Generation of Active Protein Phosphatase 2A Is Coupled to Holoenzyme Assembly

Protein phosphatase 2A (PP2A) is a prime example of the multisubunit architecture of protein serine/threonine phosphatases. Until substrate-specific PP2A holoenzymes assemble, a constitutively active, but nonspecific, catalytic C subunit would constitute a risk to the cell. While it has been assumed that the severe proliferation impairment of yeast lacking the structural PP2A subunit, TPD3, is due to the unrestricted activity of the C subunit, we recently obtained evidence for the existence of the C subunit in a low-activity conformation that requires the RRD/PTPA proteins for the switch into the active conformation. To study whether and how maturation of the C subunit is coupled with holoenzyme assembly, we analyzed PP2A biogenesis in yeast. Here we show that the generation of the catalytically active C subunit depends on the physical and functional interaction between RRD2 and the structural subunit, TPD3. The phenotype of the tpd3Δ strain is therefore caused by impaired, rather than increased, PP2A activity. TPD3/RRD2-dependent C subunit maturation is under the surveillance of the PP2A methylesterase, PPE1, which upon malfunction of PP2A biogenesis, prevents premature generation of the active C subunit and holoenzyme assembly by counteracting the untimely methylation of the C subunit. We propose a novel model of PP2A biogenesis in which a tightly controlled activation cascade protects cells from untargeted activity of the free catalytic PP2A subunit

Anti-RAINBOW dye-specific antibodies as universal tools for the visualization of prestained protein molecular weight markers in Western blot analysis

Western blotting is one of the most widely used techniques in molecular biology and biochemistry. Prestained proteins are used as molecular weight standards in protein electrophoresis. In the chemiluminescent Western blot analysis, however, these colored protein markers are invisible leaving researchers with the unsatisfying situation that the signal for the protein of interest and the signal for the markers are not captured simultaneously and have to be merged in an error-prone step. To allow the simultaneous detection of marker proteins we generated monoclonal antibodies specific for the protein dyes. To elicit a dye rather than protein specific immune response we immunized mice sequentially with dye-carrier protein complexes, in which a new carrier protein was used for each subsequent immunization. Moreover, by sequentially immunizing with dye-carrier protein complexes, in which different but structurally related dyes were used, we could also generate an antibody, termed anti-RAINBOW, that cross-reacted even with structurally related dyes not used in the immunizations. Our novel antibodies represent convenient tools for the simultaneous Western blot detection of commercially available prestained marker proteins in combination with the detection of any specific protein of interest. These antibodies will render obsolete the anachronistic tradition of manually charting marker bands on film.(VLID)468841

A novel and essential mechanism determining specificity and activity of protein phosphatase 2A (PP2A) in vivo

Protein phosphatase 2A (PP2A) is an essential intracellular serine/threonine phosphatase containing a catalytic subunit that possesses the potential to dephosphorylate promiscuously tyrosine-phosphorylated substrates in vitro. How PP2A acquires its intracellular specificity and activity for serine/threonine-phosphorylated substrates is unknown. Here we report a novel and phylogenetically conserved mechanism to generate active phospho-serine/threonine-specific PP2A in vivo. Phosphotyrosyl phosphatase activator (PTPA), a protein of so far unknown intracellular function, is required for the biogenesis of active and specific PP2A. Deletion of the yeast PTPA homologs generated a PP2A catalytic subunit with a conformation different from the wild-type enzyme, as indicated by its altered substrate specificity, reduced protein stability, and metal dependence. Complementation and RNA-interference experiments showed that PTPA fulfills an essential function conserved from yeast to man

Protein Phosphatase Methyl-Esterase PME-1 Protects Protein Phosphatase 2A from Ubiquitin/Proteasome Degradation

<div><p>Protein phosphatase 2A (PP2A) is a conserved essential enzyme that is implicated as a tumor suppressor based on its central role in phosphorylation-dependent signaling pathways. Protein phosphatase methyl esterase (PME-1) catalyzes specifically the demethylation of the C-terminal Leu309 residue of PP2A catalytic subunit (PP2Ac). It has been shown that PME-1 affects the activity of PP2A by demethylating PP2Ac, but also by directly binding to the phosphatase active site, suggesting loss of PME-1 in cells would enhance PP2A activity. However, here we show that PME-1 knockout mouse embryonic fibroblasts (MEFs) exhibit lower PP2A activity than wild type MEFs. Loss of PME-1 enhanced poly-ubiquitination of PP2Ac and shortened the half-life of PP2Ac protein resulting in reduced PP2Ac levels. Chemical inhibition of PME-1 and rescue experiments with wild type and mutated PME-1 revealed methyl-esterase activity was necessary to maintain PP2Ac protein levels. Our data demonstrate that PME-1 methyl-esterase activity protects PP2Ac from ubiquitin/proteasome degradation.</p></div

PME-1 protects PP2Ac from protein degradation.

<p>(A-B) Loss of PME-1 does not affect PP2Ac mRNA expression. mRNA levels of PP2Ac α (A) and β (B) isoforms were analyzed by real-time qPCR. Quantitative data from 2 independent experiments performed in duplicate are shown. (C-D) PME-1 protects PP2Ac from protein degradation. PP2Ac degradation was analyzed by cycloheximide chase assay. Representative images (C) and quantitative data for PP2Ac protein level (D) from 5 independent experiments are shown. *: <i>P</i><0.05 vs. WT.</p

Methyl-esterase activity of PME-1 is required to maintain levels of PP2Ac.

<p>(A) PME-1 inhibitor decreases PP2Ac protein levels. WT MEFs were treated with ABL127 (5 or 10 μM) for 48 hr and levels of proteins were determined by immunoblotting. Representative images (A) and quantitative data (B) from 4 independent experiments are shown. *: <i>P</i><0.05 vs. ABL127 untreated. (C-D) Methyl esterase activity of PME-1 is required to maintain PP2Ac levels. PME-1 KO MEFs were expressed FLAG-PME-1 WT or S156A (methyl esterase dead), and levels of proteins were determined by immunoblotting. Empty vector was used as mock. Representative images (C) and quantitative data (D) from 3 independent experiments are shown. *: <i>P</i><0.05 vs. FLAG-PME-1 WT expressed KO MEFs.</p

PME-1 knockout suppresses cell proliferation.

<p>(A) Loss of PME-1 suppresses cell proliferation. Cell proliferation of wild type (WT) and PME-1 KO (KO) MEFs were determined by Cell Counting Kit-8. N = 4 *: <i>P</i><0.05 vs. WT. (B-D) Effects of PME-1 KO on ERK1/2 and Akt phosphorylation. WT and KO MEFs were stimulated with EGF (50 ng/ml) for indicated time periods and ERK1/2 and Akt phosphorylation was determined by immunoblotting. Representative images (B) from 3 independent experiments, and quantitative data for phospho-ERK1/2 (C) and phosphoT308 Akt (D) are shown. *: <i>P</i><0.05 vs. WT. (E-F) Loss of PME-1 enhances the association of PP2A AB55αC complex. FLAG-B55α or B56α were transiently expressed in WT and PME-1 KO MEFs, and immunoprecipitated with FLAG-M2 beads. Empty vector was used as mock. PP2Ac and A subunit association was detected by immunoblotting. Representative images from 3 independent experiments were shown.</p

Proposed working hypothesis.

<p>Proposed working hypothesis for the regulatory mechanism for PP2A protein level by PME-1. See the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145226#sec018" target="_blank">Discussion</a> for details.</p