Myosin light chain kinase binding to actin filaments

Smooth muscle myosin light chain kinase (MLCK) plays important roles in contractile-motile processes of a variety of cells. Three DFRxxL motifs at the kinase N-terminus (residues 2–63) are critical for high-affinity binding to actin-containing filaments [Smith et al. (1999) J. Biol. Chem. 274, 29433–29438]. A GST fusion protein containing residues 1–75 of MLCK (GST75-MLCK) bound maximally to both smooth muscle myofilaments and F-actin at 0.28 and 0.31 mol GST75-MLCK/mol actin with respective K_D values of 0.1 μM and 0.8 μM. High-affinity binding of MLCK to actin-containing filaments may be due to each DFRxxL motif binding to one actin monomer in filaments.     

Properties of long myosin light chain kinase binding to F-actin in vitro and in vivo

Short and long myosin light chain kinases (MLCKs) are Ca(2+)/calmodulin-dependent enzymes that phosphorylate the regulatory light chain of myosin II in thick filaments but bind with high affinity to actin thin filaments. Three repeats of a motif made up of the sequence DFRXXL at the N terminus of short MLCK are necessary for actin binding (Smith, L., Su, X., Lin, P., Zhi, G., and Stull, J. T. (1999) J. Biol. Chem. 274, 29433-29438). The long MLCK has two additional DFRXXL motifs and six Ig-like modules in an N-terminal extension, which may confer unique binding properties for cellular localization. Two peptides containing either five or three DFRXXL motifs bound to F-actin and smooth muscle myofilaments with maximal binding stoichiometries consistent with each motif binding to an actin monomer in the filaments. Both peptides cross-linked F-actin and bound to stress fibers in cells. Long MLCK with an internal deletion of the five DFRXXL motifs and the unique NH(2)-terminal fragment containing six Ig-like motifs showed weak binding. Cell fractionation and extractions with MgCl(2) indicate that the long MLCK has a greater affinity for actin-containing filaments than short MLCK in vitro and in vivo. Whereas DFRXXL motifs are necessary and sufficient for short MLCK binding to actin-containing filaments, the DFRXXL motifs and the N-terminal extension of long MLCK confer high affinity binding to stress fibers in cells.     

Properties of filament-bound myosin light chain kinase

Myosin light chain kinase binds to actin-containing filaments from cells with a greater affinity than to F-actin. However, it is not known if this binding in cells is regulated by Ca2+/calmodulin as it is with F-actin. Therefore, the binding properties of the kinase to stress fibers were examined in smooth muscle-derived A7r5 cells. Full-length myosin light chain kinase or a truncation mutant lacking residues 2-142 was expressed as chimeras containing green fluorescent protein at the C terminus. In intact cells, the full-length kinase bound to stress fibers, whereas the truncated kinase showed diffuse fluorescence in the cytoplasm. After permeabilization with saponin, the fluorescence from the truncated kinase disappeared, whereas the fluorescence of the full-length kinase was retained on stress fibers. Measurements of fluorescence intensities and fluorescence recovery after photobleaching of the full-length myosin light chain kinase in saponin-permeable cells showed that Ca2+/calmodulin did not dissociate the kinase from these filaments. However, the filament-bound kinase was sufficient for Ca2+-dependent phosphorylation of myosin regulatory light chain and contraction of stress fibers. Thus, dissociation of myosin light chain kinase from actin-containing thin filaments is not necessary for phosphorylation of myosin light chain in thick filaments. We note that the distance between the N terminus and the catalytic core of the kinase is sufficient to span the distance between thin and thick filaments.     

Localization of an actin binding domain in smooth muscle myosin light chain kinase

Phosphorylation of the regulatory light chain of myosin II by myosinlight chain kinase is important for regulating many contractile processes.Smooth muscle myosin light chain kinase has been shown to be associated withboth actin and myosin filaments in vitro and in vivo. In this report wedefine an actin binding region by using molecular deletions to generaterecombinant mutant proteins that were analyzed by co-sedimentation withF-actin. An actin binding region restricted to residues 2-42 in the animoterminus of the rabbit smooth muscle myosin light chain kinase wasidentified.     

Molluscan catch muscle myorod and its N-terminal peptide bind to F-actin and myosin in a phosphorylation-dependent manner

Myorod is expressed exclusively in molluscan catch muscle and localizes on the surface of thick filaments together with twitchin and myosin. This protein is an alternatively spliced product of the myosin heavy-chain gene containing the C-terminal rod part of myosin and a unique N-terminal domain. We have recently reported that this unique domain is a target for phosphorylation by gizzard smooth muscle myosin light chain kinase (MLCK) and molluscan twitchin, which contains a MLCK-like domain. To elucidate the role of myorod phosphorylation in catch muscle, a peptide corresponding to the specific N-terminal region of the protein was synthesized in phosphorylated and unphosphorylated form. We report, for the first time, that unphosphorylated full-length myorod and its unphosphorylated N-terminal synthetic peptide are able to interact with rabbit F-actin and thin filaments from molluscan catch muscle. The binding between thin filaments and the peptide was Ca²⁺-dependent. In addition, we found that phosphorylated N-terminal peptide of myorod has higher affinity for myosin compared to the unphosphorylated peptide. Together, these observations suggest the direct involvement of the N-terminal domain of myorod in the regulation of molluscan catch muscle.     

The calmodulin binding domain of chicken smooth muscle myosin light chain kinase contains a pseudosubstrate sequence

Smooth muscle myosin light chain kinase contains a 64 residue sequence that binds calmodulin in a Ca2+-dependent manner (Guerriero, V., Jr., Russo, M. A., and Means, A. R. (1987) Biochemistry, in press). Within this region is a sequence with homology to the corresponding sequence reported for the calmodulin binding region of skeletal muscle myosin light chain kinase (Blumenthal, D. K., Takio, K., Edelman, A. M., Charbonneau, H., Titani, L., Walsh, K. A., and Krebs, E. G. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 3187-3191). Inspection of these sequences reveals that they both share a similar number and spatial arrangement of basic residues with those present in the myosin light chain substrate. We have synthesized a 22-residue peptide corresponding to residues 480-501 (determined from the cDNA) of the smooth muscle myosin light chain kinase. This peptide, Ala-Lys-Lys-Leu-Ser-Lys-Asp-Arg-Met-Lys-Lys-Tyr-Met-Ala-Arg-Arg-Lys-Trp- Gln-Lys-Thr-Gly, inhibited calmodulin-dependent activation of the smooth muscle myosin light chain kinase with an IC50 of 46 nM. At saturating concentrations of calmodulin, the 22-residue peptide inhibited myosin light chain and synthetic peptide substrate phosphorylation competitively with IC50 values of 2.7 and 0.9 microM, respectively. An 11-residue synthetic peptide analog, corresponding to part of the calmodulin-binding sequence in skeletal muscle myosin light chain kinase, Lys-Arg-Arg-Trp-Lys-Lys-Asn-Phe-Ile-Ala-Val, also competitively inhibited synthetic peptide substrate phosphorylation with a Ki of 1 microM. The competitive inhibitory activity of the calmodulin binding regions is similar to the apparent Km of 2.7 microM for phosphorylation of the 23-residue peptide analog of the smooth muscle myosin light chain and raises the possibility that the calmodulin binding region of the myosin light chain kinase may act as a pseudosubstrate inhibitor of the enzyme.     

Phosphorylation of synthetic peptides by skeletal muscle myosin light chain kinases

Substrate determinants for rabbit and chicken skeletal muscle myosin light chain kinases were examined with synthetic peptides. Both skeletal muscle myosin light chain kinases had similar phosphorylation kinetics with synthetic peptide substrates. Average kinetic constants for skeletal muscle myosin light chain heptadecapeptide, (formula; see text) where S(P) is phosphoserine, were Km, 2.3 microM and Vmax, 0.9 mumol/min/mg of enzyme. Km values were 122 and 162 microM for skeletal muscle peptides containing A-A for basic residues at positions 2-3 and 6-7, respectively. Average kinetic constants for smooth muscle myosin light chain peptide, (formula; see text), were Km, 1.4 microM and Vmax 27 mumol/min/mg of enzyme. Average Km values for the smooth muscle peptide, residues 11-23, were 10 microM which increased 6- and 11-fold with substitutions of alanine at residues 12 and 13, respectively. Vmax values decreased and Km values increased markedly by substitution of residue 16 with glutamate in the 11-23 smooth muscle tridecapeptide. Basic residues located 3 and 6-7 residues toward the NH2 terminus from phosphoserine in smooth muscle myosin light chain and 6-8 and 10-11 residues toward the NH2 terminus from phosphoserine in skeletal muscle myosin light chain appear to be important substrate determinants for skeletal muscle myosin light chain kinases. These properties are different from myosin light chain kinase from smooth muscle.     

Microfilament-binding properties of N-terminal extension of the isoform of smooth muscle long myosin light chain kinase

Myosin light chain kinases （MLCK） phosphorylate the regulatory light chain of myosin II in thick filaments and bind to F-actin-containing thin filaments with high affinity. The ability of short myosin light chain kinase （S-MLCK） to bind F-actin is structurally attributed to the DFRXXL regions in its N-terminus. The long myosin light chain kinase （L-MLCK） has two additional DFRXXL motifs and six Ig-like modules in its N-terminal extension. The six Ig-like modules are capable of binding to stress fibers independently. Our results from the imaging analysis demonstrated that the first two intact Ig-like modules （2Ig） in N-terminal extension of L-MLCK is the minimal binding module required for microfilament binding. Binding assay confirmed that F-actin was able to bind 2Ig. Stoichiometries of 2Ig peptide were similar for myofilament or pure F-actin. The binding affinities were slightly lower than 5DFRXXL peptide as reported previously. Similar to DFRXXL peptides, the 2Ig peptide also caused efficient F-actin bundle formation in vitro. In the living cell, over-expression of 2Ig fragment increased ＂spike＂-like protrusion formation with over-bundled F-actin. Our results suggest that L-MLCK may act as a potent F-actin bundling protein via its DFRXXL region and the 2Ig region, implying that L-MLCK plays a role in cytoskeleton organization.     

Substrate specificity of myosin light chain kinases.

Skeletal muscle myosin light chain kinase can phosphorylate myosin light chains isolated from skeletal or smooth muscle. In contrast, smooth muscle myosin light chain kinase specifically phosphorylates light chains isolated from smooth muscle. In this study, we have identified residues within the rabbit smooth and skeletal muscle myosin light chain kinases which may interact with the basic residues that are important substrate determinants in the light chains. Mutation of aspartic acid 270 amino-terminal of the catalytic core of the skeletal muscle myosin light chain kinase increased the Km value for both smooth and skeletal muscle light chains. Although deletions of the analogous region of the smooth muscle myosin light chain kinase (residues 663-678) markedly increased the Km value for light chain, mutation of any single acidic residue within this region did not have a similar effect. Mutation of single residues within the catalytic core of the skeletal muscle (E377 and E421) and smooth muscle (E777 and E821) myosin light chain kinases increased Km values for the smooth muscle light chain at least 35- and 100-fold, respectively. It is proposed that these residues may form ionic interactions with the arginine that is 3 residues amino-terminal of the phosphorylatable serine in the smooth muscle light chain.     

Sites phosphorylated in myosin light chain in contracting smooth muscle

Purified smooth muscle myosin light chain can be phosphorylated at multiple sites by myosin light chain kinase and protein kinase C. We have determined the sites phosphorylated on myosin light chain in intact bovine tracheal smooth muscle. Stimulation with 10 microM carbachol resulted in 66 +/- 5% monophosphorylated and 11 +/- 2% diphosphorylated myosin light chain after 1 min, and 47 +/- 4% monophosphorylated and 5 +/- 2% diphosphorylated myosin light chain after 30 min. Myosin heavy chain contained 0.06 +/- 0.01 mol of phosphate/mol of protein which did not change with carbachol. At both 1 and 30 min the monophosphorylated myosin light chain contained only phosphoserine whereas the diphosphorylated myosin light chain contained both phosphoserine and phosphothreonine. Two-dimensional peptide mapping of tryptic digests of monophosphorylated and diphosphorylated myosin light chain obtained from carbachol-stimulated tissue was similar to the peptide maps of purified light chain monophosphorylated and diphosphorylated, respectively, by myosin light chain kinase; these maps were distinct from the map obtained with tracheal light chain phosphorylated by protein kinase C. Phosphorylation of tracheal smooth muscle myosin light chain by myosin light chain kinase yields the tryptic phosphopeptide ATSNVFAMFDQSQIQEFK with S the phosphoserine in the monophosphorylated myosin light chain and TS the phosphotreonine and phosphoserine in the diphosphorylated myosin light chain. Thus, stimulation of tracheal smooth muscle with a high concentration of carbachol results in formation of both monophosphorylated and diphosphorylated myosin light chain although the amount of diphosphorylated light chain is substantially less than monophosphorylated light chain. In the intact muscle, myosin light chain is phosphorylated at sites corresponding to myosin light chain kinase phosphorylation.     

Binding of hormones and neuropeptides by calmodulin

Calmodulin exhibits high-affinity, calcium-dependent binding of 1 mol/mol of the vasoactive intestinal peptide (VIP), secretin, and either the 42- or 43-residue gastric inhibitory peptide (GIP) with dissociation constants of 0.05-0.14 microM. The affinity of VIP for calmodulin approaches its affinity for the cell-surface VIP receptors. These peptides compete with both smooth muscle myosin light chain kinase and glucagon in calmodulin binding. Calculation of amino acid frequencies for eight calmodulin binding peptides (VIP, GIP, secretin, ACTH, beta-endorphin, substance P, glucagon, and dynorphin [Malencik, D. A., & Anderson, S. R. (1982) Biochemistry 21, 3480]) shows a below-average incidence of glutamyl residues, above-average incidence of glutaminyl residues, and average incidence of both aspartyl and asparaginyl residues. Predictions of structure from sequence suggest that the bound peptides contain strongly basic turns and coils in close association with regions having above-average beta-sheet potential. The temperature dependence of glucagon binding by calmodulin shows that the association is enthalpy driven.     

Association of melittin with the isolated myosin light chains

Melittin is a 26-residue peptide which undergoes high-affinity calcium-dependent binding by calmodulin [Barnette, M.S., Daly, R., & Weiss, B. (1983) Biochem. Pharmacol. 32, 2929; Comte, M., Maulet, Y., & Cox, J.A. (1983) Biochem. J. 209, 269; Anderson, S.R., & Malencik, D.A. (1986) Calcium Cell Funct. 6, 1]. The results in this paper show that three different types of myosin light chain--the smooth muscle regulatory light chain, the smooth muscle essential light chain, and the skeletal muscle regulatory 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) light chain--also associate with melittin. The resulting complexes have dissociation constants ranging from 1.1 to 2.5 microM in the presence of 0.10 M NaCl and from approximately 50 to approximately 130 nM in solutions of 20 mM 3-(N-morpholino)propanesulfonic acid alone. The regulatory smooth muscle myosin light chain exhibits two equivalent melittin binding sites while each of the others displays only one. The myosin light chains evidently contain elements of structure related to the macromolecular interaction sites present in calmodulin and troponin C but not in parvalbumin. The association of melittin and other peptides with the light chains requires consideration whenever assays of the calmodulin-dependent activity of myosin light chain kinase are used to determine peptide binding by calmodulin. The binding measurements performed on the DTNB light chain and melittin necessitated derivation of the equation relating complex formation to the observed fluorescence anisotropy of a solution containing three fluorescent components. This analysis is generally applicable to equilibria involving the association of two fluorescent molecules emitting in the same wavelength range.     

Functional assembly of fragments from bisected smooth muscle myosin light chain kinase

The C-terminal regulatory segment of smooth muscle myosin light chain kinase folds back on its catalytic core to inhibit kinase activity. This regulatory segment consists of autoinhibitory residues linking the catalytic core to the calmodulin-binding sequence and perhaps additional C-terminal residues including an immunoglobulin-like motif. However, mutational and biochemical analyses showed no specific involvement of residues C-terminal to the calmodulin-binding sequence. To obtain additional insights on the proposed mechanisms for autoinhibition and Ca(2+)/calmodulin activation of the kinase, the polypeptide backbone chain of myosin light chain kinase was cleaved by genetic means to produce N- and C-terminal protein fragments. The N-terminal fragment containing the catalytic core was catalytically inactive when expressed alone. Co-expression of the N-terminal fragment with the C-terminal fragment containing the regulatory segment restored kinase activity. Deletion of the autoinhibitory linker residues without or with the calmodulin-binding sequence prevented restoration of kinase activity. In the presence or absence of Ca(2+)/calmodulin, regulatory segment binding occurred through the linker region connecting the catalytic core to the calmodulin-binding sequence. Collectively, these results indicate that residues C-terminal to the calmodulin-binding sequence (including the immunoglobulin-like motif) are not functional components of the regulatory segment. Furthermore, the principal autoinhibitory motif is contained in the sequence linking the catalytic core of myosin light chain kinase to the calmodulin-binding sequence.     

Localisation of light chain and actin binding sites on myosin

A gel overlay technique has been used to identify a region of the myosin S-1 heavy chain that binds myosin light chains (regulatory and essential) and actin. The 125I-labelled myosin light chains and actin bound to intact vertebrate skeletal or smooth muscle myosin, S-1 prepared from these myosins and the C-terminal tryptic fragments from them (i.e. the 20-kDa or 24-kDa fragments of skeletal muscle myosin chymotryptic or Mg2+/papain S-1 respectively). MgATP abolished actin binding to myosin and to S-1 but had no effect on binding to the C-terminal tryptic fragments of S-1. The light chains and actin appeared to bind to specific and distinct regions on the S-1 heavy chain, as there was no marked competition in gel overlay experiments in the presence of 50-100 molar excess of unlabelled competing protein. The skeletal muscle C-terminal 24-kDa fragment was isolated from a tryptic digest of Mg2+/papain S-1 by CM-cellulose chromatography, in the presence of 8 M urea. This fragment was characterised by retention of the specific label (1,5-I-AEDANS) on the SH1 thiol residue, by its amino acid composition, and by N-terminal and C-terminal sequence analyses. Electron microscopical examination of this S-1 C-terminal fragment revealed that: it had a strong tendency to form aggregates with itself, appearing as small 'segment-like' structures that formed larger aggregates, and it bound actin, apparently bundling and severing actin filaments. Further digestion of this 24-kDa fragment with Staphylococcus aureus V-8 protease produced a 10-12-kDa peptide, which retained the ability to bind light chains and actin in gel overlay experiments. This 10-12-kDa peptide was derived from the region between the SH1 thiol residue and the C-terminus of S-1. It was further shown that the C-terminal portion, but not the N-terminal portion, of the DTNB regulatory light chain bound this heavy chain region. Although at present nothing can be said about the three-dimensional arrangement of the binding sites for the two kinds of light chain (regulatory and essential) and actin in S-1, it appears that these sites are all located within a length of the S-1 heavy chain of about 100 amino acid residues.     

Myosin phosphatase-Rho interacting protein. A new member of the myosin phosphatase complex that directly binds RhoA

Regulation of vascular smooth muscle cell contractile state is critical for the maintenance of blood vessel tone. Abnormal vascular smooth muscle cell contractility plays an important role in the pathogenesis of hypertension, blood vessel spasm, and atherosclerosis. Myosin phosphatase, the key enzyme controlling myosin light chain dephosphorylation, regulates smooth muscle cell contraction. Vasoconstrictor and vasodilator pathways inhibit and activate myosin phosphatase, respectively. G-protein-coupled receptor agonists can inhibit myosin phosphatase and cause smooth muscle cell contraction by activating RhoA/Rho kinase, whereas NO/cGMP can activate myosin phosphatase and cause smooth muscle cell relaxation by activation of cGMP-dependent protein kinase. We have used yeast two-hybrid screening to identify a 116-kDa human protein that interacts with both myosin phosphatase and RhoA. This myosin phosphatase-RhoA interacting protein, or M-RIP, is highly homologous to murine p116RIP3, is expressed in vascular smooth muscle, and is localized to actin myofilaments. M-RIP binds directly to the myosin binding subunit of myosin phosphatase in vivo in vascular smooth muscle cells by an interaction between coiled-coil and leucine zipper domains in the two proteins. An adjacent domain of M-RIP directly binds RhoA in a nucleotide-independent manner. M-RIP copurifies with RhoA and Rho kinase, colocalizes on actin stress fibers with RhoA and MBS, and is associated with Rho kinase activity in vascular smooth muscle cells. M-RIP can assemble a complex containing both RhoA and MBS, suggesting that M-RIP may play a role in myosin phosphatase regulation by RhoA.     

Regulation of embryonic smooth muscle myosin by protein kinase C

Phosphorylation of the 20-kDa light chain regulates adult smooth muscle myosin; phosphorylation by the Ca2+/calmodulin-dependent enzyme myosin light chain kinase stimulates the actomyosin ATPase activity of adult smooth muscle myosin; the simultaneous phosphorylation of a separate site on the 20-kDa light chain by the Ca2+/phospholipid-dependent enzyme protein kinase C attenuates the myosin light chain kinase-induced increase in the actomyosin ATPase activity of adult myosin. Fetal smooth muscle myosin, purified from 12-day-old fertilized chicken eggs, is structurally different from adult smooth muscle myosin. Nevertheless, phosphorylation of a single site on the 20-kDa light chain of fetal myosin by myosin light chain kinase results in stimulation of the actomyosin ATPase activity of this myosin. Protein kinase C, in contrast, phosphorylates three sites on the fetal myosin 20-kDa light chain including a serine or threonine residue on the same peptide phosphorylated by myosin light chain kinase. Interestingly, phosphorylation by protein kinase C stimulates the actomyosin ATPase activity of fetal myosin. Moreover, unlike adult myosin, there is no attenuation of the actomyosin ATPase activity when fetal myosin is simultaneously phosphorylated by myosin light chain kinase and protein kinase C. These data demonstrate, for the first time, the in vitro activation of a smooth muscle myosin by another enzyme besides myosin light chain kinase and raise the possibility of alternate pathways for regulating smooth muscle myosin in vivo.     

Phosphorylation of smooth muscle myosin light chain kinase by the catalytic subunit of adenosine 3': 5'-monophosphate-dependent protein kinase.

Turkey gizzard smooth muscle light chain kinase was purified by affinity chromatography on calcium dependent regulator weight of 125,000 +/- 5,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When myosin light chain kinase is incubated with the catalytic subunit of cyclic AMP-dependent protein kinase, 1 mol of phosphate is incorporated per mol of myosin kinase. Brief tryptic digestion of the 32P-labeled myosin kinase liberates a single radioactive peptide with a molecular weight of approximately 22,000. Phosphorylation of myosin kinase results in a 2-fold decrease in the rate at which the enzyme phosphorylates the 20,000-dalton light chain of smooth muscle myosin. These results suggest that cyclic AMP has a direct effect on actin-myosin interaction in smooth muscle.     

平滑肌肌球蛋白磷酸化与肌动球蛋白功能调节

在有Ｃａ２＋和钙调蛋白存在时,肌球蛋白轻链激酶催化肌球蛋白磷酸化,促使肌动蛋白激活的肌球蛋白（肌动球蛋白）Ｍｇ２＋－ＡＴＰ酶活性显著增加．然而,肌球蛋白磷酸化水平与Ｍｇ２＋－ＡＴＰ酶之间的关系是非线性的,原肌球蛋白可以进一步增加Ｍｇ２＋－ＡＴＰ酶的活性,但仍不改变它们之间的非线性关系．肌球蛋白轻链激酶的合成肽抑制剂抑制了肌球蛋白磷酸化和Ｍｇ２＋－ＡＴＰ酶活性,并导致平滑肌去膜肌纤维的等长收缩张力与速度的降低．结果提示肌球蛋白轻链激酶参与脊椎动物平滑肌收缩的调节过程,肌球蛋白轻链磷酸化作用会引起平滑肌收缩     

Biochemical properties of chimeric skeletal and smooth muscle myosin light chain kinases.

The molecular and biochemical properties of myosin light chain kinases from chicken skeletal and smooth muscle were investigated by recombinant DNA techniques. Deletion of the amino-terminal region of either the smooth or skeletal muscle myosin light chain kinase resulted in a decrease in Vmax with no significant change in Km values for light chain substrates. Skeletal/smooth muscle chimeric kinases were inactive when a 65-residue region amino-terminal of the catalytic core was exchanged between the two forms. Changing alanine 494 to glutamic acid within this region in the chicken skeletal muscle myosin light chain kinase increased the Km values for light chains 10-fold. These results are consistent with the hypothesis that the region amino-terminal of the catalytic core in myosin light chain kinases is involved in light chain recognition. A skeletal muscle kinase which contained the smooth muscle calmodulin binding domain remained regulated by Ca2+/calmodulin. Thus, the calmodulin binding domains of smooth and skeletal muscle myosin light chain kinases share structural elements necessary for regulation.     

A variant derived from rabbit aortic smooth muscle: phenotype modulation and restoration of smooth muscle characteristics in cells in culture

We examined the relationship between growth arrest of smooth muscle cells and structural changes in microfilament bundles, and also that between the structural changes and the actions of contractile agonist using a multipassagable variant cell line (SM-3) derived from rabbit aortic smooth muscle cells. The content of smooth muscle type alpha-actin increased with density-dependent growth arrest of the SM-3 cells, but was attenuated in the logarithmically growing cultures. As assessed cytochemically, the growth-arrested cells contained longitudinally oriented bundles of actin-containing microfilament and myosin-based filaments visualized with rhodamine-phalloidin and antibody against myosin light chain 20, respectively, whereas both actin- and myosin-containing structures in logarithmically growing cells showed slight, shortened, or diffused patterns. Electron microscopic examination of the growth-arrested cells revealed that the cells contained numerous and conspicuous microfilament bundles associated with many compact electron-dense bodies. In addition, pinocytotic vesicles were often found near the plasma membrane in the growth-arrested cells. SM-3 cells in the growth-arrested phase responded to prostaglandin F2 alpha (3-30 microM) and rat endothelin (0.1-1.0 microM) with a reversible contractile response, in association with monophosphorylation and/or diphosphorylation of the myosin light chain 20. However, the influence of the contractile agonists was greatly reduced during logarithmic growth. These results suggest that in the SM-3 cells in the growth-arrested phase, there is a restoration of the contractile architecture and the myosin light chain phosphorylation system. Thus, this SM-3 cell line is expected to serve as a useful model for examining biochemical and physiological phenomena of smooth muscle.