Science, Vol. 314, p 294~297 (2006)

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

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

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

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

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

6. FOXO (Forkhead box O) FOXO proteins are transcription factors P-FOXO binds 14-3-3 and is retained in the cytoplasm 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. . 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.

7. Increased expression of these pro-apoptotic proteins is required for cell death to be induced by the DNA damaging agent camptothecin or its derivatives 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.

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

10. These results indicate that CDK2 directly phosphorylates the FOXO1 protein in vitro. Reconstitution exp: Purify bacterially produced GST-CDK2  + GST-cyclin E or GST-cyclin A  In vitro kinase assay (substrate: GST-FOXO1 fragments) The kinase activity were abolished when the CDK inhibitor p27KIP1 was included.

11. Supporting data: S/T-P

12. Supporting data: Reconstituted cyclin E/CDK2 kinase assay: CDK2 phosphorylates the FOXO1 protein at Ser249 and Ser298, with a preference at Ser249

13. Q2: Whether FOXO1 is phosphorylated at Ser249 in vivo? generated a phosphorylationspecific antibody against a peptide containing the phosphorylated Ser249. LNCaP cells  transfect with Flag-FOXO1 or S249A mutant  IP: anti-Flag  IB: anti-FOXO1 or anti-S249-p • The antibody specifically recognized the wild-type FOXO1 but not the Ser249Ala249 (S249A) mutant.

14. LNCaP cells 1. Silencing of endogenousCDK2 by siRNAs. 2. Transfect with CDK2 active mutant (CDK2-AF). transfect with CDK2-specific or a non-specific controlsiRNA  IB transfect V5-CDK2-AF with WT or S249A FOXO1  IP: anti-FOXO1  IB: anti-S249-p

15. these data indicate thatCDK2 phosphorylates FOXO1 at Ser249 invivo. M phase LNCaP –FOC4 cells  treat with nocodazole for 24 hr (arrest in M phase)  release from arrest  IB: a-FOXO1 & a-S249-p FACS analysis phosphorylation of Ser249 was low during the G1 phase and increased as cells progressed through the S phase.

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

17. FOXO1-AAA: three Akt phosphorylation sites mutant * FOXO1-S249D: ~ mimic Ser249 phosphorylation Roscovitine: CDK2 inhibitor these results suggest that CDK2-induced inhibition of transcriptional activity of FOXO1 is mediated primarily by the phosphorylation of Ser249.

18. Q4: How CDK2-mediated Ser249 phosphorylation inhibit the transcriptional activity of FOXO1? Ser249 was adjacent to the three-arginine motif, which is critical for nuclear localization of FOXO proteins To observe cellular localization of ectopically expressed wild-type or S249A FOXO1 in DU145 cells. (PTEN-positive cell)

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

20. Q5: What’s the role of FOXO1 in response to DNA 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. DU145 cells  Treat with (+) or without (-) Camptothecin (CPT) for 16 hr   IP: anti-FOXO1CDK2 kinase  assay IB: anti-S249-p LNCaP cells  cotransfect Flag-FOXO1 & control or chk1 siRNA  treat with or without CPT for 16 hr  IP: anti-FOXO1  IB: anti-S249-p p53-independent

22. sr: silencing resistant

23. Summary cytosol

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