EGF signaling induces ACK degradation.
Our previous studies observed that interaction of endogenous ACK with EGFR in response to EGF stimulation occurred at a late stage (30 to 60 min after the addition of EGF) of the stimulation, while exogenously expressed ACK interacted with EGFR immediately upon EGF stimulation (as fast as 1 min) (32
). We speculated that EGF stimulation might affect the expression of ACK as it affects that of Mig-6 (13
). Thus, we examined the expression of endogenous ACK in response to EGF stimulation in HeLa and COS7 cells. As shown in Fig. , unexpectedly, EGF treatment significantly reduced the expression of ACK accompanied by degradation of EGFR. This EGF-induced reduction of ACK was dependent on EGFR kinase activity. When the kinase activity of EGFR was inhibited by treatment with AG1478, a specific kinase inhibitor of EGFR, the reduction of expression of ACK in HeLa or COS7 cells upon EGF stimulation was eliminated (Fig. ), accompanied by inhibition of EGFR degradation (Fig. , lanes 5 to 8).
FIG. 1. EGF induces degradation of ACK. HeLa or COS7 cells were cultured to 90% confluence and starved overnight (12 h) in either 0.1% FBS medium (A, C, and E, HeLa cells) or serum-free medium (B and D, COS7 cells). The cells were stimulated with (more ...)
There are two possible causes for EGF-induced reduction of ACK expression in HeLa or COS7 cells. One is suppression of biosynthesis of ACK; the other is induction of degradation of ACK. To determine the cause, we pretreated HeLa cells before EGF stimulation with the protein translation inhibitor cycloheximide to eliminate protein biosynthesis and then examined the effect of EGF stimulation on ACK expression. As shown in Fig. , cycloheximide pretreatment did not prevent EGF-induced reduction of ACK level and EGFR degradation (lanes 6 to 8), indicating that EGF-induced reduction of ACK is protein synthesis independent. As a control, cycloheximide alone did not cause significant change in ACK expression level (lanes 1 to 4). Thus, we conclude that EGF induces ACK degradation and that this EGF-induced degradation of ACK is EGFR kinase activity dependent.
ACK binds to Nedd4 and is ubiquitinated upon EGF stimulation.
To elucidate the molecular mechanism underlying EGF-induced degradation of ACK, we first examined the ubiquitination of ACK in response to EGF stimulation. However, we found it difficult to detect the ubiquitination of endogenous ACK by immunoblotting the immunoprecipitated ACK with antiubiquitin antibody because of the low level of endogenous ACK. To overcome this difficulty, we developed a GST-ACK-Uba pulldown assay for detection of ubiquitinated ACK, based on our previous studies showing that the ACK-Uba domain has a high binding affinity for ubiquitinated proteins (32
). The GST-ACK-Uba precipitates only ubiquitinated proteins from cell lysates; thus it is able to enrich ubiquitinated ACK for detection.
With the GST-ACK-Uba pulldown assay in HeLa cells, EGFR ubiquitination was detected after 10 min stimulation with EGF (Fig. , top panel, lane 2). The EGFR ubiquitination declined rapidly, due to either degradation or dissociation of Cbl, the E3 ubiquitin ligase for EGFR. ACK was persistently ubiquitinated in response to EGF stimulation (Fig. , second panel from top, lanes 2 to 4), suggesting that ubiquitination of ACK might be catalyzed by a different E3 ubiquitin ligase from that for EGFR ubiquitination, which is consistent with our previous data that ACK was not able to complex with Cbl (32
). To further characterize EGF-induced ubiquitination of ACK, we overexpressed Myc-tagged ACK1 (Myc-ACK1) along with Cdc42Q61L, a GTPase-defective mutant of Cdc42 that activates ACK (44
), in HEK293 cells and examined the ubiquitination of Myc-ACK1 immunoprecipitation complex upon EGF stimulation (Fig. ). We found that the HECT family E3 ubiquitin ligase Nedd4 was coprecipitated with ACK upon EGF stimulation (Fig. , third panel, lanes 2 and 3), suggesting that ACK1 and Nedd4 are in complex in response to EGFR signaling. We also observed that Grb2 was associated with ACK1 upon EGF stimulation (Fig. , fourth panel). EGF stimulation rapidly induced ubiquitination of the ACK1 immunoprecipitation complex at 180 kDa (Fig. , top panel), similar to the ubiquitinated ACK band in Fig. . To confirm that this EGF-induced ubiquitination is on ACK1, not the coprecipitated proteins, we transiently expressed His-tagged ACK1 alone or with Cdc42Q61L in HEK293 cells, stimulated the cells with EGF, and denatured the cell lysates with 6 M urea to dissemble any protein complex. Then we precipitated denatured ACK1 with nickel-NTA beads and detected ubiquitination of precipitated ACK1 by immunoblotting with anti-ubiquitin antibody. As shown in Fig. , EGF stimulation induced ubiquitination of ACK (lanes 2 and 3). Coexpression with Cdc42Q61L enormously enhanced EGF-induced ubiquitination of ACK1 (lanes 5 and 6). Taken together, these data indicate that EGF stimulation promotes the association of ACK with E3 ubiquitin ligase Nedd4 and therefore the ubiquitination of ACK.
FIG. 2. ACK is ubiquitinated in response to EGF stimulation. (A) HeLa cells were starved overnight (12 h) in 0.1% FBS medium and treated with MG-132 (10 μM) 30 min before EGF stimulation. After EGF stimulation for the indicated time, the cells (more ...)
To confirm that Nedd4 could ubiquitinate ACK in cells, we cotransfected human Nedd4-1 with ACK in HEK293 cells and examined the ubiquitination of ACK (Fig. ). As expected, overexpression of Nedd4-1 caused extensive ubiquitination of ACK (Fig. , top panel, lane 2). We also examined in vitro ubiquitination of ACK by Nedd4-1. Incubation of purified Nedd4-1 with ACK1 immunoprecipitated from HEK293 cells caused strong ubiquitination of ACK1 (Fig. , top panel, lane 2). No ubiquitination was detected in samples that had no addition of Nedd4-1 or/and ACK1 (Fig. , top panel, lanes 1, 3, and 4), indicating that the ubiquitination is both ACK1 and Nedd4-1 dependent. In addition, nonubiquitinated ACK1 protein was significantly reduced by incubation with Nedd4-1 (Fig. , bottom panel, compare lane 2 with lane 4), presumably resulting from an ubiquitination-caused gel shift, implicating that ACK1 is ubiquitinated by Nedd4-1 in vitro. Taken together, these data suggest that Nedd4 is the E3 ubiquitin ligase ubiquitinating ACK in response to EGF signaling.
ACK interacts with Nedd4 via a conserved WW domain-binding motif.
We next characterized the interaction of ACK with Nedd4. Nedd4 belongs to the WW domain-containing family of E3 ubiquitin ligases (17
) and has three or four type I WW domains that interact with the PPXY motif in its ubiquitination substrates or regulatory proteins (36
). The peptide sequence of ACK contains a PPXY motif located in a proline-rich region from amino acids 647 to 650 in mouse ACK1 (Fig. ). This PPXY motif is also present in epithelial sodium channel subunits, which are known Nedd4 substrates (12
) (Fig. ). We designated this region the WW-binding domain (WWBD). To confirm that WWBD is the Nedd4 binding site, we constructed a GST-ACK-WWBD fusion protein for a Nedd4 pulldown assay. As shown in lane 2 of Fig. , GST-ACK-WWBD precipitated Nedd4 from COS7 cell lysates, whereas GST (lane 4) or GSH beads (lane 1) did not, indicating that Nedd4 interacts with the WWBD. To confirm interaction of ACK with Nedd4 in cells, Myc-tagged ACK1 was expressed in HEK293 cells. Endogenous Nedd4 was coimmunoprecipitated with ACK after immunoprecipitation of ACK with anti-Myc antibody (Fig. ). To determine whether the PPXY motif is the site in ACK interacting with Nedd4, we mutated Y650 to alanine, changing PPAY to PPAA, and then examined coimmunoprecipitation with Nedd4-1. As shown in Fig. , while wild-type ACK coprecipitated Nedd4-1 (lane 2 of the right panel), the Y650A mutation eliminated the interaction of ACK with Nedd4-1 (lane 3 of the right panel), indicating that Y650 is required for ACK to bind to Nedd4-1. To confirm that interaction between ACK and Nedd4-1 is direct and no other protein is needed, we performed an in vitro
binding assay with purified Nedd4-1 and ACK-WWBD proteins. As shown in Fig. , purified His-tagged Nedd4-1 was coprecipitated with purified GST-WWBD (lane 3) but not with GST-WWBD-Y650A, the Nedd4-binding-defective mutant (lane 2), or GST (control) (lane 1), demonstrating a direct interaction between the ACK-WWBD and Nedd4-1. Furthermore, in a GST-ACK-Uba pulldown assay, abolishing the binding of ACK to Nedd4-1 by mutation of Y650 reduced the ubiquitination of ACK by 90% (Fig. , lane 3), suggesting that the binding is required for the ubiquitination.
FIG. 3. ACK interacts with Nedd4 through a conserved PPXY WW-binding motif. (A) Alignment of the PPXY WW-binding motif of ACK with the known Nedd4-binding site of ENaC subunits. The boxed residues are the conserved WW-binding motif. (B) Glutathione (GSH)-conjugated (more ...) The WW3 domain of human Nedd4-1 interacts with ACK and is required for ubiquitination of ACK.
To determine the region in Nedd4 that interacts with ACK, we subcloned each of four WW domains of human Nedd4-1 into a GST fusion protein expression vector (Fig. ) and performed the GST fusion protein pulldown assay by incubating purified bead-bound GST-WW domain fusion proteins with ACK-overexpressed HEK293 cell lysates. As shown in Fig. , the bead-bound GST-WW3 precipitated Myc-ACK1 from the cell lysates (lane 4, top panel), while the GST-WW1, GST-WW2, and GST-WW4 did not (lanes 2, 3, and 5, top panel), indicating that WW3 is the domain binding to ACK1.
FIG. 4. ACK1 interacts with the WW3 domain of Nedd4-1. (A) Schematic representation of GST-human Nedd4-1 WW domain constructs. (B) The bead-bound GST or GST-human Nedd4-1 WW domain was incubated with Myc-ACK1-expressed HEK293 cell lysates and precipitated by (more ...) Nedd4-1 is the E3 ubiquitin ligase for ubiquitination of ACK.
There are two members in the Nedd4 family, Nedd4-1 and Nedd4-2 (also Nedd4L), in mammalian cells (5
). Both Nedd4-1 and Nedd4-2 have a similar domain structure, with a C2 domain at the N terminus, followed by four WW domains and the HECT domain at the C terminus. Human Nedd4-1 and Nedd4-2 are about 65% identical in primary sequence. The most divergent region is the segment between the WW1 and the WW3 domains. Despite their structural similarity, Nedd4-1 and Nedd4-2 recognize distinct substrates, for example, ion channels and membrane transporters for Nedd4-2 (8
) and endocytic proteins for Nedd4-1 (1
To distinguish the specificity of Nedd4-1 and Nedd4-2 in ubiquitination of ACK, we first examined the binding of Nedd4-1 and Nedd4-2 to ACK1. HA-tagged Nedd4-1 or Nedd4-2 was cotransfected with Myc-ACK1 into HEK293 cells. ACK1 was immunoprecipitated with anti-Myc antibody, and coimmunoprecipitated Nedd4-1 or Nedd4-2 was detected by immunoblotting with anti-HA antibody. As shown in Fig. , both Nedd4-1 and Nedd4-2 were coprecipitated with ACK1 (lanes 2 and 3, middle panel).
FIG. 5. Nedd4-1 is the E3 ubiquitin ligase for ACK ubiquitination. (A and B) HA-tagged human Nedd4-1, Nedd4-2, or vector was coexpressed with Myc-ACK1 in HEK293 cells. In panel A, Myc-ACK1 was immunoprecipitated with anti-Myc antibody (top panel). Coprecipitated (more ...)
Although both Nedd4-1 and Nedd4-2 bind to ACK, a significant difference in ubiquitination of ACK1 was observed between Nedd4-1 and Nedd4-2. Using a GST-ACK-Uba pulldown assay, we found that coexpression with Nedd4-1 resulted in ubiquitination of ACK1 that was more than 10-fold higher than coexpression with Nedd4-2 (Fig. , lanes 2 to 4). A difference in ubiquitination of endogenous ACK between Nedd4-1 and Nedd4-2 was also observed (Fig. ). Upon overexpression of Nedd4-1 or Nedd4-2 followed by treatment with the proteasomal inhibitor MG-132 for accumulation of ubiquitinated ACK, we detected that overexpression of Nedd4-1 significantly enhanced ubiquitination of endogenous ACK (Fig. , lanes 1 to 4). However, overexpression of Nedd4-2 did not yield any increase in ubiquitination of ACK; rather, it reduced ubiquitination of ACK, which might result from competing with endogenous Nedd4-1 (compare lane 5 with lane 3). We sometimes observed a significant gel shift of endogenous ACK in SDS-PAGE with expression of Nedd4-1 (Fig. , compare lane 1 with lane 2 in middle panel). These data suggest that Nedd4-1, not Nedd4-2, is the E3 ubiquitin ligase for ubiquitination of ACK in cells.
To confirm that Nedd4-1 is the E3 ubiquitin ligase for ACK, we also performed Nedd4-1 RNAi knockdown experiments. We transfected the Nedd4-1 RNAi oligonucleotides into either HEK293 cells or human non-small cell lung cancer (NSCLC) A549 cells for 48 to 72 h and examined the knockdown effect of Nedd4-1 on ACK protein level by immunoblotting of the cell lysates (Fig. and E). The Nedd4-1 protein level was reduced more than 50% in both HEK293 and A549 cells by Nedd4-1 RNAi knockdown over that of the control RNAi (Fig. , lanes 1 to 3 in the middle panel, and E, lanes 1 and 2 in the third panel). Knockdown of Nedd4-1 dramatically increased ACK protein levels in both cell lines compared to those of the controls (Fig. and E, top panel). A significant increase of EGFR expression by Nedd4-1 knockdown was also observed in A549 cells (Fig. , top panel, lane 2).
We further examined the effect of Nedd4-2 knockdown on the ACK expression level in A549 cells. We could not find a suitable commercial anti-Nedd4-2 antibody for detection of endogenous Nedd4-2 in A549 cells. However, the anti-Nedd4-1 antibody (Upstate Biotech) used in our studies was raised against an antigen region in Nedd4-1 that is partially conserved in Nedd4-2. We tested the anti-Nedd4-1 in detection of exogenously expressed HA-tagged Nedd4-2 (HA-Nedd4-2) and estimated the difference in detection sensitivity between HA-Nedd4-1 and HA-Nedd4-2 by comparison with anti-HA antibody. We found that the anti-Nedd4-1 was about 10-fold less sensitive in detection of HA-Nedd4-2 than detection of HA-Nedd4-1 (data not shown). As expected, we detected endogenous Nedd4-2 expression with the anti-Nedd4-1 in A549 cells (Fig. , third panel, lanes 1 to 3) and estimated that expression of Nedd4-2 is approximately 2-fold higher than that of Nedd4-1 in A549 cells after rectifying the detection sensitivity. We observed that more than 80% of endogenous Nedd4-2 was diminished by Nedd4-2 RNAi knockdown (Fig. , lane 3). To confirm this knockdown, we examined the effect of Nedd4-2 RNAi oligonucleotide on exogenous expression of HA-Nedd4-2 in HEK293 cells and demonstrated that the Nedd4-2 RNAi depleted more than 80% of exogenously expressed Nedd4-2 (Fig. , lane 7), which is consistent with the effect on knockdown of endogenous Nedd4-2. By knockdown of Nedd4-2, expression level of both ACK and EGFR was only slightly enhanced (Fig. , two top panels, lanes 2 and 3), probably due to more Nedd4-1 occupied by dual substrates upon the depletion of Nedd4-2. In summary, the RNAi knockdown data confirm that Nedd4-1, not Nedd4-2, is the E3 ubiquitin ligase regulating ACK and EGFR degradation in vivo.
The effects of the SAM and the Uba domains on the ubiquitination of ACK.
ACK contains a sterile alpha motif (SAM) domain at the N terminus and an ubiquitin association (Uba) domain at the carboxyl terminus (11
). Deletion of the SAM domain by truncating the first 89 amino acid residues (ACK1Δ89) resulted in a dramatic reduction of ubiquitination of ACK1 when coexpressed with Nedd4-1 (Fig. ), suggesting that the SAM domain is required for Nedd4-1-catalyzed ubiquitination of ACK1. How deletion of the SAM domain eliminates the ubiquitination is not known. Our speculation is that the SAM domain may contain major ubiquitination sites of ACK, as the SAM domain has a lysine-rich region (46
). On the other hand, deletion of the Uba domain (ACK1ΔUba) remarkably enhanced ubiquitination (compare lane 4 with lane 2 in the top panel of Fig. ). This enhancement of ubiquitination seems to result from increased binding to Nedd4-1, since substantially more Nedd4-1 was coimmunoprecipitated with ACK1ΔUba than with ACK1 (Fig. , compare lane 4 with lane 2 in the second panel from the top).
FIG. 6. The effects of the SAM and the Uba domains on the ubiquitination of ACK. (A) Myc-tagged ACK1 or ACK1Δ89 was cotransfected with HA-tagged Nedd4-1 or the vector into HEK293 cells. Myc-tagged ACK1 or ACK1Δ89 was immunoprecipitated and immunoblotted (more ...)
To test whether the enhanced ubiquitination caused by deletion of the Uba domain occurs in the lysine-rich region of the SAM domain, we compared the ubiquitination catalyzed by Nedd4-1 between ACK1 ΔUba and ACK1Δ89ΔUba. As shown in Fig. , deletion of the SAM domain in ACK1ΔUba significantly reduced the ubiquitination catalyzed by Nedd4-1 (lane 2), supporting the hypothesis that the lysine-rich region in the SAM domain may contain the majority of the ubiquitination sites of ACK.
Interestingly, when Cdc42 Q61L, a GTPase-defective mutant of Cdc42, was cotransfected with ACK1ΔUba and Nedd4-1, the ubiquitination of ACK was further enhanced (Fig. , compare lane 4 with lane 2 in the top panel). This is consistent with the effect of Cdc42 on EGF-stimulated ubiquitination of ACK (Fig. ). Cdc42 is a member of the Rho family GTPase and an activator of ACK. These data suggest that activated ACK may be a preferential ubiquitination substrate for Nedd4-1.
EGF-induced degradation of ACK is processed by lysosomes, not proteasomes.
Ubiquitination-mediated protein degradation can occur via proteasomes or lysosomes. It has been demonstrated that ubiquitination-mediated EGFR degradation is mediated by the lysosomal pathway (21
). To determine the mechanism of EGF-induced ACK degradation, we used inhibitors of proteasomal (MG-132, PSI, Velcade [bortezomib], lactacystin) or lysosomal (bafilomycin and NH4
Cl) pathways to assess the degradation route for ACK in COS7, HeLa, and A549 cells. As shown in Fig. , in COS7 cells, treatment with bafilomycin (lanes 7 to 9) or MG-132 (lanes 10 to 12) significantly inhibited EGF-induced degradation of both EGFR and ACK (top and middle panels). However, MG-132 is known to inhibit calpain in addition to proteasomes, and thus it is not a specific proteasomal inhibitor (6
). The specific proteasome inhibitor Velcade (bortezomib) only slightly blocked the degradation of EGFR and ACK (Fig. , top and middle panels, compare lane 6 with lane 3). Both MG-132 and Velcade activated p38 kinase (phosphorylation of Hsp27) to the same extent (Fig. , second panel from the bottom, lanes 4 to 6 and 10 to 12), suggesting that both effectively inhibited proteasomes. Therefore, inhibition of EGFR and ACK degradation by MG-132 in COS7 cells might result from a nonproteasomal inhibitory activity.
FIG. 7. EGF-induced degradation of ACK is mediated by lysosomes. COS7 (A), HeLa (B and C) or A549 (D) cells were cultured to 90% confluence, followed by serum starvation (0.1% FBS for HeLa cells, serum-free for COS7 and A549 cells) for 12 h. The (more ...)
As shown in Fig. , MG-132 treatment did not significantly affect EGF-induced degradation of either EGFR or ACK (top and bottom panels) or the binding of ACK to EGFR (middle panel) in HeLa cells. Similar results were observed with the proteasomal inhibitor lactacystin (Fig. , top and bottom panels, compare lane 9 with lane 3). Treatment with NH4Cl, an inhibitor of lysosomes, significantly blocked EGF-induced degradation of EGFR and ACK (Fig. , top and bottom panels, lane 6). These data demonstrate that EGF-induced degradation of ACK is processed by lysosomes.
Similar results were observed in A549 cells. The proteasomal inhibitors MG-132 and PSI did not inhibit degradation of EGFR or ACK (Fig. , two top panels, lanes 3 and 4 and lanes 11 and 12). The lysosomal inhibitors, NH4Cl and bafilomycin, blocked 70 to 90% of the EGF-induced degradation of EGFR and ACK (Fig. , two top panels, lanes 5 and 6 and lanes 9 and 10).
It has been reported that p38 kinase activity was required for ligand-induced EGFR degradation via regulation of tyrosine phosphorylation of Y1045 of EGFR, which is the binding site for Cbl, the E3 ubiquitin ligase for EGFR (10
). The p38 kinase inhibitor SB203850 blocked EGF-induced degradation of EGFR (Fig. , top panel, compare lanes 13 to 15 with lanes 1 to 3) and ACK (Fig. , second panel from top, compare lanes 13 to 15 with lanes 1 to 3). This result suggests that EGF-induced ACK degradation is dependent on EGFR degradation, likely via cotransporting with EGFR to lysosomes.
Nedd4-1, not Nedd4-2, is required for EGF-induced ACK and EGFR degradation.
To address the role of Nedd4-1 and Nedd4-1-catalyzed ubiquitination of ACK in regulation of EGF-induced degradation of EGFR and ACK, we compared the degradation of EGFR and ACK in Nedd4-1 knockdown cells with that in Nedd4-2 knockdown and control cells (Fig. ). Knockdown of Nedd4-1 significantly increased both EGFR and ACK expression levels (Fig. , two top panels, lanes 1 and 3), while knockdown of Nedd4-2 caused little change in expression levels of ACK and EGFR (Fig. , two top panels, lanes 5 and 7). Quantification of the data from three independent experiments shows that knockdown of 70 to 80% of Nedd4-1 caused a 2- to 5-fold increase in ACK1 expression and a 3- to 10-fold increase in EGFR expression in A549 cells, whereas knockdown of more than 80% of Nedd4-2 had little effect on expression of both ACK and EGFR (Fig. ). EGF-induced degradation of ACK and EGFR was significantly inhibited by Nedd4-1 knockdown. While about 65% of EGFR and ACK was degraded upon 60 min EGF stimulation in control RNAi-transfected cells, only about 20% of EGFR or ACK was degraded upon 60 min EGF stimulation in Nedd4-1 knockdown cells (Fig. and D). Thus, Nedd4-1 knockdown yielded inhibition of about 70% of the degradation of both EGFR and ACK. Again, knockdown of Nedd4-2 caused little effect on EGF-induced degradation of ACK and EGFR (Fig. and D). These data indicate that Nedd4-1, not Nedd4-2, plays an important role in regulation of EGF-induced degradation of EGFR and ACK.
FIG. 8. Knockdown of Nedd4-1 by RNAi enhances the expression level of EGFR and ACK and inhibits EGF-induced degradation of EGFR and ACK. (A) Nedd4-1 RNAi-B, Nedd4-2 RNAi, or the luciferase RNAi (control RNAi) was transfected into A549 cells for 60 h, followed (more ...)
We further assessed the role of Nedd4-1-catalyzed ubiquitination of ACK in regulation of EGFR degradation. We first confirmed the role of endogenous ACK in regulation of EGFR degradation by depletion of ACK with RNAi. As shown in Fig. , knockdown of about 70% of ACK1 in A549 cells inhibited EGF-induced degradation of EGFR (top panel, lanes 5 to 6). We next examined the effects of overexpression of the Nedd4-1-binding-defective mutant ACK1Y650A and the ubiquitination-defective mutant ACK1Δ89 on ligand-induced EGFR degradation in HEK293 cells. With overexpression of the Nedd4-1-binding-defective mutant ACK1Y650A, EGFR degradation upon 60 min EGF stimulation was reduced to 45% from 80% in wild-type ACK1-overexpressed or vector-transfected cells (Fig. and C), indicating that about 45% of EGF-induced degradation of EGFR was inhibited by overexpression of ACK1Y650A. Similar inhibition of ligand-induced EGFR degradation was observed with overexpression of the ubiquitination-defective mutant ACK1Δ89 in HEK293 cells (Fig. and E). Overexpression of ACK1Δ89 caused inhibition of EGF-induced degradation by about 40% compared with that in the vector-transfected cells (Fig. ). These data suggest that Nedd4-1-catalyzed ubiquitination of ACK is required for ACK in the regulation of ligand-induced degradation of EGFR.
FIG. 9. Overexpression of the Nedd4-1-binding-defective mutant ACK1Y650A and the ubiquitination-defective mutant ACK1Δ89 inhibits EGF-induced degradation of EGFR. (A) ACK1 RNAi or luciferase RNAi (control RNAi) was transfected into A549 cells for 60 h (more ...)