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USP17 is required for clathrin mediated endocytosis of epidermal growth factor receptor.

Jaworski J, de la Vega M, Fletcher SJ, McFarlane C, Greene MK, Smyth AW, Van Schaeybroeck S, Johnston JA, Scott CJ, Rappoport JZ, Burrows JF - Oncotarget (2014)

Bottom Line: Previously we have shown that expression of the deubiquitinating enzyme USP17 is required for cell proliferation and motility.More recently we reported that USP17 deubiquitinates RCE1 isoform 2 and thus regulates the processing of 'CaaX' motif proteins.In addition, we show that USP17 is required for the endocytosis of transferrin, an archetypal substrate for clathrin mediated endocytosis, and that USP17 depletion impedes plasma membrane recruitment of the machinery required for clathrin mediated endocytosis.

View Article: PubMed Central - PubMed

Affiliation: School of Pharmacy, Queen's University Belfast, Belfast, UK.

ABSTRACT
Previously we have shown that expression of the deubiquitinating enzyme USP17 is required for cell proliferation and motility. More recently we reported that USP17 deubiquitinates RCE1 isoform 2 and thus regulates the processing of 'CaaX' motif proteins. Here we now show that USP17 expression is induced by epidermal growth factor and that USP17 expression is required for clathrin mediated endocytosis of epidermal growth factor receptor. In addition, we show that USP17 is required for the endocytosis of transferrin, an archetypal substrate for clathrin mediated endocytosis, and that USP17 depletion impedes plasma membrane recruitment of the machinery required for clathrin mediated endocytosis. Thus, our data reveal that USP17 is necessary for epidermal growth factor receptor and transferrin endocytosis via clathrin coated pits, indicate this is mediated via the regulation of the recruitment of the components of the endocytosis machinery and suggest USP17 may play a general role in receptor endocytosis.

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(a) HeLa cells were transfected as indicated. 72 hrs post transfection the cells were starved in serum free medium for 30 min prior to incubation with 25 μg/ml transferrin Alexa Fluor 568. After 15 min the cells were fixed and the nuclei stained with DAPI. The cells were then stained using an anti-TfR antibody and assessed for transferrin Alexa Fluor 568 (red) and TfR (green) internalisation in brightfield and fluorescent images taken using confocal microscopy. The right panels are enlarged images of the indicated area in the merged panels. Scale bar = 25 μm. (b) HeLa cells were transfected as indicated and 72 hrs post transfection the cells were starved in serum free medium for 3 hrs prior to incubation with 25 μg/ml transferrin Alexa Fluor 568. After 45 mins the cells were acid washed, fixed and the nuclei were stained with DAPI. Transferrin Alexa Fluor 568 (red) internalisation was then assessed in brightfield and fluorescent images taken using confocal microscopy. The lower panels are enlarged images of the indicated area in the upper panels. Scale bar = 10 μm.
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Figure 5: (a) HeLa cells were transfected as indicated. 72 hrs post transfection the cells were starved in serum free medium for 30 min prior to incubation with 25 μg/ml transferrin Alexa Fluor 568. After 15 min the cells were fixed and the nuclei stained with DAPI. The cells were then stained using an anti-TfR antibody and assessed for transferrin Alexa Fluor 568 (red) and TfR (green) internalisation in brightfield and fluorescent images taken using confocal microscopy. The right panels are enlarged images of the indicated area in the merged panels. Scale bar = 25 μm. (b) HeLa cells were transfected as indicated and 72 hrs post transfection the cells were starved in serum free medium for 3 hrs prior to incubation with 25 μg/ml transferrin Alexa Fluor 568. After 45 mins the cells were acid washed, fixed and the nuclei were stained with DAPI. Transferrin Alexa Fluor 568 (red) internalisation was then assessed in brightfield and fluorescent images taken using confocal microscopy. The lower panels are enlarged images of the indicated area in the upper panels. Scale bar = 10 μm.

Mentions: We next examined the localisation of transferrin along with TfR using an anti-TfR antibody. In control cells we observed the majority of TfR on internal vesicular structures (Fig. 5A, top panels) and upon transferrin treatment we observed transferrin and TfR co-localised to these internal vesicular structures (Fig. 5A, top right panels). In contrast, USP17 depleted cells showed prominent TfR staining on the plasma membrane, which co-localized with transferrin and suggested TfR and transferrin both failed to internalise (Fig. 5A, bottom panels). Again, to confirm this was due to an internalisation block, and not a delay, we extended the incubation period with transferrin out to 45 minutes and still observed no internalisation (Fig. 5B). This now indicated that USP17 plays a more general role in CME as both EGFR (ligand triggered endocytosis) and TfR (constitutively recycles) require USP17 expression to internalise via CME.


USP17 is required for clathrin mediated endocytosis of epidermal growth factor receptor.

Jaworski J, de la Vega M, Fletcher SJ, McFarlane C, Greene MK, Smyth AW, Van Schaeybroeck S, Johnston JA, Scott CJ, Rappoport JZ, Burrows JF - Oncotarget (2014)

(a) HeLa cells were transfected as indicated. 72 hrs post transfection the cells were starved in serum free medium for 30 min prior to incubation with 25 μg/ml transferrin Alexa Fluor 568. After 15 min the cells were fixed and the nuclei stained with DAPI. The cells were then stained using an anti-TfR antibody and assessed for transferrin Alexa Fluor 568 (red) and TfR (green) internalisation in brightfield and fluorescent images taken using confocal microscopy. The right panels are enlarged images of the indicated area in the merged panels. Scale bar = 25 μm. (b) HeLa cells were transfected as indicated and 72 hrs post transfection the cells were starved in serum free medium for 3 hrs prior to incubation with 25 μg/ml transferrin Alexa Fluor 568. After 45 mins the cells were acid washed, fixed and the nuclei were stained with DAPI. Transferrin Alexa Fluor 568 (red) internalisation was then assessed in brightfield and fluorescent images taken using confocal microscopy. The lower panels are enlarged images of the indicated area in the upper panels. Scale bar = 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4196176&req=5

Figure 5: (a) HeLa cells were transfected as indicated. 72 hrs post transfection the cells were starved in serum free medium for 30 min prior to incubation with 25 μg/ml transferrin Alexa Fluor 568. After 15 min the cells were fixed and the nuclei stained with DAPI. The cells were then stained using an anti-TfR antibody and assessed for transferrin Alexa Fluor 568 (red) and TfR (green) internalisation in brightfield and fluorescent images taken using confocal microscopy. The right panels are enlarged images of the indicated area in the merged panels. Scale bar = 25 μm. (b) HeLa cells were transfected as indicated and 72 hrs post transfection the cells were starved in serum free medium for 3 hrs prior to incubation with 25 μg/ml transferrin Alexa Fluor 568. After 45 mins the cells were acid washed, fixed and the nuclei were stained with DAPI. Transferrin Alexa Fluor 568 (red) internalisation was then assessed in brightfield and fluorescent images taken using confocal microscopy. The lower panels are enlarged images of the indicated area in the upper panels. Scale bar = 10 μm.
Mentions: We next examined the localisation of transferrin along with TfR using an anti-TfR antibody. In control cells we observed the majority of TfR on internal vesicular structures (Fig. 5A, top panels) and upon transferrin treatment we observed transferrin and TfR co-localised to these internal vesicular structures (Fig. 5A, top right panels). In contrast, USP17 depleted cells showed prominent TfR staining on the plasma membrane, which co-localized with transferrin and suggested TfR and transferrin both failed to internalise (Fig. 5A, bottom panels). Again, to confirm this was due to an internalisation block, and not a delay, we extended the incubation period with transferrin out to 45 minutes and still observed no internalisation (Fig. 5B). This now indicated that USP17 plays a more general role in CME as both EGFR (ligand triggered endocytosis) and TfR (constitutively recycles) require USP17 expression to internalise via CME.

Bottom Line: Previously we have shown that expression of the deubiquitinating enzyme USP17 is required for cell proliferation and motility.More recently we reported that USP17 deubiquitinates RCE1 isoform 2 and thus regulates the processing of 'CaaX' motif proteins.In addition, we show that USP17 is required for the endocytosis of transferrin, an archetypal substrate for clathrin mediated endocytosis, and that USP17 depletion impedes plasma membrane recruitment of the machinery required for clathrin mediated endocytosis.

View Article: PubMed Central - PubMed

Affiliation: School of Pharmacy, Queen's University Belfast, Belfast, UK.

ABSTRACT
Previously we have shown that expression of the deubiquitinating enzyme USP17 is required for cell proliferation and motility. More recently we reported that USP17 deubiquitinates RCE1 isoform 2 and thus regulates the processing of 'CaaX' motif proteins. Here we now show that USP17 expression is induced by epidermal growth factor and that USP17 expression is required for clathrin mediated endocytosis of epidermal growth factor receptor. In addition, we show that USP17 is required for the endocytosis of transferrin, an archetypal substrate for clathrin mediated endocytosis, and that USP17 depletion impedes plasma membrane recruitment of the machinery required for clathrin mediated endocytosis. Thus, our data reveal that USP17 is necessary for epidermal growth factor receptor and transferrin endocytosis via clathrin coated pits, indicate this is mediated via the regulation of the recruitment of the components of the endocytosis machinery and suggest USP17 may play a general role in receptor endocytosis.

Show MeSH
Related in: MedlinePlus