Ectopically expressed Ankrd 13A, 13B, and 13D bind to EGF-activated EGFR directly in a UIM-dependent manner
To study the involvement of Ankrd 13 proteins in the endocytosis of ubiquitinated cell surface proteins, we first examined the interaction between Ankrd 13 proteins and EGFR in EGF-stimulated cells. HeLa cells were transfected with Ankrd 13 proteins tagged with a FLAG epitope at the N-terminus. After treatment with EGF for 5 or 30 min, EGFR was immunoprecipitated from the cells and analyzed by immunoblotting. Blotting with anti-EGFR and anti-Ub antibodies detected ubiquitinated EGFR after 5 min of EGF stimulation (, asterisks). Blotting with anti-FLAG antibody showed that Ankrd 13A, 13B, and 13D, but not 13C, bind to EGFR upon EGF treatment (). The binding reached its maximal level within 5 min after the stimulation and decreased in 30 min, correlating with the level of EGFR ubiquitination.
To determine the region required for EGFR binding in Ankrd 13A, we examined the binding of truncated mutants lacking the three ankyrin repeats (ΔAR) or four UIMs (ΔUIM; ). Whereas deletion of the ankyrin repeats had no effect on EGFR binding, that of the UIMs completely abolished it (). These results suggested that Ankrd 13A, 13B, and 13D bind via the UIMs either directly to the Ub moiety of ligand-activated EGFR or to another ubiquitinated protein associated with activated EGFR. To discriminate between the possibilities, we performed a pull-down experiment using purified Ankrd 13A and EGFR. FLAG-Ankrd 13A–transfected HeLa cells were lysed at 100°C in the presence of 1% SDS to strip associated ubiquitinated proteins from FLAG-Ankrd 13A (a hot-lysis method; see ). FLAG-Ankrd 13A was then precipitated with anti-FLAG antibody from the lysate and eluted with the FLAG-competing peptide. Similarly, untreated and EGF-treated (5 min) HeLa cells were lysed with the hot-lysis method, and endogenous EGFR was precipitated with anti-EGFR antibody coupled to protein A beads. The EGFR-immobilized beads were incubated with eluted FLAG-Ankrd 13A, and, after washing of the beads, bound Ankrd 13A was detected by anti-FLAG immunoblotting. As shown in , binding of Ankrd 13A to EGFR from untreated cells, as well as its nonspecific binding to EGFR-free beads, was undetectable. However, Ankrd 13A was pulled down by EGFR when EGFR was isolated from EGF-treated cells. These results suggested that Ankrd 13 proteins bind to ubiquitinated EGFR directly.
FIGURE 3: Ankrd 13A undergoes monoubiquitination. (A–H) HeLa cells were cotransfected with FLAG-Ankrd 13A and HA-Ub and treated with (+) or without (−) EGF for 5 min. Lysates of the cells were precipitated and blotted with indicated antibodies. (more ...)
Endogenous Ankrd 13A and 13D bind to EGF-activated EGFR
We raised antisera against specific regions of individual Ankrd 13 proteins and next examined the interaction between endogenous Ankrd 13 and EGFR. Expression of endogenous Ankrd 13A, 13C, and 13D, but not 13B, was detected with the antibodies in untransfected HeLa cells, whereas they all detected overexpressed FLAG-tagged versions strongly (). Because expression of Ankrd 13B mRNA was detected by reverse transcriptase–PCR experiments (unpublished data), the antibody was probably not sensitive enough to detect endogenous Ankrd 13B expressed at a low level. To examine the interaction between endogenous Ankrd 13 proteins and EGFR, untransfected HeLa cells were treated with EGF for 5 or 30 min, and EGFR was immunoprecipitated from the cells. Blotting of the precipitates with the anti–Ankrd 13 antibodies showed that endogenous Ankrd 13A and 13D, but not 13C, bind to EGFR upon EGF treatment with the same time course as transfected Ankrd 13 proteins ().
FIGURE 2: Endogenous Ankrd 13A, 13B, and 13D bind to EGF-activated EGFR. (A) Lysates of untransfected HeLa cells (−) and those transfected with indicated FLAG-tagged Ankrd 13 proteins were blotted with indicated antibodies. Closed and open arrowheads indicate (more ...)
Ankrd 13A undergoes monoubiquitination
When FLAG-tagged Ankrd 13A, 13B, and 13D, but not 13C, were expressed in HeLa cells, minor bands that migrate slightly more slowly than the major bands were detected with anti-FLAG antibody (, open arrowheads). Because several other UIM-bearing proteins undergo monoubiquitination (Polo et al., 2002
), we examined whether the minor bands represent monoubiquitinated forms of Ankrd 13 proteins. HeLa cells were cotransfected with FLAG-Ankrd 13A and HA-tagged Ub, and their lysates were precipitated with anti-FLAG or anti-EGFR antibody. Again, blotting of the anti-FLAG precipitates with anti-FLAG antibody produced major (, closed arrowhead) and minor (, open arrowhead) bands. Blotting with anti-HA antibody gave the minor band (, open arrowhead), which was still detected when cell lysates were prepared using a hot-lysis method to strip associated proteins from Ankrd 13A (), suggesting that it represents monoubiquitinated Ankrd 13A. The Ub-positive, high–molecular weight smear that was coprecipitated with Ankrd 13A (, asterisk) represents endogenous ubiquitinated proteins that bound to the UIMs of overexpressed Ankrd 13A, because it was not precipitated from cell lysates prepared using the hot-lysis method (). Blotting with anti-EGFR antibody showed that FLAG-Ankrd 13A mainly coprecipitates ubiquitinated, but not unmodified, EGFR, indicating that Ankrd 13A specifically binds to ubiquitinated EGFR (). Blotting of the anti-EGFR precipitates with anti-FLAG antibody produced only unmodified Ankrd 13A, indicating that when monoubiquitinated, Ankrd 13A loses the ability to bind to ubiquitinated EGFR (). As with other UIM-bearing proteins (Polo et al., 2002
), monoubiquitination of Ankrd 13A did not occur when UIMs were deleted ().
The UIMs of Ankrd 13A, 13B, and 13D bind to Lys-63–linked Ub chains
A previous mass spectrometric analysis demonstrated that EGFR mainly undergoes Lys-63–linked polyubiquitination (Huang et al., 2006
). We therefore examined whether Ankrd 13 proteins bind to Lys-63–linked Ub chains. COS-7 cells were transfected with FLAG-Ankrd 13 proteins and lysed with a hot-lysis method to strip Ankrd 13–bound ubiquitinated proteins. FLAG-Ankrd 13 proteins were then precipitated with anti-FLAG antibody coupled to protein A beads, and the Ankrd 13-immobilized beads were incubated with Lys-48– or Lys-63–linked Ub oligomers (dimer [Ub2
] to heptamer [Ub7
]). After washing the beads, bound Ub chains were detected by anti-Ub immunoblotting. As shown in , Ankrd 13A, 13B, and 13D, but not 13C, pulled down Lys-63–linked, but not Lys-48–linked, Ub chains. The binding affinity was much higher for Ub4-7
than for Ub3
, and the binding to Ub2
was undetectable (). Incubation of the Ankrd 13–bound beads with Lys-63–linked Ub chains caused a substantial decrease in the amount of Ankrd 13 proteins bound to the beads, suggesting that for some reason, binding of Ub chains to FLAG-Ankrd 13 proteins destabilizes their binding with anti-FLAG antibody (, bottom). In addition, Ankrd 13A-ΔUIM did not pull down Ub chains, indicating that UIMs are responsible for the binding ().
FIGURE 4: Ankrd 13A, 13B, and 13D bind to Lys-63–linked Ub chains. (A) FLAG-tagged Ankrd 13 proteins were precipitated from transfected COS-7 cells with anti-FLAG antibody–immobilized protein A beads using a hot-lysis method. The beads were incubated (more ...)
UIMs in the C-terminal side are important for Ub binding of Ankrd 13A
Ankrd 13A has four potential UIMs, designated here as UIMs 1–4 from the N-terminal side (). To examine the contribution of each UIM to the Ub binding of Ankrd 13A, we generated a series of Ankrd 13A mutants with point mutations in the individual UIMs. In these mutants, the invariant Ala/Val and Ser residues (, indicated with a dot in the consensus sequence) were replaced with Gly and Ala, respectively, in each UIM. When UIM 1, 2, or 3 was individually mutated, Ub binding of Ankrd 13A was not significantly reduced compared with that of the wild-type protein (, Δ1, Δ2, Δ3). By contrast, the Ub-binding ability was drastically affected when UIM 4 was mutated (, Δ4). Although a combined mutation in UIMs 1 and 2 did not severely affect the binding (, Δ1Δ2), that in UIMs 2 and 3 considerably reduced it (, Δ2Δ3). A combined mutation in UIMs 3 and 4 resulted in almost a complete loss of Ub binding (, Δ3Δ4). These results suggested that UIM 4 plays a central role in the Ub binding of Ankrd 13A, whereas UIM 3 and, to a lesser extent, UIM 2 are required for maximal binding.
To examine the requirement of individual UIMs for the EGFR binding of Ankrd 13A in living cells, we performed a coimmunoprecipitation experiment in HeLa cells transfected with Ankrd 13A UIM mutants and treated with EGF for 5 min. Immunoprecipitation of the cell lysates with anti-EGFR antibody followed by anti-FLAG immunoblotting showed that UIMs 3 and 4 are important for EGFR binding of Ankrd 13A (), in good correlation with the mutants' affinity for Ub ().
Ankrd 13A, 13B, and 13D colocalize with EGFR on the plasma membrane
Because Ankrd 13A, 13B, and 13D bound maximally to EGFR when cells were treated with EGF for 5 min (), they should colocalize with EGFR in these cells. We tested this possibility by immunofluorescence staining of HeLa cells expressing FLAG-Ankrd 13 proteins. On double staining with anti-FLAG and anti-EGFR antibodies at 5 min after EGF treatment, colocalization of Ankrd 13A, 13B, and 13D with EGFR was observed at the cell periphery in a proportion of the cells (, arrowheads). By contrast, the distribution of Ankrd 13 proteins and EGFR never overlapped intracellularly (). These results suggested that Ankrd 13 proteins bind to EGFR on the plasma membrane before EGFR undergoes internalization. Because this colocalization was readily observed in unstimulated cells at similar frequency (unpublished data), EGF-enhanced recruitment of Ankrd 13 proteins to the plasma membrane, which will be demonstrated biochemically later (see ), was undetectable by immunofluorescence staining.
FIGURE 5: Ankrd 13A, 13B, and 13D colocalize with EGFR on the plasma membrane. HeLa cells were transfected with FLAG-tagged Ankrd 13A (A–A′′), 13B (B–B′′), or 13D (C–C′′), treated with EGF (more ...)
FIGURE 6: Ankrd 13A, 13B, and 13D are anchored to the plasma membrane. HeLa cells were transfected with indicated FLAG-Ankrd 13 constructs (A, C) or not (B), incubated with (+) or without (−) EGF for 5 min, and labeled with biotin on the cell surface on (more ...)
FLAG-tagged Ankrd 13A, 13B, and 13D were also distributed intracellularly (). Although they exhibited different intracellular localization patterns, 13A and 13B colocalized considerably with a late endosome marker—cation-independent mannose 6-phosphate receptor (CI-M6PR)—in the perinuclear region (Supplemental Figure S1, A–B′′). Ankrd 13D marginally colocalized with CI-M6PR (Supplemental Figure S1, C–C′′). Ankrd 13B, but not 13A or 13D, also colocalized with an early endosome marker—early endosome antigen 1 (EEA1; Supplemental Figure S1, D–F′′). On the other hand, the cytoplasmic reticular distribution pattern of Ankrd 13A and 13D did not overlap with that of CD3δ, a marker for the endoplasmic reticulum, at detectable levels (Supplemental Figure S1, G–I′′). In addition, immunostaining of Ankrd 13–transfected cells with FK2—an anti-Ub antibody that recognizes Ub-protein conjugates but not free Ub (Fujimuro and Yokosawa, 2005
)—showed that ubiquitinated proteins are highly accumulated at all the Ankrd 13–positive sites, suggesting that overexpressed Ankrd 13A, 13B, and 13D bind to ubiquitinated proteins also intracellularly (Supplemental Figure S2).
Ankrd 13A, 13B, and 13D are anchored to the plasma membrane
To biochemically demonstrate that Ankrd 13 proteins localize to the plasma membrane, we examined whether they interact with biotinylated cell surface molecules. HeLa cells were transfected with FLAG-Ankrd 13 proteins and treated with or without EGF for 5 min. The cell surface was then labeled with biotin on ice, and biotinylated molecules were precipitated from the cell lysates with streptavidin beads. Immunoblotting of the precipitates with anti-FLAG antibody showed that FLAG-Ankrd 13A, 13B, and 13D, but not 13C, were coprecipitated with biotinylated molecules, suggesting that Ankrd 13A, 13B, and 13D are bound to a cell surface molecule(s) (). Ankrd 13 proteins were readily coprecipitated from unstimulated cells, indicating that they bind to a molecule other than ubiquitinated EGFR. However, since the amount of coprecipitated Ankrd 13 increased upon EGF stimulation, binding to ubiquitinated EGFR probably enhances Ankrd 13 association with the plasma membrane. Similarly, endogenous Ankrd 13A and 13D, but not 13C, were coprecipitated with biotinylated molecules from untransfected cells, and the amounts of plasma membrane-bound endogenous 13A and 13D were elevated after 5 min of EGF stimulation (). It should be noted that the amount of ubiquitinated EGFR on the cell surface was higher in cells overexpressing Ankrd 13A, 13B, or 13D than in mock-transfected cells after 5 min of EGF treatment (, asterisk). The significance of this observation will be further studied later ().
FIGURE 7: Overexpression of Ankrd 13A, 13B, or 13D inhibits EGFR internalization. (A, B) HeLa cells were untreated (A) or treated with EGF for 5 min (B) and stained with anti-EGFR antibody (green). Nuclei were also stained in blue. (C–E′′) (more ...)
FIGURE 8: Overexpression of truncated Ankrd 13A mutants inhibits EGFR internalization. HeLa cells were transfected with FLAG-tagged Ankrd 13A-ΔAR (A–A′′), ΔUIM (B–B′′), ΔARΔUIM (C–C′′), (more ...)
FIGURE 9: Internalization of ubiquitinated EGFR is inhibited by Ankrd 13A overexpression. (A) HeLa cells were transfected with or without FLAG-Ankrd 13A, incubated with (+) or without (−) EGF for 5 min, and labeled with biotin on the cell surface on ice. (more ...)
Whereas FLAG-Ankrd 13A was still precipitated with streptavidin beads when the ankyrin repeats were deleted (, ΔAR), the ankyrin repeats alone were not (, AR). By contrast, the UIMs alone bound to biotinylated molecules (, UIM) and deletion of the UIMs from Ankrd 13A drastically reduced the binding (, ΔUIM). Like full-length Ankrd 13A, the binding of two UIM-bearing mutants—ΔAR and UIM—to biotinylated molecules was enhanced by EGF treatment, again suggesting that the binding to cell surface–ubiquitinated EGFR contributes to the plasma membrane association of Ankrd 13A. Because two mutants bearing the central region but not the UIMs (ΔUIM and ΔARΔUIM) retained the ability to bind to biotinylated molecules and the binding was not enhanced by EGF treatment, the central region of Ankrd 13A participates in the steady-state plasma membrane association through Ub-independent interaction with an unidentified cell surface molecule.
Overexpression of Ankrd 13A, 13B, or 13D inhibits rapid EGFR internalization
To elucidate the biological significance of Ankrd 13 interaction with EGFR, we examined the effect of Ankrd 13 overexpression on the endocytic trafficking of EGFR in HeLa cells. Immunofluorescence staining of FLAG-Ankrd 13–transfected cells with anti-FLAG and anti-EGFR antibodies showed that when unstimulated, transfection of FLAG-Ankrd 13 proteins does not affect the distribution of EGFR at the cell surface (Supplemental Figure S3). Following EGF treatment for 5 min, EGFR was internalized and transported to the early endosome in untransfected cells (). In striking contrast, EGFR was mostly retained at the surface in cells overexpressing FLAG-tagged Ankrd 13A, 13B, or 13D (′). To exclude the possibility that the effect was due to the tagging of the FLAG epitope, we overexpressed Ankrd 13A bearing no epitope tag and treated the cells with EGF for 5 min. Costaining with anti-Ankrd 13A and anti-EGFR antibodies showed that overexpression of untagged Ankrd 13A similarly inhibits EGFR internalization (′). We also examined the effect of FLAG-Ankrd 13A overexpression on the uptake of a fluorescence-labeled EGF ligand. Transfected HeLa cells were incubated with Alexa Fluor 488–conjugated EGF for 10 min, fixed, and stained with anti-FLAG antibody. Internalization of the fluorescence-labeled EGF to the early endosome, which was clearly observed in untransfected cells, was drastically inhibited in Ankrd 13A-overexpressing cells (′). Because the cell surface EGFR level was not reduced by overexpression of Ankrd 13A, 13B, or 13D ( and Supplemental Figure S3), these results suggested that Ankrd 13 overexpression inhibits the ligand-induced internalization of EGFR. It should be noted, however, that after 1 h of EGF treatment, the EGFR in cells overexpressing Ankrd 13A, 13B, or 13D was internalized and exhibited the same subcellular distribution as that in untransfected cells (′, and Supplemental Figure S4), suggesting that Ankrd 13 overexpression does not lead to the complete inhibition of EGFR internalization.
An inhibitory effect on rapid EGFR internalization was similarly observed when Ankrd 13A-ΔAR (′), ΔUIM (′), ΔARΔUIM (′), or just the UIMs (′) were overexpressed, but the ankyrin repeats alone had no effect (′). In addition, the uptake of Alexa Fluor 488–conjugated transferrin was also substantially suppressed by Ankrd 13A overexpression (Supplemental Figure S5). These results are considered in the Discussion.
Internalization of ubiquitinated EGFR is inhibited by Ankrd 13A overexpression
To provide further evidence that Ankrd 13 overexpression inhibits EGFR internalization, we detected cell surface EGFR biochemically. HeLa cells were transfected with FLAG-Ankrd 13A and treated with or without EGF for 5 min. The cell surface was labeled with biotin on ice, and EGFR was precipitated from their lysates with anti-EGFR antibody. Biotinylated EGFR was then detected with streptavidin () and the intensity of the bands corresponding to unmodified (open arrowhead) and ubiquitinated (asterisk) EGFR in the blotting membrane was quantified. The surface level of unmodified EGFR was scarcely affected by Ankrd 13A overexpression in both untreated and EGF-treated cells (). Ankrd 13A overexpression also did not drastically affect the surface level of ubiquitinated EGFR in untreated cells (, left). However, it was elevated by ~2.2-fold at 5 min after EGF treatment (, right). This must be an underestimate because in our transfection experiments, FLAG-Ankrd 13A was overexpressed in at most 50% of the cells (unpublished data). These results suggested that the internalization of ubiquitinated EGFR is inhibited by Ankrd 13A overexpression. As noted before, the same results were obtained, although somewhat less clearly, in a converse experiment in which biotinylated proteins were first precipitated with streptavidin and the precipitates were blotted with anti-EGFR antibody (, asterisk).
Overexpression of Ankrd 13A, 13B, or 13D does not inhibit EGFR degradation
Degradation of EGFR was not yet observed in HeLa cells at 5 or 30 min after EGF stimulation, and overexpression of wild-type or truncated Ankrd 13 proteins did not affect the amount of EGFR at these time points (, top). To examine whether the inhibition of rapid EGFR internalization by Ankrd 13 overexpression affects the rate of EGFR degradation at later time points, we incubated HeLa cells overexpressing FLAG-Ankrd 13 proteins for 1 or 3 h in the presence of EGF and examined the total cellular level of EGFR by immunoblotting of the lysates. The EGFR level was comparable between untransfected cells and those overexpressing each of the Ankrd 13 proteins throughout the duration of EGF treatment (Supplemental Figure S6). These results were consistent with the observation that EGFR is normally internalized in Ankrd 13–overexpressing cells after 1 h of EGF treatment (′, and Supplemental Figure S4). The rate of EGFR degradation was also unaffected by overexpression of Ankrd 13A-ΔAR, ΔUIM, or ΔARΔUIM (Supplemental Figure S6).