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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 -
The Journal of Cell Biology Dec 2018As a dividing cell exits mitosis and daughter cells enter interphase, many proteins must be dephosphorylated. The protein phosphatase 2A (PP2A) with its B55 regulatory...
As a dividing cell exits mitosis and daughter cells enter interphase, many proteins must be dephosphorylated. The protein phosphatase 2A (PP2A) with its B55 regulatory subunit plays a crucial role in this transition, but the identity of its substrates and how their dephosphorylation promotes mitotic exit are largely unknown. We conducted a maternal-effect screen in to identify genes that function with PP2A-B55/Tws in the cell cycle. We found that eggs that receive reduced levels of Tws and of components of the nuclear envelope (NE) often fail development, concomitant with NE defects following meiosis and in syncytial mitoses. Our mechanistic studies using cells indicate that PP2A-Tws promotes nuclear envelope reformation (NER) during mitotic exit by dephosphorylating BAF and suggests that PP2A-Tws targets additional NE components, including Lamin and Nup107. This work establishes as a powerful model to further dissect the molecular mechanisms of NER and suggests additional roles of PP2A-Tws in the completion of meiosis and mitosis.
Topics: Animals; Aquaporins; DNA-Binding Proteins; Drosophila Proteins; Drosophila melanogaster; Lamins; Mitosis; Models, Biological; Nuclear Envelope; Nuclear Proteins; Phosphoprotein Phosphatases
PubMed: 30309980
DOI: 10.1083/jcb.201804018 -
The Journal of Biological Chemistry Apr 2005Cofilin, an essential regulator of actin filament dynamics, is inactivated by phosphorylation at Ser-3 and reactivated by dephosphorylation. Although cofilin undergoes...
Cofilin, an essential regulator of actin filament dynamics, is inactivated by phosphorylation at Ser-3 and reactivated by dephosphorylation. Although cofilin undergoes dephosphorylation in response to extracellular stimuli that elevate intracellular Ca2+ concentrations, signaling mechanisms mediating Ca2+-induced cofilin dephosphorylation have remained unknown. We investigated the role of Slingshot (SSH) 1L, a member of a SSH family of protein phosphatases, in mediating Ca2+-induced cofilin dephosphorylation. The Ca2+ ionophore A23187 and Ca2+-mobilizing agonists, ATP and histamine, induced SSH1L activation and cofilin dephosphorylation in cultured cells. A23187- or histamine-induced SSH1L activation and cofilin dephosphorylation were blocked by calcineurin inhibitors or a dominant-negative form of calcineurin, indicating that calcineurin mediates Ca2+-induced SSH1L activation and cofilin dephosphorylation. Importantly, knockdown of SSH1L expression by RNA interference abolished A23187- or calcineurin-induced cofilin dephosphorylation. Furthermore, calcineurin dephosphorylated SSH1L and increased the cofilin-phosphatase activity of SSH1L in cell-free assays. Based on these findings, we suggest that Ca2+-induced cofilin dephosphorylation is mediated by calcineurin-dependent activation of SSH1L.
Topics: Actin Depolymerizing Factors; Adenosine Triphosphate; Calcimycin; Calcineurin; Calcium; Cell Line; Cell-Free System; Genes, Dominant; HeLa Cells; Histamine; Humans; Ionophores; Microfilament Proteins; Phosphoprotein Phosphatases; Phosphorylation; Plasmids; RNA Interference; RNA, Small Interfering; Recombinant Proteins; Signal Transduction; Time Factors
PubMed: 15671020
DOI: 10.1074/jbc.M411494200 -
American Journal of Cancer Research 2022Transforming Growth Factor beta (TGF-β) is a multifunctional cytokine that regulates cell proliferation, differentiation, and apoptosis. Dysregulation of the TGF-β...
Transforming Growth Factor beta (TGF-β) is a multifunctional cytokine that regulates cell proliferation, differentiation, and apoptosis. Dysregulation of the TGF-β signaling is one of the major mechanisms underlying tumor progression. We have previously reported that anaplastic lymphoma kinase (ALK) phosphorylates Smad4 at Tyr95, which compromises the DNA-binding ability of Smad4 and thus renders ALK-positive cancer cells resistant to TGF-β tumor-suppressive action. In this study, we demonstrated that tyrosine phosphatase PTPN2 positively regulated TGF-β signaling through dephosphorylating Smad4 at the Tyr95 site. Both and cell-based assays revealed that PTPN2 bound to and dephosphorylated Smad4, thereby preserving the DNA-binding ability of Smad4. Furthermore, overexpression of PTPN2 restored TGF-β transcriptional and growth inhibitory responses in ALK-positive cancer cells. Consistently, Spermidine, an activator of PTPN2, also promoted TGF-β-induced gene expression, apoptosis, and anti-proliferation effect. Taken together, we revealed that PTPN2 functioned as a tumor suppressor to antagonize the inhibitory effect of tyrosine phosphorylation of Smad4 and to ensure the proper TGF-β growth inhibitory signaling in cancer cells.
PubMed: 36628288
DOI: No ID Found -
PloS One Apr 2011The cyclin-dependent kinase CDK9/cyclin T1 induces HIV-1 transcription by phosphorylating the carboxyterminal domain (CTD) of RNA polymerase II (RNAPII). CDK9 activity...
The cyclin-dependent kinase CDK9/cyclin T1 induces HIV-1 transcription by phosphorylating the carboxyterminal domain (CTD) of RNA polymerase II (RNAPII). CDK9 activity is regulated by protein phosphatase-1 (PP1) which was previously shown to dephosphorylate CDK9 Thr186. Here, we analyzed the effect of PP1 on RNAPII phosphorylation and CDK9 activity. The selective inhibition of PP1 by okadaic acid and by NIPP1 inhibited phosphorylation of RNAPII CTD in vitro and in vivo. Expression of the central domain of NIPP1 in cultured cells inhibited the enzymatic activity of CDK9 suggesting its activation by PP1. Comparison of dephosphorylation of CDK9 phosphorylated by ((32)P) in vivo and dephosphorylation of CDK9's Thr186 analyzed by Thr186 phospho-specific antibodies, indicated that a residue other than Thr186 might be dephosphorylated by PP1. Analysis of dephosphorylation of phosphorylated peptides derived from CDK9's T-loop suggested that PP1 dephosphorylates CDK9 Ser175. In cultured cells, CDK9 was found to be phosphorylated on Ser175 as determined by combination of Hunter 2D peptide mapping and LC-MS analysis. CDK9 S175A mutant was active and S175D--inactive, and dephosphorylation of CDK9's Ser175 upregulated HIV-1 transcription in PP1-dependent manner. Collectively, our results point to CDK9 Ser175 as novel PP1-regulatory site which dephosphorylation upregulates CDK9 activity and contribute to the activation of HIV-1 transcription.
Topics: Amino Acid Sequence; Cell Line; Cyclin-Dependent Kinase 9; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; HIV-1; Humans; Mass Spectrometry; Molecular Sequence Data; Mutation; Peptide Mapping; Phosphorylation; Protein Phosphatase 1; Serine; Transcription, Genetic
PubMed: 21533037
DOI: 10.1371/journal.pone.0018985 -
Cell Adhesion & Migration 2015Altered phosphorylation status of the C-terminal Thr residues of Ezrin/Radixin/Moesin (ERM) is often linked to cell shape change. To determine the role of phophorylated...
Altered phosphorylation status of the C-terminal Thr residues of Ezrin/Radixin/Moesin (ERM) is often linked to cell shape change. To determine the role of phophorylated ERM, we modified phosphorylation status of ERM and investigated changes in cell adhesion and morphology. Treatment with Calyculin-A (Cal-A), a protein phosphatase inhibitor, dramatically augmented phosphorylated ERM (phospho-ERM). Cal-A-treatment or expression of phospho-mimetic Moesin mutant (Moesin-TD) induced cell rounding in adherent cells. Moreover, reattachment of detached cells to substrate was inhibited by either treatment. Phospho-ERM, Moesin-TD and actin cytoskeleton were observed at the plasma membrane of such round cells. Augmented cell surface rigidity was also observed in both cases. Meanwhile, non-adherent KG-1 cells were rather rich in phospho-ERM. Treatment with Staurosporine, a protein kinase inhibitor that dephosphorylates phospho-ERM, up-regulated the integrin-dependent adhesion of KG-1 cells to substrate. These findings strongly suggest the followings: (1) Phospho-ERM inhibit cell adhesion, and therefore, dephosphorylation of ERM proteins is essential for cell adhesion. (2) Phospho-ERM induce formation and/or maintenance of spherical cell shape. (3) ERM are constitutively both phosphorylated and dephosphorylated in cultured adherent and non-adherent cells.
Topics: Cell Adhesion; Cell Line; Cell Membrane; Cell Shape; Cytoskeletal Proteins; Cytoskeleton; Humans; Marine Toxins; Membrane Proteins; Microfilament Proteins; Oxazoles; Phosphorylation
PubMed: 26555866
DOI: 10.1080/19336918.2015.1113366 -
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 -
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 -
Cell Cycle (Georgetown, Tex.) Feb 2020The phosphoinositide-3-kinase like kinases (PIKK) such as ATM and ATR play a key role in initiating the cellular DNA damage response (DDR). One key ATM target is the...
The phosphoinositide-3-kinase like kinases (PIKK) such as ATM and ATR play a key role in initiating the cellular DNA damage response (DDR). One key ATM target is the cyclin-dependent kinase inhibitor p27 that promotes G1 arrest. ATM activates p27-induced arrest in part through phosphorylation of p27 at Serine 140. Here we show that this site is dephosphorylated by the type 2C serine/threonine phosphatase, WIP1 (Wildtype p53-Induced Phosphatase-1), encoded by the gene. WIP1 has been shown to dephosphorylate numerous ATM target sites in DDR proteins, and its overexpression and/or mutation has often been associated with oncogenesis. We demonstrate that wildtype, but not phosphatase-dead WIP1, efficiently dephosphorylates p27 Ser140 both in vitro and in cells and that this dephosphorylation is sensitive to the WIP1-specific inhibitor GSK 2830371. Increased expression of wildtype WIP1 reduces stability of p27 while increased expression of similar amounts of phosphatase-dead WIP1 has no effect on p27 protein stability. Overexpression of wildtype p27 reduces cell proliferation and colony forming capability relative to the S140A (constitutively non-phosphorylated) form of p27. Thus, WIP1 plays a significant role in homeostatic modulation of p27 activity following activation by ATM.
Topics: Ataxia Telangiectasia Mutated Proteins; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p27; HEK293 Cells; Humans; MCF-7 Cells; Mutation; Phosphopeptides; Phosphorylation; Protein Phosphatase 2C; Protein Stability; Reproducibility of Results; Serine; Tumor Stem Cell Assay
PubMed: 31959038
DOI: 10.1080/15384101.2020.1717025 -
Nature Communications Aug 2019Very little is known about how Ser/Thr protein phosphatases specifically recruit and dephosphorylate substrates. Here, we identify how the Na/H-exchanger 1 (NHE1), a key...
Very little is known about how Ser/Thr protein phosphatases specifically recruit and dephosphorylate substrates. Here, we identify how the Na/H-exchanger 1 (NHE1), a key regulator of cellular pH homeostasis, is regulated by the Ser/Thr phosphatase calcineurin (CN). NHE1 activity is increased by phosphorylation of NHE1 residue T779, which is specifically dephosphorylated by CN. While it is known that Ser/Thr protein phosphatases prefer pThr over pSer, we show that this preference is not key to this exquisite CN selectivity. Rather a combination of molecular mechanisms, including recognition motifs, dynamic charge-charge interactions and a substrate interaction pocket lead to selective dephosphorylation of pT779. Our data identify T779 as a site regulating NHE1-mediated cellular acid extrusion and provides a molecular understanding of NHE1 substrate selection by CN, specifically, and how phosphatases recruit specific substrates, generally.
Topics: Acids; Calcineurin; Cell Membrane; Crystallography, X-Ray; Homeostasis; Humans; Hydrogen-Ion Concentration; MCF-7 Cells; Molecular Docking Simulation; Nuclear Magnetic Resonance, Biomolecular; Phosphorylation; Protein Binding; Recombinant Proteins; Sodium-Hydrogen Exchanger 1; Substrate Specificity
PubMed: 31375679
DOI: 10.1038/s41467-019-11391-7