The Myosin-binding Protein C Motif Binds to F-actin in a Phosphorylation-sensitive Manner
The Myosin-binding Protein C Motif Binds to F-actin in a Phosphorylation-sensitive Manner
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DOI：
10.1074/jbc.m808850200
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发表时间：
2009-05-01
影响因子：
4.8
通讯作者：
Harris, Samantha P.
中科院分区：
文献类型：
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作者：
Shaffer, Justin F.;Kensler, Robert W.;Harris, Samantha P.
心肌肌球蛋白结合蛋白C(cMyBP-C)是一种在心肌肌瘤中表达的调节蛋白，可与肌球蛋白、肌动蛋白和肌动蛋白相互作用。CMyBP-C以磷酸化依赖的方式调节肌球蛋白的相互作用，但目前尚不清楚这些作用是否需要与肌球蛋白、肌动蛋白或肌动蛋白的相互作用。在这里，我们用共构筑结合分析表明，小鼠cMyBP-C的4个N-末端结构域(即C_0-C_1-m-C_2)与F-肌动蛋白结合的解离常数(K-d)接近10微米，摩尔结合比(B-max)接近1.0，表明与肌动蛋白的结合为1：1(摩尔/摩尔)。电子显微镜和光散射分析表明，这些结构域与F-肌动蛋白细丝发生交叉连接，意味着与肌动蛋白有多个相互作用部位。MyBP-C调节基序或m结构域的磷酸化减少了与肌动蛋白的结合(减少了Bmax)，并消除了肌动蛋白的交叉连接。这些结果表明，cMyBP-C的N端通过多个不同的结合位点与F-肌动蛋白相互作用，一个或多个位点的结合被磷酸化减少。与肌动蛋白的可逆相互作用可能有助于cMyBP-C增加跨桥循环的效果。
Cardiac myosin-binding protein C (cMyBP-C) is a regulatory protein expressed in cardiac sarcomeres that is known to interact with myosin, titin, and actin. cMyBP-C modulates actomyosin interactions in a phosphorylation-dependent way, but it is unclear whether interactions with myosin, titin, or actin are required for these effects. Here we show using cosedimentation binding assays, that the 4 N-terminal domains of murine cMyBP-C (i. e. C0-C1-m-C2) bind to F-actin with a dissociation constant (K-d) of similar to 10 mu M and a molar binding ratio (B-max) near 1.0, indicating 1: 1 (mol/mol) binding to actin. Electron microscopy and light scattering analyses show that these domains cross-link F-actin filaments, implying multiple sites of interaction with actin. Phosphorylation of the MyBP-C regulatory motif, or m-domain, reduced binding to actin (reduced Bmax) and eliminated actin cross-linking. These results suggest that the N terminus of cMyBP-C interacts with F-actin through multiple distinct binding sites and that binding at one or more sites is reduced by phosphorylation. Reversible interactions with actin could contribute to effects of cMyBP-C to increase crossbridge cycling.