Dual phosphorylation of protein phosphatase PPM1H promotes dephosphorylation of Smad1 in cellulo
Dual phosphorylation of protein phosphatase PPM1H promotes dephosphorylation of Smad1 in cellulo
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DOI：
10.1016/j.bbrc.2020.05.082
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发表时间：
2020-09-24
影响因子：
3.1
通讯作者：
Sueyoshi, Noriyuki
中科院分区：
文献类型：
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作者：
Osawa, Jin;Akizuki, Kazutoshi;Sueyoshi, Noriyuki
已知蛋白磷酸酶PPM1H参与多种生物学或病理生理机制。然而，其调控的分子机制却鲜为人知。在本研究中，我们对直接磷酸化PPM1H的蛋白激酶进行了研究，确定它们为环腺苷酸依赖性蛋白激酶（PKA）和钙/钙调蛋白依赖性蛋白激酶I（CaMKI）。体外和计算机模拟分析表明，PKA和CaMKI对PPM1H的磷酸化位点分别为丝氨酸123和丝氨酸210。细胞中PPM1H的磷酸化状态呈现出激酶激活剂和抑制剂依赖性变化。在小鼠神经母细胞瘤Neuro2a细胞中，当用离子霉素处理时，磷酸化模拟突变体（S123D）中丝氨酸210的磷酸化程度远高于不可磷酸化突变体（S123A）。这表明在这些类神经元细胞中存在一种分级磷酸化，即丝氨酸123的初始磷酸化促进丝氨酸210的后续磷酸化。此外，在基于细胞的实验中，PPM1H（S123A/S210A）双突变体几乎不能使Smad1去磷酸化，Smad1是一种已知的PPM1H内源性底物转录因子。这些结果表明，环腺苷酸和钙/钙调蛋白通过PPM1H在丝氨酸123和丝氨酸210的双重磷酸化来调节Smad1的去磷酸化。（C）2020爱思唯尔公司。保留所有权利。
Protein phosphatase PPM1H is known to participate in various biological or pathophysiological mechanisms. However, little is known about the molecular mechanisms of its regulation. In this study, we investigated the protein kinases that directly phosphorylate PPM1H, identifying them as cAMPdependent protein kinase (PKA) and Ca2+/calmodulin-dependent protein kinase I (CaMKI). In vitro and in silico analyses showed that the phosphorylation sites of PPM1H by PKA and CaMKI were Ser-123 and Ser-210, respectively. The phosphorylation state of PPM1H in cells exhibited the kinase activator-and inhibitor-dependent changes. In mouse neuroblastoma Neuro2a cells, phosphorylation of Ser-210 was much higher in the phospho-mimetic mutant (S123D) than in the non-phosphorylatable mutant (S123A) when they were treated with ionomycin. This suggests that a hierarchical phosphorylation, with initial phosphorylation of Ser-123 promoting subsequent phosphorylation of Ser-210, occurs in these neuron like cells. Moreover, in cell-based assay a PPM1H(S123A/S210A) double mutant barely dephosphorylated Smad1, a transcription factor known as an endogenous substrate of PPM1H. These results suggest that cAMP and Ca2+/calmodulin regulate dephosphorylation of Smad1 through the dual phosphorylation of PPM1H at Ser-123 and Ser-210 (C) 2020 Elsevier Inc. All rights reserved.