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Oncogene (2002) 21, 1556 ± 1562 2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00 www.nature.com/onc
Phosphorylation of forkhead transcription factors by erythropoietin and stem cell factor prevents acetylation and their interaction with coactivator p300 in erythroid progenitor cells Dolores L Mahmud2, Maaza G-Amlak2, Dilip K Deb2, Leonidas C Platanias2, Shahab Uddin1 and Amittha Wickrema*,1 1
Section of Hematology/Oncology, University of Chicago, Chicago, Illinois, USA; 2The University of Illinois Medical Center, Chicago, Illinois, USA
The mammalian forkhead transcription factors, FOXO3a (FKHRL1), FOXO1a (FKHR) and FOXO4 (AFX) are negatively regulated by PKB/Akt kinase. In the present study we examined the engagement of forkhead family of transcription factors in erythropoietin (Epo)- and stem cell factor (SCF)-mediated signal transduction. Our data show that all three forkhead family members, FOXO3a, FOXO1a and FOXO4 are phosphorylated in human primary erythroid progenitors. Experiments performed to determine various upstream signaling pathways contributing to phosphorylation of forkhead family members show that only PI-3-kinase pathway is required for inactivation of FOXO3a. Our data also demonstrate that during Epo deprivation FOXO3a interacts with the transcriptional coactivator p300 and such interaction is disrupted by stimulation of cells with Epo. To determine the domains in FOXO3a, mediating its interaction with p300, we performed GST pull-down assays and found that the Nterminus region containing the ®rst 52 amino acids was sucient for binding p300. Finally, our data demonstrate that FOXO3a and FOXO1a are acetylated during growth factor deprivation and such acetylation is reversed by stimulation with Epo. Thus mammalian forkhead transcription factors are involved in Epo and SCF signaling in primary erythroid progenitors and may play a role in the induction of apoptotic and mitogenic signals. Oncogene (2002) 21, 1556 ± 1562. DOI: 10.1038/sj/ onc/1205230 Keywords: forkhead transcription factors; erythropoietin; stem cell factor; acetylation; p300 Introduction Erythropoietin (Epo), Stem Cell Factor (SCF) and Insulin-like Growth Factor-1 (IGF-1) mediate prolif-
*Correspondence: A Wickrema, Hematology/Oncology Section, MC 2115, University of Chicago, 5841 South Maryland Avenue, Chicago, Illinois, IL 60637, USA; E-mail:
[email protected] Received 13 July 2001; revised 27 November 2001; accepted 5 December 2001
eration, dierentiation and cell survival functions in erythroid progenitors (Krantz, 1991; Muta et al., 1994; Koury and Bondurant, 1990). The key signaling pathway responsible for maintaining the viability of erythroid cells is protein kinase B/Akt kinase (PKB/ Akt) which is activated by both Epo and SCF (Miura et al., 1991; Bao et al., 1999; Uddin et al., 2000). During engagement of Epo and SCF to their speci®c receptors, several major signaling cascades are activated although very little is known with respect to downstream elements responsible for controlling the cell survival function in these cells. In many cell types Akt phosphorylates multiple substrates that initiate the activation of downstream signaling molecules that provide the cell survival signals. These factors include Bcl2 family member, BAD (Datta et al., 1997), Caspase 9 (Cardone et al., 1998), GSK3 (Park et al., 1999) and ASK1 (Kim et al., 2001). Recent studies have identi®ed forkhead family of proteins as another target for phosphorylation by Akt kinase in several cell types including erythroid cells (Brunet et al., 1999; Rena et al., 1999; Nakae et al., 1999; Biggs et al., 1999; Medema et al., 2000; Nasrin et al., 2000; Kashii et al., 2000; Uddin et al., 2000). In contrast to many signaling events, in which protein phosphorylation ultimately results in gene transcription, in the case of forkhead family of proteins, phosphorylation inhibits their function and negatively regulates gene transcription. The mammalian forkhead (FH) family of proteins are similar in sequence to DAF-16 transcription factor in the nematode Caenorhabditis elegans. In C. elegans DAF-16 is phosphorylated by Akt kinase (Ogg et al., 1997; Paradis and Ruvkun, 1998). The three forkhead proteins FOXO3a, FOX1a and FOXO4 contain the forkhead DNA-binding domain and the conserved Akt phosphorylation sites. Recently, a new nomenclature for the forkhead transcription factors have been adopted and according to this uni®ed nomenclature these three members have been classi®ed as belonging to the `O' subfamily (Kaestner et al., 2001). In response to growth factors, FOXO3a, FOXO1a and FOXO4 are phosphorylated at several serine and one threonine site and negatively regulates the transcriptional activity (Brunet et al., 1999; Medema et al., 2000). It is unclear whether all three members of this family have distinct
Forkhead transcription factors in erythroid cells DL Mahmud et al
or redundant functions and whether they are expressed in the same cell type. Previous studies have shown that during growth factor withdrawal, FOXO3a activates the Fas ligand (FasL) promoter and induces apoptosis (Brunet et al., 1999). Further in mouse ®broblast and B cell lines it has been demonstrated that both FOXO4 and FOXO1a can arrest cells by transcribing the cell cycle gene p27 (Medema et al., 2000; Dijkers et al., 2000). A more recent study has implicated the transcriptional coactivator CBP in the regulation of transcription by DAF-16 and FOXO1a in HepG2 hepatoma cells although the mechanism of such eects remains unknown (Nasrin et al., 2000). Both CBP and its close relative p300 function to either activate or suppress the activity of many transcription factors including p53, p73, cJun, ATF-2, GATA1, SCL and EKLF, among others (Avantaggiati et al., 1997; Lee et al., 1996; Blobel et al., 1998; Blobel, 2000; Zeng et al., 2000; Huang et al., 1999; Hung et al., 1999). However, it is not known whether, in erythroid cells CPB or p300 interact with any of the forkhead transcription factors to regulate their activity. CBP and p300 are acetyl transferases and have been shown to modulate the activity of their targets by acetylation. The erythroid speci®c transcription factor GATA1 and the tumor suppressor p53 are acetylated by p300 and such acetylation upregulates the transcription by these factors (Gu and Roeder 1997; Boyes et al., 1998). It remains to be determined if FOXO3a, FOXO1a or FOXO4 are also acetylated by p300 or CBP, in a manner similar to GATA1 and p53 to modulate their activity. Recent studies by Kashii et al. (2000) and us (Uddin et al., 2000) demonstrated the involvement of FOXO3a in Epo signaling, but these studies did not address whether other FH family members are also engaged during Epo signaling. Furthermore, it was not determined whether FH proteins are speci®cally phosphorylated by Epo or whether SCF is also capable of inactivating FH family members by phosphorylation in erythroid cells. Our present study demonstrates that all three FH proteins are expressed in primary erythroid cells and are phosphorylated in an Epo and SCF dependent manner. We also demonstrate that during growth factor deprivation, FOXO3a associates with the p300 coactivator and is acetylated in intact cells. Finally, we have localized the interaction domain of FOXO3a with p300 and show that the interaction between p300 and FOXO3a is disrupted during stimulation of cells with Epo. Results We initially sought to determine whether all three members of the mammalian forkhead family of proteins are expressed and phosphorylated in response to Epo and SCF in primary erythroid progenitors. Primary cells were treated in the presence or absence of Epo or SCF, the cells were lysed and phosphorylation of FH family
members was determined by immunoblotting with phospho-speci®c anti-FH antibodies. Our results show that all three subfamily members FOXO3a, FOXO1a and FOXO4 are phosphorylated in response to Epo and SCF in dierentiating primary erythroid progenitor cells (Figure 1). All three members display similar kinetics of phosphorylation in response to Epo although the intensity of the phosphorylation of FOXO4 protein is clearly less than the other two members (Figure 1a). We then sought to determine which of the major signaling cascades contribute to the phosphorylation and inactivation of forkhead family of protein. Experiments were performed using speci®c inhibitors for Erk 1/2, p38, protein kinase A, protein kinase C, calmodulin kinase, and PI3-kinase (PD98059, SB203580, H-89, Bisindolylmaleimide 1 (Bis1), KN93 and LY 294002, respectively). As shown in Figure 2, the phosphorylation of FOXO3a was only blocked by pre-treatment of the cells with the PI3-kinase inhibitor LY294002 but not with the other inhibitors. Based on these ®ndings, it appears that from the dierent signaling cascades activated by the EpoR, only activation of the PI3-kinase pathway is required for downstream engagement of FOXO3a. In order to ascertain the mechanisms involved in the negative regulation of FOXO3a by Epo we performed experiments to identify proteins that may associate with forkhead family members, to mediate downstream signals. We found that during growth factor deprivation, FOXO3a associates with the transcriptional coactivator p300 in primary erythroid progenitors. Furthermore, our results showed that nuclear lysates prepared from cells stimulated with Epo after growth factor deprivation exhibit a greatly diminished level of association between FOXO3a and p300 (Figure 3a, lane 3). On the other hand, stimulation of cells with Epo allows the association between FOXO3a and protein 14.3.3 in the cytoplasm (Figure 3b) similar to what has been demonstrated in several other types of cells (Brunet et al., 1999). In order to identify the domains in FOXO3a, mediating its interaction with p300, we performed GST pull-down assays using a GST-fusion protein for wild type FOXO3a or a GSTfusion protein for the N-terminus region of FOXO3a. The N-terminus domain contains threonine phosphorylation site but lacks the DNA binding motif and the two serine phosphorylation sites (Figure 4a). Our results show that the region of interaction within FOXO3a is located in the N-terminal domain and such interaction is disrupted when the cells are stimulated with Epo following growth factor deprivation (Figure 4b). The control sample that was cultured with Epo showed only a slight interaction with p300 as expected. Therefore, it is likely that p300 protein itself undergoes a conformational change in response to Epo, preventing further association with FOXO3a. Since p300 protein is known to be an acetyltransferase we next sought to determine whether FOXO3a is acetylated during growth factor deprivation. Experiments were performed using nuclear lysates from HCD57 erythroid cells to immunoprecipitate FH
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Figure 1 Erythropoietin (Epo) and stem cell factor (SCF) induce phosphorylation of forkhead family members FOXO3a, FOXO1a and FOXO4 in primary erythroid progenitors. Primary erythroid progenitors that are at colony-forming unit-erythroid (CFU-E) stage of dierentiation (day 7 of culture) were deprived of growth factors for 4 h and subsequently incubated in the presence or absence of (a) Epo or (b) SCF as indicated. Cell lysates were analysed by SDS ± PAGE and immunoblotted with an antiphospho forkhead antibody that recognize the phosphorylated form of FOXO1a (Thr24), FOXO3a (Thr32) and FOXO4 (Thr28). The same blot was stripped and reprobed with an anti-FOXO3a antibody to control for protein loading
Figure 2 Determination of upstream signaling pathways contributing to phosphorylation of forkhead transcription factors. Epo responsive mouse erythroid cell line HCD57 cells were growth factor deprived for 16 h and subsequently incubated for 1 h with 10 mM concentration of each inhibitor as indicated. Cells were then stimulated with or without Epo as indicated and cell lysates were subjected to SDS ± PAGE and immunoblotted with an anti-phospho FOXO3a antibody. The same blot was stripped and reprobed with an anti-FOXO3a antibody to con®rm the presence of FOXO3a protein
transcription factors in the presence and absence of Epo. These immunocomplexes were then analysed to determine the acetylation of the proteins by probing with an anti-acetyllysine speci®c antibody. Our studies demonstrate that two forkhead family members, FOXO3a and FOXO1a are acetylated during growth factor deprivation but is deacetylated when cells are stimulated with Epo (Figure 5a). In addition, experiments were performed to determine whether FOXO1a is acetylated in vivo. Puri®ed FOXO1a-GST (FKHRGST) or GST proteins were incubated in reactions containing histone acetyl transferase (HAT) domain of p300, acetyl CoA and HAT buer and the products subsequently were analysed by SDS ± PAGE and probed with an anti-acetyllysine antibody (Figure 5b). Consistent with the results in HCD57 cells, we found that a 105 kDa size protein corresponding to the full Oncogene
Figure 3 In vivo association of forkhead family of protein, FOXO3a with transcriptional co-activator p300 and protein 14.3.3. (a) Primary erythroid progenitors were deprived of growth factors for the indicated times or stimulated with Epo for 0.5 h following growth factor deprivation. Nuclear lysates were immunoprecipitated with an anti-p300 antibody and immunoblotted with an anti-FOXO3a antibody. (b) Primary erythroid progenitors were stimulated with Epo after growth factor deprivation and total cell lysates were immunoprecipitated with anti-14.3.3 or rabbit IgG (RIgG) as indicated. Top panel, antiFOXO3a immunoblot. Bottom panel, the same blot was stripped and immunoblotted with an anti-14.3.3 antibody
length FOXO1a-GST fusion protein is acetylated (Figure 5b, lane 2) whereas no such acetylation is observed in the GST protein alone (Figure 5b, lane 1). Thus FH family members FOXO1a and FOXO3a are likely targets for acetylation by p300 coactivator in hematopoietic cells.
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Figure 4 Identi®cation of the domain of interaction between the forkhead family member FOXO3a and transcriptional coactivator p300. (a) Schematic diagram showing domains of FOXO3a protein and the two glutathione S-transferase (GST) fusion protein constructs used to identify the interaction domains of FOXO3a with p300. (b) Nuclear lysates from HCD57 cells were growth factor deprived for 16 and 24 h or stimulated with Epo following the 16 h starvation were collected. Lysates from unstarved cells were also collected as a control. GST pull-down assays were performed by incubating the nuclear lysates with either wild type GST-FOXO3a or N-terminal portion of the FOXO3a protein and the protein complexes were separated on SDS ± PAGE and immunoblotted with an anti-p300 antibody
Figure 5 Acetylation of forkhead transcription factors, FOXO3a and FOXO1a during growth factor deprivation. (a) HCD57 erythroid cells were deprived of growth factors for 16 h and either stimulated with Epo for 30 min or left untreated as indicated. Nuclear lysates were immunoprecipitated with an anti-FOXO3a antibody and immunoblot analysis performed with an antiacetyllysine antibody. (b) Puri®ed GST or FOXO1a-GST proteins were utilized in in vivo acetylation assays and the samples were separated by SDS ± PAGE and analysed by immunoblotting with an anti-acetyllysine antibody Oncogene
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Discussion Recent studies have shown that mammalian forkhead transcription factors are downstream targets of phosphorylation by kinases activated by various growth factors. It has been demonstrated that insulin/ insulin-like growth factor-1, epidermal growth factor and nerve growth factor phosphorylate one or multiple members of the forkhead transcription factors suggesting that these proteins play important roles in the induction of biological responses for various cytokines (Brunet et al., 1999; Medema et al., 2000). In hematopoietic cells, it has been demonstrated that FOXO3a is phosphorylated through the PI3-kinase/ Akt kinase pathway by Epo and thrombopoietin (Kashii et al., 2000; Uddin et al., 2000; Tanaka et al., 2001) although it is not known whether the other two members, FOXO1a and FOXO4 are also expressed and phosphorylated in these cells. On the other hand, it has been unclear whether SCF engages FH proteins in its signaling pathway. Our present study demonstrates that FOXO3a, FOXO1a and FOXO4 are expressed in primary erythroid progenitors and undergo phosphorylation in response to Epo and SCF stimulation. To our knowledge this is the ®rst report that shows that all three mammalian FH transcription factors are expressed and phosphorylated in normal primary hematopoietic cells. It is possible that FOXO1a, FOXO3a and FOXO4 have distinct and redundant functions in hematopoietic cells. Our present study shows that FOXO4 is a much better substrate for phosphorylation by SCF as compared to Epo suggesting the possibility that this FH-family member may be primarily mediating induction of mitogenic rather than an anti apoptotic response. In erythroid cells, it is well established that Epo acts primarily as a viability factor (Koury and Bondurant, 1990), whereas SCF acts as a mitogenic factor (Muta et al., 1994) and it is therefore likely that each of the three FH transcription factors regulates genes involved in either cell cycle and/or apoptosis. One recent study has implicated transcriptional activation of Fas ligand promoter by FOXO3a, whereas several other studies have shown cell cycle arrest and p27 activation by FOXO1a and FOXO4 in ®broblasts cells (Brunet et al., 1999; Medema et al., 2000; Dijkers et al., 2000). Although it is likely that many other genes are regulated by these transcription factors, our results are consistent with the functions of Epo and SCF in hematopoietic cells. Our previous studies showed that inhibition of PI3kinase/Akt kinase pathway by the PI3 kinase inhibitor LY294002 greatly reduced the level of FKHRL1 phosphorylation in normal primary erythroid progenitors although could not be completely abolished (Uddin et al., 2000). This observation suggested that other upstream kinases may also contribute to phosphorylation and inactivation of FH transcription factors. In our present study examination of the involvement of MAP kinase (Erk1/2), protein kinase A, protein kinase C, calmodulin kinase and p38 MAP kinases indicates that none of them is required for the
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phosphorylation of FOXO3a and that only activation of the PI3-kinase pathway is required. In most cells, binding of growth factors to their receptors activates many downstream signaling cascades by phosphorylation of signaling proteins. In the case of FH transcription factors such phosphorylation inactivates its transcriptional function by sequestration of the protein within the cytoplasm by binding to protein 14.3.3 (Brunet et al., 1999). Our present studies con®rm this ®nding in primary erythroid progenitors as we are able to observe an association of FOXO3a with 14.3.3 during Epo stimulation. Furthermore, such interaction was disrupted when growth factors were withdrawn. A recent study had shown that C. elegans protein, DAF-16 recruits the transcriptional co-activator p300/CBP to the insulin-like growth factor binding protein 1 promoter, thereby regulating its activity (Nasrin et al., 2000). Based on these observations, we examined whether FH protein FOXO3a associates with p300 during Epo deprivation of erythroid cells. Indeed, we observed such association between FOXO3a and p300 during Epo and SCF deprivation which was reversible by the treatment of cells with Epo. In order to de®ne the domain of interaction within FOXO3a we performed GST pulldown assays using nuclear lysates from HCD57 erythroid cells. Our results show that the interaction between FOXO3a and p300 occurs only in nuclear lysates that were obtained during Epo starvation of cells, and is not observed in lysates from cells that were stimulated with Epo. Further, our data strongly suggest that Epo is responsible for bringing about a conformational change in p300 itself, that allows the disruption of the association between the two proteins in the nucleus. Therefore, it is likely that a conformational change, possibly phosphorylation of p300, contributes to the dissociation of FOXO3a from p300 in addition to phosphorylation of FOXO3a itself by Epo. In a recent study (Guo et al., 2001), it was shown that PKB/Akt kinase phosphorylates p300 at ser1834 site in response to insulin suggesting that other growth factors such as Epo and SCF may also phosphorylate p300 to modulate the interaction with transcription factors such as FH proteins. In order to determine the domain of interaction between p300 and FOXO3a we performed GST pull-down assays using the most proximate N-terminal region (1 ± 52 aa) of the FOXO3a in our binding assays. Our results show that the N-terminal region containing the phosphorylation site thr32 binds p300 indicating that neither the winged helix domain nor the two phospho serine sites (ser253 and ser315) are required for such interaction. Since p300 is known to have acetyltransferase enzyme activity and has been shown to acetylate many transcription factors we sought to determine whether FH family members are acetylated during growth factor deprivation in erythroid cells. Using HCD57 cells we demonstrate that both FOXO1a and FOXO3a are acetylated during growth factor deprivation which is greatly diminished by stimulation of cells with Epo. These data are consistent with our observations where
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association between FOXO3a and p300 occurred during Epo starvation of cells. Therefore, it is likely that the physical interaction between p300 and FH family members is required for p300-mediated acetylation of FOXO3a and FOXO1a. Although further work is required to de®ne the speci®c genes that are regulated by each of the three FH transcription factors, our data strongly suggest that these proteins play an important function in primary hematopoietic cells and provide evidence for a direct link between growth factor receptor tyrosines and FH transcription proteins required for signal transmission. Furthermore, our study indicates FH transcription factors may be a target of acetylation by p300, and the functional consequence of such acetylation remains to be de®ned in future studies.
HCD57 erythroid cell line was cultured in IMDM containing 25% fetal calf serum, 100 units/ml penicillin, 100 mg/ml streptomycin and 2 units/ml of Epo at 378C in a 5% Co2 environment. During growth factor deprivation primary erythroid progenitors were maintained in a serumfree media for 4 h whereas HCD57 cells were maintained in a serum containing media without Epo for 16 ± 24 h. In experiments involving the treatment with various inhibitors cells were pre-incubated for 30 ± 60 min with the inhibitor prior to stimulation with Epo.
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Isolation of nuclei Nuclear and cytoplasmic isolations were performed using the nuclei EZ prep isolation buer from Sigma-Aldrich Chemicals. Immunoblotting Immunoblotting and immunoprecipitations were performed as previously described (Wickrema et al., 1999).
Materials and methods
GST binding assays
Reagents and plasmid constructs 24
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Antibody against phospho FOXO1a (Thr )/FOXO3a (Thr ) (FOXO1a/3a) that also reacts with phospho FOXO4 (Thr28) was a gift from Dr Jing Lee at Cell Signaling Technologies (Beverly, MA, USA). Antibodies against FOXO1a (FKHR) and FOXO3a (FKHRL1) were purchased from Cell Signaling Technologies or generated by the Research Resources Center at University of Illinois, respectively. Antibodies against p300 and 14.3.3 were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Antibody against acetyllysine, puri®ed FOXO1a-GST (FKHR-GST) protein that was approximately 10% full length, p300 HAT domain and HAT buer were purchased from Upstate Biotechnology Inc. (Lake Placid, NY, USA). FOXO3a-GST (FKHRL1-GST) plasmid constructs were provided by Dr Michael Greenberg (Harvard University, USA). LY294002, PD98058, H-89, KN93, Bis1 and SB203580 were purchased from Calbiochem (LaJolla, CA, USA). Tris-Glycine (4 ± 20% gels were from Invitrogen (Carlsbad, CA, USA). Isolation and culture of erythroid cells Primary human erythroid progenitors were derived by in vivo culture of CD34+ cells isolated from peripheral blood. Growth factor mobilized peripheral blood collected from normal donors was purchased from AllCells, LLC (Berkeley, CA, USA). These blood samples were separated over ®collhypaque (1.077 g/ml) to obtain mononuclear cells. CD34 positive cells were isolated from the mononuclear cells by using the antibody-coated paramagnetic microbeads in the CliniMACS2 cell isolation devise (Miltenyi Biotec, Inc., Auburn, CA, USA). The selected cells were greater than 95% positive for CD34 as determined by ¯ow cytometry and were cultured for 7 ± 8 days to obtain highly puri®ed erythroid progenitors that were at the colony-forming unit-erythroid (CFU-E) stage of dierentiation. The cell culture medium contained 15% fetal calf serum, 15% human AB serum, Iscove's modi®ed Dulbecco's medium (IMDM), 100 units/ml penicillin, 100 mg/ml streptomycin, 10 ng/ml interleukin-3 (IL-3), 2 units/ml Epo, 50 ng/ml stem cell factor (SCF) and 50 ng/ml Insulin-like growth factor-1 (IGF-1). Greater than 90% of these cells were positive for CD71 and glycophorin A as determined by ¯ow cytometry and produced erythroid colonies when plated in methylcellulose.
FOXO3a-GST (WT) and FOXO3a-GST (1 ± 52 aa) fusion proteins were generated in E. coli. Nuclear lysates collected from cells with or without treatment with Epo were incubated with each of the FOXO3a-GST fusion proteins which were previously prepared by binding with sepharose beads for 2 h. The bound proteins were resolved by SDS ± PAGE electrophoresis and transferred to Immobilon-P2 membranes. The blots were then probed with an anti-p300 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) to detect the p300 protein. In vitro acetyltransferase assay In vivo acetyl transferase reactions contained either 6 mg of puri®ed FOXO1a-GST or GST protein, 8 units of p300, HAT domain, 200 mM acetyl CoA and HAT buer (50 mM Tris-HCl, pH 8.0, 10% glycerol, 0.1 mM EDTA, 1 mM dithiothreitol) in a total volume of 25 ml. The reactions were incubated at 308C for 1 h and then terminated by adding the sample loading buer and subsequently analysed by SDS ± PAGE and probed with an anti-acetyllysine antibody.
Abbreviations FH, forkhead transcription factor; CBP, CREB-binding protein; HAT, histone acetyl transferase; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; CFU-E, colony forming unit-erythroid; GSK; glucocorticoid-inducible kinase; ASK1, apoptosis signal-regulating kinase.
Acknowledgments We thank Michael Greenberg for FKHRL1 plasmid constructs, Jing Lee for phospho speci®c FKHRL1/FkHR antibodies and Stephen Sawyer for HCD57 cell line. We also wish to thank Toni Clark for her technical assistance. This work was supported in part by an American Cancer Society Grants to A Wickrema and S Uddin and the Wendy Will Case Cancer Fund to A Wickrema. Oncogene
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