Phosphorylation and dephosphorylation of Ca2+/calmodulin-dependent protein kinase kinase β at Thr144 in HeLa cells
Phosphorylation and dephosphorylation of Ca2+/calmodulin-dependent protein kinase kinase β at Thr144 in HeLa cells
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HeLa 细胞中 Ca2+/钙调蛋白依赖性蛋白激酶激酶 β Thr144 的磷酸化和去磷酸化
DOI：
10.1016/j.bbrc.2020.02.056
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发表时间：
2020
影响因子：
3.1
通讯作者：
Tokumitsu Hiroshi
中科院分区：
文献类型：
--
作者：
Takabatake Shota;Fukumoto Yusei;Ohtsuka Satomi;Kanayama Naoki;Magari Masaki;Sakagami Hiroyuki;Hatano Naoya;Tokumitsu Hiroshi
钙/钙调蛋白依赖的蛋白激酶β(CaMKKK1)是一种调节蛋白激酶，它能磷酸化并激活包括CaMK1、CaMKIV、5‘AMP激活的蛋白激酶和蛋白激酶B在内的多种下游蛋白，从而调节各种正常和病理条件下的钙依赖的生理反应。CaMKKβ受钙/钙调蛋白结合、自磷酸化和转磷酸化等多种蛋白激酶的调节，其中包括cAMP依赖的蛋白激酶。在本报告中，我们发现在HeLa细胞中，CaMKKβ的磷酸化受蛋白磷酸酶/激酶系统的动态调节。整体蛋白组学分析显示，未刺激的HeLa细胞中，大鼠CaMKKThr144的N-末端调节域中的8个Ser残基(Ser128、132和136)发生了结构性的磷酸化，而在磷酸酶抑制剂冈田酸(Okadaic acid，OA)处理的细胞中，β发生了可诱导的磷酸化。在转染的HeLa细胞中，OA以时间和剂量依赖的方式快速诱导CaMKKβ中Thr144的磷酸化，这表明CaMKKβ中的Thr144在蛋白磷酸酶(S)的作用下保持非磷酸化状态。我们还发现，在蛋白磷酸酶2A和1的体外去磷酸化过程中，蛋白磷酸酶(S)显著地诱导了β细胞裂解物和完整细胞中的CaMKpThr144磷酸化活性，然而，这种活性不太可能是由先前发现的Thr144-β酶催化的。综上所述，这些结果表明，CaMKKβ中Thr144的磷酸化和去磷酸化受到多个激酶/磷酸酶信号的动态调节，从而导致酶性质的微调。
Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ) acts as a regulatory kinase that phosphorylates and activates multiple downstream kinases including CaMKI, CaMKIV, 5′AMP-activated protein kinase (AMPK) and protein kinase B (PKB), resulting in regulation of wide variety of Ca2+-dependent physiological responses under normal and pathological conditions. CaMKKβ is regulated by Ca2+/calmodulin-binding, autophosphorylation, and transphosphorylation by multiple protein kinases including cAMP-dependent protein kinase (PKA). In this report, we found that phosphorylation of CaMKKβ is dynamically regulated by protein phosphatase/kinase system in HeLa cells. Global phosphoproteomic analysis revealed the constitutive phosphorylation at 8 Ser residues including Ser128, 132, and 136 in the N-terminal regulatory domain of rat CaMKKβ in unstimulated HeLa cells as well as inducible phosphorylation of Thr144 in the cells treated with a phosphatase inhibitor, okadaic acid (OA). Thr144 phosphorylation in CaMKKβ has shown to be rapidly induced by OA treatment in a time- and dose-dependent manner in transfected HeLa cells, indicating that Thr144 in CaMKKβ is maintained unphosphorylated state by protein phosphatase(s). We confirmed thatin vitrodephosphorylation of pThr144 in CaMKKβ by protein phosphatase 2A and 1. We also found that the pharmacological inhibition of protein phosphatase(s) significantly induces CaMKKβ-phosphorylating activity (at Thr144) in HeLa cell lysates as well as in intact cells; however, it was unlikely that this activity was catalyzed by previously identified Thr144-kinases, such as AMPK and PKA. Taken together, these results suggest that the phosphorylation and dephosphorylation of Thr144 in CaMKKβ is dynamically regulated by multiple kinases/phosphatases signaling resulting in fine-tuning of the enzymatic property.