Myosin light chain kinase binding to a unique site on F-actin revealed by three-dimensional image reconstruction

Department of Physiology and Biophysics, Boston University School of Medicine, Boston, MA 02118-2526, USA.
The Journal of Cell Biology (Impact Factor: 9.83). 09/2001; 154(3):611-7. DOI: 10.1083/jcb.200105079
Source: PubMed


Ca2+-calmodulin-dependent phosphorylation of myosin regulatory light chains by the catalytic COOH-terminal half of myosin light chain kinase (MLCK) activates myosin II in smooth and nonmuscle cells. In addition, MLCK binds to thin filaments in situ and F-actin in vitro via a specific repeat motif in its NH2 terminus at a stoichiometry of one MLCK per three actin monomers. We have investigated the structural basis of MLCK-actin interactions by negative staining and helical reconstruction. F-actin was decorated with a peptide containing the NH2-terminal 147 residues of MLCK (MLCK-147) that binds to F-actin with high affinity. MLCK-147 caused formation of F-actin rafts, and single filaments within rafts were used for structural analysis. Three-dimensional reconstructions showed MLCK density on the extreme periphery of subdomain-1 of each actin monomer forming a bridge to the periphery of subdomain-4 of the azimuthally adjacent actin. Fitting the reconstruction to the atomic model of F-actin revealed interaction of MLCK-147 close to the COOH terminus of the first actin and near residues 228-232 of the second. This unique location enables MLCK to bind to actin without interfering with the binding of any other key actin-binding proteins, including myosin, tropomyosin, caldesmon, and calponin.

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    • "A ''parking problem'' on filaments might therefore result in restricting binding associations on actin, particularly given the limited binding surface on actin. This consideration has been partially addressed by EM reconstruction, mapping the F-actin surface associated with smooth muscle tropomyosin (Hodgkinson et al. 1997a, b; Lehman et al. 2000, 2009), the actin-binding domain of smooth muscle myosin light chain kinase (Hatch et al. 2001), full-length and expressed caldesmon and its actin-binding fragments (Vibert et al. 1993; Hodgkinson et al. 1997b; Lehman et al. 1997; Foster et al. 2004), full-length calponin (Hodgkinson et al. 1997b; Galkin et al. 2006), and engineered constructs representing paired calponin-homology (CH) domains derived from diverse actin-binding partners such as a-actinin (McGough et al. 1994, 2003), dystrophin (Sutherland-Smith et al. 2003), fimbrin (Hanein et al. 1997, 1998; Galkin et al. 2008) and plectin (Galkin et al. 2008). These and other studies suggest that many actin-binding proteins bind to consensus ''hot-spots'' on actin (Fig. 3). "
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    ABSTRACT: The thin filaments of differentiated smooth muscle cells are composed of actin and tropomyosin isoforms and numerous ancillary actin-binding proteins that assemble together into distinct thin filament classes. These different filament classes are segregated in smooth muscle cells into structurally and functionally separated contractile and cytoskeletal cellular domains. Typically, thin filaments in smooth muscle cells have been considered to be relatively stable structures like those in striated cells. However, recent efforts have shown that smooth muscle thin filaments indeed are dynamic and that remodeling of the actin cytoskeleton, in particular, regulates smooth muscle function. Thus, the cytoskeleton of differentiated smooth muscle cells appears to function midway between that of less dynamic striated muscle cells and that of very plastic proliferative cells such as fibroblasts. Michael and Kate Bárány keenly followed and participated in some of these studies, consistent with their broad interest in actin function and smooth muscle mechanisms. As a way of honoring the memory of these two pioneer members of the muscle research community, we review data on distribution and remodeling of thin filaments in smooth muscle cells, one of the many research topics that intrigued them.
    Journal of Muscle Research and Cell Motility 02/2012; 33(6). DOI:10.1007/s10974-012-9283-z · 2.09 Impact Factor
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    • "Apart from titin and obscurin, whose cytoskeletal integration is obvious and which, in the case of titin, combines the function of an elastic link between actin and myosin filaments with kinase signalling, similar linker functions emerge for other kinases. Non-muscle MLCK is firmly attached to actin and myosin filaments via an N-terminal nebulin-like actin-binding motif and the C-terminal myosin-binding telokin Ig domain [42, 48, 62, 114, 137, 138], and it could be interesting to investigate whether MLCK activity might therefore, in addition to the well-established CaM regulation, be subject to mechanical modulation that might contribute to stretch-induced MLCK activity [7, 67]. "
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    ABSTRACT: Titin, the giant elastic ruler protein of striated muscle sarcomeres, contains a catalytic kinase domain related to a family of intrasterically regulated protein kinases. The most extensively studied member of this branch of the human kinome is the Ca(2+)-calmodulin (CaM)-regulated myosin light-chain kinases (MLCK). However, not all kinases of the MLCK branch are functional MLCKs, and about half lack a CaM binding site in their C-terminal autoinhibitory tail (AI). A unifying feature is their association with the cytoskeleton, mostly via actin and myosin filaments. Titin kinase, similar to its invertebrate analogue twitchin kinase and likely other "MLCKs", is not Ca(2+)-calmodulin-activated. Recently, local protein unfolding of the C-terminal AI has emerged as a common mechanism in the activation of CaM kinases. Single-molecule data suggested that opening of the TK active site could also be achieved by mechanical unfolding of the AI. Mechanical modulation of catalytic activity might thus allow cytoskeletal signalling proteins to act as mechanosensors, creating feedback mechanisms between cytoskeletal tension and tension generation or cellular remodelling. Similar to other MLCK-like kinases like DRAK2 and DAPK1, TK is linked to protein turnover regulation via the autophagy/lysosomal system, suggesting the MLCK-like kinases have common functions beyond contraction regulation.
    Pflügers Archiv - European Journal of Physiology 03/2011; 462(1):119-34. DOI:10.1007/s00424-011-0946-1 · 4.10 Impact Factor
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    • "Although it has already been established in the original study (Sobieszek 1977) that phosphorylation of myosin is required for the actin activation of myosin, the localization of the enzyme involved is still controversial. From the earlier studies on binding, it has been concluded that MLCK and its activator (CaM) is localized or bound to the actin filaments (Dabrowska et al. 1982; Ikebe et al. 1987; Hatch et al. 2001) but their affinity for F-actin is rather low (Sobue et al. 1982; Kanoh et al. 1993; Smith et al. 2002). In contrast, it has been previously demonstrated that MLCK and CaM are tightly associated with the myosin component because they are present in apparently homogenous filamentous myosin preparations (Sobieszek 1985, 1990). "
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    ABSTRACT: Smooth muscle myosin copurifies with myosin light chain kinase (MLCK) and calmodulin (CaM) as well as with variable amounts of myosin phosphatase. Therefore, myosin filaments formed in vitro also contain relatively high levels of these enzymes. Thus these filaments may be considered to be native-like because they are similar to those expected to exist in vivo. These endogenous enzymes are present at high concentrations relative to myosin, sufficient for rapid phosphorylation and dephosphorylation of the filaments at rates comparable to those observed for contraction and relaxation in intact muscle strips. The phosphorylation by MLCK/CaM complex appears to exhibit some directionality and is not governed by a random diffusional process. For the mixtures of myosin filaments with and without the endogenous MLCK/CaM complex, the complex preferentially phosphorylates its own parent filament at a higher rate than the neighboring filaments. This selective or vectorial-like activation is lost or absent when myosin filaments are dissolved at high ionic strength. Similar vectorial-like activation is exhibited by the reconstituted filament suspensions, but the soluble systems composed of isolated regulatory light chain or soluble myosin head subfragments exhibit normal diffusional kinetic behavior. At physiological concentrations, kinase related protein (telokin) effectively modulates the activation process by reducing the phosphorylation rate of the filaments without affecting the overall phosphorylation level. This results from telokin-induced liberation of the active MLCK/CaM complex from the filaments, so that the latter can also activate the neighboring filaments via a slower diffusional process. When this complex is bound at insufficient levels, this actually results in acceleration of the initial phosphorylation rates. In short, I suggest that in smooth muscle, telokin plays a chaperone role for myosin and its filaments.
    Canadian Journal of Physiology and Pharmacology 11/2005; 83(10):899-912. DOI:10.1139/y05-053 · 1.77 Impact Factor
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