Abstract
The reversible phosphorylation of proteins is a key mechanism whereby signalling cascades involved in the response to extracellular stimuli bring about changes in cellular function. These proteins include the kinases/phosphatases that form such signaling pathways as well as the transcription factors involved in inducible changes in gene expression (1). Phosphorylation induces changes in the function of these proteins either by induction of allosteric conformational changes in the protein itself or in the regulation of its interaction with other cellular factors.
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References
Hunter T. (1995) Protein kinase and phosphatases: the yin and yang of protein phosphorylation and signalling. Cell 80, 225–236
Van der Geer P., Luo K., Sefton B. M., and Hunter T. (1993) Phosphopeptide mapping and phosphoamino acid analysis on cellulose thin-layer plates, in Protein Phosphorylation (Hardie G., ed.), IRL, Oxford, pp. 31–58.
Yeargin J. and Haas M. (1995) Elevated levels of wild-type p53 induced by radiolabeling of cells leads to apoptosis or sustained growth arrest. Curr. Biol 5. 423–431.
Dover R., Jayaram Y., Patel K., and Chinery R. (1994) p53 expression in cultured cells following radioisotope labelling. J. CeU. Sci. 107, 1181–1184.
Bond J. A., Webley K., Wyllie F. S., Jones C. J., Craig A., Hupp T., and Wynford-Thomas D. (1999) p53-Dependent growth arrest and altered p53-immunoreactivity following metabolic labelling with 32P ortho-phosphate in human fibroblasts. Oncogene 18, 3788–3792.
Czernik A. J., Girault J.-A., Nairm A. C, Chen J., Snyder G., Kebabian J., and Greengard P. (1991) Production of phosphorylation state-specific antibodies, in Methods inEnzymology Vol. 201, Academic Press, London, pp. 264–283.
Ueno Y., Makino S., Kitagawa M., Nishimura S., Taya Y., and Hata T. (1995) Chemical synthesis of phosphopeptides using the arylthio group for protection of phosphate: application to identification of cdc2 kinase phosphorylation sites. Int. J. Peptide Protein Res. 46, 106–112
Alberts A. S., Arias J., Hagiwara M., Montminy M. R., and Feramisco J. R. (1994) Recombinant cyclic-AMP response element-binding protein (creb) phosphorylated on ser-133 is transcription ally active upon its introduction into fibroblast nuclei. J. Biol Chem. 269, 7623–7630
Weng Q. P., Kozlowski M., Belham C, Zhang A., Comb M., and Avruch J. (1998) Regulation of the p70S6 kinase by phosphorylation in vivo. J. BioL Chem. 273, 16,621–16,629.
Kitagawa M., Higashi H., Junag H.-K., Suzuki-Takahashi I., Ikeda M., Tamai. K., Kato J.-Y., Segawa K., Yoshida E., Nishimura S., and Taya Y. (1996) The consensus motif for phosphorylationby cyclinDl-cdk4 is different from that for phosphorylation by cyclin A/E-cdk2. EMBO J. 15, 7060–7069.
Blaydes J. P. and Hupp T. R. (1998) DNA damage triggers DRB-resistant phosphorylation of human p53 at the CK2 site. Oncogens 17, 1045–1052.
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.
Kapoor M. and Lozano G. (1998) Functional activation of p53 via phosphorylation following DNA damage by UV but not y radiation. Proc. Natl. Acad. Sci. USA 95, 2834–2837.
Waterman M. J., Stavridi E. S, Waterman J. L., and Halazonetis T. D. (1998) ATM-dependent activation of p53 involves dephosphorylation and association with 14-3-3 proteins. Nat. Genet. 19, 175–178.
Shieh S.-Y., Ikeda M., Taya Y., and Prives C. (1997) DNA dam age-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. (1997) DNA damage induces phosphorylation of the amino terminus of p53. Genes Dev. 11, 3471–3481.
Harlow E. and Lane D. P. (1988) Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, New York.
Atherton E. and Sheppard R. C. (1989) Solid-Phase Pep tide Synthesis. A Practical Approach. IRL Press.
Stephen C. W., Helminen P., and Lane D. P. (1995) Characterisation of epitopes on human p53 using phage-displayed peptide libraries: insights into antibodypeptide interactions. J. Mol. BioL 248, 58–78.
Ravera M. W., Carcamo J., Brissette R., Alam-Moghe A., Dedova O., Cheng W., Hsiao K. C., Klebanov D., Shen H., Tang P., Blume A., and Mandecki W. (1998) Identification of an allosteric binding site on the transcription factor p53 using a phage-displayed peptide library. Oncogene 16, 1993–1999.
Stephen C. and Lane D. P. (1992) Mutant conformation of p53: Precise epitope mapping using a filamentous phage epitope library. J. Mol. BioL 225, 577–583.
Vojtesek B., Dolezalova H., Lauerova L., Svitakova M., Havlis P., Kovarik J. Midgley C. A., and Lane D. P. (1995) Conformational changes in p53 analysed using new antibodies to the core DNA binding domain of the protein. Oncogene 10, 389–393.
Wade-Evans A. and Jenkins J. R. (1985) Precise epitope mapping of the murine transformation-associated protein, p53. EMBO J. 4, 699–706.
Hupp T. R. and Lane D. P. (1994) Allosteric activation of latent p53 tetramers. Curr. Biol 4, 865–875.
Craig A. L., Burch L., Vojtesek B., Mikutowska J., Thompson A., and Hupp T. R. (1999) Novel phosphorylation sites of human tumour suppressor protein p53 at Ser20 and Thr18 that disrupt the binding of MDM2 (mouse double minute 2) protein are modified in human cancers. Biochem. J. 342, 133–141.
Craig A. L., Blaydes J. P., Burch L. R., Thompson A. M., and Hupp T. R. (1999) Dephosphorylation of p53 at Ser20 after exposure to low levels of nonionizing radiation. Oncogens 18, 6305–6312.
Meek D. W., Simon S., Kikkawa U. and Eckhart W. (1990) Thep53tumour suppressor Proteinisphosphorylatedatserine389bycaseinkinaseII. EMSO J. 9, 3252–3260.
Hupp T. R., Meek D. W., Midgely C. A., and Lane D. P. (1992) Regulation of the specific DNA binding function of p53. Cell 71, 875–886.
Sakaguchi K., Sakamoto H., Lewis M. S., Anderson C. W., Erickson J. W. Appella E., and Xie D. (1992) Phosphorylation of serine-392 stabilizes the tetramer formation oftumor-suppressorprotein-p53. Biochemistry 36, 10,117–10,124.
Wang Y. and Roach P. J. (1993) Purification and assay of mammalian protein (serine/threonine) kinases, in Protein Phosphorylation (Hardie G., ed.), IRL., Oxford, pp. 121–142.
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Blaydes, J.P., Vojtesek, B., Bloomberg, G.B., Hupp, T.R. (2000). The Development and Use of Phospho-Specific Antibodies to Study Protein Phosphorylation. In: Walker, J.M., Keyse, S.M. (eds) Stress Response. Methods in Molecular Biology™, vol 99. Humana Press. https://doi.org/10.1385/1-59259-054-3:177
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DOI: https://doi.org/10.1385/1-59259-054-3:177
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