Yura Yoshimitsu, Amano Mutsuki, Takefuji Mikito, Bando Tomohiro, Suzuki Kou, Kato Katsuhiro, Hamaguchi Tomonari, Hasanuzzaman Shohag Md, Takano Tetsuya, Funahashi Yasuhiro, Nakamuta Shinichi, Kuroda Keisuke, Nishioka Tomoki, Murohara Toyoaki, Kaibuchi Kozo
Department of Cell Pharmacology, Graduate School of Medicine, Nagoya University.
Cell Struct Funct. 2016 Aug 23;41(2):105-20. doi: 10.1247/csf.16011. Epub 2016 Jun 23.
Protein phosphorylation plays an important role in the physiological regulation of cardiac function. Myocardial contraction and pathogenesis of cardiac diseases have been reported to be associated with adaptive or maladaptive protein phosphorylation; however, phosphorylation signaling in the heart is not fully elucidated. We recently developed a novel kinase-interacting substrate screening (KISS) method for exhaustive screening of protein kinase substrates, using mass spectrometry and affinity chromatography. First, we examined protein phosphorylation by extracellular signal-regulated kinase (ERK) and protein kinase A (PKA), which has been relatively well studied in cardiomyocytes. The KISS method showed that ERK and PKA mediated the phosphorylation of known cardiac-substrates of each kinase such as Rps6ka1 and cTnI, respectively. Using this method, we found about 330 proteins as Rho-kinase-mediated substrates, whose substrate in cardiomyocytes is unknown. Among them, CARP/Ankrd1, a muscle ankyrin repeat protein, was confirmed as a novel Rho-kinase-mediated substrate. We also found that non-phosphorylatable form of CARP repressed cardiac hypertrophy-related gene Myosin light chain-2v (MLC-2v) promoter activity, and decreased cell size of heart derived H9c2 myoblasts more efficiently than wild type-CARP. Thus, focused proteomics enable us to reveal a novel signaling pathway in the heart.
蛋白质磷酸化在心脏功能的生理调节中起着重要作用。据报道，心肌收缩和心脏疾病的发病机制与适应性或非适应性蛋白质磷酸化有关；然而，心脏中的磷酸化信号传导尚未完全阐明。我们最近开发了一种新型的激酶相互作用底物筛选（KISS）方法，用于使用质谱和亲和色谱法详尽筛选蛋白激酶底物。首先，我们研究了细胞外信号调节激酶（ERK）和蛋白激酶A（PKA）介导的蛋白质磷酸化，这两种激酶在心肌细胞中已有相对充分的研究。KISS方法表明，ERK和PKA分别介导了各自激酶已知的心脏底物（如Rps6ka1和cTnI）的磷酸化。使用这种方法，我们发现约330种蛋白质是Rho激酶介导的底物，其在心肌细胞中的底物尚不清楚。其中，一种肌肉锚蛋白重复蛋白CARP/Ankrd1被确认为一种新的Rho激酶介导的底物。我们还发现，CARP的非磷酸化形式比野生型CARP更有效地抑制心脏肥大相关基因肌球蛋白轻链-2v（MLC-2v）的启动子活性，并减小心脏来源的H9c2成肌细胞的细胞大小。因此，聚焦蛋白质组学使我们能够揭示心脏中的一条新信号通路。
