Yoichi Taya
Radiobiology Division, National Cancer Center Research Institute, Tsukiji 5-1-1, Chuo-ku, Tokyo 104-0045, Japan
(Tel: 81-3-3542-2511 ext. 4655 Fax: 81-3-5565-0727 E-mail: ytaya@gan2.ncc.go.jp)
Upon DNA damage, p53 protein accumulates rapidly through a posttranscriptional mechanism(s) and is also activated as a transcription factor, which then leads to growth arrest or apoptosis. We have generated antibodies which recognize most of the potential phosphorylation (13 sites) and acetylation sites (3 sites) of p53, and using these antibodies, we have shown that phosphorylation of human p53 at serine 15 and 20 occurs after DNA damage and that this leads to reduced interaction of p53 with its negative regulator, MDM21)2). Subsequently, we have also shown that ATM protein has intrinsic protein kinase activity and that it phosphorylates Ser15 of p533)4).
It is an important question how two different pathways, apoptosis and G1-arrest, are selected by p53. We have now found that phosphorylation of Ser46 regulates apoptosis-inducing ability of p535).
A newly found transcriptional activation domain (43-63) and the proline-rich domain (64-92) have been suggested to be needed for apoptosis induction by p53 because deletion of either of these two domains abolishes this activity. In order to test the possibility that phosphorylation of a specific site in these domains regulates apoptosis-inducing ability of p53, we generated antibodies to recognize 3 potential phosphorylation sites in these domains. We now found that phosphorylation of Ser46 is induced after DNA damage. Particularly, phosphorylation of this site was observed only at the late stage after DNA damage by several different agents, and high dose of gamma-ray or UV was needed compared to Ser15 or Ser20. Moreover, the time course of phosphorylation of Ser46 and apoptosis induction was similar. When p53 mutants which were changed at Ser46 were transfected or microinjected into H1299 cells, apoptosis-inducing ability of S46A or S46del was greatly reduced. However, there were no difference in induction of p21waf1 between wild-type and these mutants.
These results suggested an intriguing possibility that phosphorylation of Ser46 regulates the transcriptional activation of apoptosis-inducing genes by p53. Dr. Yusuke Nakamura's lab. has isolated such a candidate apoptosis-inducing gene which is regulated by phosphorylation of Ser46 of p53. It is p53AlP15) (p53-regulated Apoptosis Inducing Protein 1). Its expression is induced by wild-type p53 and its protein is localized in mitochondria. Ectopic expression of p53AIP1 induces apoptosis through dissipation of mitochondrial membrane potential. Interestingly, only the expression of p53AIP1, among many p53-target genes, was consistent to induction of phosphorylation of Ser46 in p53 and apoptosis of cells. Thus, it appears that swich between promoters of Gl-arrest and apoptosis genes occurs by Ser46-phosphorylation. We also found that another novel protein which is induced by p53 is complexed with Ser46-kinase to activate it. Moreover, we are approaching to identification of the Ser46-kinase. It is not p38 MAP-kinase.
References:
1) 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.
2) Shieh, S-Y., Ahn, J., Tamai, K., Taya, Y. and Prives, C. (2000) The human homologues of checkpoint kinases Chk1 and Cds 1 (Chk2) phosphorylate p53 at multiple DNA damage inducible sites. Genes & Dev., 14: 289-300.
3) Banin, S., Moyal, L., Shieh, S.-Y., Taya, Y., Chessa, L., Snorodinsky, N.1., Prives, C., Reiss, R., Shiloh, Y., & Ziv, Y. ( 1998) Enhanced phosphorylation of p53 by ATM in response to DNA damage. Science, 281: 1674-1677.
4) Canman, C.E., Lim, D.-S., Cimprich, K.A., Taya, Y., Tamai, K., Sakaguchi, K., Appella, E., Kastan, M.B. & Siliciano, J.D. ( 1998) Activation of the ATM kinase by ionizing radiation and phosphorylation of p53. Science, 281: 1677-1679.
5) Oda, K., Arakawa, H.,Tanaka, T., Matsuda, K., Tanikawa, C., Mori, T., Nishimori, H., Tamai, K., Tokino, T., Nakamura, Y. & Taya, Y. (2000) p53AIP1, a potential mediator of p53-dependent apoptosis, and its regulation by Ser46-phosphorylated p53. Cell, 102: 849-862.
Yoichi Taya Ph.D. 1969 B.Sc. University of Tokyo, Faculty of Science
1974 Ph.D. University of Tokyo, Faculty of Science
1974 Research Associate, Biology Division, National Cancer Center Research Institute Tokyo, Japan
1980-82 Visiting Scientist, Ghent University, Belgium
1991 Section Chief, Biology Division, National Cancer Center Research Institute
2001 Chief, Radiobiology Division, National Cancer Center Research Institute