Spatial control of protein phosphatase 2A (de)methylation.

Reversible methylation of the protein phosphatase 2A catalytic subunit (PP2AC11Abbreviations used are: AA, amino acids; EGFP, enhanced green fluorescent protein; LCMT1, leucine carboxyl methyltransferase 1; NLS, nuclear localization signal; PME-1, protein phosphatase methylesterase 1; PP2A, protein phosphatase 2A; PP2AC, catalytic subunit of PP2A; PP2AD, core dimer of PP2A; PP2Ai, inactive PP2A; PP2AT55, PP2A trimer with PR55/B as third subunit; PTPA, phosphatase two a phosphatase activator.) is an important regulatory mechanism playing a crucial role in the selective recruitment of regulatory B subunits. Here, we investigated the subcellular localization of leucine carboxyl methyltransferase (LCMT1) and protein phosphatase methylesterase (PME-1), the two enzymes catalyzing this process. The results show that PME-1 is predominantly localized in the nucleus and harbors a functional nuclear localization signal, whereas LCMT1 is underrepresented in the nucleus and mainly localizes to the cytoplasm, Golgi region and late endosomes. Indirect immunofluorescence with methylation-sensitive anti-PP2AC antibodies revealed a good correlation with the methylation status of PP2AC, demethylated PP2AC being substantially nuclear. Throughout mitosis, demethylated PP2AC is associated with the mitotic spindle and during cytokinesis with the cleavage furrow. Overexpression of PME-1, but not of an inactive mutant, results in increased demethylation of PP2AC in the nucleus, whereas overexpression of a cytoplasmic PME-1 mutant lacking the NLS results in increased demethylation in the cytoplasm—in all cases, however, without any obvious functional consequences. PME-1 associates with an inactive PP2A population, regardless of its esterase activity or localization. We propose that stabilization of this inactive, nuclear PP2A pool is a major in vivo function of PME-1.