CaM kinase phosphatase (CaMKP/PPM1F/POPX2) is specifically inactivated through gallate-mediated protein carbonylation
CaM kinase phosphatase (CaMKP/PPM1F/POPX2) is specifically inactivated through gallate-mediated protein carbonylation
复制标题
CaM 激酶磷酸酶 (CaMKP/PPM1F/POPX2) 通过没食子酸介导的蛋白质羰基化而特异性失活
DOI：
10.1016/j.abb.2022.109170
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发表时间：
2022
影响因子：
3.9
通讯作者：
Ishida Atsuhiko
中科院分区：
文献类型：
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作者：
Akizuki Kazutoshi;Ishikawa Shun;Obatake Rika;Ozaki Hana;Shimoda Nao;Nehira Tatsuo;Yamazaki Takeshi;Kinumi Tomoya;Osawa Jin;Sueyoshi Noriyuki;Kameshita Isamu;Shigeri Yasushi;Ishida Atsuhiko
CaMK磷酸酶（CaMKP/PPM 1F/POPX 2）是一种Mn 2+依赖性、calyculin A/冈田酸不敏感的Ser/Thr蛋白磷酸酶，属于PPM家族。CaMKP被认为不仅参与调节各种蛋白激酶，如CaM激酶和p21活化蛋白激酶，而且还参与调节由磷酸化调节的细胞蛋白。化学库的大规模筛选鉴定了没食子酸及其一些烷基酯作为对CaMKP高度特异性的新型CaMKP抑制剂。令人惊讶的是，它们引起CaMKP的特异性羰基化，导致其失活。在相同条件下，当使用与CaMKP属于同一家族的PPM 1A和λ-磷酸酶时，未观察到羰基化或失活。羰基化反应被SH化合物如半胱胺以剂量依赖性方式抑制，伴随着没食子酸乙酯对CaMKP抑制的降低。没食子酸酯的邻苯三酚结构是没食子酸酯介导的CaMKP羰基化反应所必需的。导致磷酸酶活性受损的CaMKP点突变并不显著影响没食子酸介导的羰基化。没食子酸乙酯导致几乎完全抑制CaMKP的条件下，其中羰基化水平几乎相同的CaMKP羰基化通过金属催化氧化与抗坏血酸/FeSO 4，这导致CaMKP的仅部分抑制。没食子酸盐介导的CaMKP羰基化反应需要Mn ~（2+）、Cu ~（2+）、Co ~（2+）和Fe ~（2+）等二价阳离子参与，磷酸肽底物能显著增强羰基化反应。当用没食子酸乙酯处理瞬时表达CaM激酶I（CaMKP底物）的MDA-MB-231细胞时，观察到CaM激酶I的磷酸化显著增强，表明没食子酸乙酯可以渗透到细胞内以激活细胞内的CaMKP。所有呈现的数据强烈支持CaMKP通过与没食子酸烷基酯和二价金属阳离子孵育而经历其特定氨基酸残基的羰基化，导致CaMKP特异性失活的假设。
CaMK phosphatase (CaMKP/PPM1F/POPX2) is a Mn2+-dependent, calyculin A/okadaic acid-insensitive Ser/Thr protein phosphatase that belongs to the PPM family. CaMKP is thought to be involved in regulation of not only various protein kinases, such as CaM kinases and p21-activated protein kinase, but also of cellular proteins regulated by phosphorylation. A large-scale screening of a chemical library identified gallic acid and some of its alkyl esters as novel CaMKP inhibitors highly specific to CaMKP. Surprisingly, they caused specific carbonylation of CaMKP, leading to its inactivation. Under the same conditions, no carbonylation nor inactivation was observed when PPM1A, which is affiliated with the same family as CaMKP, and λ-phosphatase were used. The carbonylation reaction was inhibited by SH compounds such as cysteamine in a dose-dependent manner with a concomitant decrease in CaMKP inhibition by ethyl gallate. The pyrogallol structure of gallate was necessary for the gallate-mediated carbonylation of CaMKP. Point mutations of CaMKP leading to impairment of phosphatase activity did not significantly affect the gallate-mediated carbonylation. Ethyl gallate resulted in almost complete inhibition of CaMKP under the conditions where the carbonylation level was nearly identical to that of CaMKP carbonylation via metal-catalyzed oxidation with ascorbic acid/FeSO4, which resulted in only a partial inhibition of CaMKP. The gallate-mediated carbonylation of CaMKP absolutely required divalent cations such as Mn2+, Cu2+, Co2+and Fe2+, and was markedly enhanced by a phosphopeptide substrate. When MDA-MB-231 cells transiently expressing CaM kinase I, a CaMKP substrate, were treated by ethyl gallate, significant enhancement of phosphorylation of CaM kinase I was observed, suggesting that ethyl gallate can penetrate into cells to inactivate cellular CaMKP. All the presented data strongly support the hypothesis that CaMKP undergoes carbonylation of its specific amino acid residues by incubation with alkyl gallates and the divalent metal cations, leading to inactivation specific to CaMKP.