Article

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: 9.69). 09/2001; 154(3):611-7. DOI: 10.1083/jcb.200105079
Source: PubMed

ABSTRACT 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.

0 Followers
 · 
85 Views
  • Source
  • [Show abstract] [Hide abstract]
    ABSTRACT: The major motor protein in all hollow organs, except the heart, is smooth-muscle myosin II (SmM) and the emphasis of this chapter is on the function of SmM in differentiated smooth muscle. A sliding-filament mechanism is assumed for smooth muscle, as in striated muscle, but there are differences in smooth-muscle thick filaments with respect to assembly and stability. Various isoforms of SmM are expressed and are discussed. SmM differs from its striated muscle counterparts in the requirement for phosphorylation of the regulatory light chains to regulate motor activity. The components of myosin structure required for phosphorylation dependence are outlined. The level of SmM phosphorylation is controlled by opposing actions of the Ca2+-calmodulin-dependent myosin-light-chain kinase and myosin phosphatase. Both are subject to regulation and putative mechanisms are presented. The focus of recent research has been on regulation of myosin phosphatase and inhibition and activation are proposed. Several signaling pathways converge at the myosin phosphatase target subunit to regulate activity. Two pathways that work in opposition are inhibition via the RhoA/Rho-kinase couple and activation by cyclic nucleotides, particularly cGMP. Each is vital in smooth-muscle function and both pathways have important clinical application and are argeted bypharmacological intervention in treatment of smooth-muscle disorders.
    11/2007: pages 171-222;
  • [Show abstract] [Hide abstract]
    ABSTRACT: From the extracellular matrix to the cytoskeleton, a network of molecular links connects cells to their environment. Molecules in this network transmit and detect mechanical forces, which subsequently determine cell behavior and fate. Here, we reconstruct the mechanical pathway followed by these forces. From matrix proteins to actin through integrins and adaptor proteins, we review how forces affect the lifetime of bonds and stretch or alter the conformation of proteins, and how these mechanical changes are converted into biochemical signals in mechanotransduction events. We evaluate which of the proteins in the network can participate in mechanotransduction and which are simply responsible for transmitting forces in a dynamic network. Besides their individual properties, we also analyze how the mechanical responses of a protein are determined by their serial connections from the matrix to actin, their parallel connections in integrin clusters and by the rate at which force is applied to them. All these define mechanical molecular pathways in cells, which are emerging as key regulators of cell function alongside better studied biochemical pathways.
    Journal of Cell Science 07/2012; 125(Pt 13):3025-38. DOI:10.1242/jcs.095794 · 5.33 Impact Factor

Full-text (2 Sources)

Download
21 Downloads
Available from
Jun 2, 2014