A calcium-dependent protein kinase can inhibit a calmodulin-stimulated Ca2+ pump (ACA2) located in the endoplasmic reticulum of Arabidopsis
A calcium-dependent protein kinase can inhibit a calmodulin-stimulated Ca2+ pump (ACA2) located in the endoplasmic reticulum of Arabidopsis
复制标题
DOI：
10.1073/pnas.97.11.6224
复制
发表时间：
2000-05-23
影响因子：
11.1
通讯作者：
Harper, JF
中科院分区：
文献类型：
--
作者：
Hwang, I;Sze, H;Harper, JF
胞质Ca 2+释放的幅度和持续时间可以通过改变Ca 2+流出的速率来改变。在植物细胞中，Ca ~（2+）从细胞质中流出是由H ~+/Ca ~（2+）-反向转运蛋白和两种Ca ~（2+）-ATP酶介导的。ACA 2是一种位于内质网的钙调素调节的钙泵，在此，我们发现其N端调节结构域被一种钙依赖性蛋白激酶磷酸化，（CDPK同种型CPK 1），抑制基础活性（约10%）和钙调素刺激（约75%），如用酵母中表达的重组酶进行的Ca 2 +-转运测定所示。将CDPK磷酸化位点定位于钙调蛋白结合位点附近的Ser（45），使用含有N-末端结构域的融合蛋白作为重组CPK 1的体外底物。在全长酶中，Ala取代Ser（45）（S45/A）完全阻断了所观察到的CDPK对基础活性和钙调素刺激活性的抑制。Asp取代（S45/D）模拟磷酸化抑制，表明该位置的负电荷足以解释磷酸化抑制。有趣的是，钙调蛋白的预先结合阻断了磷酸化。这表明，一旦ACA 2结合钙调蛋白，其活化状态变得对磷酸化抑制有抗性。这些结果支持的假设，即ACA 2活性调节的初始动力学之间的平衡，钙调素刺激和CDPK抑制，提供了一个例子，在植物中的一个潜在的点之间的串扰两个不同的Ca 2+信号通路。
The magnitude and duration of a cytosolic Ca2+ release can potentially be altered by changing the rate of Ca2+ efflux. In plant cells, Ca2+ efflux from the cytoplasm is mediated by H+/Ca2+-antiporters and two types of Ca2+-ATPases. ACA2 was recently identified as a calmodulin-regulated Ca2+-pump located in the endoplasmic reticulum, Here, we show that phosphorylation of its N-terminal regulatory domain by a Ca2+-dependent protein kinase (CDPK isoform CPK1), inhibits both basal activity (approximate to 10%) and calmodulin stimulation (approximate to 75%), as shown by Ca2+-transport assays with recombinant enzyme expressed in yeast, A CDPK phosphorylation site was mapped to Ser(45) near a calmodulin binding site, using a fusion protein containing the N-terminal domain as an in vitro substrate for a recombinant CPK1. In a full-length enzyme, an Ala substitution for Ser(45) (S45/A) completely blocked the observed CDPK inhibition of both basal and calmodulin-stimulated activities. An Asp substitution (S45/D) mimicked phosphoinhibition, indicating that a negative charge at this position is sufficient to account for phosphoinhibition. Interestingly, prior binding of calmodulin blocked phosphorylation, This suggests that, once ACA2 binds calmodulin, its activation state becomes resistant to phosphoinhibition. These results support the hypothesis that ACA2 activity is regulated as the balance between the initial kinetics of calmodulin stimulation and CDPK inhibition, providing an example in plants for a potential point of crosstalk between two different Ca2+-signaling pathways.