p53-dependent inhibition of FKHRL1in response to DNA damage through protein kinase SGK1

Han You,YingJu Jang,Annick Itie You-Ten,Hitoshi Okada,Jennifer Liepa,Andrew Wakeham,Kathrin Zaugg,

and Tak W.Mak*

Institute for Breast Cancer Research,University Health Network,620University Avenue,Suite706,Toronto,ON,Canada M5G2C1;and Department of Medical Biophysics and Immunology,University of Toronto,Toronto,ON,Canada M5G2C1

Contributed by Tak W.Mak,August25,2004

FKHRL1(FOXO3a)and p53are two potent stress-response regula-tors.Here we show that these two transcription factors exhibit ‘‘crosstalk’’in vivo.In response to DNA damage,p53activation led to FKHRL1phosphorylation and subcellular localization change, which resulted in inhibition of FKHRL1transcription activity.AKT was dispensable for p53-dependent suppression of FKHRL1.By contrast,serum-and glucocorticoid-inducible kinase1(SGK1)was signi?cantly induced in a p53-dependent manner after DNA dam-age,and this induction was through extracellular signal-regulated kinase1?2-mediated posttranslational regulation.Furthermore, inhibition of SGK1expression by a small interfering RNA knock-down experiment signi?cantly decreased FKHRL1phosphorylation in response to DNA damage.Taken together,our observations reveal previously unrecognized crosstalk between p53and FKHRL1.Moreover,our?ndings suggest a new pathway for understanding aging and the age dependency of human diseases governed by these two transcription factors.

H uman longevity depends on genome stability.Several

mouse models have revealed that an age-related decrease of DNA repair or an increase in DNA damage plays a role in mammalian aging(1–3).The idea that cellular responses to stress may be important in aging is supported by studies of p53,the functions of which are critical for both the apoptotic and senescence responses to DNA damage,telomeric shortening, and oxidative stress(4–6).

The mammalian FOXO family of forkhead transcription factors(including FKHR,FKHRL1,and AFX)have been proposed as antiaging factors based on evidence from their orthologues,DAF-16in Caenorhabditis elegans and dFOXO in Drosophila melanogaster,which regulate longevity in response to reduced insulin?insulin-like growth factor I(IGF-I)signaling or by overexpressing constitutively active FOXO(7,8).Growth factor signaling to FOXO family members through phosphati-dylinositol3-kinase(PI3K)and its downstream kinase,Akt,has been found to be evolutionarily conserved for FOXO phosphor-ylation,subcellular translocation,and inhibition of its transcrip-tional activity(9).However,the role of FOXO in response to DNA damage,as well as the signaling pathway upstream of FOXO,is,as yet,unclear.Overexpression of FKHRL1can protect cells from oxidative stress-induced cell death,as de-scribed in refs.10–12.Induction of a number of antioxidant enzymes and stress-related gene products has been proposed as a potential mechanism(9,13–15).The fact that the precise biological consequences of FOXO activation are cell-type-specific and stress-type-dependent suggests that there might be crosstalk between FOXO and other stress regulators.How FOXO communicates and coordinates with other signaling pathways in response to genotoxic stress remains unknown. The opposing functions of p53and FKHRL1with regard to the aging process suggest that a regulatory mechanism might exist to integrate these two signaling pathways.We sought to elucidate the mechanism underlying this crosstalk and its regu-lation in the hopes of better understanding how these two transcription factors communicate and coordinate with each other in controlling cell fate in response to genotoxic stress. Experimental Procedures

Cell Culture.E1A?H-ras-V12-transformed p53???and p53???mouse embryonic fibroblasts(MEFs)were kind gifts from Scott Lowe(Cold Spring Harbor Laboratory,Cold Spring Harbor, NY).p53QS primary MEFs were a gift of Geoffrey Wahl(The Salk Institute for Biological Studies,San Diego).MEFs were cultured in DMEM(GIBCO)supplemented with10%FCS (GIBCO).Primary MEFs were transformed with pLPC ras?E1A (a gift from Athena Lin,Roswell Park Cancer Institute,Buffalo, NY)and then subjected to puromycin selection.Pharmacolog-ical inhibitors PD98059(50?M),LY294002(10?M),and SB203580(20?M)were purchased from Calbiochem.

Constructs and Antibodies.The pSIRIPP retroviral small interfer-ing RNA(siRNA)vector(a gift from Tyler Jacks,Massachusetts Institute of Technology,Cambridge)is described in ref.16. Oligonucleotide sequences for murine serum-and glucocorti-coid-inducible kinase1(SGK1)siRNA are available on request. The pBabe Akt-KD,human WT p53,p53R175H,and p53R273H expression plasmids were kindly provided by Arnold Levine (Institute for Advanced Study,Princeton).pECE-HA-FKHRL1,HA-FKHRL1-TM expression plasmids,and the anti-SGK antibody were generously provided by Michael Greenberg (Harvard Medical School,Boston).Anti-p53antibody(FL393, Santa Cruz Biotechnology),anti-p21and anti-p27antibodies (BD Biosciences),antibodies against total FKHRL1or phospho-Thr-32FKHRL1(Upstate Biotechnology,Lake Placid,NY), anti-total-Akt or phospho-Akt antibodies(Cell Signaling Tech-nology,Beverly,MA),and anti-total-extracellular signal-regulated kinase(ERK)1?2or phospho-ERK1?2antibodies (Cell Signaling Technology)were purchased commercially.

Retroviral Infection.Phoenix cells were transfected with indicated retroviral constructs by calcium chloride transfection.At24h posttransfection,conditioned medium recovered from cultures of transfected Phoenix cells was filtered through a0.45-?m filter and added to the MEF cultures.Spin infection was performed at 1,400?g for90min.Cells were then cultured for?20h and then subjected to puromycin selection.

Chromatin Immunoprecipitation(ChIP)Assays.ChIP assays were performed by using an Acetyl-Histone H3Immunoprecipitation Assay Kit(17-245,Upstate Biotechnology).Briefly,107cells were fixed with1%formaldehyde and then washed,harvested in

Abbreviations:Akt-DN,kinase-dead form of Akt;ChIP,chromatin immunoprecipitation; CISK,cytokine-independent survival kinase;ERK,extracellular signal-regulated kinase, IGF-I,insulin-like growth factor I;MEF,mouse embryonic?broblast;PI3K,phosphatidyl-inositol3-kinase;SGK1,serum-and glucocorticoid-inducible kinase1;siRNA,small inter-fering RNA;TNF-?,tumor necrosis factor?.

*To whom correspondence should be addressed.E-mail:tmak@uhnres.utoronto.ca.

?2004by The National Academy of Sciences of the USA

http://www.doczj.com/doc/d753260b6c85ec3a87c2c56c.html?cgi?doi?10.1073?pnas.0406286101PNAS?September28,2004?vol.101?no.39?14057–14062B I O C H E M I S T R Y