scholarly journals Insulin-induced formation of macromolecular complexes involved in activation of cyclic nucleotide phosphodiesterase 3B (PDE3B) and its interaction with PKB

2007 ◽  
Vol 404 (2) ◽  
pp. 257-268 ◽  
Author(s):  
Faiyaz Ahmad ◽  
Rebecka Lindh ◽  
Yan Tang ◽  
Marie Weston ◽  
Eva Degerman ◽  
...  
Keyword(s):  
Kinase Inhibitor ◽  
Gel Filtration ◽  
Heat Shock Protein 90 ◽  
Pleckstrin Homology Domain ◽  
Ph Domain ◽  
Structural Determinants ◽  
Macromolecular Complexes ◽  
Pi3k Inhibitor Ly294002 ◽  
Signalling Molecules ◽  
Phosphodiesterase 3B

Fractionation of 3T3-L1 adipocyte membranes revealed that PDE3B (phosphodiesterase 3B) was associated with PM (plasma membrane) and ER (endoplasmic reticulum)/Golgi fractions, that insulin-induced phosphorylation/activation of PDE3B was greater in internal membranes than PM fractions, and that there was no significant translocation of PDE3B between membrane fractions. Insulin also induced formation of large macromolecular complexes, separated during gel filtration (Superose 6 columns) of solubilized membranes, which apparently contain phosphorylated/activated PDE3B and signalling molecules potentially involved in its activation by insulin, e.g. IRS-1 (insulin receptor substrate-1), IRS-2, PI3K p85 [p85-subunit of PI3K (phosphoinositide 3-kinase)], PKB (protein kinase B), HSP-90 (heat-shock protein 90) and 14-3-3. Expression of full-length recombinant FLAG-tagged murine (M) PDE3B and M3BΔ604 (MPDE3B lacking N-terminal 604 amino acids) indicated that the N-terminal region of MPDE3B was necessary for insulin-induced activation and recruitment of PDE3B. siRNA (small interfering RNA) knock-down of PDE3B indicated that PDE3B was not required for formation of insulin-induced complexes. Wortmannin inhibited insulin-induced assembly of macromolecular complexes, as well as phosphorylation/activation of PKB and PDE3B, and their co-immunoprecipitation. Another PI3K inhibitor, LY294002, and the tyrosine kinase inhibitor, Genistein, also inhibited insulin-induced activation of PDE3B and its co-immunoprecipitation with PKB. Confocal microscopy indicated co-localization of PDE3B and PKB. Recombinant MPDE3B co-immunoprecipitated, and co-eluted during Superose 12 chromatography, to a greater extent with recombinant pPKB (phosphorylated/activated PKB) than dephospho-PKB or p-ΔPKB [pPKB lacking its PH domain (pleckstrin homology domain)]. Truncated recombinant MPDE3B proteins and pPKB did not efficiently co-immunoprecipitate, suggesting that structural determinants for their interaction reside in, or are regulated by, the N-terminal portion of MPDE3B. Recruitment of PDE3B in macromolecular complexes may be critical for regulation of specific cAMP pools and signalling pathways by insulin, e.g. lipolysis.

scholarly journals Role of Phosphatidylinositol 3′ Kinase and a Downstream Pleckstrin Homology Domain–Containing Protein in Controlling Chemotaxis inDictyostelium

2001 ◽  
Vol 153 (4) ◽  
pp. 795-810 ◽  
Author(s):  
Satoru Funamoto ◽  
Kristina Milan ◽  
Ruedi Meili ◽  
Richard A. Firtel
Keyword(s):  
Actin Polymerization ◽  
Adaptor Protein ◽  
Leading Edge ◽  
Membrane Lipid ◽  
Lipid Binding ◽  
Pleckstrin Homology Domain ◽  
Ph Domain ◽  
Wild Type ◽  
Pi3k Inhibitor Ly294002 ◽  
Effector Pathways

We show that cells lacking two Dictyostelium class I phosphatidylinositol (PI) 3′ kinases (PI3K and pi3k1/2-null cells) or wild-type cells treated with the PI3K inhibitor LY294002 are unable to properly polarize, are very defective in the temporal, spatial, and quantitative regulation of chemoattractant-mediated filamentous (F)-actin polymerization, and chemotax very slowly. PI3K is thought to produce membrane lipid-binding sites for localization of PH domain–containing proteins. We demonstrate that in response to chemoattractants three PH domain–containing proteins do not localize to the leading edge in pi3k1/2-null cells, and the translocation is blocked in wild-type cells by LY294002. Cells lacking one of these proteins, phdA-null cells, exhibit defects in the level and kinetics of actin polymerization at the leading edge and have chemotaxis phenotypes that are distinct from those described previously for protein kinase B (PKB) (pkbA)-null cells. Phenotypes of PhdA-dominant interfering mutations suggest that PhdA is an adaptor protein that regulates F-actin localization in response to chemoattractants and links PI3K to the control of F-actin polymerization at the leading edge during pseudopod formation. We suggest that PKB and PhdA lie downstream from PI3K and control different downstream effector pathways that are essential for proper chemotaxis.

Evidence that the tandem-pleckstrin-homology-domain-containing protein TAPP1 interacts with Ptd(3,4)P2 and the multi-PDZ-domain-containing protein MUPP1 in vivo

2002 ◽  
Vol 361 (3) ◽  
pp. 525-536 ◽  
Author(s):  
Wendy A. KIMBER ◽  
Laura TRINKLE-MULCAHY ◽  
Peter C. F. CHEUNG ◽  
Maria DEAK ◽  
Louisa J. MARSDEN ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Kinase Inhibitor ◽  
Pdz Domain ◽  
Pleckstrin Homology Domain ◽  
Adapter Proteins ◽  
Ph Domain ◽  
Pleckstrin Homology ◽  
Junction Protein

PtdIns(3,4,5)P3 is an established second messenger of growth-factor and insulin-induced signalling pathways. There is increasing evidence that one of the immediate breakdown products of PtdIns(3,4,5)P3, namely PtdIns(3,4)P2, whose levels are elevated by numerous extracellular agonists, might also function as a signalling molecule. Recently, we identified two related pleckstrin-homology (PH)-domain-containing proteins, termed ‘tandem-PH-domain-containing protein-1’ (TAPP1) and TAPP2, which interacted in vitro with high affinity with PtdIns(3,4)P2, but did not bind PtdIns(3,4,5)P3 or other phosphoinositides. In the present study we demonstrate that stimulation of Swiss 3T3 or 293 cells with agonists that stimulate PtdIns(3,4)P2 production results in the marked translocation of TAPP1 to the plasma membrane. This recruitment is dependent on a functional PtdIns(3,4)P2-binding PH domain and is inhibited by wortmannin, a phosphoinositide 3-kinase inhibitor that prevents PtdIns(3,4)P2 generation. A search for proteins that interact with TAPP1 identified the multi-PDZ-containing protein termed ‘MUPP1’, a protein possessing 13 PDZ domains and no other known modular or catalytic domains [PDZ is postsynaptic density protein (PSD-95)/Drosophila disc large tumour suppressor (dlg)/tight junction protein (ZO1)]. We demonstrate that immunoprecipitation of endogenously expressed TAPP1 from 293-cell lysates results in the co-immunoprecipitation of endogenous MUPP1, indicating that these proteins are likely to interact with each other physiologically. We show that TAPP1 and TAPP2 interact with the 10th and 13th PDZ domain of MUPP1 through their C-terminal amino acids. The results of the present study suggest that TAPP1 and TAPP2 could function in cells as adapter proteins to recruit MUPP1, or other proteins that they may interact with, to the plasma membrane in response to signals that elevate PtdIns(3,4)P2.

Stratifin, a keratinocyte specific 14–3-3 protein, harbors a pleckstrin homology (PH) domain and enhances protein kinase C activity

1995 ◽  
Vol 108 (11) ◽  
pp. 3569-3579
Author(s):  
E. Dellambra ◽  
M. Patrone ◽  
B. Sparatore ◽  
A. Negri ◽  
F. Ceciliani ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Terminal Differentiation ◽  
Pleckstrin Homology Domain ◽  
Ph Domain ◽  
Pleckstrin Homology ◽  
Protein Database ◽  
Kinase C ◽  
Human Keratinocyte ◽  
Protein Kinase C Activity

The intrinsic signal(s) responsible for the onset of human keratinocyte terminal differentiation is not yet fully understood. Evidence has been recently accumulated linking the phospholipase-mediated activation of protein kinase C to the coordinate changes in gene expression occurring during keratinocyte terminal differentiation. Here we report the purification of a keratinocyte-derived protein enhancing protein kinase C enzymatic activity. The stimulator eluted as a peak with estimated molecular mass of approximately 70 kDa, while analysis by SDS-PAGE showed a 30 kDa protein migrating as a distinct doublet, suggesting the formation of a 30 kDa homodimer. The amino acid sequence analysis allowed the unambigous identification of the protein kinase C stimulator as a mixture of the highly homologous sigma (stratifin) and zeta isoforms of 14–3-3 proteins, which are homodimers of identical 30 kDa subunits. Mono Q anion exchange chromatography and immunoblot analysis further confirmed that stratifin enhances protein kinase C activity. Stratifin was originally sequenced from a human keratinocyte protein database, but its function was unknown. The pleckstrin homology domain has been recently related to protein translocation to the cell membrane as well as to functional interactions of intracellular proteins involved in signal transduction. We show here that stratifin (and 14–3-3 zeta) harbors a pleckstrin homology domain, and the consequent functional implications will be discussed.

TAPP2 links phosphoinositide 3-kinase signaling to B-cell adhesion through interaction with the cytoskeletal protein utrophin: expression of a novel cell adhesion-promoting complex in B-cell leukemia

Blood ◽  
2009 ◽  
Vol 114 (21) ◽  
pp. 4703-4712 ◽  
Author(s):  
Jennifer L. Costantini ◽  
Samuel M. S. Cheung ◽  
Sen Hou ◽  
Hongzhao Li ◽  
Sam K. Kung ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
B Cells ◽  
B Cell ◽  
Cell Activation ◽  
Cell Types ◽  
Potential Interaction ◽  
Cytoskeletal Proteins ◽  
Lymphocytic Leukemia ◽  
Pleckstrin Homology Domain ◽  
Ph Domain

Abstract Tandem pleckstrin homology domain proteins (TAPPs) are recruited to the plasma membrane via binding to phosphoinositides produced by phosphoinositide 3-kinases (PI3Ks). Whereas PI3Ks are critical for B-cell activation, the functions of TAPP proteins in B cells are unknown. We have identified 40 potential interaction partners of TAPP2 in B cells, including proteins involved in cytoskeletal rearrangement, signal transduction and endocytic trafficking. The association of TAPP2 with the cytoskeletal proteins utrophin and syntrophin was confirmed by Western blotting. We found that TAPP2, syntrophin, and utrophin are coexpressed in normal human B cells and B-chronic lymphocytic leukemia (B-CLL) cells. TAPP2 and syntrophin expression in B-CLL was variable from patient to patient, with significantly higher expression in the more aggressive disease subset identified by zeta-chain–associated protein kinase of 70 kDa (ZAP70) expression and unmutated immunoglobulin heavy chain (IgH) genes. We examined whether TAPP can regulate cell adhesion, a known function of utrophin/syntrophin in other cell types. Expression of membrane-targeted TAPP2 enhanced B-cell adhesion to fibronectin and laminin, whereas PH domain–mutant TAPP2 inhibited adhesion. siRNA knockdown of TAPP2 or utrophin, or treatment with PI3K inhibitors, significantly inhibited adhesion. These findings identify TAPP2 as a novel link between PI3K signaling and the cytoskeleton with potential relevance for leukemia progression.

scholarly journals Role of TAPP1 and TAPP2 adaptor binding to PtdIns(3,4)P2 in regulating insulin sensitivity defined by knock-in analysis

2011 ◽  
Vol 434 (2) ◽  
pp. 265-274 ◽  
Author(s):  
Stephan Wullschleger ◽  
David H. Wasserman ◽  
Alex Gray ◽  
Kei Sakamoto ◽  
Dario R. Alessi
Keyword(s):  
Insulin Sensitivity ◽  
Insulin Signalling ◽  
Adaptor Proteins ◽  
Whole Body ◽  
Pleckstrin Homology Domain ◽  
Ph Domain ◽  
Enhanced Production ◽  
Pi3k Signalling ◽  
Inositol Phosphatases ◽  
Phosphoinositide 3 Kinase

Insulin sensitivity is critically dependent on the activity of PI3K (phosphoinositide 3-kinase) and generation of the PtdIns(3,4,5)P3 second messenger. PtdIns(3,4,5)P3 can be broken down to PtdIns(3,4)P2 through the action of the SHIPs (Src-homology-2-domain-containing inositol phosphatases). As PtdIns(3,4)P2 levels peak after those of PtdIns(3,4,5)P3, it has been proposed that PtdIns(3,4)P2 controls a negative-feedback loop that down-regulates the insulin and PI3K network. Previously, we identified two related adaptor proteins termed TAPP [tandem PH (pleckstrin homology)-domain-containing protein] 1 and TAPP2 that specifically bind to PtdIns(3,4)P2 through their C-terminal PH domain. To determine whether TAPP1 and TAPP2 play a role in regulating insulin sensitivity, we generated knock-in mice that express normal endogenous levels of mutant TAPP1 and TAPP2 that are incapable of binding PtdIns(3,4)P2. These homozygous TAPP1R211L/R211LTAPP2R218L/R218L double knock-in mice are viable and exhibit significantly enhanced activation of Akt, a key downstream mediator of insulin signalling. Consistent with increased PI3K and Akt activity, the double knock-in mice display enhanced whole body insulin sensitivity and disposal of glucose uptake into muscle tissues. We also generated wild-type and double TAPP1R211L/R211LTAPP2R218L/R218L knock-in embryonic fibroblasts and found that insulin triggered enhanced production of PtdIns(3,4,5)P3 and Akt activity in the double knock-in fibroblasts. These observations provide the first genetic evidence to support the notion that binding of TAPP1 and TAPP2 adap-tors to PtdIns(3,4)P2 function as negative regulators of the insulin and PI3K signalling pathways.

scholarly journals Dual Phosphorylation of Btk by Akt/Protein Kinase B Provides Docking for 14-3-3ζ, Regulates Shuttling, and Attenuates both Tonic and Induced Signaling in B Cells

2013 ◽  
Vol 33 (16) ◽  
pp. 3214-3226 ◽  
Author(s):  
Dara K. Mohammad ◽  
Beston F. Nore ◽  
Alamdar Hussain ◽  
Manuela O. Gustafsson ◽  
Abdalla J. Mohamed ◽  
...  
Keyword(s):  
Protein Kinase ◽  
B Cell ◽  
Tyrosine Phosphorylation ◽  
Nuclear Translocation ◽  
Kinase Inhibitor ◽  
Protein Kinase B ◽  
Negative Regulator ◽  
Pleckstrin Homology Domain ◽  
Loss Of Function ◽  
Interaction Sites

Bruton's tyrosine kinase (Btk) is crucial for B-lymphocyte activation and development. Mutations in theBtkgene cause X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (Xid) in mice. Using tandem mass spectrometry, 14-3-3ζ was identified as a new binding partner and negative regulator of Btk in both B-cell lines and primary B lymphocytes. The activated serine/threonine kinase Akt/protein kinase B (PKB) phosphorylated Btk on two sites prior to 14-3-3ζ binding. The interaction sites were mapped to phosphoserine pS51 in the pleckstrin homology domain and phosphothreonine pT495 in the kinase domain. The double-alanine, S51A/T495A, replacement mutant failed to bind 14-3-3ζ, while phosphomimetic aspartate substitutions, S51D/T495D, caused enhanced interaction. The phosphatidylinositol 3-kinase (PI3-kinase) inhibitor LY294002 abrogated S51/T495 phosphorylation and binding. A newly characterized 14-3-3 inhibitor, BV02, reduced binding, as did the Btk inhibitor PCI-32765 (ibrutinib). Interestingly, in the presence of BV02, phosphorylation of Btk, phospholipase Cγ2, and NF-κB increased strongly, suggesting that 14-3-3 also regulates B-cell receptor (BCR)-mediated tonic signaling. Furthermore, downregulation of 14-3-3ζ elevated nuclear translocation of Btk. The loss-of-function mutant S51A/T495A showed reduced tyrosine phosphorylation and ubiquitination. Conversely, the gain-of-function mutant S51D/T495D exhibited intense tyrosine phosphorylation, associated with Btk ubiquitination and degradation, likely contributing to the termination of BCR signaling. Collectively, this suggests that Btk could become an important new candidate for the general study of 14-3-3-mediated regulation.

Molecular modelling and site-directed mutagenesis of the inositol 1,3,4,5-tetrakisphosphate-binding pleckstrin homology domain from the Ras GTPase-activating protein GAP1IP4BP

2000 ◽  
Vol 349 (1) ◽  
pp. 333-342 ◽  
Author(s):  
Gyles COZIER ◽  
Richard SESSIONS ◽  
Joanna R. BOTTOMLEY ◽  
Jon S. REYNOLDS ◽  
Peter J. CULLEN
Keyword(s):  
Crystal Structure ◽  
Binding Site ◽  
Inositol Phosphate ◽  
Site Directed Mutagenesis ◽  
Pleckstrin Homology Domain ◽  
Directed Mutagenesis ◽  
Ph Domain ◽  
Pleckstrin Homology ◽  
Gtpase Activating Protein ◽  
Ras Gtpase

GAP1IP4BP is a Ras GTPase-activating protein (GAP) that in vitro is regulated by the cytosolic second messenger inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. We have studied Ins(1,3,4,5)P4 binding to GAP1IP4BP, and shown that the inositol phosphate specificity and binding affinity are similar to Ins(1,3,4,5)P4 binding to Bruton's tyrosine kinase (Btk), evidence which suggests a similar mechanism for Ins(1,3,4,5)P4 binding. The crystal structure of the Btk pleckstrin homology (PH) domain in complex with Ins(1,3,4,5)P4 has shown that the binding site is located in a partially buried pocket between the β1/β2- and β3/β4-loops. Many of the residues involved in the binding are conserved in GAP1IP4BP. Therefore we generated a model of the PH domain of GAP1IP4BP in complex with Ins(1,3,4,5)P4 based on the Btk-Ins(1,3,4,5)P4 complex crystal structure. This model had the typical PH domain fold, with the proposed binding site modelling well on the Btk structure. The model has been verified by site-directed mutagenesis of various residues in and around the proposed binding site. These mutations have markedly reduced affinity for Ins(1,3,4,5)P4, indicating a specific and tight fit for the substrate. The model can also be used to explain the specificity of inositol phosphate binding.

Inhibition and flare patterns of metabolic response to the heat shock protein 90 (Hsp90) inhibitor IPI-504 visualized by FDG-PET in patients (pts) with advanced gastrointestinal stromal tumors (GIST) resistant to tyrosine kinase inhibitor (TKI) therapy

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 3530-3530 ◽  
Author(s):  
A. D. Van den Abbeele ◽  
J. T. Yap ◽  
D. S. Grayzel ◽  
J. Walker ◽  
G. D. Demetri
Keyword(s):  
Fdg Pet ◽  
Kinase Inhibitor ◽  
Metabolic Response ◽  
Clinical Investigation ◽  
Heat Shock Protein 90 ◽  
Hsp90 Inhibitor ◽  
Resistance Mutations ◽  
Hsp90 Inhibition ◽  
Standardized Uptake Values ◽  
Time Point

3530 Background: We have previously demonstrated the inhibition and rebound of GIST glycolytic metabolism with FDG-PET while pts were on or off TKI in prior trials of imatinib and sunitinib. We tested the same principle in a phase I trial of IPI-504, a novel potent inhibitor of Hsp90 (a chaperone for protein homeostasis) that results in selective destruction of the mutated KIT kinase in human GIST cell lines regardless of TKI-resistance mutations. Methods: Twenty-one patients with metatastic and/or unresectable GIST following failure of prior therapy with TKI were treated with IPI-504. Serial FDG-PET imaging was performed at baseline, during the 1st cycle after at least 2 doses (C1, days 4–11, “ON”, n = 18), and at the end of the 10-day off-treatment period prior to the start of the 2nd cycle (C1, “OFF”, n = 20). A subset of 5 pts also had FDG-PET at the end of the 3rd cycle (C3, day 11, “ON”). Maximum standardized uptake values (SUVmax) were measured in up to 3 lesions/pt with the greatest FDG uptake, and the SUVmax of all lesions was summed at each time point. Percent change in the summed mean SUVmax was calculated at each time point relative to the previous scan. Temporal changes were evaluated in those pts showing more than a 10% decrease in SUVmax during C1 “ON” compared to baseline. Results: We observed a >10% reduction in SUVmax (mean = - 28%) during the 1st cycle (C1, “ON”) in 8/18 pts. All these pts showed an increase in SUVmax (mean = +29%) when off therapy (C1, “OFF”). Three of these 8 pts had a scan during cycle 3. All demonstrated a decrease in SUVmax (mean = -30%) while on the drug (C3, “ON”). Conclusion: These preliminary findings suggest that: (1) tumor metabolic response as measured with FDG-PET parallels the intermittent pattern of IPI-504 administration in this study as early as after the 2nd dose administration; and, (2) IPI-504 has a rapid downstream effect on glucose metabolism similar to that observed with TKIs despite the very different mechanism of action of IPI-504. The pattern of response to Hsp90 inhibition seen in this heavily pretreated population strongly supports further clinical investigation. No significant financial relationships to disclose.

Assessment of the heat shock protein 90 (HSP90) inhibitor IPI504 alone or in combination with the tyrosine kinase inhibitor (TKI) imatinib in mice carrying xenografts of human gastrointestinal stromal tumors (GIST)

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. 10534-10534 ◽  
Author(s):  
P. Schoffski ◽  
G. Floris ◽  
R. Sciot ◽  
C. Stefan ◽  
A. Wozniak ◽  
...  
Keyword(s):  
Kinase Inhibitor ◽  
Single Agent ◽  
Substantial Reduction ◽  
Heat Shock Protein 90 ◽  
Dose Schedule ◽  
Hsp90 Inhibitor ◽  
Antitumor Effects ◽  
New Strategy ◽  
Grade 3 ◽  
Anti Tumor Activity

10534 Background: Inhibition of HSP90 is a new strategy for treatment of GIST. IPI-504 is a potent i.v. HSP90 inhibitor. We assessed the activity of IPI-504 in imatinib-sensitive GIST xenografts, alone/combined with the TKI. Methods: Human GIST882 cells (KIT exon 13 mut.) were grafted in 43 nude mice, who were randomized to 4 groups: A (n=13; control); B (n=18; IPI-504 100 mg/kg 3x/wk p.o.); C (n=8; imatinib 50 mg/kg 2x/d p.o.); D (n=4; IPI-504+imatinib, dose/schedule as above) for 2 weeks. Histopathological assessment was done by H&E and KIT immunostaining, histological response (HR) was defined by magnitude of necrosis and myxoid degeneration [grade 1 (0–10%), 2 (>10% - ≤ 50%), 3 (> 50% - ≤90%), 4 (>90%)]. Expression/activation of KIT and its signaling (AKT, S6, MAPK) was assessed by Western blot. Results: IPI-504 alone significantly reduced tumor volume (41%) at day 14, further enhanced by adding imatinib (58%). Imatinib alone led to only a 5% reduction. IPI-504 induced grade 2 HR in 17/36 tumors, while the majority of tumors (13/15) treated with imatinib showed grade 1 HR. Grade 3/4 HRs were only seen with the combination. Mitosis decreased 3.3-fold, apoptosis increased 1.3-fold with IPI-504. Despite substantial reduction in mitotic activity in C and D (8.2- and 82-fold), apoptosis was virtually unaffected. In the IPI-504 arm, KIT levels were partially downregulated. Imatinib alone had no effect on KIT expression, while the combination produced a marked suppression of total KIT, accompanied by complete downregulation of the signaling. Loss of KIT in the combination arm was confirmed by immunostaining. Although KIT was not completely degraded with IPI-504, measurable effects on activation of AKT, S6 and MAPK were observed. Conclusions: IPI-504 has consistent antitumor effects in GIST xenografts, both histologically and on the molecular level. Combining IPI-504 with imatinib substantially enhances anti-tumor activity, providing a strong rationale for clinical trials of IPI-504 in GIST as single agent and in combination. [Table: see text]

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