Two new modes of smooth muscle myosin regulation by the interaction between the two regulatory light chains, and by the S2 domain

Previous studies indicated that single-headed smooth muscle myosin and S1 ( a single head fragment) are not regulated through phosphorylation of the re gulatory light chain (RLC). To investigate the importance of the double-hea dedness of myosin and of the S2 region for the phosphorylation-dependent re gulation, we made three types of recombinant mutant smooth muscle HMMs with one intact head and an N-terminally truncated head. The truncated head of Delta MD lacked the motor domain, that of Delta (MD+ELC) lacked the motor a nd essential light chain binding domains, and single-headed HMM had one int act head alone. The basal ATPase activities of the three mutants decreased as the KCl concentration became less than 0.1 M, Such a decrease was not ob served for S1, which had no S2 region, suggesting that S2 is necessary for this myosin behavior. This activity decrease also disappeared when RLCs of Delta MD and Delta (MD+ELC), but that of single-headed HMM, were phosphoryl ated. When their RLCs were unphosphorylated, the three mutants exhibited si milar actin-activated ATPase levels. However, when they were phosphorylated , the actin-activated ATPase activities of Delta MD and Delta (MD+ELC) incr eased to the S1 level, while that of single-headed HMM remained unchanged. Even in the phosphorylated state, the actin-activated ATPase activities of the three mutants and S1 were much lower than that of wild-type HMM. We pro pose that S2 has an inhibitory function that is canceled by an interaction between two phosphorylated RLCs, We also propose that a cooperative interac tion between two motor domains is required for a higher level of actin acti vation.