p53 The Master Guardian

1. p53 The Master Guardian

2. Cell cycle control involves several checkpoints and checkpoint (molecular breaking) mechanisms R point

3. Three lectures in one slide p53 Mutations in p53 can be found in half of all tumors

4. Discovery of p53 - SV40 DNA tumor virus transforms monkey kidney cells into cancerous cells - SV40 DNA tumor virus can also transform murine cells in culture - The virus is injected into hamsters and the serum is used to precipitate the oncogenic protein: Large T antigen - … and another protein, ~53kD - Both T antigen and p53 are expressed only upon viral infection

5. Antibodies specific to the large T protein show that T co-immunoprecipitates 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-expressionof p53 and the latter acts as an oncogene

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

8. - p53 is a transcription factor that acts to inhibit tumor progression - T antigen oncogenic activity was carried out by sequesteringp53 when the latter was supposed to inhibit tumor progression - T antigen binds also to pRb, sequestering it from negatively regulating cell cycle progression

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

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

11. 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 However, missense mutations and not nonsense/frameshift are the common p53 mutations in cancer patients

12. 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%)

13. How does p53 act?

14. # 1) Li-Fraumeni syndrome Inherited germ-line mutations in p53 cause predisposition for distinct cancers in variable ages

15. # 2) Homozygote mutant mice can complete embryogenesis (but die shortly after)

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

17. # 1) Inherited mutations cause distinct cancers # 2) Homozygote mutant mice can complete embryogenesis (but die shortly after) # 3) Low levels of p53 expression in normal cells

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

19. 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 the ubiquitine system

20. p53 protein is ubiquitinated by the E3 ligase MDM2

21. p53 mutations in human tumors are found with high frequency in the DNA binding domain Missense mutations can inhibit p53 from binding to its target genes, including mdm2 (think about expression levels of p53 in these mutants)

22. Some p53 mutants show over expression of inertp53 protein

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

24. Subtle DNA damage is sensed by sensor proteins and repaired by the DNA repair machinery 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. ATRis recruited to single - stranded DNA ATR Chk2 (inactive) Rad9 - ATR-dependent phosphorylation of the Rad9 adaptor protein is needed for activating Chk2 Chk2 (active) - Activated Chk2is released to phosphorylate its effectors

27. Phosphorylation of p53 (by ATM/ATR and/or Chk2) makes it insusceptible to MDM2 binding 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. DNA damage Hyperproliferative stress p53 e.g. high activity of E2Fs E2Fs

30. Hyperproliferative stress response is mediated through the ARFprotein - 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. ARFstands for Alternative Reading Frame - Found in the same locus of the p16 gene - Uses an alternative promoter

33. Two reactions for the price of one

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

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

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

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