Identification of novel protein kinase A phosphorylation sites in the M-domain of human and murine cardiac myosin binding protein-C using mass spectrometry analysis.
Identification of novel protein kinase A phosphorylation sites in the M-domain of human and murine cardiac myosin binding protein-C using mass spectrometry analysis.
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DOI：
10.1021/pr901006h
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发表时间：
2010-04-05
影响因子：
4.4
通讯作者：
Leary, Julie A.
中科院分区：
文献类型：
--
作者：
Jia, Weitao;Shaffer, Justin F.;Harris, Samantha P.;Leary, Julie A.
关键词：
心肌肌球蛋白结合蛋白-C（cMyBP-C）是一种与横纹肌肌节中的肌球蛋白粗丝结合的多结构域辅助蛋白。它在肌肉收缩的调节中起着重要作用，编码cMyBP-C的基因突变是家族性肥厚型心肌病的常见原因，家族性肥厚型心肌病是年轻人心脏性猝死的主要原因。包括C 0、C1、cMyBP-C基序和C2结构域的N-末端结构域以磷酸化依赖性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）中起关键作用。cMyBP-C基序或“M-结构域”是cMyBP-C的N-末端中的高度保守的接头结构域，其含有三至五个蛋白激酶A（PKA）磷酸化位点，这取决于物种。对于人同种型，先前鉴定了三个PKA位点（Ser 275、Ser 284和Ser 304），而鼠同种型中存在三个同源位点（Ser 273、Ser 282和Ser 302）。鼠cMyBP-C同种型含有一个额外的保守的共有位点，Ser 307，这在人同种型中不存在。在这项研究中，我们通过用PKA处理重组蛋白C 0 C2（~50 KDa，含有N-末端C 0，C1，M和C2结构域）和C1 C2（~35 KDa，含有C1，M和C2结构域），并使用带有ProQ Diamond染色的SDS-PAGE和强大的混合质谱分析评估磷酸化状态，研究了鼠和人cMyBP-C的PKA磷酸化位点。使用高准确度自下而上和完整蛋白质质谱方法的测量来确定有或没有PKA处理的C 0 C2和C1 C2蛋白的磷酸化状态。在此，我们首次报告，有四个PKA磷酸化位点在小鼠和人的M-域，小鼠Ser 307和一种新的人Ser 311可以在体外磷酸化PKA。需要进一步研究小鼠和人cMyBP-C在体内的磷酸化状态。
Cardiac myosin binding protein-C (cMyBP-C) is a large multi-domain 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. 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 (Ser275, Ser284, and Ser304), while three homologous sites exist in the murine isoform (Ser273, Ser282, and Ser302). The murine cMyBP-C isoform contains an additional conserved consensus site, Ser307 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 (~50 KDa, which contains the N-terminal C0, C1, M, and C2 domains) and C1C2 (~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 Ser307 and a novel human Ser311 can be phosphorylated in vitro by PKA. Future studies are needed to investigate the phosphorylation state of murine and human cMyBP-C in vivo.
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