Favre B, Zolnierowicz S, Turowski P, Hemmings B A
Friedrich Miescher-Institut, Basel, Switzerland.
J Biol Chem. 1994 Jun 10;269(23):16311-7.
We have used polyclonal antibodies against an internal peptide (residues 169 to 182; Ab169/182) and a peptide corresponding to the carboxyl terminus (residues 299 to 309; Ab299/309) to look for in vivo modifications of protein phosphatase 2A catalytic (PP2Ac) subunit. Treatment of extracts from human breast cancer (MCF7) cells with either alkali or ethanol increased immunoreactivity of PP2Ac subunit severalfold on Western blots with Ab299/309, but did not apparently change molecular weight or isoelectric point of the protein. In contrast, immunoreactivity with Ab169/182 was unchanged by these treatments. Subsequently, we demonstrated that the increase in PP2Ac subunit recognition by Ab299/309 coincides with the demethylation of this protein at the carboxyl-terminal leucine (Leu309). Methylation of PP2Ac subunit, in vitro, increases its activity toward both phosphorylase a and a phosphopeptide. The carboxyl-terminal sequence (TPDYFL) of PP2Ac subunit is completely conserved between mammals, yeast, fruit fly, and plants which suggests that regulation of this enzyme activity by carboxyl-terminal methylation has been conserved during evolution.
我们使用了针对内部肽段（第169至182位氨基酸残基；Ab169/182）和对应羧基末端的肽段（第299至309位氨基酸残基；Ab299/309）的多克隆抗体，来寻找蛋白磷酸酶2A催化亚基（PP2Ac）在体内的修饰情况。用碱或乙醇处理人乳腺癌（MCF7）细胞提取物后，在使用Ab299/309进行的蛋白质印迹实验中，PP2Ac亚基的免疫反应性增加了数倍，但该蛋白的分子量或等电点并未明显改变。相比之下，这些处理并未改变Ab169/182的免疫反应性。随后，我们证明了Ab299/309对PP2Ac亚基识别的增加与该蛋白羧基末端亮氨酸（Leu309）的去甲基化同时发生。在体外，PP2Ac亚基的甲基化会增加其对磷酸化酶a和磷酸肽的活性。PP2Ac亚基的羧基末端序列（TPDYFL）在哺乳动物、酵母、果蝇和植物中完全保守，这表明通过羧基末端甲基化对该酶活性的调节在进化过程中得以保留。
