Demonstration of a direct role for myosin light chain kinase in fibroblast-populated collagen lattice contraction (original) (raw)
Related papers
Circulation Research, 1991
We have used an immunological approach to investigate the role of myosin light chain phosphorylation (MLC-Pi) in the control of contractility in smooth muscle. Our aim was to specifically inhibit myosin light chain kinase (MLCK) in the presence of physiologically activating levels of Ca2+ so that other putative Ca2(+)-dependent regulatory systems could be unmasked. Fab fragments were prepared by papain digestion of immunoglobulin G (IgG) molecules obtained from goats immunized with turkey gizzard MLCK. Anti-MLCK Fab was then purified by chromatography on an MLCK-Sepharose 4B column. These affinity-purified Fab fragments inhibit the activity of MLCK purified from turkey gizzard smooth muscle and interact monospecifically with MLCK in various mammalian smooth muscles as demonstrated by a Western blot analysis. The effect of these Fab fragments on the contractile properties was tested in guinea pig taenia coli made permeable (skinned) using Triton X-100. Skinned fibers, approximately 1...
Kinetics of Myosin Light Chain Kinase Activation of Smooth Muscle Myosin in an in Vitro Model System
Biochemistry, 2013
During activation of smooth muscle contraction, one myosin light chain kinase (MLCK) molecule rapidly phosphorylates many smooth muscle myosin (SMM) molecules, suggesting that muscle activation rates are influenced by the kinetics of MLCK-SMM interactions. To determine the ratelimiting step underlying activation of SMM by MLCK, we measured the kinetics of calciumcalmodulin (Ca 2+-CaM)-MLCK-mediated SMM phosphorylation and the corresponding initiation of SMM-based F-actin motility in an in vitro system with SMM attached to a coverslip surface. Fitting the time course of SMM phosphorylation to a kinetic model gave an initial phosphorylation rate, k p o , of ~1.17 heads s −1 •MLCK −1. Also we measured the dwell time of single QD-labeled MLCK molecules interacting with surface-attached SMM and phosphorylated SMM using total internal reflection fluorescence microscopy. From these data, the dissociation rate constant from phosphorylated SMM was 0.80 s −1 , which was similar to k p o mentioned above and with rates measured in solution. This dissociation rate was essentially independent of the phosphorylation state of SMM. From calculations using our measured dissociation rates and K d s, and estimates of [SMM] and [MLCK] in muscle, we predict that the dissociation of MLCK from phosphorylated SMM is rate-limiting and that the rate of the phosphorylation step is faster than this dissociation rate. Also, association to SMM (11-46 s −1) would be much faster than to pSMM (<0.1-0.2 s −1). This suggests that the probability of MLCK interacting with unphosphorylated versus pSMM is 55-460 times greater. This would avoid sequestering MLCK to unproductive interactions with previously phosphorylated SMM, potentially leading to faster rates of phosphorylation in muscle.
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.
Biochemistry of smooth muscle myosin light chain kinase
Archives of Biochemistry and Biophysics, 2011
The smooth muscle isoform of myosin light chain kinase (MLCK) is a Ca 2+-calmodulin-activated kinase that is found in many tissues. It is particularly important for regulating smooth muscle contraction by phosphorylation of myosin. This review summarizes selected aspects of recent biochemical work on MLCK that pertains to its function in smooth muscle. In general, the focus of the review is on new findings, unresolved issues, and areas with the potential for high physiological significance that need further study. The review includes a concise summary of the structure, substrates, and enzyme activity, followed by a discussion of the factors that may limit the effective activity of MLCK in the muscle. The interactions of each of the many domains of MLCK with the proteins of the contractile apparatus, and the multi-domain interactions of MLCK that may control its behaviors in the cell are summarized. Finally, new in vitro approaches to studying the mechanism of phosphorylation of myosin are introduced.
Regulation of cytoskeletal mechanics and cell growth by myosin light chain phosphorylation
The American journal of physiology, 1998
The role of myosin light chain phosphorylation in regulating the mechanical properties of the cytoskeleton was studied in NIH/3T3 fibroblasts expressing a truncated, constitutively active form of smooth muscle myosin light chain kinase (tMK). Cytoskeletal stiffness determined by quantifying the force required to indent the apical surface of adherent cells showed that stiffness was increased twofold in tMK cells compared with control cells expressing the empty plasmid (Neo cells). Cytoskeletal stiffness quantified using magnetic twisting cytometry showed an approximately 1.5-fold increase in stiffness in tMK cells compared with Neo cells. Electronic volume measurements on cells in suspension revealed that tMK cells had a smaller volume and are more resistant to osmotic swelling than Neo cells. tMK cells also have smaller nuclei, and activation of mitogen-activated protein kinase (MAP kinase) and translocation of MAP kinase to the nucleus are slower in tMK cells than in control cells....
Correlation of myosin light chain phosphorylation with isometric contraction of fibroblasts
The Journal of biological chemistry, 1993
In vitro studies have indicated that the enzymatic activity of myosin II from non-muscle cells is controlled by phosphorylation of its regulatory light chain (LC20). We have studied one likely functional consequence of phosphorylating LC20 in living chick embryo fibroblasts (CEF) by measuring contractile force developed by these cells. Using a recently developed method, we recorded quantitative changes in isometric force generated by a population of cells following mitogenic stimulation. Fetal bovine serum, thrombin, and lysophosphatidic acid stimulate rapid isometric contraction of CEF. Cells stimulated with thrombin develop maximal force within 5-10 min. Force development correlates temporally with a 3-5-fold increase in the overall fraction of LC20 phosphorylated and with the fractions of LC20 in both the monophosphorylated and diphosphorylated states. Unloaded shortening velocity also increases after thrombin stimulation. Although both force and phosphorylation begin to decline ...
Shape and substructure of skeletal muscle myosin light chain kinase
Biochemistry, 1983
Abbreviations: MLCK, myosin light chain kinase; CM, calmodulin; Ca2+-CM, Ca2+-saturated CM; LC2, light chain 2; MLCK-CM, 1:l molar ratio complex of MLCK and Ca2+-CM; FH,, head fragment, spontaneously proteolytic; FH,, head fragment, tryptic; FH,.CM, 1 : 1 molar ratio complex of FH, and Ca2+-CM; FH,.CM, 1:l molar ratio complex of FH, and Ca*+-CM; FT, tail fragment; MRW, mean residue weight; EDTA, ethylenediaminetetraacetic acid; DTE, dithioerythritol; PMSF, phenylmethanesulfonyl fluoride; NaDodS04, sodium dodecyl sulfate; TPCK, tosyl-L-phenylalanine chloromethyl ketone; Cl,CCOOH, trichloroacetic acid; GdmCI, guanidinium chloride; EGTA, ethylene glycol bis(&aminoethyl ether)-N,N,N',N'-tetraacetic acid.
Biochemistry, 1990
The relationship between the light-chain phosphorylation and the actin-activated ATPase activity of pig urinary bladder myosin was either linear or nonlinear depending on the free Mg2+ concentration. Varying the free [Mg2'] in the presence of 50 m M ionic strength (I) had a biphasic effect on the actinactivated ATPase. In 100 m M I, the activity increased on raising the free [Mg2+]. The activity of the phosphorylated myosin was 3-23-fold higher than that of the unphosphorylated myosin at all concentrations of free Mg2+, pH, and temperature used in this study. The increase in the turbidity and sedimentability of both phosphorylated and unphosphorylated myosins on raising the free [Mg2'] was associated with a rise in the actin-activated ATPase activity. However, myosin light-chain phosphorylation still had a remarkable effect on the actin activation. The myosin polymers formed under these conditions were sedimented by centrifugation. Experiments performed with myosin polymers formed in mixtures of unphosphorylated and phosphorylated myosins showed that the presence of phosphorylated myosin in these mixtures had a slight effect on the sedimentation of the unphosphorylated myosin but it had no effect on the actin-activated ATP hydrolysis. Electron microscopy showed that the unphosphorylated myosin formed unorganized aggregates while phosphorylated myosin molecules assembled into bipolar filaments with tapered ends. These data show that although the unphosphorylated and phosphorylated myosins have the same level of sedimentability and turbidity, the filament assembly present only with the phosphorylated myosin can be associated