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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 starved in serum free medium for 3 hrs prior to incubation with 0.32 nM EGF Alexa Fluor 555 and mRNA and protein samples were harvested at the time points indicated. USP17 and GAPDH (loading control) mRNA were then assessed by RT-PCR. USP17 protein levels were assessed by immunoblotting and tubulin was used as a loading control. (b) HeLa cells were transfected as indicated. 72 hrs post transfection mRNA and protein samples were harvested and USP17 mRNA and protein levels assessed as above. (c) HeLa cells were transfected with the constructs 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. After 15 min the cells were acid washed, fixed and the nuclei were stained with DAPI. EGF Alexa Fluor 555 (red) internalisation was then assessed in brightfield and fluorescent images taken using confocal microscopy. The bottom panels are enlarged images of the indicated area in the top panels. Scale bar = 20 μm. (d) At least 50 cells per condition were blindly scored for three separate experiments based on the presence of EGF Alexa Fluor 555 internalisation. (e) A549 cells were transfected as indicated and 72 hrs post transfection the cells were starved in serum free medium for 3hrs prior to incubation with 0.32nM EGF Alexa Fluor 555. After 10 mins the cells were either acid washed or washed with PBS. Subsequently they were fixed and the nuclei were stained with DAPI. EGF Alexa Fluor 555 (red) internalisation was then assessed in brightfield and fluorescent images taken using confocal microscopy. The middle panels are enlarged images of the indicated area in the upper panels. Scale bar = 25 μm. (f) 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 0.32nM EGF Alexa Fluor 555. After 45 mins the cells were acid washed, fixed and the nuclei were stained with DAPI. EGF Alexa Fluor 555 (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. ** p<0.01
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Figure 1: (a) HeLa cells were starved in serum free medium for 3 hrs prior to incubation with 0.32 nM EGF Alexa Fluor 555 and mRNA and protein samples were harvested at the time points indicated. USP17 and GAPDH (loading control) mRNA were then assessed by RT-PCR. USP17 protein levels were assessed by immunoblotting and tubulin was used as a loading control. (b) HeLa cells were transfected as indicated. 72 hrs post transfection mRNA and protein samples were harvested and USP17 mRNA and protein levels assessed as above. (c) HeLa cells were transfected with the constructs 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. After 15 min the cells were acid washed, fixed and the nuclei were stained with DAPI. EGF Alexa Fluor 555 (red) internalisation was then assessed in brightfield and fluorescent images taken using confocal microscopy. The bottom panels are enlarged images of the indicated area in the top panels. Scale bar = 20 μm. (d) At least 50 cells per condition were blindly scored for three separate experiments based on the presence of EGF Alexa Fluor 555 internalisation. (e) A549 cells were transfected as indicated and 72 hrs post transfection the cells were starved in serum free medium for 3hrs prior to incubation with 0.32nM EGF Alexa Fluor 555. After 10 mins the cells were either acid washed or washed with PBS. Subsequently they were fixed and the nuclei were stained with DAPI. EGF Alexa Fluor 555 (red) internalisation was then assessed in brightfield and fluorescent images taken using confocal microscopy. The middle panels are enlarged images of the indicated area in the upper panels. Scale bar = 25 μm. (f) 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 0.32nM EGF Alexa Fluor 555. After 45 mins the cells were acid washed, fixed and the nuclei were stained with DAPI. EGF Alexa Fluor 555 (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. ** p<0.01

Mentions: To determine if EGF induced USP17 expression we stimulated HeLa cells with EGF (0.32 nM) and observed a strong induction of both USP17 mRNA and protein (Fig. 1A) indicating that USP17 expression was indeed induced by EGF and this led us to further probe the role of USP17 in EGFR signaling.


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 starved in serum free medium for 3 hrs prior to incubation with 0.32 nM EGF Alexa Fluor 555 and mRNA and protein samples were harvested at the time points indicated. USP17 and GAPDH (loading control) mRNA were then assessed by RT-PCR. USP17 protein levels were assessed by immunoblotting and tubulin was used as a loading control. (b) HeLa cells were transfected as indicated. 72 hrs post transfection mRNA and protein samples were harvested and USP17 mRNA and protein levels assessed as above. (c) HeLa cells were transfected with the constructs 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. After 15 min the cells were acid washed, fixed and the nuclei were stained with DAPI. EGF Alexa Fluor 555 (red) internalisation was then assessed in brightfield and fluorescent images taken using confocal microscopy. The bottom panels are enlarged images of the indicated area in the top panels. Scale bar = 20 μm. (d) At least 50 cells per condition were blindly scored for three separate experiments based on the presence of EGF Alexa Fluor 555 internalisation. (e) A549 cells were transfected as indicated and 72 hrs post transfection the cells were starved in serum free medium for 3hrs prior to incubation with 0.32nM EGF Alexa Fluor 555. After 10 mins the cells were either acid washed or washed with PBS. Subsequently they were fixed and the nuclei were stained with DAPI. EGF Alexa Fluor 555 (red) internalisation was then assessed in brightfield and fluorescent images taken using confocal microscopy. The middle panels are enlarged images of the indicated area in the upper panels. Scale bar = 25 μm. (f) 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 0.32nM EGF Alexa Fluor 555. After 45 mins the cells were acid washed, fixed and the nuclei were stained with DAPI. EGF Alexa Fluor 555 (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. ** p<0.01
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 1: (a) HeLa cells were starved in serum free medium for 3 hrs prior to incubation with 0.32 nM EGF Alexa Fluor 555 and mRNA and protein samples were harvested at the time points indicated. USP17 and GAPDH (loading control) mRNA were then assessed by RT-PCR. USP17 protein levels were assessed by immunoblotting and tubulin was used as a loading control. (b) HeLa cells were transfected as indicated. 72 hrs post transfection mRNA and protein samples were harvested and USP17 mRNA and protein levels assessed as above. (c) HeLa cells were transfected with the constructs 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. After 15 min the cells were acid washed, fixed and the nuclei were stained with DAPI. EGF Alexa Fluor 555 (red) internalisation was then assessed in brightfield and fluorescent images taken using confocal microscopy. The bottom panels are enlarged images of the indicated area in the top panels. Scale bar = 20 μm. (d) At least 50 cells per condition were blindly scored for three separate experiments based on the presence of EGF Alexa Fluor 555 internalisation. (e) A549 cells were transfected as indicated and 72 hrs post transfection the cells were starved in serum free medium for 3hrs prior to incubation with 0.32nM EGF Alexa Fluor 555. After 10 mins the cells were either acid washed or washed with PBS. Subsequently they were fixed and the nuclei were stained with DAPI. EGF Alexa Fluor 555 (red) internalisation was then assessed in brightfield and fluorescent images taken using confocal microscopy. The middle panels are enlarged images of the indicated area in the upper panels. Scale bar = 25 μm. (f) 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 0.32nM EGF Alexa Fluor 555. After 45 mins the cells were acid washed, fixed and the nuclei were stained with DAPI. EGF Alexa Fluor 555 (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. ** p<0.01
Mentions: To determine if EGF induced USP17 expression we stimulated HeLa cells with EGF (0.32 nM) and observed a strong induction of both USP17 mRNA and protein (Fig. 1A) indicating that USP17 expression was indeed induced by EGF and this led us to further probe the role of USP17 in EGFR signaling.

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