An increase or a decrease in myosin II phosphorylation inhibits macrophage motility

Myosin II purified from mammalian non-muscle cells is phosphorylated on the 20-kD light chain subunit (MLC20) by the Ca2+/calmodulin-dependent enzyme myosin light chain kinase (MLCK). The importance of MLC20 phosphorylation in regulating cell motility was investigated by introducing either antibodies to MLCK (MK-Ab) or a Ca2+/calmodulin-independent, constitutively active form of MLCK (MK-) into macrophages. The effects of these proteins on cell motility were then determined using a quantitative chemotaxis assay. Chemotaxis is significantly diminished in macrophages containing MK-Ab compared to macrophages containing control antibodies. Moreover, there is an inverse relationship between the number of cells that migrate and the amount of MK-Ab introduced into cells. Interestingly, there is also an inverse relationship between the number of cells that migrate and the amount of MK- introduced into cells. Other experiments demonstrated that MK-Ab decreased intracellular MLC20 phosphorylation while MK- increased MLC20 phosphorylation. MK- also increased the amount of myosin associated with the cytoskeleton. These data demonstrate that the regulation of MLCK is an important aspect of cell motility and suggest that MLC20 phosphorylation must be maintained within narrow limits during translational motility by mammalian cells.

[1]  J. White,et al.  Cortical flow in animal cells. , 1988, Science.

[2]  J. George,et al.  Random phosphorylation of the two heads of thymus myosin and the independent stimulation of their actin-activated ATPases. , 1985, The Journal of biological chemistry.

[3]  S. Schuster,et al.  Transfer of monoclonal antibodies into mammalian cells by electroporation. , 1989, The Journal of biological chemistry.

[4]  J. Fox,et al.  Role of phosphorylation in mediating the association of myosin with the cytoskeletal structures of human platelets. , 1982, The Journal of biological chemistry.

[5]  U. Zimmermann,et al.  Electrical breakdown, electropermeabilization and electrofusion. , 1986, Reviews of physiology, biochemistry and pharmacology.

[6]  J. Scholey,et al.  Thymus myosin. Isolation and characterization of myosin from calf thymus and thymic lymphocytes, and studies on the effect of phosphorylation of its Mr = 20,000 light chain. , 1982, The Journal of biological chemistry.

[7]  Y. Fukui,et al.  Myosin I is located at the leading edges of locomoting Dictyostelium amoebae , 1989, Nature.

[8]  J. Kendrick‐Jones,et al.  Light-chain phosphorylation controls the conformation of vertebrate non-muscle and smooth muscle myosin molecules , 1983, Nature.

[9]  K. Weber,et al.  Calcium control of actin-myosin based contraction in triton models of mouse 3T3 fibroblasts is mediated by the myosin light chain kinase (MLCK)-calmodulin complex. , 1983, Experimental cell research.

[10]  R. Snyderman,et al.  Molecular and cellular mechanisms of leukocyte chemotaxis. , 1981, Science.

[11]  T. Pollard,et al.  Binding of myosin I to membrane lipids , 1989, Nature.

[12]  R. Adelstein,et al.  Regulation of contractile proteins by phosphorylation. , 1983, The Journal of clinical investigation.

[13]  J. Horwitz,et al.  Evaluation of the electroinjection method for introducing proteins into living cells. , 1991, The American journal of physiology.

[14]  J. Davies,et al.  Molecular Biology of the Cell , 1983, Bristol Medico-Chirurgical Journal.

[15]  E Neumann,et al.  Electric field mediated gene transfer. , 1982, Biochemical and biophysical research communications.

[16]  D A Knecht,et al.  Antisense RNA inactivation of myosin heavy chain gene expression in Dictyostelium discoideum. , 1987, Science.

[17]  R. Adelstein,et al.  Increased phosphorylation of myosin light chain kinase after an increase in cyclic AMP in intact smooth muscle. , 1984, Science.

[18]  R. Adelstein,et al.  Phosphorylation of platelet myosin increases actin-activated myosin ATPase activity , 1975, Nature.

[19]  R. Snyderman,et al.  Humoral mediators of chemotaxis of mononuclear leukocytes. , 1972, The Journal of infectious diseases.

[20]  A. K. Wilson,et al.  Okadaic acid, a phosphatase inhibitor, decreases macrophage motility. , 1991, The American journal of physiology.

[21]  W. Falk,et al.  A 48-well micro chemotaxis assembly for rapid and accurate measurement of leukocyte migration. , 1980, Journal of immunological methods.

[22]  J. Trotter,et al.  Macrophage myosin. Regulation of actin-activated ATPase, activity by phosphorylation of the 20,000-dalton light chain. , 1979, The Journal of biological chemistry.

[23]  P. Greengard.  Phosphorylated proteins as physiological effectors. , 1978, Science.

[24]  W. T. Chen.  Mechanism of retraction of the trailing edge during fibroblast movement , 1981, The Journal of cell biology.

[25]  R. Ezzell,et al.  Evidence for regulation of lamellipodial and tail contraction of glycerinated chicken embryonic fibroblasts by myosin light chain kinase. , 1986, Cell motility and the cytoskeleton.

[26]  R. Adelstein,et al.  Immunological properties of myosin light-chain kinases. , 1987, Biochimica et biophysica acta.

[27]  J. Feramisco,et al.  Characterization of antibodies to smooth muscle myosin kinase and their use in localizing myosin kinase in nonmuscle cells. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[28]  G F Oster,et al.  On the crawling of cells. , 1984, Journal of embryology and experimental morphology.

[29]  H. Ishikawa,et al.  THE POLYMERIZATION OF ACTIN: ITS ROLE IN THE GENERATION OF THE ACROSOMAL PROCESS OF CERTAIN ECHINODERM SPERM , 1973, Journal of Cell Biology.

[30]  J. Hammer,et al.  Myosins of nonmuscle cells. , 1988, Annual review of biophysics and biophysical chemistry.

[31]  J. Spudich,et al.  Disruption of the Dictyostelium myosin heavy chain gene by homologous recombination. , 1987, Science.