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Kidney International Feb 2022Kidney tubular epithelial cells are high energy-consuming epithelial cells that depend mainly on fatty acid oxidation for an energy supply. AMP-activated protein kinase...
Kidney tubular epithelial cells are high energy-consuming epithelial cells that depend mainly on fatty acid oxidation for an energy supply. AMP-activated protein kinase (AMPK) is a key regulator of energy production in most cells, but the function of AMPK in tubular epithelial cells in acute kidney disease is unclear. Here, we found a rapid decrease in Thr172-AMPKα phosphorylation after ischemia/reperfusion in both in vivo and in vitro models. Mice with kidney tubular epithelial cell-specific AMPKα deletion exhibited exacerbated kidney impairment and apoptosis of tubular epithelial cells after ischemia/reperfusion. AMPKα deficiency was accompanied by the accumulation of lipid droplets in the kidney tubules and the elevation of ceramides and free fatty acid levels following ischemia/reperfusion injury. Mechanistically, ischemia/reperfusion triggered ceramide production and activated protein phosphatase PP2A, which dephosphorylated Thr172-AMPKα. Decreased AMPK activity repressed serine/threonine kinase ULK1-mediated autophagy and impeded clearance of the dysfunctional mitochondria. Targeting the PP2A-AMPK axis by the allosteric AMPK activator C24 restored fatty acid oxidation and reduced tubular cell apoptosis during ischemia/reperfusion-induced injury, by antagonizing PP2A dephosphorylation and promoting the mitophagy process. Thus, our study reveals that AMPKα plays an important role in protecting against tubular epithelial cell injury in ischemia/reperfusion-induced acute kidney injury. Hence, activation of AMPK could be a potential therapeutic strategy for acute kidney injury treatment.
Topics: AMP-Activated Protein Kinases; Acute Kidney Injury; Animals; Apoptosis; Ischemia; Kidney; Mice; Mitochondria; Reperfusion; Reperfusion Injury
PubMed: 34774556
DOI: 10.1016/j.kint.2021.10.028 -
Cell Reports Aug 2023Mitochondrial morphology is regulated by the post-translational modifications of the dynamin family GTPase proteins including mitofusin 1 (MFN1), MFN2, and...
Mitochondrial morphology is regulated by the post-translational modifications of the dynamin family GTPase proteins including mitofusin 1 (MFN1), MFN2, and dynamin-related protein 1 (DRP1). Mitochondrial phosphatase phosphoglycerate mutase 5 (PGAM5) is emerging as a regulator of these post-translational modifications; however, its precise role in the regulation of mitochondrial morphology is unknown. We show that PGAM5 interacts with MFN2 and DRP1 in a stress-sensitive manner. PGAM5 regulates MFN2 phosphorylation and consequently protects it from ubiquitination and degradation. Further, phosphorylation and dephosphorylation modification of MFN2 regulates its fusion ability. Phosphorylation enhances fission and degradation, whereas dephosphorylation enhances fusion. PGAM5 dephosphorylates MFN2 to promote mitochondrial network formation. Further, using a Drosophila genetic model, we demonstrate that the MFN2 homolog Marf and dPGAM5 are in the same biological pathway. Our results identify MFN2 dephosphorylation as a regulator of mitochondrial fusion and PGAM5 as an MFN2 phosphatase.
Topics: GTP Phosphohydrolases; Phosphoric Monoester Hydrolases; Phosphoglycerate Mutase; Mitochondrial Dynamics; Mitochondrial Proteins; Dynamins
PubMed: 37498743
DOI: 10.1016/j.celrep.2023.112895 -
Cell Research Mar 2023Emerging evidence demonstrates that some metabolic enzymes that phosphorylate soluble metabolites can also phosphorylate a variety of protein substrates as protein...
Emerging evidence demonstrates that some metabolic enzymes that phosphorylate soluble metabolites can also phosphorylate a variety of protein substrates as protein kinases to regulate cell cycle, apoptosis and many other fundamental cellular processes. However, whether a metabolic enzyme dephosphorylates protein as a protein phosphatase remains unknown. Here we reveal the gluconeogenic enzyme fructose 1,6-biphosphatase 1 (FBP1) that catalyzes the hydrolysis of fructose 1,6-bisphosphate (F-1,6-BP) to fructose 6-phosphate (F-6-P) as a protein phosphatase by performing a high-throughput screening of metabolic phosphatases with molecular docking followed by molecular dynamics (MD) simulations. Moreover, we identify IκBα as the substrate of FBP1-mediated dephosphorylation by performing phosphoproteomic analysis. Mechanistically, FBP1 directly interacts with and dephosphorylates the serine (S) 32/36 of IκBα upon TNFα stimulation, thereby inhibiting NF-κB activation. MD simulations indicate that the catalytic mechanism of FBP1-mediated IκBα dephosphorylation is similar to F-1,6-BP dephosphorylation, except for higher energetic barriers for IκBα dephosphorylation. Functionally, FBP1-dependent NF-κB inactivation suppresses colorectal tumorigenesis by sensitizing tumor cells to inflammatory stresses and preventing the mobilization of myeloid-derived suppressor cells. Our finding reveals a previously unrecognized role of FBP1 as a protein phosphatase and establishes the critical role of FBP1-mediated IκBα dephosphorylation in colorectal tumorigenesis.
Topics: Humans; Fructose-Bisphosphatase; NF-kappa B; NF-KappaB Inhibitor alpha; Molecular Docking Simulation; Carcinogenesis; Phosphoric Monoester Hydrolases; Cell Transformation, Neoplastic; Fructose; Colorectal Neoplasms
PubMed: 36646759
DOI: 10.1038/s41422-022-00773-0 -
Molecular Cell Nov 2019The PTEN tumor suppressor is frequently mutated or deleted in cancer and regulates glucose metabolism through the PI3K-AKT pathway. However, whether PTEN directly...
The PTEN tumor suppressor is frequently mutated or deleted in cancer and regulates glucose metabolism through the PI3K-AKT pathway. However, whether PTEN directly regulates glycolysis in tumor cells is unclear. We demonstrate here that PTEN directly interacts with phosphoglycerate kinase 1 (PGK1). PGK1 functions not only as a glycolytic enzyme but also as a protein kinase intermolecularly autophosphorylating itself at Y324 for activation. The protein phosphatase activity of PTEN dephosphorylates and inhibits autophosphorylated PGK1, thereby inhibiting glycolysis, ATP production, and brain tumor cell proliferation. In addition, knockin expression of a PGK1 Y324F mutant inhibits brain tumor formation. Analyses of human glioblastoma specimens reveals that PGK1 Y324 phosphorylation levels inversely correlate with PTEN expression status and are positively associated with poor prognosis in glioblastoma patients. This work highlights the instrumental role of PGK1 autophosphorylation in its activation and PTEN protein phosphatase activity in governing glycolysis and tumorigenesis.
Topics: Adenosine Triphosphate; Animals; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Female; Glioblastoma; Glucose; Glycolysis; HEK293 Cells; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; PTEN Phosphohydrolase; Phosphoglycerate Kinase; Phosphorylation; Prognosis; Signal Transduction; Time Factors; Tumor Burden; Tyrosine
PubMed: 31492635
DOI: 10.1016/j.molcel.2019.08.006 -
Cell Death & Disease Jun 2022Ferroptosis is a newly identified form of regulated cell death (RCD) characterized by the iron-dependent lipid reactive oxygen species (ROS) accumulation, but its...
Ferroptosis is a newly identified form of regulated cell death (RCD) characterized by the iron-dependent lipid reactive oxygen species (ROS) accumulation, but its mechanism in gliomas remains elusive. Acyl-coenzyme A (CoA) synthetase long-chain family member 4 (Acsl4), a pivotal enzyme in the regulation of lipid biosynthesis, benefits the initiation of ferroptosis, but its role in gliomas needs further clarification. Erastin, a classic inducer of ferroptosis, has recently been found to regulate lipid peroxidation by regulating Acsl4 other than glutathione peroxidase 4 (GPX4) in ferroptosis. In this study, we demonstrated that heat shock protein 90 (Hsp90) and dynamin-related protein 1 (Drp1) actively regulated and stabilized Acsl4 expression in erastin-induced ferroptosis in gliomas. Hsp90 overexpression and calcineurin (CN)-mediated Drp1 dephosphorylation at serine 637 (Ser637) promoted ferroptosis by altering mitochondrial morphology and increasing Acsl4-mediated lipid peroxidation. Importantly, promotion of the Hsp90-Acsl4 pathway augmented anticancer activity of erastin in vitro and in vivo. Our discovery reveals a novel and efficient approach to ferroptosis-mediated glioma therapy.
Topics: Coenzyme A Ligases; Dynamins; Ferroptosis; Glioma; Humans; Lipids; Serine
PubMed: 35697672
DOI: 10.1038/s41419-022-04997-1 -
Cell Discovery Mar 2023Metabolic reprogramming is a hallmark of cancer. However, it is not well known how metabolism affects cancer progression. We identified that metabolic enzyme acyl-CoA...
Metabolic reprogramming is a hallmark of cancer. However, it is not well known how metabolism affects cancer progression. We identified that metabolic enzyme acyl-CoA oxidase 1 (ACOX1) suppresses colorectal cancer (CRC) progression by regulating palmitic acid (PA) reprogramming. ACOX1 is highly downregulated in CRC, which predicts poor clinical outcome in CRC patients. Functionally, ACOX1 depletion promotes CRC cell proliferation in vitro and colorectal tumorigenesis in mouse models, whereas ACOX1 overexpression inhibits patient-derived xenograft growth. Mechanistically, DUSP14 dephosphorylates ACOX1 at serine 26, promoting its polyubiquitination and proteasomal degradation, thereby leading to an increase of the ACOX1 substrate PA. Accumulated PA promotes β-catenin cysteine 466 palmitoylation, which inhibits CK1- and GSK3-directed phosphorylation of β-catenin and subsequent β-Trcp-mediated proteasomal degradation. In return, stabilized β-catenin directly represses ACOX1 transcription and indirectly activates DUSP14 transcription by upregulating c-Myc, a typical target of β-catenin. Finally, we confirmed that the DUSP14-ACOX1-PA-β-catenin axis is dysregulated in clinical CRC samples. Together, these results identify ACOX1 as a tumor suppressor, the downregulation of which increases PA-mediated β-catenin palmitoylation and stabilization and hyperactivates β-catenin signaling thus promoting CRC progression. Particularly, targeting β-catenin palmitoylation by 2-bromopalmitate (2-BP) can efficiently inhibit β-catenin-dependent tumor growth in vivo, and pharmacological inhibition of DUSP14-ACOX1-β-catenin axis by Nu-7441 reduced the viability of CRC cells. Our results reveal an unexpected role of PA reprogramming induced by dephosphorylation of ACOX1 in activating β-catenin signaling and promoting cancer progression, and propose the inhibition of the dephosphorylation of ACOX1 by DUSP14 or β-catenin palmitoylation as a viable option for CRC treatment.
PubMed: 36878899
DOI: 10.1038/s41421-022-00515-x -
Cell Death and Differentiation Sep 2023Impaired transcription factor EB (TFEB) function and deficient autophagy activity have been shown to aggravate intervertebral disc (IVD) degeneration (IDD), yet the...
Impaired transcription factor EB (TFEB) function and deficient autophagy activity have been shown to aggravate intervertebral disc (IVD) degeneration (IDD), yet the underlying mechanisms remain less clear. Protein posttranslational modifications (PTMs) are critical for determining TFEB trafficking and transcriptional activity. Here, we demonstrate that TFEB activity is controlled by protein methylation in degenerated nucleus pulposus cells (NPCs), even though TFEB itself is incapable of undergoing methylation. Specifically, protein phosphatase 1 catalytic subunit alpha (PPP1CA), newly identified to dephosphorylate TFEB, contains a K141 mono-methylated site. In degenerated NPCs, increased K141-methylation of PPP1CA disrupts its interaction with TEFB and subsequently blocks TEFB dephosphorylation and nuclear translocation, which eventually leads to autophagy deficiency and NPC senescence. In addition, we found that the PPP1CA-mediated targeting of TFEB is facilitated by the protein phosphatase 1 regulatory subunit 9B (PPP1R9B), which binds with PPP1CA and is also manipulated by K141 methylation. Further proteomic analysis revealed that the protein lysine methyltransferase suppressor of variegation 3-9 homologue 2 (SUV39H2) is responsible for the K141 mono-methylation of PPP1CA. Targeting SUV39H2 effectively mitigates NPC senescence and IDD progression, providing a potential therapeutic strategy for IDD intervention.
Topics: Humans; Methylation; Lysine; Intervertebral Disc Degeneration; Protein Phosphatase 1; Proteomics; Autophagy; Histone-Lysine N-Methyltransferase; Protein Processing, Post-Translational; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors
PubMed: 37605006
DOI: 10.1038/s41418-023-01210-4 -
The Plant Cell Sep 2023Catalase (CAT) is often phosphorylated and activated by protein kinases to maintain hydrogen peroxide (H2O2) homeostasis and protect cells against stresses, but whether...
Catalase (CAT) is often phosphorylated and activated by protein kinases to maintain hydrogen peroxide (H2O2) homeostasis and protect cells against stresses, but whether and how CAT is switched off by protein phosphatases remains inconclusive. Here, we identified a manganese (Mn2+)-dependent protein phosphatase, which we named PHOSPHATASE OF CATALASE 1 (PC1), from rice (Oryza sativa L.) that negatively regulates salt and oxidative stress tolerance. PC1 specifically dephosphorylates CatC at Ser-9 to inhibit its tetramerization and thus activity in the peroxisome. PC1 overexpressing lines exhibited hypersensitivity to salt and oxidative stresses with a lower phospho-serine level of CATs. Phosphatase activity and seminal root growth assays indicated that PC1 promotes growth and plays a vital role during the transition from salt stress to normal growth conditions. Our findings demonstrate that PC1 acts as a molecular switch to dephosphorylate and deactivate CatC and negatively regulate H2O2 homeostasis and salt tolerance in rice. Moreover, knockout of PC1 not only improved H2O2-scavenging capacity and salt tolerance but also limited rice grain yield loss under salt stress conditions. Together, these results shed light on the mechanisms that switch off CAT and provide a strategy for breeding highly salt-tolerant rice.
Topics: Catalase; Oryza; Hydrogen Peroxide; Protein Phosphatase 1; Salt Tolerance; Homeostasis; Plant Proteins
PubMed: 37325884
DOI: 10.1093/plcell/koad167 -
Cell Death & Disease Nov 2021Cervical cancer is the leading cause of cancer-related deaths in women, and treatment for cervical cancer is very limited. Emerging evidence suggests that targeting...
Cervical cancer is the leading cause of cancer-related deaths in women, and treatment for cervical cancer is very limited. Emerging evidence suggests that targeting ferroptosis is a promising way to treat cancer. Here, we investigated the role of ferroptosis in cervical cancer, with a focus on the Cdc25A/PKM2/ErbB2 axis. Cervical cancer cells were treated with sorafenib to induce ferroptosis. Cellular MDA/ROS/GSH/iron detection assays were used to measure ferroptosis. MTT assays were performed to assess cell viability. qRT-PCR, western blot, and immunostaining assays were performed to measure the levels of proteins. Autophagy was monitored by fluorescence microscopy. Nuclear and cytosolic fractions were isolated to examine the location of PKM2 modifications. Co-IP experiments were conducted to determine the Cdc25A/PKM2 interaction. ChIP assays were performed to measure the binding affinity between H3K9Ac and the ErbB3 promoter, and a dual luciferase assay was performed to examine the transcriptional activity of ErbB2. A nude mouse xenograft model was used to examine the effects of the Cdc25A/ErbB2 axis on tumour growth in vivo. Cdc25A was elevated in human cervical cancer tissues but was reduced during sorafenib-induced ferroptosis of cervical cancer cells. Overexpression of Cdc25A inhibited sorafenib-induced ferroptosis by dephosphorylating nuclear PKM2 and suppressing autophagy. Cdc25A regulated autophagy-induced ferroptosis by increasing ErbB2 levels via the PKM2-pH3T11-H3K9Ac pathway. Cdc25A increased the resistance of cervical cancer to sorafenib, while knockdown of ErbB2 blocked these effects. Cdc25A suppressed autophagy-dependent ferroptosis in cervical cancer cells by upregulating ErbB2 levels through the dephosphorylation of PKM2. These studies revealed that Cdc25A/PKM2/ErbB2 pathway-regulated ferroptosis could serve as a therapeutic target in cervical cancer.
Topics: Animals; Autophagy; Carrier Proteins; Cell Line, Tumor; Female; Ferroptosis; Gene Expression Regulation, Neoplastic; Humans; Male; Membrane Proteins; Mice, Nude; Phosphorylation; RNA, Messenger; Receptor, ErbB-2; Signal Transduction; Sorafenib; Thyroid Hormones; Up-Regulation; Uterine Cervical Neoplasms; cdc25 Phosphatases; Thyroid Hormone-Binding Proteins; Mice
PubMed: 34743185
DOI: 10.1038/s41419-021-04342-y -
Theranostics 2023As a key endogenous negative regulator of ferroptosis, glutathione peroxidase 4 (GPX4) can regulate its antioxidant function through multiple post-translational...
Protein phosphatase 2A-B55β mediated mitochondrial p-GPX4 dephosphorylation promoted sorafenib-induced ferroptosis in hepatocellular carcinoma via regulating p53 retrograde signaling.
As a key endogenous negative regulator of ferroptosis, glutathione peroxidase 4 (GPX4) can regulate its antioxidant function through multiple post-translational modification pathways. However, the effects of the phosphorylation/dephosphorylation status of GPX4 on the regulation of inducible ferroptosis in hepatocellular carcinoma (HCC) remain unclear. To investigate the effects and molecular mechanism of GPX4 phosphorylation/dephosphorylation modification on ferroptosis in HCC cells. Sorafenib (Sora) was used to establish the ferroptosis model in HCC cells . Using the site-directed mutagenesis method, we generated the mimic GPX4 phosphorylation or dephosphorylation HCC cell lines at specific serine sites of GPX4. The effects of GPX4 phosphorylation/dephosphorylation modification on ferroptosis in HCC cells were examined. The interrelationships among GPX4, p53, and protein phosphatase 2A-B55β subunit (PP2A-B55β) were also explored. To explore the synergistic anti-tumor effects of PP2A activation on Sora-administered HCC, we established PP2A-B55β overexpression xenograft tumors in a nude mice model . In the Sora-induced ferroptosis model of HCC , decreased levels of cytoplasmic and mitochondrial GPX4, mitochondrial dysfunction, and enhanced p53 retrograde signaling occurred under Sora treatment. Further, we found that mitochondrial p53 retrograded remarkably into the nucleus and aggravated Sora-induced ferroptosis. The phosphorylation status of GPX4 at the serine 2 site (GPX4) revealed that mitochondrial p-GPX4 dephosphorylation was positively associated with ferroptosis, and the mechanism might be related to mitochondrial p53 retrograding into the nucleus. In HCC cells overexpressing PP2A-B55β, it was found that PP2A-B55β directly interacted with mitochondrial GPX4 and promoted Sora-induced ferroptosis in HCC. Further, PP2A-B55β reduced the interaction between mitochondrial GPX4 and p53, leading to mitochondrial p53 retrograding into the nucleus. Moreover, it was confirmed that PP2A-B55β enhanced the ferroptosis-mediated tumor growth inhibition and mitochondrial p53 retrograde signaling in the Sora-treated HCC xenograft tumors. Our data uncovered that the PP2A-B55β/p-GPX4/p53 axis was a novel regulatory pathway of Sora-induced ferroptosis. Mitochondrial p-GPX4 dephosphorylation triggered ferroptosis via inducing mitochondrial p53 retrograding into the nucleus, and PP2A-B55β was an upstream signal modulator responsible for mitochondrial p-GPX4 dephosphorylation. Our findings might serve as a potential theranostic strategy to enhance the efficacy of Sora in HCC treatment through the targeted intervention of p-GPX4 dephosphorylation via PP2A-B55β activation.
Topics: Animals; Humans; Mice; Carcinoma, Hepatocellular; Cell Nucleus; Down-Regulation; Drug Resistance, Neoplasm; Ferroptosis; Heterografts; Liver Neoplasms; Mice, Inbred BALB C; Mice, Nude; Mitochondria; Neoplasm Transplantation; Phospholipid Hydroperoxide Glutathione Peroxidase; Phosphorylation; Signal Transduction; Sorafenib; Protein Phosphatase 2
PubMed: 37554285
DOI: 10.7150/thno.82132