Investigation on the functional significance of myosin light chain kinase and myosin light chain phosphorylation

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Myosin light chain kinase (MLCK) is a multifunctional protein with kinase domain in the middle, actin-binding domain in the amino-terminus, and myosin binding domain in the carboxyl-terminus of the molecule. In smooth muscles, MLCK phosphorylates the regulatory light chain (RLC) to activate myosin ATPase activity and start muscle shortening. In cardiac muscles, however, MLCK also phosphorylates RLC and has a modulatory effect on muscle contraction, i.e. phorphorylation of RLC increases calcium sensitivity of isometric force production. Recently, Kohama et al. reported that MLCK could enhance molecular function by directly binding to smooth muscle myosin molecules without phosphorylating RLC. Although similar MLCK isoform is expressed and localized on the myosin filaments in cardiac muscles, the functional role of MLCK is not well elucidated. Therefore, we investigated the effect of MLCK on the molecular function of cardiac myosin in vitro.An amino-terminal MLCK fragment (MF) containing … More myosin-binding region but lacking the kinase domain was expressed in E.coli using bovine stomach MLCK cDNA and purified. Myosin was purified from rat cardiac muscles. Actin-activated ATPase activity and actin filament velocity in the in vitro motility assay were measured in the presence and absence of 5mmol/L of MF. The filament velocity was 22% higher in the presence of MF compared to the control condition (5.6±0.5 v.s. 4.6±0.4 mm/sec, p<0.01). The ATPase activity was not different between these conditions. On the urea polyacrylamide gels, the phosphorylation level was not affected by the addition of MF, either.The MLCK fragment studied in the present study is expressed in smooth muscle cells as an independent protein, telokin. In smooth muscles, telokin is proposed to bind to the S-1-S-2 junction of myosin molecule and induce a conformational change. Taken together, the present results suggest that MLCK may modulate cardiac function not only by phosphorylating RLC to increase calcium sensitivity but also by directly binding to the myosin molecule to change conformation. Less