A novel assay for protein phosphatase 2A (PP2A) complexes in vivo reveals differential effects of covalent modifications on different Saccharomyces cerevisiae PP2A heterotrimers.
Protein phosphatase 2A (PP2A) catalytic subunit can be covalently modified at its carboxy terminus by phosphorylation or carboxymethylation. Determining the effects of these covalent modifications on the relative amounts and functions of different PP2A heterotrimers is essential to understanding how these modifications regulate PP2A-controlled cellular processes. In this study we have validated and used a novel in vivo assay for assessing PP2A heterotrimer formation in Saccharomyces cerevisiae: the measurement of heterotrimer-dependent localization of green fluorescent protein-PP2A subunits. This assay relies on the fact that the correct cellular localization of PP2A requires that it be fully assembled. Thus, reduced localization would occur as the result of the inability to assemble a stable heterotrimer. Using this assay, we determined the effects of PP2A C-subunit phosphorylation mimic mutations and reduction or loss of PP2A methylation on the formation and localization of PP2A(B/Cdc55p) and PP2A(B'/Rts1p) heterotrimers. Collectively, our findings demonstrate that phosphorylation and methylation of the PP2A catalytic subunit can influence its function both by regulating the total amount of specific PP2A heterotrimers within a cell and by altering the relative proportions of PP2A(B/Cdc55p) and PP2A(B'/Rts1p) heterotrimers up to 10-fold. Thus, these posttranslational modifications allow flexible, yet highly coordinated, regulation of PP2A-dependent signaling pathways that in turn modulate cell growth and function.
Gentry
Matthew S
MS
Department of Biochemistry (RRC4125), Emory University School of Medicine, 1510 Clifton Rd., Atlanta, GA 30322, USA.
Li
Yikun
Y
Wei
Huijun
H
Syed
Farhana F
FF
Patel
Sameer H
SH
Hallberg
Richard L
RL
Pallas
David C
DC
D004591:Electrophoresis, Polyacrylamide Gel; D015966:Gene Expression Regulation, Fungal; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D018386:Kinetochores / Q000378:metabolism; D008745:Methylation; D008954:Models, Biological; D009154:Mutation; D010749:Phosphoprotein Phosphatases / Q000032:analysis* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D054648:Protein Phosphatase 2; D011499:Protein Processing, Post-Translational; D021122:Protein Subunits; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2