Activation of skeletal muscle myosin light chain kinase by calcium(2+) and calmodulin

Abstract

Many biological processes are now known to be regulated by Ca2+ via calmodulin (CM). Although a general mechanistic model by which Ca2+ and calmodulin modulate many of these activities has been proposed, an accurate quantitative model is not available. A detailed analysis of skeletal muscle myosin light chain kinase activation was undertaken in order to determine the stoichiometries and equilibrium constants of Ca2+, calmodulin, and enzyme catalytic subunit in the activation process. The analysis indicates that activation is a sequential, fully reversible process requiring both Ca2+ and calmodulin. The first step of the activation process appears to require binding of Ca2+ to all four divalent metal binding sites on calmodulin for form the complex, Ca42+-calmodulin. This complex then interacts with the inactive catalytic subunit of the enzyme to form the active holoenzyme complex, Ca42+-calmodulin-enzyme. Formation of the holoenzyme follows simply hyperbolic kinetics, indicating 1:1 stoichiometry of Ca42+-calmodulin to catalytic subunit. The rate equation derived from the mechanistic model was used to determine the values of KCa2+ and KCM, the intrinsic activation constants for each step of the activation process. KCa2+ and KCM were found to have values of 10 microM and 0.86 nM, respectively, at 10 mM Mg2+. The rate equation using these equilibrium constants accurately predicts the extent of enzyme activation over a wide range of Ca2+ and calmodulin concentrations. The kinetic model and analytical techniques employed herein may be generally applicable to other enzymes with similar regulatory schemes.

Inhibitor‐2 functions like a chaperone to fold three expressed isoforms of mammalian protein phosphatase‐1 into a conformation with the specificity and regulatory properties of the native enzyme

“…The catalytic subunit of PP1 (PPlC; Cohen et al, 1988 a, purified by Dr D. Schelling), thc glycogen-associated form of PP1 (PPlG) (Hubbard and Cohen, 1989a), glycogen phosphorylase (Fischer and Krebs, 1958; purified by Dr C. MacKintosh), phosphorylase kinase (Cohen, 1973 ; purified by Mr F. B. Caudwell), inhibitor-1 ; purified by Dr M. J. Hubbard), heavy meromyosin (Margossian and Lowey 1982; purified by Dr L. K. MacDougall), myosin light-chain kinase (Blumenthal and Stull, 1980; purified by Dr P. Dent), skeletal-muscle protein phosphatase-lM (Dent et al, 1992; Dr L. K. MacDougall), GSK3 ; purified by Dr C. Smythe) and glycogen (Smythe et al, 1988; purified by Dr P. Dent) were prepared from rabbit skeletal muscle by other members of the Unit. Smooth-muscle protein phosphatase-1M was purified to homogeneity from chicken gizzard myofibrils (Alessi et al, 1992).…”

Smooth muscle myosin phosphatase inhibition and force enhancement by black sponge toxin

“…The mechanical response was not altered when ethanol was used instead of DMSO. The isometric tension was recorded using an AME 801 force transducer (SensoNor, Horten, Norway) [7].…”

“…Protein phosphatase activity was measured by a modification of the isotope method [9], using isolated bovine heart light chains [7] which had been 32P-phosphorylated [lo] by chicken gizzard MLCK [ 1 l] as substrate. MLCK activity was determined by measuring the incorporation of 32P from [T-~~P]ATP into the bovine heart light chains [l 11.…”

Phosphorylation of skeletal muscle myosin light chain kinase by the catalytic subunit of cAMP‐dependent protein kinase

“…Although highly specific for myosin as compared with other possible protein substrates, MLCK from a given muscle type preferentially phosphorylates the light chain from the same muscle type [7,8 ]. By SDS gel electrophoresis, the apparent Mr-values of the purified enzymes were reported to be Abbreviations: EGTA, ethylene glycol bis-(#-aminoethyl) ether-N,N,N',N'-tetraacetic acid; SDS, sodium dodecyl sulfate; CaM, calmodulin; MES, 2 [N-morpholino ] ethanesulfonic acid; DTT, dithiothreitol; PIPES, piperazine-N,N'-bis-(2-ethane sulfonic acid); cAMP, adenosine 3' :5'-cyclic monophosphate; LC2, 18 500 M r phosphorylatable myosin light chain; MLCK, myosin light chain kinase 77 000-90 000 in skeletal muscle [9][10][11][12][13], 94 000 in cardiac muscle [14], but 130 000 in smooth muscle [8]. Several reports have suggested that the enzyme is the same size in all muscle types (M r 130 000-160 000) [15,16].…”