Membrane-bound Trafficking Regulates Nuclear Transport of Integral Epidermal Growth Factor Receptor (EGFR) and ErbB-2^*

Materials

The following antibodies and chemicals were purchased commercially: anti-EGFR (sc-03, Santa Cruz Biotechnology, CA; Ab-13, Thermo Scientific, Fremont, CA); anti-importin β, anti-Sp1, anti-LAMP1, anti-calregulin, and anti-calnexin (Santa Cruz Biotechnology); anti-actin and human recombinant EGF (Sigma); anti-ErbB-2 (Ab-3) and digitonin (Calbiochem, San Diego, CA); anti-CD44 and anti-emerin (NeoMarkers); anti-FGFR-1 (sc-121, Santa Cruz Biotechnology; ab-10646, Abcam, Cambridge, MA); anti-Rab5 (BD Biosciences, Franklin Lakes, NJ); and FGF-2 (PeproTech, Rocky Hill, NJ; Sigma). The secondary antibodies were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA).

Cell Culture

A431 human epidermoid carcinoma cells, Her-5 cells (derived from Swiss 3T3 cells by stable transfection with the human EGFR expression vector), Swiss 3T3 mouse fibroblasts, and MDA-MB-468 and MDA-MB-453 human breast carcinoma cells were maintained in DMEM/F-12 supplemented with 10% FCS and 100 μg/ml penicillin and streptomycin.

Permeabilization by Digitonin

Cells were washed in a transport buffer (20 mm HEPES pH 7.0, 110 mm KOAc, 5 mm NaOAc, 2 mm Mg[OAc]₂, 1 mm egtazic acid, 2 mm dithiothreitol, and 1 mg/ml each aprotinin and leupeptin) and permeabilized with 75 μm digitonin at 4 °C for 6 min in a transport buffer. Cells were then treated with EGF at 37 °C for 30 min in a transport buffer containing 1 mm ATP (Roche, San Francisco, CA).

Biotinylation of Cell Surface Proteins

Cell surface proteins in MDA-MB-453 cells were biotinylated using 1 mm Sulfo-NHS-LC-Biotin (Pierce) at room temperature for 30 min, and the cells were further incubated at 37 °C for 15 min. Swiss 3T3 cells maintained in a medium containing 0.5% FCS for 48 h were biotinylated on the cell surface at 4 °C for 30 min and then treated with or without FGF-2 (50 ng/ml) at 37 °C for 60 min. The biotinylation reaction was quenched with PBS containing 100 mm glycine.

Cellular Fractionation, ER Purification, INM Purification, and Cytosolic/Membrane Fractionation

Cellular fractionation of non-nuclear/nuclear fractions (7) and ER purification (19) were performed as described previously. For INM purification (19), the isolated nuclei were extracted using cellular fractionation and suspended in buffer A (0.25 m sucrose, 50 mm Tris-HCl, pH 7.4, 10 mm MgCl₂, 1 mm dithiothreitol, and protease inhibitor mixture (Sigma)). After incubation with 1% (w/v) sodium citrate at 4 °C for 30 min, the suspended nuclear pellet was centrifuged at 500 × g for 15 min. The resulting pellet was digested with 250 mg/ml DNase I (Sigma) at 4 °C for 14 h. After centrifugation at 10,000 × g for 2 h, the supernatant was collected as a nucleoplasm (NP) portion. The digested pellet was submitted to recentrifugation at 100,000 × g for 20 min on a sucrose gradient (INM-sucrose), and the purified INM fraction was then collected at the 0.25–1.60 m sucrose interface. In addition, another set of digested pellet was resuspended in NETN buffer (150 mm NaCl, 1 mm EDTA, 20 mm Tris, pH 8.0, 0.5% Nonidet P-40, and protease inhibitor mixture) and then sonicated (Sonics Vibra-Cell, amplitude 30; Sonics & Materials, Newtown, CT). After centrifugation at maximum speed, the INM portion was collected. Purification of the cytosolic, organelle membrane, and nucleic fractions was performed using a ProteoExtract Subcellular Proteome Extraction Kit (Calbiochem) with a slight modification. The organelle membrane-enrich fraction was further isolated by centrifugation at 20,000 × g for 30 min to remove mitochondria and other large organelles such as cell membrane portion (26, 27) after sequential extraction according to the manufacturer's instructions.

Confocal Microscopy and Immuno Electron Microscopy (Immuno-EM)

Confocal microscopy and Immuno-EM were performed as previously described (19).

EGF-dependent Nuclear Transport of EGFR Involves Membrane-bound Trafficking

To compare the nuclear translocation of the INTERNET mechanism for EGFR (19) with that of the INFS pathway for FGFR-1 (24), we used the digitonin permeabilization assay, which is known to selectively wash away cytosolic nuclear transporters (28) such as soluble importin β from the cytoplasm. Nuclear translocation of FGFR-1 is regulated by soluble importin β, as shown by digitonin pretreatment (22). By quantifying the percentage of cells with nuclear localization of FGFR-1, we confirmed that FGF-2-dependent nuclear transport of FGFR-1 (ab-10646, Abcam) was substantially blocked after digitonin permeabilization in Swiss 3T3 cells (Fig. 1, lower panel, 78% versus 7%), which express endogenous FGFR-1 (supplemental Fig. S1). To investigate whether EGFR uses the same mechanism, we then performed the digitonin permeabilization assay in MDA-MB-468 cells harboring endogenous EGFR but not ErbB-2 or FGFR-1 (supplemental Fig. S1). Interestingly, we found that EGFR can translocate to the nucleus through membrane-bound trafficking, which is different from the classic nuclear import pathways involving cytosolic proteins, such as the soluble form of importin β (Fig. 2).

Digitonin permeabilizes the cholesterol-rich plasma membrane but leaves the NE and other intracellular membrane organelles structurally intact (28). Thus, in MDA-MB-468 cells, most of the soluble proteins in the non-nuclear fraction are selectively released from the cells during digitonin permeabilization (Fig. 2A, lanes 5 and 6 versus lanes 1 and 2). To determine whether the ER membrane was intact after digitonin permeabilization, we further performed confocal immunofluorescence of calregulin, which resides in the ER lumen. The signal from calregulin was not detected in the digitonin-permeabilized cells but was readily detectable when the ER membrane was permeabilized with Triton X-100, suggesting that the structure of the ER remained intact during digitonin treatment, as expected (Fig. 2B). After validating the properties of digitonin-permeabilized cells, we were surprised to observe that, in contrast to FGFR-1 (Fig. 1), the nuclear transport of EGFR (sc-03, Santa Cruz Biotechnology) still occurred after EGF stimulation for 30 min following digitonin pretreatment in MDA-MB-468 cells (Fig. 2C), and we detected no marked difference in the percentage of cells with EGFR nuclear translocation with (66%) or without (58%) digitonin permeabilization (Fig. 2D). We obtained similar results from the experiments performed in EGFR stable transfectant Her-5 cells (29) (Fig. 2E, lower panel, 52% versus 51%; supplemental Fig. S1). We further used different antibodies to confirm the subnuclear localization of EGFR (Ab-13, Thermo Scientific) and FGFR-1 (sc-121, Santa Cruz Biotechnology) in the digitonin permeabilization systems by examining sequential photosections of nuclei in MDA-MB-468 (supplemental Fig. S2) and Swiss 3T3 (supplemental Fig. S3) cells in multiple planes. Consistently, the results of serial focal sections clearly showed that digitonin pretreatment did not affect the processes of nuclear transport of EGFR (supplemental Fig. S2, A versus B). However, in the case of FGFR-1, FGF-2 strongly induced nuclear FGFR-1 as evident from apparent white signals in the merged images representing localization of FGFR-1 (green) in the nucleus (blue) in the absence of digitonin (supplemental Fig. S3A) while no nuclear FGFR-1 was detectable in the presence of digitonin (supplemental Fig. S3B). In addition, we performed the digitonin permeabilization assay in HeLa cells transfected with an exogenous construct of either GFP-EGFR (supplemental Fig. S4, A and B) or GFP-FGFR-1 (supplemental Fig. S4, C and D) and examined sequential photosections of a nucleus of a HeLa cell in multiple planes by green fluorescence detection. Similar to the results of endogenous EGFR and FGFR-1 in response to digitonin pretreatment, EGF-dependent nuclear transport of GFP-EGFR was still detectable following digitonin pretreatment, whereas FGF-2-induced nuclear transport of GFP-FGFR-1 was severely impaired after digitonin permeabilization. Together, these results suggest that membrane-associated proteins that cannot be washed away by digitonin pretreatment may be involved in the mechanism underlying EGFR transport into the nucleus, thus supporting the possibility that membrane-bound trafficking regulates the nuclear transport of EGFR (19), which is different from FGFR-1 nuclear translocation and from the classic nuclear import pathways involving soluble proteins.

ER Membrane-associated Importin β Is Responsible for Membrane-bound Trafficking of EGFR Nuclear Transport

As a low concentration of importin β can be detected around the nucleus in the digitonin-permeabilized cells (30) and importin β is required for nuclear translocation of EGFR (31) and ErbB-2 (23), we asked whether importin β could associate with intracellular membranes. Indeed, we detected a small pool of remaining importin β in the non-nuclear extracts of the digitonin-permeabilized cells, which contained mainly membrane-associated proteins (Fig. 3A, lanes 1 and 2 versus lanes 3 and 4). In addition, importin β in the nucleus was unchanged during digitonin treatment as digitonin was unable to permeabilize the nuclear membrane (Fig. 3A, lanes 5 and 6 versus lanes 7 and 8). The purity of the non-nuclear and nuclear fractions was validated as evident from the absence of the ER marker calregulin in the nuclear fraction (Fig. 3A, lanes 5–8) and the nuclear marker lamin B in the non-nuclear fraction (Fig. 3A, lanes 1–4).

Saksena et al. (32) demonstrated that importin-α-16, a membrane-associated isoform of Spodoptera frugiperda importin-α, associates with the ER membranes and is involved in membrane protein trafficking. To determine whether the remaining importin β also localizes with the ER membrane after digitonin permeabilization, we used an OptiPrep gradient technique, which is an established method to isolate the ER fraction as described under “Experimental Procedures.” Immunoblotting analysis of the purified ER fractions with an anti-importin β antibody showed that digitonin pretreatment did not affect the level of the ER-associated importin β (Fig. 3B, lane 4 versus lane 3), whereas we observed an apparent decrease in importin β level in the non-nuclear/non-microsomal fraction containing cytosolic proteins (Fig. 3B, lane 2 versus lane 1). Therefore, the results suggest that soluble importin β in the cytoplasm is released from the cells after digitonin treatment, while a portion of importin β is associated with the ER membrane and unaffected by digitonin permeabilization, similar to the previous report (30). Consistently, co-localization (yellow signals in the merged images) of the remaining importin β (red) and the ER membrane protein calnexin (green) in MDA-MB-468 cells was detectable by confocal microscopy even after digitonin permeabilization (Fig. 3C, left panel, arrows), whereas importin β signals in the cytoplasmic portion (Fig. 3C, inset 2 versus inset 1) but not the nuclear portion (Fig. 3C, inset 4 versus inset 3) were clearly diminished. Furthermore, quantification of the percentage of cells with co-localization showed that digitonin did not inhibit the co-localization of importin β and calnexin (Fig. 3C, right panel, 91% versus 85%). Together with the previous findings that importin β is involved in the nuclear transport of EGFR and ErbB-2 (23, 31) and that importin β further regulates INTERNET membrane trafficking of EGFR nuclear transport to the INM (19), these results suggest that the ER membrane-associated importin β is responsible for membrane-bound trafficking of EGFR to the nucleus, favoring the membrane-bound INTERNET pathway but not the ERAD pathway involving cytosolic EGFR (19, 25).

Interestingly, in addition to MDA-MB-468 cells used in EGFR experiments, importin β signals in the cytoplasmic portion was also diminished but remained in the ER membrane in Swiss 3T3 cells we used for the FGFR-1 experiments (Fig. 3D). We found that yellow signals in the merged images representing co-localization of the remaining importin β and calnexin were still detectable following digitonin pretreatment (Fig. 3D, inset 3 versus insets 1 and 2, arrows); meanwhile, importin β signals in the cytoplasmic portion were obviously decreased (Fig. 3D, inset 6 versus insets 4 and 5, arrowheads), similar to the results shown in Fig. 3C. Together with the previous findings indicating that digitonin permeabilization results in different outcomes of nuclear transport of EGFR (Fig. 2 and supplemental Fig. S2) and FGFR-1 (Fig. 1 and supplemental Fig. S3), our data suggest that the soluble importin β regulates FGFR-1 nuclear transport via the INFS model and the ER membrane-associated importin β mediates nuclear transport of EGFR through the membrane-bound INTERNET pathway.

Sec61β Regulates Nuclear Transport of ErbB-2 from the INM to the NP

Researchers have proposed that nuclear transport of ErbB-2 is similar to that of EGFR (23, 31, 33). Thus, we next asked whether ErbB-2 is also trafficked into the nucleus from the cell surface to the INM through the membrane-bound INTERNET mechanism. To this end, we analyzed ErbB-2 proteins in the INM using two cellular fractionation methods, as described under “Experimental Procedures,” in MDA-MB-453 cells in which ErbB-2 is readily detectable but EGFR and FGFR-1 were undetectable, compared with those in other cells, including MDA-MB-468, Her-5, and Swiss 3T3 cells (supplemental Fig. S1). In brief, cell surface proteins were labeled with biotin, and then the biotinylated proteins were biochemically separated into various fractions, including the non-nuclear, outer nuclear membrane (ONM), NP, and INM pellet. The INM pellet was subjected to centrifugation on a sucrose gradient (INM-sucrose) and immunoblotted with emerin, an INM marker, indicating the recovery of INM in fraction 7 (Fig. 4A, upper panel). In fraction 7, representing INM, we detected biotinylated cell surface ErbB-2 precipitated by streptavidin-agarose beads in MDA-MB-453 cells (Fig. 4A, lower panel, lane 2), indicating that cell surface ErbB-2 was localized to the INM. In addition, ultrastructural studies using immuno-EM further supported the localization of ErbB-2 inside the NE (Fig. 4B, arrows) and NP (Fig. 4B, arrowheads). We also performed confocal microscopy to examine the subnuclear localization of ErbB-2 (Ab-3, Calbiochem) in the digitonin pretreatment assay in MDA-MB-453 cells and found that nuclear transport of ErbB-2 was still detected after digitonin permeabilization (supplemental Fig. S5). These results strongly suggest that cell surface ErbB-2 translocates to the nucleus through the membrane-bound mechanism, similar to that of EGFR (19). However, the distribution of FGFR-1 in the purified INM fractions was undetectable after precipitation using streptavidin-agarose beads (Fig. 4C, lower panel, lanes 1 and 2), indicating that nuclear transport of FGFR-1 is distinct from that of ErbB-2 and does not involve membrane-bound trafficking to the INM. In addition to the above-mentioned biochemical assays, we further demonstrated the INM localization of EGFR and ErbB-2, but not FGFR-1, using confocal microscopy (supplemental Fig. S6), further supporting the nuclear transport of EGFR and ErbB-2 is entirely membrane-bound. HeLa cells were transfected with an exogenous construct of GFP-EGFR (supplemental Fig. S6A), GFP-ErbB-2 (supplemental Fig. S6B), and GFP-FGFR-1 (supplemental Fig. S6C), and detected co-localization of the green fluorescence and the INM marker emerin. We observed the yellow signals representing co-localization of GFP-EGFR/emerin (supplemental Fig. S6, A, insets 2 and 3, arrows) and GFP-ErbB-2/emerin (supplemental Fig. S6, B, insets 1 and 2, arrows), but no obvious yellow signals were detected in GFP-FGFR-1-expressed HeLa cells after FGF-2 stimulation (supplemental Fig. S6C, inset 2), indicating both of EGFR and ErbB-2, but not FGFR-1, can be targeted to the INM.

Because the translocon Sec61β has been shown to be localized in the INM, where it associates with EGFR and regulates EGFR trafficking to the NP (19), we wanted to further study whether Sec61β is also involved in and interacts with the INM-localized ErbB-2. To answer this question of ErbB-2-Sec61β association, we subjected another set of biotinylated lysates to subsequent subnuclear fractionation to obtain INM portions in NETN buffer because INM-sucrose fractions containing sucrose as a solvent were more challenging in a co-immunoprecipitation assay. First, we validated the purity of various portions by their specific markers (Fig. 5A). We did not detect any cross-contamination with the process of cellular fractionation in the INM portions (Fig. 5A, lanes 5 and 6) as evident from the presence of the INM marker LAP2 accompanying the absence of the ER markers calnexin and calregulin, the early endosome protein Rab5, the late endosome protein LAMP1, and the nuclear protein Sp1 in the INM portions. We then detected biotinylated cell surface ErbB-2 precipitated with streptavidin-agarose beads both in the INM portion (Fig. 5B, lower panel, lane 2), which was consistent results shown in Fig. 4A, and in the NP portion (Fig. 5B, lower panel, lane 4), as expected (23). Interestingly, we further showed the interaction of Sec61β with biotinylated ErbB-2 in the INM (Fig. 5B, lower panel, lane 2) but not in the NP (Fig. 5B, lower panel, lane 4) and detected no expression of Sec61β in the NP portions in MDA-MB-453 cells (Fig. 5B, upper panel). In addition, knocking down Sec61β expression by two individual small interfering RNAs (siRNA) targeting Sec61β, siRNA-Sec61β (Fig. 5C) and siRNA-Sec61β-2 (data not shown), increased ErbB-2 expression level in the INM (Fig. 5C, lane 2 versus lane 1) and decreased it in the NP (Fig. 5C, lane 4 versus lane 3), similar to previous results of EGFR in MDA-MB-468 cells (19). These results indicate that cell surface ErbB-2 is transported to the INM, where it associates with Sec61β, through membrane-bound trafficking, which is similar to the INTERNET trafficking of EGFR, and further suggest that the INM-localized Sec61β also assists in the release of membrane-embedded ErbB-2 from the lipid bilayer of the INM to the nucleus.

Nuclear Transport of EGFR Is Distinct from that of FGFR

To strengthen the notion that nuclear translocation of the membrane-bound INTERNET mechanism for EGFR is different from that of the INFS pathway for FGFR-1, we further performed a cellular fractionation assay using ProteoExtract® Subcellular Proteome Extraction kit (Fig. 6). Briefly, MDA-MB-468 (Fig. 6A) and Swiss 3T3 cells (Fig. 6B) were biochemically separated into various fractions, including the cytosolic, organelle membrane, and nucleic fractions upon their individual ligands treatment. We found that both of nuclear transport of EGFR (Fig. 6A, lanes 7–9) and FGFR-1 (Fig. 6B, lanes 7–9) occurred in a ligand-dependent manner; nevertheless, different expression ratios of EGFR and FGFR-1 were detected in the cytosolic and organelle membrane-enriched fractions. The results showed that EGFR was mainly expressed in the organelle membrane-enriched fraction and increased in response to EGF (Fig. 6A, lanes 4–6), whereas level of EGFR in the cytosolic fraction was very low and had no significant induction after EGF stimulation (Fig. 6A, lanes 1–3), favoring the membrane-bound INTERNET pathway for EGFR nuclear transport. In contrast, FGFR-1 was mainly detected in the cytosolic fraction and cytosolic FGFR-1 was increased upon FGF-2 treatment in a time-dependent manner (Fig. 6B, lanes 1–3), however, only a small amount of FGFR-1 was detected in the organelle membrane fraction and not to respond to ligand stimulation (Fig. 6B, lanes 4–6), supporting the INFS model proposed for FGFR-1 nuclear transport in which FGFR-1 is released from the cytoplasmic membrane into the cytosol and then transported into the nucleus.