Identification of phosphorylation and other post-translational modifications in the central C4-C5 domains of murine cardiac myosin binding protein C

Cardiac myosin binding protein C (cMyBPC) is a critical multidomain protein that modulates myosin cross bridge behavior and cardiac contractility. cMyBPC is principally regulated by phosphorylation of the residues within the M-domain of its N-terminus. However, not much is known about the phosphorylation or other post-translational modification (PTM) landscape of the central C4C5 domains. In this study, the presence of phosphorylation outside the M-domain was confirmed in vivo using mouse models expressing cMyBPC with nonphosphorylatable serine (S) to alanine substitutions. Purified recombinant mouse C4C5 domain constructs were incubated with 13 different kinases, and samples from the 6 strongest kinases were chosen for mass spectrometry analysis. A total of 26 unique phosphorylated peptides were found, representing 13 different phosphorylation sites including 10 novel sites. Parallel reaction monitoring and subsequent mutagenesis experiments revealed that the S690 site (UniProtKB O70468) was the predominant target of PKA and PKG1. We also report 6 acetylation and 7 ubiquitination sites not previously described in the literature. These PTMs demonstrate the possibility of additional layers of regulation and potential importance of the central domains of cMyBPC in cardiac health and disease. Data are available via ProteomeXchange with identifier PXD031262

心肌肌球蛋白结合蛋白C（Cardiac myosin binding protein C，cMyBPC）是一类关键的多结构域蛋白，可调控肌球蛋白横桥行为与心肌收缩能力。cMyBPC主要通过其N端M结构域内的残基磷酸化实现调控，但目前对于中央C4C5结构域的磷酸化及其他翻译后修饰（post-translational modification，PTM）谱仍知之甚少。本研究通过构建携带丝氨酸（S）突变为丙氨酸的不可磷酸化cMyBPC突变体的小鼠模型，在体内证实了M结构域外存在磷酸化修饰。将纯化的重组小鼠C4C5结构域构建体与13种不同激酶共孵育后，选取与6种活性最强的激酶反应后的样品进行质谱分析。最终共鉴定得到26条独特的磷酸化肽段，对应13个不同的磷酸化位点，其中10个为全新发现的位点。平行反应监测及后续诱变实验表明，S690位点（UniProtKB数据库编号O70468）是蛋白激酶A（Protein Kinase A，PKA）与蛋白激酶G1（Protein Kinase G1，PKG1）的主要作用靶点。本研究还报道了6个乙酰化位点与7个泛素化位点，均为此前文献未记载的全新位点。上述翻译后修饰提示，cMyBPC中央结构域可能存在多层调控机制，且其在心脏健康与疾病进程中或具有重要作用。相关数据集已通过ProteomeXchange数据库公开，标识符为PXD031262