PDF(Pubmed)
Abstract：
In the heart, alternative splicing of the igf-I gene produces two isoforms: IGF-IEa and IGF-IEc, (Mechano-growth factor, MGF). The sequence divergence between their E-domain regions suggests differential isoform function. To define the biological actions of MGF\'s E-domain, we performed in silico analysis of the unique C-terminal sequence and identified a phosphorylation consensus site residing within a putative 14-3-3 binding motif. To test the functional significance of Ser 18 phosphorylation, phospho-mimetic (S/E18) and phospho-null (S/A18) peptides were delivered to mice at different doses for 2 weeks. Cardiovascular function was measured using echocardiography and a pressure-volume catheter. At the lowest (2.25 mg/kg/day) and highest (9 mg/kg/day) doses, the peptides produced a depression in systolic and diastolic parameters. However, at 4.5 mg/kg/day the peptides produced opposing effects on cardiac function. Fractional shortening analysis also showed a similar trend, but with no significant change in cardiac geometry. Microarray analysis discovered 21 genes (FDR p < 0.01), that were expressed accordant with the opposing effects on contractile function at 4.5 mg/kg/day, with the nuclear receptor subfamily 4 group A member 2 (Nr4a2) identified as a potential target of peptide regulation. Testing the regulation of the Nr4a family, showed the E-domain peptides modulate Nr4a gene expression following membrane depolarization with KCl in vitro. To determine the potential role of 14-3-3 proteins, we examined 14-3-3 isoform expression and distribution. 14-3-3γ localized to the myofilaments in neonatal cardiac myocytes, the cardiac myocytes and myofilament extracts from the adult heart. Thermal shift analysis of recombinant 14-3-3γ protein showed the S/A18 peptide destabilized 14-3-3γ folding. Also, the S/A18 peptide significantly inhibited 14-3-3γ\'s ability to interact with myosin binding protein C (MYPC3) and phospholamban (PLN) in heart lysates from dobutamine injected mice. Conversely, the S/E18 peptide showed no effect on 14-3-3γ stability, did not inhibit 14-3-3γ\'s interaction with PLN but did inhibit the interaction with MYPC3. Replacing the glutamic acid with a phosphate group on Ser 18 (pSer18), significantly increased 14-3-3γ protein stability. We conclude that the state of Ser 18 phosphorylation within the 14-3-3 binding motif of MGF\'s E-domain, modulates protein-protein interactions within the 14-3-3γ interactome, which includes proteins involved in the regulation of contractile function.
摘要：
在心中,igf-I基因的选择性剪接产生两种亚型：IGF-IEa和IGF-IEc，(机械生长因子，MGF）。它们的E结构域区域之间的序列差异表明不同的同工型功能。为了定义MGF的E域的生物学作用，我们对独特的C端序列进行了计算机模拟分析,并鉴定了一个位于推定的14-3-3结合基序内的磷酸化共有位点.为了测试Ser18磷酸化的功能意义，将磷酸模拟肽(S/E18)和无磷酸肽(S/A18)以不同剂量递送至小鼠2周。使用超声心动图和压力容积导管测量心血管功能。在最低（2.25毫克/千克/天）和最高（9毫克/千克/天）剂量，这些肽在收缩和舒张参数方面产生了抑制作用。然而,在4.5mg/kg/天时，肽对心脏功能产生相反的作用。分数缩短分析也显示出类似的趋势，但心脏几何形状没有明显变化。微阵列分析发现21个基因（FDRp<0.01），这与4.5mg/kg/天对收缩功能的相反影响一致，核受体亚家族4组A成员2(Nr4a2)被鉴定为肽调节的潜在靶标。测试Nr4a家族的规定，显示E结构域肽在体外用KCl进行膜去极化后调节Nr4a基因表达。为了确定14-3-3蛋白的潜在作用，我们检查了14-3-3亚型的表达和分布。14-3-3γ定位于新生儿心肌细胞肌丝，从成人心脏中提取的心肌细胞和肌丝。重组14-3-3γ蛋白的热移位分析显示S/A18肽使14-3-3γ折叠不稳定。此外，S/A18肽显着抑制了多巴酚丁胺注射小鼠心脏裂解物中14-3-3γ与肌球蛋白结合蛋白C（MYPC3）和磷蛋白（PLN）相互作用的能力。相反，S/E18肽对14-3-3γ稳定性无影响，不抑制14-3-3γ与PLN的相互作用，但抑制与MYPC3的相互作用。用Ser18（pSer18）上的磷酸基团代替谷氨酸，显著提高14-3-3γ蛋白的稳定性。我们得出结论，在MGF的E域的14-3-3结合基序内Ser18磷酸化的状态，调节14-3-3γ相互作用组中的蛋白质-蛋白质相互作用，其中包括参与收缩功能调节的蛋白质。
