The human papillomavirus type 16 E5 oncoprotein inhibits epidermal growth factor trafficking independently of endosome acidification

Abstract

The human papillomavirus type 16 E5 oncoprotein (16E5) enhances acute, ligand-dependent activation of the epidermal growth factor receptor (EGFR) and concomitantly alkalinizes endosomes, presumably by binding to the 16-kDa "c" subunit of the V-ATPase proton pump (16K) and inhibiting V-ATPase function. However, the relationship between 16K binding, endosome alkalinization, and altered EGFR signaling remains unclear. Using an antibody that we generated against 16K, we found that 16E5 associated with only a small fraction of endogenous 16K in keratinocytes, suggesting that it was unlikely that E5 could significantly affect V-ATPase function by direct inhibition. Nevertheless, E5 inhibited the acidification of endosomes, as determined by a new assay using a biologically active, pH-sensitive fluorescent EGF conjugate. Since we also found that 16E5 did not alter cell surface EGF binding, the number of EGFRs on the cell surface, or the endocytosis of prebound EGF, we postulated that it might be blocking the fusion of early endosomes with acidified vesicles. Our studies with pH-sensitive and -insensitive fluorescent EGF conjugates and fluorescent dextran confirmed that E5 prevented endosome maturation (acidification and enlargement) by inhibiting endosome fusion. The E5-dependent defect in vesicle fusion was not due to detectable disruption of actin, tubulin, vimentin, or cytokeratin filaments, suggesting that membrane fusion was being directly affected rather than vesicle transport. Perhaps most importantly, while bafilomycin A(1) (like E5) binds to 16K and inhibits endosome acidification, it did not mimic the ability of E5 to inhibit endosome enlargement or the trafficking of EGF. Thus, 16E5 alters EGF endocytic trafficking via a pH-independent inhibition of vesicle fusion.

FIG. 1.

Significant 16E5 binding to 16K occurs only when both proteins are highly overexpressed. (A) Antibodies recognizing both the HA epitope tag and 16K immunoprecipitate a 16-kDa polypeptide, which is HA positive on immunoblots specifically from COS cells transfected with HA epitope-tagged 16K DNA. Molecular mass markers (in kilodaltons) are shown on the right. IP, immunoprecipitation; IB, immunoblotting. (B) Association of 16E5 and 16K in metabolically labeled COS cells transfected with AU1 epitope-tagged 16E5 (AU1-16E5), AU1-16E5 and HA epitope-tagged 16K (HA-16K), or the empty pJS55 expression vector (JS55). Immunoprecipitations (IP) were performed with anti-16K antiserum (16K), preimmune serum from the same rabbit (P.I. 16K), or anti-AU1 antibody (AU1). (C) Immunoprecipitates (IP) from lysates of metabolically labeled HFKs stably expressing AU1-tagged 16E5 or harboring the empty pLXSN expression vector. The autoradiography exposure time was identical to that in panel B. (D) Threefold longer exposure of panel C. In all cases, immunoprecipitations were performed on cell lysates containing equal amounts of protein.

FIG. 2.

16E5 does not generally inhibit organelle acidification. (A) Acidic compartments in EGF-starved HFKs stably expressing 16E5 or harboring the empty pLXSN expression vector were labeled with LysoTracker Red for 60 min at 37°C. Where indicated, LXSN-HFKs were treated with 0.33 μM bafilomycin A₁ during labeling (+BfA). Nuclei were costained with Hoechst dye 33342 (DNA). Cells were imaged, using a fluorescence microscope. Scale bar, 20 μm. (B) Flow cytometry of EGF-starved 16E5- and LXSN-HFKs labeled with LysoTracker Yellow as described in Materials and Methods. Where indicated, cells were treated with 0.33 μM bafilomycin A₁ during labeling (+BfA). The geometric mean fluorescence values were as follows: 37 (16E5-HFKs), 39 (LXSN-HFKs), 11 (16E5-HFKs + BfA), 12 (LXSN-HFKs + BfA), 7 (unlabeled 16E5-HFKs), and 8 (unlabeled LXSN-HFKs).

FIG. 3.

16E5 and bafilomycin A₁ enhance ligand-dependent EGFR activation. (A) Recombinant human EGF (100 ng/ml; Invitrogen) was prebound to EGF-starved HFKs expressing 16E5 (or harboring the empty pLXSN expression vector) for 60 min at 4°C. The levels of EGFR tyrosine phosphorylation subsequently were determined before (0 min) and up to 150 min after warming to 37°C using anti-phosphotyrosine immunoblots (IB) of EGFR immunoprecipitates (IP). For LXSN (+BfA), 0.33 μM bafilomycin A₁ was added to LXSN-HFKs as they were shifted to 37°C. Molecular mass marker (in kilodaltons) is shown on the right. (B) Quantitative analysis of panel A using densitometry.

FIG. 4.

pHrodo-EGF measures the acidification of EGF-containing endosomes in live cells. (A) Design of pHrodo-EGF. (B) Trafficking and acidification of endosomes in unfixed EGF-starved LXSN-HFKs labeled with Alexa Fluor 488-EGF and pHrodo-EGF at 4°C before (0 min) and up to 60 min after warming to 37°C. Where indicated, 0.33 μM bafilomycin A₁ (BfA) was added to cells after 50 min at 37°C, followed by imaging 10 min later. Scale bar, 20 μm. (C) Fluorescent EGF conjugates are biologically active. Anti-phosphotyrosine immunoblot of EGFR immunoprecipitates (pY-EGFR) from EGF-starved HFKs that were treated for 5 min at 37°C with recombinant human EGF, Alexa Fluor 488-EGF, or pHrodo-EGF (all at a concentration of 960 nM). The immunoblot subsequently was stripped and relabeled to detect total EGFR in the immunoprecipitates.

FIG. 5.

16E5 inhibits endosome acidification and trafficking. Trafficking and acidification of endosomes in unfixed EGF-starved HFKs expressing 16E5 (or empty pLXSN vector) after labeling with Alexa Fluor 488-EGF and pHrodo-EGF at 4°C and warming to 37°C for up to 70 min was examined. Where indicated, 0.33 μM bafilomycin A₁ was added to cells as they were shifted to 37°C (+BfA). Scale bar, 20 μm.

FIG. 6.

16E5 does not alter surface EGFR expression or EGF binding capacity. (A) Flow cytometry of Alexa Fluor 488-EGF bound to the surface of growing (KGM) and EGF-starved (KSFM) 16E5- and LXSN-HFKs. The geometric mean fluorescence values were as follows: 21 (16E5-HFKs in KGM), 21 (LXSN-HFKs in KGM), 113 (16E5-HFKs in KSFM), and 102 (LXSN-HFKs in KSFM). (B) Flow cytometry of growing (KGM) and EGF-starved (KSFM) 16E5- and LXSN-HFKs labeled with an anti-EGFR antibody conjugated to R-phycoerythrin (PE). The geometric mean fluorescence values were as follows: 62 (16E5-HFKs in KGM), 74 (LXSN-HFKs in KGM), 195 (16E5-HFKs in KSFM), and 160 (LXSN-HFKs in KSFM).

FIG. 7.

16E5 does not slow the initial endocytosis of prebound EGF. Trafficking of Alexa Fluor 488-EGF bound to the surface of EGF-starved LXSN-HFKs, 16E5-HFKs (with or without the AU1 epitope tag), and 16E5(−20)-HFKs (which express a 20-amino-acid C-terminal 16E5 deletion mutant) at 4°C (0 min) and after warming to 37°C for 5 min or 30 min. Cells were fixed at the indicated times to allow high-resolution imaging using a 63× oil immersion objective lens. Scale bar, 20 μm. Images taken at 30 min were analyzed using Kodak MI software to determine the size of EGF-containing endosomes. Bar graphs indicate the percentages of endosomes that fall within a defined range of sizes: 0 to 165 pixels (group 1), 166 to 331 pixels (group 2), or >331 pixels (group 3).

FIG. 8.

16E5 inhibits endosome fusion. Alexa Fluor 488-EGF (green) was prebound to EGF-starved LXSN-HFKs and 16E5-HFKs at 4°C and was internalized for 10 min at 37°C before fixable Alexa Fluor 594-dextran (red) was added to the culture medium for an additional 10 min. The cells were moved to ice, washed to remove noninternalized dextran, and shifted back to 37°C for 10 or 25 min before fixation. The fusion of endosomes containing EGF with endosomes containing dextran is evidenced by merging of the green and red fluorescence signals (yellow). Scale bar, 20 μm.

FIG. 9.

16E5 does not disrupt cytoskeletal filament networks. Immunofluorescence microscopy showing actin, tubulin, vimentin, and cytokeratin filaments (green) in COS cells 24 h after transfection with AU1 epitope-tagged 16E5 or the empty pJS55 expression vector. Scale bar, 20 μm. The cells were colabeled with anti-AU1 antibody (red) to demonstrate 16E5 expression (insets).