PhD. Cell Biology, Baylor College of Medicine
The tumor suppressor protein p53 play an important role in maintaining genome stability. The significance is fully demonstrated by its high frequency (over 50%) of deletion or mutation in tumors. Under normal condition, the cellular p53 protein is unstable and barely detectable. However, upon genotoxic stress such as ionizing radiation, UV, hypoxia, or even nutrient depletion, p53 is stabilized and activated, which then leads to either G1/G2 growth arrest or apoptosis. Although the phenomenon has been known for years, how p53 receives the stress signal remains elusive. Our main interest is to decipher the signal transduction pathway and the molecular mechanisms underlying p53 induction.
l DNA damage-induced p53 phosphorylation
Post-translational modification of proteins, in particular, phosphorylation, has been shown to mediate a variety of signal transduction events. The possibility that phosphorylation may play a role in the signaling to p53 has been a subject of intense investigation. In fact, we and others have discovered that DNA damage induces p53 phosphorylation at multiple sites in the N-terminus and the C-terminus of the protein. However, the connection between these phosphorylation events and p53 induction remains to be established. We have recently identified two cell cycle checkpoint kinases, hCHK1 and CHK2, as p53 N-terminal kinases that phosphorylate DNA damage-inducible sites in p53 in vitro. The possibility that other cell cycle checkpoint players may also participate in the signal transduction process upstream of CHKs is now under investigation. Using yeast two-hybrid screen and biochemical purification coupled with proteomic analysis, we have identifie several potential CHKs-interacting targets. Further characterization is underway.
l Molecular mechanism underlying p53 stabilization
DNA damage induces p53 through post-transcriptional mechanism, most likely through increasing the stability of p53 protein. Several proteins have been shown to regulate the half-life of p53 either directly or indirectly. One of them, the oncoprotein MDM2, binds the N-terminus of p53, and as a result, inhibits transcription activation by p53 and promotes p53 degradation through ubiquitin / proteasome pathway. Although p53 N-terminal phosphorylation can modulate MDM2-p53 interaction, some stress-related stabilization of p53 does not appear to involve phosphorylation. Other lines of evidence also suggest that different genotoxic agents may signal to p53 through different pathways. We are interested in locating the responsible p53 domain(s) that can mediate these stabilizing effects, as well as potential proteins that interact with these domains.
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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.
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de Stanchina, E., McCurrach, M. E., Zindy, F., Shieh, S.-Y., Ferbeyre, G., Samuelson, A. V., Prives, C., Roussel, M. F., Sherr, C. J., and Lowe, S. W. (1998) E1A signaling to p53 involves the p19ARF tumor suppressor. Genes & Dev. , 12: 2434-2442.
Banin, S., Moyal, R., Shieh, S.-Y., Taya, Y., Anderson, C. W., Chessa, L., Smorodinsky, N., Prives, C., Reiss, Y., Shiloh, Y., and Ziv, Y. (1998) Enhanced phosphorylation of p53 by ATM in response to DNA damage. Science 281: 1674-1677.
Tibbetts, R. S., Brumbaugh, K. M., Williams, J. M., Sarkaria, J. N., Cliby, W. A., Shieh, S.-Y., Taya, 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., Taya, Y., and Prives, C. (1999) DNA damage-inducible phosphorylation of p53 at N-terminal sites including a novel site, serine 20, requires tetramerization. EMBO J. 18: 1815-1823.
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.
Gottifredi, V., Shieh, S. -Y., and Prives, C. (2000) Regulation of p53 after different forms of stress and at different cell cycle stage. Cold Spring Harbor Symp. Quant. Biol. LXV: 483-487.
Gottifredi, V., Karni-Schmidt, O., Shieh, S.-Y., and Prives, C. (2001) p53 down-regulates CHK1 through p21 and the retinoblastoma protein. Mol. Cell. Biol. 21: 1066-1076.
Gottifredi, V., Shieh, S.-Y., Taya, Y., and Prives, C. (2001) p53 accumulates but is functionally impaired when DNA synthesis is blocked. Proc. Natl. Acad. Sci. USA 98: 1036-1041.
Ou, Y-H., Chung, P.-H., Sun, T.-P., and Shieh, S.-Y. (2005) p53 C-terminal phosphorylation by CHK1 and CHK2 participates in the regulation of DNA damage-induced C-terminal acetylation. Mol. Biol. Cell, 16:1684-1695.