J Biol Chem. 2001 Jan 12;276(2):1570-7.

Carboxymethylation of the PP2A catalytic subunit in Saccharomyces cerevisiae is required for efficient interaction with the B-type subunits Cdc55p and Rts1p.

Wei H, Ashby DG, Moreno CS, Ogris E, Yeong FM, Corbett AH, Pallas DC.

Source

Department of Biochemistry and Winship Cancer Center, Emory University School of Medicine, Atlanta, Georgia 30322, USA.

Abstract

Protein phosphatase 2A (PP2A) is an essential eukaryotic serine/threonine phosphatase known to play important roles in cell cycle regulation. Association of different B-type targeting subunits with the heterodimeric core (A/C) enzyme is known to be an important mechanism of regulating PP2A activity, substrate specificity, and localization. However, how the binding of these targeting subunits to the A/C heterodimer might be regulated is unknown. We have used the budding yeast Saccharomyces cerevisiae as a model system to investigate the hypothesis that covalent modification of the C subunit (Pph21p/Pph22p) carboxyl terminus modulates PP2A complex formation. Two approaches were taken. First, S. cerevisiae cells were generated whose survival depended on the expression of different carboxyl-terminal Pph21p mutants. Second, the major S. cerevisiae methyltransferase (Ppm1p) that catalyzes the methylation of the PP2A C subunit carboxyl-terminal leucine was identified, and cells deleted for this methyltransferase were utilized for our studies. Our results demonstrate that binding of the yeast B subunit, Cdc55p, to Pph21p was disrupted by either acidic substitution of potential carboxyl-terminal phosphorylation sites on Pph21p or by deletion of the gene for Ppm1p. Loss of Cdc55p association was accompanied in each case by a large reduction in binding of the yeast A subunit, Tpd3p, to Pph21p. Moreover, decreased Cdc55p and Tpd3p binding invariably resulted in nocodazole sensitivity, a known phenotype of CDC55 or TPD3 deletion. Furthermore, loss of methylation also greatly reduced the association of another yeast B-type subunit, Rts1p. Thus, methylation of Pph21p is important for formation of PP2A trimeric and dimeric complexes, and consequently, for PP2A function. Taken together, our results indicate that methylation and phosphorylation may be mechanisms by which the cell dynamically regulates PP2A complex formation and function.

PMID:: 11038366; [PubMed - indexed for MEDLINE]
PMCID:: PMC3508460

Free PMC Article

Images from this publication.See all images (7)Free text

Fig. 2

Substitution of threonine 364 or tyrosine 367 with an acidic residue abrogates C subunit interaction with Cdc55p and decreases its interaction with Tpd3p, whereas substitution with more conservative residues does not

A, characterization of the Cdc55p antibody. 12CA5 immunoprecipitates were prepared from H328 cells expressing untagged Pph21p (Pph21p) or HA-tagged Pph21p (HA-Pph21p) and analyzed along with cell lysates from W303a (wt) and ADR496 (Δ CDC55) by immunoblotting with anti-Cdc55p rabbit polyclonal antibody (1:5000). All lanes are from the same gel but were not all originally adjacent. The dark exposure shown reveals no trace of Cdc55p signal in the deletion strain. B, characterization of the Tpd3p antibody. Immunoblots of cell lysates from W303a cells lacking Tpd3p (Δ TPD3) and from W303a cells (wt) were probed with anti-Tpd3p rabbit polyclonal antibody (1:5000). C, Cdc55p coimmunoprecipitates with T364A and Y367F but not with T364D or Y367E. H328 cells expressing the indicated Pph21p proteins were grown in glucose-containing medium. 12CA5 immunopre-cipitates prepared from lysates of these cells were immunoblotted with anti-Cdc55p and anti-Tpd3p antibodies (IPs). Even on long exposures, no Cdc55p could be seen coimmunoprecipitating with Y364D and Y367E, whereas a small amount of Tpd3p was detected. Pph21p mutants migrate as doublets in these gels, but whether double or single bands are seen can vary. This pattern of migration in SDS-polyacrylamide gel electrophoresis has been noted previously for endogenous and epitope-tagged mammalian PP2A C subunits (10, 25, 34) and does not appear to be due to degradation. D, Cdc55p and Tpd3p are still present in H328 cells expressing mutant Pph21p proteins. Lysates from H328 cells expressing the various wt and mutant Pph21p proteins (same order of lanes as in panel C) were immunoblotted for Cdc55p and Tpd3p.

Carboxymethylation of the PP2A Catalytic Subunit in Saccharomyces cerevisiae Is Required for Efficient Interaction with the B-type Subunits Cdc55p and Rts1p

J Biol Chem. 2001 January 12;276(2):1570-1577.

Fig. 5

PPM1 encodes the major PP2A methyltransferase in S. cerevisiae

A, 1d6 monoclonal antibody specifically recognizes unmethylated PP2A carboxyl-terminal peptide. A C subunit carboxyl-terminal nonapeptide carboxymethylated on leucine (described under “Materials and Methods”) was synthesized and high performance liquid chromatography-purified. 0.25 μg of this peptide was demethylated by treatment with 0.5 M NaOH (+) for 5 min on ice and then neutralized, whereas another 0.25 μg of peptide (−) was treated with an equivalent amount of preneutralized solution. The unmethylated and methylated aliquots of the peptides were then spotted onto nitrocellulose, and the membrane was probed with 1d6 monoclonal antibody. B, deletion of PPM1 results in nearly complete loss of PP2A methylation. Lysates were prepared from wt (BY4741), Δ PPM1, Δ PPM2, and Δ PPM1Δ PPM2 cells in the presence of 200 nM okadaic acid to prevent further methylation and demethylation (19). Twenty μl of each lysate (+) was placed on ice and demethylated by the addition of 10 μl of 0.5 M NaOH. After a 5-min incubation, the samples were neutralized with an equivalent volume of 0.5 M HCl and one-half volume of 2 M Tris, pH 6.8. Another 20 μl of each lysate (−) was combined with an equivalent amount of preneutralized base solution. Then the samples were analyzed by 10% SDS-polyacrylamide gel electrophoresis and immunoblotted with methylation-sensitive C subunit monoclonal antibody (1d6; 1:10,000). Because 1d6 almost exclusively recognizes unmethylated Pph21p and Pph22p, the ratio of signal intensity between the −base lane (in vivo amount of unmethylated C subunit) and the +base lane (100% unmethylated) indicates the percentage of C subunit that is unmethylated in vivo. The percent unmethylated C subunits was subtracted from 100 to obtain the percentage of methylated C subunits (see values under “Results”). S. cerevisiae C subunits migrated as tight doublets in this gel, but whether double or single bands are seen can vary.

Carboxymethylation of the PP2A Catalytic Subunit in Saccharomyces cerevisiae Is Required for Efficient Interaction with the B-type Subunits Cdc55p and Rts1p

J Biol Chem. 2001 January 12;276(2):1570-1577.