Abstract
p21WAF1 was originally identified as a protein that binds and inhibits cyclin-dependent kinases (CDKs). p21WAF1 is recognized to have at least two separate roles—first as a CDK inhibitor, and second as an inhibitor of PCNA, an accessory protein of DNA polymerase δ. p21WAF1 plays a critical role in the cellular response to DNA damage. Additionally, p21WAF1 plays a role in DNA repair, apoptosis, cellular senescence, terminal differentiation, and cell cycle arrest upon extracellular signaling. p21WAF1 protein levels are regulated both by transcriptional control by p53 and by factors other than p53, as well as by posttranscriptional regulation. Although the role of p21WAF1 has been explained so far only by its interaction with CDKs and with PCNA, it has several other binding partners. The ability of p21WAF1 to participate in several cellular functions has been widely studied by transfection of cells with p21WAF1 vectors. We describe here procedures for analysis of p21WAF1 function in mammalian cells after transfection of p21 plasmids. The procedures include inhibition of DNA synthesis, cellular localization, association with binding partners, and half-life measurements.
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References
Boulaire, J., Fotedar, A., and Fotedar, R. (2000) The functions of cdk—cyclin kinase inhibitor p21. Pathol. Biol. 48, 192–202.
Harper, J. W., Adami, G. R., Wei, N., Keyomarsi, K., and Elledge, S. J. (1993) The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell 75, 805–816.
Gu, Y., Turck, C. W., and Morgan, D. O. (1993) Inhibition of CDK2 activity in vivo by an associated 20K regulatory subunit. Nature 366, 707–710.
Xiong, Y., Hannon, G. J., Zhang, H., Casso, D., Kobayashi, R., and Beach, D. (1993) p21 is a universal inhibitor of cyclin kinases. Nature 366, 701–704.
Waga, S., Hannon, G. J., Beach, D., and Stillman, B. (1994) The p21 inhibitor of cyclin-dependent kinases controls DNA replication by interaction with PCNA Nature 369, 574–578.
Chen, J., Jackson, P. K., Kirschner, M. W., and Dutta, A. (1995) Separate domains of p21 involved in the inhibition of Cdk kinase and PCNA. Nature 374, 386–388.
Goubin, F., and Ducommun, B. (1995) Identification of binding domains on the p21Cip1 cyclin-dependent kinase inhibitor. Oncogene 10, 2281–2287.
Harper, J. W., Elledge, S. J., Keyomarsi, K., Dynlacht, B., Tsai, L. H., Zhang, P., et al. (1995) Inhibition of cyclin-dependent kinases by p21. Mol Biol Cell 6, 387–400.
Luo, Y., Hurwitz, J., and Massague, J. (1995) Cell-cycle inhibition by independent CDK and PCNA binding domains in p21Cip1. Nature 375, 159–161.
Nakanishi, M., Robetorye, R. S., Pereira-Smith, O. M., and Smith, J. R. (1995) The C-terminal region of p21SDI1/WAF1/CIP1 is involved in proliferating cell nuclear antigen binding but does not appear to be required for growth inhibition. J. Biol. Chem. 270, 17060–17063.
Adams, P. D., Sellers, W. R., Sharma, S. K., Wu, A. D., Nalin, C. M., and Kaelin, W. G., Jr. (1996) Identification of a cyclin—cdk2 recognition motif present in substrates and p21-like cyclin-dependent kinase inhibitors. Mol. Cell. Biol. 16, 6623–6633.
Fotedar, R., Fitzgerald, P., Rousselle, T., et al. (1996) p21 contains independent binding sites for cyclin and cdk2: both sites are required to inhibit cdk2 kinase activity. Oncogene 12, 2155–2164.
Russo, A. A., Jeffrey, P. D., and Pavletich, N. P. (1996) Structural basis of cyclin-dependent kinase activation by phosphorylation. Nat. Struct. Biol. 3, 696–700.
Ball, K. L., Lain, S., Fahraeus, R., Smythe, C., and Lane, D. P. (1997) Cell-cycle arrest and inhibition of Cdk4 activity by small peptides based on the carboxy-terminal domain of p21WAF1. Curr. Biol. 7, 71–80.
Rousseau, D., Cannella, D., Boulaire, J., Fitzgerald, P., Fotedar, A., and Fotedar, R. (1999) Growth inhibition by CDK—cyclin and PCNA binding domains of p21 occurs by distinct mechanisms and is regulated by ubiquitin-proteasome pathway. Oncogene 18, 4313–4325.
El-Deiry, W. S., Harper, J. W., O’Connor, P. M., et al. (1994) WAF1/CIP1 is induced in p53-mediated G1 arrest and apoptosis. Cancer. Res. 54, 1169–1174.
Bates, S., Ryan, K. M., Phillips, A. C., and Vousden, K. H. (1998) Cell cycle arrest and DNA endoreduplication following p21Waf1/Cip1 expression. Oncogene 17, 1691–1703.
Medema, R. H., Klompmaker, R., Smits, V. A., and Rijksen, G. (1998) p21waf1 can block cells at two points in the cell cycle, but does not interfere with processive DNA-replication or stress-activated kinases. Oncogene 16, 431–441.
Niculescu, A. B. R., Chen, X., Smeets, M., Hengst, L., Prives, C., and Reed, S. I. (1998) Effects of p21(Cip1/Waf1) at both the G1/S and the G2/M cell cycle transitions: pRb is a critical determinant in blocking DNA replication and in preventing endo-reduplication. Mol. Cell. Biol. 18, 629–643.
Taylor W. R., Schonthal A. H., Galante J., and Stark G. R. (2001) p130/E2F4 binds to and represses the cdc2 promoter in response to p53. J. Biol. Chem. 276, 1998–2006.
Di Leonardo, A., Linke, S. P., Clarkin, K., and Wahl, G. M. (1994) DNA damage triggers a prolonged p53-dependent G1 arrest and long-term induction of Cip1 in normal human fibroblasts. Genes Dev. 8, 2540–2551.
Macleod, K. F., Sherry, N., Hannon, G., et al. (1995) p53-dependent and independent expression of p21 during cell growth, differentiation, and DNA damage. Genes Dev. 9, 935–944.
Brugarolas, J., Chandrasekaran, C., Gordon, J. I., Beach, D., Jacks, T., and Hannon, G. J. (1995) Radiation-induced cell cycle arrest compromised by p21 deficiency. Nature 377, 552–557.
Deng, C., Zhang, P., Harper, J. W., Elledge, S. J., and Leder, P. (1995) Mice lacking p21CIP1/WAF1 undergo normal development, but are defective in G1 checkpoint control. Cell 82, 675–684.
Bunz, F., Dutriaux, A., Lengauer, C., et al. (1998) Requirement for p53 and p21 to sustain G2 arrest after DNA damage. Science 282, 1497–1500.
Harrington, E. A., Bruce, J. L., Harlow, E., and Dyson, N. (1998) pRB plays an essential role in cell cycle arrest induced by DNA damage. Proc. Natl. Acad. Sci. USA 95, 11,945–11,950.
Blagosklonny, M. V., Wu, G. S., Omura, S., and El-Deiry, W. S. (1996) Proteasome-dependent regulation of p21WAF1/CIP1 expression. Biochem. Biophys. Res. Commun. 227, 564–569.
Maki, C. G. and Howley, P. M. (1997) Ubiquitination of p53 and p21 is differentially affected by ionizing and UV radiation. Mol. Cell. Biol. 17, 355–363.
Cayrol, C., Knibiehler, M., and Ducommun, B. (1998) p21 binding to PCNA causes G1 and G2 cell cycle arrest in p53-deficient cells. Oncogene 16, 311–320.
Sheaff, R. J., Singer, J. D., Swanger, J., Smitherman, M., Roberts, J. M., and Clurman, B. E. (2000) Proteasomal turnover of p21Cip1 does not require p21Cip1 ubiquitination. Mol. Cell 5, 403–410.
Brenot-Bosc, F., Gupta, S., Margolis, R. L., and Fotedar, R. (1995) Changes in the subcellular localization of replication initiation proteins and cell cycle proteins during G1-to S-phase transition in mammalian cells. Chromosoma 103, 517–527.
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Fotedar, R., Bendjennat, M., Fotedar, A. (2004). Functional Analysis of CDK Inhibitor p21WAF1 . In: Schönthal, A.H. (eds) Checkpoint Controls and Cancer. Methods in Molecular Biology, vol 281. Humana Press. https://doi.org/10.1385/1-59259-811-0:055
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DOI: https://doi.org/10.1385/1-59259-811-0:055
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