STRUCTURAL REQUIREMENTS FOR PHOSPHORYLATION OF MYOSIN REGULATORY LIGHT-CHAIN FROM SMOOTH-MUSCLE

Site-directed and chimeric mutations of myosin regulatory light chains were used to identify residues important for phosphorylation of Ser(1 9) by smooth muscle myosin light chain kinase. Arg(16) and hydrophobic residues C-terminal of Ser(19) in smooth muscle light chain were impo rtant substrate determinants in the intact protein. However, changes i n the kinetic properties with mutations in the light chain were substa ntially smaller than results reported with structurally similar synthe tic peptide substrates. These results together with the low V-max valu e for short peptide substrates containing the consensus phosphorylatio n sequence suggest that there may be additional sites of interactions between the kinase and protein substrate. Chimeras of skeletal and smo oth muscle light chains were constructed with exchanges at the N termi nus and subdomains I, II, III, and IV. Analysis of results obtained on the kinetic properties for phosphorylation showed that subdomains I a nd II contribute to high V-max values. Thus, a region distant from the consensus phosphorylation sequence in smooth muscle light chain is al so an important substrate determinant for myosin light chain kinase.