1. 1 Science, Vol. 314, p 294~297 (2006) 講者 : 賴金美 日期 : 2007. 1. 8. CDK2-Dependent Phosphorylation of FOXO1 as an Apoptotic Response to DNA Damage Haojie Huang, Kevin M. Regan, Zhenkun Lou, Junjie Chen, Donald J. Tindall 生科四論文選讀 ( 示範 )

2. 2 CDK and cyclin together drive the cell from one cell cycle phase to the next. The pairing between individual cyclins and Cdks is highly specific, and only certain combinations are found. Unlike yeast cells, which have a single Cdk, mammalian cells produce several different versions of Cdks.

3. 3 G2 M G1 S CDK4-6 Cyclin D CDK1 Cyclin A CDK2 CDK2 Cyclin E CDK1 Cyclin B Cell cycle “checkpoints” * A checkpoint is a critical control point where stop and go-adhead signals can regulate the cycle.

4. 4 Checkpoint pathways monitor for DNA replication, chromosome-to-spindle attachments, and DNA damage p53 protein plays a central role in DNA damage checkpoints pathways. 1.repair 2.trigger apoptosis * Cancer cells exhibit a loss of restriction point control. 1.2. 3. p53 accumulates and triggers cell cycle arrest

5. 5 ATM, ATR: ~ sensors that recognize DNA damage or cellular abnormalities checkpoint kinase: Chk1, Chk2 / E DNA replication However, it is not clear whether CDK2 plays a role in the regulation of DNA damage-induced cell death.

6. 6 Activation of FOXO transcription factors induces apoptosis by up-regulating a number of cell death genes, including those encoding the ligand for the death receptor known as Fas or CD95, the Bcl-2–interacting mediator (Bim) of cell death, and the tumor necrosis factor–related apoptosis- inducing ligand. FOXO proteins are transcription factors P-FOXO binds 14-3-3 and is retained in the cytoplasm FOXO (Forkhead box O). The FOXO transcription factors, FKHR (FOXO1), FKHRL1 (FOXO3a), and AFX (FOXO4), share DNA binding specificity to a core consensus site and are targets of PI3-K signaling, which regulates their activity via phosphorylation mediated by protein kinase B/Akt and serum and glucocorticoid–induced kinase.

7. 7 Induction of apoptosis in 9-nitrocamptothecin-treated DU145 human prostate carcinoma cells correlates with de novo synthesis of CD95 and CD95 ligand and down-regulation of c-FLIP(short). Cancer Res. 2001 Oct 1;61(19):7148-54. Activation of multidomain and BH3-only pro-apoptotic Bcl-2 family members in p53-defective cells. Apoptosis. 2004 Nov;9(6):815-31. Increased expression of these pro-apoptotic proteins is required for cell death to be induced by the DNA damaging agent camptothecin or its derivatives

8. 8 Because CDK2 is a key mediator of most checkpoint functions The authors hypothesized that the activities of FOXO proteins might be regulated by DNA damage signals through functional interactions with CDK2.

9. 9 Q1: Whether CDK2 could phosphorylate the FOXO1 protein? NIH3T3 cells  IP endogenous CDK2  In vitro kinase assay (substrate: GST-FOXO1)

10. 10 Purify bacterially produced GST-CDK2  + GST-cyclin E or GST-cyclin A  In vitro kinase assay (substrate: GST-FOXO1 fragments) Reconstitution exp: The kinase activity were abolished when the CDK inhibitor p27 KIP1 was included. These results indicate that CDK2 directly phosphorylates the FOXO1 protein in vitro.

11. 11 Supporting data: S/T-P

12. 12 Supporting data: CDK2 phosphorylates the FOXO1 protein at Ser 249 and Ser 298, with a preference at Ser 249 Reconstituted cyclin E/CDK2 kinase assay:

13. 13  The antibody specifically recognized the wild-type FOXO1 but not the Ser 249  Ala 249 (S249A) mutant. but not the Ser 249  Ala 249 (S249A) mutant. Q2: Whether FOXO1 is phosphorylated at Ser 249 in vivo? generated a phosphorylationspecific antibody against a peptide containing the phosphorylated Ser 249. LNCaP cells  transfect with Flag-FOXO1 or S249A mutant  IP: anti-Flag  IB: anti-FOXO1 or anti-S249-p

14. 14 transfect V5-CDK2-AF with WT or S249A FOXO1  IP: anti-FOXO1  IB: anti-S249-p transfect with CDK2-specific or a non-specific control siRNA IB 1. Silencing of endogenous CDK2. by siRNAs. 2. Transfect with CDK2 active mutant (CDK2-AF). (CDK2-AF). LNCaP cells

15. 15 LNCaP –FOC4 cells  treat with nocodazole for 24 hr (arrest in M phase)  release from arrest  IB:  -FOXO1 &  -S249-p FACS analysis M phase phosphorylation of Ser 249 was low during the G1 phase and increased as cells progressed through the S phase. and increased as cells progressed through the S phase. these data indicate that CDK2 phosphorylates FOXO1 at Ser 249 in vivo.

16. 16 Q3: What’s the effect account for CDK2-mediated FOXO1 phosphorylation? FOXO1 phosphorylation? To examine the effect on the transcriptional activity of FOXO1. LNCaP cells  Co-transfect FOXO1 luciferase reporter with FOXO1 or other as indicated  measure luciferase activities. 3 x IRS luciferase FOXO1 PTEN  inhibit Akt activity

17. 17 FOXO1-AAA: three Akt phosphorylation sites mutant Roscovitine: CDK2 inhibitor * FOXO1-S249D: ~ mimic Ser 249 phosphorylation ~ mimic Ser 249 phosphorylation these results suggest that CDK2-induced inhibition of transcriptional activity of FOXO1 is mediated primarily by the phosphorylation of Ser 249.

18. 18 Q4: How CDK2-mediated Ser 249 phosphorylation inhibit the transcriptional activity of FOXO1? the transcriptional activity of FOXO1? To observe cellular localization of ectopically expressed wild-type or S249A FOXO1 in DU145 cells. (PTEN-positive cell) Ser 249 was adjacent to the three-arginine motif, which is critical for nuclear localization of FOXO proteins

19. 19 C: cytoplasm N: nucleus Trafficking of FOXO1 from the nucleus to the cytoplasm is affected by CDK2-mediated Ser249 phosphorylation.

20. 20 Q5: What’s the role of FOXO1 in response to DNA damage? damage? Treating cells with DNA damaging agent, camptothecin. Camptothecin is a plant secondary metabolite used as an anti-cancer drug that damages DNA, leading to the destruction of the cell. Camptothecin affects the activity of the enzyme topoisomerase I, whose normal action is to cleave, unwind, and religate DNA. activation of the DNA double-strand break checkpoint pathways and inhibition of CDK2 activity.

21. 21 LNCaP cells  cotransfect Flag-FOXO1 & control or chk1 siRNA  treat with or without CPT for 16 hr  IP: anti-FOXO1  IB: anti-S249-p DU145 cells  Treat with (+) or without (-) Camptothecin (CPT) for 16 hr   IP: anti-FOXO1 CDK2 kinase IP: anti-FOXO1 CDK2 kinase  assay  assay IB: anti-S249-p p53-independent

22. 22 sr: silencing resistant

23. 23 cytosol

24. 24 SCIENCE VOL 314, p261~262 (2006)

25. 25