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

講者講者 : : 賴金美賴金美

日期日期 : 2007. 1. : 2007. 1. 8.8.

CDK2-Dependent Phosphorylation of CDK2-Dependent Phosphorylation of

FOXO1 as an Apoptotic Response to DNA FOXO1 as an Apoptotic Response to DNA

DamageDamageHaojie Huang, Kevin M. Regan, Zhenkun Lou, Haojie Huang, Kevin M. Regan, Zhenkun Lou,

Junjie Chen, Donald J. TindallJunjie Chen, Donald J. Tindall

生科四論文選讀 ( 示範 )

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CDK and cyclin together drive the cell from CDK and cyclin together drive the cell from

one cell cycle phase to the next.one cell cycle phase to the next.

The pairing between The pairing between individual cyclins and individual cyclins and Cdks is highly specific, Cdks is highly specific, and only certain and only certain combinations are found.combinations are found.

Unlike yeast cells, which have a single Cdk, Unlike yeast cells, which have a single Cdk, mammalian cells produce mammalian cells produce several different several different versions of Cdksversions of Cdks..

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G2G2

MM

G1G1

SS

CDK4-6CDK4-6Cyclin DCyclin D

CDK1CDK1Cyclin ACyclin ACDK2CDK2

Cyclin ACyclin A

CDK2CDK2Cyclin ECyclin E

CDK1CDK1Cyclin BCyclin B

Cell cycle “checkpoints”

* A checkpoint is a critical control point where stop and go-adhead signals can regulate the cycle.

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Checkpoint pathwaysCheckpoint pathways monitor for DNA replication, monitor for DNA replication, chromosome-to-spindle attachments, and DNA chromosome-to-spindle attachments, and DNA damagedamage

p53p53 protein protein plays a central plays a central role in DNA damage role in DNA damage checkpoints pathways.checkpoints pathways.

1.1. repairrepair2.2. trigger apoptosistrigger apoptosis

* * Cancer cells exhibit a loss of restriction point control.Cancer cells exhibit a loss of restriction point control.

1.1. 2.2.

3.3.

p53 accumulates and p53 accumulates and triggers cell cycle arresttriggers cell cycle arrest

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ATM, ATR:ATM, ATR:~ ~ sensorssensors that recognize DNA damage or cellular that recognize DNA damage or cellular abnormalitiesabnormalities

chcheckpoint eckpoint kkinase: Chk1, Chk2inase: Chk1, Chk2

/ E/ E

DNA replicationDNA replication

However, it is not clear whether CDK2 plays a role However, it is not clear whether CDK2 plays a role

in the regulation of DNA damage-induced cell in the regulation of DNA damage-induced cell

death.death.

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Activation of FOXO transcription factors induces Activation of FOXO transcription factors induces apoptosisapoptosis by up-regulating a number of cell death by up-regulating a number of cell death genes, including those encoding the ligand for the genes, including those encoding the ligand for the death receptor known as death receptor known as Fas or CD95Fas or CD95, the , the Bcl-2–Bcl-2–interacting mediator (Bim)interacting mediator (Bim) of cell death, and the of cell death, and the tumor tumor necrosis factor–related apoptosis-inducing ligandnecrosis factor–related apoptosis-inducing ligand..

FOXO proteins are FOXO proteins are transcription factors transcription factors

P-FOXO P-FOXO binds 14-3-3binds 14-3-3 and is retained in the cytoplasm and is retained in the cytoplasm

FOXO (Forkhead box O)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.

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Induction of apoptosis in 9-nitrocamptothecin-treated DU145 Induction of apoptosis in 9-nitrocamptothecin-treated DU145 human prostate carcinoma cells correlates with de novo human prostate carcinoma cells correlates with de novo synthesis of CD95 and CD95 ligand and down-regulation of synthesis of CD95 and CD95 ligand and down-regulation of c-FLIP(short).c-FLIP(short).

Cancer Res. 2001 Oct 1;61(19):7148-54. Cancer Res. 2001 Oct 1;61(19):7148-54. Activation of multidomain and BH3-only pro-apoptotic Bcl-2 Activation of multidomain and BH3-only pro-apoptotic Bcl-2 family members in p53-defective cells.family members in p53-defective cells.

Apoptosis. 2004 Nov;9(6):815-31. 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

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Because CDK2 is a key mediator of most checkpoint functions

The authors hypothesized that the activities of The authors hypothesized that the activities of

FOXO proteins might be regulated by DNA damage FOXO proteins might be regulated by DNA damage

signals through functional interactions with CDK2.signals through functional interactions with CDK2.

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Q1: Q1: Whether CDK2 could phosphorylate the FOXO1 protein?Whether CDK2 could phosphorylate the FOXO1 protein?

NIH3T3 cellsNIH3T3 cells

IP IP endogenous endogenous CDK2CDK2

In vitro kinase assayIn vitro kinase assay(substrate: GST-FOXO1)(substrate: GST-FOXO1)

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Purify Purify bacterially produced GST-CDK2bacterially produced GST-CDK2

+ GST-cyclin E or GST-cyclin A+ GST-cyclin E or GST-cyclin A

In vitro kinase assayIn vitro kinase assay(substrate: GST-FOXO1 fragments)(substrate: GST-FOXO1 fragments)

Reconstitution exp:Reconstitution exp:

The kinase activity were abolished The kinase activity were abolished when the CDK inhibitor p27when the CDK inhibitor p27KIP1KIP1 was was included. included.

These results indicate that CDK2 directly These results indicate that CDK2 directly

phosphorylates the FOXO1 protein in vitro.phosphorylates the FOXO1 protein in vitro.

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Supporting data:Supporting data:

S/T-PS/T-P

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Supporting data:Supporting data:

CDK2 phosphorylates the FOXO1 protein at CDK2 phosphorylates the FOXO1 protein at SerSer249249 and Ser and Ser298298, with a preference at , with a preference at SerSer249249

Reconstituted cyclin Reconstituted cyclin E/CDK2 kinase assay:E/CDK2 kinase assay:

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The antibody specifically recognized the wild-type The antibody specifically recognized the wild-type FOXO1 FOXO1 but not the Serbut not the Ser249249AlaAla249249 (S249A) mutant. (S249A) mutant.

Q2: Q2: Whether FOXO1 is phosphorylated at SerWhether FOXO1 is phosphorylated at Ser249249 in vivo? in vivo?

generated a phosphorylationspecific antibody generated a phosphorylationspecific antibody against a peptide containing the phosphorylated against a peptide containing the phosphorylated SerSer249249..

LNCaP cellsLNCaP cells

transfect with transfect with Flag-FOXO1 or S249A mutantFlag-FOXO1 or S249A mutant

IP: anti-FlagIP: anti-Flag

IB: anti-FOXO1 orIB: anti-FOXO1 or

anti-S249-panti-S249-p

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transfect transfect V5-CDK2-AF V5-CDK2-AF with with WT or S249A FOXO1WT or S249A FOXO1

IP: anti-FOXO1 IP: anti-FOXO1 IB: anti-S249-p IB: anti-S249-p

transfect with transfect with CDK2-specificCDK2-specific or or a non-specific controla non-specific control siRNAsiRNA

IBIB

1. Silencing of endogenous CDK2 by siRNAs..

2. Transfect with CDK2 active 2. Transfect with CDK2 active mutantmutant (CDK2-AF).(CDK2-AF).

LNCaP cellsLNCaP cells

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LNCaP –FOC4 cells LNCaP –FOC4 cells

treat with nocodazole for 24 hr treat with nocodazole for 24 hr ((arrest in M phasearrest in M phase))

release from arrestrelease from arrest

IBIB: : -FOXO1 & -FOXO1 & -S249-p-S249-p

FACS analysisFACS analysis

M phaseM phase

phosphorylation of Serphosphorylation of Ser249249 was low during the G1 was low during the G1 phasephase and increased as cells progressed through the S and increased as cells progressed through the S phase.phase.

these data indicate that CDK2 phosphorylates FOXO1 at Ser249 in vivo.

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Q3: Q3: What’s the effect account for CDK2-mediated What’s the effect account for CDK2-mediated FOXO1 phosphorylation?FOXO1 phosphorylation?

To examine the effect on the To examine the effect on the transcriptional transcriptional activity of FOXO1.activity of FOXO1.

LNCaP cellsLNCaP cells

Co-transfect Co-transfect FOXO1 luciferase reporterFOXO1 luciferase reporterwith with FOXO1 or other as indicatedFOXO1 or other as indicated

measure luciferase activities.measure luciferase activities.

3 3 xx IRS IRSluciferasluciferasee

FOXO1FOXO1

PTEN PTEN inhibit Akt activity inhibit Akt activity

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FOXO1-AAAFOXO1-AAA: three Akt phosphorylation sites mutant: three Akt phosphorylation sites mutant

Roscovitine: CDK2 inhibitorRoscovitine: CDK2 inhibitor

* * FOXO1-S249DFOXO1-S249D: : ~ mimic Ser~ mimic Ser249249 phosphorylation phosphorylation

these results suggest that these results suggest that CDK2-induced inhibition of CDK2-induced inhibition of transcriptional activity of FOXO1transcriptional activity of FOXO1 is mediated primarily is mediated primarily by the by the phosphorylation of Serphosphorylation of Ser249249..

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Q4: Q4: How CDK2-mediated SerHow CDK2-mediated Ser249249 phosphorylation phosphorylation inhibit inhibit the transcriptional activity of FOXO1?the transcriptional activity of FOXO1?

To observe To observe cellular localizationcellular localization of of ectopically expressed wild-type or S249A ectopically expressed wild-type or S249A

FOXO1 in DU145 cells. (PTEN-positive cell)FOXO1 in DU145 cells. (PTEN-positive cell)

SerSer249249 was adjacent to the three-arginine motif, was adjacent to the three-arginine motif, which is critical for nuclear localization of FOXO which is critical for nuclear localization of FOXO proteinsproteins

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C: cytoplasmC: cytoplasmN: nucleusN: nucleus

Trafficking of FOXO1 from the nucleus to the cytoplasmTrafficking of FOXO1 from the nucleus to the cytoplasm is affected by CDK2-mediated Ser249 phosphorylation.is affected by CDK2-mediated Ser249 phosphorylation.

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Q5: Q5: What’s the role of FOXO1 in response to DNA What’s the role of FOXO1 in response to DNA damage?damage?

Treating cells with DNA damaging agent, Treating cells with DNA damaging agent, camptothecin.camptothecin.

CamptothecinCamptothecin is a plant is a plant

secondary metabolite used as secondary metabolite used as

an an anti-cancer drug that anti-cancer drug that

damages DNA, leading to the damages DNA, leading to the

destruction of the cell.destruction of the cell.

Camptothecin affects the Camptothecin affects the

activity of the enzyme activity of the enzyme

topoisomerase Itopoisomerase I, whose normal , whose normal

action is to cleave, unwind, and action is to cleave, unwind, and

religate DNA. religate DNA. activation of the DNA double-strand break activation of the DNA double-strand break checkpoint pathways and inhibition of CDK2 activity.checkpoint pathways and inhibition of CDK2 activity.

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LNCaP cellsLNCaP cells

cotransfect Flag-FOXO1 & cotransfect Flag-FOXO1 & control control or or chk1 siRNAchk1 siRNA

treat with or without CPT for 16 hrtreat with or without CPT for 16 hr

IP: anti-FOXO1 IP: anti-FOXO1 IB: anti-S249-p IB: anti-S249-p

DU145 cellsDU145 cells

Treat with (+) or without (-)Treat with (+) or without (-)Camptothecin (CPT) Camptothecin (CPT) for 16 hrfor 16 hr

IP: anti-FOXO1IP: anti-FOXO1 CDK2 kinase CDK2 kinase

assayassayIB: anti-S249-p IB: anti-S249-p

p53-independentp53-independent

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sr: silencing resistantsr: silencing resistant

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cytosolcytosol

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

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