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p45SKP2 promotes p27Kip1 degradation and induces S phase in quiescent cells

Abstract

The F-box protein p45SKP2 is the substrate-targeting subunit of the ubiquitin–protein ligase SCFSKP2 and is frequently overexpressed in transformed cells. Here we report that expression of p45SKP2 in untransformed fibroblasts activates DNA synthesis in cells that would otherwise growth-arrest. Expression of p45SKP2 in quiescent fibroblasts promotes p27Kip1 degradation, allows the generation of cyclin-A-dependent kinase activity and induces S phase. Coexpression of a degradation-resistant p27Kip1 mutant suppresses p45SKP2-induced cyclin-A-kinase activation and S-phase entry. We propose that p45SKP2 is important in the progression from quiescence to S phase and that the ability of p45SKP2 to promote p27Kip1 degradation is a key aspect of its S-phase-inducing function. In transformed cells, p45SKP2 may contribute to deregulated initiation of DNA replication by interfering with p27Kip1 function.

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Figure 1: Effects of overexpression of p45SKP2 on growth of Rat-1 cells in low concentrations of serum.
Figure 2: Production of p45SKP2 in serum-starved fibroblasts induces S-phase entry, activation of kinases associated with cyclin A/E and proteasomal elimination of p27Kip1.
Figure 3: A degradation-resistant mutant of p27Kip1 suppresses S-phase induction by p45SKP2.
Figure 4: Analysis of p45SKP2 mutants defective in forming SCF complexes and in binding to cyclin-A-associated kinase activities.
Figure 5: S-phase induction by p45SKP2 in quiescent cells is followed by apoptosis.
Figure 6: p45SKP2 induces quiescent primary MEFs to enter S phase.

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References

  1. Nigg, E. A. Cyclin-dependent protein kinases: key regulators of the eukaryotic cell cycle . Bioessays 17, 471–480 (1995).

    Article  CAS  Google Scholar 

  2. Sherr, C. J. Cancer cell cycles. Science 274, 1672– 1677 (1996).

    Article  CAS  Google Scholar 

  3. Heichman, K. A. & Roberts, J. M. Rules to replicate by. Cell 79, 557–562 (1994).

    Article  CAS  Google Scholar 

  4. Pines, J. & Hunter, T. Human cyclin A is adenovirus E1A-associated protein p60 and behaves differently from cyclin B. Nature 346, 760–763 (1990).

    Article  CAS  Google Scholar 

  5. Cardoso, M. C., Leonhardt, H. & Nadal-Ginard, B. Reversal of terminal differentiation and control of DNA replication: cyclin A and Cdk2 specifically localize at subnuclear sites of DNA replication. Cell 74, 979– 992 (1993).

    Article  CAS  Google Scholar 

  6. Krude, T., Jackman, M., Pines, J. & Laskey, R. A. Cyclin/Cdk-dependent initiation of DNA replication in a human cell-free system. Cell 88, 109–119 ( 1997).

    Article  CAS  Google Scholar 

  7. Pagano, M., Pepperkok, R., Verde, F., Ansorge, W. & Draetta, G. Cyclin As required at two points in the human cell cycle . EMBO J. 11, 961–971 (1992).

    Article  CAS  Google Scholar 

  8. Zindy, F. et al. Cyclin A is required in S phase in normal epithelial cells. Biochem. Biophys. Res. Commun. 182, 1144– 1154 (1992).

    Article  CAS  Google Scholar 

  9. Girard, F., Strausfeld, U., Fernandez, A. & Lamb, N. J. Cyclin A is required for the onset of DNA replication in mammalian fibroblasts . Cell 67, 1169–1179 (1991).

    Article  CAS  Google Scholar 

  10. Russo, A. A., Jeffrey, P. D., Patten, A. K., Massague, J. & Pavletich, N. P. Crystal structure of the p27Kip1 cyclin-dependent-kinase inhibitor bound to the cyclin A-Cdk2 complex. Nature 382, 325– 331 (1996).

    Article  CAS  Google Scholar 

  11. Schulman, B. A., Lindstrom, D. L. & Harlow, E. Substrate recruitment to cyclin-dependent kinase 2 by a multipurpose docking site on cyclin A. Proc. Natl Acad. Sci. USA 95, 10453–10458 ( 1998).

    Article  CAS  Google Scholar 

  12. Resnitzky, D., Hengst, L. & Reed, S. I. Cyclin A-associated kinase activity is rate-limiting for entrance into S phase and is negatively regulated in G1 by p27Kip1. Mol. Cell. Biol. 15, 4347– 4352 (1995).

    Article  CAS  Google Scholar 

  13. 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).

    Article  CAS  Google Scholar 

  14. Bai, C. et al. SKP1 connects cell cycle regulators to the ubiquitin proteolysis machinery through a novel motif, the F-box. Cell 86 , 263–274 (1996).

    Article  CAS  Google Scholar 

  15. Koepp, D. M., Harper, J. W. & Elledge, S. J. How the cyclin became a cyclin: regulated proteolysis in the cell cycle. Cell 97, 431– 434 (1999).

    Article  CAS  Google Scholar 

  16. Patton, E. E., Willems, A. R. & Tyers, M. Combinatorial control in ubiquitin-dependent proteolysis: don’t Skp the F-box hypothesis. Trends Genet. 14, 236–243 (1998).

    Article  CAS  Google Scholar 

  17. Lisztwan, J. et al. Association of human CUL-1 and ubiquitin-conjugating enzyme CDC34 with the F-box protein p45SKP2: evidence for evolutionary conservation in the subunit composition of the CDC34-SCF pathway. EMBO J. 17, 368–383 ( 1998).

    Article  CAS  Google Scholar 

  18. Lyapina, S. A., Correll, C. C., Kipreos, E. T. & Deshaies, R. J. Human CUL1 forms an evolutionarily conserved ubiquitin ligase complex (SCF) with SKP1 and an F-box protein. Proc. Natl Acad. Sci. USA 95, 7451–7456 (1998).

    Article  CAS  Google Scholar 

  19. Michel, J. J. & Xiong, Y. Human Cul-1, but not other cullin family members selectively interacts with SKP1 to form a complex with SKP2 and Cyclin A. Cell Growth Differ. 9, 435 –449 (1998).

    CAS  PubMed  Google Scholar 

  20. Yu, Z. K., Gervais, J. L. M. & Zhang, H. Human CUL-1 associates with the SKP1/SKP2 complex and regulates p21(CIP1/WAF1) and cyclin D proteins. Proc. Natl Acad. Sci. USA 95, 11324–11329 (1998).

    Article  CAS  Google Scholar 

  21. Marti, A., Wirbelauer, C., Scheffner, M. & Krek, W. Interaction between the ubiquitin-protein ligase SCFSKP2 and E2F-1 underlies the regulation of E2F-1 degradation. Nature Cell Biol. 1, 14–19 ( 1999).

    Article  CAS  Google Scholar 

  22. Kato, J., Matsuoka, M., Polyak, K., Massague, J. & Cherr, C. J. Cyclic-AMP-induced G1 arrest mediated by an inhibitor, p27(Kip1), of cyclin-dependent kinase 4 activation. Cell 79, 487–496 (1994).

    Article  CAS  Google Scholar 

  23. Nourse, J. et al. Interleukin-2-mediated elimination of the p27Kip1 cyclin-dependent kinase inhibitor prevented by rapamycin. Nature 372, 570–573 ( 1994).

    Article  CAS  Google Scholar 

  24. Pagano, M. et al. Role of the ubiquitin-proteasome pathway in regulating abundance of the cyclin-dependent kinase inhibitor p27. Science 269, 682–685 (1995).

    Article  CAS  Google Scholar 

  25. Coats, S., Flanagan, W. M., Nourse, J. & Roberts, J. M. Requirement of p27Kip1 for restriction point control of the fibroblast cell cycle. Science 272, 877– 880 (1996).

    Article  CAS  Google Scholar 

  26. Polyak, K. et al. Cloning of p27Kip1, a cyclin-dependent kinase inhibitor and a potential mediator of extracellular antimitogenic signals . Cell 78, 59–66 (1994).

    Article  CAS  Google Scholar 

  27. Vlach, J., Hennecke, S., Alevizopoulos, K., Conti, D. & Amati, B. Growth arrest by the cyclin-dependent kinase inhibitor p27Kip1 is abrogated by c-Myc. EMBO J. 15, 6595–6604 ( 1996).

    Article  CAS  Google Scholar 

  28. Sheaff, R. J., Groudine, M., Gordon, M., Roberts, J. M. & Clurman, B. E. Cyclin E-CDK2 is a regulator of p27Kip1. Genes Dev 11, 1464–1478 (1997).

    Article  CAS  Google Scholar 

  29. Vlach, J., Hennecke, S. & Amati, B. Phosphorylation-dependent degradation of the cyclin-dependent kinase inhibitor p27Kip1. EMBO J. 16 , 5334–5344 (1997).

    Article  CAS  Google Scholar 

  30. Montagnoli, A. et al. Ubiquitination of p27 is regulated by cdk-dependent phosphorylation and trimeric complex formation. Genes Dev. 13, 1181–1189 (1999).

    Article  CAS  Google Scholar 

  31. Nguyen, H., Gitig, D. M. & Koff, A. Cell-free degradation of p27Kip1, a G1 cyclin-dependent kinase inhibitor, is dependent on CDK2 activity and the proteasome . Mol. Cell. Biol. 19, 1190– 1201 (1999).

    Article  CAS  Google Scholar 

  32. Shirane, M. et al. Down-regulation of p27(Kip1) by two mechanisms ubiquitin-mediated degradation and proteolytic processing. J. Biol. Chem. 274, 13886–13893 (1999).

    Article  CAS  Google Scholar 

  33. Johnson, D. G., Schwarz, J. K., Cress, W. D. & Nevins, J. R. Expression of transcription factor E2F-1 induced quiescent cells to enter S phase. Nature 365, 349–352 (1993).

  34. Eilers, M., Schirm, S. & Bishop, J. M. The MYC protein activates transcription of the alpha-prothymosin gene. EMBO J. 10, 133–141 (1991).

    Article  CAS  Google Scholar 

  35. Connell-Crowley, L., Elledge, S. J. & Harper, J. W. G1 cyclin-dependent kinases are sufficient to initiate DNA synthesis in quiescent human fibroblasts. Curr. Biol. 8, 65–68 (1998).

    Article  CAS  Google Scholar 

  36. Porter, P. L. et al. Expression of the cell-cycle regulators p27Kip1 and cyclin E, alone and in combination, correlate with survival in young breast cancer patients. Nature Med. 3, 222– 226 (1997).

    Article  CAS  Google Scholar 

  37. Loda, M. et al. Increased proteasome-dependent degradation of the cyclin-dependent kinase inhibitor p27 in aggessive colorectal carcinomas. Nature Med. 3, 231–234 ( 1997).

    Article  CAS  Google Scholar 

  38. Catzavelos, C. et al. Decreased levels of the cell cycle inhibitor p27Kip1 protein: Prognostic implications in primary breast cancer. Nature Med. 3, 227–230 ( 1997).

    Article  CAS  Google Scholar 

  39. Fero, M. L., Randel, E., Gurley, K. E., Roberts, J. M. & Kemp, C. J. The murine gene p27Kip1 is haplo-insufficient for tumor suppression. Nature 396, 177–180 (1998).

    Article  CAS  Google Scholar 

  40. Hurford, R. K., Cobrinik, D., Lee, M. H. & Dyson, N. pRB and p107/p130 are required for the regulated expression of different sets of E2F responsive genes. Genes Dev. 11, 1447– 1463 (1997).

    Article  CAS  Google Scholar 

  41. Hardy, S., Kitamura, M., Harris-Stansil, T., Dai, Y. & Phipps, M. L. Construction of adenovirus vectors through Cre-lox recombination. J. Virol. 71, 1842–1849 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Krek, W., Xu, G. & Livingston, D. M. Cyclin A-kinase regulation of E2F-1DNA binding function underlies suppression of an S phase checkpoint. Cell 83, 1149–1158 (1995).

    Article  CAS  Google Scholar 

  43. Ludin, B., Doll, T., Meili, R., Kaech, S. & Matus, A. Application of novel vectors for GFP-tagging of proteins to study microtubule-associated proteins. Gene 173, 107–111 (1996).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank members of our laboratory for helpful discussions; D. Cobrinik for MEFs; B. Amati for retroviral plasmids for p27Kip1; members of the Matus laboratory, in particular B. Ludin and S. Käch, for help in GFP analysis; and B. Amati and B. Hemmings for critical reading of the manuscript. This work was supported by a postdoctoral fellowship to H.S. from the Erwin Schrödinger Society, Austria. E.C. is supported by a grant from the Swiss National Science Foundation. A.M. and C.W. are supported by the Novartis Research Foundation. W.K. is a START Fellow and is supported by the Swiss National Science Foundation and the Novartis Research Foundation.

Correspondence and requests for material should be addressed to W.K.

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Correspondence to Wilhelm Krek.

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Sutterlüty, H., Chatelain, E., Marti, A. et al. p45SKP2 promotes p27Kip1 degradation and induces S phase in quiescent cells. Nat Cell Biol 1, 207–214 (1999). https://doi.org/10.1038/12027

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