Department of Molecular and Cellular Biology, University of California, Davis, One Shields Avenue, Davis, California 95616, USA.
J Proteome Res. 2010 Apr 5;9(4):1843-53. doi: 10.1021/pr901006h.
Cardiac myosin binding protein-C (cMyBP-C) is a large multidomain accessory protein bound to myosin thick filaments in striated muscle sarcomeres. It plays an important role in the regulation of muscle contraction, and mutations in the gene encoding cMyBP-C are a common cause of familial hypertrophic cardiomyopathy, the leading cause of sudden cardiac death in young people. (1) The N-terminal domains including the C0, C1, cMyBP-C motif, and C2 domains play a crucial role in maintaining and modulating actomyosin interactions (keeping normal cardiac function) in a phosphorylation-dependent manner. The cMyBP-C motif or "M-domain" is a highly conserved linker domain in the N-terminus of cMyBP-C that contains three to five protein kinase A (PKA) phosphorylation sites, depending on species. For the human isoform, three PKA sites were previously identified (Ser(275), Ser(284), and Ser(304)), while three homologous sites exist in the murine isoform (Ser(273), Ser(282), and Ser(302)). The murine cMyBP-C isoform contains an additional conserved consensus site, Ser(307) that is not present in the human isoform. In this study, we investigated sites of PKA phosphorylation of murine and human cMyBP-C by treating the recombinant protein C0C2 ( approximately 50 KDa, which contains the N-terminal C0, C1, M, and C2 domains) and C1C2 (approximately 35 KDa, contains C1, M, and C2 domains) with PKA and assessing the phosphorylation states using SDS-PAGE with ProQ Diamond staining, and powerful hybrid mass spectrometric analyses. Both high-accuracy bottom-up and measurements of intact proteins mass spectrometric approaches were used to determine the phosphorylation states of C0C2 and C1C2 proteins with or without PKA treatment. Herein, we report for the first time that there are four PKA phosphorylation sites in both murine and human M-domains; both murine Ser(307) and a novel human Ser(311) can be phosphorylated in vitro by PKA. Future studies are needed to investigate the phosphorylation state of murine and human cMyBP-C in vivo.
心肌肌球蛋白结合蛋白 C（cMyBP-C）是一种结合在横纹肌肌节肌球蛋白粗丝上的大型多功能辅助蛋白。它在肌肉收缩的调节中发挥着重要作用，编码 cMyBP-C 的基因突变是家族性肥厚型心肌病的常见原因，这是年轻人心脏性猝死的主要原因。（1）N 端结构域包括 C0、C1、cMyBP-C 结构域和 C2 结构域，通过依赖于磷酸化的方式，以一种关键的方式在维持和调节肌动球蛋白相互作用（保持正常心脏功能）中发挥作用。cMyBP-C 结构域或“M 结构域”是 cMyBP-C N 端的一个高度保守的连接结构域，包含三个到五个蛋白激酶 A（PKA）磷酸化位点，具体取决于物种。对于人类同工型，以前已经鉴定出三个 PKA 位点（Ser（275）、Ser（284）和 Ser（304）），而在鼠同工型中存在三个同源位点（Ser（273）、Ser（282）和 Ser（302））。鼠 cMyBP-C 同工型含有一个额外的保守共识位点 Ser（307），该位点不存在于人类同工型中。在这项研究中，我们通过用 PKA 处理重组蛋白 C0C2（约 50 kDa，包含 N 端 C0、C1、M 和 C2 结构域）和 C1C2（约 35 kDa，包含 C1、M 和 C2 结构域）来研究鼠和人 cMyBP-C 的 PKA 磷酸化位点，并使用 SDS-PAGE 与 ProQ Diamond 染色，以及强大的杂交质谱分析来评估磷酸化状态。使用高精度从头测序和完整蛋白质质谱测量的两种方法来确定经或未经 PKA 处理的 C0C2 和 C1C2 蛋白的磷酸化状态。在此，我们首次报道在鼠和人 M 结构域中都有四个 PKA 磷酸化位点；鼠 Ser（307）和新的人 Ser（311）都可以在体外被 PKA 磷酸化。未来的研究需要研究体内鼠和人 cMyBP-C 的磷酸化状态。
