UBPY Overexpression Reduces the Level of Ligand-induced EGFR Ubiquitination
We first examined whether overexpression of UBPY affects the ubiquitination level of EGFR in EGF-stimulated cells. COS-7 cells were transfected with HA-tagged UBPY or its mutant, together with EGFR, c-Cbl, and FLAG-tagged Ub to enhance the receptor ubiquitination, and stimulated with EGF for 15 or 30 min. Ubiquitination of EGFR was detected by immunoprecipitation of EGFR from these cells followed by immunoblotting with anti-FLAG antibody (, top). In mock-transfected cells, EGFR ubiquitination was induced by 15 min of EGF stimulation. In cells overexpressing wild-type UBPY, the level of EGFR ubiquitination was significantly reduced at both 15 and 30 min after EGF treatment. No such effect was observed on overexpression of UBPYC748A
, a catalytically inactive mutant in which cysteine 748 in the Cys box is replaced by alanine (Naviglio et al., 1998
), indicating that the Ub isopeptidase activity of UBPY is required for the effect.
Figure 1. Effect of UBPY overexpression on EGFR down-regulation. (A) COS-7 cells were transfected with mock, HA-UBPY, HA-UBPYC748A, or HA-AMSH expression vector together with EGFR, c-Cbl, and FLAG-Ub, and stimulated with EGF for 15 or 30 min. EGFR was immunoprecipitated (more ...)
In addition to UBPY, the SH3 domain of STAM binds to AMSH (associated molecule with the SH3 domain of STAM), a protein that contains the JAB1/MPN/Mov34 metalloenzyme (JAMM) motif (Tanaka et al., 1999
). Recently, AMSH was shown to possess deubiquitinating activity toward EGFR in vitro (McCullough et al., 2004
). We therefore examined whether AMSH affects the ubiquitination level of EGFR. In cells overexpressing HA-tagged AMSH, however, EGFR was ubiquitinated to a similar level to that in mock-transfected cells after EGF stimulation (, top). EGFR was not drastically degraded by 30 min of EGF treatment (, middle). Different UBPY constructs and AMSH were expressed at similar levels (, bottom).
UBPY Overexpression Delays the Rate of Ligand-induced EGFR Degradation
To examine whether the reduced ubiquitination of ligand-activated EGFR in UBPY-overexpressing cells affects the down-regulation of the receptor, COS-7 cells were transfected with FLAG-tagged UBPY or UBPYC748A together with EGFR and c-Cbl and then stimulated with EGF for 1 or 3 h. EGFR was immunoprecipitated with anti-EGFR antibody and detected by immunoblotting with the same antibody (, top). After 3 h of EGF stimulation, EGFR was mostly degraded in mock- as well as in UBPYC748A-transfected cells. In contrast, a significant amount of EGFR was still detectable in cells transfected with wild-type UBPY, indicating that the reduced EGFR ubiquitination in these cells resulted in delayed down-regulation of the receptor.
UBPY Deubiquitinates EGFR Directly
Next, we examined using an in vitro assay whether the reduced ubiquitination of EGFR in UBPY-overexpressing cells is due to a direct action of UBPY on EGFR. FLAG-tagged UBPY, UBPYC748A, and AMSH were expressed in COS-7 cells, immunoprecipitated with anti-FLAG antibody, and eluted with the FLAG competing peptide. The purity of the recombinant proteins in eluted fractions was assessed by CBB staining after SDS-PAGE. In addition to a major band that corresponds to intact UBPY (, top, closed arrowhead), two smaller minor bands were detected in the UBPY and UBPYC748A fractions (, top, asterisks). They most likely represent degradation products of UBPY because they were not detected in the mock and AMSH fractions and one of them was detected by anti-FLAG immunoblotting (, bottom, asterisks). In the AMSH fraction, no protein but intact AMSH was detected by CBB staining as well as immunoblotting with anti-FLAG antibody (, open arrowheads). No band was detected in the fraction prepared from mock-transfected cells (). Using purified BSA as a standard in CBB staining, the yield of the recombinant proteins was roughly estimated to be 2.5 μg for UBPY and AMSH and 1 μg for UBPYC748A from transfected cells in a 6-cm dish (our unpublished data).
Figure 2. Deubiquitination of EGFR by UBPY in vitro. (A) FLAG-UBPY, FLAG-UBPYC748A, and FLAG-AMSH were expressed in COS-7 cells, immunoprecipitated with anti-FLAG antibody, and eluted with an excess amount of the FLAG peptide. Proteins in the eluates were separated (more ...)
In poly-Ub chains, Ub is mainly conjugated to lysine 48 (K48) or lysine 63 (K63) of another Ub molecule by an isopeptide bond. It has been shown that bacterially expressed glutathione S
-transferase (GST)-UBPY fusion protein cleaves the isopeptide bonds in K48-linked Ub chains (McCullough et al., 2004
). When K48- and K63-linked Ub chains (0.5 μg) were incubated with our immunopurified UBPY fraction (~0.3 μM UBPY), the amounts of Ub chains (Ub2
) were reduced and the amount of Ub monomer (Ub1
) was increased, compared with their levels when Ub chains were incubated with the mock fraction, indicating that the UBPY fraction retains the Ub isopeptidase activity (). The UBPYC748A
fraction cleaved neither K48- nor K63-linked Ub chains, excluding the possibility that the enzymatic activity in the wild-type UBPY fraction is due to other UBPY-associated or contaminating proteases (). As reported for bacterially expressed GST-AMSH fusion protein (McCullough et al., 2004
), the AMSH fraction exhibited isopeptidase activity toward K63-linked but not K48-linked Ub chains (). It is unclear why more K63-linked Ub dimers (Ub2
) than monomers (Ub1
) were accumulated after incubation with the AMSH fraction (, bottom).
We then examined the deubiquitinating activity of these fractions toward EGFR. COS-7 cells were transfected with EGFR, c-Cbl, and FLAG-Ub and stimulated with EGF for 15 min. Ubiquitinated EGFR was immunoprecipitated from these cells and incubated with the UBPY and AMSH fractions (~0.3 μM recombinant protein) for 1 h. Whereas the wild-type UBPY fraction completely deubiquitinated EGFR, the UBPYC748A and AMSH fractions exhibited undetectable deubiquitinating activity on EGFR in this assay (, top). The amount of EGFR was unchanged by the incubation (, bottom).
UBPY Binds to Ligand-activated EGFR in an Hrs- and STAM-dependent Manner
The deubiquitination of EGFR by UBPY suggested that UBPY might bind to ubiquitinated EGFR stably. We examined this possibility by coimmunoprecipitation experiments. Endogenous EGFR was immunoprecipitated from HeLa cells stimulated with EGF for up to 3 h. Immunoblotting of the precipitates with anti-UBPY antibody showed that EGF induced the binding of endogenous UBPY to EGFR (, top). The binding was detectable after 30 min of stimulation and reached a maximal level in ~1 to 2 h.
Figure 3. UBPY binding to EGFR. (A) HeLa cells were stimulated with EGF for indicated periods. EGFR was immunoprecipitated from the cells and immunoblotted with anti-UBPY (top) or anti-EGFR (middle) antibody. The expression level of UBPY was assessed by immunoblotting (more ...)
We next examined the binding of the mutants, UBPYC748A
, to EGFR. UBPYΔSBM
harbors mutations in the two STAM-binding motifs (SBMs) composed of nine amino acids PX(V/I)(D/N)RXXKP and lacks the ability to interact with the Hrs–STAM complex (Kato et al., 2000
). HeLa cells were transfected with FLAG-tagged UBPY constructs and stimulated with EGF for 1 h. EGFR was immunoprecipitated from these cells, and bound UBPY proteins were detected by anti-FLAG immunoblotting. UBPYC748A
bound to EGFR more efficiently than the wild-type protein after EGF treatment (, top). UBPYΔSBM
, in contrast, did not bind to EGFR (, top).
To test whether the binding of UBPY to EGFR requires the Hrs–STAM complex, we examined the effect of RNAi-mediated depletion of Hrs on the binding. We have previously shown that the Hrs depletion results in the mislocalization of STAM proteins to the cytoplasm and in the reduction of their levels within cells, leading to the depletion of the Hrs–STAM complex on the endosomal membrane (Mizuno et al., 2004
). In Hrs siRNA-transfected cells, EGF-induced binding of UBPY to EGFR was drastically reduced (, top). The levels of EGFR (, second panel from the top) and UBPY (, bottom) were unchanged by Hrs depletion. These results, together with the inability of UBPYΔSBM
to bind to EGFR (), suggest that the interaction with the Hrs–STAM complex is required for UBPY to bind to EGFR within cells.
UBPY Is Not Stably Associated with the Hrs–STAM Complex
The Hrs–STAM complex binds to ubiquitinated EGFR (Morino et al., 2004
; Sigismund et al., 2005
), raising the possibility that UBPY and EGFR are not directly associated but are linked by the Hrs–STAM complex. To test this possibility, lysates of HeLa cells stimulated with EGF for up to 60 min were immunoprecipitated with antibody against STAM1, one of the two STAM family proteins with redundant function (Komada and Kitamura, 2005
). However, coprecipitation of endogenous UBPY with endogenous STAM1 was undetectable irrespective of EGF stimulation (Figure S1A, top). Hrs, in contrast, was continuously associated with STAM1 (Figure S1A, middle). In a converse experiment, anti-UBPY antibody did not coprecipitate STAM1 from unstimulated or EGF-stimulated cells (Figure S1B). The failure to coprecipitate UBPY and STAM1 is consistent with the low affinity of the UBPY SBMs to the STAM SH3 domain (Kd
= 27 μM; Kaneko et al., 2003
). These results therefore exclude the possibilities that UBPY is stably associated with the Hrs–STAM complex and that the UBPY-EGFR interaction is mediated by the presence of the Hrs–STAM complex between them.
UBPY Undergoes EGF-induced Ubiquitination
When HA-tagged UBPYC748A, but not wild-type UBPY, was expressed in HeLa cells together with FLAG-Ub, a faint band that migrates slightly more slowly than the major band was detected by anti-HA immunoblotting (, left, closed and open arrowheads). We examined whether this shift-up is due to the ubiquitination of UBPYC748A. Immunoprecipitation of HA-UBPYC748A followed by immunoblotting with anti-FLAG antibody showed that UBPYC748A was indeed ubiquitinated to some extent in unstimulated cells (, right). Moreover, stimulation of the cells with EGF for 15 min elevated the ubiquitination level of the protein (, right). Ubiquitinated UBPYC748A was mainly detected as a single band (, right, arrowhead) with the same mobility as the faint band detected by anti-HA antibody, suggesting that UBPY is mostly monoubiquitinated. Smeared bands detected above monoubiquitinated UBPYC748A by anti-FLAG antibody (, right, asterisk) could represent polyubiquitinated UBPY or UBPY-associated ubiquitinated proteins. In contrast, ubiquitination of wild-type UBPY was undetectable in unstimulated or EGF-stimulated cells (, right), suggesting a possibility that UBPY deubiquitinates itself, thereby regulating its own function.
Figure 4. Ubiquitination of UBPY. HeLa cells were transfected with HA-UBPY or HA-UBPYC748A together with FLAG-Ub and stimulated with or without EGF for 15 min. Lysates of the cells were immunoblotted with anti-HA antibody (left), or immunoprecipitated with anti-HA (more ...)
UBPY Functions at Endosomes
To elucidate the subcellular localization of UBPY, we performed immunofluorescence staining of HeLa cells with anti-UBPY antibody. As a negative control, we used cells in which UBPY was depleted by RNAi. In cells transfected with an siRNA expression vector that targets human UBPY, UBPY expression was significantly reduced (, third panel from the top). Staining of mock-transfected cells with anti-UBPY antibody exhibited a cytoplasmic localization pattern that was not characteristic of any single organelle (). This staining was mostly lost in siRNA-transfected cells (), indicating that it represents the UBPY localization but not background staining. However, when HA-tagged Hrs was exogenously expressed, UBPY colocalized with Hrs on enlarged endosomes generated by Hrs overexpression (, arrowheads). SKD1 is a mammalian orthologue of yeast Vps4, an AAA-type ATPase that also participates in the endosomal sorting of ubiquitinated membrane proteins (Katzmann et al., 2002
). Overexpression of SKD1E235Q
, a dominant-negative SKD1 mutant lacking ATPase activity, causes the accumulation of endosomal proteins on morphologically aberrant endosomes (Bishop and Woodman, 2000
; Yoshimori et al., 2000
). In SKD1E235Q
-overexpressing cells, UBPY also localized to the SKD1E235Q
-positive aberrant endosomes (, arrowheads).
Figure 7. Effect of UBPY depletion on EGFR down-regulation. (A) HeLa cells were transfected with mock or UBPY siRNA expression vector together with FLAG-Ub and stimulated with EGF for indicated periods. EGFR was immunoprecipitated from the cells and immunoblotted (more ...)
Figure 5. Subcellular localization of endogenous UBPY. (A and B) HeLa cells were transfected with mock (A) or UBPY siRNA (B) expression vector and stained with anti-UBPY antibody. (C–C″) HeLa cells were transfected with HA-tagged Hrs and double-stained (more ...)
Exogenously expressed UBPY localized diffusely in the cytoplasm but was similarly accumulated on aberrant endosomes when Hrs or SKD1E235Q was overexpressed (Figure S2, A–A″ and C–C″). In contrast, HA-UBPYC748A localized on punctate structures even in the absence of overexpressed Hrs or SKD1E235Q () and overlapped mostly with endogenous Hrs () and partially with a late endosome marker LAMP1 (). EGFR localized on the cell surface in these cells when they were unstimulated (). After 60 min of EGF stimulation, however, clear colocalization of endocytosed EGFR with UBPYC748A was observed on the punctate structures (). These results suggest that UBPY partially localizes and deubiquitinates EGFR on endosomes.
Figure 6. Subcellular localization of UBPYC748A. (A–A″, B–B″) HeLa cells were transfected with HA-UBPYC748A and stained with anti-HA antibody (A and B), together with anti-Hrs (A′) or anti-LAMP1 (B′) antibody. (C–C″, (more ...)
Endosomal Localization of UBPY Is Not Mediated by the Hrs–STAM Complex
In cells overexpressing Hrs or SKD1E235Q, UBPYΔSBM localized on aberrant endosomes (Figure S2, B–B″ and D–D″). This localization pattern was essentially the same as that observed for wild-type UBPY (Figure S2, A–A″ and C–C″), suggesting that the endosomal localization of UBPY is independent of its interaction with the Hrs–STAM complex. To further test this possibility, we examined the effect of Hrs depletion, which results in the depletion of the endosomal Hrs–STAM complex, on the localization of UBPYC748A. In Hrs siRNA-transfected cells, UBPYC748A exhibited a punctate localization (), which was similar to that in untransfected cells (). The UBPYC748A-positive punctate structures in Hrs-depleted cells were mostly positive for an early endosome marker EEA1 (Figure S3, A–A″) and partially positive for LAMP1 (Figure S3, B–B″), strongly suggesting that the endosomal localization of UBPY is regulated by an Hrs- and STAM-independent mechanism.
UBPY Depletion Elevates the Level of EGFR Ubiquitination and Accelerates Its Down-Regulation
We last examined the effects of RNAi-mediated UBPY depletion on EGFR down-regulation as well as the cytoplasmic free Ub level. HeLa cells were transfected with the mock or UBPY siRNA expression vector together with FLAG-Ub, and stimulated with EGF for up to 60 min. Immunoblot analysis of the total cell lysates with anti-UBPY antibody showed that transfection of the siRNA resulted in a significant reduction in the UBPY level (, third panel from the top). EGFR was immunoprecipitated from these cells and immunoblotted with anti-FLAG antibody. The ubiquitination level of ligand-activated EGFR was higher in siRNA-transfected cells than in mock-transfected cells in all periods, especially at 30 and 60 min, after EGF stimulation, suggesting that UBPY deubiquitinates ligand-activated EGFR in normal cells (, top). The level of EGFR was similar between these cells until 60 min of stimulation (, second panel from the top). To examine the rate of EGFR down-regulation in UBPY-depleted cells, cells were stimulated with EGF for longer periods. After 2 and 3 h of stimulation, less EGFR molecules remained in siRNA-transfected cells than in mock-transfected cells (, top). Quantification of the intensity of bands corresponding to intact EGFR indicated that 69% (mock) versus 56% (siRNA) of EGFR remained after 2 h stimulation, and 41% (mock) versus 22% (siRNA) remained after 3 h stimulation. This experiment was repeated three times, and the mean ± SD of the relative EGFR levels remaining in EGF-stimulated cells are shown in . At 3 h after stimulation, the level of EGFR in UBPY-depleted cells was consistently reduced to ~50% of that in mock-transfected cells, suggesting that the EGFR down-regulation is accelerated in the absence of UBPY.
Doa4, a yeast deubiquitinating enzyme homologous to mammalian UBPY, is implicated in maintaining the level of free Ub in the cytoplasm by recycling Ub molecules from proteasome- and vacuole-targeted ubiquitinated proteins (Swaminathan et al., 1999
; Amerik et al., 2000
). We therefore examined the effect of depleting or overexpressing UBPY on the cytoplasmic free Ub level in unstimulated cells as well as in cells stimulated with EGF for 15 min. However, immunoblot analysis of the lysates of cells transfected with the mock, UBPY siRNA, and FLAG-UBPY expression vectors with anti-Ub antibody showed that the level of Ub monomer was similar among these cells irrespective of EGF stimulation ().