Diphosphorylated MRLC is Required for Organization of Stress Fibers in Interphase Cells and the Contractile Ring in Dividing Cells.

Abstract

ABSTRACT. Activity of nonmuscle myosin II is regulated by phosphorylation of its regulatory light chain (MRLC). Phosphoryration of MRLC at both Thr18 and Ser19 (diphosphorylation) results in higher MgATPase activity and in promotion of the assembly of myosin II filaments than does that of MRLC at Ser19 (monophosphorylation) in vitro. To determine the roles of the diphosphorylated MRLC in vivo, we transfected three kinds of MRLC mutants, unphosphorylated, monophosphorylated and diphosphorylated forms (MRLC2 T18AS19A , substitution of both Ser19 and Thr18 by Ala; MRLC2 T18AS19D , Ser19 by Asp and Thr18 by Ala; and MRLC2 T18DS19D , both Ser19 and Thr18 by Asp, respectively), into HeLa cells. Cells overexpressing the mutant MRLC2 T18DS19D contained a larger number of actin filament bundles than did those overexpressing the mutant MRLC2 T18AS19D . Moreover, cells overexpressing the nonphosphorylatable mutant MRLC2 T18AS19A showed a decrease in the number of actin filament bundles. Taken together, our data suggest that diphosphorylation of MRLC plays an important role in regulating actin filament assembly and reorganization in nonmuscle cells. Key words: phosphorylation/myosin II filament/myosin II regulatory light chain/cytokinesisMyosin II is an essential protein to generate force for cell locomotion (Berlot et al., 1985(Berlot et al., , 1987 and cytokinesis (Mabuchi and Okuno, 1977;De Lozanne and Spudich, 1987). In smooth muscle and vertebrate nonmuscle cells, myosin II activity is both negatively and positively regulated by phosphorylation of its regulatory light chain (MRLC). The phosphorylation sites of MRLC by Ca 2+ /calmodulindependent myosin light chain kinase (MLCK) are mainly Ser19 (monophosphorylation) and, under some conditions, both Ser19 and Thr18 (diphosphorylation) in vitro (Ikebe and Hartshorne, 1985;Ikebe et al., 1986). Phosphorylation of MRLC by MLCK enhances the actin activated Mg ATPase activity (Sellers et al., 1981;Trybus, 1989) and promotes the assembly of myosin II filaments (Suzuki et al., 1978). These effects are more effective when MRLC is diphosphorylated (Ikebe et al., 1988). On the other hand, protein kinase C (PKC) phosphorylates MRLC at Ser1/2 and Thr9. Phosphorylation of MRLC by PKC down-regulate the MgATPase activity of myosin II phosphorylated by MLCK (Nishikawa et al., 1984;Ikebe et al., 1987).It has been reported that substitution of amino acid residues on phosphorylation sites of MRLC affects the biochemical properties of myosin II in vitro. Substitution of both Ser19 and Thr18 to Asp, which mimics the diphosphorylated MRLC, exerted the actin activated MgATPase activity of myosin II and formation of myosin II filaments (Kamisoyama et al., 1994;Sweeney et al., 1994).In this study, we attempted to determine the roles of diphosphorylated MRLC in vivo. To address this, we transfected three kinds of MRLC mutants, which mimic nonphosphorylated, monophosphorylated and diphosphorylated MRLC, into HeLa cells and compared their localization in both interphase and mitotic cells. Materials ...

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“…5B). The existence of multiple MLC isoforms has been reported in other cell types as well (16,40). Accordingly, on urea-glycerol PAGE, MLC obtained from LLC-PK1 cells could be separated into three doublets (6 bands): the two uppermost bands corresponded to the nonphosphorylated forms, the middle bands to the monophosphorylated forms (pMLC), and the lowermost bands to the diphosphorylated forms (ppMLC).…”

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