PKA-dependent phosphorylation of serum response factor inhibits smooth muscle-specific gene expression.
PKA-dependent phosphorylation of serum response factor inhibits smooth muscle-specific gene expression.
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DOI：
10.1161/atvbaha.109.197285
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发表时间：
2009-12
期刊：
影响因子：
--
通讯作者：
Mack CP
中科院分区：
文献类型：
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作者：
Blaker AL;Taylor JM;Mack CP
我们的目标是确定磷酸化位点，调节血清反应因子（SRF）的活动，以更好地了解信号转导机制，调节SRF的参与平滑肌细胞（SMC）特异性和早期反应基因的表达。通过在SRF −/− ES细胞中筛选磷酸化缺陷和模拟突变，我们确定T159是一个磷酸化位点，可显著抑制SMC分化的ES细胞模型中SMC特异性基因表达。该残基与高度保守的cAMP依赖性蛋白激酶（PKA）位点一致，体外和体内标记研究表明它被PKA磷酸化。凝胶位移和染色质免疫沉淀试验的结果表明，T159磷酸化抑制SRF结合SMC特异性CArG元素。有趣的是，在某些条件下，心肌蛋白因子至少可以部分地挽救T159 D突变的影响，但这种反应是启动子特异性的。最后，PKA信号对c-fos启动子活性和SRF与c-fos CArG结合的影响要小得多。我们的研究结果表明，PKA磷酸化SRF抑制SMC特异性转录，这表明一种新的信号转导机制控制SMC表型。
Our goal was to identify phosphorylation sites that regulate serum response factor (SRF) activity to gain a better understanding of the signaling mechanisms that regulate SRF’s involvement in smooth muscle cell (SMC)-specific and early response gene expression. By screening phosphorylation deficient and mimetic mutations in SRF −/− ES cells, we identified T159 as a phosphorylation site that significantly inhibits SMC-specific gene expression in an ES cell model of SMC differentiation. This residue conforms to a highly conserved consensus cAMP-dependent protein kinase (PKA) site, and in vitro and in vivo labeling studies demonstrated that it was phosphorylated by PKA. Results from gel shift and chromatin immunoprecipitation assays demonstrated that T159 phosphorylation inhibited SRF binding to SMC-specific CArG elements. Interestingly, the myocardin factors could at least partially rescue the effects of the T159D mutation under some conditions, but this response was promoter specific. Finally, PKA signaling had much less of an effect on c-fos promoter activity and SRF binding to the c-fos CArG. Our results indicate that phosphorylation of SRF by PKA inhibits SMC-specific transcription suggesting a novel signaling mechanism for the control of SMC phenotype.