SITES OF INTERACTION BETWEEN KINASE-RELATED PROTEIN AND SMOOTH-MUSCLEMYOSIN

Kinase-related protein, also known as KRP or telokin, is an independen tly expressed protein product derived from a gene within the gene for myosin light chain kinase (MLCK). KRP binds to unphosphorylated smooth muscle myosin filaments and stabilizes them against ATP-induced depol ymerization in vitro. KRP competes with MLCK for binding to myosin, su ggesting that both proteins bind to myosin by the KRP domain (Shirinsk y, V. P., Vorotnikov, A. V., Birukov, K. G., Nanaev, A. K., Collinge, M., Lukas, T. J., Sellers, J. R., and Watterson, D. M. (1993) J. Biol. Chem. 268, 16578-16583). In this study, we investigated which regions of myosin and KRP interact in vitro. Using cosedimentation assays, we determined that HRP binds to unphosphorylated myosin with a stoichiom etry of 1 mol of KRP/1 mol of myosin and an affinity of 5.5 mu m. KRP slows the rate of proteolytic cleavage of the head-tail junction of he avy meromyosin by papain and chymotrypsin, suggesting it is binding to this region of myosin. In addition, competition experiments, using so luble headless fragments of nonmuscle myosin, confirmed that KRP inter acts with the regulatory light chain binding region of myosin. The reg ions important for KRP's binding to myosin were investigated using bac terially expressed KRP truncation mutants. We determined that the acid -rich sequence between Gly(138) and Asp(151) of KRP is required for hi gh affinity myosin binding, and that the amino terminus and beta-barre l regions weakly interact with myosin. All KRP truncations, at concent rations comparable to their K-D values, exhibited some stabilization o f myosin filaments against ATP depolymerization in vitro, suggesting t hat KRP's ability to stabilize myosin filaments is commensurate with i ts myosin binding affinity. HRP weakened the K-m but not the V-max of phosphorylation of myosin by MLCK, demonstrating that bound HRP does n ot prevent MLCK from activating myosin.