Carboxyl methylation of the phosphoprotein phosphatase 2A catalytic subunit promotes its functional association with regulatory subunits in vivo

Abstract

The phosphoprotein phosphatase 2A (PP2A) catalytic subunit contains a methyl ester on its C-terminus, which in mammalian cells is added by a speci®c carboxyl methyltransferase and removed by a speci®c carboxyl methylesterase. We have identi®ed genes in yeast that show signi®cant homology to human carboxyl methyltransferase and methylesterase. Extracts of wild-type yeast cells contain carboxyl methyltransferase activity, while extracts of strains deleted for one of the methyltransferase genes, PPM1, lack all activity. Mutation of PPM1 partially disrupts the PP2A holoenzyme in vivo and ppm1 mutations exhibit synthetic lethality with mutations in genes encoding the B or B¢ regulatory subunit. Inactivation of PPM1 or overexpression of PPE1, the yeast gene homologous to bovine methylesterase, yields phenotypes similar to those observed after inactivation of either regulatory subunit. These phenotypes can be reversed by overexpression of the B regulatory subunit. These results demonstrate that Ppm1 is the sole PP2A methyltransferase in yeast and that its activity is required for the integrity of the PP2A holoenzyme.

PP2A holoenzyme assembly: in cauda venenum (the sting is in the tail)

“…Within the B-type subunit pool, Rts1p (ROX three suppressor 1; the yeast homolog of PR61/B 0 ), is 12Â as abundant as Cdc55p (cell-division-cycle mutant 55; the yeast homolog of PR55/B), indicating that, at any given time, there will always be a free pool of Rts1p and that an as yet undefined mechanism must operate to permit competition between Cdc55p and Rts1p for binding to PP2A D . The same study also confirmed earlier reports demonstrating that, in yeast, subunit stability is not linked to heterotrimer formation [20][21][22][23][24]. However, in Drosophila melanogaster Schneider cells, the absence of all B-type subunits promotes the degradation of the A and C subunits, and vice versa [25,26], which indicates that PP2A enzymes are obligate trimers in this system.…”

“…However, a variety of experimental approaches both in yeast and mammalian cells demonstrate that PP2A C methylation has a crucial role in B-type-subunit binding, specifically in the recruitment of PR55/B subunits [20][21][22][23]44,[51][52][53][54][55]. By contrast, the recruitment of A [44,[51][52][53]55], PR61/B 0 [21][22][23]55], PR72/B 00 [54,55] and striatin [44] does not strictly require PP2A C methylation in vivo, and binding of polyoma middle T [44,51] and a4 [20,56] is increased in the absence of methylation. In yeast, the deletion of PPM1 (protein phosphatase methyltransferase 1, which encodes the yeast LCMT1 ortholog) or the expression of PP2A C harboring a Leu309!Ala substitution, inhibits Cdc55p binding [20][21][22][23], whereas Tpd3p (tRNA-processing-deficient mutant 3; the yeast A subunit homolog) and Rts1p binding is only slightly diminished [20][21][22][23].…”

“…By contrast, the recruitment of A [44,[51][52][53]55], PR61/B 0 [21][22][23]55], PR72/B 00 [54,55] and striatin [44] does not strictly require PP2A C methylation in vivo, and binding of polyoma middle T [44,51] and a4 [20,56] is increased in the absence of methylation. In yeast, the deletion of PPM1 (protein phosphatase methyltransferase 1, which encodes the yeast LCMT1 ortholog) or the expression of PP2A C harboring a Leu309!Ala substitution, inhibits Cdc55p binding [20][21][22][23], whereas Tpd3p (tRNA-processing-deficient mutant 3; the yeast A subunit homolog) and Rts1p binding is only slightly diminished [20][21][22][23]. Similarly, when PP2A C methylation is substantially reduced in mammalian cells following RNAi-mediated knockdown of LCMT1, PP2A T61 or PP2A T72 can recruit demethylated PP2A C , whereas PP2A T55 still exclusively recruits methylated PP2A C [55].…”

Dysregulation of protein phosphatase 2A in parkinson disease and dementia with lewy bodies

“…Since the main dephosphorylating enzyme of α ‐synuclein is the B55 α containing heterotrimeric isoform of PP2A,13 and methylation of the C subunit of PP2A enhances the incorporation of the regulatory B55 α subunit into the catalytically active holoenzyme,3, 4, 5 the markedly decreased expression of the methylating enzyme LCMT‐1 in these conditions, and increased expression of the demethylating enzyme PME‐1, lead to a striking decrease in the methylated form of PP2A. These dysregulatory changes are not only particularly prominent in the substantia nigra pars compacta but also present in the cortex.…”

“…In neuronal cells, most of the C subunit is associated with a conserved scaffold‐like A subunit, and one of several different regulatory B subunits that confer different substrate specificities on the resulting trimeric holoenzymes. The binding of different B subunits to the AC dimer is regulated by reversible carboxyl methylation of the C subunit 3, 4, 5. Thus, PP2A methylation is critical for its selective phosphatase activity toward different phospho‐protein substrates.…”