Methylated C-terminal leucine residue of PP2A catalytic subunit is important for binding of regulatory Bα subunit
Methylated C-terminal leucine residue of PP2A catalytic subunit is important for binding of regulatory Bα subunit
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DOI：
10.1042/0264-6021:3390241
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发表时间：
1999-04-15
影响因子：
4.1
通讯作者：
Wadzinski, BE
中科院分区：
文献类型：
--
作者：
Bryant, JC;Westphal, RS;Wadzinski, BE
已知蛋白丝氨酸/苏氨酸磷酸酶2A催化亚基（PP 2A（c））的C-末端亮氨酸残基（Leu（309））的甲基化在体外调节催化活性，但在细胞背景下这种翻译后修饰的功能后果仍不清楚。碱诱导的PP 2A（c）在纯化的PP 2A异源三聚体（AB α C）中的去甲基化，但在纯化的PP 2A异源二聚体（AC）中没有，表明在ABaC中比在AC中更大部分的PP 2A被羧甲基化。为探讨Leu（309）在PP 2A全酶组装中的作用，在COS细胞中瞬时表达了表位标记的PP 2A催化亚基（HA-PP 2A）和IIA-PP突变体（HA-PP 2A-L309 A）。这两种重组蛋白表现出丝氨酸/苏氨酸磷酸酶活性时，免疫分离COS细胞提取物。HA-PP 2A，但不是HA-PP 2A-L309 A，羧甲基化ii？体外细胞提取物的色谱分析表明，大多数内源性PP 2A（c）和HA-PP 2A与PP 2A的A和Ba调节亚基共洗脱，而大多数HA-PP 2A-L309 A似乎与A亚基作为较小的复合物或作为游离催化（C）亚基共洗脱。A亚基与两种标记的蛋白质共免疫分离;然而，与HA-PP 2A相比，与HA-PP 2A-L309 A共免疫分离的B α亚基显著较少。这些结果表明，PP 2 A的可逆甲基化C-末端亮氨酸残基对于B α调节亚基结合是重要的。此外，这些结果为PP 2A（c）、羧甲基化和PP 2A全酶组装之间的相互关系提供了证据。
Methylation of the C-terminal leucine residue (Leu(309)) of protein serine/threonine phosphatase 2A catalytic subunit (PP2A(c)) is known to regulate catalytic activity in vitro, but the functional consequence(s) of this post-translational modification in the context of the cell remain unclear. Alkali-induced demethylation of PP2A(c) in purified PP2A heterotrimer (AB alpha C), but not in purified PP2A heterodimer (AC), indicated that a larger fraction of PP2A, is carboxymethylated in ABaC than in AC. To explore the role of Leu(309) in PP2A holoenzyme assembly, epitope-tagged PP2A catalytic subunit (HA-PP2A) and a mutant of IIA-PP,A containing an alanine residue in place of Leu309 (HA-PP2A-L309A) were transiently expressed in COS cells. Both recombinant proteins exhibited serine/threonine phosphatase activity when immunoisolated from COS cell extracts. HA-PP2A, but not HA-PP2A-L309A, was carboxymethylated ii? vitro. A chromatographic analysis of cell extracts indicated that most endogenous PP2A(c) and HA-PP2A were co-eluted with the A and Ba regulatory subunits of PP2A, whereas most HA-PP2A-L309A seemed to elute with the A subunit as a smaller complex or, alternatively, as free catalytic (C) subunit. The A subunit co-immunoisolated with both tagged proteins; however, substantially less B alpha subunit co-immunoisolated with HA-PP2A-L309A than with HA-PP2A. These results demonstrate that the reversibly methylated C-terminal leucine residue of PP2A, is important for B alpha regulatory subunit binding. Furthermore, the results provide evidence for an interrelationship between PP2A(c), carboxymethylation and PP2A holoenzyme assembly.