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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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Colocalization of eIF3f, DTX, and ΔE.U2OS cells were transfected with expression vectors as indicated on the left. eIF3f, DTX, and ΔE were detected using mouse anti-HA, rabbit anti-VSV, and goat anti-myc, respectively (with CY3-coupled, Alexa 488-coupled, and Alexa 647-coupled secondary antibodies, respectively). Insets represent enlargements (4-fold) of the boxed region, arrows indicate colocalizations, and left panels are merges of the three adjacent panels.
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pbio-1000545-g004: Colocalization of eIF3f, DTX, and ΔE.U2OS cells were transfected with expression vectors as indicated on the left. eIF3f, DTX, and ΔE were detected using mouse anti-HA, rabbit anti-VSV, and goat anti-myc, respectively (with CY3-coupled, Alexa 488-coupled, and Alexa 647-coupled secondary antibodies, respectively). Insets represent enlargements (4-fold) of the boxed region, arrows indicate colocalizations, and left panels are merges of the three adjacent panels.

Mentions: In order to identify the site of action and the target of eIF3f in the Notch activation cascade, we first overexpressed ΔE and murine eIF3f in U2OS cells. We observed by immunofluorescence a low frequency of vesicular colocalization of the two proteins (Figure 4, Panels A and B). As eIF3f might require a cofactor or target an intermediate protein to regulate Notch ubiquitination, we tested several components of the Notch signaling pathway that could be associated with trafficking. Some DUBs are known to be recruited to their substrate indirectly via an E3 ubiquitin ligase [25],[26], so we particularly focused on the E3 ubiquitin ligases of the Notch pathway known to be associated with the endocytic machinery: Itch/AIP4 and Deltex1 (hereafter designated as DTX) ([27] and therein). In contrast to Itch/AIP4, DTX significantly colocalized with eIF3f when both proteins were coexpressed (Figure 4, Panel C). In addition, the presence of DTX strikingly increased the colocalization of ΔE and eIF3f, the three proteins being associated to the same vesicles (Figure 4, Panel D). These results suggest that DTX could recruit eIF3f to activated 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)

Colocalization of eIF3f, DTX, and ΔE.U2OS cells were transfected with expression vectors as indicated on the left. eIF3f, DTX, and ΔE were detected using mouse anti-HA, rabbit anti-VSV, and goat anti-myc, respectively (with CY3-coupled, Alexa 488-coupled, and Alexa 647-coupled secondary antibodies, respectively). Insets represent enlargements (4-fold) of the boxed region, arrows indicate colocalizations, and left panels are merges of the three adjacent panels.
© Copyright Policy
Related In: Results  -  Collection

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

pbio-1000545-g004: Colocalization of eIF3f, DTX, and ΔE.U2OS cells were transfected with expression vectors as indicated on the left. eIF3f, DTX, and ΔE were detected using mouse anti-HA, rabbit anti-VSV, and goat anti-myc, respectively (with CY3-coupled, Alexa 488-coupled, and Alexa 647-coupled secondary antibodies, respectively). Insets represent enlargements (4-fold) of the boxed region, arrows indicate colocalizations, and left panels are merges of the three adjacent panels.
Mentions: In order to identify the site of action and the target of eIF3f in the Notch activation cascade, we first overexpressed ΔE and murine eIF3f in U2OS cells. We observed by immunofluorescence a low frequency of vesicular colocalization of the two proteins (Figure 4, Panels A and B). As eIF3f might require a cofactor or target an intermediate protein to regulate Notch ubiquitination, we tested several components of the Notch signaling pathway that could be associated with trafficking. Some DUBs are known to be recruited to their substrate indirectly via an E3 ubiquitin ligase [25],[26], so we particularly focused on the E3 ubiquitin ligases of the Notch pathway known to be associated with the endocytic machinery: Itch/AIP4 and Deltex1 (hereafter designated as DTX) ([27] and therein). In contrast to Itch/AIP4, DTX significantly colocalized with eIF3f when both proteins were coexpressed (Figure 4, Panel C). In addition, the presence of DTX strikingly increased the colocalization of ΔE and eIF3f, the three proteins being associated to the same vesicles (Figure 4, Panel D). These results suggest that DTX could recruit eIF3f to activated 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