Smooth muscle myosin light chain kinase, supramolecular organization, modulation of activity, and related conformational changes

Abstract

It has recently been suggested that activation of smooth muscle myosin light chain kinase (MLCK) can be modulated by formation of supramolecular structures (Sobieszek, A. 1991. Regulation of smooth muscle myosin light chain kinase. Allosteric effects and co-operative activation by CaM. J. Mol. Biol. 220:947-957). The present light scattering data demonstrate that the inactive (calmodulin-free) MLCK apoenzyme exists in solution as a mixture of oligomeric (2% by weight), dimeric (53%), and monomeric (45%) species at physiological ionic strength (160 mM salt). These long-living assemblies, the lifetime of which was measured by minutes, were in equilibrium with each other. The most likely form of the oligomer was a spiral-like hexamer, the dimensions of which fit very well the helical structure of self-assembled myosin filaments (Sobieszek, A. 1972. Cross-bridges on self-assembled smooth muscle myosin filaments. J. Mol. Biol. 70:741-744). After activation of the kinase by calmodulin (CaM) we could not detect any appreciable changes in the distribution of the kinase species either when the kinase was saturated with CaM or when its molar concentration exceeded that of CaM. Our fluorescent measurements suggest that the earlier observed inhibition of kinase at substoichiometric amounts of CaM (Sobieszek, A., A. Strobl, B. Ortner, and E. Babiychuk. 1993. Ca2+-calmodulin-dependent modification of smooth-muscle myosin light chain kinase leading to its co-operative activation by calmodulin. Biochem. J. 295:405-411) is associated with slow conformational change(s) of the activated (CaM-bound) kinase molecules. Such conformational rearrangements also took place with equimolar kinase to CaM; however, in this case there was no decrease in MLCK activity. The nature of these conformational changes, which are accompanied by reduction of the kinase for CaM affinity, is discussed.

Insights on the vasorelaxant mode of action of malbrancheamide

“…myosin light‐chain kinase (MLCK). The contractile activity in smooth muscle is initiated by a Ca 2+ ‐CaM‐dependent self‐phosphorylation of MLCK; upon CaM binding to MLCK, it self‐phosphorylates and becomes active [22]. Thus, any molecule with the ability to prevent the formation of CaM‐Ca 2+ ‐MLCK complex will lead to an inactivation of MLCK and in turn to a smooth muscle relaxant effect.…”

“…The contractile activity in smooth muscle is initiated by a Ca 2+ -CaMdependent self-phosphorylation of MLCK; upon CaM binding to MLCK, it self-phosphorylates and becomes active. [22] Thus, any molecule with the ability to prevent the formation of CaM-Ca 2+ -MLCK complex will lead to an inactivation of MLCK and in turn to a smooth muscle relaxant effect. In this context, González-Andrade et al demonstrated by means of a CaM fluorescent biosensor that malbrancheamide (1) prevented the formation of the complex CaM-Ca 2+ -MLCK.…”

Importance of the interaction protein–protein of the CaM–PDE1A and CaM–MLCK complexes in the development of new anti‐CaM drugs

“…In the absence of Ca 2+ and/or CaM, MLCK remains inactive because it binds to its own pseudosubstrate domain, which has sequence homology to the myosin light chain, its physiological substrate. However, upon CaM binding, this domain is removed from the active site, and the enzyme becomes active and self-phosphorylates (Filenko et al, 1997). As with the complex Ca 2+ -CaM-PDE1, the Ca 2+ -CaM-MLCK complex could be the target of novel drugs.…”

“…In this study, we analyzed both experimentally and theoretically the effect of the CaM inhibitor malbrancheamide (MBC) on three Ca 2+ -CaM target protein or peptide complexes, namely, Ca 2+ -CaM-PDE1A, Ca 2+ -CaM-MLCK, and Ca 2+ -CaMpeptide 1 derived from the human aII-spectrin (aII-spec). All these were carried out using the fluorescent biosensor hCaM M124C-mBBr (González-Andrade et al, 2009). In the first two cases, chlorpromazine (CPZ), a classical CaM inhibitor was also analyzed.…”

Modulation of myosin filament activation by telokin in smooth muscle

“…Our studies on the oligomeric suprastructure of MLCK [33] and the effects of telokin on its oligomeric properties [20] as well as a very slow phosphorylation of telokin by this kinase [34] provide explanation for at least some of these controversial observations. The inhibition of myosin phosphorylation was shown to result from dimerization (or monomerization) of the kinase by telokin because only the oligomers appeared to be tightly bound to myosin filaments.…”