National Heart and Lung Institute, Imperial College London, London, SW3 6LY, UK.
J Mol Cell Cardiol. 2010 Dec;49(6):1003-11. doi: 10.1016/j.yjmcc.2010.09.007. Epub 2010 Sep 17.
A unique feature of MyBP-C in cardiac muscle is that it has multiple phosphorylation sites. MyBP-C phosphorylation, predominantly by PKA, plays an essential role in modulating contractility as part of the cellular response to β-adrenergic stimulation. In vitro studies indicate MyBP-C can be phosphorylated at Serine 273, 282, 302 and 307 (mouse sequence) but little is known about the level of MyBP-C phosphorylation or the sites phosphorylated in heart muscle. Since current methodologies are limited in specificity and are not quantitative we have investigated the use of phosphate affinity SDS-PAGE together with a total anti MyBP-C antibody and a range of phosphorylation site-specific antibodies for the main sites (Ser-273, -282 and -302). With these newly developed methods we have been able to make a detailed quantitative analysis of MyBP-C phosphorylation in heart tissue in situ. We have found that MyBP-C is highly phosphorylated in non-failing human (donor) heart or mouse heart; tris and tetra-phosphorylated species predominate and less than 10% of MyBP-C is unphosphorylated (0, 9.3 ± 1%: 1P, 13.4 ± 2.7%: 2P, 10.5 ± 3.3%: 3P, 28.7 ± 3.7%: 4P, 36.4 ± 2.7%, n=21). Total phosphorylation was 2.7 ± 0.07 mol Pi/mol MyBP-C. In contrast in failing heart and in myectomy samples from HCM patients the majority of MyBP-C was unphosphorylated. Total phosphorylation levels were 23% of normal in failing heart myofibrils (0, 60.1 ± 2.8%: 1P, 27.8 ± 2.8%: 2P, 4.8 ± 2.0%: 3P, 3.7 ± 1.2%: 4P, 2.8 ± 1.3%, n=19) and 39% of normal in myectomy samples. The site-specific antibodies showed a distinctive distribution pattern of phosphorylation sites in the multiple phosphorylation level species. We found that phosphorylated Ser-273, Ser-282 and Ser-302 were all present in the 4P band of MyBP-C but none of them were significant in the 1P band, indicating that there must be at least one other site of MyBP-C phosphorylation in human heart. The pattern of phosphorylation at the three sites was not random, but indicated positive and negative interactions between the three sites. Phosphorylation at Ser-282 was not proportional to the number of sites available. The 2P band contained 302 but not 273; the 3P band contained 273 but not 302.
肌球蛋白结合蛋白 C（MyBP-C）在心肌中的一个独特特征是它具有多个磷酸化位点。MyBP-C 的磷酸化，主要由蛋白激酶 A（PKA）介导，在细胞对β-肾上腺素刺激的反应中作为调节收缩性的一部分，发挥着重要作用。体外研究表明，MyBP-C 可以在丝氨酸 273、282、302 和 307 位（鼠序列）被磷酸化，但关于心肌中 MyBP-C 的磷酸化水平或磷酸化位点知之甚少。由于目前的方法在特异性和定量方面存在局限性，我们研究了使用磷酸化亲和 SDS-PAGE 与总抗 MyBP-C 抗体和一系列磷酸化位点特异性抗体（主要是 Ser-273、-282 和 -302 位）相结合的方法。使用这些新开发的方法，我们能够对心脏组织中 MyBP-C 的磷酸化进行详细的定量分析。我们发现，非衰竭人类（供体）心脏或小鼠心脏中的 MyBP-C 高度磷酸化；三磷酸化和四磷酸化形式占主导地位，不到 10%的 MyBP-C 未磷酸化（0，9.3±1%：1P，13.4±2.7%：2P，10.5±3.3%：3P，28.7±3.7%：4P，36.4±2.7%，n=21）。总磷酸化水平为 2.7±0.07 mol Pi/mol MyBP-C。相比之下，在衰竭心脏和肥厚型心肌病患者的心肌切除术样本中，大多数 MyBP-C 未磷酸化。衰竭心肌肌原纤维中的总磷酸化水平为正常的 23%（0，60.1±2.8%：1P，27.8±2.8%：2P，4.8±2.0%：3P，3.7±1.2%：4P，2.8±1.3%，n=19），心肌切除术样本中的总磷酸化水平为正常的 39%。磷酸化特异性抗体显示出在多种磷酸化水平的 MyBP-C 中磷酸化位点的独特分布模式。我们发现磷酸化的 Ser-273、Ser-282 和 Ser-302 均存在于 MyBP-C 的 4P 带中，但在 1P 带中均不显著，表明在人心脏中，MyBP-C 肯定还有至少一个其他磷酸化位点。三个位点的磷酸化模式不是随机的，而是表明三个位点之间存在正、负相互作用。Ser-282 的磷酸化与可用位点的数量不成比例。2P 带包含 302，但不包含 273；3P 带包含 273，但不包含 302。
