1. Volume 7, Issue 3, Pages 871-882 (May 2014)
Impeded Nedd4-1-Mediated Ras Degradation Underlies Ras-Driven Tumorigenesis 
Taoling Zeng, Qun Wang, Jieying Fu, Qi Lin, Jing Bi, Weichao Ding, Yikai Qiao, Sheng Zhang, Wenxiu Zhao, Huayue Lin, Meilin Wang, Binfeng Lu, Xianming Deng, Dawang Zhou, Zhenyu Yin, Hong-Rui Wang 
Cell Reports 
Volume 7, Issue 3, Pages (May 2014)
DOI: /j.celrep
Copyright © 2014 The Authors Terms and Conditions

2. Cell Reports 2014 7, 871-882DOI: (10.1016/j.celrep.2014.03.045)
Copyright © 2014 The Authors Terms and Conditions

3. Figure 1 Nedd4-1 Interacts with Ras Proteins
(A) K-Ras associates with Nedd4-1. Halo-tagged Nedd4-1 C867A (Halo/Nedd4-1 C867A) or Halo-tag control transiently expressed in HEK293T cells was purified and applied to SDS-PAGE. The proteins were visualized by silver staining, and indicated spots were analyzed by mass spectrometry.
(B) Interaction of endogenous Nedd4-1 and Ras proteins. Cell lysates from HEK293T were subjected to IP with specific antibodies to K-Ras, H-Ras, or N-Ras followed by immunoblotting (IB) to detect endogenous Nedd4-1.
(C) In vitro interaction between Nedd4-1 and Ras proteins. Bacterially expressed and purified FLAG-tagged K-Ras (F/K-Ras), H-Ras (F/H-Ras), or N-Ras (F/N-Ras) and GST-tagged Nedd4-1 (GST/Nedd4-1) were subjected to GST pull-down assays as indicated. Associated Ras proteins were detected with anti-FLAG. GST and GST/Nedd4-1 were determined by Ponceau S staining.
(D) Nedd4-1 colocalizes with Ras proteins at plasma membrane. HeLa cells were cotransfected with different FLAG-tagged Ras and HA-tagged Nedd4-1 C867A (HA/Nedd4-1 C867A) as indicated. Localization of Ras (red) and Nedd4-1 (green) was detected by immunofluorescence. The nuclei were stained with DAPI (blue). The scale bars indicate 5 μm.
Cell Reports 2014 7, DOI: ( /j.celrep )
Copyright © 2014 The Authors Terms and Conditions

4. Figure 2
Nedd4-1 Targets Ras Proteins for Ubiquitination and Degradation
(A) Nedd4-1 overexpression decreases endogenous levels of Ras proteins. HEK293T cells were transiently transfected with FLAG-tagged Nedd4-1 (F/Nedd4-1) or Nedd4-2 (F/Nedd4-2), WT or catalytically inactive mutants C867A or C942A as indicated. Total cell lysates were subjected to immunoblotting to determine the protein levels. Levels of GAPDH were used as a loading control.
(B) Knocking down Nedd4-1 increases endogenous Ras protein levels. HEK293T cells were transfected with control shRNA (sh-con) or Nedd4-1 shRNA (sh-N4-1-A or sh-N4-1-B), and the endogenous protein levels of Ras and Nedd4-1 were detected by immunoblotting.
(C) Nedd4-1 promotes ubiquitination of K-Ras. After overnight treatment of 120 μM chloroquine, HEK293T cells transfected with indicated combinations of F/K-Ras, Myc-tagged Nedd4-1 (Myc/Nedd4-1) (WT or C867A), and HA-tagged ubiquitin (HA/Ub) were subjected to anti-FLAG IP, eluted by boiling in 1% SDS, and then reprecipitated with anti-FLAG (2× IP). The ubiquitin-conjugated K-Ras ((Ub)n-K-Ras) was detected with anti-HA.
(D) Direct ubiquitination of K-Ras mediated by Nedd4-1 in vitro. GST/Nedd4-1 (WT or C867A) and F/K-Ras purified from bacteria were subjected to an in vitro ubiquitination assay. Ubiquitinated F/K-Ras was detected with anti-Ub.
(E) Nedd4-1 regulates turnover rates of K-Ras. HEK293T cells were transfected with combinations of F/K-Ras, F/Nedd4-1, control shRNA, and Nedd4-1 shRNA as indicated. The levels of F/K-Ras at different time points after cycloheximide (CHX) treatment were determined by immunoblotting the total cell lysates and quantification using Image Lab software (Bio-Rad) with GAPDH as a loading control. Results plotted are the amounts of F/K-Ras at each time point relative to the level at time 0.
Cell Reports 2014 7, DOI: ( /j.celrep )
Copyright © 2014 The Authors Terms and Conditions

5. Figure 3 Nedd4-1 Suppresses Tumorigenesis by Targeting Ras
(A) Knockdown of Nedd4-1 induces transformation of NIH 3T3 cells. NIH 3T3 cells transduced with lentivirus encoding control shRNA or Nedd4-1 shRNA (sh-N4-1-A or sh-N4-1-B) were subjected to soft agar assay. The scale bars indicate 100 μm. Viable colonies formed were quantitated and plotted as mean ± SD of six independent experiments.
(B) Simultaneously silencing Ras inhibits Nedd4-1 knockdown-induced transformation of NIH 3T3 cells. NIH 3T3 cells transduced with indicated combinations of lentivirus encoding Nedd4-1 shRNA and Ras shRNA were applied to soft agar assay. Viable colonies formed were quantitated and plotted as mean ± SD of three independent experiments.
(C and D) Knockdown of Nedd4-1 induces tumorigenicity of NIH 3T3 cells in nude mice. Nude mice were subcutaneously injected with NIH 3T3 cells stably expressing control shRNA (left flank) or Nedd4-1 shRNA (right flank) to observe tumor development (C), and volumes of tumor were determined and plotted as mean ± SD of six independent experiments (D).
(E) Nedd4-1 and K-Ras present inverse correlations in some human colon cancer specimens. Protein levels of Nedd4-1 and K-Ras were determined by immunoblotting the protein extracts from tumor tissues (T) and their matched surrounding normal mucosal tissues (N). GAPDH was used as a loading control.
(F) Increase of K-Ras is not due to upregulation of its transcription. The mRNA levels of K-Ras in tumor (T) and matching normal tissues (N) were measured by real-time quantitative PCR using GAPDH as an internal control. Data are presented as mean ± SD of three independent experiments.
(G) The protein levels of Nedd4-1 and K-Ras in colorectal carcinomas. The protein levels of Nedd4-1 (blue line) and K-Ras (red line) observed in a series of 46 colorectal carcinomas were plotted in the order of increasing Nedd4-1 protein levels.
(H) Correlation between Nedd4-1 and K-Ras protein levels in colorectal cancer. A total of 46 samples of human colorectal cancer were classified into 2 groups based on the protein levels of Nedd4-1 in tumor tissues compared to surrounding normal tissues (low Nedd4-1, 22 samples; high Nedd4-1, 24 samples). The correlation coefficient r value was obtained by Spearman’s rank correlation analysis.
Cell Reports 2014 7, DOI: ( /j.celrep )
Copyright © 2014 The Authors Terms and Conditions

6. Figure 4
Ras Evades Nedd4-1-Mediated Degradation via Site Mutation or Consecutive Activation
(A) G12V mutation restrains Nedd4-1-mediated K-Ras degradation. HEK293T cells transfected with F/K-Ras (WT or V12) and F/Nedd4-1 (WT or C867A) as indicated were subjected to immunoblotting.
(B) Nedd4-1 overexpression decreases levels of WT K-Ras but not V12 mutant. Endogenous levels of WT or V12 K-Ras in HT-29 or SW480 cells overexpressing F/Nedd4-1 (WT or C867A) were examined by immunoblotting.
(C) Knocking down Nedd4-1 increases endogenous levels of WT K-Ras but not V12 mutant. HT-29 cells or SW480 cells were transduced with lentivirus encoding control shRNA or Nedd4-1 shRNA, and endogenous levels of WT K-Ras in HT-29 cells and V12 mutant in SW480 cells were examined by immunoblotting.
(D) EGF treatment inhibits Nedd4-1-mediated Ras degradation. HEK293T cells overexpressing F/Nedd4-1 (WT or C867A) were treated with or without EGF (50 ng/ml) for the indicated time and then subjected to immunoblotting.
(E) Overexpression of EGFR prevents Nedd4-1-mediated Ras degradation. Endogenous protein levels of total Ras in HEK293T cells transduced with indicated combinations of lentivirus encoding WT or constitutive active form (L813R) C-terminal HA-tagged EGFR (EGFR/HA) and F/Nedd4-1 (WT or C867A) were determined by immunoblotting the total cell lysates.
(F) EGF signaling-caused increase of Ras levels is not through upregulating Ras transcription. The mRNA levels of K-Ras, H-Ras, and N-Ras in HEK293T cells treated with or without EGF (50 ng/ml) for the indicated hours, or transfected with WT or L813R EGFR, were measured by real-time RT-PCR using GAPDH as an internal control. Data are presented as mean ± SD of three independent experiments.
Cell Reports 2014 7, DOI: ( /j.celrep )
Copyright © 2014 The Authors Terms and Conditions

7. Figure 5
Oncogenic Mutation or Constant Activation Protects K-Ras by Attenuating Nedd4-1-Mediated Ubiquitination
(A) G12V mutation or EGF treatment does not inhibit binding of K-Ras to Nedd4-1. HEK293T cells transfected with F/K-Ras (WT or V12) and HA/Nedd4-1 C867A as indicated were treated with or without EGF (50 ng/ml for 24 hr) as indicated before being subjected to coimmunoprecipitation assay.
(B) Nedd4-1 interacts with both GDP- and GTPγS-bound forms of K-Ras in vitro. Nucleotide-free F/K-Ras obtained by EDTA treatment was loaded with GDP or GTPγS and then subjected to GST pull-down assay as indicated. GST and GST/Nedd4-1 were detected by Ponceau S staining.
(C) G12V mutation attenuates Nedd4-1-mediated K-Ras ubiquitination. HEK293T cells transfected with indicated combinations of F/K-Ras (WT or V12), Myc/Nedd4-1 (WT or C867A), and HA/Ub were subjected to ubiquitination assay.
(D) EGF treatment attenuates Nedd4-1-mediated K-Ras ubiquitination. HEK293T cells transfected with indicated combinations of F/K-Ras, Myc/Nedd4-1 (WT or C867A), and HA/Ub were treated with or without EGF (50 ng/ml) for 24 hr and then subjected to ubiquitination assay.
(E) Overexpression of EGFR diminishes Nedd4-1-mediated K-Ras ubiquitination. HEK293T cells transfected with indicated combinations of F/K-Ras, Myc/Nedd4-1 (WT or C867A), EGFR/HA (WT or L813R), and HA/Ub were subjected to ubiquitination assay.
(F) Load of GTPγS inhibits Nedd4-1-mediated K-Ras ubiquitination. Nucleotide-free F/K-Ras was loaded with GDP or GTPγS and then subjected to in vitro ubiquitination assay as indicated.
Cell Reports 2014 7, DOI: ( /j.celrep )
Copyright © 2014 The Authors Terms and Conditions

8. Figure 6 Ras Activation Upregulates Nedd4-1 Transcription
(A) Overexpressing Ras increases endogenous Nedd4-1 levels. Nedd4-1 levels in HEK293T cells transfected with F/K-Ras (WT or V12) were examined by immunoblotting.
(B) Treatment of EGF increases endogenous levels of Nedd4-1. HEK293T cells were treated with different doses of EGF as indicated for 36 hr before being applied to western blot to determine the endogenous levels of Nedd4-1.
(C) Ras overexpression enhances Nedd4-1 transcription. The mRNA levels of Nedd4-1 in HEK293T, HeLa, BGC-823, or HT-29 cells transfected with K-Ras (WT or V12) were measured by real-time RT-PCR using GAPDH as an internal control. Data are presented as mean ± SD of three independent experiments.
(D) EGF treatment upregulates Nedd4-1 transcription. The mRNA levels of Nedd4-1 in HEK293T, HeLa, BGC-823, or HT-29 cells treated 2 hr with different doses of EGF as indicated were measured by real-time quantitative PCR using GAPDH as an internal control. Data are presented as mean ± SD of three independent experiments.
(E) The mRNA levels of K-Ras and Nedd4-1 in colorectal carcinomas. The mRNA levels of K-Ras (blue line) and Nedd4-1 (red line) observed in a series of 56 colorectal carcinomas were plotted in the order of increasing K-Ras expression. The mRNA data were obtained from the Oncomine database (
(F) Positive correlation between mRNA levels of K-Ras and Nedd4-1. The correlation between Nedd4-1 and K-Ras mRNA expression in 56 human colon cancer tissues was analyzed by Pearson’s correlation analysis with a coefficient of 0.82 (p < 0.001).
(G) The mRNA levels of EGFR and Nedd4-1 in colorectal carcinomas. Same as in (E), except that mRNA levels of EGFR instead of K-Ras were used.
(H) Positive correlation between mRNA levels of EGFR and Nedd4-1. Same as in (F), except that mRNA levels of EGFR instead of K-Ras were used. Pearson’s correlation coefficient (r) was 0.84 (p < 0.001).
(I) Positive correlation between protein levels of Nedd4-1 and phospho-EGFR in colorectal tumor samples. The protein levels of Nedd4-1 and phospho-EGFR (pY1068) were determined by immunoblotting the protein extracts from tumor tissues (T) and their matched surrounding normal mucosal tissues (N). GAPDH was used as a loading control.
(J) Positive correlation between expression of Nedd4-1 and phospho-EGFR in colorectal cancer. The expression of Nedd4-1 in tumor tissue (T) and surrounding normal mucosal tissue (N) in typical colorectal tumor samples with (bottom) or without (top) upregulation of phospho-EGFR (pY1068) was detected by immunohistochemistry assay. The scale bars indicate 200 μm.
Cell Reports 2014 7, DOI: ( /j.celrep )
Copyright © 2014 The Authors Terms and Conditions

9. Figure 7
Ras Oversignaling Promotes PTEN Degradation through Upregulating Nedd4-1
(A) Overexpression of K-Ras decreases PTEN levels. Endogenous levels of Nedd4-1 and PTEN in HeLa cells overexpressing F/K-Ras (WT or V12) were examined by immunoblotting.
(B) EGF treatment decreases PTEN levels in HeLa cells. HeLa cells were treated overnight with or without indicated doses of EGF and then subjected to western blot.
(C) Overexpressing Nedd4-1 decreases PTEN levels. Endogenous PTEN levels in HeLa cells overexpressing F/Nedd4-1 (WT or C867A) were determined by immunoblotting.
(D) Knocking down Nedd4-1 increases PTEN levels. HeLa cells expressing control or Nedd4-1 shRNA were subjected to immunoblotting to determine the protein levels.
(E) Activation of Ras signaling does not affect PTEN transcription. The mRNA levels of PTEN in HeLa cells transduced with lentivirus encoding K-Ras (WT or V12) or treated with indicated doses of EGF were measured by real-time quantitative PCR using GAPDH as an internal control. Data are presented as mean ± SD of three independent experiments.
(F) Ras overexpression-induced PTEN degradation is dependent on Nedd4-1. HeLa cells expressing indicated combinations of F/K-Ras V12 and control or Nedd4-1 shRNA were subjected to immunoblotting to examine the endogenous levels of Nedd4-1 and PTEN.
(G) EGF treatment-induced decrease of PTEN is dependent on Nedd4-1. HeLa cells transduced with lentivirus encoding control shRNA or Nedd4-1 shRNAs were treated overnight with or without EGF (50 ng/ml) as indicated. The endogenous Nedd4-1, PTEN, and Ras levels were determined by western blot.
(H) K-Ras and PTEN are reversely correlated in human colorectal cancer samples with high levels of Ras and Nedd4-1. Protein levels of PTEN, Nedd4-1, and K-Ras were determined by immunoblotting the protein extracts from tumor tissues (T) and their matched surrounding normal mucosal tissues (N). GAPDH was used as a loading control.
(I) Correlation between protein levels of K-Ras and PTEN. A total of 46 samples of human colorectal cancer were classified into 4 groups based on the protein levels of Nedd4-1 and K-Ras in tumor tissues compared to surrounding normal tissues. Indicated correlation coefficient r value was calculated by Spearman’s rank correlation analysis.
Cell Reports 2014 7, DOI: ( /j.celrep )
Copyright © 2014 The Authors Terms and Conditions