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The translation initiation factor 3f (eIF3f) exhibits a deubiquitinase activity regulating Notch activation.

Moretti J, Chastagner P, Gastaldello S, Heuss SF, Dirac AM, Bernards R, Masucci MG, Israël A, Brou C - PLoS Biol. (2010)

Bottom Line: Knocking down eIF3f leads to an accumulation of monoubiquitinated forms of activated Notch, an effect counteracted by murine WT eIF3f but not by a catalytically inactive mutant.Our results support two new and provocative conclusions: (1) The activated form of Notch needs to be deubiquitinated before being processed by the gamma-secretase activity and entering the nucleus, where it fulfills its transcriptional function. (2) The enzyme accounting for this deubiquitinase activity is eIF3f, known so far as a translation initiation factor.These data improve our knowledge of Notch signaling but also open new avenues of research on the Zomes family and the translation initiation factors.

View Article: PubMed Central - PubMed

Affiliation: Institut Pasteur, Unité de Signalisation Moléculaire et Activation Cellulaire and CNRS URA 2582, rue du Dr. Roux, Paris, France.

ABSTRACT
Activation of the mammalian Notch receptor after ligand binding relies on a succession of events including metalloprotease-cleavage, endocytosis, monoubiquitination, and eventually processing by the gamma-secretase, giving rise to a soluble, transcriptionally active molecule. The Notch1 receptor was proposed to be monoubiquitinated before its gamma-secretase cleavage; the targeted lysine has been localized to its submembrane domain. Investigating how this step might be regulated by a deubiquitinase (DUB) activity will provide new insight for understanding Notch receptor activation and downstream signaling. An immunofluorescence-based screening of an shRNA library allowed us to identify eIF3f, previously known as one of the subunits of the translation initiation factor eIF3, as a DUB targeting the activated Notch receptor. We show that eIF3f has an intrinsic DUB activity. Knocking down eIF3f leads to an accumulation of monoubiquitinated forms of activated Notch, an effect counteracted by murine WT eIF3f but not by a catalytically inactive mutant. We also show that eIF3f is recruited to activated Notch on endocytic vesicles by the putative E3 ubiquitin ligase Deltex1, which serves as a bridging factor. Finally, catalytically inactive forms of eIF3f as well as shRNAs targeting eIF3f repress Notch activation in a coculture assay, showing that eIF3f is a new positive regulator of the Notch pathway. Our results support two new and provocative conclusions: (1) The activated form of Notch needs to be deubiquitinated before being processed by the gamma-secretase activity and entering the nucleus, where it fulfills its transcriptional function. (2) The enzyme accounting for this deubiquitinase activity is eIF3f, known so far as a translation initiation factor. These data improve our knowledge of Notch signaling but also open new avenues of research on the Zomes family and the translation initiation factors.

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eiF3f DUB activity.(A) eIF3f exhibits a DUB activity in a bacterial Ub-GFP assay. Bl21 bacteria were transformed with Ub-GFP-S-Tag plasmid and either GST-fusions or His-fusions as indicated. After protein expression induction, they were lysed by sonication. WCE were analyzed by Western blot using the antibodies indicated under each panel to visualize GFP release from Ub-GFP (WB S-Tag) and to control protein expression (WB GST and His). The GFP product resulting from the DUB activity and the Ub-GFP substrate are indicated by the arrows. (B) eIF3f binds ubiquitin in an eukaryotic Ub-VS assay. HeLa cells were transfected with expression vectors encoding BPLF1 WT or CM (Lanes A–D, J, K), or meIF3f WT or Mut (Lanes E–H, M, N) as indicated. 36 h after transfection, 10 µg of WCE were incubated (or not) with Ub-VS probe (Lanes A to H). Samples were finally analyzed by Western blot as indicated under the panels to monitor Ub-VS binding (Lanes A to H) and to check protein expression (Lanes I to N). β-Actin was used as a loading control. The apparent molecular weights of BPLF1 and meIF3f are 32 kDa and 50 kDa, respectively.
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pbio-1000545-g003: eiF3f DUB activity.(A) eIF3f exhibits a DUB activity in a bacterial Ub-GFP assay. Bl21 bacteria were transformed with Ub-GFP-S-Tag plasmid and either GST-fusions or His-fusions as indicated. After protein expression induction, they were lysed by sonication. WCE were analyzed by Western blot using the antibodies indicated under each panel to visualize GFP release from Ub-GFP (WB S-Tag) and to control protein expression (WB GST and His). The GFP product resulting from the DUB activity and the Ub-GFP substrate are indicated by the arrows. (B) eIF3f binds ubiquitin in an eukaryotic Ub-VS assay. HeLa cells were transfected with expression vectors encoding BPLF1 WT or CM (Lanes A–D, J, K), or meIF3f WT or Mut (Lanes E–H, M, N) as indicated. 36 h after transfection, 10 µg of WCE were incubated (or not) with Ub-VS probe (Lanes A to H). Samples were finally analyzed by Western blot as indicated under the panels to monitor Ub-VS binding (Lanes A to H) and to check protein expression (Lanes I to N). β-Actin was used as a loading control. The apparent molecular weights of BPLF1 and meIF3f are 32 kDa and 50 kDa, respectively.

Mentions: In order to test whether eIF3f exhibits a deubiquitinase activity, we first used a functional in bacteria assay using GFP fused to ubiquitin (Ub-GFP) [24]. The peptide bond between ubiquitin and GFP can be cleaved by ubiquitin- or UbL-deconjugase activities, which are absent from the bacterial genome. Bl21 bacteria were transfected with plasmids encoding GST alone, GST-fused to human WT eIF3f or WT MPN domain, or His-tagged murine eIF3f WT or mutant MPN domain (Mut), together with a vector encoding Ub-GFP fused to an S-Tag at its C-terminus (Ub-GFP-S-Tag) (Figure 3A). As control DUB, we used BPLF1 WT (an EBV DUB of the cysteine-protease family [24]) and its catalytically inactive form BPLF1 CM. After induction of protein expression, the bacteria were lysed and cleavage of Ub-GFP was assessed in Western blot by the appearance of free GFP-S-Tag using anti-S-Tag antibody (Figure 3A, upper panel). Anti-GST or anti-His antibodies were used to verify protein expression (Figure 3A, bottom panel). As expected, we observed released GFP with BPLF1 WT but not its CM mutant (compare Lanes A–C). Interestingly, we also detected protease activity with heIF3f WT, heIF3f MPN WT, and meIF3f MPN WT (Lanes D, E, and F, respectively) but not with the mutant meIF3f MPN Mut (Lane G).


The translation initiation factor 3f (eIF3f) exhibits a deubiquitinase activity regulating Notch activation.

Moretti J, Chastagner P, Gastaldello S, Heuss SF, Dirac AM, Bernards R, Masucci MG, Israël A, Brou C - PLoS Biol. (2010)

eiF3f DUB activity.(A) eIF3f exhibits a DUB activity in a bacterial Ub-GFP assay. Bl21 bacteria were transformed with Ub-GFP-S-Tag plasmid and either GST-fusions or His-fusions as indicated. After protein expression induction, they were lysed by sonication. WCE were analyzed by Western blot using the antibodies indicated under each panel to visualize GFP release from Ub-GFP (WB S-Tag) and to control protein expression (WB GST and His). The GFP product resulting from the DUB activity and the Ub-GFP substrate are indicated by the arrows. (B) eIF3f binds ubiquitin in an eukaryotic Ub-VS assay. HeLa cells were transfected with expression vectors encoding BPLF1 WT or CM (Lanes A–D, J, K), or meIF3f WT or Mut (Lanes E–H, M, N) as indicated. 36 h after transfection, 10 µg of WCE were incubated (or not) with Ub-VS probe (Lanes A to H). Samples were finally analyzed by Western blot as indicated under the panels to monitor Ub-VS binding (Lanes A to H) and to check protein expression (Lanes I to N). β-Actin was used as a loading control. The apparent molecular weights of BPLF1 and meIF3f are 32 kDa and 50 kDa, respectively.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2990700&req=5

pbio-1000545-g003: eiF3f DUB activity.(A) eIF3f exhibits a DUB activity in a bacterial Ub-GFP assay. Bl21 bacteria were transformed with Ub-GFP-S-Tag plasmid and either GST-fusions or His-fusions as indicated. After protein expression induction, they were lysed by sonication. WCE were analyzed by Western blot using the antibodies indicated under each panel to visualize GFP release from Ub-GFP (WB S-Tag) and to control protein expression (WB GST and His). The GFP product resulting from the DUB activity and the Ub-GFP substrate are indicated by the arrows. (B) eIF3f binds ubiquitin in an eukaryotic Ub-VS assay. HeLa cells were transfected with expression vectors encoding BPLF1 WT or CM (Lanes A–D, J, K), or meIF3f WT or Mut (Lanes E–H, M, N) as indicated. 36 h after transfection, 10 µg of WCE were incubated (or not) with Ub-VS probe (Lanes A to H). Samples were finally analyzed by Western blot as indicated under the panels to monitor Ub-VS binding (Lanes A to H) and to check protein expression (Lanes I to N). β-Actin was used as a loading control. The apparent molecular weights of BPLF1 and meIF3f are 32 kDa and 50 kDa, respectively.
Mentions: In order to test whether eIF3f exhibits a deubiquitinase activity, we first used a functional in bacteria assay using GFP fused to ubiquitin (Ub-GFP) [24]. The peptide bond between ubiquitin and GFP can be cleaved by ubiquitin- or UbL-deconjugase activities, which are absent from the bacterial genome. Bl21 bacteria were transfected with plasmids encoding GST alone, GST-fused to human WT eIF3f or WT MPN domain, or His-tagged murine eIF3f WT or mutant MPN domain (Mut), together with a vector encoding Ub-GFP fused to an S-Tag at its C-terminus (Ub-GFP-S-Tag) (Figure 3A). As control DUB, we used BPLF1 WT (an EBV DUB of the cysteine-protease family [24]) and its catalytically inactive form BPLF1 CM. After induction of protein expression, the bacteria were lysed and cleavage of Ub-GFP was assessed in Western blot by the appearance of free GFP-S-Tag using anti-S-Tag antibody (Figure 3A, upper panel). Anti-GST or anti-His antibodies were used to verify protein expression (Figure 3A, bottom panel). As expected, we observed released GFP with BPLF1 WT but not its CM mutant (compare Lanes A–C). Interestingly, we also detected protease activity with heIF3f WT, heIF3f MPN WT, and meIF3f MPN WT (Lanes D, E, and F, respectively) but not with the mutant meIF3f MPN Mut (Lane G).

Bottom Line: Knocking down eIF3f leads to an accumulation of monoubiquitinated forms of activated Notch, an effect counteracted by murine WT eIF3f but not by a catalytically inactive mutant.Our results support two new and provocative conclusions: (1) The activated form of Notch needs to be deubiquitinated before being processed by the gamma-secretase activity and entering the nucleus, where it fulfills its transcriptional function. (2) The enzyme accounting for this deubiquitinase activity is eIF3f, known so far as a translation initiation factor.These data improve our knowledge of Notch signaling but also open new avenues of research on the Zomes family and the translation initiation factors.

View Article: PubMed Central - PubMed

Affiliation: Institut Pasteur, Unité de Signalisation Moléculaire et Activation Cellulaire and CNRS URA 2582, rue du Dr. Roux, Paris, France.

ABSTRACT
Activation of the mammalian Notch receptor after ligand binding relies on a succession of events including metalloprotease-cleavage, endocytosis, monoubiquitination, and eventually processing by the gamma-secretase, giving rise to a soluble, transcriptionally active molecule. The Notch1 receptor was proposed to be monoubiquitinated before its gamma-secretase cleavage; the targeted lysine has been localized to its submembrane domain. Investigating how this step might be regulated by a deubiquitinase (DUB) activity will provide new insight for understanding Notch receptor activation and downstream signaling. An immunofluorescence-based screening of an shRNA library allowed us to identify eIF3f, previously known as one of the subunits of the translation initiation factor eIF3, as a DUB targeting the activated Notch receptor. We show that eIF3f has an intrinsic DUB activity. Knocking down eIF3f leads to an accumulation of monoubiquitinated forms of activated Notch, an effect counteracted by murine WT eIF3f but not by a catalytically inactive mutant. We also show that eIF3f is recruited to activated Notch on endocytic vesicles by the putative E3 ubiquitin ligase Deltex1, which serves as a bridging factor. Finally, catalytically inactive forms of eIF3f as well as shRNAs targeting eIF3f repress Notch activation in a coculture assay, showing that eIF3f is a new positive regulator of the Notch pathway. Our results support two new and provocative conclusions: (1) The activated form of Notch needs to be deubiquitinated before being processed by the gamma-secretase activity and entering the nucleus, where it fulfills its transcriptional function. (2) The enzyme accounting for this deubiquitinase activity is eIF3f, known so far as a translation initiation factor. These data improve our knowledge of Notch signaling but also open new avenues of research on the Zomes family and the translation initiation factors.

Show MeSH
Related in: MedlinePlus