Abstract
Skp2 is an F-box protein that forms the SCF complex with Skp1 and Cullin-1 to constitute an E3 ligase for ubiquitylation. Ubiquitylation and degradation of the p27 are critical for Skp2-mediated entry to the cell cycle, and overexpression and cytosolic accumulation of Skp2 have been clearly associated with tumorigenesis, although the functional significance of the latter is still unknown. Here we show that Akt/protein kinase B (PKB) interacts with and directly phosphorylates Skp2. We find that Skp2 phosphorylation by Akt triggers SCF complex formation and E3 ligase activity. A phosphorylation-defective Skp2 mutant is drastically impaired in its ability to promote cell proliferation and tumorigenesis. Furthermore, we show that Akt-mediated phosphorylation triggers 14-3-3β-dependent Skp2 relocalization to the cytosol, and we attribute a specific role to cytosolic Skp2 in the positive regulation of cell migration. Finally, we demonstrate that high levels of activation of Akt correlate with the cytosolic accumulation of Skp2 in human cancer specimens. Our results therefore define a novel proto-oncogenic Akt/PKB-dependent signalling pathway.
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References
Bloom, J. & Pagano, M. Deregulated degradation of the cdk inhibitor p27 and malignant transformation. Semin. Cancer Biol. 13, 41–47 (2003).
Nakayama, K. I. & Nakayama, K. Regulation of the cell cycle by SCF-type ubiquitin ligases. Semin. Cell Dev. Biol. 16, 323–333 (2005).
Amati, B. & Vlach, J. Kip1 meets SKP2: new links in cell-cycle control. Nature Cell Biol. 1, E91–E93 (1999).
Carrano, A. C., Eytan, E., Hershko, A. & Pagano, M. SKP2 is required for ubiquitin-mediated degradation of the CDK inhibitor p27. Nature Cell Biol. 1, 193–199 (1999).
Tsvetkov, L. M., Yeh, K. H., Lee, S. J., Sun, H. & Zhang, H. p27Kip1 ubiquitination and degradation is regulated by the SCFSkp2 complex through phosphorylated Thr187 in p27. Curr. Biol. 9, 661–664 (1999).
Kossatz, U. et al. Skp2-dependent degradation of p27kip1 is essential for cell cycle progression. Genes Dev. 18, 2602–2607 (2004).
Sutterlüty, H. et al. p45SKP2 promotes p27Kip1 degradation and induces S phase in quiescent cells. Nature Cell Biol. 1, 207–214 (1999).
Nakayama, K. et al. Targeted disruption of Skp2 results in accumulation of cyclin E and p27Kip1, polyploidy and centrosome overduplication. EMBO J. 19, 2069–2081 (2000).
Nakayama, K. et al. Skp2-mediated degradation of p27 regulates progression into mitosis. Dev. Cell 6, 661–672 (2004).
Gstaiger, M. et al. Skp2 is oncogenic and overexpressed in human cancers. Proc. Natl Acad. Sci. USA 98, 5043–5048 (2001).
Latres, E. et al. Role of the F-box protein Skp2 in lymphomagenesis. Proc. Natl Acad. Sci. USA 98, 2515–2520 (2001).
Shim, E. H. et al. Expression of the F-box protein SKP2 induces hyperplasia, dysplasia, and low-grade carcinoma in the mouse prostate. Cancer Res. 63, 1583–1588 (2003).
Zhang, H., Kobayashi, R., Galaktionov, K. & Beach, D. p19Skp1 and p45Skp2 are essential elements of the cyclin A-CDK2 S phase kinase. Cell 82, 915–925 (1995).
Bilodeau, M. et al. Skp2 induction and phosphorylation is associated with the late G1 phase of proliferating rat hepatocytes. FEBS Lett. 452, 247–253 (1999).
Dan, H. C. et al. Phosphatidylinositol 3-kinase/Akt pathway regulates tuberous sclerosis tumor suppressor complex by phosphorylation of tuberin. J. Biol. Chem. 277, 35364–35370 (2002).
Inoki, K., Li, Y., Zhu, T., Wu, J. & Guan, K. L. TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling. Nature Cell Biol. 4, 648–657 (2002).
Manning, B. D., Tee, A. R., Logsdon, M. N., Blenis, J. & Cantley, L. C. Identification of the tuberous sclerosis complex-2 tumor suppressor gene product tuberin as a target of the phosphoinositide 3-kinase/akt pathway. Mol. Cell 10, 151–162 (2002).
Potter, C. J., Pedraza, L. G. & Xu, T. Akt regulates growth by directly phosphorylating Tsc2. Nature Cell Biol. 4, 658–665 (2002).
Yaffe, M. B. et al. A motif-based profile scanning approach for genome-wide prediction of signaling pathways. Nature Biotechnol. 19, 348–353 (2001).
Posewitz, M. C. & Tempst, P. Immobilized gallium(III) affinity chromatography of phosphopeptides. Anal. Chem. 71, 2883–2892 (1999).
Malek, N. P. et al. A mouse knock-in model exposes sequential proteolytic pathways that regulate p27Kip1 in G1 and S phase. Nature 413, 323–327 (2001).
Drobnjak, M. et al. Altered expression of p27 and Skp2 proteins in prostate cancer of African-American patients. Clin. Cancer Res. 9, 2613–2619 (2003).
Li, Q., Murphy, M., Ross, J., Sheehan, C. & Carlson, J. A. Skp2 and p27kip1 expression in melanocytic nevi and melanoma: an inverse relationship. J. Cutan. Pathol. 31, 633–642 (2004).
Lim, M. S. et al. Expression of Skp2, a p27Kip1 ubiquitin ligase, in malignant lymphoma: correlation with p27Kip1 and proliferation index. Blood 100, 2950–2956 (2002).
Radke, S., Pirkmaier, A. & Germain, D. Differential expression of the F-box proteins Skp2 and Skp2B in breast cancer. Oncogene 24, 3448–3458 (2005).
Cantley, L. C. & Neel, B. G. New insights into tumor suppression: PTEN suppresses tumor formation by restraining the phosphoinositide 3-kinase/AKT pathway. Proc. Natl Acad. Sci. USA 96, 4240–4245 (1999).
Di Cristofano, A. & Pandolfi, P. P. The multiple roles of PTEN in tumor suppression. Cell 100, 387–390 (2000).
Brazil, D. P., Park, J. & Hemmings, B. A. PKB binding proteins. Getting in on the Akt. Cell 111, 293–303 (2002).
Datta, S. R., Brunet, A. & Greenberg, M. E. Cellular survival: a play in three Akts. Genes Dev. 13, 2905–2927 (1999).
Gao, D. et al. Phosphorylation by Akt1 promotes Skp2 cytoplasmic localization and impairs APC/Cdh1-mediated Skp2 destruction. Nature Cell Biol. 11, doi: 10.1038/ncb1847 (2009).
Hermeking, H. The 14-3-3 cancer connection. Nature Rev. Cancer 3, 931–943 (2003).
Brunet, A. et al. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 96, 857–868 (1999).
Brunet, A. et al. 14-3-3 transits to the nucleus and participates in dynamic nucleocytoplasmic transport. J. Cell Biol. 156, 817–828 (2002).
Fujita, N., Sato, S., Katayama, K. & Tsuruo, T. Akt-dependent phosphorylation of p27Kip1 promotes binding to 14-3-3 and cytoplasmic localization. J. Biol. Chem. 277, 28706–28713 (2002).
Sekimoto, T., Fukumoto, M. & Yoneda, Y. 14-3-3 suppresses the nuclear localization of threonine 157-phosphorylated p27Kip1. EMBO J. 23, 1934–1942 (2004).
Mamillapalli, R. et al. PTEN regulates the ubiquitin-dependent degradation of the CDK inhibitor p27KIP1 through the ubiquitin E3 ligase SCFSKP2. Curr Biol 11, 263–267 (2001).
Rodier, G., Coulombe, P., Tanguay, P. L., Boutonnet, C. & Meloche, S. Phosphorylation of Skp2 regulated by CDK2 and Cdc14B protects it from degradation by APCCdh1 in G1 phase. EMBO J. 27, 679–691 (2008).
Zheng, N. et al. Structure of the Cul1–Rbx1–Skp1–F boxSkp2 SCF ubiquitin ligase complex. Nature 416, 703–709 (2002).
Lin, H. K., Bergmann, S. & Pandolfi, P. P. Cytoplasmic PML function in TGF-β signalling. Nature 431, 205–211 (2004).
Lin, H. K., Wang, L., Hu, Y. C., Altuwaijri, S. & Chang, C. Phosphorylation-dependent ubiquitylation and degradation of androgen receptor by Akt require Mdm2 E3 ligase. EMBO J. 21, 4037–4048 (2002).
Ma, L., Chen, Z., Erdjument-Bromage, H., Tempst, P. & Pandolfi, P. P. Phosphorylation and functional inactivation of TSC2 by Erk: implications for tuberous sclerosis and cancer pathogenesis. Cell 121, 179–193 (2005).
Sebastiaan Winkler, G. et al. Isolation and mass spectrometry of transcription factor complexes. Methods 26, 260–269 (2002).
Kim, S. Y., Herbst, A., Tworkowski, K. A., Salghetti, S. E. & Tansey, W. P. Skp2 regulates Myc protein stability and activity. Mol. Cell 11, 1177–1188 (2003).
Lin, H. K., Yeh, S., Kang, H. Y. & Chang, C. Akt suppresses androgen-induced apoptosis by phosphorylating and inhibiting androgen receptor. Proc. Natl Acad. Sci. USA 98, 7200–7205 (2001).
Acknowledgements
We thank D. Bohmann, P. Jackson, W. Wei, M. Pagano and M. H. Lee for reagents. We are also grateful to M. Asherov and I. Linkov in the Immunohistochemistry Pathology Core Laboratory, T. Matos for immunohistochemistry technical assistance, P. Bonner for data management, L. Lacomis for help with mass spectrometry, X. H. Zhu for technical advice, and S. Clohessy for flow cytometry analysis. We also thank M. C. Hung and L. Cantley for insightful comments and suggestions, and W. Wei for discussion and for sharing experimental results. Special thanks are extended to B. Carver and L. DiSantis for editing and critical reading of the manuscript, as well as to all the members of the Pandolfi laboratory for comments and discussion. This work was supported by NIH grants RO1 CA-71692 and CA-74031 to P.P.P. and by M. D. Anderson Cancer Center Trust Scholar funds to H.K.L. The Microchemistry & Proteomics Core is supported by NIH grant P30 CA-08748.
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H.K.L. and P.P.P. designed the experiments and wrote the manuscript. H.K.L., G.W. Z.C., Y.L., C.H.C. and W.L.Y. performed the experiments. J.T. performed the immunohistochemistry and analysed the data. K.I.N. provided the Skp2−/− mice. S.N. provided valuable suggestions. H.E. and P.T. performed the mass spectrometry analysis.
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Lin, HK., Wang, G., Chen, Z. et al. Phosphorylation-dependent regulation of cytosolic localization and oncogenic function of Skp2 by Akt/PKB. Nat Cell Biol 11, 420–432 (2009). https://doi.org/10.1038/ncb1849
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DOI: https://doi.org/10.1038/ncb1849
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