Proteolytic processing of cGMP-dependent protein kinase I mediates nuclear cGMP signaling in vascular smooth muscle cells.
Proteolytic processing of cGMP-dependent protein kinase I mediates nuclear cGMP signaling in vascular smooth muscle cells.
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cGMP 依赖性蛋白激酶 I 的蛋白水解过程介导血管平滑肌细胞中的核 cGMP 信号传导。
DOI：
10.1161/circresaha.108.176321
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发表时间：
2008
影响因子：
20.1
通讯作者：
RobertsJr,JesseD
中科院分区：
文献类型：
--
作者：
Sugiura,Takahiro;Nakanishi,Hidehiko;RobertsJr,JesseD
环 GMP 部分通过刺激 cGMP 依赖性蛋白激酶 I (PKGI) 和转录因子的磷酸化来调节血管平滑肌细胞 (SMC) 中的基因表达。在某些细胞中，cGMP 会增加 PKGI 的核转位以及转录调节因子的 PKGI 依赖性磷酸化；然而，这些观察结果各不相同，介导核 PKGI 易位的机制尚不完全清楚。我们测试了以下假设：cGMP 刺激的 PKGI 核区划和转录因子磷酸化需要 PKGI 的蛋白水解裂解。我们在 SMC 的胞质和内质网中检测到具有亮氨酸拉链结构域免疫反应性的 NH2 末端 PKGI 片段，但在高尔基体 (GA) 和核质中仅观察到包含催化区域（现在称为 PKGIγ）的 COOH 末端 PKGI 片段。 GA 中的翻译后 PKGI 处理对于 PKGIγ 的核区划至关重要，因为用诺考达唑或布雷菲德素 A 破坏 GA 会抑制 PKGIγ 核定位。 PKGIγ 免疫反应性在间期 SMC 的核仁中特别丰富，其中它与核仁致密原纤维成分蛋白 fibrillarin 的共定位与核仁组装的水平密切匹配。纯化的核仁 PKGIγ 酶活性对 cGMP 刺激不敏感，这与其缺乏 NH2 末端自抑制结构域一致。 PKGI 中假定的蛋白水解切割区域的突变抑制了 cGMP 介导的 cAMP 反应元件结合蛋白的磷酸化、cAMP 反应元件依赖性转录和 PKGIγ 的核定位。这些观察结果表明 PKGI 的翻译后修饰严重影响 SMC 中 PKGI 的核转位和 cGMP 活性。
Cyclic GMP modulates gene expression in vascular smooth muscle cells (SMCs) in part by stimulating cGMP-dependent protein kinase I (PKGI) and the phosphorylation of transcription factors. In some cells, cGMP increases nuclear translocation of PKGI and PKGI-dependent phosphorylation of transcription regulators; however, these observations have been variable, and the mechanisms mediating nuclear PKGI translocation are incompletely understood. We tested the hypothesis that proteolytic cleavage of PKGI is required for cGMP-stimulated nuclear compartmentation of PKGI and phosphorylation of transcription factors. We detected an NH2-terminal PKGI fragment with leucine zipper domain immunoreactivity in the cytosol and endoplasmic reticulum of SMCs, but only a COOH-terminal PKGI fragment containing the catalytic region (now termedPKGIγ) was observed in the Golgi apparatus (GA) and nucleoplasm. Posttranslational PKGI processing in the GA was critical for nuclear compartmentation of PKGIγ because GA disruption with nocodazol or brefeldin A inhibited PKGIγ nuclear localization. PKGIγ immunoreactivity was particularly abundant in the nucleolus of interphase SMCs where its colocalization with the nucleolar dense fibrillar component protein fibrillarin closely matched the level of nucleolar assembly. Purified nucleolar PKGIγ enzyme activity was insensitive to cGMP stimulation, which is consistent with its lack of the NH2-terminal autoinhibitory domain. Mutation of a putative proteolytic cleavage region in PKGI inhibited cGMP-mediated phosphorylation of cAMP response element-binding protein, cAMP response element-dependent transcription, and nuclear localization of PKGIγ. These observations suggest that posttranslational modification of PKGI critically influences the nuclear translocation of PKGI and activities of cGMP in SMCs.