Molecular Screen Identifies Cardiac Myosin-Binding Protein-C as a Protein Kinase G-Iα Substrate.
Molecular Screen Identifies Cardiac Myosin-Binding Protein-C as a Protein Kinase G-Iα Substrate.
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分子筛选将心脏肌球蛋白结合蛋白-C鉴定为蛋白激酶G-Iα底物。
DOI：
10.1161/circheartfailure.115.002308
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发表时间：
2015-11
期刊：
影响因子：
--
通讯作者：
Blanton RM
中科院分区：
文献类型：
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作者：
Thoonen R;Giovanni S;Govindan S;Lee DI;Wang GR;Calamaras TD;Takimoto E;Kass DA;Sadayappan S;Blanton RM
cgmp依赖性蛋白激酶I （PKGI）的药理学激活已成为人类心力衰竭的治疗策略。然而，PKG激活药物受到PKG诱导的血管舒张引起的低血压的限制。心肌特异性的PKGIα抗重塑底物可能提供了规避这一限制的靶点，但目前对其知之甚少。我们筛选了与PKGIα亮氨酸拉链（LZ）结合域相互作用的心肌蛋白，以鉴定心肌特异性PKGI抗重塑底物。我们的筛选确定了心肌肌球蛋白结合蛋白C (cMyBP-C)，这是一种心肌细胞特异性蛋白，已被证明在磷酸化状态下抑制心脏重塑，当突变导致人类肥厚性心肌病。gst下拉和cgmp结合珠沉淀证实心肌裂解物中PKGIα-cMyBP-C相互作用。体外研究表明，纯化的PKGIα使cMyBP-C m结构域Ser-273、Ser-282和Ser-302磷酸化。在转染PKGIα的COS细胞中，cGMP诱导这些残基上的cMyBP-C磷酸化，而在转染LZ突变体PKGIα的细胞中，cGMP诱导的这些残基上的cMyBP-C磷酸化不会破坏LZ底物结合。在左室压力过载的小鼠中，与未治疗的小鼠相比，西地那非激活PKGI增加了Ser-273位点的cMyBP-C磷酸化。cGMP还能诱导离体心肌细胞cMyBP-C磷酸化。综上所述，这些数据支持PKGIα和cMyBP-C在心脏中相互作用，cMyBP-C是一种抗重构PKGIα激酶底物。该研究首次鉴定了心肌特异性PKGIα LZ依赖性抗重构底物，并支持进一步探索PKGIα心肌LZ底物作为心力衰竭的潜在治疗靶点。
Pharmacologic activation of cGMP-dependent protein kinase I (PKGI) has emerged as a therapeutic strategy for humans with heart failure. However, PKG activating drugs have been limited by hypotension arising from PKG-induced vasodilation. PKGIα anti-remodeling substrates specific to the myocardium might provide targets to circumvent this limitation, but currently remain poorly understood. We performed a screen for myocardial proteins interacting with the PKGIα leucine zipper (LZ) binding domain to identify myocardial-specific PKGI anti-remodeling substrates. Our screen identified cardiac myosin binding protein C (cMyBP-C), a cardiac myocyte-specific protein, which has been demonstrated to inhibit cardiac remodeling in the phosphorylated state, and when mutated leads to hypertrophic cardiomyopathy in humans. GST-pulldowns and precipitations with cGMP-conjugated beads confirmed the PKGIα-cMyBP-C interaction in myocardial lysates. In vitro studies demonstrated that purified PKGIα phosphorylates the cMyBP-C M-domain at Ser-273, Ser-282, and Ser-302. cGMP induced cMyBP-C phosphorylation at these residues in COS cells transfected with PKGIα, but not in cells transfected with LZ mutant PKGIα containing mutations to disrupt LZ substrate binding. In mice subjected to LV pressure overload, PKGI activation with sildenafil increased cMyBP-C phosphorylation at Ser-273 compared with untreated mice. cGMP also induced cMyBP-C phosphorylation in isolated cardiac myocytes. Taken together these data support that PKGIα and cMyBP-C interact in the heart, and that cMyBP-C is an anti-remodeling PKGIα kinase substrate. This study provides the first identification of a myocardial specific PKGIα LZ-dependent anti-remodeling substrate and supports further exploration of PKGIα myocardial LZ substrates as potential therapeutic targets for heart failure.