University of South Florida Scholar Commons Integrative Biology Faculty and Staff ubP lications Integrative Biology 2005 Suppression of FOXO1 Activity by FHL2 through SIRT1 Mediated deacetylation Yonghua Yang University of South Florida Huayan Hou University of South Florida Edward M. Haller University of South Florida, [email protected] Santo V. Nicosia University of South Florida Wenlong Bai University of South Florida Follow this and additional works at: https://scholarcommons.usf.edu/bin_facpub Scholar Commons Citation Yang, Yonghua; Hou, Huayan; Haller, Edward M.; Nicosia, Santo V.; and Bai, Wenlong, "Suppression of FOXO1 Activity by FHL2 through SIRT1 Mediated deacetylation" (2005). Integrative Biology Faculty and Staff Publications. 265. https://scholarcommons.usf.edu/bin_facpub/265 This Article is brought to you for free and open access by the Integrative Biology at Scholar Commons. 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The EMBO Journal (2005) 24, 1021–1032 | & 2005 European Molecular Biology Organization | All Rights Reserved 0261-4189/05 www.embojournal.org TTHEH E EEMBOMBO JJOURNALOURN AL Suppression of FOXO1 activity by FHL2 through SIRT1-mediated deacetylation Yonghua Yang, Huayan Hou, FOXO4 (AFX, acute-lymphocytic-leukemia-1) (Parry et al, Edward M Haller, Santo V Nicosia 1994), and the recently cloned FOXO6 (Jacobs et al, 2003). and Wenlong Bai* As demonstrated by genetic and biochemical analysis of lower organisms and mammalian systems, the FOXO factors Departments of Pathology and Interdisciplinary Oncology, University of South Florida College of Medicine and Programs of Molecular Oncology, are critical regulators of stress responses, insulin sensitivity, Drug Discovery and Experimental Therapeutics, H Lee Moffitt Cancer longevity, and oncogenesis. For example, in Caenorhabditis Center, Tampa, FL, USA elegans, the FOXO-related protein Daf-16 increases longevity and signals formation of the dauer larva, which is a stage of Forkhead box class O (FOXO) proteins are transcription developmental arrest and reduced metabolic activity that factors that function downstream of the PTEN tumor enhances survival during periods of environmental stress suppressor and directly control the expression of genes (Kenyon et al, 1993). Mutation of the insulin/IGF1 receptor involved in apoptosis, cell cycle progression, and stress homolog Daf-2 or of Age-1, the catalytic subunit of phosphoi- responses. In the present study, we show that FOXO1 nositide 3-kinase (PI3K), mimics the effects of Daf-16, thus interacts with four and a half LIM 2 (FHL2) in prostate suggesting that the insulin/IGF receptor–PI3K pathway nega- cancer cells. This interaction occurred in the nucleus and tively regulates Daf-16 function. Consistent with this premise, was enhanced by lysophosphatic acid. FHL2 decreased the Daf-16 and its human homologs contain multiple conserved transcriptional activity of FOXO1 and the expression of phosphorylation sites for members of the protein kinase B known FOXO target genes and inhibited FOXO1-induced family (AKT-1, AKT-2, AKT3, and SGK-1), all of which func- apoptosis. Interestingly, SIRT1, a mammalian homolog of tion downstream of PI3K (Brunet et al, 1999). yeast Sir2, bound to and deacetylated FOXO1 and inhibited Phosphorylation of the FOXO factors by PKB results in their its transcriptional activity. FHL2 enhanced the interaction nuclear export, their interaction with the 14-3-3 proteins in of FOXO1 and SIRT1 and the deacetylation of FOXO1 by the cytosol, and consequently in the abrogation of their Sirtuin-1 (SIRT1). Overall, our data show that FHL2 in- transcriptional activity. Recent studies suggest that kinases hibits FOXO1 activity in prostate cancer cells by promoting in addition to PKB inhibit FOXO activity (Brunet et al,2001; the deacetylation of FOXO1 by SIRT1. Woods et al, 2001; Hu et al, 2004), as do nuclear hormone The EMBO Journal (2005) 24, 1021–1032. doi:10.1038/ receptors (Schuur et al, 2001; Zhao et al, 2001; Dowell et al, sj.emboj.7600570; Published online 3 February 2005 2003; Li et al, 2003) and other forkhead factors (Seoane et al, Subject Categories: chromatin & transcription; molecular 2004). biology of disease The PI3K–PKB pathway is active in and enhances the Keywords: acetylation; FKHR; forkhead; prostate cancer; growth and survival of many types of human cancer cells. SIR2 Conversely, the FOXO factors, which function downstream of the PI3K antagonist PTEN in cancer cells, inhibit cell cycle progression and promote cell death. These factors accomplish these actions by modulating the expression of genes encoding apoptosis and growth regulatory proteins by two methods: Introduction (1) direct binding to the insulin response sequence (IRS) in The forkhead transcription factors are winged helical proteins gene promoters and (2) ‘tethering’ to other transcription first known for their role in development. Each of the more factors (Ramaswamy et al, 2002). Examples of genes regu- than 90 members of the forkhead family contains a conserved lated by direct binding of the FOXO factors to DNA include DNA binding domain with a butterfly-shaped structure of those encoding apoptotic proteins such as Bim and Fas three a-helices and two characteristic large loops or ‘wings’ ligand, and examples of genes regulated by ‘tethering’ in- (Kaufmann and Kno¨chel, 1996). clude those encoding the cell cycle regulators such as cyclin Members of the forkhead family, as classified by phyloge- G2 and cyclin D1. Both wild-type FOXO and FOXO mutants netic analysis of the amino-acid sequence of the DNA binding that cannot bind DNA modulate the expression of cyclin D1 domain, include the forkhead box class O (FOXO) factors. and cyclin G2 (Ramaswamy et al, 2002). There are four FOXO factors: FOXO1 (FKHR, Forkhead in FHL2 (four and a half LIM 2) was first identified as a rhabdomyosarcoma) (Galili et al, 1993; Kaestner et al, 2000), protein differentially expressed in normal human myoblasts FOXO3a (FKHRL1, FKHR-like 1) (Anderson et al, 1998), and their malignant counterparts, rhabdomyosarcoma cells; thus, it is also named DRAL (Downregulated in *Corresponding author. Department of Pathology, University of South Rhabdomyosarcoma LIM Protein) (Genini et al, 1997). Florida, College of Medicine, 12901 Bruce B Downs Blvd, MDC 11, FHL2 is a LIM-only protein with five LIM domains whereby Tampa, FL 33612-4799, USA. Tel.: þ 1 813 974 0563; the LIM domain in the amino terminus consists only of the Fax: þ 1 813 974 5536; E-mail: [email protected] second half of the consensus motif. Although not required for Received: 6 September 2004; accepted: 7 January 2005; published normal heart development (Chu et al, 2000), FHL2 may online: 3 February 2005 control the hypertrophic response to stress in cardiomyocytes, &2005 European Molecular Biology Organization The EMBO Journal VOL 24 | NO 5 | 2005 1021 SIRT1-dependent inhibition of FOXO1 activity by FHL2 YYanget al where it is enriched (Kong et al, 2001, Purcell et al, 2004). human prostate cancer. b-Galactosidase (b-gal)-based screen- The present study shows that FHL2 interacts with FOXO1 ing identified several positive clones, and sequencing of both in vitro and in prostate cancer cells. Binding of FHL2 to cDNA identified one of the positive clones as full-length FOXO1 inhibits the transcriptional activity of FOXO1 by FHL2 fused in-frame to the VP16 activation domain. facilitating its deacetylation through Sirtuin-1 (SIRT1), the To determine whether FHL2 and FOXO1 interact in mam- mammalian homolog of the yeast Sir2 deacetylase. Our data malian cells, we ectopically expressed Myc-FHL2 and Flag- raise the interesting possibility that interaction of FHL2 with FOXO1 in DU145 cells, which are human prostate tumor cells. FOXO1 and SIRT1 may promote prostate tumorigenesis in Cell extracts were immunoprecipitated and immunoblotted response to increased stress during aging. with antibodies to the tags. In cells expressing both proteins, antibody to Myc co-precipitated FOXO1, and antibody to Flag Results co-precipitated Myc-FHL2 (Figure 1A). Co-precipitation did not occur in cells expressing Myc-FHL2 or Flag-FOXO1 alone, FHL2 interacts with the amino terminus of FOXO1 and thus is not the result of antibody crossreactivity. and inhibits the transcriptional activity of FOXO1 To substantiate the interaction data, the subcellular dis- The functions of the amino terminus of FOXO1, in contrast to tribution of FHL2 and FOXO1 in DU145 cells was determined that of the carboxyl-terminal activation domain and of the by immunofluorescence microscopy. As shown in Figure 1B, central DNA binding domain, are unclear. The amino termi- a significant amount of ectopic FOXO1 was nuclear (panel nus interacts with transcriptional coactivators (Puigserver B2). This finding is consistent with the presence of functional et al, 2003) and thus may contain an activation function. To PTEN, and thus of relatively low amounts of PKB activity, in better define its function and to identify additional binding DU145 cells (Ramaswamy et al, 1999). Some cytoplasmic partners, we used the amino terminus (amino acids 1–150) of staining was detected, presumably because the cells were in FOXO1 as bait in a two-hybrid screen of a cDNA library from medium containing 5% serum, which may activate PKB. AC− ++− Flag-FOXO1 FOXO1(1−150) −−++Myc-FHL2 IP: Anti-Flag Interaction with FOXO1 IB: Ant-Myc Input IP: Anti-Myc GST-FHL2 GST 1/2 LIM 1 LIM 2 LIM 3 LIM 4 + IB: Anti-Flag GST-LIM (3&1/2) GST 1/2 LIM 1 LIM 2 LIM 3 + IP: Anti-Flag GST-LIM (2&1/2) GST 1/2 LIM 1 LIM 2 + IB: M2 GST-LIM (1&1/2) GST 1/2 LIM 1 − IP: Anti-Myc IB: Anti-FHL2 GST-LIM (1/2) GST 1/2 − GST GST − BDB1. DAPI B2. FOXO1 B3. FHL2 B4. 2-Color B5: 3-Color 120 100 80 60 40 B6: 2-Color confocal 20 deconvolution Luciferase activity (RLU) image 0 Gal4-FOXO1(1−150) 0 0.05 0.05 0.05 0.05 0 (µg) FHL2 0 0 0.2 0.4 1 0.2 (µg) Figure 1 FHL2 interacts with FOXO1 and inhibits the transcriptional activity of the amino terminus of FOXO1.