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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: 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.

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 3 hrs prior to incubation with 0.32 nM EGF Alexa Fluor 555 where indicated. After 15 min the cells were acid washed, where indicated, fixed and the nuclei stained with DAPI. The cells were then stained using an anti-EGFR antibody and EGF Alexa Fluor 555 (red) and EGFR (green) internalisation was assessed in brightfield and fluorescent images taken using confocal microscopy. The right panels are enlarged images of the indicated area in the left panels. Scale bar = 10 μm. (b) At least 50 cells per condition were blindly scored for three separate experiments based on the presence of EGF Alexa Fluor 555 and EGFR co-staining post acid wash. (c) HeLa cells were transfected as indicated. 72 hrs post transfection the cells were starved in serum free medium for 3 hrs prior to incubation with 0.32 nM EGF. Whole cell lysates were harvested and levels of phosphorylated ERK1/2, ERK1/2, EGFR and tubulin were assessed by immuno-blotting using anti-pERK1/2, anti-ERK1/2, anti-EGFR and anti-tubulin antibodies. ** p<0.01
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Figure 2: (a) HeLa cells were transfected as indicated. 72 hrs post transfection the cells were starved in serum free medium for 3 hrs prior to incubation with 0.32 nM EGF Alexa Fluor 555 where indicated. After 15 min the cells were acid washed, where indicated, fixed and the nuclei stained with DAPI. The cells were then stained using an anti-EGFR antibody and EGF Alexa Fluor 555 (red) and EGFR (green) internalisation was assessed in brightfield and fluorescent images taken using confocal microscopy. The right panels are enlarged images of the indicated area in the left panels. Scale bar = 10 μm. (b) At least 50 cells per condition were blindly scored for three separate experiments based on the presence of EGF Alexa Fluor 555 and EGFR co-staining post acid wash. (c) HeLa cells were transfected as indicated. 72 hrs post transfection the cells were starved in serum free medium for 3 hrs prior to incubation with 0.32 nM EGF. Whole cell lysates were harvested and levels of phosphorylated ERK1/2, ERK1/2, EGFR and tubulin were assessed by immuno-blotting using anti-pERK1/2, anti-ERK1/2, anti-EGFR and anti-tubulin antibodies. ** p<0.01

Mentions: This indicated that USP17 was necessary for EGF entry. However, it could have also been explained by USP17 depletion resulting in the trafficking of EGFR away from the plasma membrane. Therefore, to investigate if USP17 was altering the location of EGFR we examined the localisation of EGF, along with EGFR, using an anti-EGFR antibody. In control HeLa cells we observed EGFR on the plasma membrane prior to stimulation, and upon EGF treatment, EGF and EGFR co-localised to internal vesicular structures which were more prominent after cells were stripped of external EGF using an acid wash (Figs 2A, top panels, 2B). In USP17 depleted cells we again observed prominent EGFR plasma membrane staining (Fig. 2A, bottom left panels). However, when EGF was added, both the EGF and EGFR remained at the plasma membrane and the EGF was lost upon acid wash demonstrating it had failed to enter the cell (Figs 2A, bottom middle and bottom right panels, 2B).


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 3 hrs prior to incubation with 0.32 nM EGF Alexa Fluor 555 where indicated. After 15 min the cells were acid washed, where indicated, fixed and the nuclei stained with DAPI. The cells were then stained using an anti-EGFR antibody and EGF Alexa Fluor 555 (red) and EGFR (green) internalisation was assessed in brightfield and fluorescent images taken using confocal microscopy. The right panels are enlarged images of the indicated area in the left panels. Scale bar = 10 μm. (b) At least 50 cells per condition were blindly scored for three separate experiments based on the presence of EGF Alexa Fluor 555 and EGFR co-staining post acid wash. (c) HeLa cells were transfected as indicated. 72 hrs post transfection the cells were starved in serum free medium for 3 hrs prior to incubation with 0.32 nM EGF. Whole cell lysates were harvested and levels of phosphorylated ERK1/2, ERK1/2, EGFR and tubulin were assessed by immuno-blotting using anti-pERK1/2, anti-ERK1/2, anti-EGFR and anti-tubulin antibodies. ** p<0.01
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: (a) HeLa cells were transfected as indicated. 72 hrs post transfection the cells were starved in serum free medium for 3 hrs prior to incubation with 0.32 nM EGF Alexa Fluor 555 where indicated. After 15 min the cells were acid washed, where indicated, fixed and the nuclei stained with DAPI. The cells were then stained using an anti-EGFR antibody and EGF Alexa Fluor 555 (red) and EGFR (green) internalisation was assessed in brightfield and fluorescent images taken using confocal microscopy. The right panels are enlarged images of the indicated area in the left panels. Scale bar = 10 μm. (b) At least 50 cells per condition were blindly scored for three separate experiments based on the presence of EGF Alexa Fluor 555 and EGFR co-staining post acid wash. (c) HeLa cells were transfected as indicated. 72 hrs post transfection the cells were starved in serum free medium for 3 hrs prior to incubation with 0.32 nM EGF. Whole cell lysates were harvested and levels of phosphorylated ERK1/2, ERK1/2, EGFR and tubulin were assessed by immuno-blotting using anti-pERK1/2, anti-ERK1/2, anti-EGFR and anti-tubulin antibodies. ** p<0.01
Mentions: This indicated that USP17 was necessary for EGF entry. However, it could have also been explained by USP17 depletion resulting in the trafficking of EGFR away from the plasma membrane. Therefore, to investigate if USP17 was altering the location of EGFR we examined the localisation of EGF, along with EGFR, using an anti-EGFR antibody. In control HeLa cells we observed EGFR on the plasma membrane prior to stimulation, and upon EGF treatment, EGF and EGFR co-localised to internal vesicular structures which were more prominent after cells were stripped of external EGF using an acid wash (Figs 2A, top panels, 2B). In USP17 depleted cells we again observed prominent EGFR plasma membrane staining (Fig. 2A, bottom left panels). However, when EGF was added, both the EGF and EGFR remained at the plasma membrane and the EGF was lost upon acid wash demonstrating it had failed to enter the cell (Figs 2A, bottom middle and bottom right panels, 2B).

Bottom Line: 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.

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