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Cyclin-dependent kinases regulate the antiproliferative function of Smads

Nature volume 430, pages 226–231 (2004)Cite this article

Abstract

Transforming growth factor-β (TGF-β) potently inhibits cell cycle progression at the G1 phase1,2. Smad3 has a key function in mediating the TGF-β growth-inhibitory response. Here we show that Smad3 is a major physiological substrate of the G1 cyclin-dependent kinases CDK4 and CDK2. Except for the retinoblastoma protein family3,4, Smad3 is the only CDK4 substrate demonstrated so far. We have mapped CDK4 and CDK2 phosphorylation sites to Thr 8, Thr 178 and Ser 212 in Smad3. Mutation of the CDK phosphorylation sites increases Smad3 transcriptional activity, leading to higher expression of the CDK inhibitor p15. Mutation of the CDK phosphorylation sites of Smad3 also increases its ability to downregulate the expression of c-myc. Using Smad3-/- mouse embryonic fibroblasts and other epithelial cell lines, we further show that Smad3 inhibits cell cycle progression from G1 to S phase and that mutation of the CDK phosphorylation sites in Smad3 increases this ability. Taken together, these findings indicate that CDK phosphorylation of Smad3 inhibits its transcriptional activity and antiproliferative function. Because cancer cells often contain high levels of CDK activity5,6, diminishing Smad3 activity by CDK phosphorylation may contribute to tumorigenesis and TGF-β resistance in cancers.

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Figure 1: CDK4 and CDK2 can phosphorylate Smad3 and Smad2 in vitro.
Figure 2: Smad3 is phosphorylated by endogenous G1 CDKs in vivo.
Figure 3: Mutation of CDK phosphorylation sites in Smad3 leads to an increased p15 level and reduced c-myc expression in reporter gene assays.
Figure 4: Mutation of the CDK phosphorylation sites in Smad3 leads to increased antiproliferative activities.

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Acknowledgements

We are very grateful to X.-F. Wang for Smad3 mutant mice; E. P. Reddy, R. V. Mettus and H. Kiyokawa for CDK4 mutant mice; J. Massagué for reagents and continued support; C.-X. Deng for reagents and communications before publication; M. M. Shen and members of his laboratory for assistance with mouse work; C. Abate-Shen, M. Cobb, X.-F. Feng, J. Germino, G. J. Hannon, S.-J. Kim, M. Kretzschmar, H. Lee, M.-H. Lee, E. Lees, X. Liu, H. L. Moses, G. Nolan, A. Rabson, D. Reinberg, M. Reiss, Y. Shi, N. Walworth and W. Xie for reagents and/or suggestions; and numerous colleagues for discussions. This work was supported by the 1999 American Association for Cancer Research–National Foundation for Cancer Research Career Development Award, a Burroughs Wellcome Fund New Investigator Award, a Kimmel Scholar Award from the Sidney Kimmel Foundation for Cancer Research, the Emerald Foundation, the New Jersey Commission on Cancer Research, and the NIH (to F.L.).

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Author notes

  1. Natalia G. Denissova and Guannan Wang: These authors contributed equally to this work

Authors and Affiliations

  1. Center for Advanced Biotechnology and Medicine, Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, New Jersey, 08854, USA

    Isao Matsuura, Natalia G. Denissova, Guannan Wang, Dongming He, Jianyin Long & Fang Liu

  2. Susan Lehman Cullman Laboratory for Cancer Research, Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, New Jersey, 08854, USA

    Isao Matsuura, Natalia G. Denissova, Guannan Wang, Dongming He, Jianyin Long & Fang Liu

  3. Cancer Institute of New Jersey, New Brunswick, New Jersey, 08903, USA

    Isao Matsuura, Natalia G. Denissova, Guannan Wang, Dongming He, Jianyin Long & Fang Liu

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  1. Isao Matsuura
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Correspondence to Fang Liu.

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The authors declare that they have no competing financial interests.

Supplementary information

Supplementary Information

Supplementary notes, references, and figure legends (DOC 36 kb)

Supplementary Figure S1 and Table S1

Smad3 is an excellent substrate for CDK4 in vitro. (PDF 131 kb)

Supplementary Figure S2

Smad3 contains potential CDK phosphorylation sites. (PDF 76 kb)

Supplementary Figure S3

The T8, T178, S212, S203 and S207 in Smad3 are phosphorylated by CDK4 and CDK2 in vitro. (PDF 76 kb)

Supplementary Figure S4

The pT8, pT178, pS212, pS203 and pS207 phosphopeptide antibodies have very good specificities towards phosphorylated versus unphosphorylated Smad3. (PDF 104 kb)

Supplementary Figure S5

The T8, T178, and S212 in Smad3 are phosphorylated by CDK4 and CDK2 in vivo. (PDF 215 kb)

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Matsuura, I., Denissova, N., Wang, G. et al. Cyclin-dependent kinases regulate the antiproliferative function of Smads. Nature 430, 226–231 (2004). https://doi.org/10.1038/nature02650

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