Abstract
Kinase-related protein, also known as KRP or telokin, is an independently 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 depolymerization in vitro. KRP competes with MLCK for binding to myosin, suggesting that both proteins bind to myosin by the KRP domain (Shirinsky, 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 KRP binds to unphosphorylated myosin with a stoichiometry of 1 mol of KRP/1 mol of myosin and an affinity of 5.5 microM. KRP slows the rate of proteolytic cleavage of the head-tail junction of heavy meromyosin by papain and chymotrypsin, suggesting it is binding to this region of myosin. In addition, competition experiments, using soluble headless fragments of nonmuscle myosin, confirmed that KRP interacts with the regulatory light chain binding region of myosin. The regions important for KRP's binding to myosin were investigated using bacterially expressed KRP truncation mutants. We determined that the acid-rich sequence between Gly138 and Asp151 of KRP is required for high affinity myosin binding, and that the amino terminus and beta-barrel regions weakly interact with myosin. All KRP truncations, at concentrations comparable to their KD values, exhibited some stabilization of myosin filaments against ATP depolymerization in vitro, suggesting that KRP's ability to stabilize myosin filaments is commensurate with its myosin binding affinity. KRP weakened the Km but not the Vmax of phosphorylation of myosin by MLCK, demonstrating that bound KRP does not prevent MLCK from activating myosin.
Highlights
In resting smooth muscle, myosin exists in a predominantly dephosphorylated and inactive state [1] and contraction is initiated by a calcium-calmodulin-dependent phosphorylation of the regulatory light chain (RLC)1 of myosin by myosin light chain kinase (MLCK) [2]
MLCK has two other IgG repeats, in addition to the kinase-related protein (KRP) IgG repeat, which are located amino-terminal to the catalytic core [26]
Okagawi et al [31] compared the primary sequences of the myosin binding IgG motif in MyBP-C and the carboxyl-terminal IgG motif of MLCK, and predicted 15 residues within the KRP -barrel that might be important for binding to myosin
Summary
Vol 272, No 40, Issue of October 3, pp. 25353–25359, 1997 Printed in U.S.A. Debra L. KRP binds to unphosphorylated smooth muscle myosin filaments and stabilizes them against ATP-induced depolymerization in vitro. The discrepancy between myosin’s filamentous state in vivo and in vitro may be explained by the presence of regulatory proteins, which stabilize unphosphorylated smooth muscle myosin filaments in vivo. One candidate for such a protein is an abundant myosin-binding protein, termed kinase-related protein (KRP) and referred to as telokin. KRP binds to unphosphorylated myosin filaments in vitro and stabilizes them against MgATP-dependent depolymerization [10]. We demonstrate that the acidic carboxyl terminus of KRP is important for high affinity myosin binding and subsequent stabilization of myosin filaments. Some of these data have been presented in preliminary form [17]
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