Jake Leon Department of Biology and Microbiology California State University Los Angeles
description
Transcript of Jake Leon Department of Biology and Microbiology California State University Los Angeles
![Page 1: Jake Leon Department of Biology and Microbiology California State University Los Angeles](https://reader037.fdocuments.us/reader037/viewer/2022110104/568159e7550346895dc731ed/html5/thumbnails/1.jpg)
Jake LeonDepartment of Biology and Microbiology California State University Los Angeles
5151 State University DriveLos Angeles, CA 90032
May 25, 2006MOLECULAR DIAGNOSTICS
Dr. Sandra Sharp
Structural Basis of the DNA binding Domain of the p53 tumor suppressor
protein
![Page 2: Jake Leon Department of Biology and Microbiology California State University Los Angeles](https://reader037.fdocuments.us/reader037/viewer/2022110104/568159e7550346895dc731ed/html5/thumbnails/2.jpg)
Normal Cells are able to prevent cancer by activating a naturaldefense mechanism
Cancer:
• DNA damage
• DNA damage activates gene expression
• Genes code proteins
• Proteins participate in response to DNA damage
• p53 acts as a transcription factor that transactivates genes to stop tumors by causing apoptosis, repairing DNA, or by preventing cell proliferation.
![Page 3: Jake Leon Department of Biology and Microbiology California State University Los Angeles](https://reader037.fdocuments.us/reader037/viewer/2022110104/568159e7550346895dc731ed/html5/thumbnails/3.jpg)
Role of p53 in tumor suppression
p53 knockout mice develop tumors spontaneously(Donehower et al., 1992)
p53 binds to specific sequences on the DNA (Bargonetti et al., 1991; El-Deiry et al., 1992)
p53 activates transcription of genes(Levine, 1997; Giaccia and Kastan, 1998)
In human cancers, p53 is frequently observed to showmutations that inhibit its ability to bind to DNA
![Page 4: Jake Leon Department of Biology and Microbiology California State University Los Angeles](https://reader037.fdocuments.us/reader037/viewer/2022110104/568159e7550346895dc731ed/html5/thumbnails/4.jpg)
Outline of Next Series of Slides
• Normal p53 in the cell when there is no detectable DNA damage
• Normal p53 in response to DNA damage– 2 slides – repairable damage
• Phopshorylation of p53 blocking its degradation and• Causing p53 to function as a transcription factor to stop
the cell cycle
– 2 slides – irreparable damage• Phosphorylation of p53 blocking its degradation and• Causing p53 to function as a transcription factor to
induce apoptosis
![Page 5: Jake Leon Department of Biology and Microbiology California State University Los Angeles](https://reader037.fdocuments.us/reader037/viewer/2022110104/568159e7550346895dc731ed/html5/thumbnails/5.jpg)
p53
Latent p53
Half-life: ~20 min
Nucleus
MDM2
Murine Double Minute 2
Momand et al., 2005
26 S Proteosome
This normally rapid turnover prevents normal cells from entering into cell cycle arrest
or undergoing apoptosis
![Page 6: Jake Leon Department of Biology and Microbiology California State University Los Angeles](https://reader037.fdocuments.us/reader037/viewer/2022110104/568159e7550346895dc731ed/html5/thumbnails/6.jpg)
ATM
Ataxia Telangiectasia Mutagenesis
ATM
p53 CHK2 Checkpoint Kinase 2
S15
S20
ATP
ADP
ATP
ADP
MDM2
![Page 7: Jake Leon Department of Biology and Microbiology California State University Los Angeles](https://reader037.fdocuments.us/reader037/viewer/2022110104/568159e7550346895dc731ed/html5/thumbnails/7.jpg)
p53p53p53p53TAF70
TAF31RNA
polymerase
p21GADD45
![Page 8: Jake Leon Department of Biology and Microbiology California State University Los Angeles](https://reader037.fdocuments.us/reader037/viewer/2022110104/568159e7550346895dc731ed/html5/thumbnails/8.jpg)
DNA-PK
DNA-dependent protein kinase
p53
S15
ATP
ADP
![Page 9: Jake Leon Department of Biology and Microbiology California State University Los Angeles](https://reader037.fdocuments.us/reader037/viewer/2022110104/568159e7550346895dc731ed/html5/thumbnails/9.jpg)
p53p53p53p53TAF70
TAF31RNA
polymerase
BaxNOXAPUMAKILLER/DR5Fas/Apo1
These genes participate in the activation ofAPOPTOSIS
![Page 10: Jake Leon Department of Biology and Microbiology California State University Los Angeles](https://reader037.fdocuments.us/reader037/viewer/2022110104/568159e7550346895dc731ed/html5/thumbnails/10.jpg)
Apoptosis Induction by p53
• P53 also responds to unrepaired DNA damage by inducing expression of genes that trigger apoptosis (programmed cell death) of the injured cell.– This ultimately leads to cell death.
• DNA-PKc (catalytic subunit) is part of the enzymatic machinery for– VDJ rearrangement– Non-homologous end joining
![Page 11: Jake Leon Department of Biology and Microbiology California State University Los Angeles](https://reader037.fdocuments.us/reader037/viewer/2022110104/568159e7550346895dc731ed/html5/thumbnails/11.jpg)
Apoptosis – in response to irreparable DNA damage
Bax
Note the role of tumor suppressor p53.
![Page 12: Jake Leon Department of Biology and Microbiology California State University Los Angeles](https://reader037.fdocuments.us/reader037/viewer/2022110104/568159e7550346895dc731ed/html5/thumbnails/12.jpg)
What is p53?
• A protein of ~53 kilodaltons• A nuclear phosphoprotein
![Page 13: Jake Leon Department of Biology and Microbiology California State University Los Angeles](https://reader037.fdocuments.us/reader037/viewer/2022110104/568159e7550346895dc731ed/html5/thumbnails/13.jpg)
DNA viruses and their oncogenes.• E6 is produced by Human Papilloma Virus (HPV) and can
contribute to cervical cancer.• E1b is produced by Adenovirus.
– Human cells are permissive for adenovirus.• Causes the common cold.
– Adenovirus transforms rodent (non-permissive) cells
• Human virus JC is similar to SV40 and may be associated with certain cancers, but a causative role has yet to be confirmed. – JC virus T antigen causes tumors in nude mice.
• All these proteins are products of “early” genes in their viral replication cycles.
• A productive infection of these viruses leads to lysis of the host cell. A non-productive infection allows the cells to live.
![Page 14: Jake Leon Department of Biology and Microbiology California State University Los Angeles](https://reader037.fdocuments.us/reader037/viewer/2022110104/568159e7550346895dc731ed/html5/thumbnails/14.jpg)
What is p53?• Transcriptional regulator
– Binds to 12 bp recognition sequence in the promoters (regulatory regions) of the genes it regulates
– Activates transcription by interacting with RNA polymerase complex
![Page 15: Jake Leon Department of Biology and Microbiology California State University Los Angeles](https://reader037.fdocuments.us/reader037/viewer/2022110104/568159e7550346895dc731ed/html5/thumbnails/15.jpg)
What is p53?
• Acts as a tetramer– Individual molecules associate at
tetramerization region– Oligomerization of mutated p53 with wt p53
inactive p53 complex
![Page 16: Jake Leon Department of Biology and Microbiology California State University Los Angeles](https://reader037.fdocuments.us/reader037/viewer/2022110104/568159e7550346895dc731ed/html5/thumbnails/16.jpg)
What is p53?• Detection of damaged DNA by p53 causes p53 to be
stabilized and accumulate in the cell.• DNA damage activates the kinase ATM, which
phosphorylates p53. • When damaged DNA is not present, p53 is turned over
rapidly and does not accumulate because – the protein MDM2 binds to the transcription-activation region of p53
and targets p53 for degradation by a proteosome.
Note: MDM2 binds when the TAD is LESS phosphorylated.
(TAD)
![Page 17: Jake Leon Department of Biology and Microbiology California State University Los Angeles](https://reader037.fdocuments.us/reader037/viewer/2022110104/568159e7550346895dc731ed/html5/thumbnails/17.jpg)
p53 as a transcriptional regulator• If DNA damage is detected by binding of DNA fragments to the
non-specific DNA binding region of p53, p53 stops DNA synthesis until the damage is repaired.
• If DNA damage is detected, then– p53 is phosphorylated by a protein known as ATM– MDM2 is released from being bound to the transcriptional activation
domain of p53 and– p53 is able to act as a transcriptional activator and turn on genes for
• cyclin dependent kinase inhibitor p21, which– stops or prevents DNA synthesis
• DNA repair – Example: GADD45
• If DNA damage is extensive and can not be repaired, p53 induces genes for apoptosis (programmed cell death).
![Page 18: Jake Leon Department of Biology and Microbiology California State University Los Angeles](https://reader037.fdocuments.us/reader037/viewer/2022110104/568159e7550346895dc731ed/html5/thumbnails/18.jpg)
p53 as a transcriptional regulator
• p53 activates the gene for MDM2– MDM2
• targets p53 for degradation and prevents inappropriate build up
• prevents transcriptional activation by p53
– So, it’s a negative feedback loop!
• p53 also turns expression of some genes off.
![Page 19: Jake Leon Department of Biology and Microbiology California State University Los Angeles](https://reader037.fdocuments.us/reader037/viewer/2022110104/568159e7550346895dc731ed/html5/thumbnails/19.jpg)
How does p53 inhibit DNA synthesis? Let’s work backwards.
• E2F transcription factor turns on transcription of genes for DNA synthesis.
• E2F can’t turn on genes if it is bound to Rb1, a tumor suppressor.
• Rb1 can’t bind E2F if it is heavily phosphorylated.
• Rb1 is phosphorylated by cyclin-dependent kinases (CDKs).
![Page 20: Jake Leon Department of Biology and Microbiology California State University Los Angeles](https://reader037.fdocuments.us/reader037/viewer/2022110104/568159e7550346895dc731ed/html5/thumbnails/20.jpg)
How does p53 inhibit DNA synthesis?
• Cylin dependent kinases can be inhibited by cyclin dependent kinase inhibitors (CDKIs). If CDKs are inhibited– Rb1 won’t be phosphorylated– E2F will be bound by Rb1– DNA synthesis genes will not be transcribed
• And remember . . . . P53 induces expression of CDKI p21, a
cyclin dependent kinase inhibitor!• Check out the next slide for a visual of these
pathways.
![Page 21: Jake Leon Department of Biology and Microbiology California State University Los Angeles](https://reader037.fdocuments.us/reader037/viewer/2022110104/568159e7550346895dc731ed/html5/thumbnails/21.jpg)
Phosphorylation of Rb
![Page 22: Jake Leon Department of Biology and Microbiology California State University Los Angeles](https://reader037.fdocuments.us/reader037/viewer/2022110104/568159e7550346895dc731ed/html5/thumbnails/22.jpg)
Figure legend on next slide.
![Page 23: Jake Leon Department of Biology and Microbiology California State University Los Angeles](https://reader037.fdocuments.us/reader037/viewer/2022110104/568159e7550346895dc731ed/html5/thumbnails/23.jpg)
![Page 24: Jake Leon Department of Biology and Microbiology California State University Los Angeles](https://reader037.fdocuments.us/reader037/viewer/2022110104/568159e7550346895dc731ed/html5/thumbnails/24.jpg)
p53 Mutations - where are they?
![Page 25: Jake Leon Department of Biology and Microbiology California State University Los Angeles](https://reader037.fdocuments.us/reader037/viewer/2022110104/568159e7550346895dc731ed/html5/thumbnails/25.jpg)
This magnification of mutations in the DNA binding region of p53 gives more information regarding how the mutation affects p53. Note particularly that some mutations cause p53 to be misfolded (denatured) and others do not.
![Page 26: Jake Leon Department of Biology and Microbiology California State University Los Angeles](https://reader037.fdocuments.us/reader037/viewer/2022110104/568159e7550346895dc731ed/html5/thumbnails/26.jpg)
Have you figured it out?For our assay, the samples are cell extracts from two mouse cell lines,
BC3H1 and C2C12.
One line is wild type for p53; one is mutant.One accumulates detectable levels of p53; one doesn’t.
Based on this lecture and your assay results, have you figured out which cell line does what?
Have you thought about why?There is one explanation confirmed in the literature and at least one
additional plausible contribution to what you observe.
(Hint: P53 is not accumulating in either of these cell lines in response to DNA damage. DNA damage is a temporary condition which is repaired immediately. If it is not repaired, the cell soon dies as a result of apoptosis. Mutations may be the result of incorrect repair of DNA damage, but they are no longer considered damage because they are perfectly base-paired.)
![Page 27: Jake Leon Department of Biology and Microbiology California State University Los Angeles](https://reader037.fdocuments.us/reader037/viewer/2022110104/568159e7550346895dc731ed/html5/thumbnails/27.jpg)
Zn
Cho, Y., Gorina, S., Jeffrey, P.D. and Pavletich, N.P. (1994) Science 265: 346-355
p53 DBD Folding
![Page 28: Jake Leon Department of Biology and Microbiology California State University Los Angeles](https://reader037.fdocuments.us/reader037/viewer/2022110104/568159e7550346895dc731ed/html5/thumbnails/28.jpg)
Cho, Y., Gorina, S., Jeffrey, P.D. and Pavletich, N.P. (1994) Science 265: 346-355
Crystal Structure of p53 DBD-DNA Complex PDB file 1tsr
![Page 29: Jake Leon Department of Biology and Microbiology California State University Los Angeles](https://reader037.fdocuments.us/reader037/viewer/2022110104/568159e7550346895dc731ed/html5/thumbnails/29.jpg)
Cho, Y., Gorina, S., Jeffrey, P.D. and Pavletich, N.P. (1994) Science 265: 346-355
PDB file 1tsr Crystal Structure of p53 DBD-DNA Complex
![Page 30: Jake Leon Department of Biology and Microbiology California State University Los Angeles](https://reader037.fdocuments.us/reader037/viewer/2022110104/568159e7550346895dc731ed/html5/thumbnails/30.jpg)
Cho, Y., Gorina, S., Jeffrey, P.D. and Pavletich, N.P. (1994) Science 265: 346-355
PDB file 1tsr Crystal Structure of p53 DBD-DNA Complex
![Page 31: Jake Leon Department of Biology and Microbiology California State University Los Angeles](https://reader037.fdocuments.us/reader037/viewer/2022110104/568159e7550346895dc731ed/html5/thumbnails/31.jpg)
![Page 32: Jake Leon Department of Biology and Microbiology California State University Los Angeles](https://reader037.fdocuments.us/reader037/viewer/2022110104/568159e7550346895dc731ed/html5/thumbnails/32.jpg)
• American Cancer Society: American Cancer Society: www. cancer.org• Bargonetti, J., Friedman, P., Kern, S., Vogelstein, B. and Prives, C. A. (1991) Cell 65, 1083-1091• Bertram, J.S. (2001) Mol. Aspects. Med. 21, 167-223• Buzek, J., Latonen, L., Kurki, S., Peltonen, K. and Laiho, M. (2002) Nucleic Acids Res 30, 2340-2348• Chehab, N. H., Malikzay, A., Appel, M. and Halazonetis, T. D. (2000) Gene Dev. 14, 278-288.• Chehab, N. H., Malikzay, A., Stavridi, E. S. and Halazonetis, T. D. (1999) Proc. Natl. Acad. Sci. U S A. 96, 13777-13782.• Chene, P. (1999) Biochem Biophys Res Commun 263, 1-5 • Cho, Y., Gorina, S., Jeffrey, P. D. and Pavletich, N. P. (1994) Crystal structure of a p53 tumor suppressor-DNA complex: understanding tumorigenic mutations. Science
265:346-355,.• Delphin, C., Cahen, P., Lawrence J. J, and Baudier, J. (1994) Eur. J. Biochem. 223, 683-692• Donehower, L. A., Harvey, M., Slagle, B. L., McArthur, M. J., Montgomery, C. A., Butel, J. S. and Bradley, A. (1992) Nature 356, 215-221• El-Deiry, W. S., Kinzler, S. E., Pietenpol, J. A., Kinzler, K.W., Vogelstein B. (1992) Nat Genet 1, 45• Furuta, S., Ortiz, F., Sun, X., Wu, H., Mason, A., Momand, J. (2002) Biochem. J. 365, 639-648.• Gaskel, S. (1997) Journal of Mass Spectrometry 32, 677-688• Giaccia, A. J. and Kastan, M. B. (1998) Genes Dev. 12, 2973-2983• Hainaut, P. and Milner, J. (1993) Cancer Res. 53, 4469-4473,. • Hainaut, P., Rolley, N., Davies, M. and Milner, J. (1995) Oncogene 10, 27-32• Hirao, A., Kong, Y.Y., Matsuoka, S., Wakeham, A., Ruland, J., Yoshida, H., Liu, D., Elledge, S.J., and Mak, T.W. (2000) Science 287, 1824-1827 • Jayaraman, L., Murthy, K. G., Zhu, C., Curran, T., Xanthudakis, S., Prives, C. (1997) Genes Dev 11, 558-570• Kaeser, M., Iggo, R. (2002) PNAS 99, 95-100.• Kim, S. T., Lim, D. S., Canman, C. E. and Kastan, M. B. (1999) J. Biol. Chem. 274, 37538-43• Klein, C., Planker, E., Diercks, T., Kessler, H., Kunkele, K., Lang, K., Hansen, S., Schwaiger, M. (2001) The Journal of Biological Chemistry 276, 49020-49027.• Lee, S., Yang, K., Kwon, J., Lee, C., Jeong, W., Rhee, S., Reversible Inactivation of the Tumor Suppressor PTEN by H2O2. The Journal of Biological Chemistry 274,
20336-20342.• Levine, A. J. (1990) Virology 177, 419-426.• Levine, A. J. (1997) Cell 88, 323-331• Levine, A. J., Momand, J. and Finlay, C. A. (1991) Nature 351, 453-456• Makmura, L., Hamman, M., Areopagita, A., Furuta, S., Muñoz, A. and Momand, J. (2001) Antioxid Redox Signal 3, 1105-1118• Margalioth, E. J., Schenker, J. and Chevion, M. (1983) Cancer 52, 868-872• Mary, J., Vougier, S., Picot, C., Perichon, M., Petropoulos, I., Friguet, B. (2004) Experimental Gerontology 39, 1117-1123• Matsuoka, S., Rotman, G., Ogawa, A., Shiloh, Y., Tamai, K., and Elledge, S.J. (2000) Proc. Natl. Acad. Sci. U S A. 97, 10389-394• McLure, K., Lee, P., (1998) The EMBO Journal 17, 3342-3350.• Momand, J., Wu, H., Dasgupta, G. (2000) Gene 242, 15-29• Narayanan, V., Fitch, C., Levenson, C. (2001). Journal Nutrition 131, 1427-1432.• Oren, M. (1999) The Journal of Biological Chemistry 274, 36031-36034• Peng, Y., Chen, L., Li, C., Lu, W., Agrawal, S., Chen, J. (2001) The Journal of Biological Chemistry 276, 6874-6878.• Protein Data Bank (PDB): http//:www.rcsb.org/pdb• Rainwater, R., Parks, D., Anderson, M. E., Tegtmeyer, P. and Mann K. (1995) Mol. Cell. Biol. 15, 3892-3903 • Siliciano, J., Canman, C., Taya, Y., Sakaguchi, K., Appella E., Kastan, M. (1997) Genes &Development 11, 3471-3481• Smith, M., Ford, J., Hollander, M., Bortnick, R., Amundson, S., Seo, Y., Deng, C., Hanawalt, P., Fornace, A. (2000) Molecular and Cellular Biology 20, 3705-3714• Smith, M.L., Ford, J. M., Hollander, M. C., Bortnick, R.A., Amundson, S. A., Seo Y. R., Deng, C.X., Hanawalt, P. C., and Fornace A. J. Jr. (2000). Mol. Cell. Biol. 20,
3705-3714• Standing, K. (2003) Current Opinion in Structural Biology 13, 595-601• Sun, X., Vinci, C., Makmura, L., Han, S., Tran, D., Nguyen, J., Hamann, M., Grazziani, S., Sheppard, S., Gutova, M., Zhou, F., Thomas, J. and Momand, J. (2003)
Antioxidants & Redox Signaling 5, 655-665.• Wang, P., Sait, F., Winter, G. (2001) Oncogene 20, 2318-2324.• Wang, S., Guo, M., Ouyang, H., Li, Z., Cordon-Cardo, C., Kurimasa, A., Chen, D. J., Fuks, Z., Ling, C.C., and Li, G.C. (2000) Proc. Natl. Acad. Sci. U S A. 97, 1584-
1588• Wu, H. H., Sherman, M., Yuan, Y. C. and Momand, J. (1999) Gene Ther. Mol. Biol. 4, 119-132• Wu, H., Thomas, J. and Momand, J. (2000) Biochem. J. 351, 87-93.• Xiao, G., Chicas, A., Oliver, M., Taya, Y., Tyagi, S., Kramer, F.R., and Bargonetti, J. (2000) Cancer Res. 60, 1711-1719• Yang, H., Wen, Y., Chen, C., Lozano, G., Lee, M. (2003) Mol. Cell. Biol. 23, 7096-7107.