Skip to main content

Generation and Application of Phospho-specific Antibodies for p53 and pRB

  • Protocol
Tumor Suppressor Genes

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 223))

Abstract

The functions of many proteins are likely to be regulated by phosphorylation. Thus, antibodies that can recognize specifically phosphorylated sites on proteins have a wide variety of uses for studying the function and regulation of phosphoproteins. We have improved methods for generation of phosphorylation site-specific antibodies and have successfully obtained antibodies for the analysis of most of the phosphorylation sites on p53 and RB proteins.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Taya, Y. (1997) RB-kinases and RB-binding proteins: new points of view. Trends Biochem. Sci., 22, 14–17.

    Article  PubMed  CAS  Google Scholar 

  2. Sherr, C. J. (1994) The ins and outs of RB: coupling gene expression to the cell cycle clock. Trends Cell Biol. 4, 15–18.

    Article  PubMed  CAS  Google Scholar 

  3. Weinberg, R. A. (1995) The retinoblastoma protein and cell cycle control. Cell 81, 323–330.

    Article  PubMed  CAS  Google Scholar 

  4. Wakamiya, T., Saruta, K., Kusumoto, S., et al. (1993) An effcient procedure for synthesis of phosphopeptides through the benzyl phosphate-protection by the Boc-mode solid-phase method. Chem. Lett. 1993, 401–1404T

    Google Scholar 

  5. Wakamiya, T., Saruta, K., Kusumoto, S., Aimoto, S., Kumagaye, K.-Y., and Nakajima, K. (1994) Synthetic study of phosphopeptides by solid-phase method in Peptide Chemistry 1993, Okada, Y., ed.). Protein Research Foundation, Osaka, Japan, pp. 17–20.

    Google Scholar 

  6. Ueno, Y., Suda, F., Taya, Y., Noyori, R., Hayakawa, H., and Hata, T. (1995) Allyl derivative synthesis: a synthetic study of phosphopeptides by solid-phase. Bioorg. Med. Chem. Lett. 5, 823–826.

    Article  CAS  Google Scholar 

  7. Kitas, E., Knorr, R., Treciak, A., and Bannwarth, W. (1991) Alternative strategies for Fmoc solid-phase synthesis of O-phospho-l-tyrosine-containing peptides. Helv. Chim. Acta 74, 1314–1328.

    Article  CAS  Google Scholar 

  8. Wakamiya, T., Togashi, R., Nishida, T., et al. (1997) Synthetic study of phosphopeptides related to heat shock protein HSP27. Bioorg. Med. Chem. 5, 135–145.

    Article  PubMed  CAS  Google Scholar 

  9. Prives, C. (1998) Signaling to p53: breaking the MDM2-p53 circuit. Cell 95, 5–8.

    Article  PubMed  CAS  Google Scholar 

  10. Lyungman, M. (2000) Dial 9-1-1 for p53: mechanism of p53 activation by cellular stress. Neoplasia 2, 208–225.

    Google Scholar 

  11. Shieh, S.-Y., Ikeda, M., Taya, Y., and Prives, C. (1997) DNA damage-induced phosphorylation of p53 alleviates inhibition by MDM2. Cell 91, 325–334.

    Article  PubMed  CAS  Google Scholar 

  12. Siliciano, J.D., Canman, C.E., Taya, Y., Sakaguchi, K., Appella, E., and Kastan, M. B. (1997) DNA damage induces phosphorylation of the amino terminus of p53. Genes Dev. 11, 3471–3481.

    Article  PubMed  CAS  Google Scholar 

  13. Banin, S., Moyal, S., Shieh, S. Y., et al. (1998) Enhanced phosphorylation of p53 by ATM in response to DNA damage. Science 281, 1674–1677.

    Article  PubMed  CAS  Google Scholar 

  14. Canman, C. E., Lim, D. S., Cimprich, K. A., et al. (1998) Activation of the ATM kinase by ionizing radiation and phosphorylation of p53. Science 281, 1677–1679.

    Article  PubMed  CAS  Google Scholar 

  15. Nakagawa, K., Taya, Y., Tamai, K., and Yamaizumi, M. (1999) Requirement of ATM in the phosphorylation of the human p53 protein at serine 15 following DNA double-strand breaks. Mol. Cell. Biol. 19, 2828–2834.

    PubMed  CAS  Google Scholar 

  16. Tibbetts, R. S., Williams, J. M., Taya, Y., Shieh, S.-Y., Prives, C., and Abraham, R. T. (1999) A role for ATR in the DNA damage-induced phosphorylation of p53. Genes Dev. 13, 152–157.

    Article  PubMed  CAS  Google Scholar 

  17. Shieh, S.-Y., Ahn, J., Tamai, K., Taya, Y., and Prives, C. (2000) The human homologues of checkpoint kinases Chk1 and Cds1 (Chk2) phosphorylate p53 at multiple DNA damage inducible sites. Genes Dev. 14, 289–300.

    PubMed  CAS  Google Scholar 

  18. Ko, L. J., Chen, X., Shieh, S.-Y., et al. (1997) p53 is phosphorylated by CDK7/cyclin H in a p36/MAT1 dependent manner. Mol. Cell. Biol. 17, 7220–7229.

    PubMed  CAS  Google Scholar 

  19. Takekawa, M., Adachi, M., Nakahata, A., et al. (2000) p53-inducible Wip1 phosphatasemediates a negative feedback regulation of p38 MAPK-p53 signaling in response to UV radiation. EMBO J. 19, 6517–6521.

    Article  PubMed  CAS  Google Scholar 

  20. Kishi, H., Nakagawa, K., Matsumoto, M., et al. (2001) Osmotic shock induces G1-arrest through p53 phosphorylation at Ser33 by activated p38MAPK without phosphorylation at Ser15 and Ser20. J. Biol. Chem. 276, 39115–391122.

    Article  PubMed  CAS  Google Scholar 

  21. Oda, K., Arakawa, H., Tanaka, T., et al. (2000) p53AIP1, a potential mediator of p53-dependent apoptosis, and its regulation by Ser46-phosphorylated p53. Cell 102, 849–862.

    Article  PubMed  CAS  Google Scholar 

  22. Okamura, S., Arakawa, H., Tanaka, T., et al. (2001) p53DINP1, a novel p53-inducible gene, regulates p53-dependent apoptosis. Mol. Cell 8, 85–94.

    Article  PubMed  CAS  Google Scholar 

  23. Lu, H., Taya, Y., Ikeda, M., and Levine, A. J. (1998) Ultraviolet radiation, but not γ radiation or etoposide-induced DNA damage, results in the phosphorylation of the murine p53 protein at serine-389. Proc. Natl. Acad. Sci. USA 95, 6399–6402.

    Article  PubMed  CAS  Google Scholar 

  24. Kitagawa, M., Higashi, H., Jung, H.-K., et al. (1996) The consensus motif for phosphorylation by cyclin D1-Cdk4 is different from that for phosphorylation by cyclin A/E-Cdk2. EMBO J. 15, 7060–7069.

    PubMed  CAS  Google Scholar 

  25. Adams, P. D., Li, X., Sellers, W. R., et al. (1999) The retinoblastoma protein contains a C-terminal motif that targets it for phosphorylation by cyclin/cdk2 complexes. Mol. Cell. Biol. 19, 1068–1080.

    PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2003 Humana Press Inc.

About this protocol

Cite this protocol

Taya, Y., Nakajima, K., Yoshizawa-Kumagaye, K., Tamai, K. (2003). Generation and Application of Phospho-specific Antibodies for p53 and pRB. In: El-Deiry, W.S. (eds) Tumor Suppressor Genes. Methods in Molecular Biology™, vol 223. Humana Press. https://doi.org/10.1385/1-59259-329-1:17

Download citation

  • DOI: https://doi.org/10.1385/1-59259-329-1:17

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-987-2

  • Online ISBN: 978-1-59259-329-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics