Programmed cell death 4 (Pdcd4) is a tumor suppressor protein that interacts with eukaryotic initiation factor 4A and inhibits protein synthesis. Pdcd4 also suppresses the transactivation of activator protein-1 (AP-1)–responsive promoters by c-Jun. The Akt (protein kinase B) serine/threonine kinase is a key mediator of phosphoinositide 3-kinase pathway involved in the regulation of cell proliferation, survival, and growth. Because Pdcd4 has two putative Akt phosphorylation sites at Ser67 and Ser457, we investigated whether Akt phosphorylates and regulates Pdcd4. Our results show that Akt specifically phosphorylates Ser67 and Ser457 residues of Pdcd4 in vitro and in vivo. We further show that phosphorylation of Pdcd4 by Akt causes nuclear translocation of Pdcd4. Using luciferase assay, we show that phosphorylation of Pdcd4 by Akt also causes a significant decrease of the ability of Pdcd4 to interfere with the transactivation of AP-1–responsive promoter by c-Jun. (Cancer Res 2005; 65(24): 11282-6)
- tumor suppressor
The expression of programmed cell death 4 (PDCD4) gene is strongly induced during apoptosis in a number of cell types (reviewed in ref. 1). PDCD4 encodes a tumor suppressor protein whose expression is lost in progressed carcinomas of lung, breast, colon, and prostate (2). In mouse keratinocytes, Pdcd4 acts as a transformation suppressor (3), and epidermal expression of Pdcd4 in transgenic mice suppresses tumorigenesis (4). It was also shown that Pdcd4 binds and inhibits the helicase activity of eukaryotic translation initiation factor 4A, a component of translation initiation complex (5). In addition, Pdcd4 expression inhibits transactivation and transformation mediated by the transcription factor activator protein-1 (AP-1; refs. 6, 7). It was shown that Pdcd4 interferes with the phosphorylation and transactivation of c-Jun by Jun NH2-terminal kinase (JNK) and blocks the recruitment of the coactivator p300 by c-Jun (8). The Pdcd4 protein mostly localizes in the nuclei of confluent or quiescent normal fetal lung fibroblasts (9). In another study, Pdcd4 was shown to be predominantly nuclear protein that can be exported from the nucleus to the cytoplasm by a leptomycin B–sensitive mechanism upon serum withdrawal (10). The serine/threonine kinase Akt (protein kinase B) regulates a number of normal cellular processes, including cell proliferation, survival, growth, and motility, through phosphorylation of multiple downstream targets (reviewed in refs. 11, 12). Akt is activated by a phosphoinositide 3-kinase (PI3K)–dependent translocation to the cell membrane and subsequent phosphorylation at Thr308 and Ser473 (13, 14). Aberrant activation of the PI3K/Akt pathway has been widely implicated in many cancers (15). Previously, we showed that Akt phosphorylates and regulates Nur77, a transcription factor regulating expression of proapoptotic genes in T cells (16). We also identified Tal1 oncoprotein as a novel target of Akt phosphorylation (17). Our analysis of Pdcd4 sequence revealed two putative Akt phosphorylation sites surrounding Ser67 and Ser457. In this study, we investigated possible phosphorylation of Pdcd4 by Akt.
Materials and Methods
DNA constructs, transfection, Western blotting, and immunofluorescence. Full-length human PDCD4 open reading frame was cloned into a pcDNA4-HisMaxC vector (Omni-PDCD4; Invitrogen, Carlsbad, CA) using standard protocols. Omni-PDCD4 (S67A), Omni-PDCD4 (S457A), and Omni-PDCD4 (S67,457A) constructs were created using standard PCR-based mutagenesis. A glutathione S-transferase (GST)–encoding DNA fragment was cloned between Omni-tag and PDCD4 cDNA sequence of Omni-PDCD4, Omni-PDCD4 (S67A), Omni-PDCD4 (S457A), and Omni-PDCD4 (S67,457A) constructs creating Omni-GST-PDCD4, Omni-GST-PDCD4 (S67A), Omni-GST-PDCD4 (S457A), and Omni-GST-PDCD4 (S67,457A) constructs. myc-AKT1 construct was purchased from Upstate Biotechnology (Lake Placid, NY). Dual-Luciferase Reporter Assay System and Renilla luciferase reporter vector pRL-TK were purchased from Promega (Madison, WI). The AP-1 reporter construct (pAP1-Luc) and positive control construct (pFC-MEKK) were purchased from Stratagene (La Jolla, CA). HEK293 and NIH-3T3 cells were grown in RPMI 1640 with 10% fetal bovine serum (FBS) and 25 μg/mL gentamicin at 37°C in a humidified atmosphere of 5% CO2. FuGene 6 transfection reagent and protease inhibitor cocktail tablets were obtained from Roche (Indianapolis, IN). Transfections, except luciferase assay experiments (see below), cell lysate preparations, and Western blot analysis were carried out as previously described (18). To investigate whether Pdcd4 phosphorylation is PI3K dependent, HEK293 cells were starved overnight in RPMI 1640 with 0% FBS, cells were then treated with insulin (5 μg/mL) for 30 minutes or with wortmannin (200 nmol/L) for 30 minutes followed by insulin for 30 minutes and lysed. Antibodies used were monoclonal anti-GST (BabCO, Richmond, CA); anti-Akt, anti-phospho-Ser473-Akt, and polyclonal anti-phospho-Akt substrate (Cell Signaling Technology, Beverly, MA); and anti-Omni and anti-myc (Santa Cruz Biotechnology, Santa Cruz, CA). Immunofluorescence was carried out as previously described using Zeiss LCM 510 confocal microscope (18).
In vitro phosphorylation. The DNA segments encoding parts of Pdcd4 protein (amino acids 11-105 for site 1 construct and amino acids 401-467 for site 2 construct) were cloned into a pGEX-4T-1 vector (Amersham Biosciences, Piscataway, NJ). GST fusion proteins were isolated according to the manufacturer's recommendations. Activated Akt was purchased from Upstate Biotechnology. In vitro phosphorylation was carried out using an Akt kinase assay kit (Cell Signaling Technology) with the following modifications: 200 ng of activated Akt and 500 ng of GST fusion proteins were used in each reaction. Western blot detection was carried out using polyclonal anti-phospho-Akt substrate antibody (Cell Signaling Technology).
Luciferase assay. HEK293 cells were transfected with the indicated constructs. Firefly and Renilla luciferase activities were assayed with the dual luciferase assay system (Promega), and firefly luciferase activity was normalized to Renilla luciferase activity, as suggested by manufacturer. All experiments were carried out in triplicate.
Akt phosphorylates Pdcd4 in vitro and in vivo and in a phosphoinositide 3-kinase–dependent manner. Because Pdcd4 contains two putative Akt phosphorylation sites (RXRXXS/T; ref. 14) at Ser67 (RLRKNS) and Ser457 (RKRFVS), we investigated whether Akt phosphorylates these residues in vitro. For these experiments, we used two GST fusion proteins containing domains of Pdcd4 surrounding putative Akt phosphorylation sites. GST-S67 wild-type (WT) fusion protein contained amino acids 11 to 105 of Pdcd4, and GST-S457 WT fusion protein included amino acids 401 to 467 of Pdcd4 (Fig. 1). In addition, we used two mutant GST fusion proteins (GST-S67A and GST-S457A). Figure 1 shows that Akt specifically phosphorylates Ser67 and Ser457 of Pdcd4 WT but not GST-S67A or GST-S457A GST fusions.
Because Akt phosphorylates Pdcd4 in vitro, we proceeded to determine if this phosphorylation also occurs in vivo. We first transfected HEK293 cells with Omni-PDCD4 WT (Fig. 2A, lane 1) or empty vector (Fig. 2A, lane 2) and detected phosphorylated proteins in these lysates using anti-phospho-Akt substrate antibody. This antibody detects proteins containing phosphorylated (R/KXR/KXXS/T) motifs. We identified a ∼55-kDa band present in lane 1 but absent in lane 2 (Fig. 2A). Because molecular weight of this band corresponds to that of Omni-Pdcd4, we concluded that Pdcd4 contains (R/KXR/KXXS/T) motif(s) phosphorylated by Akt. We then cotransfected HEK293 cells with Omni-GST-PDCD4 WT, or single and double mutants of PDCD4, and myc-AKT. Lysates were used for GST pool downs. We detected phosphorylated forms of Pdcd4 using anti-phospho-Akt substrate antibody (Fig. 2B, top); the expression of Pdcd4 was detected using anti-Omni antibody (Fig. 2B, middle). Our results show that WT Pdcd4 is indeed phosphorylated by Akt (Fig. 2B, lane 1, top). The levels of phosphorylated Pdcd4 in S67A and S457A mutants were lower than in WT Pdcd4 (lanes 2 and 3 versus lane 1), indicating that both serine residues are phosphorylated by Akt. As expected, no phosphorylation signal was detected in the cells expressing S67,457A Pdcd4 double mutant (Fig. 2B, lane 4). This suggests that Akt phosphorylates Ser67 and Ser457 of Pdcd4 in vivo.
The activation of Akt by various survival and growth factors, such as insulin and platelet-derived growth factor, involves a PI3K-dependent membrane translocation and a phosphorylation of Thr308 and Ser473 of Akt mediated by PDK-1 (13, 14). Wortmannin, a PI3K inhibitor, inhibits this activation of Akt (19). The activation of Akt by treatment of 293 cells with insulin is a model system often used to assay phosphorylation of various Akt targets (14, 17). Thus, we proceeded to determine whether Akt phosphorylates Pdcd4 in a PI3K manner. HEK293 cells were transfected alternatively with Omni-PDCD4, Omni-PDCD4 S67A, Omni-PDCD4 S457A, or Omni-PDCD4 S67,457A and starved overnight in 0% FBS. Cells were then treated with insulin or wortmannin, and insulin and phosphorylated Pdcd4 was detected with anti-phospho-Akt substrate antibody. Figure 2C (top) shows that insulin treatment significantly increases the phosphorylation of WT Pdcd4 by endogenous Akt (lane 2), and that treatment with wortmannin completely inhibits this effect (lane 3). Phosphorylation of Pdcd4 by endogenous Akt was significantly less efficient when Ser67 of Pdcd4 was mutated to alanine (lane 5). Effect of Ser457 to Ala457 mutation of Pdcd4 on its phosphorylation by Akt was minimal (lane 8). No phosphorylation signal was detected in the cells expressing S67,457A Pdcd4 double mutant (Fig. 2C, lane 11, top left), confirming that we identified the correct band corresponding to phosphorylated Pdcd4 (Fig. 2A). As expected, in experiments with all Pdcd4 constructs, Akt was activated (i.e., phosphorylated at Ser473) by insulin, and this effect was inhibited by wortmannin (Fig. 2C, middle bottom). Consistent amounts of Pdcd4 and endogenous Akt were present in all experiments, as determined by Western blot analysis with anti-Omni and anti-Akt antibodies, respectively. Thus, Pdcd4 is phosphorylated by endogenous Akt in HEK293 cells at Ser67 and Ser457 in a PI3K-dependent manner.
Akt regulates the intracellular localization of Pdcd4. The Pdcd4 protein has predominantly nuclear intracellular localization under normal growth conditions but is exported from the nucleus upon serum withdrawal (10). Akt, on the other hand, is primarily localized in the cytoplasm, but some nuclear presence was also observed (19). To investigate whether phosphorylation of Pdcd4 affects its intracellular localization, we transfected NIH-3T3 cells with Omni-PDCD4, Omni-PDCD4 S67A, Omni-PDCD4 S457A, or Omni-PDCD4 S67,457A constructs and studied the location of Pdcd4 by immunofluorescence. As expected, Pdcd4 WT under normal growth conditions showed clear nuclear localization (Fig. 3, top left). In starved cells (0% FBS), Pdcd4 localization became mostly cytoplasmic, confirming previously published results (Fig. 3, top middle; ref. 10). To assess the effect of Akt phosphorylation on the intracellular distribution of Pdcd4, cells expressing Pdcd4 WT were treated with wortmannin to block the phosphorylation of Pdcd4 by Akt. Figure 3 (top right) shows that under these conditions, Pdcd4 was redistributed to the cytoplasm. This indicates that phosphorylation by Akt causes nuclear translocation of Pdcd4. To confirm this finding, we studied the intracellular localization of Pdcd4 S67A, S457A, and S67,457A mutants. Figure 3 (bottom) shows that S457A mutation leads to cytoplasmic localization of Pdcd4, indicating that phosphorylation of Pdcd4 by Akt at S457 regulates its intracellular localization. Interestingly, S67A mutation did not affect nuclear localization of Pdcd4 (Fig. 3). Pdcd4 S67,457A mutant showed cytoplasmic localization confirming the importance of S457A mutation (Fig. 3).
Akt inhibits the ability of Pdcd4 to interfere with the transactivation of activator protein-1–responsive promoters by c-Jun. Recent studies have shown that Pdcd4 suppresses the transactivation of AP-1-responsive promoters by c-Jun, suggesting that tumor suppressor activity of Pdcd4 may be, at least in part, due to the inhibition of c-Jun activity (5, 8). To investigate whether phosphorylation of Pdcd4 by Akt affects this function of Pdcd4, we used pAP1-Luc construct containing the luciferase reporter gene under the control several AP-1-binding sites and minimal TATA box from thymidine kinase promoter (Stratagene) and carried out reporter gene assay experiments in HEK293 cells. In these assays, we used Omni-PDCD4, Omni-PDCD4 S67A, Omni-PDCD4 S457A, or Omni-PDCD4 S67,457A constructs. Because basic promoter activity of pAP1-Luc construct without additional activation of c-Jun is very low, we used pFC-MEKK construct (Stratagene), expressing upstream activator of c-Jun, mitogen-activated protein kinase kinase kinase 1 (MEKK1; MAP3K1) in all experiments. HEK293 cells were cotransfected with the reporter construct, pFC-MEKK, and each of four PDCD4 constructs (Fig. 4). As expected, expression of Pdcd4 WT significantly inhibited promoter activity when compared with empty vector. Pdcd4 S67A and Pdcd4 S457A mutants showed more potent inhibition of the promoter activity. Most interestingly, Pdcd4 S67,457A double mutant showed 2-fold decrease of promoter activity when compared with Pdcd4 WT (Fig. 4). These results suggest that phosphorylation of Ser67 and Ser457 by Akt inhibits the function of Pdcd4 as a repressor of AP-1-responsive promoters.
In this report, we show that Pdcd4 is a novel target of Akt protein kinase. Our results show that Akt phosphorylates Pdcd4 in vitro and in vivo in a PI3K-dependent manner, causes nuclear translocation of Pdcd4, and inactivates Pdcd4 in its function as an inhibitor of AP-1-mediated transcription. Akt phosphorylates and regulates a number of targets, and this regulation is most often associated with the distribution of prosurvival and antiapoptotic signals (reviewed in refs. 11, 12). This direction of regulation is also apparent in the case of Pdcd4: phosphorylation of Pdcd4 by Akt inhibits the tumor suppressor function of Pdcd4 by decreasing the ability of Pdcd4 to interfere with the transactivation of AP-1-responsive promoter by c-Jun.
As mentioned above, Pdcd4 shuttles between nucleus and cytoplasm (10). Our results (Fig. 3) suggest that phosphorylation of S457 but not S67 causes nuclear translocation of Pdcd4. On the other hand, because Akt phosphorylates these both residues, it is possible that both residues are phosphorylated at the same time; therefore, any phosphorylation of Pdcd4 by Akt results in the nuclear translocation of Pdcd4. S67A and S547A mutants have different intracellular location (S67A mutant is located in the nucleus, whereas S567A mutant is located in the cytoplasm; Fig. 3). On the other hand, these both mutants show more potent inhibition of AP-1-mediated transcription than Pdcd4 WT (Fig. 4), predominantly located in the nucleus (Fig. 3). This suggests that this inhibition of AP-1-mediated transcription by Pdcd4 can occur in both the nucleus and cytoplasm. Recent report have shown that Pdcd4 interacts with both c-Jun and JNK-1 and inhibits AP-1 function by inhibiting phosphorylation of c-Jun by JNK-1 (8). Because c-Jun is mostly nuclear protein and JNK-1 is located in both the nucleus and cytoplasm (20), it is possible that nuclear Pdcd4 inhibits c-Jun-mediated transactivation by direct interaction with c-Jun, and cytoplasmic Pdcd4 inhibits JNK-1 in the cytoplasm and prevents it from phosphorylation of c-Jun. Additional studies are necessary to define the exact mechanism of this inhibition.
Grant support: NIH grant CA76259 (C.M. Croce and Y. Pekarsky).
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Received September 28, 2005.
- Accepted October 27, 2005.
- Revision received October 25, 2005.