1. p53 The Master Guardian of the Genome

2. p53 gene mutations in human tumors Greenblatt et al. (1995) Cancer Res. 54:4855 50%

3. p53 (low) p53 (high) Cell cycle arrest Apoptosis Genotoxic Stress (e.g. DNA damage) The Basic Paradigm of p53 Function INPUTS OUTPUTS Proliferative Stress (e.g. oncogenes)

4. The Discovery of p53

5. SV40 large T protein binds to p53

6. Hypothesis Large T antigen and p53 are oncogenes - p53, a proto-oncogene, is expressed in low concentrations in normal cells - T antigen oncogenic activity leads to over- expression of p53 and the latter acts as an oncogene WRONG!!

7. Cloning of the p53 gene, followed by successive experiments showed that it is actually a tumor suppressor gene Moshe Oren Arnold Levine

8. Autosomal Dominant Li-Fraumeni syndrome Inherited germ-line mutations in p53 cause predisposition for distinct cancers in variable ages

9. p53 Mutant Mice Develop Cancer

10. p53 is a transcription factor, active only as a homotetramer

11. p53 acts only as a tetramer Imagine a scenario: - One normal copy - One lof copy, encoding a mutated protein that can still bind to its partners

12. Does this mean that +/- heterozygotes do not need a second mutation for tumor progression? Not quite, even 1/16 of p53 molecules have some activity Missense mutations and not nonsense/frameshift are the common p53 mutations in cancer patients

13. p53 Mutations in Human Tumors are Found with High Frequency In the DNA Binding Domain In 143 families reported: point mutations (85%) deletions (9%) splice mutations (3.5%) insertions (2%)

14. Ribbon ModelSpace Filling Model p53 Binds DNA The most common mutation changes arginine 248, colored red here. Notice how it snakes into the minor groove of the DNA (shown in blue and green), forming a strong stabilizing interaction. When mutated to another amino acid, this interaction is lost. Other key sites of mutation are shown in pink, including arginine residues 175, 249, 273 and 282, and glycine 245.

15. p53 (low) p53 (high) Cell cycle arrest Apoptosis Genotoxic Stress (e.g. DNA damage) The Basic Paradigm of p53 Function INPUTS OUTPUTS Proliferative Stress (e.g. oncogenes)

16. Low levels of p53 expression in normal cells p53 protein levels increase upon exposure to UV (and many other agents) Campbell et al. Biochemical Society Transactions (2001)

17. - p53 is a transcription factor, acting as a homotetramer Summary - Expressed when cells gone awry - Two mutated copies in tumors, first is usually a dominant-negative mutation - Acts as a tumor suppressor gene

18. In normal cells we find only low concentrations of the p53 protein - p53 protein is actually synthesized all the time, but is degraded very fast via ubiquitin mediated proteolysis

19. p53 protein is ubiquitinated by the E3 ligase MDM2

20. Genetic Evidence that Mdm2 Inhibits p53 p53-/- mdm2-/- p53-/- mdm2-/-

21. Mdm2 is a p53 Target Gene p53 control of Mdm2 transcription is a negative feedback loop

22. Some p53 mutants show over expression of inert p53 protein p53 control of Mdm2 transcription is a negative feedback loop

23. p53 DNA damage Hyperproliferative stress Many agents induce p53 activity Grouped into two classes

24. Extensive DNA damage recruits the DNA damage response machinery Two key players: The protein kinases ATM and ATR

25. ATM and ATR are recruited to distinct sites and phosphorylate downstream effectors

26. ATR is recruited to single - stranded DNA - ATR-dependent phosphorylation of the Rad9 adaptor protein is needed for activating Chk1 - Activated Chk1 is released to phosphorylate its effectors ATR Rad9 Chk1 (inactive) Chk1 (active)

27. Phosphorylation of p53 (by ATM/ATR and/or Chk2) makes it unable to bind MDM2 Phosphorylated p53 acts as a transcription factor

28. DNA damage response activates p53 by stabilizing the protein via phosphorylation (and additional mechanisms) Additional inhibitory phosphorylation of MDM2

29. p53 DNA damage Hyperproliferative stress E2Fs e.g. high activity of E2Fs

30. Hyperproliferative stress response is mediated through the ARF protein - E2Fs induce transcription of the ARF gene - ARF binds to and sequesters MDM2 - p53 is stabilized

31. Over activity of oncogenes stimulates apoptosis through ARF

32. ARF stands for Alternative Reading Frame - Found in the same locus of the p16 gene - Uses an alternative promoter

33. p53 and Ink4a are the two most frequently mutated genes in human tumors

34. - p53 is a transcription factor, acting as a homotetramer Summary - Transcribed constitutively, but has a very short half life - DNA damage and a stalled replication fork induce p53 phosphorylation and activation - Hyperproliferative stress (e.g. oncogenic signaling, hypoxia) activates p53 via ARF - Ubiquitinated by the E3 ligase MDM2

35. p53 DNA damage Cell cycle arrestApoptosis What about outputs? Hyperproliferative stress

36. (p16) (p21) p53 activates transcription of the CKI p21

38. p53 DNA damage Cell cycle arrestApoptosis What about outputs? Hyperproliferative stress

39. Programmed cell death: aka Apoptosis Specific cells are dying in a programmed manner

40. http://mathematica.edublogs.org/files/2011/03/C-elegans-2kbu264.JPG Worms are transparent C. elegans One can follow the individual lineage of each of the cells <1000 cells

41. http://www.imsc.res.in/~sitabhra/research/neural/celegans/

42. Deciphering the cell lineage of C. elegans

43. Cell death is also a “cell fate”

44. Use genetics to identify mutants Epistasis analysis and biochemical studies allow the scientists to build a pathway

45. Genetic control of programmed cell death CED-4 No death signal CED-4 is inactive Death signal CED-9 Activates CED-3 caspase for the killing CED-9 CED-4 ced-9 Cell death ced-3ced-4

46. Gain of function mutations in bcl-2 were associated with human cancers (bcl= b cell lymphoma resulting from a translocation event) Our body uses the same apoptotic mechanisms used in PCD, for killing “bad” or unwanted cells

47. http://herkules.oulu.fi/isbn9514266676/html/i267425.html

48. Lineage and programmed cell death Bob Horvitz John Sulston Sydney Brenner Physiology and Medicine 2002 Nobel Prize

49. - Pro-apoptotic signals open up the channels, allowing cyt C to be released to the cytoplasm

50. A simplified representation of apoptosis Noxa Proapoptotic (BH3-only) Proapoptotic (multidomain) Bax Bcl-2 Cytochrome C Apaf Procaspase Caspase Mitochondria All the regulators are from the same protein family (containing BH domains) Antiapoptotic

51. http://www.weizmann.ac.il/home/ligivol/apoptosis_project/apoptotic_pathways.html p53 triggers apoptosis