Research ArticleCell Biology

Cycles of Ubiquitination and Deubiquitination Critically Regulate Growth Factor–Mediated Activation of Akt Signaling

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Science Signaling  08 Jan 2013:
Vol. 6, Issue 257, pp. ra3
DOI: 10.1126/scisignal.2003197
  • Fig. 1

    CYLD is a DUB for Akt. (A) Cellular ubiquitination assays performed in HEK293T cells transfected with His-ubiquitin (His-Ub), hemagglutinin (HA)–Akt, along with various DUB constructs. Ni–nitrilotriacetic acid (Ni-NTA), nickel bead precipitate; WCE, whole-cell extracts. (B) Cellular ubiquitination assays performed in HEK293T cells transfected with HA-Akt, His-Ub, and Flag-TRAF6, along with Flag-CYLD-WT or Flag-CYLD-C/A. WT, wild type; C/A, enzyme-dead mutant (C601A). (C) Immunoblot analysis of Akt immunoprecipitates from HEK293T cells transfected with Akt and Flag-CYLD, along with HA-Ub K48 (K48-only ubiquitin) or HA-Ub K63 (K63-only ubiquitin). IB, immunoblot; IP, immunoprecipitation. (D) Cellular ubiquitination assays performed in HEK293T cells transfected with HA-Akt, His-Ub, and Xp-Skp2, along with Flag-CYLD-WT or Flag-CYLD-C/A. (E) In vitro deubiquitination assays with purified ubiquitinated Akt proteins incubated with purified Flag-CYLD-C/A or Flag-CYLD-WT proteins. A representative blot from three independent experiments (n = 3) is shown for each panel.

  • Fig. 2

    CYLD interacts with Akt and inhibits the ubiquitination of endogenous Akt. (A) Immunoblot analysis of Akt immunoprecipitates from Cyld+/+ and Cyld−/− MEFs that were serum-starved and treated with or without IGF-1. (B) Immunoblot analysis of Akt immunoprecipitates from control or CYLD stable knockdown PC-3 or DU-145 cells that were serum-starved and treated with or without IGF-1. (C) Immunoblot analysis of HA immunoprecipitates from HEK293T cells transfected with HA-Akt along with Flag-CYLD-WT or Flag-CYLD-C/A. (D) Immunoblot analysis of CYLD or Akt immunoprecipitates from serum-starved PC-3 cells. (E) Immunoblot analysis of Akt immunoprecipitates from PC-3 cells that were serum-starved and treated with IGF-1 at various time points. (F) Immunoblot analysis of HA immunoprecipitates from HEK293T cells transfected with HA-Akt along with Flag-CYLD or various amounts of Flag-TRAF6. A representative blot from three independent experiments is shown for each panel. The quantification data for (E) and (F) are presented as the means ± SD of triplicate measurements in table S1.

  • Fig. 3

    PI3K activity is not required for ubiquitination and deubiquitination of Akt. (A) Immunoblot analysis of Flag immunoprecipitates from HEK293T cells transfected with Flag-CYLD, HA-Akt, His-Ub, and TRAF6. (B) Immunoblot analysis of HA immunoprecipitates from HEK293T cells transfected with Flag-CYLD and His-Ub, along with HA-Akt-WT or HA-Akt K8R. (C) Immunoblot analysis of HA immunoprecipitates from serum-starved PC-3 cells that were pretreated with or without the PI3K inhibitor LY294002 and treated with or without IGF-1. (D) Cellular ubiquitination assays performed in HEK293T cells transfected with HA-Akt, His-Ub, and Flag-CYLD and pretreated with or without LY294002. (E) Immunoblot analysis of HEK293T cells transfected with HA-Akt-WT, HA-Akt-E17K, or HA-Akt-K8R and treated with or without LY294002. A representative blot from three independent experiments (n = 3) is shown for each panel.

  • Fig. 4

    CYLD deficiency facilitates growth factor–mediated membrane recruitment and phosphorylation of Akt. (A) Immunoblot analysis of Cyld+/+ and Cyld−/− MEFs that were serum-starved and treated with IGF-1 at various time points. (B) Immunoblot analysis of control or CYLD stable knockdown PC-3 or DU-145 cells that were serum-starved and treated with IGF-1 at various time points. (C) Immunoblot analysis of control or two different CYLD-knockdown PC-3 cell lines that were serum-starved and treated with EGF at various time points. (D) Immunoblot analysis of CYLD-knockdown PC-3 cells that were transfected with mock, Flag-CYLD-WT, or Flag-CYLD-C/A, serum-starved, and treated with IGF-1 at various time points. (E) Immunoblot analysis of membrane and cytosolic fractions of Cyld+/+ and Cyld−/− MEFs that were serum-starved and treated with IGF-1 at various time points. (F) Immunoblot analysis of membrane and cytosolic fractions of control or CYLD stable knockdown PC-3 or DU-145 cells that were serum-starved and treated with IGF-1 at various time points. (G) Immunoblot analysis of membrane and cytosolic fractions of control or two CYLD-knockdown PC-3 cell lines that were serum-starved and treated with EGF at various time points. A representative blot from three independent experiments (n = 3) is shown for each panel. The quantification data for (A), (B), (C), and (E) are presented as the means ± SD of triplicate measurements in table S1.

  • Fig. 5

    CYLD inhibits cancer cell proliferation, cell survival, and glucose uptake. (A) Cell proliferation in control or CYLD stable knockdown PC-3 or DU-145 cells, presented as means ± SD from three biological replicates. **P < 0.01, ***P < 0.001 for all pairwise comparisons by one-way analysis of variance (ANOVA) and post hoc intergroup comparisons with Sidak test. (B) Cell apoptosis as determined by annexin V staining and flow cytometric analysis in control or CYLD stable knockdown PC-3 or DU-145 cells that were treated with vehicle or cisplatin. Results are presented as mean percentages from three biological replicates. *P < 0.05, **P < 0.01 for all pairwise comparisons by Pearson χ2 test. (C) Cell proliferation in control or CYLD-knockdown PC-3 or DU-145 cells treated with vehicle, LY294002 (LY), or wortmannin (Wort), presented as means ± SD from three biological replicates. ***P < 0.001 for all pairwise comparisons by one-way ANOVA and post hoc intergroup comparisons with Sidak test. (D) Cell apoptosis as determined as in (B) in control or CYLD stable knockdown PC-3 or DU-145 cells that were pretreated with vehicle, LY294002 (LY), or wortmannin (Wort), then with vehicle or cisplatin. Results are presented as mean percentages from three biological replicates. *P < 0.05, **P < 0.01 for all pairwise comparisons by Pearson χ2 test. (E) Immunoblot analysis of Glut1 and Glut4 in cytosolic or membrane fractions of Cyld+/+ and Cyld−/− MEFs or PC-3 cells with control or CYLD knockdown that were treated with IGF-1 for the indicated time intervals. A representative blot from three independent experiments (n = 3) is shown. (F) Analysis of glucose uptake ratios in Cyld+/+ and Cyld−/− MEFs or PC-3 cells with control or CYLD knockdown, treated with or without IGF-1 and grown in the presence of the fluorescent glucose analog 2-NBDG. Results are presented as mean percentages from three biological replicates. N.S., nonsignificant; ***P < 0.001 for all pairwise comparisons by Pearson χ2 test.

  • Fig. 6

    CYLD suppresses prostate cancer development. (A) PC-3 or DU-145 cells with control or CYLD knockdown were injected into nude mice (n = 6 for each group), and the increase of tumor volume in mice was monitored every week. Results are presented as means ± SD; *P < 0.05 for all pairwise comparisons by one-way ANOVA and post hoc intergroup comparisons with Sidak test. (B) Immunohistochemistry in representative primary nonmetastatic prostate cancer samples. A panel of images showing hematoxylin and eosin (H&E) staining, Akt phosphorylated at Ser473, and CYLD staining in early- and advanced-stage prostate cancer samples. Scale bar, 50 μm. (C) Histological score (H-score) graph showing a negative correlation between phosphorylation of Akt at Ser473 and CYLD abundance in prostate cancer samples. P < 0.001 by using Spearman’s correlation and Mann-Whitney U test. (D) Associations of H-score between phosphorylation of Akt at Ser473 and CYLD abundance and different tumor stages. Results are presented as means ± SD; P values were obtained with Spearman’s correlation and Mann-Whitney U test.

  • Fig. 7

    Model for growth factor–mediated ubiquitination and activation of Akt. In serum-starved cells (left panel), CYLD interacts with inactive Akt and keeps it in a hypoubiquitinated state. Growth factor signaling (dashed lines in right panel) may promote the dissociation of CYLD from Akt, thereby enabling the E3 ligase TRAF6 or Skp2 to interact with and ubiquitinate Akt, in turn facilitating its membrane recruitment, phosphorylation, and activation. Activation of Akt therefore contributes to tumorigenesis by promoting cancer cell proliferation, survival, and glucose metabolism.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/6/257/ra3/DC1

    Fig. S1. CYLD, but not USP7 and USP22, promotes deubiquitination of Akt.

    Fig. S2. CYLD deubiquitinates and interacts with Akt isoforms.

    Fig. S3. Phosphorylation of Akt does not affect its interaction with CYLD.

    Fig. S4. Cyld−/− MEFs show enhanced basal membrane localization of Akt.

    Fig. S5. The role of PI3K activity in the membrane recruitment of Akt in wild-type and Cyld−/− MEFs.

    Fig. S6. CYLD deficiency enhances cell proliferation in primary MEFs and breast cancer cells.

    Table S1. Quantification of the indicated immunoblotting signals.

  • Supplementary Materials for:

    Cycles of Ubiquitination and Deubiquitination Critically Regulate Growth Factor–Mediated Activation of Akt Signaling

    Wei-Lei Yang, Guoxiang Jin, Chien-Feng Li, Yun Seong Jeong, Asad Moten, Dazhi Xu, Zizhen Feng, Wei Chen, Zhen Cai, Bryant Darnay, Wei Gu, Hui-Kuan Lin*

    *To whom correspondence should be addressed. E-mail: hklin{at}mdanderson.org

    This PDF file includes:

    • Fig. S1. CYLD, but not USP7 and USP22, promotes deubiquitination of Akt.
    • Fig. S2. CYLD deubiquitinates and interacts with Akt isoforms.
    • Fig. S3. Phosphorylation of Akt does not affect its interaction with CYLD.
    • Fig. S4. Cyld–/– MEFs show enhanced basal membrane localization of Akt.
    • Fig. S5. The role of PI3K activity in the membrane recruitment of Akt in wild-type and Cyld–/– MEFs.
    • Fig. S6. CYLD deficiency enhances cell proliferation in primary MEFs and breast cancer cells.
    • Table S1. Quantification of the indicated immunoblotting signals.

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    Citation: W.-L. Yang, G. Jin, C.-F. Li, Y. S. Jeong, A. Moten, D. Xu, Z. Feng, W. Chen, Z. Cai, B. Darnay, W. Gu, H.-K. Lin, Cycles of Ubiquitination and Deubiquitination Critically Regulate Growth Factor–Mediated Activation of Akt Signaling. Sci. Signal. 6, ra3 (2013).

    © 2013 American Association for the Advancement of Science

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