This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wohlschlegel, J. A.
Right arrow Articles by Dutta, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wohlschlegel, J. A.
Right arrow Articles by Dutta, A.

 Previous Article  |  Next Article 

Molecular and Cellular Biology, August 2001, p. 4868-4874, Vol. 21, No. 15
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.15.4868-4874.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Mutational Analysis of the Cy Motif from p21 Reveals Sequence Degeneracy and Specificity for Different Cyclin-Dependent Kinases

James A. Wohlschlegel, Brian T. Dwyer, David Y. Takeda, and Anindya Dutta*

Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115

Received 16 March 2001/Returned for modification 26 April 2001/Accepted 2 May 2001

Inhibitors, activators, and substrates of cyclin-dependent kinases (cdks) utilize a cyclin-binding sequence, known as a Cy or RXL motif, to bind directly to the cyclin subunit. Alanine scanning mutagenesis of the Cy motif of the cdk inhibitor p21 revealed that the conserved arginine or leucine (constituting the conserved RXL sequence) was important for p21's ability to inhibit cyclin E-cdk2 activity. Further analysis of mutant Cy motifs showed, however, that RXL was neither necessary nor sufficient for a functional cyclin-binding motif. Replacement of either of these two residues with small hydrophobic residues such as valine preserved p21's inhibitory activity on cyclin E-cdk2, while mutations in either polar or charged residues dramatically impaired p21's inhibitory activity. Expressing p21N with non-RXL Cy sequences inhibited growth of mammalian cells, providing in vivo confirmation that RXL was not necessary for a functional Cy motif. We also show that the variant Cy motifs identified in this study can effectively target substrates to cyclin-cdk complexes for phosphorylation, providing additional evidence that these non-RXL motifs are functional. Finally, binding studies using p21 Cy mutants demonstrated that the Cy motif was essential for the association of p21 with cyclin E-cdk2 but not with cyclin A-cdk2. Taking advantage of this differential specificity toward cyclin E versus cyclin A, we demonstrate that cell growth inhibition was absolutely dependent on the ability of a p21 derivative to inhibit cyclin E-cdk2.

* Corresponding author. Mailing address: Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115. Phone: (617) 278-0468. Fax: (617) 732-7449. E-mail: adutta{at}rics.bwh.harvard.edu.

Molecular and Cellular Biology, August 2001, p. 4868-4874, Vol. 21, No. 15
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.15.4868-4874.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Park, E. A., MacAlpine, D. M., Orr-Weaver, T. L. (2007). Inaugural Article: Drosophila follicle cell amplicons as models for metazoan DNA replication: A cyclinE mutant exhibits increased replication fork elongation. Proc. Natl. Acad. Sci. USA 104: 16739-16746 [Abstract] [Full Text]  
  • Giono, L. E., Manfredi, J. J. (2007). Mdm2 Is Required for Inhibition of Cdk2 Activity by p21, Thereby Contributing to p53-Dependent Cell Cycle Arrest. Mol. Cell. Biol. 27: 4166-4178 [Abstract] [Full Text]  
  • Heron-Milhavet, L., Franckhauser, C., Rana, V., Berthenet, C., Fisher, D., Hemmings, B. A., Fernandez, A., Lamb, N. J. C. (2006). Only Akt1 Is Required for Proliferation, while Akt2 Promotes Cell Cycle Exit through p21 Binding. Mol. Cell. Biol. 26: 8267-8280 [Abstract] [Full Text]  
  • Churchman, M. L., Brown, M. L., Kato, N., Kirik, V., Hulskamp, M., Inze, D., De Veylder, L., Walker, J. D., Zheng, Z., Oppenheimer, D. G., Gwin, T., Churchman, J., Larkin, J. C. (2006). SIAMESE, a Plant-Specific Cell Cycle Regulator, Controls Endoreplication Onset in Arabidopsis thaliana. Plant Cell 18: 3145-3157 [Abstract] [Full Text]  
  • Cheng, K.-Y., Noble, M. E. M., Skamnaki, V., Brown, N. R., Lowe, E. D., Kontogiannis, L., Shen, K., Cole, P. A., Siligardi, G., Johnson, L. N. (2006). The Role of the Phospho-CDK2/Cyclin A Recruitment Site in Substrate Recognition. J. Biol. Chem. 281: 23167-23179 [Abstract] [Full Text]  
  • Surjit, M., Liu, B., Chow, V. T. K., Lal, S. K. (2006). The Nucleocapsid Protein of Severe Acute Respiratory Syndrome-Coronavirus Inhibits the Activity of Cyclin-Cyclin-dependent Kinase Complex and Blocks S Phase Progression in Mammalian Cells. J. Biol. Chem. 281: 10669-10681 [Abstract] [Full Text]  
  • Takeda, D. Y., Parvin, J. D., Dutta, A. (2005). Degradation of Cdt1 during S Phase Is Skp2-independent and Is Required for Efficient Progression of Mammalian Cells through S Phase. J. Biol. Chem. 280: 23416-23423 [Abstract] [Full Text]  
  • Yoshizawa-Sugata, N., Ishii, A., Taniyama, C., Matsui, E., Arai, K.-i., Masai, H. (2005). A Second Human Dbf4/ASK-related Protein, Drf1/ASKL1, Is Required for Efficient Progression of S and M Phases. J. Biol. Chem. 280: 13062-13070 [Abstract] [Full Text]  
  • Dubourg, B., Kamphausen, T., Weiwad, M., Jahreis, G., Feunteun, J., Fischer, G., Modjtahedi, N. (2004). The Human Nuclear SRcyp Is a Cell Cycle-regulated Cyclophilin. J. Biol. Chem. 279: 22322-22330 [Abstract] [Full Text]  
  • Wilmes, G. M., Archambault, V., Austin, R. J., Jacobson, M. D., Bell, S. P., Cross, F. R. (2004). Interaction of the S-phase cyclin Clb5 with an 'RXL' docking sequence in the initiator protein Orc6 provides an origin-localized replication control switch. Genes Dev. 18: 981-991 [Abstract] [Full Text]  
  • Major, M. L., Lepe, R., Costa, R. H. (2004). Forkhead Box M1B Transcriptional Activity Requires Binding of Cdk-Cyclin Complexes for Phosphorylation-Dependent Recruitment of p300/CBP Coactivators. Mol. Cell. Biol. 24: 2649-2661 [Abstract] [Full Text]  
  • Stevenson-Lindert, L. M., Fowler, P., Lew, J. (2003). Substrate Specificity of CDK2-Cyclin A: WHAT IS OPTIMAL?. J. Biol. Chem. 278: 50956-50960 [Abstract] [Full Text]  
  • Prokhorova, T. A., Mowrer, K., Gilbert, C. H., Walter, J. C. (2003). DNA replication of mitotic chromatin in Xenopus egg extracts. Proc. Natl. Acad. Sci. USA 100: 13241-13246 [Abstract] [Full Text]  
  • Goda, T., Ishii, T., Nakajo, N., Sagata, N., Kobayashi, H. (2003). The RRASK Motif in Xenopus Cyclin B2 Is Required for the Substrate Recognition of Cdc25C by the Cyclin B-Cdc2 Complex. J. Biol. Chem. 278: 19032-19037 [Abstract] [Full Text]  
  • Mendoza, N., Fong, S., Marsters, J., Koeppen, H., Schwall, R., Wickramasinghe, D. (2003). Selective Cyclin-dependent Kinase 2/Cyclin A Antagonists that Differ from ATP Site Inhibitors Block Tumor Growth. Cancer Res. 63: 1020-1024 [Abstract] [Full Text]  
  • Wohlschlegel, J. A., Kutok, J. L., Weng, A. P., Dutta, A. (2002). Expression of Geminin as a Marker of Cell Proliferation in Normal Tissues and Malignancies. Am. J. Pathol. 161: 267-273 [Abstract] [Full Text]  
  • Leng, X., Noble, M., Adams, P. D., Qin, J., Harper, J. W. (2002). Reversal of Growth Suppression by p107 via Direct Phosphorylation by Cyclin D1/Cyclin-Dependent Kinase 4. Mol. Cell. Biol. 22: 2242-2254 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»