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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 recombinant EGF. After 1 min the cells were fixed and stained using an anti-PIP2 antibody (green) and PIP2 localisation was assessed in brightfield and fluorescent images taken using confocal microscopy. Bottom panels are enlarged images of the indicated area in the upper panels. Scale bar = 10 μm. (b) At least 50 cells per condition were blindly scored for two separate experiments based on the presence of PIP2 at the plasma membrane. (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 recombinant EGF. After 1 min the cells were fixed and stained using an anti-FLAG antibody (green) and PIP5K beta localisation was assessed in brightfield and fluorescent images. Bottom panels are enlarged images of the indicated area in the upper panels. Scale bar = 10 μm. (d) At least 50 cells per condition were blindly scored for two separate experiments based on the presence of PIP5K beta at the plasma membrane. * p<0.05, ** p<0.01, *** p<0.001.
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Figure 7: (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 recombinant EGF. After 1 min the cells were fixed and stained using an anti-PIP2 antibody (green) and PIP2 localisation was assessed in brightfield and fluorescent images taken using confocal microscopy. Bottom panels are enlarged images of the indicated area in the upper panels. Scale bar = 10 μm. (b) At least 50 cells per condition were blindly scored for two separate experiments based on the presence of PIP2 at the plasma membrane. (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 recombinant EGF. After 1 min the cells were fixed and stained using an anti-FLAG antibody (green) and PIP5K beta localisation was assessed in brightfield and fluorescent images. Bottom panels are enlarged images of the indicated area in the upper panels. Scale bar = 10 μm. (d) At least 50 cells per condition were blindly scored for two separate experiments based on the presence of PIP5K beta at the plasma membrane. * p<0.05, ** p<0.01, *** p<0.001.

Mentions: Phosphoinositol-4, 5-phosphate (PIP2) is a lipid whose production at the plasma membrane allows the recruitment of many of the components of the CME machinery [19]. We also looked at the impact of USP17 knockdown on the localisation of PIP2, using an anti-PIP2 antibody, and we observed that USP17 loss resulted in a significant reduction in the PIP2 observed around the periphery of the cell upon EGF treatment (Figs 7A, middle and right panels, 7B). A number of enzymes are capable of producing PIP2 at the plasma membrane, however phosphoinositol-5-phosphate kinase beta (PIP5Kβ) has previously been shown to be necessary for EGFR and TfR endocytosis [20, 21]. Therefore, we looked at the localisation of a carboxy terminal FLAG-tagged PIP5Kβ and found that USP17 loss also resulted in a significant decrease in plasma membrane localisation of PIP5Kβ (Figs 7C, middle and right panels, 7D).


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 recombinant EGF. After 1 min the cells were fixed and stained using an anti-PIP2 antibody (green) and PIP2 localisation was assessed in brightfield and fluorescent images taken using confocal microscopy. Bottom panels are enlarged images of the indicated area in the upper panels. Scale bar = 10 μm. (b) At least 50 cells per condition were blindly scored for two separate experiments based on the presence of PIP2 at the plasma membrane. (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 recombinant EGF. After 1 min the cells were fixed and stained using an anti-FLAG antibody (green) and PIP5K beta localisation was assessed in brightfield and fluorescent images. Bottom panels are enlarged images of the indicated area in the upper panels. Scale bar = 10 μm. (d) At least 50 cells per condition were blindly scored for two separate experiments based on the presence of PIP5K beta at the plasma membrane. * p<0.05, ** p<0.01, *** p<0.001.
© Copyright Policy - open-access
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Figure 7: (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 recombinant EGF. After 1 min the cells were fixed and stained using an anti-PIP2 antibody (green) and PIP2 localisation was assessed in brightfield and fluorescent images taken using confocal microscopy. Bottom panels are enlarged images of the indicated area in the upper panels. Scale bar = 10 μm. (b) At least 50 cells per condition were blindly scored for two separate experiments based on the presence of PIP2 at the plasma membrane. (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 recombinant EGF. After 1 min the cells were fixed and stained using an anti-FLAG antibody (green) and PIP5K beta localisation was assessed in brightfield and fluorescent images. Bottom panels are enlarged images of the indicated area in the upper panels. Scale bar = 10 μm. (d) At least 50 cells per condition were blindly scored for two separate experiments based on the presence of PIP5K beta at the plasma membrane. * p<0.05, ** p<0.01, *** p<0.001.
Mentions: Phosphoinositol-4, 5-phosphate (PIP2) is a lipid whose production at the plasma membrane allows the recruitment of many of the components of the CME machinery [19]. We also looked at the impact of USP17 knockdown on the localisation of PIP2, using an anti-PIP2 antibody, and we observed that USP17 loss resulted in a significant reduction in the PIP2 observed around the periphery of the cell upon EGF treatment (Figs 7A, middle and right panels, 7B). A number of enzymes are capable of producing PIP2 at the plasma membrane, however phosphoinositol-5-phosphate kinase beta (PIP5Kβ) has previously been shown to be necessary for EGFR and TfR endocytosis [20, 21]. Therefore, we looked at the localisation of a carboxy terminal FLAG-tagged PIP5Kβ and found that USP17 loss also resulted in a significant decrease in plasma membrane localisation of PIP5Kβ (Figs 7C, middle and right panels, 7D).

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