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 specific carboxyl methyltransferase and removed by a specific carboxyl methylesterase. We have identified genes in yeast that show significant 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.

Fig. 1. Sequence relationship of putative human, S.cerevisiae, S.pombe, D.melanogaster and C.elegans PP2A carboxyl methyltransferases. Predicted amino acid sequences of human methyltransferases (hsmt1, AAF18267; hsmt2, BAA25473) and those of related proteins predicted from the genomic sequences of S.cerevisiae (PPM1, YDR435c; PPM2, YOL141w), S.pombe (spmt1, CAA21793; spmt2, CAA19576), D.melanogaster (dmmt, AAF53483) and C.elegans (cemt, CAA84295) are aligned using the PILEUP program from the University of Wisconsin GCG.

Fig. 2. PPM1 and PPE1 encode a PP2A carboxyl methyltransferase and a PP2A carboxyl methylesterase, respectively. (A) Extracts from W303-1A (PPM1 PPM2), Y2752 (ppm1), Y2739 (ppm2) and Y2740 (ppm1 ppm2) were incubated at 30°C for 30 min with or without bovine AC dimers and [³H]AdoMet, and then subjected to SDS–PAGE. The C subunit was analyzed for incorporation of [³H]methyl esters as described in Materials and methods. The data shown are means ± SD of duplicates from two separate experiments. (B) Extracts from Y2762 (pph21 pph22, lanes 1–4), Y2760 (pph21 pph22 ppm1, lane 5) or Y2761 (pph21 pph22 ppe1, lane 6) expressing triple HA-tagged Pph22 containing the indicated mutations were separated by SDS–PAGE and then transferred to PDVF membranes. The membranes were probed with a monoclonal antibody against HA, monoclonal antibody 6A3 (which recognizes both methylated and unmethylated C subunit) and monoclonal antibody 4D9 (which recognizes only methylated C subunit). Extracts were run as separate sets of tracks for each antibody.

Fig. 3. Loss of Ppm1 activity affects the interaction of PP2A regulatory subunits with the C subunits. Extracts from Y2480 [pph22:: (HA)₃PPH22] and Y2734 [ppm1 pph22:: (HA)₃PPH22] cells were immunoprecipitated with anti-HA epitope monoclonal antibodies. Samples of the precipitates (‘IP’), extracts before precipitation (‘Ext’) and extracts after precipitation (‘Sup’) were separated by SDS–PAGE and then transferred to PVDF membranes. The membranes were probed separately with anti-HA, anti-Tpd3, anti-Cdc55 (A) and anti-Tap42 (B) polyclonal antibodies.

Fig. 4. Genetic interactions between Ppm1 and PP2A B regulatory subunit genes. Exponentially growing cultures of W303-1A (wild type), Y2752 (ppm1), Y2739 (ppm2), Y2740 (ppm1 ppm2), Y2483 (cdc55), Y2745 (ppm1 cdc55), Y2741 (ppm2 cdc55), Y2742 (ppm1 ppm2 cdc55), Y2736 (rts1), Y2737 (ppm1 rts1), Y2738 (ppm2 rts1) and Y2744 (ppm1 ppm2 rts1) were 10-fold serially diluted and spotted on to YEPD plates. Growth is shown after 2 days at 30 or 37°C.

Fig. 5. ppm1 cells are resistant to and supersensitive to benomyl. Exponentially growing cultures of wild-type cells, Y2752 (ppm1), Y2739 (ppm2), Y2483 (cdc55), Y2736 (rts1), Y2745 (ppm1 cdc55) and Y2737 (ppm1 rts1) were 10-fold serially diluted and spotted on to YEPD plates containing 100 nM rapamycin (A) or 12 µg/ml benomyl (B). Growth is shown after incubation at 30°C for 2 days without drug or 4 days in the presence of rapamycin and benomyl.

Fig. 6. ppm1, cdc55 and rts1 are required for spindle assembly checkpoint activity. Exponentially growing cultures of W303-1A (wild type), Y2752 (ppm1), Y2739 (ppm2), Y2483 (cdc55) and Y2736 (rts1) were incubated in the presence of α-factor for 3 h at 30°C and then transferred into fresh YEPD medium containing 12 µg/ml nocodazole. At different times after release from cell cycle arrest, cells were removed, sonicated, counted and then plated on to YEPD plates. Viable colonies were counted after 2 days. Squares, wild type; diamonds, cdc55; circles, rts1; upward pointing triangles, ppm1; downward pointing triangles, ppm2.

Fig. 7. Overexpression of PPE1 induces rapamycin resistance. Exponentially growing cultures of strain W303-1A containing plasmid pRS425 (2µ), pJW131 (2µ PPM1) or pJW141 (2µ PPE1) and strain Y2752 (ppm1) containing plasmid pRS425 or pJW141 and strain Y2746 (ppe1) containing plasmid pRS425 or pJW131 were 10-fold serially diluted and spotted on to SD – Leu plates without rapamycin or with 100 nM rapamycin. Growth is shown after 2 days at 30°C in the absence of rapamycin or after 4 days in the presence of rapamycin.

Fig. 8. Overexpression of Cdc55 restores rapamycin sensitivity to ppm1 strains. Exponentially growing cultures of strains W303-1A and Y2752 (ppm1) containing either pRS426 (2µ) or YEpCDC55 (2µ CDC55) were 10-fold serially diluted and spotted on to SD – Ura plates containing no rapamycin or 100 nM rapamycin. Growth is shown after incubation at 30°C for 2 days in the absence of rapamycin or after 4 days in the presence of rapamycin.