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Journal of Cancer 2023Endometrial carcinoma is one of the most common female malignancies worldwide. Based on our preliminary investigation, DUSP1 was identified as a potential biomarker for...
Endometrial carcinoma is one of the most common female malignancies worldwide. Based on our preliminary investigation, DUSP1 was identified as a potential biomarker for endometrial carcinoma prognosis, but its function and mechanism remained unclear. In this study, genes highly correlated with DUSP1 in endometrial cancer were found through correlation analysis, and the promoter sequence of DUSP1 was analyzed by PROMO program. Next-generation phosphorylation mass spectrometry was used to explore new downstream target proteins and pathways of DUSP1 in endometrial carcinoma. The mRNA and protein expression levels were detected by real-time quantitative PCR, immunohistochemistry and Western blotting. The cell survival and proliferation were analyzed by CCK8 assay, cell apoptosis was analyzed by Annexin-V-APC and PI dual staining assay, and the cell invasion was analyzed by Transwell method. (1) There was a high correlation between the expression of DUSP1 and the genes involved in AP-1 complex and its co-expression network. (2) Promoter sequence analysis predicted that the members of AP-1 complex might be the upstream transcriptional regulators of DUSP1. (3) Transfection experiments proved DUSP1 can inhibit tumor growth and invasion, and promote apoptosis by regulating ERK pathway. (4) The results of phosphorylation mass spectrometry showed that overexpression of DUSP1 mainly dephosphorylated EPHA2 in endometrial carcinoma, and co-immunoprecipitation verified the protein interaction between DUSP1 and EPHA2. (5) Overexpression or knockdown of EPHA2 significantly changed the phosphorylation level of EPHA2. (6) The expression of EPHA2 protein was high in patients with more aggressive endometrial cancer. (7) Using EPHA2 inhibitor could significantly slow down the growth rate of tumor cells. (1) There exists a mutual regulation relationship between DUSP1 and AP-1 co-expression network in endometrial carcinoma. (2) It is reported for the first time that DUSP1 phosphatase acts on the ser899 site of EphA2 in endometrial carcinoma. (3) DUSP1 can inhibit tumor growth and invasion, and promote apoptosis by regulating MAPK pathway through directly dephosphorylating ERK, or by dephosphorylating EPHA2.
PubMed: 37057290
DOI: 10.7150/jca.81069 -
Molecular Oncology Mar 2024Homologous recombination (HR) repair for DNA double-strand breaks (DSBs) is critical for maintaining genome stability and conferring the resistance of tumor cells to...
Homologous recombination (HR) repair for DNA double-strand breaks (DSBs) is critical for maintaining genome stability and conferring the resistance of tumor cells to chemotherapy. Nuclear PTEN which contains both phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and protein phosphatase plays a key role in HR repair, but the underlying mechanism remains largely elusive. We find that SUMOylated PTEN promotes HR repair but represses nonhomologous end joining (NHEJ) repair by directly dephosphorylating TP53-binding protein 1 (53BP1). During DNA damage responses (DDR), tumor suppressor ARF (p14ARF) was phosphorylated and then interacted efficiently with PTEN, thus promoting PTEN SUMOylation as an atypical SUMO E3 ligase. Interestingly, SUMOylated PTEN was subsequently recruited to the chromatin at DSB sites. This was because SUMO1 that was conjugated to PTEN was recognized and bound by the SUMO-interacting motif (SIM) of breast cancer type 1 susceptibility protein (BRCA1), which has been located to the core of 53BP1 foci on chromatin during S/G2 stage. Furthermore, these chromatin-loaded PTEN directly and specifically dephosphorylated phosphothreonine-543 (pT543) of 53BP1, resulting in the dissociation of the 53BP1 complex, which facilitated DNA end resection and ongoing HR repair. SUMOylation-site-mutated PTEN mice also showed decreased DNA damage repair in vivo. Blocking the PTEN SUMOylation pathway with either a SUMOylation inhibitor or a p14ARF(2-13) peptide sensitized tumor cells to chemotherapy. Our study therefore provides a new mechanistic understanding of PTEN in HR repair and clinical intervention of chemoresistant tumors.
Topics: Animals; Mice; BRCA1 Protein; Chromatin; DNA; DNA Damage; DNA End-Joining Repair; DNA Repair; Neoplasms; Tumor Suppressor Protein p14ARF
PubMed: 38060346
DOI: 10.1002/1878-0261.13563 -
The Journal of Biological Chemistry Jan 2009Gln3, the major activator of nitrogen catabolite repression (NCR)-sensitive transcription, is often used as an assay of Tor pathway regulation in Saccharomyces...
Gln3, the major activator of nitrogen catabolite repression (NCR)-sensitive transcription, is often used as an assay of Tor pathway regulation in Saccharomyces cerevisiae. Gln3 is cytoplasmic in cells cultured with repressive nitrogen sources (Gln) and nuclear with derepressive ones (Pro) or after treating Gln-grown cells with the Tor inhibitor, rapamycin (Rap). In Raptreated or Pro-grown cells, Sit4 is posited to dephosphorylate Gln3, which then dissociates from a Gln3-Ure2 complex and enters the nucleus. However, in contrast with this view, Sit4-dependent Gln3 dephosphorylation is greater in Gln than Pro. Investigating this paradox, we show that PP2A (another Tor pathway phosphatase)-dependent Gln3 dephosphorylation is regulated oppositely to that of Sit4, being greatest in Pro- and least in Gln-grown cells. It thus parallels nuclear Gln3 localization and NCR-sensitive transcription. However, because PP2A is not required for nuclear Gln3 localization in Pro, PP2A-dependent Gln3 dephosphorylation and nuclear localization are likely parallel responses to derepressive nitrogen sources. In contrast, Rap-induced nuclear Gln3 localization absolutely requires all four PP2A components (Pph21/22, Tpd3, Cdc55, and Rts1). In pph21Delta22Delta, tpd3Delta, or cdc55Delta cells, however, Gln3 is dephosphorylated to the same level as in Rap-treated wild-type cells, indicating Rap-induced Gln3 dephosphorylation is insufficient to achieve nuclear localization. Finally, PP2A-dependent Gln3 dephosphorylation parallels conditions where Gln3 is mostly nuclear, while Sit4-dependent and Rap-induced dephosphorylation parallels those where Gln3 is mostly cytoplasmic, suggesting the effects of these phosphatases on Gln3 may occur in different cellular compartments.
Topics: Active Transport, Cell Nucleus; Cell Cycle Proteins; Cytoplasm; Gene Expression Regulation, Enzymologic; Glutamine; Mutation; Nitrogen; Phosphorylation; Protein Phosphatase 2; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sirolimus
PubMed: 19015262
DOI: 10.1074/jbc.M806162200 -
Journal of Fungi (Basel, Switzerland) Jan 2021Protein phosphorylation cascades are universal in cell signaling. While kinome diversity allows specific phosphorylation events, relatively few phosphatases...
Protein phosphorylation cascades are universal in cell signaling. While kinome diversity allows specific phosphorylation events, relatively few phosphatases dephosphorylate key signaling proteins. Fungal mitogen activated protein kinases (MAPK), in contrast to their mammalian counterparts, often show detectable basal phosphorylation levels. Dephosphorylation, therefore, could act as a signal. In , the Dothideomycete causing Southern corn leaf blight, ferulic acid (FA)-an abundant phenolic found in plant host cell walls-acts as a signal to rapidly dephosphorylate the stress-activated MAP kinase Hog1 (High Osmolarity Glycerol 1). In order to identify the protein phosphatases responsible, we constructed mutants in Hog1 phosphatases predicted from the genome by homology to yeast and other species. We found that mutants lacking PtcB, a member of the PP2C family, exhibited altered growth, sporulation, and attenuated dephosphorylation in response to FA. The loss of the dual-specificity phosphatase CDC14 led to slow growth, decreased virulence, and attenuated dephosphorylation. Mutants in two predicted tyrosine phosphatase genes PTP1 and PTP2 showed normal development and virulence. Our results suggest that a network of phosphatases modulate Hog1's dual phosphorylation levels. The mutants we constructed in this work provide a starting point to further unravel the signaling hierarchy by which exposure to FA leads to stress responses in the pathogen.
PubMed: 33530602
DOI: 10.3390/jof7020083 -
Frontiers in Immunology 2022In this study, we investigated the effects of intestinal alkaline phosphatase (IAP) in controlled intestinal inflammation and alleviated associated insulin resistance...
In this study, we investigated the effects of intestinal alkaline phosphatase (IAP) in controlled intestinal inflammation and alleviated associated insulin resistance (IR). We also explored the possible underlying molecular mechanisms, showed the preventive effect of IAP on IR , and verified the dephosphorylation of IAP for the inhibition of intestinal inflammation . Furthermore, we examined the preventive role of IAP in IR induced by a high-fat diet in mice. We found that an IAP + IAP enhancer significantly ameliorated blood glucose, insulin, low-density lipoprotein, gut barrier function, inflammatory markers, and lipopolysaccharide (LPS) in serum. IAP could dephosphorylate LPS and nucleoside triphosphate in a pH-dependent manner . Firstly, LPS is inactivated by IAP and IAP reduces LPS-induced inflammation. Secondly, adenosine, a dephosphorylated product of adenosine triphosphate, elicited anti-inflammatory effects by binding to the A receptor, which inhibits NF-κB, TNF, and PI3K-Akt signalling pathways. Hence, IAP can be used as a natural anti-inflammatory agent to reduce intestinal inflammation-induced IR.
Topics: Alkaline Phosphatase; Animals; Inflammation; Insulin Resistance; Lipopolysaccharides; Mice; Phosphatidylinositol 3-Kinases
PubMed: 35958560
DOI: 10.3389/fimmu.2022.927272 -
Biochimica Et Biophysica Acta Jul 2011Cardiac sarcoplasmic reticulum (SR) Ca(2+) ATPase (SERCA2a) promotes Ca(2+) uptake in the SR. Dephosphorylated phospholamban (PLB) inhibits SERCA2a activity. We found a...
Cardiac sarcoplasmic reticulum (SR) Ca(2+) ATPase (SERCA2a) promotes Ca(2+) uptake in the SR. Dephosphorylated phospholamban (PLB) inhibits SERCA2a activity. We found a distinct dephosphorylation of PLB at Thr(17) and Ser(16) after 20-30min of ischemia produced by coronary artery occlusion in rats. The aim of the study was to investigate how PLB is dephosphorylated in ischemia and to determine whether PLB dephosphorylation causes myocardial hypercontraction and calpain activation through Ca(2+) overload in reperfusion. Protein inhibitor-1 (I-1) specifically inhibits protein phosphatase 1 (PP1), the predominant PLB phosphatase in heart. A Ca(2+)-dependent phosphatase calcineurin may also induce PLB dephosphorylation. Ischemia for 30min induced PKC-α translocation, resulting in inactivation of I-1 through PKC-α-dependent phosphorylation at Ser(67). The PP1 activation following I-1 inactivation was thought to induce PLB dephosphorylation in ischemia. Ischemia for 30min activated calcineurin, and pre-treatment with a calcineurin inhibitor, cyclosporine A (CsA), inhibited PKC-α translocation, I-1 phosphorylation at Ser(67), and PLB dephosphorylation in ischemia. Reperfusion for 5min following 30min of ischemia induced spreading of contraction bands (CBs) and proteolysis of fodrin by calpain. Both CsA and an anti-PLB antibody that inhibits binding of PLB to SERCA2a reduced the CB area and fodrin breakdown after reperfusion. These results reveal a novel pathway via which ischemia induces calcineurin-dependent activation of PKC-α, inactivation of I-1 through PKC-α-dependent phosphorylation at Ser(67), and PP1-dependent PLB dephosphorylation. The pathway contributes to the spreading of CBs and calpain activation through Ca(2+) overload in early reperfusion.
Topics: Animals; Blotting, Western; Calcineurin; Calcium; Calcium-Binding Proteins; Ischemia; Male; Phosphorylation; Protein Kinase C-alpha; Protein Phosphatase 1; Rats; Rats, Sprague-Dawley; Reperfusion
PubMed: 21447388
DOI: 10.1016/j.bbadis.2011.03.014 -
The Journal of Biological Chemistry 2021The insulin receptor (INSR) binds insulin to promote body growth and maintain normal blood glucose levels. While it is known that steroid hormones such as estrogen and...
The insulin receptor (INSR) binds insulin to promote body growth and maintain normal blood glucose levels. While it is known that steroid hormones such as estrogen and 20-hydroxyecdysone counteract insulin function, the molecular mechanisms responsible for this attenuation remain unclear. In the present study, using the agricultural pest lepidopteran Helicoverpa armigera as a model, we proposed that the steroid hormone 20-hydroxyecdysone (20E) induces dephosphorylation of INSR to counteract insulin function. We observed high expression and phosphorylation of INSR during larval feeding stages that decreased during metamorphosis. Insulin upregulated INSR expression and phosphorylation, whereas 20E repressed INSR expression and induced INSR dephosphorylation in vivo. Protein tyrosine phosphatase 1B (PTP1B, encoded by Ptpn1) dephosphorylated INSR in vivo. PTEN (phosphatase and tensin homolog deleted on chromosome 10) was critical for 20E-induced INSR dephosphorylation by maintaining the transcription factor Forkhead box O (FoxO) in the nucleus, where FoxO promoted Ptpn1 expression and repressed Insr expression. Knockdown of Ptpn1 using RNA interference maintained INSR phosphorylation, increased 20E production, and accelerated pupation. RNA interference of Insr in larvae repressed larval growth, decreased 20E production, delayed pupation, and accumulated hemolymph glucose levels. Taken together, these results suggest that a high 20E titer counteracts the insulin pathway by dephosphorylating INSR to stop larval growth and accumulate glucose in the hemolymph.
Topics: Animals; Ecdysterone; Estrogens; Forkhead Box Protein O1; Gene Expression Regulation; Humans; Insulin; Metamorphosis, Biological; Moths; PTEN Phosphohydrolase; Phosphorylation; Protein Tyrosine Phosphatase, Non-Receptor Type 1; RNA Interference; Receptor, Insulin; Signal Transduction
PubMed: 33484713
DOI: 10.1016/j.jbc.2021.100318 -
IUBMB Life Jan 2002Protein tyrosine phosphatases (PTPs) are a large and structurally diverse family of enzymes that are found in eukaryotes, prokaryotes, viruses, and plants. PTPs catalyse... (Review)
Review
Protein tyrosine phosphatases (PTPs) are a large and structurally diverse family of enzymes that are found in eukaryotes, prokaryotes, viruses, and plants. PTPs catalyse the dephosphorylation of tyrosyl phosphorylated proteins and can either antagonise or potentiate protein tyrosine kinase signalling. PTPs regulate fundamental cellular processes and have been implicated in the etiology and pathogenesis of various human diseases. The epidermal growth factor receptor (EGFR) is a widely distributed protein tyrosine kinase that regulates both normal development and plays a role in pathological conditions such as cancer. This review discusses the structure and function of PTPs and focuses on the PTPs that have been implicated in the dephosphorylation of the EGFR and the consequent suppression of EGFR signalling.
Topics: Animals; ErbB Receptors; Humans; Mice; Mice, Knockout; Mice, Transgenic; Molecular Structure; Phosphorylation; Protein Tyrosine Phosphatases; Signal Transduction
PubMed: 12018405
DOI: 10.1080/15216540210811 -
Plant Physiology Mar 2022Plasma membrane (PM) H+-ATPase in guard cells is activated by phosphorylation of the penultimate residue, threonine (Thr), in response to blue and red light, promoting...
Plasma membrane (PM) H+-ATPase in guard cells is activated by phosphorylation of the penultimate residue, threonine (Thr), in response to blue and red light, promoting stomatal opening. Previous in vitro biochemical investigation suggested that Mg2+- and Mn2+-dependent membrane-localized type 2C protein phosphatase (PP2C)-like activity mediates the dephosphorylation of PM H+-ATPase in guard cells. PP2C clade D (PP2C.D) was later demonstrated to be involved in PM H+-ATPase dephosphorylation during auxin-induced cell expansion in Arabidopsis (Arabidopsis thaliana). However, it is unclear whether PP2C.D phosphatases are involved in PM H+-ATPase dephosphorylation in guard cells. Transient expression experiments using Arabidopsis mesophyll cell protoplasts revealed that all PP2C.D isoforms dephosphorylate the endogenous PM H+-ATPase. We further analyzed PP2C.D6/8/9, which display higher expression levels than other isoforms in guard cells, observing that pp2c.d6, pp2c.d8, and pp2c.d9 single mutants showed similar light-induced stomatal opening and phosphorylation status of PM H+-ATPase in guard cells as Col-0. In contrast, the pp2c.d6/9 double mutant displayed wider stomatal apertures and greater PM H+-ATPase phosphorylation in response to blue light, but delayed dephosphorylation of PM H+-ATPase in guard cells; the pp2c.d6/8/9 triple mutant showed similar phenotypes to those of the pp2c.d6/9 double mutant. Taken together, these results indicate that PP2C.D6 and PP2C.D9 redundantly mediate PM H+-ATPase dephosphorylation in guard cells. Curiously, unlike auxin-induced cell expansion in seedlings, auxin had no effect on the phosphorylation status of PM H+-ATPase in guard cells.
Topics: Arabidopsis; Arabidopsis Proteins; Cell Membrane; Light; Phosphoprotein Phosphatases; Phosphorylation; Protein Phosphatase 2C; Proton-Translocating ATPases
PubMed: 34894269
DOI: 10.1093/plphys/kiab571 -
The Journal of Biological Chemistry Jun 2009Neurodegenerative tauopathies, including Alzheimer disease, are characterized by abnormal hyperphosphorylation of the microtubule-associated protein Tau. One group of...
Neurodegenerative tauopathies, including Alzheimer disease, are characterized by abnormal hyperphosphorylation of the microtubule-associated protein Tau. One group of tauopathies, known as frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), is directly associated with mutations of the gene tau. However, it is unknown why mutant Tau is highly phosphorylated in the patient brain. In contrast to in vivo high phosphorylation, FTDP-17 Tau is phosphorylated less than wild-type Tau in vitro. Because phosphorylation is a balance between kinase and phosphatase activities, we investigated dephosphorylation of mutant Tau proteins, P301L and R406W. Tau phosphorylated by Cdk5-p25 was dephosphorylated by protein phosphatases in rat brain extracts. Compared with wild-type Tau, R406W was dephosphorylated faster and P301L slower. The two-dimensional phosphopeptide map analysis suggested that faster dephosphorylation of R406W was due to a lack of phosphorylation at Ser-404, which is relatively resistant to dephosphorylation. We studied the effect of the peptidyl-prolyl isomerase Pin1 or microtubule binding on dephosphorylation of wild-type Tau, P301L, and R406W in vitro. Pin1 catalyzes the cis/trans isomerization of phospho-Ser/Thr-Pro sequences in a subset of proteins. Dephosphorylation of wild-type Tau was reduced in brain extracts of Pin1-knockout mice, and this reduction was not observed with P301L and R406W. On the other hand, binding to microtubules almost abolished dephosphorylation of wild-type and mutant Tau proteins. These results demonstrate that mutation of Tau and its association with microtubules may change the conformation of Tau, thereby suppressing dephosphorylation and potentially contributing to the etiology of tauopathies.
Topics: Amino Acid Substitution; Animals; Base Sequence; Brain; DNA Primers; Humans; In Vitro Techniques; Kinetics; Mice; Mice, Knockout; Microtubules; Models, Neurological; Mutation; NIMA-Interacting Peptidylprolyl Isomerase; Peptide Mapping; Peptidylprolyl Isomerase; Phosphorylation; Protein Conformation; Rats; Recombinant Proteins; Tauopathies; tau Proteins
PubMed: 19401603
DOI: 10.1074/jbc.M109.003277