Modulation of Actin-Myosin Interaction by N-terminal Unique Domain of Myorod of Molluscan Catch Muscle

Abstract

Myorod, a thick filament protein of molluscan smooth muscle, is an alternative product of the myosin heavy chain gene. It contains the rod domain identical to that of the rod portion of the myosin molecule and a unique N-terminal domain (NMR). We previously reported that myorod is phosphorylated within NMR at Thr141 by vertebrate smooth muscle myosin light chain kinase (Sobieszek et al., Arch. Biochem. Biophys., 454: 197-205, 2006). To investigate whether phosphorylation of NMR may affect the actin-myosin interaction, two peptides were synthesized with sequence corresponding to this domain. One of two peptides included a phosphorylated Thr141 (NMR-P) and the other not (NMR-unP). We found that the latter peptide interacted with rabbit and molluscan F-actin causing an aggregation and sedimentation of F-actin at low-speed centrifugation while NMR-P had no effect on the distribution of F-actin in the supernatant and pellet fractions. Co-sedimentation of NMR-unP with isolated molluscan thin filaments revealed that in this case the interaction was Ca2+-dependent. NMR-unP slightly inhibited the Mg2+-ATPase activity of actomyosin reconstructed from molluscan myosin and rabbit F-actin. In contrast, NMR-P as well as intact phosphorylated myorod increased actomyosin Mg2+-ATPase activity of about 1.5-3 fold depending on the experimental conditions. This finding was supported by a 3-fold higher binding affinity of NMR-P for myosin filaments with comparison of that of NMR-unP. Taken together these results implicate that myorod, a thick filament protein of molluscan catch muscle, can modulate actin-myosin interaction in a phosphorylation-dependent manner.