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Address correspondence to William Lehman, Department of Physiology and Biophysics, Boston University School of Medicine, 715 Albany St., Boston, MA 02118-2526. Tel.: (617) 638-4397. Fax: (617) 638-4273. E-mail: wlehman{at}bu.edu
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Abstract |
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Key Words: actin; electron microscopy; myosin; myosin light chain kinase; phosphorylation
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Introduction |
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Myosin regulatory light chains are phosphorylated by Ca2+calmodulin-dependent myosin light chain kinase (MLCK)* and dephosphorylated by a distinct form of phosphatase type 1 (for reviews see Kamm and Stull, 1985, 2001; Sellers and Adelstein, 1986; Erdödi et al., 1996; Somlyo and Somlyo, 2000). There is no troponin in smooth muscle, and here, as in nonmuscle cells, Ca2+ binds to its target protein, calmodulin, as cytoplasmic Ca2+ concentration increases after cell stimulation. The Ca2+calmodulin complex then binds to MLCK, leading to its activation, light chain phosphorylation, increased actin-activated myosin ATPase activity, cross-bridge cycling on thin filaments, and contraction. Both MLCK and phosphatase are modulated by phosphorylation and dephosphorylation, reactions mediated by signaling networks (Stull et al., 1996, 1997; Somlyo and Somlyo, 2000; MacDonald et al., 2001).
There are two genes that express a skeletal muscle specific MLCK and a smooth muscle MLCK, respectively. The smooth muscle MLCK is ubiquitously expressed (Herring et al., 2000) and consists of short and long isoforms due to multiple promoters in the gene (Kamm and Stull, 2001). Short MLCK is present in smooth muscle cells at 4 µM (Hartshorne, 1987; Tansey et al., 1994) in an approximate molar ratio to myosin of 1:10 and to actin in filaments of 1:100. The short MLCK isoform found in smooth muscle and some nonmuscle cells possesses an NH2-terminal actin-binding extension (Kanoh et al., 1993; Gallagher and Stull, 1997; Ye et al., 1997), not present in skeletal muscle MLCK (Nunnally and Stull, 1984; Lin et al., 1997), that localizes the kinase to thin filaments, restricting its cellular mobility (Guerriero et al., 1981; Dabrowska et al., 1982; Sellers and Pato, 1984). The long MLCK is the same as the short MLCK with additional actin-binding motifs and structural modules at the NH2 terminus. The long MLCK has a greater affinity for F-actin and is found primarily in nonmuscle cells (Kamm and Stull, 2001), where myosin IIbased motility is also regulated by light chain phosphorylation. The resulting compartmentalization of MLCK may be involved in defining contractile regions in both smooth muscle and nonmuscle cells.
The NH2-terminal extensions contain a novel high-affinity actin-binding sequence, DFRXXL, which repeats three times in short MLCK, leading to binding saturation of one MLCK to three actin monomers along F-actin in vitro (Smith et al., 1999; Smith and Stull, 2000). Mutating the binding motifs significantly decreases association of MLCK with myofilaments in vitro and with actin-containing filaments in intact smooth muscle cells (Smith et al.., 1999). The full-length short kinase is an elongated molecule, and intervening PEVK, Ig-like, and fibronectin-like repeats between NH2-terminal and more COOH-terminal catalytic domains are thought to provide sufficient extension for the catalytic core to reach thick filaments abutting thin filaments (Stull et al., 1996; Lin et al., 1999; Smith and Stull, 2000), thus allowing effective light chain phosphorylation of cross-bridges. A COOH-terminal low-affinity myosin-binding fragment in MLCK (Sellers and Pato, 1984) may direct the kinase towards myosin filaments, increasing the probability of myosin phosphorylation during activity, whereas the NH2 terminus binds with sufficient high affinity to anchor the kinase to actin filaments. Additionally, since MLCK apparently is not dissociated from thin filaments by Ca2+ or Ca2+calmodulin (Lin et al., 1999), the enzyme will localize close to thin and thick filaments both at the onset and during contractile activity.
Actin, the core of the thin filament, serves as a molecular track for the myosin cross-bridge motor. Tropomyosin, also present on thin filaments, ensures that onoff switching of myosin ATPase and consequently contractility occurs cooperatively, narrowing the Ca2+ concentration range required for regulation (Lehman et al., 2000). Smooth muscle and nonmuscle thin filaments also contain the protein caldesmon and smooth muscle filaments calponin, both often implicated in the regulation of thin filaments but of uncertain function despite extensive biochemical characterization (Marston and Huber, 1996; Chalovich et al., 1998; Marston et al., 1998; Winder et al., 1998). Structural information on the interactions of each of these proteins with F-actin has been extracted from three-dimensional (3-D) reconstructions (Vibert et al., 1993; Whittaker et al., 1995; Hodgkinson et al., 1997a,b; Lehman et al., 1997; Volkmann et al., 2000). Together, these proteins and myosin cover and/or move over much of the outer face of actin, leaving little room free for extra binding proteins, hence raising the possibility of a structural competition and functional clash with actin-bound MLCK. In the current report, the interactions of MLCK on F-actin were examined structurally. The site of MLCK binding was determined by electron microscopy and 3-D reconstruction to assess if MLCK is specifically and uniquely localized on F-actin. We found that MLCK associates between laterally adjacent actin monomers on F-actin, over an unoccupied patch far from the myosin-binding site and from sites occupied by tropomyosin, caldesmon, and calponin on actin. Hence, MLCK binding should not interfere sterically with myosin cross-bridge cycling, cooperative activation by tropomyosin, or the operation of caldesmon or calponin.
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Results and discussion |
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Materials and methods |
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Rabbit skeletal muscle F-actin was purified by standard methods (Spudich and Watt, 1971). The F-actin used was stable and did not aggregate, an advantage for the structural studies performed. Different actin isoforms are highly conserved (we used skeletal muscle -actin in our studies); isoform variability, in fact, occurs mainly at the acidic NH2-terminal six amino acids of actin (Vanderkerckhove and Weber, 1978; Lehman et al., 1996) whose position is distal from the site of MLCK-147 binding (see Results and discussion). There were no significant differences in binding affinity of NH2-terminal MLCK fragments to skeletal muscle F-actin and detergent washed native smooth muscle myofilaments; the stoichiometry of binding was the same (Smith and Stull, 2000; unpublished data).
Electron microscopy and helical reconstruction
F-actin (1 µM) was decorated with excess MLCK-147 (2 µM) in solutions of 50 mM KCl, 1 mM EGTA, 1 mM MgCl2, 0.2 mM ATP, 1 mM DTT, 10 mM imidazole buffer (pH 7.2) at room temperature (25°C). Reconstituted filaments were immediately applied to carbon-coated electron microscope grids and negatively stained as previously described (Moody et al., 1990). Prolonged decoration was avoided, since F-actin and MLCK-147 incubated together for as short as 5 min formed very large and unusable filament aggregates. Electron micrograph images of decorated filaments were recorded on a Philips CM120 electron microscope at 60,000x magnification under low-dose conditions (
12e-/Å2). Micrographs were digitized using a ZEISS SCAI scanner at a pixel size corresponding to 0.7 nm in the filaments. Well-stained regions of filaments were selected and straightened as previously described (Egelman, 1986; Hodgkinson et al., 1997a). Helical reconstruction was carried out by standard methods (DeRosier and Moore, 1970; Amos and Klug, 1975; Owen et al., 1996) as previously described (Vibert et al., 1993, 1997). Two independently analyzed sets of filaments totaling 28 filaments were chosen for averaging, based on their relative phase agreement. The reconstruction had a resolution (Owen and DeRosier, 1993) of 2.53.0 nm; the positional accuracy of the method is
0.5 nm (Milligan et al., 1990). Difference density analysis to define MLCK position on actin was carried out as previously described (Xu et al., 1999), and differences between maps were evaluated statistically using a Student's t test (Milligan and Flicker, 1987; Trachtenberg and DeRosier, 1987). Fitting of reconstructions to the atomic resolution structure of F-actin (Lorenz et al., 1993) was carried out as previously described (McGough and Way, 1995; Vibert et al., 1997; Xu et al., 1999) using the program O (Jones et al., 1991).
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Footnotes |
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* Abbreviations used in this paper: 3-D, three-dimensional; MLCK, myosin light chain kinase; MLCK-147; peptide containing the NH2-terminal 147 residues of MLCK.
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Acknowledgments |
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Submitted: 15 May 2001
Revised: 13 June 2001
Accepted: 3 July 2001
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References |
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