Insulin-like Growth Factor-1-induced Phosphorylation of the Forkhead Family Transcription Factor FKHRL1 Is Mediated by Akt Kinase in PC12 Cells*

The Forkhead family transcription factor FKHRL1, a mammalian homolog of DAF16 in the nematodeCaenorhabditis elegans, is an inducer of apoptosis in its unphosphorylated form and was recently reported as a substrate of Akt kinases. Insulin-like growth factor (IGF-1) is a potent stimulant of Akt kinase, leading to inhibition of the apoptotic pathway. In this study, we characterized the phosphorylation of FKHRL1 induced by IGF-1 in PC12 cells and various neuronal cell types and examined the potential role of Akt in this regard. IGF-1 rapidly induced the phosphorylation of Akt and FKHRL1 in PC12 cells. The phosphorylation of Akt and FKHRL1 induced by 10 nm IGF-1 was inhibited by the phosphatidylinositide 3-kinase (PI3K) inhibitors wortmannin (0.25–2 μm) and LY294002 (12.5–100 μm), but not by the MEK inhibitor PD98059 (50 μm) or the p70 S6 kinase pathway inhibitor rapamycin (50 nm), suggesting that the phosphorylation of FKHRL1 induced by IGF-1 is mediated by the PI3K pathway. As observed for IGF-1, an in vitro kinase assay with purified active Akt kinase demonstrated that the kinase is capable of directly phosphorylating FKHRL1 at Thr32 and Ser253, leading to inhibition of its pro-apoptotic properties. Moreover, transient expression of constitutively active Akt (MS-Akt, where MS is a myristylation signal) increased the phosphorylation of FKHRL1, whereas the expression of kinase-dead Akt (M179A Akt) attenuated the phosphorylation of FKHRL1 induced by 10 nm IGF-1 in PC12 cells. Interestingly, FKHRL1 co-immunoprecipitated with Akt in PC12 cells, indicating that these two proteins can associate in these cells. As IGF-1 also induced the phosphorylation of FKHRL1 in primary cortical and cerebellar neuronal cultures, these data, taken together, demonstrate that IGF-1, acting via the PI3K/Akt kinase pathway, can regulate the phosphorylation of FKHRL1, leading to inhibition of this apoptotic transcription factor in neuronal cells.

Insulin-like growth factor-1 (IGF-1) 1 is a polypeptide tropic factor that has important roles in the survival and differentiation of both neuronal and non-neuronal cells (1). The biological actions of IGF-1 are mediated by a heterotetrameric tyrosine kinase receptor, the IGF-1 receptor, which is similar to the insulin receptor both in structure and functions (1,2). Binding of IGF-1 to its receptor induces receptor autophosphorylation and the activation of intrinsic tyrosine kinase, which phosphorylates a host of intracellular substrates, including insulin receptor substrate-1 and Shc (2)(3)(4), leading to activation of the mitogen-activated protein kinase (MAPK; also called extracellular signal-regulated kinase (ERK)) and the phosphatidylinositide 3-kinase (PI3K)/Akt pathways (1)(2)(3)(4)(5)(6).
Stimulation of cells with survival factors also causes the nuclear translocation of Akt in target cells (26). However, the nuclear targets of Akt have yet to be fully established. Among them, the Forkhead family of transcription factors is generating great interest. Three members of the mammalian Forkhead family of transcription factors termed FKHRL1, FKHR, and AFX have been isolated thus far (27)(28)(29)(30). This subfamily of transcription factors demonstrates sequence homologies to DAF16 expressed in Caenorhabditis elegans (21-25, 31, 32). DAF16 is a transcription factor and a candidate target of Akt in the insulin/IGF-1-induced Akt cascade that stimulates metabolism and regulates the longevity signal of this nematode (31,32).
Three putative Akt-sensitive phosphorylation sites are present in DAF16 and its human counterparts, including FKHRL1 (21)(22)(23)(24)(25)(31)(32)(33). It has recently been shown that Akt can phosphorylate Thr 32 , Ser 253 , and Ser 315 of FKHRL1 in the Chinese hamster lung fibroblast cell line CCL39 (21). The phosphorylation of Thr 32 and Ser 253 by Akt inhibits the stimulatory effect of FKHRL1 on the transcription of genes that encode deathactivating proteins such as the Fas ligand, leading to the survival of target cells. However, very little information is currently available on the effect of IGF-1 and Akt on the phosphorylation of FKHRL1 in neuronal cells. Accordingly, the major aim of this study was to investigate if IGF-1, acting via the PI3K/Akt kinase pathway, was able to induce the phosphorylation of FKHRL1 in neuronal cells, possibly leading to enhanced cell survival. Rat pheochromocytoma cells (PC12) were used in addition to primary rat cortical and cerebellar neuronal cultures. IGF-1 was found to be a potent stimulant of FKHRL1 phosphorylation in these cells.

EXPERIMENTAL PROCEDURES
Materials-Human recombinant IGF-1 was obtained as a gift from Genentech Inc. (San Francisco, CA). LY294002, PD98059, rapamycin, and GO6983 were purchased from Calbiochem (Bad Soden, Germany), and wortmannin, leupeptin, aprotinin, sodium vanadate, and phorbol 12-myristate 13-acetate (PMA) were from Sigma. U0126 was purchased from Promega (Madison, WI). Anti-FKHRL1(Ser 253 ), anti-FKHRL1, and anti-Akt antibodies were obtained from Upstate Biotechnology Inc. (Lake placid, NY), and anti-FKHRL1(Thr 32 ) antibody was a gift from this company. Anti-FKHRL1 antibody detects FKHRL1 or closely related proteins, whereas anti-FKHRL1(Thr 32 ) and anti-FKHRL1(Ser 253 ) antibodies recognize phosphorylated FKHRL1 Thr 32 and phosphorylated FKHRL1 Ser 253 , respectively. The full selectivity of these antibodies to distinguish between the various Forkhead family transcription factors remains to be established. Anti-phospho-Akt, anti-phospho-ERK, anti-phospho-GSK3␣/␤ antibodies and GSK3␣ fusion proteins were from New England Biolabs Inc. (Beverly, MA) and anti-GSK3␣/␤ is also a gift from Dr. A. Nelsbach in the same company. Anti-ERK and anti-hemagglutinin A (HA) antibodies and all secondary antibodies conjugated with horseradish peroxidase were from Santa Cruz Biotechnology (Santa Cruz, CA). Cell culture reagents and LipofectAMINE TM 2000 were purchased from Life Technologies, Inc., and all other reagents were from Sigma or Fisher. The CMV6, CMV6-PH-Akt-HA, and CMV6-M179A-Akt-HA plasmids were kindly provided by Drs. Sandeep S. R. Datta and M. E. Greenberg (Harvard Medical School, Boston, MA), and HA-PKB-CAAX (where PKB is protein kinase B and A is aliphatic amino acid) was given to us by Dr. B. M. Burgering (Utrecht University, Utrecht, The Netherlands) (34). The ␤-galactosidase staining kit containing the CMV-LacZ control vector was purchased from Invitrogen (Carlsbad, CA).
Cell Culture-PC12 and NIH 3T3 cells were kindly provided by Dr. Gordon Guroff (NICHD, National Institutes of Health, Bethesda, MD) and cultured as described before (35,36). In brief, PC12 cells, human kidney 293 cells (kindly provided by Dr. Y. Dumont, Douglas Hospital Research Center), and NIH 3T3 cells were maintained in 75-cm 2 flasks in high glucose Dulbecco's modified Eagle's medium supplemented with 5% (v/v) fetal bovine serum, 5% horse serum, 100 g/ml streptomycin, and 100 units/ml penicillin (for NIH 3T3 and 293 cells with 5% fetal bovine serum). Cells were incubated at 37°C in a 5% CO 2 humidified atmosphere. The stock culture was routinely subcultured at a 1:5 ratio at 1-week intervals.
Transient Transfection of PC12 Cells with Constitutively Active Akt and Kinase-dead Akt-Transient transfections of PC12 cells with Akt were performed as suggested by Life Technologies, Inc. with Lipo-fectAMINE 2000. Briefly, PC12 cells were plated in 24-well plates at 2.5 ϫ 10 5 cells/well in 0.5 ml of Dulbecco's modified Eagle's medium containing 5% fetal bovine serum and 5% horse serum without antibiotics. On the following day, 0.8 g of DNA in 50 l of Opti-MEM ® reduced serum medium was mixed with 2 l of LipofectAMINE 2000 prediluted in 50 l of the same medium for each well. After incubation at room temperature for 20 min to allow DNA-LipofectAMINE 2000 complexes to form, 100 l of mixture was added to each well. Then, the plate was shaken gently, and cells were incubated at 37°C in a 5% CO 2 incubator for 24 -48 h before the assay was performed. Transfection efficiency was determined by cotransfection with a CMV-LacZ control vector at a 1:10 control vector/DNA ratio, followed by in situ staining with the ␤-galactosidase staining kit as suggested by Invitrogen. The expression of Akt in transfected cells was confirmed by Western blotting using anti-HA antibody.
Cortical and Cerebellar Neuronal Cultures-Cortical and cerebellar cultured neurons were prepared from fetuses (embryonic day 19) obtained from pregnant Harlan Sprague-Dawley rats (Charles River Breeding Laboratories, St. Constant, Quebec, Canada) and cultured as described previously (37) with minor modifications. Animal care was according to protocols and guidelines approved by the McGill University Animal Care Committee and the Canadian Council for Animal Care. Neurons were plated at density of 5-8 ϫ 10 5 cells/well in 12-well plates (coated with 10 g/ml poly-D-lysine) under serum-free conditions in neurobasal medium supplemented with B27 (Life Technologies, Inc.). On the third day of plating, the medium was replaced with neurobasal supplemented with N2 (Life Technologies, Inc.), and the experimental treatment was performed on the seventh day of plating.
Treatments-Before each experiment, cells were detached using 5 mM EDTA in Hanks' balanced buffer and seeded in 12-or 6-well plates (coated with 10 g/ml poly-D-lysine) at a density of 4 -8 ϫ10 5 cells/well in 2% serum medium for 24 h. The culture medium was replaced with Dulbecco's modified Eagle's medium (for primary cultures, the medium was replaced with neurobasal medium) 2 h before the desired reagents were added. To study the effect of IGF-1 on the phosphorylation of FKHRL1 and Akt, cells were treated with 10 nM (100 nM for primary cultures) IGF-1 for 10 min. Alternatively, cells were pretreated with wortmannin (0.5 M, 20 min), LY294002 (50 M, 20 min), rapamycin (50 nM, 20 min), GO6983 (0.5 M, 20 min), and PD98059 (50 M, 40 min), followed by stimulation with 10 nM IGF-1. For the experiments with protein kinase C, 400 nM PMA was added to cells 2.5 min prior to IGF-1 stimulation. To evaluate the effect of peroxovanadate, 50 M peroxovanadate for 10 min was used. Peroxovanadate was prepared fresh by incubating 12 mM sodium vanadate and 12 mM H 2 O 2 in 40 mM Hepes (pH 7.4) for 20 min at 25°C prior to use as described (38).
Western Blotting-Western blotting was performed as described earlier with some modifications (35,36). Briefly, treated cells from different experimental conditions were rinsed twice with ice-cold Hanks' balanced buffer and lysed in either sample buffer (62.5 mM Tris-HCl (pH 6.8), 2% (w/v) SDS, 1% glycerol, 50 mM dithiothreitol, and 0.1% (w/v) bromphenol blue) or RIPA buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1% Igepal CA-630, 0.1% SDS, 50 mM NaF, 1 mM NaVO 3, 5 mM phenylmethylsulfonyl fluoride, 10 g/ml leupeptin, and 50 g/ml aprotinin). Samples with equal amounts of protein were then separated by 4 -20% polyacrylamide gel electrophoresis, and the resolved proteins were electrotransferred to Hybond-C nitrocellulose. Membranes were incubated with 5% nonfat milk in TBST (10 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.2% Tween 20) for 1 h at room temperature and incubated with the appropriate primary antibody at 4°C overnight. Membranes were then washed twice with TBST and probed with the corresponding secondary antibodies (anti-rat/mouse/ rabbit/goat) conjugated with horseradish peroxidase at room temperature for 1 h. Membranes were finally washed several times with TBST to remove unbound secondary antibodies and visualized using an ECL detection kit (Amersham Pharmacia Biotech, Toronto, Ontario, Canada). A part of the SDS gel was stained with Coomassie Blue to ensure the use of equal amounts of protein.
The respective phosphorylation of Akt, MAPK, and FKHRL1 was determined by Western blotting using anti-phospho-Akt, anti-phospho-ERK, or a mixture of anti-phospho-FKHRL1(Thr 32 ) and anti-phospho-FKHRL1(Ser 253 ) antibodies, respectively. To establish the phosphorylation of FKHRL1 at Thr 32 or Ser 253 , anti-phospho-FKHRL1(Thr 32 ) or anti-phospho-FKHRL1(Ser 253 ) antibody used, respectively. Blots were stripped and reprobed with anti-Akt or anti-ERK antibodies to ensure that equal amounts of Akt were present. In the case of FKHRL1, blots were stripped and reprobed with anti-␤-actin antibody as a control. In some experiments, two parallel running gels loaded with identical samples were used. One of them was used to evaluate the phosphorylation of FKHRL1, whereas the other was used to determine FKHRL1 levels in cell extracts. The effect of IGF-1 was determined by comparing the phosphorylation of FKHRL1 and the FKHRL1 level in cell extracts determined as described above. Quantification of the blots was performed using the MCID image analysis system (Imaging Research Inc., St. Catharines, Ontario). The levels of phosphorylation of FKHRL1 and Akt were normalized to the amount of ␤-actin or Akt present in each band, and IGF-1-stimulated FKHRL1/Akt phosphorylation was calculated as the level of FKHRL1/Akt phosphorylation seen in IGF-1-stimulated cells versus untreated cells.
Determination of the Phosphorylation of FKHRL1 by Active Akt in Vitro-PC12 cells pretreated with 0.5 M wortmannin were rinsed with cold phosphate-buffered saline. Cells were lysed in pre-cooled RIPA buffer and incubated on ice for 20 min. Cell lysates were then pelleted at 13,000 ϫ g for 10 min, and the concentration of protein in each sample (supernatant) was determined using the Bio-Rad dye-binding method with bovine serum as a standard. The supernatant (with an equal amount of protein) was incubated overnight at 4°C with anti-FKHRL1 antibody. Formed immunocomplexes were isolated by protein A/G PLUS-agarose (Santa Cruz Biotechnology). The immunoprecipitates were then washed four times with RIPA buffer and once with kinase buffer (25 mM Tris-HCl (pH 7.5), 5 mM ␤-glycerophosphate, 2 mM dithiothreitol, 0.1 mM Na3VO 4 , and 10 mM MgCl 2 ). Next, an in vitro kinase reaction was carried out in 40 l of kinase buffer containing precipitated FKHRL1 with or without 0.2 g of purified active Akt␣ and 200 M ATP. In the positive control tubes, precipitated FKHRL1 was replaced by 0.2 g of GSK3␣ fusion protein as substrate. Following a 30-min incubation at 33°C, the reaction was stopped by the addition of 10 l of 5ϫ reduced SDS sample buffer. Phosphorylation of FKHRL1 at Thr 32 and Ser 253 was determined by Western blotting using anti-phospho-FKHRL1(Thr 32 ) and anti-FKHRL1(Ser 253 ) antibodies, respectively. Akt activity was determined by Western blotting by measuring the phosphorylation of the GSK3␣ fusion protein with anti-phospho-GSK3␣/␤ antibody.
Detection of the Association of Akt and FKHRL1 in PC12 Cells by Immunoprecipitation-PC12 cells treated with or without 10 nM IGF-1 for 10 min were lysed in pre-cooled RIPA buffer, and Akt was isolated by immunoprecipitation using anti-Akt antibody as described above. Precipitated Akt was separated by 4 -20% SDS gel electrophoresis, and then the association with FKHRL1 was determined by Western blotting. Blots were stripped and reprobed with anti-Akt antibody to ensure the presence of equal amounts of Akt proteins. Quantification of the blots was performed as described above, and the amount of FKHRL1 associated with Akt was normalized to total Akt present in each lane.
Statistical Analysis-Data are expressed as means Ϯ S.E. A one-way analysis of variance with the Newman-Keul test was used to establish statistical significance set at p Ͻ 0.05.

IGF-1 Rapidly Induces the Phosphorylation of Akt and FKHRL1 in PC12
Cells-PC12 cells were treated with 10 nM IGF-1 for different times (0 -40 min), and the phosphorylation of FKHRL1, Akt, and MAPK/ERK kinase was evaluated. As shown in Fig. 1 (A and C), IGF-1 rapidly induced the phosphorylation of FKHRL1 and Akt in PC12 cells. The phosphorylation of both FKHRL1 and Akt was evident at 5 min and peaked at about the same time to remain stable for at least 40 min. IGF-1 also induced the phosphorylation of MAPK/ERK kinase. However, the activation of MAPK was transient, peaking at 5 min to decrease thereafter. Additionally, Fig. 1 (B and D) demonstrates that the effect of IGF-1 on the phosphorylation of FKHRL1 was concentration-dependent, with a significant effect observed at 0.11 nM, reaching a maximal level at ϳ1 nM IGF-1.
Phosphorylation of FKHRL1 by IGF-1 Is Mediated by a PI3K/Akt Kinase Pathway-Having established that IGF-1 can induce the phosphorylation of FKHRL1, we studied the signaling pathways mediating the action of IGF-1. Fig. 2A (lane 5 versus lane 1) demonstrates that 10 nM IGF-1 caused a 3-6-fold increase in the phosphorylation of FKHRL1 and Akt. Pretreatment of the cells with the PI3K inhibitor wortmannin (0.5 M) blocked IGF-1-induced activation of Akt and FKHRL1 (Fig. 2A, lane 6 versus lane 5). In contrast, the MEK inhibitor PD98059 (50 M) (Fig. 2A, lane 7 versus lane 5) and the p70 S6 kinase pathway inhibitor rapamycin (50 nM) (lane 8 versus lane 5) failed to significantly alter IGF-1-induced FKHRL1 and Akt phosphorylation. Similar results were obtained in NIH 3T3 cells (Fig. 2B). Additional studies revealed that the inhibitory effect of the PI3K inhibitors wortmannin and LY294002 on IGF-1-induced Akt and FKHRL1 phosphorylation were concentrationdependent (Fig. 3). These data suggest that PI3K/Akt kinase is upstream of FKHRL1 in IGF-1 receptor-mediated phosphorylation.
To investigate the phosphorylation of FKHRL1 in greater detail, the effect of IGF-1 on the phosphorylation of Thr 32 and Ser 253 was studied. Fig. 4 shows that in PC12 cells, the phosphorylation of FKHRL1 at these two sites was induced by 10 nM Akt Can Directly Phosphorylate FKHRL1-Our data indicated that the phosphorylation of FKHRL1 induced by IGF-1 was mediated by the PI3K/Akt kinase pathway. However, it is not known if Akt can directly phosphorylate FKHRL1 in PC12 cells. Therefore, an in vitro kinase assay with a purified recombinant active form of Akt was performed. Fig. 5 (upper and lower panels, lane 5 versus lane 6) shows that incubation of purified GSK3␣ fusion protein (used as a positive control) with active Akt increased the phosphorylation of GSK3␣, whereas a control reaction without active Akt had no effect. These results demonstrated the functionality of the Akt assay. In parallel assays with immunoprecipitated FKHRL1 from PC12 cells, significant increases in the phosphorylation of its Thr 32 and Ser 253 residues were observed in the reaction with purified enzyme, whereas the control reaction had no effect on FKHRL1 (Fig. 5, upper and lower panels, lane 1 versus lane 2). Similar results were obtained with immunoprecipitated FKHRL1 from HEK 293 cells (Fig. 5, upper and lower panels, lane 3 versus  lane 4). These results demonstrate that Akt is able to directly phosphorylate FKHRL1 at Thr 32 and Ser 253 .

Expression of Constitutively Active Akt Increases the Phosphorylation of FKHRL1, whereas Expression of Kinase-dead Akt Attenuates IGF-1-induced Effects on This Transcription
Factor-PC12 and HEK 293 cells were transfected with the CMV6 control plasmid, constitutively active Akt (MS-Akt), and kinase-dead Akt (M179A Akt or HA-Akt-CAAX). Transfection efficiency was determined by the cotransfection of a CMV-LacZ plasmid, followed by ␤-galactosidase staining. Following an overnight serum starvation, transfected cells were treated with 10 nM IGF-1, and the phosphorylation of FKHRL1 was determined. Fig. 6A shows that both constitutively active Akt (lane 2) and kinase-dead Akt (lanes 3 and 5) were successfully expressed in PC12 cells. The expression of the constitutively active form of Akt increased the phosphorylation of FKHRL1 (Fig. 6A, lane 2 versus lane 1), whereas the expression of kinase-dead Akt attenuated the IGF-1-induced phosphorylation of FKHRL1 (lane 5 versus lane 6). Similar results were obtained in transfected HEK 293 cells (data not shown). To investigate the effect of kinase-dead Akt on the IGF-1-induced phosphorylation of FKHRL1 in greater detail, PC12 cells were transfected with different amounts of DNA encoding HA-PKB-CAAX (another kinase-dead Akt) (34) or control DNA in quadruplicate and then treated with IGF-1. The phosphorylation of FKHRL1 was measured and compared with FKHRL1 in cell extracts determined by a parallel running gel as described under "Experimental Procedures." Fig. 6B shows that kinasedead Akt dose-dependently attenuated the IGF-1-induced phosphorylation of FKHRL1 in PC12 cells. Thus, the active form of Akt is by itself able to induce the phosphorylation of FKHRL1, and the IGF-1-induced phosphorylation of this tran- scription factor is mediated by Akt kinase in PC12 cells.
Akt Can Directly Associate with FKHRL1-Since some kinases have been shown to associate with their substrates (39), we tested if this was indeed in the case for Akt and FKHRL1 in PC12 cells. Cells were treated with 10 nM IGF-1, and the Akt kinase was separated by immunoprecipitation using anti-Akt antibody. Precipitated Akt and its associated proteins were separated by gel electrophoresis, and the resolved proteins were electrotransferred to Hybond-C nitrocellulose. The association of FKHRL1 with Akt was subsequently detected by Western blotting. As shown in Fig. 7, FKHRL1 and Akt coprecipitated in PC12 cells. Moreover, stimulation with 10 nM IGF-1 slightly increased the association of FKHRL1 with Akt (Fig. 7).

Peroxovanadate Activates Akt and Increases the Phosphorylation of FKHRL1, whereas PMA Decreases IGF-1-induced Akt Activation and Attenuates IGF-1-induced FKHRL1 Phosphorylation-
We have previously shown that cotreatment of PC12 cells with the protein kinase C activator PMA attenuated IGF-1-induced activation of Akt, whereas pretreatment with the protein kinase C inhibitor GO6983 blocked the inhibitory effect of PMA (36). To investigate the effect of Akt kinase activators and inhibitors on the phosphorylation of FKHRL1 induced by IGF-1, PC12 cells were treated with the PI3K activator peroxovanadate (50 M) in addition to PMA (400 nM), IGF-1 (10 nM), and GO6983 (0.5 M). As expected, peroxovanadate stimulated Akt and induced the phosphorylation of FKHRL1 (Fig. 8A). Moreover, cotreatment of PC12 cells with PMA and IGF-1 attenuated the effect of IGF-1 on the phosphorylation of Akt and FKHRL1. The inhibitory effect of PMA was reversed by the addition of 0.5 M GO6983 (Fig. 8B). These results further support the notion that FKHRL1 is a downstream substrate of Akt in the IGF-1 receptor signaling pathway.
IGF-1 Induces the Phosphorylation of Akt and FKHRL1 in Primary Neuronal Cultures-It has been reported that the intracellular signaling pathways of various growth factors may be cell type-specific (19,20). In addition to PC12 cells, we have shown that IGF-1 can induce the phosphorylation of FKHRL1 in NIH 3T3 (Fig. 2B) and HEK 293 cells (data not shown). Moreover, IGF-1 (100 nM) induced the phosphorylation of Akt and FKHRL1 in primary cortical and cerebellar neuronal cultures (Fig. 9), demonstrating the relevance of data obtained in PC12 cells. DISCUSSION This study reveals that IGF-1 can stimulate the phosphorylation of FKHRL1 in PC12 and primary neuronal cultured cells. It also provides evidence indicating that Akt mediates this action of IGF-1. Moreover, Akt kinase can directly associate with and phosphorylate FKHRL1 in neuronal cells.
Akt is a serine/threonine kinase and a downstream target of PI3K playing an important role in the survival of various cell types (7)(8)(9). In agreement with this hypothesis, our previous work demonstrated that Akt is a downstream target of PI3K in IGF-1 receptor signaling in PC12 cells (36). However, little is currently known about the downstream signaling events mediated by Akt, especially its nuclear targets, in PC12 cells. The present results reveal that FKHRL1 is a most likely nuclear target of Akt in this cell line as well as in primary cortical and cerebellar neuronal cultures. IGF-1 rapidly induced the phos- phorylation of FKHRL1 in PC12 cells in a concentration-and time-dependent manner. The IGF-1-induced phosphorylation of FKHRL1 was inhibited by two PI3K inhibitors, namely wortmannin (40) and LY294002 (41), but not by rapamycin, a p70 S6 kinase pathway inhibitor (42). Interestingly, the basal phosphorylation of FKHRL1 is slightly increased in the presence of rapamycin. A similar phenomenon was also observed for the basal phosphorylation of FKHR in 32 P-labeled hepatocytes transfected with FKHR (24). The mechanism involved is not clear at present. As unphosphorylated FKHRL1 plays a role in the cell cycle and oncogenesis, its phosphorylation was suggested to counteract the transcriptional activity of FKHRL1. Hence, the increased phosphorylation of FKHRL1 in the presence of rapamycin is consistent with the antiproliferative effect of this drug (43). In any case, our results are consistent with a recent report showing that DAF16, a nematode homolog of FKHRL1, is a downstream target of Akt in this species (31,32), suggesting that the phosphorylation of FKHRL1 is mediated, at least in part, by the PI3K/Akt kinase pathway.
Three additional series of evidence further support this hypothesis. First, the transient expression of constitutively active Akt in PC12 and HEK 293 cells increased the phosphorylation of FKHRL1, indicating that the activation of Akt is sufficient to stimulate the phosphorylation of FKHRL1. Additional support for this notion comes from the fact that agents known to activate Akt kinase such as peroxovanadate (38) also increased the phosphorylation of FKHRL1 in PC12 cells. Second, the inhibition of IGF-1-induced activation of Akt by cotreatment with PMA attenuated the phosphorylation of FKHRL1. Conversely, the antagonism of the effect of PMA on Akt by the protein kinase C inhibitor GO6983 (44) reversed the PMA inhibition of IGF-1-induced FKHRL1 phosphorylation. Finally, the transient expression of kinase-dead Akt attenuated the IGF-1-induced phosphorylation of FKHRL1. These results demonstrate that the activation of Akt is an essential step in the IGF-1induced phosphorylation of FKHRL1.
However, we cannot rule out the possibility that other kinases are also involved, as kinase-dead Akt did not completely block the IGF-1-induced phosphorylation of FKHRL1 in PC12 cells. Despite this possibility, the following arguments support the genuine role of Akt in IGF-1-induced FKHRL1 phosphorylation. (i) The transfection efficiency of PC12 cells with kinasedead Akt is between 25 and 40%; (ii) kinase-dead Akt did not fully inhibit IGF-1-induced activation of endogenous Akt because a higher level expression of kinase-dead Akt is likely required for full inhibition; and (iii) Akt isoforms (Akt2 and Akt3) are less sensitive to kinase-dead Akt than Akt1. Accordingly, even if Akt isoforms were the only kinases involved in FKHRL1 phosphorylation, kinase-dead Akt would not fully block the IGF-1-induced phosphorylation of FKHRL1 in PC12 cells on the basis of these considerations.
The finding that IGF-1 induced the phosphorylation of Thr 32 and Ser 253 in FKHRL1 by a PI3K/Akt kinase pathway raises the possibility that the Akt kinase may directly phosphorylate this transcription factor in PC12 cells. This hypothesis was verified using an in vitro kinase assay in which purified active Akt kinase was shown to be able to potentially and directly phosphorylate immunoprecipitated FKHRL1 in PC12 cells. Moreover, FKHRL1 was found to co-immunoprecipitate with Akt in this cell line. Taken together, these results demonstrate that FKHRL1 is a downstream target and a substrate of Akt in PC12 cells.
In addition to Akt, p90 RSK also phosphorylates Ser/Thr residues in Arg-Xaa-Arg-Xaa-Xaa-(Ser/Thr) consensus sequences (21,45). Since p90 RSK is a downstream target of MAPK that can be activated by IGF-1 in PC12 cells (36), a role for MAPK in the action of IGF-1 could be envisioned. However, in contrast to PI3K inhibitors, the MEK kinase inhibitors PD98059 (46) and U1069 (47) had no effect on the phosphorylation of Akt and FKHRL1 induced by IGF-1. It is thus unlikely that the MAPK pathway is directly involved. Accordingly, our results support the role of Akt in mediating the phosphorylation of FKHRL1 induced by IGF-1 and are consistent with an earlier study suggesting that Akt directly phosphorylates FKHRL1 in Chinese hamster lung fibroblast cells (21).
The genuine functional relevance of the IGF-1-induced phosphorylation of FKHRL1 via Akt in neuronal cells is mostly unknown at this time. However, the subfamily of Forkhead transcription factors named FKHRL1, FKHR, and AFX has recently been suggested to play important roles in cell proliferation, differentiation, and apoptosis, with FKHRL1 and FKHR being mainly involved in the latter (9, 21-25, 48 -50). However, FKHR is not significantly expressed in the brain, as shown by Northern blotting (29). Hence, FKHRL1 is likely to be involved in neuronal cells. Consistent with this interpretation, our preliminary data have shown that IGF-1 has less effect on the phosphorylation of FKHR at Ser 256 in primary neuronal cultures.
Like other Forkhead transcription factors, FKHRL1 contains a DNA-binding domain that binds to consensus binding sites in the promoter of some genes such as the Fas ligand to subsequently activate the transcription of these genes, leading to cell apoptosis. The phosphorylation of FKHRL1 may block this process and lead to cell survival. This hypothesis is supported by a recent study showing that overexpression of unphosphorylated FKHRL1 in fibroblasts and cultured cerebellar neurons induces cell apoptosis, which can be inhibited by the application of Fas-Fc, a soluble fusion protein preventing the Fas ligand from binding to its receptor Fas (21). In contrast, the phosphorylation of FKHRL1 induced by survival factors like IGF-1 enhances the association of FKHRL1 with 14-3-3 protein in HEK 293 cells. This event sequesters FKHRL1 in the cytoplasm, rendering it unable to regulate its target genes in the nucleus and hence inhibiting apoptosis (21). Although this mechanism needs to be established further, the phosphorylation of FKHRL1 and other Akt targets induced by IGF-1 in neuronal cells likely plays a role in the survival effect of IGF-1.
Additional support for this notion includes the following facts. 1) The regulatory region of the promoter of the Fas ligand gene contains three insulin-responsive sequence-like sequences for FKHRL1, and the binding of FKHRL1 to these sequences enhances the transcription of the Fas ligand (51); 2) the removal of growth factors from culture medium up-regulates the expression of Fas ligand mRNA and protein and stimulates apoptosis in several cell lines, including PC12 cells and primary cultured neurons (52); and 3) the stimulation of PC12 cells with a survival factor as shown in the present study activates PI3K/Akt kinase and increases the phosphorylation FIG. 9. IGF-1-induced phosphorylation of Akt and FKHRL1 in primary cultured neurons. Cortical and cerebellar neuronal cultures were treated with IGF-1 (100 nM), and the phosphorylation of Akt and FKHRL1 was determined. As in PC12 cells, IGF-1 induced the phosphorylation of Akt and FKHRL1 in these two primary culture models. Blots represent prototypical examples of experiments replicated at least three times. p, phosphorylated. of FKHRL1, which was shown to inhibit the expression of the Fas ligand in fibroblasts (21,52). Hence, the survival effect of IGF-1 in PC12 and primary cultured neuronal cells is most likely directly related to its effect on Akt kinase and the phosphorylation of death-inducing factors, including FKHRL1 and Fas.
Interestingly, recent studies have shown that the phosphorylation, via Akt, of important components of the intrinsic cell death machinery such as Bad and caspase-9 suppresses their pro-apoptotic function and enhances the survival of target cells (17,18). Therefore, FKHRL1 is likely an additional factor involved in Akt anti-apoptotic properties, as shown in this study.
In summary, this study demonstrates that IGF-1 induces the phosphorylation of FKHRL1 via a PI3K/Akt kinase pathway in neuronal cells. This effect leads, in turn, to the inactivation of the apoptosis-promoting activities of this transcription factor. The phosphorylation of FKHRL1 is thus an additional mechanism by which IGF-1 can promote neuronal cell survival.