Association of myosin light chain kinase with lymphocyte membrane-cytoskeleton complex (original) (raw)
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Phosphorylation of myosin light chain during capping of mouse T- lymphoma cells
The Journal of Cell Biology, 1981
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).
FEBS Letters, 2003
Myosin II regulatory light chain (RLC) phosphorylation by Ca 2+ /calmodulin (CaM)-dependent myosin light chain kinase (MLCK) is implicated in many cellular actin cytoskeletal functions. We examined MLCK activation quantitatively with a £uorescent biosensor MLCK where Ca 2+-dependent increases in kinase activity were coincident with decreases in £uorescence resonance energy transfer (FRET) in vitro. In cells stably transfected with CaM sensor MLCK, increasing [Ca 2+ ] i increased MLCK activation and RLC phosphorylation coincidently. There was no evidence for CaM binding but not activating MLCK at low [Ca 2+ ] i. At saturating [Ca 2+ ] i MLCK was not fully activated probably due to limited availability of cellular Ca 2+ /CaM.
Biochemistry and Cell Biology, 1996
Smooth muscle myosin light chain kinase (MLCK) features several consensus sites of phosphorylation by prolinedirected protein serinetthreonine kinases. The phosphorylation of MLCK by two proline-directed kinases isolated from sea star oocytes, i.e., p44mpk (Mpk, a mitogen-activated protein kinase homologue) and cyclin-dependent kinase-1 (CDK1, also known as ~3 4~~~~1 , was investigated. Chicken gizzard MLCK was phosphorylated on seryl and threonyl residues by both Mpk and CDK1. Phosphorylation of MLCK to 0.6 mol P,/mol by Mpk increased the V,,, of phosphotransferase activity towards a synthetic peptide corresponding to residues 11-23 of the 20-kDa light chain of myosin by 1.6-fold. Phosphorylation of MLCK to 1.0 mol Pi/mol by CDKl increased the V,,, by 2.3-fold. Phosphorylation by either kinase had no significant effect on the concentration of calmodulin required for half-maximal activation of MLCK. Analysis of the phosphorylation of synthetic peptides containing consensus phosphorylation sites for Mpk and CDKl indicated that the major site of phosphorylation in MLCK by Mpk was Ser-834, and by CDKl was Thr-283. Both of these sites are located outside the calmodulin-binding site (residues 796-815), consistent with the observation that phosphorylation by Mpk or CDKl was unaffected by the presence of bound ~a~+/calmodulin. These results indicate that MLCK activity may be regulated by phosphorylation catalyzed by prolinedirected kinases, possibly directed at Thr-40 and Thr-43 at the amino terminus of MLCK.
Proceedings of the National Academy of Sciences, 1981
Antibodies to myosin light chain kinase, purified from turkey gizzard smooth muscle, were developed in rabbits and purified by affinity chromatography on a myosin light chain kinase-Sepharose 4B column. The purified antibodies crossreact with purified smooth muscle myosin light chain kinase but not with a variety of contractile or cytoskeletal proteins. The antibodies inhibit the catalytic activity of smooth musclet myosin light chain kinase, and there is an inverse relationship between the kinase activity and the amount ofantibody present in an assay. Half-maximal inhibition of myosin kinase activity occurs at an antibody/ myosin kinase molar ratio of 10:1. The affinity-purified antibodies to smooth muscle myosin kinase were used to study the location of myosin kinase in a variety of nonmuscle cells. Immunofluorescence studies indicate that myosin light chain kinase is localized on microfilament bundles (stress fibers) in cultured fibroblasts. The stress fiber staining pattern is abolished when the antibodies are incubated with purified smooth muscle myosin light chain kinase prior to staining cells, while the staining pattern is unaffected when the antibodies are incubated with actin, myosin, a-actinin, or tropomyosin prior to staining. Moreover, the stress fiber staining pattern is periodic in well-spread gerbil fibroma cells and experiments have demonstrated that myosin light chain kinase appears to have the same periodic distribution as myosin but an antiperiodic distribution relative to a-actinin. These data indicate that myosin light chain kinase and its substrate, myosin, are in close proximity and are consistent with the hypothesis that myosin light chain kinase regulates actin-myosin interactions in nonmuscle cells.
Phosphorylation of Calmodulin in the First Calcium-Binding Pocket by Myosin Light Chain Kinase
Archives of Biochemistry and Biophysics, 1996
ATPase and mechanisms of MLCK activation and substrate recogsubsequent contractile events. We have previously nition. ᭧ 1996 Academic Press, Inc. demonstrated that CaM phosphorylated by casein ki-Key Words: calmodulin; myosin light chain kinase; nase II fails to activate bovine platelet MLCK (Sacks phosphorylation. et al. (1992) Biochem. J. 283, 21-24). While myosin light chains are perceived as the only known substrate for MLCK phosphorylation activity, we now show that MLCK phosphorylates CaM. This phosphorylation of CaM is dependent upon the presence of basic peptides
Biochemical Journal, 2002
Ca2+/calmodulin (CaM)-dependent protein kinase I (CaM-KI), which is a member of the multifunctional CaM-K family, is thought to be involved in various Ca2+-signalling pathways. In this report, we demonstrate that CaM-KI activated by an upstream kinase (CaM-K kinase), but not unactivated CaM-KI, phosphorylates myosin II regulatory light chain (MRLC) efficiently (Kcat, 1.7s-1) and stoichiometrically (0.8mol of phosphate/mol) in a Ca2+/CaM-dependent manner in vitro. One-dimensional phosphopeptide mapping and mutational analysis of MRLC revealed that the activated CaM-KI monophosphorylates only Ser-19 in MRLC. Transient expression of the Ca2+/CaM-independent form of CaM-KI (CaM-KI1-293) in HeLa cells induced Ser-19 phosphorylation of myosin, II accompanied by reorganization of actin filaments in the peripheral region of the cells. CaM-KI-induced reorganization of actin filaments was suppressed by co-expression of non-phosphorylatable MRLC mutants (S19A and T18AS19A). Furthermore, a kin...