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The Journal of Biological Chemistry 2021Histidine phosphorylation is a posttranslational modification that alters protein function and also serves as an intermediate of phosphoryl transfer. Although...
Histidine phosphorylation is a posttranslational modification that alters protein function and also serves as an intermediate of phosphoryl transfer. Although phosphohistidine is relatively unstable, enzymatic dephosphorylation of this residue is apparently needed in some contexts, since both prokaryotic and eukaryotic phosphohistidine phosphatases have been reported. Here we identify the mechanism by which a bacterial phosphohistidine phosphatase dephosphorylates the nitrogen-related phosphotransferase system, a broadly conserved bacterial pathway that controls diverse metabolic processes. We show that the phosphatase SixA dephosphorylates the phosphocarrier protein NPr and that the reaction proceeds through phosphoryl transfer from a histidine on NPr to a histidine on SixA. In addition, we show that Escherichia coli lacking SixA are outcompeted by wild-type E. coli in the context of commensal colonization of the mouse intestine. Notably, this colonization defect requires NPr and is distinct from a previously identified in vitro growth defect associated with dysregulation of the nitrogen-related phosphotransferase system. The widespread conservation of SixA, and its coincidence with the phosphotransferase system studied here, suggests that this dephosphorylation mechanism may be conserved in other bacteria.
Topics: Bacterial Proteins; Escherichia coli; Histidine; Phosphoric Monoester Hydrolases; Phosphorylation; Signal Transduction
PubMed: 33199374
DOI: 10.1074/jbc.RA120.015121 -
The Journal of Biological Chemistry Mar 1995Microtubule-associated protein tau is abnormally hyperphosphorylated in the brain of patients with Alzheimer disease and in this form is the major protein subunit of the...
Microtubule-associated protein tau is abnormally hyperphosphorylated in the brain of patients with Alzheimer disease and in this form is the major protein subunit of the paired helical filaments (PHF), the most prominent lesion of the disease. In this study the dephosphorylation of sparingly soluble PHF, PHF II-tau by brain protein phosphatase (PP)-2A1 and PP-2B, and the resulting biochemical, biological, and structural alterations were investigated. Both of the phosphatases dephosphorylated PHF II-tau at the sites of Ser-199/Ser-202 and partially dephosphorylated it at Ser-396/Ser-404; in addition, PHF II-tau was dephosphorylated at Ser-46 by PP-2A1 and Ser-235 by PP-2B. The relative electrophoretic mobility of PHF II-tau increased after dephosphorylation by either enzyme. Divalent cations, manganese, and magnesium increased the activities of PP-2A1 and PP-2B toward PHF II-tau. Dephosphorylation both by PP-2B and PP-2A1 decreased the resistance of PHF II-tau to proteolysis by the brain calcium-activated neutral proteases (CANP). The ability of PHF II-tau to promote the in vitro microtubule assembly was restored after dephosphorylation by PP-2A1 and PP-2B. Microtubules assembled by the dephosphorylated PHF II-tau were structurally identical to those assembled by bovine tau used as a control. The dephosphorylation both by PP-2A1 and PP-2B caused dissociation of the tangles and the PHF; some of the PHF dissociated into straight protofilaments/subfilaments. Approximately 25% of the total tau was released from PHF on dephosphorylation by PP-2A1. These observations demonstrate that PHF II-tau is accessible to dephosphorylation by PP-2A1 and PP-2B, and dephosphorylation makes PHF dissociate, accessible to proteolysis by CANP, and biologically active in promoting the assembly of tubulin into microtubules.
Topics: Alzheimer Disease; Animals; Brain; Cations, Divalent; Cattle; Humans; Microscopy, Electron; Microtubules; Phosphoprotein Phosphatases; Phosphorylation; Protein Phosphatase 2
PubMed: 7876258
DOI: 10.1074/jbc.270.9.4854 -
Phosphatase Shp2 regulates biogenesis of small extracellular vesicles by dephosphorylating Syntenin.Journal of Extracellular Vesicles Mar 2021As novel mediators of cell-to-cell signalling, small extracellular vesicles (sEVs) play a critical role in physiological and pathophysiological processes. To date, the...
As novel mediators of cell-to-cell signalling, small extracellular vesicles (sEVs) play a critical role in physiological and pathophysiological processes. To date, the molecular mechanisms that support sEV generation are incompletely understood. Many kinases are reported for their roles in sEV generation or composition, whereas the involvement of phosphatases remains largely unexplored. Here we reveal that pharmacological inhibition and shRNA-mediated down-regulation of tyrosine phosphatase Shp2 significantly increases the formation of sEVs. By Co-immunoprecipitation (Co-IP) and in vitro dephosphorylation assays, we identified that Shp2 negatively controlled sEV biogenesis by directly dephosphorylating tyrosine 46 of Syntenin, which has been reported as a molecular switch in sEV biogenesis. More importantly, Shp2 dysfunction led to enhanced epithelial sEV generation in vitro and in vivo. The increase of epithelial sEVs caused by shRNA-mediated down-regulation of Shp2 promoted macrophage activation, resulting in strengthened inflammation. Our findings highlight the role of Shp2 in regulating sEV-mediated epithelial-macrophage crosstalk by controlling sEV biogenesis through dephosphorylation of Syntenin Y46. The present study determines the strengthened inflammatory characteristics of alveolar macrophages elicited by epithelial sEVs transferred intercellularly. These findings provide a basis for understanding the mechanism of sEV formation and relevant function in epithelial-macrophage crosstalk.
Topics: Animals; Cell Line; Extracellular Vesicles; Humans; Mice; Organelle Biogenesis; Phosphorylation; Protein Tyrosine Phosphatase, Non-Receptor Type 11; Syntenins
PubMed: 33732417
DOI: 10.1002/jev2.12078 -
Cellular Physiology and Biochemistry :... 2018Integrin-linked kinase-associated phosphatase (ILKAP), a serine/threonine phosphatase that belongs to the protein phosphatase 2C family, has a role in cell survival and...
BACKGROUND/AIMS
Integrin-linked kinase-associated phosphatase (ILKAP), a serine/threonine phosphatase that belongs to the protein phosphatase 2C family, has a role in cell survival and apoptosis. Hypoxia-inducible factor 1α (HIF-1α) is the key transcription factor in the response to oxygen deficiency in mammals. Direct phosphorylation and dephosphorylation of HIF-1α affect its function. The present study investigated the role of ILKAP on HIF-1α dephosphorylation and cell behavior.
METHODS
HIF-1α was induced by hypoxia. Physical binding between ILKAP and HIF-1α was demonstrated by a co-immunoprecipitation assay. HIF-1α transcriptional activity was investigated using a hypoxia-response element-containing luciferase reporter plasmid. Cell viability was evaluated by a trypan blue dye exclusion assay. ILKAP function was explored by a gain and loss assay with an overexpression plasmid and shRNA infection.
RESULTS
ILKAP physically interacted with HIF-1α and induced its dephosphorylation. Both the HIF-1α-p53 interaction and apoptosis relied on ILKAP.
CONCLUSION
The results indicated that the ILKAP directly binds and dephosphorylates HIF-1α and responsible for severe hypoxia-induced cell apoptosis.
Topics: Apoptosis; Cell Hypoxia; Cell Line, Tumor; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Phosphoprotein Phosphatases; Phosphorylation; Protein Binding; Protein Interaction Maps
PubMed: 29742494
DOI: 10.1159/000489656 -
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 -
Oncotarget Jan 2017Aberrant phosphorylation and overexpression of BCR-ABL fusion protein are responsible for the main pathogenesis in chronic myeloid leukemia (CML). Phosphorylated BCR-ABL...
Aberrant phosphorylation and overexpression of BCR-ABL fusion protein are responsible for the main pathogenesis in chronic myeloid leukemia (CML). Phosphorylated BCR-ABL Y177 recruits GRB2 adaptor and triggers leukemic RAS-MAPK and PI3K-AKT signals. In this study, we engineered a SPOA system to dephosphorylate and degrade BCR-ABL by targeting BCR-ABL Y177. We tested its effect on BCR-ABL phosphorylation and expression, as well as cell proliferation and apoptosis in CML cells. We found that SPOA remarkably dephosphorylated BCR-ABL Y177, prevented GRB2 recruitment, and uncoupled RAS-MAPK and PI3K-AKT signals. Meanwhile, SPOA degraded BCR-ABL oncoprotein in ubiquitin-independent manner and depressed the signal transduction of STAT5 and CRKL by BCR-ABL. Furthermore, SPOA inhibited proliferation and induced apoptosis in CML cells and depressed the oncogenecity of K562 cells in mice. These results provide evidence that dephosphorylating and degrading oncogenic BCR-ABL offer an alternative CML therapy.
Topics: Animals; Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Disease Models, Animal; Fusion Proteins, bcr-abl; Gene Expression Regulation; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mice; Phosphorylation; Proteasome Endopeptidase Complex; Protein Binding; Proteolysis; Signal Transduction; Ubiquitin
PubMed: 27926512
DOI: 10.18632/oncotarget.13754 -
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 Dec 1982Axenic cultures of Paramecium tetraurelia take up 32Pi and phosphorylate a number of polypeptides as determined by autoradiography following sodium dodecyl... (Comparative Study)
Comparative Study
Axenic cultures of Paramecium tetraurelia take up 32Pi and phosphorylate a number of polypeptides as determined by autoradiography following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The most heavily labeled polypeptide has an apparent Mr of approximately 65,000. Wild type cells stimulated to secrete with picric acid, the standard secretagogue for these cells, show a marked reduction in labeling of the 65,000 Mr polypeptide. There is no change in the Coomassic blue staining protein pattern after addition of picric acid. Addition of picric acid to cells solubilized in sample buffer containing 10% sodium dodecyl sulfate, significantly lowers the pH but does not induce dephosphorylation of the 65,000 Mr polypeptide. Dephosphorylation of the 65,000 Mr polypeptide is further correlated with secretion in two types of experiments. 1) Preincubation of cells in Mg2+ (no added Ca2+) inhibits both secretion and dephosphorylation in response to picric acid. 2) A temperature-sensitive mutant, nd 9, when grown at 18 degrees C (permissive temperature) has the normal intramembrane particle array (rosette) at the secretory site and secretes and dephosphorylates the 65,000 Mr polypeptide in response to picric acid, but when grown at 27 degrees C (nonpermissive temperature) does not have assembled rosettes at the secretory site, and does not secrete nor dephosphorylate the 65,000 Mr polypeptide in response to picric acid. This represents the first correlation between a phosphoprotein and a physiological activity (secretion) in Paramecium. Our results show the presence of an in vivo stimulus-sensitive phosphoprotein of Mr 65,000 which appears related to Ca2+-mediated exocytosis. Inhibition of dephosphorylation occurs when secretion is blocked, either by Mg2+ or by a mutation affecting an intramembrane particle array, the rosette.
Topics: Animals; Magnesium; Molecular Weight; Mutation; Paramecium; Phosphoproteins; Phosphorylation; Proteins; Species Specificity; Temperature
PubMed: 7142183
DOI: No ID Found -
BioRxiv : the Preprint Server For... May 2024Calcineurin (CN), the only Ca -calmodulin activated protein phosphatase, dephosphorylates substrates within membrane-associated Ca microdomains. CN binds to substrates...
Calcineurin (CN), the only Ca -calmodulin activated protein phosphatase, dephosphorylates substrates within membrane-associated Ca microdomains. CN binds to substrates and regulators via short linear motifs (SLIMs), PxIxIT and LxVP. PxIxIT binding to CN is Ca independent and affects its distribution, while LxVP associates only with the active enzyme and promotes catalysis. 31 human proteins contain one or more composite 'LxVPxIxIT' motifs, whose functional properties have not been examined. Here we report studies of calcimembrin/C16orf74 (CLMB), a largely uncharacterized protein containing a composite motif that binds and directs CN to membranes. We demonstrate that CLMB associates with membranes via N-myristoylation and dynamic S-acylation and is dephosphorylated by CN on Thr44. The LxVP and PxIxIT portions of the CLMB composite sequence, together with Thr44 phosphorylation, confer high affinity PxIxIT-mediated binding to CN (KD∼8.9 nM) via an extended, LxVPxIxITxx(p)T sequence. This binding promotes CLMB-based targeting of CN to membranes, but also protects Thr44 from dephosphorylation. Thus, we propose that CN dephosphorylates CLMB in multimeric complexes, where one CLMB molecule recruits CN to membranes via PxIxIT binding, allowing others to engage through their LxVP motif for dephosphorylation. This unique mechanism makes dephosphorylation sensitive to CLMB:CN ratios and is supported by and analyses. CLMB overexpression is associated with poor prognoses for several cancers, suggesting that it promotes oncogenesis by shaping CN signaling.
PubMed: 38798520
DOI: 10.1101/2024.05.12.593783 -
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