Targeted disruption of the PME-1 gene causes loss of demethylated PP2A and perinatal lethality in mice.
ABSTRACT: Phosphoprotein phosphatase 2A (PP2A), a major serine-threonine protein phosphatase in eukaryotes, is an oligomeric protein comprised of structural (A) and catalytic (C) subunits to which a variable regulatory subunit (B) can associate. The C subunit contains a methyl ester post-translational modification on its C-terminal leucine residue, which is removed by a specific methylesterase (PME-1). Methylesterification is thought to control the binding of different B subunits to AC dimers, but little is known about its physiological significance in vivo.Here, we show that targeted disruption of the PME-1 gene causes perinatal lethality in mice, a phenotype that correlates with a virtually complete loss of the demethylated form of PP2A in the nervous system and peripheral tissues. Interestingly, PP2A catalytic activity over a peptide substrate was dramatically reduced in PME-1(-/-) tissues, which also displayed alterations in phosphoproteome content.These findings suggest a role for the demethylated form of PP2A in maintenance of enzyme function and phosphorylation networks in vivo.
Project description:Carboxymethylation of proteins is a highly conserved means of regulation in eukaryotic cells. The protein phosphatase 2A (PP2A) catalytic (C) subunit is reversibly methylated at its carboxyl terminus by specific methyltransferase and methylesterase enzymes which have been purified, but not cloned. Carboxymethylation affects PP2A activity and varies during the cell cycle. Here, we report that substitution of glutamine for either of two putative active site histidines in the PP2A C subunit results in inactivation of PP2A and formation of stable complexes between PP2A and several cellular proteins. One of these cellular proteins, herein named protein phosphatase methylesterase-1 (PME-1), was purified and microsequenced, and its cDNA was cloned. PME-1 is conserved from yeast to human and contains a motif found in lipases having a catalytic triad-activated serine as their active site nucleophile. Bacterially expressed PME-1 demethylated PP2A C subunit in vitro, and okadaic acid, a known inhibitor of the PP2A methylesterase, inhibited this reaction. To our knowledge, PME-1 represents the first mammalian protein methylesterase to be cloned. Several lines of evidence indicate that, although there appears to be a role for C subunit carboxyl-terminal amino acids in PME-1 binding, amino acids other than those at the extreme carboxyl terminus of the C subunit also play an important role in PME-1 binding to a catalytically inactive mutant.
Project description:The serine hydrolase protein phosphatase methylesterase-1 (PME-1) regulates the methylesterification state of protein phosphatase 2A (PP2A) and has been implicated in cancer and Alzheimer's disease. We recently reported a fluorescence polarization-activity-based protein profiling (fluopol-ABPP) high-throughput screen for PME-1 that uncovered a remarkably potent and selective class of aza-?-lactam (ABL) PME-1 inhibitors. Here, we describe a distinct set of sulfonyl acrylonitrile inhibitors that also emerged from this screen. The optimized compound, 28 (AMZ30), selectively inactivates PME-1 and reduces the demethylated form of PP2A in living cells. Considering that 28 is structurally unrelated to ABL inhibitors of PME-1, these agents, together, provide a valuable set of pharmacological probes to study the role of methylation in regulating PP2A function. We furthermore observed that several serine hydrolases were sensitive to analogues of 28, suggesting that more extensive structural exploration of the sulfonyl acrylonitrile chemotype may result in useful inhibitors for other members of this large enzyme class.
Project description:National Institutes of Health (NIH)-sponsored screening centers provide academic researchers with a special opportunity to pursue small-molecule probes for protein targets that are outside the current interest of, or beyond the standard technologies employed by, the pharmaceutical industry. Here, we describe the outcome of an inhibitor screen for one such target, the enzyme protein phosphatase methylesterase-1 (PME-1), which regulates the methylesterification state of protein phosphatase 2A (PP2A) and is implicated in cancer and neurodegeneration. Inhibitors of PME-1 have not yet been described, which we attribute, at least in part, to a dearth of substrate assays compatible with high-throughput screening. We show that PME-1 is assayable by fluorescence polarization-activity-based protein profiling (fluopol-ABPP) and use this platform to screen the 300,000+ member NIH small-molecule library. This screen identified an unusual class of compounds, the aza-?-lactams (ABLs), as potent (IC(50) values of approximately 10 nM), covalent PME-1 inhibitors. Interestingly, ABLs did not derive from a commercial vendor but rather an academic contribution to the public library. We show using competitive-ABPP that ABLs are exquisitely selective for PME-1 in living cells and mice, where enzyme inactivation leads to substantial reductions in demethylated PP2A. In summary, we have combined advanced synthetic and chemoproteomic methods to discover a class of ABL inhibitors that can be used to selectively perturb PME-1 activity in diverse biological systems. More generally, these results illustrate how public screening centers can serve as hubs to create spontaneous collaborative opportunities between synthetic chemistry and chemical biology labs interested in creating first-in-class pharmacological probes for challenging protein targets.
Project description:Altered folate homeostasis is associated with many clinical and pathological manifestations in the CNS. Notably, folate-mediated one-carbon metabolism is essential for methyltransferase-dependent cellular methylation reactions. Biogenesis of protein phosphatase 2A (PP2A) holoenzyme containing the regulatory B(alpha) subunit, a major brain tau phosphatase, is controlled by methylation. Here, we show that folate deprivation in neuroblastoma cells induces downregulation of PP2A leucine carboxyl methyltransferase-1 (LCMT-1) expression, resulting in progressive accumulation of newly synthesized demethylated PP2A pools, concomitant loss of B(alpha), and ultimately cell death. These effects are further accentuated by overexpression of PP2A methylesterase (PME-1) but cannot be rescued by PME-1 knockdown. Overexpression of either LCMT-1 or B(alpha) is sufficient to protect cells against the accumulation of demethylated PP2A, increased tau phosphorylation, and cell death induced by folate starvation. Conversely, knockdown of either protein accelerates folate deficiency-evoked cell toxicity. Significantly, mice maintained for 2 months on low-folate or folate-deficient diets have brain-region-specific alterations in metabolites of the methylation pathway. Those are associated with downregulation of LCMT-1, methylated PP2A, and B(alpha) expression and enhanced tau phosphorylation in susceptible brain regions. Our studies provide novel mechanistic insights into the regulation of PP2A methylation and tau. They establish LCMT-1- and B(alpha)-containing PP2A holoenzymes as key mediators of the role of folate in the brain. Our results suggest that counteracting the neuronal loss of LCMT-1 and B(alpha) could be beneficial for all tauopathies and folate-dependent disorders of the CNS.
Project description:We have described recently the purification and cloning of PP2A (protein phosphatase 2A) leucine carboxylmethyltransferase. We studied the purification of a PP2A-specific methylesterase that co-purifies with PP2A and found that it is tightly associated with an inactive dimeric or trimeric form of PP2A. These inactive enzyme forms could be reactivated as Ser/Thr phosphatase by PTPA (phosphotyrosyl phosphatase activator of PP2A). PTPA was described previously by our group as a protein that stimulates the in vitro phosphotyrosyl phosphatase activity of PP2A; however, PP2A-specific methyltransferase could not bring about the activation. The PTPA activation could be distinguished from the Mn2+ stimulation observed with some inactive forms of PP2A, also found associated with PME-1 (phosphatase methylesterase 1). We discuss a potential new function for PME-1 as an enzyme that stabilizes an inactivated pool of PP2A.
Project description: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.
Project description:BACKGROUND AND PURPOSE:Although protein phosphatases regulate multiple cellular functions, their modulation under hypoxia remains unclear. We investigated expression of the protein phosphatase system under normoxic/hypoxic conditions and the mechanism by which hypoxia alters protein phosphatase 2A (PP2A) activity. EXPERIMENTAL APPROACH:Human cardiovascular cells were cultured in cell type specific media under normoxic or hypoxic conditions (1% O2 ). Effects on mRNA expression, phosphatase activity, post-translational modification, and involvement of hypoxia inducible factor 1? (HIF-1?) were assessed using RT-PCR, immunoblotting, an activity assay, and siRNA silencing. KEY RESULTS:All components of the protein phosphatase system studied were expressed in each cell line. Hypoxia attenuated mRNA expression of the transcripts in a cell line- and time-dependent manner. In human aortic smooth muscle cells (HASMC) and AC16 cells, hypoxia decreased PP2Ac activity and mRNA expression without altering PP2Ac abundance. Hypoxia increased demethylated PP2Ac (DPP2Ac) and phosphatase methylesterase 1 (PME-1) abundance but decreased leucine carboxyl methyltransferase 1 (LCMT-1) abundance. HIF-1? siRNA prevented the hypoxia-mediated decrease in phosphatase activity and expression of the catalytic subunit of protein phosphatase 2A (PPP2CA), independently of altering pPP2Ac, DPP2Ac, LCMT-1, or PME-1 abundance. CONCLUSION AND IMPLICATIONS:Cardiovascular cells express multiple components of the PP2A system. In HASMC and AC16 cells, hypoxia inhibits PP2A activity through HIF-1?-dependent and -independent mechanisms, with the latter being consistent with altered PP2A holoenzyme assembly. This indicates a complex inhibitory effect of hypoxia on the PP2A system, and highlights PP2A as a therapeutic target for diseases associated with dysregulated protein phosphorylation.
Project description:The pathology of Parkinson's disease (PD) is characterized by intracellular neurofibrillary tangles of phosphorylated ?-synuclein (?-syn). Protein phosphatase 2A (PP2A) is responsible for ?-syn dephosphorylation. Previous work has demonstrated that ?-syn can regulate PP2A activity. However, the mechanisms underlying ?-syn regulation of PP2A activity are not well understood. In this study, we found that ?-syn overexpression induced increased ?-syn phosphorylation at serine 129 (Ser129), and PP2A inhibition, in vitro and in vivo. ?-syn overexpression resulted in PP2A demethylation. This demethylation was mediated via downregulated leucine carboxyl methyltransferase (LCMT-1) expression, and upregulated protein phosphatase methylesterase (PME-1) expression. Furthermore, LCMT-1 overexpression, or PME-1 inhibition, reversed ?-syn-induced increases in ?-syn phosphorylation and apoptosis. In addition to post-translational modifications of the catalytic subunit, regulatory subunits are involved in the regulation of PP2A activity. We found that the levels of regulatory subunits which belong to the PPP2R2 subfamily, not the PPP2R5 subfamily, were downregulated in the examined brain regions of transgenic mice. Our work identifies a novel mechanism to explain how ?-syn regulates PP2A activity, and provides the optimization of PP2A methylation as a new target for PD treatment.
Project description:Leucine carboxyl methyltransferase-1 (LCMT1) and protein phosphatase methylesterase-1 (PME-1) are essential enzymes that regulate the methylation of the protein phosphatase 2A catalytic subunit (PP2AC). LCMT1 and PME-1 have been linked to the regulation of cell growth and proliferation, but the underlying mechanisms have remained elusive. We show here an important role for an LCMT1-PME-1 methylation equilibrium in controlling mitotic spindle size. Depletion of LCMT1 or overexpression of PME-1 led to long spindles. In contrast, depletion of PME-1, pharmacological inhibition of PME-1 or overexpression of LCMT1 led to short spindles. Furthermore, perturbation of the LCMT1-PME-1 methylation equilibrium led to mitotic arrest, spindle assembly checkpoint activation, defective cell divisions, induction of apoptosis and reduced cell viability. Thus, we propose that the LCMT1-PME-1 methylation equilibrium is critical for regulating mitotic spindle size and thereby proper cell division.
Project description:Pectin methylesterase (PME) catalyses the de-methylesterification of pectin in plant cell walls during cell elongation. (1) Pectins are mainly composed of ?(1, 4)-D-galacturonosyl acid units that are synthesised in a methylesterified form in the Golgi apparatus to prevent any interaction with Ca2+ ions during their intracellular transport. (2) The highly methylesterified pectins are then secreted into the apoplasm (3) and subsequently de-methylesterified in muro by PMEs. This can either induce the formation of pectin gels through the Ca2+ crosslinking of neighbouring non-methylesterified chains or create substrates for pectin-degrading enzymes such as polygalacturonases and pectate lyases for the initiation of cell wall loosening. (4) PMEs belong to a large multigene family. Sixty-six PME-related genes are predicted in the Arabidopsis genome. (1) Among them, we have recently shown that AtPME3 (At3g14310), a major basic PME isoform in A. thaliana, is ubiquitously expressed in vascular tissues and play a role in adventitious rooting. (5) In flax (Linum usitatissimum), three genes encoding PMEs have been sequenced so far, including LuPME3, the orthologue of AtPME3. Analysis of the LuPME3 isoform brings new insights into the processing of these proteins.