Yang Z, Sweeney H L
Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia 19104-6085, USA.
J Biol Chem. 1995 Oct 20;270(42):24646-9. doi: 10.1074/jbc.270.42.24646.
Regulation of the ATPase activity of smooth and nonmuscle myosin II involves reversible phosphorylation of the regulatory light chain (RLC). The RLC from skeletal muscle myosin (skRLC) is unable to confer regulation (myosin is locked in an inactive state) to smooth muscle myosin when substituted for the endogenous smooth RLC (smRLC). Studies of chimeric light chains comprised of the N- or C-terminal half of each skRLC and smRLC suggest that the structural basis for the loss of this regulation is within the C-terminal half of the RLC (Trybus, K.M., and Chatman, T.A. (1993) J. Biol. Chem. 268, 4412-4419). The purpose of this study is to delineate the structural elements within the C-terminal half of the smRLC that are absent in the skRLC and are necessary for regulation. By sequence comparison, six residues, Arg-103, Arg-123, Met-129, Gly-130, Arg-143, and Arg-160, which are conserved in regulated myosin RLCs but missing in nonregulated myosin RLCs, were identified in smRLC. To test whether these amino acids provide the missing structural elements necessary for phosphorylation-mediated regulation, a skRLC was engineered that replaced the corresponding skRLC amino acids (positions 100, 120, 126, 127, 140, and 157, respectively) with their smRLC counterparts. Using a newly developed RLC exchange procedure, the purified mutant protein was evaluated for its ability to regulate chicken gizzard smooth muscle myosin. Substitution of the six conserved amino acids into the skRLC completely restored phosphorylation-mediated regulation. Thus, a subset of these amino acids, including four basic arginine residues located in the E, F, G, and H helices which are missing in skRLC, may be the structural coordinates for the phosphorylserine in the N terminus. Based on this result, the regulation of glycogen phosphorylase is discussed as a model for the regulation of smooth muscle myosin.
平滑肌和非肌肉肌球蛋白II的ATP酶活性调节涉及调节性轻链（RLC）的可逆磷酸化。当用内源性平滑肌RLC（smRLC）替代时，骨骼肌肌球蛋白的RLC（skRLC）无法赋予平滑肌肌球蛋白调节能力（肌球蛋白被锁定在无活性状态）。对由每个skRLC和smRLC的N端或C端一半组成的嵌合轻链的研究表明，这种调节丧失的结构基础在于RLC的C端一半（Trybus，K.M.，和Chatman，T.A.（1993）J. Biol. Chem. 268，4412 - 4419）。本研究的目的是描绘smRLC C端一半中skRLC所没有且调节所必需的结构元件。通过序列比较，在smRLC中鉴定出六个在受调节的肌球蛋白RLC中保守但在不受调节的肌球蛋白RLC中缺失的残基，即精氨酸-103、精氨酸-123、甲硫氨酸-129、甘氨酸-130、精氨酸-143和精氨酸-160。为了测试这些氨基酸是否提供了磷酸化介导调节所必需的缺失结构元件，构建了一种skRLC，用其smRLC对应物替换了相应的skRLC氨基酸（分别位于位置100、120、126、127、140和157）。使用新开发的RLC交换程序，评估纯化的突变蛋白调节鸡砂囊平滑肌肌球蛋白的能力。将这六个保守氨基酸替换到skRLC中完全恢复了磷酸化介导的调节。因此，这些氨基酸的一个子集，包括位于skRLC中缺失的E、F、G和H螺旋中的四个碱性精氨酸残基，可能是N端磷酸丝氨酸的结构坐标。基于这一结果，讨论了糖原磷酸化酶的调节作为平滑肌肌球蛋白调节的模型。
