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.
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References
Taya, Y. (1997) RB-kinases and RB-binding proteins: new points of view. Trends Biochem. Sci., 22, 14–17.
Sherr, C. J. (1994) The ins and outs of RB: coupling gene expression to the cell cycle clock. Trends Cell Biol. 4, 15–18.
Weinberg, R. A. (1995) The retinoblastoma protein and cell cycle control. Cell 81, 323–330.
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
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.
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.
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.
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.
Prives, C. (1998) Signaling to p53: breaking the MDM2-p53 circuit. Cell 95, 5–8.
Lyungman, M. (2000) Dial 9-1-1 for p53: mechanism of p53 activation by cellular stress. Neoplasia 2, 208–225.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Okamura, S., Arakawa, H., Tanaka, T., et al. (2001) p53DINP1, a novel p53-inducible gene, regulates p53-dependent apoptosis. Mol. Cell 8, 85–94.
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.
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.
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.
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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
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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
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