ZIP kinase is responsible for the phosphorylation of myosin II and necessary for cell motility in mammalian fibroblasts

Reorganization of actomyosin is an essential process for cell migration and myosin regulatory light chain (MLC20) phosphorylation plays a key role in this process. Here, we found that zipper-interacting protein (ZIP) kinase plays a predominant role in myosin II phosphorylation in mammalian fibroblasts. Using two phosphorylation site-specific antibodies, we demonstrated that a significant portion of the phosphorylated MLC20 is diphosphorylated and that the localization of mono- and diphosphorylated myosin is different from each other. The kinase responsible for the phosphorylation was ZIP kinase because (a) the kinase in the cell extracts phosphorylated Ser19 and Thr18 of MLC20 with similar potency; (b) immunodepletion of ZIP kinase from the cell extracts markedly diminished its myosin II kinase activity; and (c) disruption of ZIP kinase expression by RNA interference diminished myosin phosphorylation, and resulted in the defect of cell polarity and migration efficiency. These results suggest that ZIP kinase is critical for myosin phosphorylation and necessary for cell motile processes in mammalian fibroblasts.

[1]  Fumio Matsumura,et al.  Distinct Roles of Rock (Rho-Kinase) and Mlck in Spatial Regulation of Mlc Phosphorylation for Assembly of Stress Fibers and Focal Adhesions in 3t3 Fibroblasts , 2000, The Journal of cell biology.

[2]  M. Ikebe,et al.  Phosphorylation of bovine platelet myosin by protein kinase C. , 1990, Biochemistry.

[3]  M. Ikebe,et al.  Dephosphorylation of the two regulatory components of myosin phosphatase, MBS and CPI17 , 2002, FEBS letters.

[4]  T. Haystead,et al.  Smooth Muscle Myosin Phosphatase-associated Kinase Induces Ca2+ Sensitization via Myosin Phosphatase Inhibition* , 2002, The Journal of Biological Chemistry.

[5]  D. Hartshorne,et al.  Identification, phosphorylation, and dephosphorylation of a second site for myosin light chain kinase on the 20,000-dalton light chain of smooth muscle myosin. , 1986, The Journal of biological chemistry.

[6]  Kozo Kaibuchi,et al.  Regulation of Myosin Phosphatase by Rho and Rho-Associated Kinase (Rho-Kinase) , 1996, Science.

[7]  D. Hartshorne,et al.  Effects of Ca2+ on the conformation and enzymatic activity of smooth muscle myosin. , 1985, The Journal of biological chemistry.

[8]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[9]  D. Hartshorne,et al.  Calyculin-A increases the level of protein phosphorylation and changes the shape of 3T3 fibroblasts. , 1991, Cell motility and the cytoskeleton.

[10]  Fumio Matsumura,et al.  Distinct roles of MLCK and ROCK in the regulation of membrane protrusions and focal adhesion dynamics during cell migration of fibroblasts , 2004, The Journal of cell biology.

[11]  Sven Christian,et al.  Cloning and characterization of Dlk, a novel serine/threonine kinase that is tightly associated with chromatin and phosphorylates core histones , 1998, Oncogene.

[12]  M. Ikebe,et al.  Boundary of the autoinhibitory region of smooth muscle myosin light-chain kinase. , 1993, Biochemistry.

[13]  Yoshiharu Matsuura,et al.  Phosphorylation and Activation of Myosin by Rho-associated Kinase (Rho-kinase)* , 1996, The Journal of Biological Chemistry.

[14]  A. Means,et al.  Proteolysis of smooth muscle myosin light chain kinase. Formation of inactive and calmodulin-independent fragments. , 1987, The Journal of biological chemistry.

[15]  M. Ikebe,et al.  Mutagenesis of the phosphorylation site (serine 19) of smooth muscle myosin regulatory light chain and its effects on the properties of myosin. , 1994, Biochemistry.

[16]  D. Hartshorne,et al.  Inhibitory Phosphorylation Site for Rho-associated Kinase on Smooth Muscle Myosin Phosphatase* , 1999, The Journal of Biological Chemistry.

[17]  Shizuo Akira,et al.  ZIP Kinase, a Novel Serine/Threonine Kinase Which Mediates Apoptosis , 1998, Molecular and Cellular Biology.

[18]  Fumio Matsumura,et al.  Phosphorylation of Myosin-Binding Subunit (Mbs) of Myosin Phosphatase by Rho-Kinase in Vivo , 1999, The Journal of cell biology.

[19]  I. Dawid,et al.  Isolation and characterization of calmodulin genes from Xenopus laevis , 1984, Molecular and cellular biology.

[20]  D. Hartshorne,et al.  Phosphorylation of the 20,000-dalton light chain of smooth muscle myosin by the calcium-activated, phospholipid-dependent protein kinase. Phosphorylation sites and effects of phosphorylation. , 1987, The Journal of biological chemistry.

[21]  R. Tuft,et al.  Effects of the Regulatory Light Chain Phosphorylation of Myosin II on Mitosis and Cytokinesis of Mammalian Cells* , 2000, The Journal of Biological Chemistry.

[22]  D. Hartshorne,et al.  Phosphorylation of smooth muscle myosin at two distinct sites by myosin light chain kinase. , 1985, The Journal of biological chemistry.

[23]  H. Hosoya,et al.  HeLa ZIP kinase induces diphosphorylation of myosin II regulatory light chain and reorganization of actin filaments in nonmuscle cells , 2001, Oncogene.

[24]  R. Adelstein,et al.  Secretion from rat basophilic RBL-2H3 cells is associated with diphosphorylation of myosin light chains by myosin light chain kinase as well as phosphorylation by protein kinase C. , 1994, The Journal of biological chemistry.

[25]  J. Sellers,et al.  Protein kinase C modulates in vitro phosphorylation of the smooth muscle heavy meromyosin by myosin light chain kinase. , 1984, The Journal of biological chemistry.

[26]  David P. Wilson,et al.  Phosphorylation of the myosin phosphatase target subunit by integrin-linked kinase. , 2002, The Biochemical journal.

[27]  H. Singer.  Protein kinase C activation and myosin light chain phosphorylation in 32P-labeled arterial smooth muscle. , 1990, The American journal of physiology.

[28]  Shuh Narumiya,et al.  Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension , 1997, Nature.

[29]  D. Hartshorne,et al.  Integrin-linked kinase phosphorylates the myosin phosphatase target subunit at the inhibitory site in platelet cytoskeleton. , 2002, The Biochemical journal.

[30]  S. Perry,et al.  An electrophoretic study of the low-molecular-weight components of myosin. , 1970, The Biochemical journal.

[31]  F. Matsumura,et al.  Specific Localization of Serine 19 Phosphorylated Myosin II during Cell Locomotion and Mitosis of Cultured Cells , 1998, The Journal of cell biology.

[32]  C. Slaughter,et al.  Sites phosphorylated in myosin light chain in contracting smooth muscle. , 1988, The Journal of biological chemistry.

[33]  J. Stull,et al.  Regulation of smooth muscle contractile elements by second messengers. , 1989, Annual review of physiology.

[34]  F. Suizu,et al.  ZIP kinase identified as a novel myosin regulatory light chain kinase in HeLa cells , 1999, FEBS letters.

[35]  T. Haystead,et al.  Identification of the endogenous smooth muscle myosin phosphatase-associated kinase , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[36]  J. Spudich,et al.  Control of nonmuscle myosins by phosphorylation. , 1992, Annual review of biochemistry.

[37]  M. Ikebe,et al.  Zipper-interacting Protein Kinase Induces Ca2+-free Smooth Muscle Contraction via Myosin Light Chain Phosphorylation* , 2001, The Journal of Biological Chemistry.

[38]  J. Koretz,et al.  Effects of phosphorylation of light chain residues threonine 18 and serine 19 on the properties and conformation of smooth muscle myosin. , 1988, The Journal of biological chemistry.

[39]  L. Johnson,et al.  Physiology of the gastrointestinal tract , 2012 .

[40]  A. Fire,et al.  Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans , 1998, Nature.

[41]  K. Itoh,et al.  Diphosphorylation of platelet myosin ex vivo in the initial phase of activation by thrombin. , 1992, Biochimica et biophysica acta.

[42]  J. Spudich,et al.  Light chain phosphorylation regulates the movement of smooth muscle myosin on actin filaments , 1985, The Journal of cell biology.

[43]  C. Sutherland,et al.  Ca2+-independent Smooth Muscle Contraction , 2001, The Journal of Biological Chemistry.

[44]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.