We are interested in the interactions of two myosin binding proteins, kinase related protein (KRP) and mts1, with myosin. KRP, also called telokin, is an independently expressed protein product derived from a gene within the gene for myosin light chain kinase (MLCK). KRP binds with a stoichiometry of 1 mol/mol and an affinity of 7 uM to unphosphorylated smooth muscle and nonmuscle myosin filaments, but not to phosphorylated myosin or skeletal muscle myosin. KRP prevents the ATP-depolymerization of myosin in vitro, by binding to the S1-S2 junction of myosin. Enzymatic digestions of this region in the presence and absence of KRP provide further evidence that this is the binding site. KRP may prevent myosin from assuming a folded 10S conformation by masking the binding site of the tail for the head region. Both protein kinase A (PK-A) and mitogen-activated-protein (MAP) kinase transferred 1 mole phosphate/mol KRP as determined by a shift in isoelectric focusing. There appears to be no significant effect of KRP phosphorylation by either kinase on myosin binding affinity or stabilization of filaments in the presence of ATP. The mts1 protein belongs to a family of s100 Ca2+ binding proteins, and is highly expressed in metastatic cells and motile cell types. Nonmetastatic mouse mammary carcinoma cells transfected with mts1 exhibit increased cell motility. Mts1 binds both actin and nonmuscle myosin II isoforms as determined by in vitro cosedimentation assays. No interactions between mts1 and nonmuscle myosin I and V are detected in gel overlay assays. The binding of mts1 to nonmuscle myosin II (Kd=13 uM) is dependent upon the presence of Ca2+. The stoichiometry of binding is about 6 mol mts1/mol myosin and the binding site on myosin appears to be in the rod. Both cosedimentation analysis and electron microscopy suggest that mts1 may promote myosin filament disassembly. In the presence of 0.2 mM Ca2+ and 10 uM F-actin, the actin-activated MgATPase activity of phosphorylated nonmuscle myosin II is inhibited when mts1 is present. In an in vitro motility assay, the sliding of F- actin by phosphorylated nonmuscle II was inhibited with increasing concentrations of mts1, in a Ca2+-dependent manner. These results support the notion that mts1 may affect cell motility by its interactions with myosin and actin.
