Allosteric Activators of Protein Phosphatase 2A Display Broad Antitumor Activity Mediated by Dephosphorylation of MYBL2.

Protein phosphatase 2A (PP2A) enzymes can suppress tumors, but they are often inactivated in human cancers overexpressing inhibitory proteins. Here, we identify a class of small-molecule iHAPs (improved heterocyclic activators of PP2A) that kill leukemia cells by allosterically assembling a specific heterotrimeric PP2A holoenzyme consisting of PPP2R1A (scaffold), PPP2R5E (B56ε, regulatory), and PPP2CA (catalytic) subunits. One compound, iHAP1, activates this complex but does not inhibit dopamine receptor D2, a mediator of neurologic toxicity induced by perphenazine and related neuroleptics. The PP2A complex activated by iHAP1 dephosphorylates the MYBL2 transcription factor on Ser241, causing irreversible arrest of leukemia and other cancer cells in prometaphase. In contrast, SMAPs, a separate class of compounds, activate PP2A holoenzymes containing a different regulatory subunit, do not dephosphorylate MYBL2, and arrest tumor cells in G1 phase. Our findings demonstrate that small molecules can serve as allosteric switches to activate distinct PP2A complexes with unique substrates.

Histone phosphorylation integrates the hepatic glucagon-PKA-CREB gluconeogenesis program in response to fasting.

The glucagon-PKA signal is generally believed to control hepatic gluconeogenesis via the CREB transcription factor. Here we uncovered a distinct function of this signal in directly stimulating histone phosphorylation for gluconeogenic gene regulation in mice. In the fasting state, CREB recruited activated PKA to regions near gluconeogenic genes, where PKA phosphorylated histone H3 serine 28 (H3S28ph). H3S28ph, recognized by 14-3-3ζ, promoted recruitment of RNA polymerase II and transcriptional stimulation of gluconeogenic genes. In contrast, in the fed state, more PP2A was found near gluconeogenic genes, which counteracted PKA by dephosphorylating H3S28ph and repressing transcription. Importantly, ectopic expression of phosphomimic H3S28 efficiently restored gluconeogenic gene expression when liver PKA or CREB was depleted. These results together highlight a different functional scheme in regulating gluconeogenesis by the glucagon-PKA-CREB-H3S28ph cascade, in which the hormone signal is transmitted to chromatin for rapid and efficient gluconeogenic gene activation.

IntS6 and the Integrator phosphatase module tune the efficiency of select premature transcription termination events.

The metazoan-specific Integrator complex catalyzes 3' end processing of small nuclear RNAs (snRNAs) and premature termination that attenuates the transcription of many protein-coding genes. Integrator has RNA endonuclease and protein phosphatase activities, but it remains unclear if both are required for complex function. Here, we show IntS6 (Integrator subunit 6) over-expression blocks Integrator function at a subset of Drosophila protein-coding genes, although having no effect on snRNAs or attenuation of other loci. Over-expressed IntS6 titrates protein phosphatase 2A (PP2A) subunits, thereby only affecting gene loci where phosphatase activity is necessary for Integrator function. IntS6 functions analogous to a PP2A regulatory B subunit as over-expression of canonical B subunits, which do not bind Integrator, is also sufficient to inhibit Integrator activity. These results show that the phosphatase module is critical at only a subset of Integrator-regulated genes and point to PP2A recruitment as a tunable step that modulates transcription termination efficiency.

Protein phosphatases in the RNAPII transcription cycle: erasers, sculptors, gatekeepers, and potential drug targets.

The transcription cycle of RNA polymerase II (RNAPII) is governed at multiple points by opposing actions of cyclin-dependent kinases (CDKs) and protein phosphatases, in a process with similarities to the cell division cycle. While important roles of the kinases have been established, phosphatases have emerged more slowly as key players in transcription, and large gaps remain in understanding of their precise functions and targets. Much of the earlier work focused on the roles and regulation of sui generis and often atypical phosphatases-FCP1, Rtr1/RPAP2, and SSU72-with seemingly dedicated functions in RNAPII transcription. Decisive roles in the transcription cycle have now been uncovered for members of the major phosphoprotein phosphatase (PPP) family, including PP1, PP2A, and PP4-abundant enzymes with pleiotropic roles in cellular signaling pathways. These phosphatases appear to act principally at the transitions between transcription cycle phases, ensuring fine control of elongation and termination. Much is still unknown, however, about the division of labor among the PPP family members, and their possible regulation by or of the transcriptional kinases. CDKs active in transcription have recently drawn attention as potential therapeutic targets in cancer and other diseases, raising the prospect that the phosphatases might also present opportunities for new drug development. Here we review the current knowledge and outstanding questions about phosphatases in the context of the RNAPII transcription cycle.

Targeted chemotherapy overcomes drug resistance in melanoma.

The emergence of drug resistance is a major obstacle for the success of targeted therapy in melanoma. Additionally, conventional chemotherapy has not been effective as drug-resistant cells escape lethal DNA damage effects by inducing growth arrest commonly referred to as cellular dormancy. We present a therapeutic strategy termed "targeted chemotherapy" by depleting protein phosphatase 2A (PP2A) or its inhibition using a small molecule inhibitor (1,10-phenanthroline-5,6-dione [phendione]) in drug-resistant melanoma. Targeted chemotherapy induces the DNA damage response without causing DNA breaks or allowing cellular dormancy. Phendione treatment reduces tumor growth of BRAFV600E-driven melanoma patient-derived xenografts (PDX) and diminishes growth of NRASQ61R-driven melanoma, a cancer with no effective therapy. Remarkably, phendione treatment inhibits the acquisition of resistance to BRAF inhibition in BRAFV600E PDX highlighting its effectiveness in combating the advent of drug resistance.

Urolithin A attenuates hexavalent chromium-induced small intestinal injury by modulating PP2A/Hippo/YAP1 pathway.

Hexavalent chromium (Cr(VI)) exposure has been linked with gastrointestinal toxicity, whereas the molecular pathways and key targets remain elusive. Computational toxicology analysis predicted the correlation between protein phosphatase 2A (PP2A) and genes regarding Cr(VI)-induced intestinal injury. Here, we generated a mouse model with intestinal epithelium-specific knock out of Ppp2r1a (encoding PP2A Aα subunit) to investigate the mechanisms underlying Cr(VI)-induced small intestinal toxicity. Heterozygous (HE) mice and matched WT littermates were administrated with Cr(VI) at 0, 5, 20, and 80 mg/l for 28 successive days. Cr(VI) treatment led to crypt hyperplasia, epithelial cell apoptosis, and intestinal barrier dysfunction, accompanied by the decline of goblet cell counts and Occludin expression in WT mice. Notably, these effects were aggravated in HE mice, indicating that PP2A Aα deficiency conferred mice with susceptibility to Cr(VI)-induced intestinal injury. The combination of data analysis and biological experiments revealed Cr(VI) exposure could decrease YAP1 phosphorylation at Ser127 but increase protein expression and activity, together with elevated transcriptional coactivator with PDZ-binding motif protein driving epithelial crypt cells proliferation following damage, suggesting the involvement of Hippo/YAP1 signaling pathway in Cr(VI)-induced intestinal toxicity. Nevertheless, the enhanced phosphorylation of YAP1 in HE mice resulted in proliferation/repair defects in intestinal epithelium, thereby exacerbating Cr(VI)-induced gut barrier dysfunction. Notably, by molecular docking and further studies, we identified urolithin A, a microbial metabolite, attenuated Cr(VI)-induced disruption of intestinal barrier function, partly by modulating YAP1 expression and activity. Our findings reveal the novel molecular pathways participated in Cr(VI)-caused small intestinal injury and urolithin A could potentially protect against environmental hazards-induced intestinal diseases.

Biochemical characterization of the Eya and PP2A-B55α interaction.

The eyes absent (Eya) proteins were first identified as co-activators of the six homeobox family of transcription factors and are critical in embryonic development. These proteins are also re-expressed in cancers after development is complete, where they drive tumor progression. We have previously shown that the Eya3 N-terminal domain (NTD) contains Ser/Thr phosphatase activity through an interaction with the protein phosphatase 2A (PP2A)-B55α holoenzyme and that this interaction increases the half-life of Myc through pT58 dephosphorylation. Here, we showed that Eya3 directly interacted with the NTD of Myc, recruiting PP2A-B55α to Myc. We also showed that Eya3 increased the Ser/Thr phosphatase activity of PP2A-B55α but not PP2A-B56α. Furthermore, we demonstrated that the NTD (∼250 amino acids) of Eya3 was completely disordered, and it used a 38-residue segment to interact with B55α. In addition, knockdown and phosphoproteomic analyses demonstrated that Eya3 and B55α affected highly similar phosphosite motifs with a preference for Ser/Thr followed by Pro, consistent with Eya3's apparent Ser/Thr phosphatase activity being mediated through its interaction with PP2A-B55α. Intriguingly, mutating this Pro to other amino acids in a Myc peptide dramatically increased dephosphorylation by PP2A. Not surprisingly, MycP59A, a naturally occurring mutation hotspot in several cancers, enhanced Eya3-PP2A-B55α-mediated dephosphorylation of pT58 on Myc, leading to increased Myc stability and cell proliferation, underscoring the critical role of this phosphosite in regulating Myc stability.

PP2A inhibitor SET promotes mTORC1 and Bmi1 signaling through Akt activation and maintains the colony-formation ability of cancer cells.

Protein phosphatase 2A (PP2A) is an essential tumor suppressor, with its activity often hindered in cancer cells by endogenous PP2A inhibitory proteins like SE translocation (SET). SET/PP2A axis plays a pivotal role in the colony-formation ability of cancer cells and the stabilization of c-Myc and E2F1 proteins implicated in this process. However, in osteosarcoma cell line HOS, SET knock-down (KD) suppresses the colony-formation ability without affecting c-Myc and E2F1. This study aimed to unravel the molecular mechanism through which SET enhances the colony-formation ability of HOS cells and determine if it is generalized to other cancer cells. Transcriptome analysis unveiled that SET KD suppressed mTORC1 signaling. SET KD inhibited Akt phosphorylation, an upstream kinase for mTORC1. PP2A inhibitor blocked SET KD-mediated decrease in phosphorylation of Akt and a mTORC1 substrate p70S6K. A constitutively active Akt restored decreased colony-formation ability by SET KD, indicating the SET/PP2A/Akt/mTORC1 axis. Additionally, enrichment analysis highlighted that Bmi-1, a polycomb group protein, is affected by SET KD. SET KD decreased Bmi-1 protein by Akt inhibition but not by mTORC1 inhibition, and exogenous Bmi-1 expression rescued the reduced colony formation by SET KD. Four out of eight cancer cell lines exhibited decreased Bmi-1 by SET KD. Further analysis of these cell lines revealed that Myc activity plays a role in SET KD-mediated Bmi-1 degradation. These findings provide new insights into the molecular mechanism of SET-regulated colony-formation ability, which involved Akt-mediated activation of mTORC1/p70S6K and Bmi-1 signaling.

Enhanced Ca2+-Dependent SK-Channel Gating and Membrane Trafficking in Human Atrial Fibrillation.

BACKGROUND: Small-conductance Ca2+-activated K+ (SK)-channel inhibitors have antiarrhythmic effects in animal models of atrial fibrillation (AF), presenting a potential novel antiarrhythmic option. However, the regulation of SK-channels in human atrial cardiomyocytes and its modification in patients with AF are poorly understood and were the object of this study. METHODS: Apamin-sensitive SK-channel current (ISK) and action potentials were recorded in human right-atrial cardiomyocytes from sinus rhythm control (Ctl) patients or patients with (long-standing persistent) chronic AF (cAF). RESULTS: ISK was significantly higher, and apamin caused larger action potential prolongation in cAF- versus Ctl-cardiomyocytes. Sensitivity analyses in an in silico human atrial cardiomyocyte model identified IK1 and ISK as major regulators of repolarization. Increased ISK in cAF was not associated with increases in mRNA/protein levels of SK-channel subunits in either right- or left-atrial tissue homogenates or right-atrial cardiomyocytes, but the abundance of SK2 at the sarcolemma was larger in cAF versus Ctl in both tissue-slices and cardiomyocytes. Latrunculin-A and primaquine (anterograde and retrograde protein-trafficking inhibitors) eliminated the differences in SK2 membrane levels and ISK between Ctl- and cAF-cardiomyocytes. In addition, the phosphatase-inhibitor okadaic acid reduced ISK amplitude and abolished the difference between Ctl- and cAF-cardiomyocytes, indicating that reduced calmodulin-Thr80 phosphorylation due to increased protein phosphatase-2A levels in the SK-channel complex likely contribute to the greater ISK in cAF-cardiomyocytes. Finally, rapid electrical activation (5 Hz, 10 minutes) of Ctl-cardiomyocytes promoted SK2 membrane-localization, increased ISK and reduced action potential duration, effects greatly attenuated by apamin. Latrunculin-A or primaquine prevented the 5-Hz-induced ISK-upregulation. CONCLUSIONS: ISK is upregulated in patients with cAF due to enhanced channel function, mediated by phosphatase-2A-dependent calmodulin-Thr80 dephosphorylation and tachycardia-dependent enhanced trafficking and targeting of SK-channel subunits to the sarcolemma. The observed AF-associated increases in ISK, which promote reentry-stabilizing action potential duration shortening, suggest an important role for SK-channels in AF auto-promotion and provide a rationale for pursuing the antiarrhythmic effects of SK-channel inhibition in humans.

Protein phosphatase 4 mediates palmitic acid-induced endothelial dysfunction by decreasing eNOS phosphorylation at serine 633 in HUVECs.

Plasma saturated free fatty acid (FFA)-induced endothelial dysfunction (ED) contributes to the pathogenesis of atherosclerosis and cardiovascular diseases. However, the mechanism underlying saturated FFA-induced ED remains unclear. This study demonstrated that palmitic acid (PA) induced ED by activating the NADPH oxidase (NOX)/ROS signaling pathway to activate protein phosphatase 4 (PP4) and protein phosphatase 2A (PP2A), thereby reducing endothelial nitric oxide synthase (eNOS) phosphorylation at Ser633 and Ser1177, respectively. Okadaic acid (OA) and fostriecin (FST), which are inhibitors of PP2A, inhibited the PA-induced decreases in eNOS phosphorylation at Ser633 and Ser1177. The antioxidants N-acetylcysteine (NAC) and apocynin (APO) or knockdown of gp91phox or p67phox (NOX subunits) restored PA-mediated downregulation of PP4R2 protein expression and eNOS Ser633 phosphorylation. Knockdown of the PP4 catalytic subunit (PP4c) specifically increased eNOS Ser633 phosphorylation, while silencing the PP2A catalytic subunit (PP2Ac) restored only eNOS Ser1177 phosphorylation. Furthermore, PA dramatically decreased the protein expression of the PP4 regulatory subunit R2 (PP4R2) but not the other regulatory subunits. PP4R2 overexpression increased eNOS Ser633 phosphorylation, nitric oxide (NO) production, cell migration and tube formation but did not change eNOS Ser1177 phosphorylation levels. Coimmunoprecipitation (Co-IP) suggested that PP4R2 and PP4c interacted with the PP4R3α and eNOS proteins. In summary, PA decreases PP4R2 protein expression through the Nox/ROS pathway to activate PP4, which contributes to ED by dephosphorylating eNOS at Ser633. The results of this study suggest that PP4 is a novel therapeutic target for ED and ED-associated vascular diseases.

Downregulation of protein phosphatase 2Aα in asthmatic airway smooth muscle.

Airway smooth muscle cell (ASM) is renowned for its involvement in airway hyperresponsiveness through impaired ASM relaxation and bronchoconstriction in asthma, which poses a significant challenge in the field. Recent studies have explored different targets in ASM to alleviate airway hyperresponsiveness, however, a sizeable portion of patients with asthma still experience poor control. In our study, we explored protein phosphatase 2 A (PP2A) in ASM as it has been reported to regulate cellular contractility by controlling intracellular calcium ([Ca2+]i), ion channels, and respective regulatory proteins. We obtained human ASM cells and lung tissues from healthy and patients with asthma and evaluated PP2A expression using RNA-Seq data, immunofluorescence, and immunoblotting. We further investigated the functional importance of PP2A by determining its role in bronchoconstriction using mouse bronchus and human ASM cell [Ca2+]i regulation. We found robust expression of PP2A isoforms in human ASM cells with PP2Aα being highly expressed. Interestingly, PP2Aα was significantly downregulated in asthmatic tissue and human ASM cells exposed to proinflammatory cytokines. Functionally, FTY720 (PP2A agonist) inhibited acetylcholine- or methacholine-induced bronchial contraction in mouse bronchus and further potentiated isoproterenol-induced bronchial relaxation. Mechanistically, FTY720 inhibited histamine-evoked [Ca2+]i response and myosin light chain (MLC) phosphorylation in the presence of interleukin-13 (IL-13) in human ASM cells. To conclude, we for the first time established PP2A signaling in ASM, which can be further explored to develop novel therapeutics to alleviate airway hyperresponsiveness in asthma.NEW & NOTEWORTHY This novel study deciphered the expression and function of protein phosphatase 2Aα (PP2Aα) in airway smooth muscle (ASM) during asthma and/or inflammation. We showed robust expression of PP2Aα in human ASM while its downregulation in asthmatic ASM. Similarly, we demonstrated reduced PP2Aα expression in ASM exposed to proinflammatory cytokines. PP2Aα activation inhibited bronchoconstriction of isolated mouse bronchi. In addition, we unveiled that PP2Aα activation inhibits the intracellular calcium release and myosin light chain phosphorylation in human ASM.

Striatin plays a major role in angiotensin II-induced cardiomyocyte and cardiac hypertrophy in mice in vivo.

The three striatins (STRN, STRN3, STRN4) form the core of STRiatin-Interacting Phosphatase and Kinase (STRIPAK) complexes. These place protein phosphatase 2A (PP2A) in proximity to protein kinases thereby restraining kinase activity and regulating key cellular processes. Our aim was to establish if striatins play a significant role in cardiac remodelling associated with cardiac hypertrophy and heart failure. All striatins were expressed in control human hearts, with up-regulation of STRN and STRN3 in failing hearts. We used mice with global heterozygote gene deletion to assess the roles of STRN and STRN3 in cardiac remodelling induced by angiotensin II (AngII; 7 days). Using echocardiography, we detected no differences in baseline cardiac function or dimensions in STRN+/- or STRN3+/- male mice (8 weeks) compared with wild-type littermates. Heterozygous gene deletion did not affect cardiac function in mice treated with AngII, but the increase in left ventricle mass induced by AngII was inhibited in STRN+/- (but not STRN3+/-) mice. Histological staining indicated that cardiomyocyte hypertrophy was inhibited. To assess the role of STRN in cardiomyocytes, we converted the STRN knockout line for inducible cardiomyocyte-specific gene deletion. There was no effect of cardiomyocyte STRN knockout on cardiac function or dimensions, but the increase in left ventricle mass induced by AngII was inhibited. This resulted from inhibition of cardiomyocyte hypertrophy and cardiac fibrosis. The data indicate that cardiomyocyte striatin is required for early remodelling of the heart by AngII and identify the striatin-based STRIPAK system as a signalling paradigm in the development of pathological cardiac hypertrophy.

Driving Mitochondrial Fission Improves Cognitive, but not Motor Deficits in a Mouse Model of Ataxia of Charlevoix-Saguenay.

Autosomal-recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is caused by loss-of-function mutation in the SACS gene, which encodes sacsin, a putative HSP70-HSP90 co-chaperone. Previous studies with Sacs knock-out (KO) mice and patient-derived fibroblasts suggested that SACSIN mutations inhibit the function of the mitochondrial fission enzyme dynamin-related protein 1 (Drp1). This in turn resulted in mitochondrial hyperfusion and dysfunction. We experimentally tested this hypothesis by genetically manipulating the mitochondrial fission/fusion equilibrium, creating double KO (DKO) mice that also lack positive (PP2A/Bß2) and negative (PKA/AKAP1) regulators of Drp1. Neither promoting mitochondrial fusion (Bß2 KO) nor fission (Akap1 KO) influenced progression of motor symptoms in Sacs KO mice. However, our studies identified profound learning and memory deficits in aged Sacs KO mice. Moreover, this cognitive impairment was rescued in a gene dose-dependent manner by deletion of the Drp1 inhibitor PKA/Akap1. Our results are inconsistent with mitochondrial dysfunction as a primary pathogenic mechanism in ARSACS. Instead, they imply that promoting mitochondrial fission may be beneficial at later stages of the disease when pathology extends to brain regions subserving learning and memory.

Far-infrared irradiation inhibits proliferation of human upper airway epithelial cells via protein phosphatase 2A-promoted dephosphorylation of p70 S6 kinase.

Far-infrared (FIR) ray, an invisible electromagnetic radiation with a wavelength of 3‒1000 µm, elicits various biological effects. Excessive proliferation of human upper airway epithelial cells (HUAEpCs) contributes to the development and exacerbation of nasal narrowing diseases, including nasal polyposis and chronic rhinosinusitis with nasal polyps (CRSwNP). Here, we investigated the molecular mechanisms through which FIR irradiation inhibits the proliferation of HUAEpCs. FIR irradiation significantly inhibited the proliferation of NCI-H292 cells without alteration in cell viability. The anti-proliferative effect of FIR radiation was accompanied by decreased phosphorylation of p70S6K at Thr389 (p-p70S6K-Thr389), without changes in the phosphorylation of mammalian target of rapamycin and adenosine monophosphate-activated protein kinase (AMPK). Overexpression of p70S6K-T389E mutant gene, not dominant negative-AMPKα1 gene, significantly reversed FIR irradiation-inhibited p-p70S6K-Thr389 and cell proliferation. Cotreatment with okadaic acid or knockdown of protein phosphatase 2A catalytic subunit (PP2Ac) gene expression significantly reversed FIR irradiation-decreased p-p70S6K-Thr389 and cell proliferation. FIR irradiation remarkably promoted the physical association of p70S6K and PP2Ac without change in total PP2Ac expression. Hyperthermal stimulus (39 °C) did not alter p-p70S6K-Thr389 and cell proliferation. In line with NCI-H292 cell results, FIR irradiation, not hyperthermal stimulus, significantly decreased p-p70S6K-Thr389 and cell proliferation in primary human nasal turbinate and polyp epithelial cells. These results demonstrated that FIR irradiation decreased the proliferation of HUAEpCs through PP2A-mediated inhibition of p70S6K phosphorylation, independent of its hyperthermal effect. Our data suggest that FIR therapy can be used to treat upper airway narrowing epithelial hyperplastic diseases, including nasal polyposis and CRSwNP.

Protein phosphatase 2A deficiency in hippocampal CA1 inhibits priming effect of morphine on conditioned place preference in mice.

Studies have shown that protein phosphorylation plays an important role in morphine abuse. However, the neurobiological mechanism of protein phosphatase 2A (PP2A) underlying the morphine-priming process is still unclear. Here we constructed T29-2-Cre; PP2Afl/fl conditional knockout mice (KO) and investigated the role of hippocampal PP2A in morphine priming. We observed that the deficit of PP2A inhibited the priming behavior of morphine and blocked the priming-induced long-term potentiation (LTP) in the hippocampus of KO mice. Moreover, the expression levels of Rack1 and the membrane GluN2B were significantly reduced in the nucleus accumbens of KO mice compared with those in the control mice, which may be attributed to the decreased HDAC4 in the hippocampus of KO mice. Consistent with it, the similar inhibited priming effects were also observed in the wild-type mice treated with sodium butyrate (NaB)-a nonspecific inhibitor of histone deacetylases-3 h after morphine administration. Taken together, our results suggest that hippocampal PP2A may be involved in morphine priming through the PP2A/HDAC4/Rack1 pathway.

Circular MTHFD2L RNA-encoded CM-248aa inhibits gastric cancer progression by targeting the SET-PP2A interaction.

The available targeted therapies for gastric cancer (GC) are still limited, so it is important to discover novel molecules as potential treatment options. Proteins or peptides encoded by circular RNAs (circRNAs) are increasingly reported to play essential roles in malignancies. The aim of the present study was to identify an undiscovered protein encoded by circRNA and explore its key role and molecular mechanism in GC progression. CircMTHFD2L (hsa_circ_0069982) was screened and validated as a downregulated circRNA with coding potential. The protein encoded by circMTHFD2L, named CM-248aa, was identified for the first time by immunoprecipitation and mass spectrometry. CM-248aa was significantly downregulated in GC, while its low expression was associated with advanced tumor-node-metastasis (TNM) stage and histopathological grade. Low expression of CM-248aa could be an independent risk factor for poor prognosis. Functionally, CM-248aa, instead of circMTHFD2L suppressed the proliferation and metastasis of GC in vitro and in vivo. Mechanistically, CM-248aa competitively targeted the acidic domain of SET nuclear oncogene (SET) and acted as an endogenous inhibitor of the SET-protein phosphatase 2A interaction to promote dephosphorylation of AKT, extracellular signal-regulated kinase, and P65. Our discovery revealed that CM-248aa could be a potential prognostic biomarker and endogenous therapeutic option for GC.

PP2A Affects Angiogenesis via Its Interaction with a Novel Phosphorylation Site of TSP1.

Alterations in angiogenic properties play a pivotal role in the manifestation and onset of various pathologies, including vascular diseases and cancer. Thrombospondin-1 (TSP1) protein is one of the master regulators of angiogenesis. This study unveils a novel aspect of TSP1 regulation through reversible phosphorylation. The silencing of the B55α regulatory subunit of protein phosphatase 2A (PP2A) in endothelial cells led to a significant decrease in TSP1 expression. Direct interaction between TSP1 and PP2A-B55α was confirmed via various methods. Truncated TSP1 constructs were employed to identify the phosphorylation site and the responsible kinase, ultimately pinpointing PKC as the enzyme phosphorylating TSP1 on Ser93. The biological effects of B55α-TSP1 interaction were also analyzed. B55α silencing not only counteracted the increase in TSP1 expression during wound closure but also prolonged wound closure time. Although B55α silenced cells initiated tube-like structures earlier than control cells, their spheroid formation was disrupted, leading to disintegration. Cells transfected with phosphomimic TSP1 S93D exhibited smaller spheroids and reduced effectiveness in tube formation, revealing insights into the effects of TSP1 phosphorylation on angiogenic properties. In this paper, we introduce a new regulatory mechanism of angiogenesis by reversible phosphorylation on TSP1 S93 by PKC and PP2A B55α.

Evaluating Novel Protein Phosphatase 2A Activators as Therapeutics for Emphysema.

The activity of PP2A (protein phosphatase 2A), a serine-threonine phosphatase, is reduced by chronic cigarette smoke (SM) exposure and α-1 antitrypsin (AAT) deficiency, and chemical activation of PP2A reduces the loss of lung function in SM-exposed mice. However, the previously studied PP2A-activator tricyclic sulfonamide compound DBK-1154 has low stability to oxidative metabolism, resulting in fast clearance and low systemic exposure. Here we compare the utility of a new more stable PP2A activator, ATUX-792, versus DBK-1154 for the treatment of SM-induced emphysema. ATUX-792 was also tested in human bronchial epithelial cells and a mouse model of AAT deficiency, Serpina1a-e-knockout mice. Human bronchial epithelial cells were treated with ATUX-792 or DBK-1154, and cell viability, PP2A activity, and MAP (mitogen-activated protein) kinase phosphorylation status were examined. Wild-type mice received vehicle, DBK-1154, or ATUX-792 orally in the last 2 months of 4 months of SM exposure, and 8-month-old Serpina1a-e-knockout mice received ATUX-792 daily for 4 months. Forced oscillation and expiratory measurements and histology analysis were performed. Treatment with ATUX-792 or DBK-1154 resulted in PP2A activation, reduced MAP kinase phosphorylation, immune cell infiltration, reduced airspace enlargements, and preserved lung function. Using protein arrays and multiplex assays, PP2A activation was observed to reduce AAT-deficient and SM-induced release of CXCL5, CCL17, and CXCL16 into the airways, which coincided with reduced neutrophil lung infiltration. Our study indicates that suppression of the PP2A activity in two models of emphysema could be restored by next-generation PP2A activators to impact lung function.

Hsa-miR-409-3p regulates endothelial progenitor senescence via PP2A-P38 and is a potential ageing marker in humans.

We explored the roles of hsa-microRNA (miR)-409-3p in senescence and signalling mechanism of human endothelial progenitor cells (EPCs). Hsa-miR-409-3p was found upregulated in senescent EPCs. Overexpression of miRNA mimics in young EPCs inhibited angiogenesis. In senescent EPCs, compared to young EPCs, protein phosphatase 2A (PP2A) was downregulated, with activation of p38/JNK by phosphorylation. Young EPCs treated with siPP2A caused inhibited angiogenesis with activation of p38/JNK, similar to findings in senescent EPCs. Time series analysis showed, in young EPCs treated with hsa-miR-409-3p mimics, PP2A was steadily downregulated for 72 h, while p38/JNK was activated with a peak at 48 hours. The inhibited angiogenesis of young EPCs after miRNA-409-3p mimics treatment was reversed by the p38 inhibitor. The effect of hsa-miR-409-3p on PP2A signalling was attenuated by exogenous VEGF. Analysis of human peripheral blood mononuclear cells (PBMCs) obtained from healthy people revealed hsa-miR-409-3p expression was higher in those older than 65 years, compared to those younger than 30 years, regardless of gender. In summary, hsa-miR-409-3p was upregulated in senescent EPCs and acted as a negative modulator of angiogenesis via targeting protein phosphatase 2 catalytic subunit alpha (PPP2CA) gene and regulating PP2A/p38 signalling. Data from human PBMCs suggested hsa-miR-409-3p a potential biomarker for human ageing.

A new paradigm for regulation of protein phosphatase 2A function via Src and Fyn kinase-mediated tyrosine phosphorylation.

Protein phosphatase 2A (PP2A) is a major phospho-Ser/Thr phosphatase and a key regulator of cellular signal transduction pathways. While PP2A dysfunction has been linked to human cancer and neurodegenerative disorders such as Alzheimer's disease (AD), PP2A regulation remains relatively poorly understood. It has been reported that the PP2A catalytic subunit (PP2Ac) is inactivated by a single phosphorylation at the Tyr307 residue by tyrosine kinases such as v-Src. However, multiple mass spectrometry studies have revealed the existence of other putative PP2Ac phosphorylation sites in response to activation of Src and Fyn, two major Src family kinases (SFKs). Here, using PP2Ac phosphomutants and novel phosphosite-specific PP2Ac antibodies, we show that cellular pools of PP2Ac are instead phosphorylated on both Tyr127 and Tyr284 upon Src activation, and on Tyr284 following Fyn activation. We found these phosphorylation events enhanced the interaction of PP2Ac with SFKs. In addition, we reveal SFK-mediated phosphorylation of PP2Ac at Y284 promotes dissociation of the regulatory Bα subunit, altering PP2A substrate specificity; the phosphodeficient Y127/284F and Y284F PP2Ac mutants prevented SFK-mediated phosphorylation of Tau at the CP13 (pSer202) epitope, a pathological hallmark of AD, and SFK-dependent activation of ERK, a major growth regulatory kinase upregulated in many cancers. Our findings demonstrate a novel PP2A regulatory mechanism that challenges the existing dogma on the inhibition of PP2A catalytic activity by Tyr307 phosphorylation. We propose dysregulation of SFK signaling in cancer and AD can lead to alterations in PP2A phosphorylation and subsequent deregulation of key PP2A substrates, including ERK and Tau.