Phosphorylation of myosin light chain during capping of mouse T-lymphoma cells.

Abstract

Colchicine induces the clustering of at least three different T-lymphoma surface antigens (T200, Thy-1, and gp 69/71) into a cap structure in the absence of any external ligand . In addition, colchicine induces the intracellular accumulation of actin and myosin directly beneath the surface cap structure . We have discovered that myosin molecules (both heavy and light chains) are closely associated with the plasma membrane of T-lymphoma cells. Most importantly, we have found that the 20,000-dalton light chain of lymphocyte myosin is both phosphorylated and preferentially accumulated in the plasma membrane of colchicine-induced capped cells. It is proposed that myosin light chain is directly involved in the activation of membrane-associated actomyosin required for the collection of surface proteins into a cap structure (analogous to muscle cell sliding filament contraction).Redistribution of lymphocyte surface receptors can be induced either with or without the addition of external ligands (15,18,23,29,30,37,41,(46)(47)(48) . During the process of receptor movement, surface components first form small clusters (patches), which then aggregate at one pole of the cell into socalled caps .In the case of ligand-dependent capping, the ligand and its receptors appear to be clustered by a cross-linking event at the cell surface that is highly dependent on the valency of the ligand (37). For example, divalent antibodies against surface immunoglobulin (Ig) are required for the induction of Ig capping; monovalent anti-Ig is unable to cause any redistribution of the Ig receptor (30) . In addition, capping induced by the lectin, concanavalin A (Con A), requires the tetrameric form of Con A (at the proper concentration) since the dimer form (e .g ., succinyl Con A) is not active in cap formation (15,18) . Information concerning ligand-independent capping is very limited . Recently, it has been found that hypertonic media, mitogens, trypsin, and cholinergic drugs are each able to induce surface receptors to form cap structures in the absence of any externally added ligand (29,(46)(47)(48). The molecular mechanisms underlying both ligand-dependent and ligand-independent capping are not yet understood .Previously, we have used a double immunofluorescence labeling technique to simultaneously determine the distribution of certain surface receptors and intracellular actin and myosin in human skin fibroblasts grown in monolayers (9) or in murine lymphoid cells grown in suspension cultures (7,10,11) . The THE JOURNAL of CELL BIOLOGY -VOLUME 91 DECEMBER 1981 889-894

MLCK-A, an unconventional myosin light chain kinase from Dictyostelium , is activated by a cGMP-dependent pathway

“…RLC phosphorylation also is increased in Dictyostelium under these circumstances (20). Con A-induced capping of cell surface proteins induces RLC phosphorylation in Dictyostelium (21) as does colchicine-induced capping in mouse lymphoma cells (17). Cytoskeletal changes during capping also bear similarity to those observed in intestinal cells during adhesion of pathogenic Escherichia coli (19).…”

“…This phosphorylation increases the actinactivated ATPase activity of myosin and is carried out by myosin light chain kinase (MLCK). In animal cells, increased RLC phosphorylation is observed during mitosis (13)(14)(15), after exposure to chemoattractant (16), during colchicine-induced capping (17), in agonist-driven shape changes (18), and in response to adsorption of pathogenic bacteria to the intestinal cell surface (19). In Dictyostelium, activating RLC phosphorylation is induced after exposure to chemoattractant (20) and during Con A-induced capping (21).…”

Molecular Cloning and Sequencing of Myosin Light Chains in Human Megakaryoblastic Leukemia Cells.

“…These data suggest that both PKC and MLCK, similar to bovine platelet myosin (Ikebe, 1989;Ikebe and Reardon, 1990) and turkey gizzard smooth muscle (Ikebe et al, 1986;Ikebe et al, 1987) could phosphorylate the sequence of five basic residues of MLC-2 in hematopoietic cells. This may reflect the importance of the primary structure in providing hematopoietic cells MLC-2, similar to the smooth muscle MLC-2, with unique properties in regulating both actin-activated myosin ATPase activity and cellular functions such as cytokinesis, receptor capping, and cell locomotion (Bourguignon et al, 1981;Berlot et al, 1985;Yamakita et al, 1994;Matsumura et al, 1998).…”

“…Phosphorylation of MLC-2 initiates the contraction of vertebrate smooth muscle (Hartshorne and Mrwa, 1982), and is presumed to play a similar role in functions of nonmuscle cells. MLC-2 phosphorylation of cytoplasmic myosin has been implicated in cytokinesis, receptor capping, and cell locomotion (Bourguignon et al, 1981;Berlot et al, 1985;Yamakita et al, 1994;Matsumura et al, 1998). In vitro, high concentrations of MLCK phosphorylate threonine (Thr)-18 in addition to serine (Ser)-19 and markedly increase actin-activated ATPase activity in both gizzard smooth muscle (Ikebe et al, 1986) and bovine platelets (Ikebe, 1989).…”

Surface CD3 expression proceeds through both myosin regulatory light chain 9 (MYL9)-dependent and MYL9-independent pathways in Jurkat cells

“…Twenty kDa RLC is phosphorylated by MLCK in the presence of calcium and calmodulin and increases actin-activated ATPase activities of myosins of both smooth muscle (Adelstein et al, 1980;Somlyo et al, 1994) and nonmuscle cells such as platelets (Ikebe, 1989;Higashihara et al, 1991) and red blood cells (Higashihara et al, 1989). RLC phosphorylation of cytoplasmic myosin has been implicated in cytokinesis, receptor capping, and cell locomotion (Bourguignon et al, 1981;Yamakita et al, 1994;Matsumura et al, 1998).…”