Ogris E, Du X, Nelson K C, Mak E K, Yu X X, Lane W S, Pallas D C
Division of Cellular and Molecular Biology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, Massachusetts 02115, USA.
J Biol Chem. 1999 May 14;274(20):14382-91. doi: 10.1074/jbc.274.20.14382.
Carboxymethylation of proteins is a highly conserved means of regulation in eukaryotic cells. The protein phosphatase 2A (PP2A) catalytic (C) subunit is reversibly methylated at its carboxyl terminus by specific methyltransferase and methylesterase enzymes which have been purified, but not cloned. Carboxymethylation affects PP2A activity and varies during the cell cycle. Here, we report that substitution of glutamine for either of two putative active site histidines in the PP2A C subunit results in inactivation of PP2A and formation of stable complexes between PP2A and several cellular proteins. One of these cellular proteins, herein named protein phosphatase methylesterase-1 (PME-1), was purified and microsequenced, and its cDNA was cloned. PME-1 is conserved from yeast to human and contains a motif found in lipases having a catalytic triad-activated serine as their active site nucleophile. Bacterially expressed PME-1 demethylated PP2A C subunit in vitro, and okadaic acid, a known inhibitor of the PP2A methylesterase, inhibited this reaction. To our knowledge, PME-1 represents the first mammalian protein methylesterase to be cloned. Several lines of evidence indicate that, although there appears to be a role for C subunit carboxyl-terminal amino acids in PME-1 binding, amino acids other than those at the extreme carboxyl terminus of the C subunit also play an important role in PME-1 binding to a catalytically inactive mutant.
蛋白质的羧甲基化是真核细胞中一种高度保守的调节方式。蛋白磷酸酶2A（PP2A）催化（C）亚基在其羧基末端被已纯化但未克隆的特定甲基转移酶和甲基酯酶可逆地甲基化。羧甲基化影响PP2A活性，且在细胞周期中有所变化。在此，我们报道在PP2A C亚基中，用谷氨酰胺替代两个假定的活性位点组氨酸中的任何一个，会导致PP2A失活，并使PP2A与几种细胞蛋白形成稳定复合物。其中一种细胞蛋白，在此命名为蛋白磷酸酶甲基酯酶-1（PME-1），被纯化并进行微量测序，其cDNA被克隆。PME-1从酵母到人类都保守，并且包含在脂肪酶中发现的一个基序，该基序具有一个以催化三联体激活的丝氨酸作为其活性位点亲核试剂。细菌表达的PME-1在体外使PP2A C亚基去甲基化，而冈田酸，一种已知的PP2A甲基酯酶抑制剂，抑制了该反应。据我们所知，PME-1是第一个被克隆的哺乳动物蛋白甲基酯酶。几条证据表明，尽管C亚基羧基末端氨基酸在PME-1结合中似乎有作用，但C亚基极端羧基末端以外的氨基酸在PME-1与催化无活性突变体的结合中也起重要作用。
