+-binding properties of light chain, LC2

The association of myosin light chains with heavy chains, i.e. the intact oligomeric structure, profoundly affects the Ca²⁺-binding properties of the light chains. The Ca²⁺-binding affinity of the light chains is more than two magnitudes higher in the presence of heavy chains than in its absence. Modification of the reactive SH₂ thiol of myosin results in an alteration in the conformation of heavy chains of the molecule that influences the Ca²⁺-binding properties of light chains and generation of tension. When the SH₂ moiety is blocked with N-ethylmaleimide the influence of the heavy chains on the Ca²⁺-binding properties of light chain LC₂ is lost; under these conditions the Ca²⁺-binding affinity value of SH₂-N-ethylmaleimide-blocked myosin (3.3×10⁴m⁻¹) decreases to near that expressed with the dissociated light chain LC₂ (0.7×10⁴m⁻¹). Conversely, the presence of actin, nucleotides or modification of either the reactive lysyl residue or SH₂ thiol does not affect Ca²⁺ binding. The native secondary and tertiary structure of myosin seem to be required for Ca²⁺ binding; binding does not occur in the presence of 6m-urea with either native myosin or the dissociated light chains. With SH₂-N-ethylmaleimide-blocked myosin normal Ca²⁺- and (Mg²⁺+actin)-stimulated ATPase activities are expressed; however, there is a loss in K⁺-stimulated ATPase activity and the synthetic actomyosin threads of such myosin express no isometric tension. There are also variances in the binding of Ca²⁺ with alterations in pH values. In the absence of Ca²⁺/EGTA buffer the biphasic Ca²⁺-binding affinity of myosin is twice as high at pH7.4 (site one: 1.2×10⁶m⁻¹ and site two: 0.4×10⁶m⁻¹) as compared with values obtained at pH6.5 (site one: 0.64×10⁶m⁻¹ and site two: 0.2×10⁶m⁻¹). The Ca²⁺-binding affinity of light chain LC₂ and S₁, where the (S-1)–(S-2) junction was absent, were not influenced by changes in pH values. Both expressed a low Ca²⁺-binding affinity, approx. 0.7×10⁴m⁻¹, whereas heavy meromyosin, where both (S-1) and (S-2) myosin subfragments were present, expressed a Ca²⁺-binding affinity value similar to that of native myosin, but was not biphasic. However, it is important to point out than in preparation of S₁ myosin subfragment light chain LC₂ was lost and thus was added back to the purified S₁ fraction. Light chain LC₂ was not, however, added to the heavy meromyosin fraction because it was not lost during preparation of the heavy meromyosin subfragment. In conclusion, it appears that the (S-1)–(S-2) junction is needed for the positioning of light chain LC₂ and thus influences its essential conformation for Ca²⁺ binding.     

Effect of calcium and F-actin on the rate of SH2 modification in skeletal myosin

The rate constant of modification of a specific thiol group, SH₂, with N-ethylmaleimide (NEM) has been used to estimate the conformational change in the local area containing SH₂ (SH₂ region) of skeletal myosin as a structural probe. The rate of Mg²⁺-ATP-induced SH₂ modification of subfragment-1 (S-l) isozymes was regulated by Ca²⁺ in the pCa range below 6.4 and was not regulated in the pCa range above 6.4. No substantial difference between S-1 containing alkali light chain, A1, (S-1(A1)) and S-1 containing alkali light chain, A2, (S-1(A2)) was observed in the Ca²⁺-dependent rate of SH₂ modification. Due to the presence of this Ca²⁺ regulation in myosin (absence in S-1 isozymes) in the pCa range above 6.4, absence of 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) light chain in S-1 isozymes, and high affinity of Ca²⁺ for DTNB light chain, this Ca²⁺ regulation in the pCa range above 6.4 is possibly related to the Ca²⁺ binding to DTNB light chain. F-Actin, which is entirely free from tropomyosin and troponin, enhanced the rate of Mg²⁺-ATP-induced SH₂ modification of S-1 isozymes equally and of myosin, and reduced the Ca²⁺ sensitivity with an increase in F-actin concentration.     

Slow type muscle cells in the earthworm gizzard with a distinct,Ca2+-regulated myosin isoform

Summary Histochemical staining of the gizzard from the earthworm,Lumbricus terrestris, reveals low ATPase and high succinic dehydrogenase activity for all muscle cells as compared to the main part of the body wall. In accordance with the presence of slow type muscle cells in the gizzard, isolated actomyosin shows an ATPase activity three times lower than the body wall actomyosin.Gizzard myosin represents an isoform, distinct from those of the body wall muscle, by comparison of the light chain pattern in isoelectric focusing. No difference was observed in the Ca²⁺-regulatory properties between gizzard and body wall actomyosin. Gizzard actomyosin is dual-regulated, and the myosin contains a regulatory light chain which is reversibly dissociated by EDTA. Isolated gizzard binds two molecules of Ca²⁺ per molecule, in the same range of free Ca²⁺ concentrations over which actomyosin is activated, suggesting that the myosin-linked regulatory system is mediated by direct binding of Ca²⁺.The molar ratios of the major contractile proteins of body wall and gizzard actomyosins differ considerably, indicating a structural diversity of fast and slow type muscle cells.Abbreviations DTNB 5,5-dithio-bis-(2-nitrobenzoic acid) - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - HC myosin heavy chain(s) - HMM heavy meromyosin (product of limited proteolytic cleavage of myosin) - IEF isoelectric focusing - LC myosin light chain(s) - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonyl fluoride - SDH succinic dehydrogenase - SDS sodium dodecyl sulfate - Tris tris(hydroxymethyl)aminomethane