Real-time monitoring of calcineurin activity in living cells:: Evidence for two distinct Ca2+-dependent pathways in fission yeast
Real-time monitoring of calcineurin activity in living cells:: Evidence for two distinct Ca2+-dependent pathways in fission yeast
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DOI：
10.1091/mbc.e06-06-0526
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发表时间：
2006-11-01
影响因子：
3.3
通讯作者：
Kuno, Takayoshi
中科院分区：
文献类型：
--
作者：
Deng, Lu;Sugiura, Reiko;Kuno, Takayoshi
在分裂酵母中，钙调磷酸酶去磷酸化并激活Prz 1转录因子。在这里，我们确定了钙调神经磷酸酶依赖性反应元件（CDRE）的prz 1（+）基因的启动子区域，并监测钙调神经磷酸酶活性在活细胞中使用不稳定的荧光素酶报告基因融合到三个串联重复的CDRE。升高的细胞外CaCl 2，引起钙调神经磷酸酶活性的增加，初始峰值，然后接近一个持续的恒定水平，在浓度依赖性的方式。在CaCl 2敏感的突变体，如三角洲pmc 1，反应显着增强，反映了其高细胞内Ca 2+。预期诱导Ca 2+内流的药物显示出不同的CDRE报告基因活性模式，表明钙调神经磷酸酶激活的不同机制。敲除yam（8+）或cch 1（+）编码Ca 2+通道的假定亚基，在暴露于各种刺激（包括高细胞外NaCl和细胞壁损伤剂）时，可消除钙调神经磷酸酶的激活。然而，敲除yam 8（+）或cch 1（+）并不影响钙调磷酸酶在细胞外Ca 2+升高刺激下的激活。Pck 2蛋白激酶C-Pmk 1丝裂原活化蛋白激酶途径是Ca 2+通过Yam 8/Cch 1介导的Ca 2+内流刺激钙调神经磷酸酶所必需的，但它不是升高的细胞外Ca 2+刺激所必需的，这表明钙调神经磷酸酶激活的两种不同途径。
In fission yeast, calcineurin dephosphorylates and activates the Prz1 transcription factor. Here, we identified the calcineurin-dependent response element (CDRE) in the promoter region of prz1(+) gene and monitored the calcineurin activity in living cells using a destabilized luciferase reporter gene fused to three tandem repeats of CDRE. Elevated extracellular CaCl2, caused an increase in calcineurin activity with an initial peak and then approached a sustained constant level in a concentration-dependent manner. In CaCl2-sensitive mutants such as Delta pmc1, the response was markedly enhanced, reflecting its high intracellular Ca2+. Agents expected to induce Ca2+ influx showed distinct patterns of the CDRE-reporter activity, suggesting different mechanisms of calcineurin activation. Knockout of yam(8+) or cch1(+) encoding putative subunits of a Ca2+ channel abolished the activation of calcineurin upon exposure to various stimuli, including high extracellular NaCl and cell wall-damaging agents. However, knockout of yam8(+) or cch1(+) did not affect the activation of calcineurin upon stimulation by elevated extracellular Ca2+. The Pck2 protein kinase C-Pmk1 mitogen-activate protein kinase pathway was required for the stimulation of calcineurin via Yam8/Cch1-mediated Ca2+ influx, but it was not required for the stimulation by elevated extracellular Ca2+, suggesting two distinct pathways for calcineurin activation.