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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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Interaction of eIF3f and activated Notch in the presence of DTX.HEK293T cells were cotransfected with vectors encoding HA-tagged forms of eIF3f (either WT or Mut in Panel A), VSV-DTX, and myc-tagged Notch constructs (ΔE, ΔE-LLFF, NIC, or FL in Panel B) as indicated above the lanes. Immunoprecipitations were performed with anti-HA antibody. In both panels, immunoprecipitates and WCE (10% of the lysates) were analyzed by Western blot as indicated. α- or β-tubulin were used as loading controls.
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pbio-1000545-g006: Interaction of eIF3f and activated Notch in the presence of DTX.HEK293T cells were cotransfected with vectors encoding HA-tagged forms of eIF3f (either WT or Mut in Panel A), VSV-DTX, and myc-tagged Notch constructs (ΔE, ΔE-LLFF, NIC, or FL in Panel B) as indicated above the lanes. Immunoprecipitations were performed with anti-HA antibody. In both panels, immunoprecipitates and WCE (10% of the lysates) were analyzed by Western blot as indicated. α- or β-tubulin were used as loading controls.

Mentions: We then performed co-immunoprecipitations in HEK293T in the presence of Notch ΔE (Figure 6A). While DTX co-immunoprecipitated with WT eIF3f but also with meIF3f Mut (Figure 6A, Lanes B–E), ΔE did not (Lanes I and J), unless DTX was cotransfected (Lanes D and E). This strongly suggests that a tripartite interaction occurs between activated Notch, DTX, and eIF3f, DTX being required for the Notch-eIF3f interaction. In addition, we verified that neither DTX nor ΔE could be co-immunoprecipitated with eIF3f from cell extracts that were transfected separately and mixed (unpublished data). We then repeated these experiments using other forms of Notch: ΔE-LLFF, NIC, or the non-activated full-length Notch (FL) (Figure 6B). Only two forms could co-immunoprecipitate with eIF3f in the presence of DTX: ΔE and ΔE-LLFF (Lanes C and D). No signal was detected with FL and NIC (Lanes E and F) even with a longer exposure. It is of note that NIC produced from ΔE (Lanes C, G) and detected by the V1744 antibody was not co-immunoprecipitated with eIF3f either. These results show that the tripartite interaction between Notch, DTX, and eIF3f occurs preferentially with activated, membrane-associated, but γ-secretase unprocessed, forms of Notch.


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)

Interaction of eIF3f and activated Notch in the presence of DTX.HEK293T cells were cotransfected with vectors encoding HA-tagged forms of eIF3f (either WT or Mut in Panel A), VSV-DTX, and myc-tagged Notch constructs (ΔE, ΔE-LLFF, NIC, or FL in Panel B) as indicated above the lanes. Immunoprecipitations were performed with anti-HA antibody. In both panels, immunoprecipitates and WCE (10% of the lysates) were analyzed by Western blot as indicated. α- or β-tubulin were used as loading controls.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2990700&req=5

pbio-1000545-g006: Interaction of eIF3f and activated Notch in the presence of DTX.HEK293T cells were cotransfected with vectors encoding HA-tagged forms of eIF3f (either WT or Mut in Panel A), VSV-DTX, and myc-tagged Notch constructs (ΔE, ΔE-LLFF, NIC, or FL in Panel B) as indicated above the lanes. Immunoprecipitations were performed with anti-HA antibody. In both panels, immunoprecipitates and WCE (10% of the lysates) were analyzed by Western blot as indicated. α- or β-tubulin were used as loading controls.
Mentions: We then performed co-immunoprecipitations in HEK293T in the presence of Notch ΔE (Figure 6A). While DTX co-immunoprecipitated with WT eIF3f but also with meIF3f Mut (Figure 6A, Lanes B–E), ΔE did not (Lanes I and J), unless DTX was cotransfected (Lanes D and E). This strongly suggests that a tripartite interaction occurs between activated Notch, DTX, and eIF3f, DTX being required for the Notch-eIF3f interaction. In addition, we verified that neither DTX nor ΔE could be co-immunoprecipitated with eIF3f from cell extracts that were transfected separately and mixed (unpublished data). We then repeated these experiments using other forms of Notch: ΔE-LLFF, NIC, or the non-activated full-length Notch (FL) (Figure 6B). Only two forms could co-immunoprecipitate with eIF3f in the presence of DTX: ΔE and ΔE-LLFF (Lanes C and D). No signal was detected with FL and NIC (Lanes E and F) even with a longer exposure. It is of note that NIC produced from ΔE (Lanes C, G) and detected by the V1744 antibody was not co-immunoprecipitated with eIF3f either. These results show that the tripartite interaction between Notch, DTX, and eIF3f occurs preferentially with activated, membrane-associated, but γ-secretase unprocessed, forms of Notch.

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