Phenothiazines Induce Apoptosis in T-Cell Acute Lymphoblastic Leukemia by Activating the Phosphatase Activity of the PP2A Tumor Suppressor
Alejandro GutiérrezAlex KentsisPan LiFrédéric BaleydierJason MarineauRuta GrebliunaiteElena KozakewichCasie ReedFrançoise PflumioSandrine PoglioBenjamin UzanPaul A. ClemonsLynn VerPlankW. Frank AnJason BurbankStephanie NortonNicola TollidayHanno SteenJames E. BradnerA. Thomas LookJon C. Aster
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Abstract: We have found that modification of rat PC12 cells with pertussis toxin resulted in an ∼50% inhibition of a protein phosphatase 2A‐like phosphatase. Protein phosphatase 2A (PP2A) is a major cellular serine/threonine‐specific protein phosphatase. Treatment of extracts from pertussis toxin‐modified PC12 cells with either immobilized alkaline phosphatase or Ca 2+ reversed this inhibition. Reactivation of the PP2A‐like phosphatase in Ca 2+ appears to result from the dephosphorylation of a protein by the Ca 2+ /calmodulin‐dependent protein phosphatase calcineurin. The PP2A‐like phosphatase in extracts from pertussis toxin‐modified PC12 cells eluted from a Mono Q column at a higher ionic strength than did the PP2A‐like phosphatase in extracts from control cells. After incubation in Ca 2+ , the PP2A‐like phosphatase in extracts from pertussis toxin‐modified cells eluted from a Mono Q column at the same ionic strength as did the PP2A‐like phosphatase in extracts from control cells. These results indicate that the effect of pertussis toxin on this PP2A‐like activity results from the phosphorylation of either one of the subunits of the PP2A‐like phosphatase or a protein that when phosphorylated binds to and inhibits this phosphatase. Pertussis toxin modification did not result in the phosphorylation of the catalytic subunit of PP2A. Because phosphorylation regulates the activities of many enzymes and cell surface receptors, a pertussis toxin‐induced decrease in PP2A activity could alter signaling pathways and other cellular processes in which G proteins are not directly involved.
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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.
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Eya genes encode a unique family of multifunctional proteins that serve as transcriptional co-activators and as haloacid dehalogenase-family Tyr phosphatases. Intriguingly, the N-terminal domain of Eyas, which does not share sequence similarity to any known phosphatases, contains a separable Ser/Thr phosphatase activity. Here, we demonstrate that the Ser/Thr phosphatase activity of Eya is not intrinsic, but arises from its direct interaction with the protein phosphatase 2A (PP2A)-B55α holoenzyme. Importantly, Eya3 alters the regulation of c-Myc by PP2A, increasing c-Myc stability by enabling PP2A-B55α to dephosphorylate pT58, in direct contrast to the previously described PP2A-B56α-mediated dephosphorylation of pS62 and c-Myc destabilization. Furthermore, Eya3 and PP2A-B55α promote metastasis in a xenograft model of breast cancer, opposing the canonical tumor suppressive function of PP2A-B56α. Our study identifies Eya3 as a regulator of PP2A, a major cellular Ser/Thr phosphatase, and uncovers a mechanism of controlling the stability of a critical oncogene, c-Myc.
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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.
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We identified an essential Saccharomyces cerevisiae protein, Tap42, that associates with Sit4, a type 2A-related protein phosphatase, and with the type 2A phosphatase catalytic subunits. The association of Tap42 with the phosphatases does not require the previously identified phosphatase subunits. Genetic analysis suggests that Tap42 functions positively with both phosphatases. Mutations in TAP42 can confer almost complete rapamycin resistance. In addition, Tap42/Sit4 and Tap42/PP2A complex formation is regulated by nutrient growth signals and the rapamycin-sensitive Tor signaling pathway. These findings, combined with the defect in translation of the tap42-11 mutant at the nonpermissive temperature, suggest that Tap42, Sit4, and PP2A are components of the Tor signaling pathway.
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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.
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Fostriecin and cytostatin are structurally related natural inhibitors of serine/threonine phosphatases, with promising antitumor activity. The total synthesis of these antitumor agents has enabled the production of structural analogs, which are useful to explore the biological significance of features contained in the parent compounds. Here, the inhibitory activity of fostriecin, cytostatin, and 10 key structural analogs were tested in side-by-side phosphatase assays to further characterize their inhibitory activity against PP1c (Ser/Thr protein phosphatase 1 catalytic subunit), PP2Ac (Ser/Thr protein phosphatase 2A catalytic subunit), PP5c (Ser/Thr protein phosphatase 5 catalytic subunit), and chimeras of PP1 (Ser/Thr protein phosphatase 1) and PP5 (Ser/Thr protein phosphatase 5), in which key residues predicted for inhibitor contact with PP2A (Ser/Thr protein phosphatase 2A) were introduced into PP1 and PP5 using site-directed mutagenesis. The data confirm the importance of the C9-phosphate and C11-alcohol for general inhibition and further demonstrate the importance of a predicted C3 interaction with a unique cysteine (Cys269) in the β12–β13 loop of PP2A. The data also indicate that additional features beyond the unsaturated lactone contribute to inhibitory potency and selectivity. Notably, a derivative of fostriecin lacking the entire lactone subunit demonstrated marked potency and selectivity for PP2A, while having substantially reduced and similar activity against PP1 and PP1/PP2A- PP5/PP2A-chimeras that have greatly increased sensitivity to both fostriecin and cytostatin. This suggests that other features [e.g., the (Z,Z,E)-triene] also contribute to inhibitory selectivity. When considered together with previous data, these studies suggest that, despite the high structural conservation of the catalytic site in PP1, PP2A and PP5, the development of highly selective catalytic inhibitors should be feasible.
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