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The Journal of Immunology, 2008, 180, 222-229
Copyright © 2008 by The American Association of Immunologists, Inc.
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Nuclear Export of NF90 to Stabilize IL-2 mRNA Is Mediated by AKT-Dependent Phosphorylation at Ser647 in Response to CD28 Costimulation1

Yuan Pei*,{dagger}, Ping Zhu*, Yongjun Dang{ddagger}, Jiaxue Wu*, Xianmei Yang*, Bo Wan*, Jun O. Liu{ddagger}, Qing Yi§ and Long Yu2,*,{dagger}

* State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences and {dagger} Institutes of Biomedical Sciences, Fudan University, Shanghai, China; {ddagger} Departments of Pharmacology and Molecular Sciences and Neuroscience, The Johns Hopkins School of Medicine, Baltimore, MD 21205; and § Department of Lymphoma and Myeloma, Center for Cancer Immunology Research, University of Texas MD Anderson Cancer Center, Houston, TX 77030


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 Disclosures
 References
 
IL-2 is one of the most important cytokines required for T cell-mediated immune responses. Costimulation of CD28 in T cells up-regulates IL-2 mRNA levels via transcription activation and mRNA stabilization. Upon T cell activation, NF90, an AU-rich element (ARE)-binding protein, translocates from the nucleus into the cytoplasm, where it binds to the ARE-containing 3' untranslated regions of IL-2 mRNA and slows down degradation of IL-2 mRNA. The translocation of NF90 is mediated through a nuclear export signal at its N terminus, but how it is triggered is still unclear. Phosphorylation of ARE-binding proteins has been reported as a signal transduction pathway to stabilize ARE-containing transcripts. In this study, we demonstrate that AKT phosphorylates NF90 on Ser647 upon CD28 costimulation. This phosphorylation is necessary for nuclear export of NF90 and IL-2 mRNA stabilization by this protein, because a mutation at Ser647 abolished both functions. We observed that treatment of cells with CD28 costimulation induced distinct increase in phosphorylation of AKT and NF90 at Ser647 concomitantly. Phosphorylation at Ser647 of NF90 up-regulated IL-2 production in response to CD28 costimulation. In vivo and in vitro data support a model in which CD28 costimulation activates AKT to phosphorylate NF90 at Ser647 and phosphorylation triggers NF90 to relocate to the cytoplasm and stabilize IL-2 mRNA.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 Disclosures
 References
 
Interleukin-2 is one of the most important cytokines required for T cell immune responses. Its expression is precisely regulated at both transcriptional and posttranscriptional levels. In addition to T cell activation, IL-2 plays significant roles in T cell proliferation as autocrine and paracrine factors, affects the signaling cascade and amplification of immune responses, and regulates T cell apoptosis (1, 2, 3, 4). Defective IL-2 secretion results in loss of T cell immune responses, whereas overexpression of IL-2 may lead to autoimmune responses (5, 6). Due to the importance of IL-2 to T cells and other cells of the immune system, the mRNA and protein levels of IL-2 are tightly regulated. The steady-state level of IL-2 mRNA is determined by its dynamic transcription and decay. In quiescent T cells, mRNA of IL-2 and other cytokines are rapidly eliminated after transcription, determined by the AU-rich elements (AREs)3 located within their 3' untranslated regions (UTR). However, the production of IL-2 increases ~100-fold upon T cell activation, due to transcription activation and increased IL-2 mRNA stability (7). This mechanism of modulation results in rapid increase of cytokine mRNAs shortly after infection, and the excessive mRNAs are eliminated via ARE-mediated decay once the infection recedes.

AREs are usually found in the 3' UTR of some rapidly induced genes, such as early immune response genes, proto-oncogenes, and some growth-regulatory genes (8, 9, 10, 11). Conserved AREs mainly function to induce rapid decay of the ARE-containing transcripts as c-fos, c-myc, c-jun, GM-CSF, IL-3, and TNF-{alpha} mRNAs (11, 12, 13, 14). AU-binding proteins (AUBP) recognize AREs. Although some of the proteins promote mRNA decay, others mediate mRNA stabilization (8, 15, 16). One example is tristetraprolin (TTP), an endotoxin-induced protein (17). TTP binds to the AREs of TNF-{alpha} mRNA to promote their rapid decay (18). It also binds to other cytokine mRNAs and promotes their decay via deadenylation. Another AUBP, butyrate response factor (BRF1), was shown to promote IL-3 mRNA decay in vitro (19). The ability of BRF1 to promote mRNA decay is weakened when Ser92 is phosphorylated by the serine/threonine kinase AKT, thereby stabilizing IL-3 mRNA. Furthermore, the mRNA decay-promoting AUBPs were shown to be able to recruit the 3' exonuclease complex and stimulate mRNA decay. However, the mRNA-stabilizing AUBPs, such as HuR, do not possess such activity. HuR is found in several cell lines, including T lymphocytes, and can stabilize IL-3 and c-fos mRNA.

CD28 belongs to the Ig superfamily and plays critical roles in T cell activation and maturation (20, 21). In the absence of CD28, immunostimulation of T cells results in anergy, and only costimulation through CD28 can avoid anergy (22, 23). The production of IL-2 increases greatly and rapidly upon CD28 costimulation, due to an increase in IL-2 transcription and stabilization of its mRNA (24, 25). Moreover, CD28 is important for T cell survival because it induces the anti-apoptotic protein Bcl-xL (26). Despite its well-known importance, the signaling pathways downstream of CD28 are still poorly elucidated. In T cells, CD28 activates AKT, an important player in PI3K pathway. Overexpression of AKT increases IL-2 transcription, overriding CD28 and other costimulation signals (27, 28). It was shown that the stabilization of IL-2 mRNA by CD28 was mediated via the JNK signaling pathway (29, 30, 31). However, another study showed that p38, but not JNK, was activated by CD28 costimulation in murine T cells (32). JNK1/2 knockout mice exhibited normal levels of IL-2 (33). There have been very few reports on RNA-binding proteins related to CD28-induced IL-2 mRNA stabilization. Even though HuR has been shown to bind IL-2 mRNA, it is not required for IL-2 mRNA stabilization in response to CD28 costimulation (34).

NF90 was first purified as a transcriptional factor associated with the NF-AT DNA binding site (35, 36). However, NF90 does not contain the DNA-binding motif present in NF-AT, but has two conserved dsRNA-binding motifs and one RGG (arginine-glycine-glycine) motif related to RNA binding. NF90 binds to dsRNA and VA RNAII, a tiny, but highly ordered adenovirus RNA (37). NF90 also interacts with several other proteins, such as NF45, DNA-dependent protein kinase, Ku, eIF2, RNA helicase A, and protein kinase R (38, 39, 40). The function of these interactions is still not clear. NF90 binds to the five AREs located in the 3' UTR of IL-2 mRNA and stabilizes it. This specific binding may be due to the double-strand structure of IL-2 mRNA. Moreover, NF90 localizes in the nucleus of quiescent Jurkat cells. In response to immune stimulation, cytoplasmic NF90 increases greatly and functions to stabilize IL-2 mRNA. The translocation of NF90 from nucleus to cytoplasm is required for IL-2 mRNA stabilization (16). Although NF90 has a nuclear export signal (NES) in its N terminus, it is unclear how the translocation is triggered.

In this study, we proposed and confirmed a phosphorylation site in NF90, and phosphorylation at this site triggers its nuclear export. We identified the interaction between AKT and NF90 and their colocalization in nucleus. AKT phosphorylates NF90-Ser647 and enhances its function in IL-2 mRNA stabilization. We also confirmed that phosphorylation of NF90-Ser647 by AKT was induced by CD28 costimulation, and led to increased production and secretion of IL-2 by the cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 Disclosures
 References
 
Cell lines and T cell stimulation

Jurkat cells (American Type Culture Collection) were maintained in RPMI 1640 cell culture medium supplemented with 10% FBS. Lipofectamine 2000 (Invitrogen Life Technologies) was used as the transfection reagent. Stimulation of T cells was performed 18 h after the cells were transfected. T cells were incubated with PMA (50 ng/ml; Sigma-Aldrich) and Ca2+ ionophore A23187 (1 µg/ml; Calbiochem) for 5 h, followed by addition of cyclosporin A (CsA, 1 µg/ml; Calbiochem). Primary human blood T cells were isolated from PBMC by CD3 MACS MicroBeads (Miltenyi Biotec) and stimulated accordingly.

Immunoprecipitation (IP)

Myc-NF90 and hemagglutinin (HA)-Akt were precipitated from cell lysates by incubating with anti-Myc mAb or anti-HA mAb (Sigma-Aldrich) bound to Protein G Plus/Protein A agarose suspension (Oncogene Research Products) overnight at 4°C. Proteins were eluted with SDS loading buffer, separated on 12% SDS-PAGE, and analyzed by Western blotting using either anti-Myc mAb (1:1000) or anti-HA Ab (1:2000) (Sigma-Aldrich) and secondary Abs linked to HRP (Calbiochem). Proteins were visualized with the ECL detection system (NEN). For endogenous IP, AKT Ab and NF90 Ab (Cell Signaling Technology) were used.

In vitro kinase assay

Phosphorylation of NF90 was analyzed using an in vitro kinase assay. Akt kinase (Cell Signaling Technology) incubated with either purified NF90500–702, NF90-Ser647A500–702, or controls in the presence of 2 µl of kinase buffer, 1 µl of [{gamma}-32P]ATP (10 mCi/ml) for 30 min at 37°C. Proteins were eluted with SDS loading buffer, separated by SDS-PAGE (12%), and analyzed by autoradiography.

Real-time RT-PCR

Total RNA was isolated using TRIzol (Invitrogen Life Technologies) and reverse transcribed into cDNA. The level of IL-2 mRNA was determined by real-time PCR following normalization to GAPDH, using the {Delta}{Delta}CT method for relative quantitation with IL-2 mRNA levels. PCR was performed on an iCycler iQTM (Bio-Rad) using SYBR Green Premix (Takara Shuzo) as a dsDNA-specific binding dye and continuous fluorescence monitoring. For detection of GAPDH, the sense primers 5'-GAAGGTGAAGGTCGGAGTC-3' and antisense primer 5'-GAAGATGGTGATGGGATTTC-3' were used; for detection of IL-2, the primers 5'-TACAACTGGAGCATTTACTG-3' (sense) and 5'-GTTTCAGATCCCTTTAGTTC-3' (antisense) were used.

Preparation of cytoplasmic and nuclear extracts

Twenty million cells were disrupted in 0.5 ml of buffer A (0.2% Nonidet P-40, 50 mM KCl, 20 mM HEPES (pH 7.9), 1.5 mM MgCl2, 1 mM DTT, 8 ng/ml aprotinin, 2 ng/ml leupeptin, and 1 mM PMSF) and centrifuged at 6500 rpm at 4°C, and the supernatant was collected as the cytoplasmic fraction. The nuclear pellet was washed twice with lysis buffer and resuspended in 0.3 ml of buffer B (20 mM HEPES (pH 7.9), 0.4 M NaCl, 25% glycerol, 1.5 mM MgCl2, 1 mM DTT, 8 ng/ml aprotinin, 2 ng/ml leupeptin, and 1 mM PMSF). After incubation on ice for 30 min, the solution was centrifuged at 12,000 rpm for 30 min, and the supernatant was collected as the nuclear extract.

IL-2 radioimmunoassay

After transfection, cells were stimulated for 24 h with anti-CD3 (clone UCHT-1) and anti-CD28 (clone CD28.2) Abs (Sigma-Aldrich) at final concentrations of 1 and 0.5 µg/ml, respectively. CsA was added to the culture, and supernatants were collected at indicated times and assayed by radioimmunoassay for IL-2 secretion.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 Disclosures
 References
 
NF90-Ser647 phosphorylation is required for its nuclear export

NF90 has been reported to play an important role in IL-2 mRNA stabilization. Upon T cell activation, NF90 accumulates in the cytoplasm and binds to AREs located in the 3' UTR of IL-2 mRNA. Translocation of NF90 is mediated through an N-terminal NES, but how it is triggered is still unclear. Phosphorylation has been shown to regulate nuclear export of some proteins, including NF-AT (41, 42). NF90 moves into the cytoplasmic compartment during mitosis and is highly phosphorylated (43). Therefore, we asked whether nuclear export of NF90 in response to CD28 costimulation is regulated through phosphorylation. The first experiment was to analyze the coding sequence of NF90 for possible phosphorylation sites by kinases downstream of CD28. Interestingly, we found Ser647 located in its RGG box to be one possible phosphorylation site by AKT family. Ser647 was embedded in a specific sequence RXRXRXXS, and this conserved sequence is generally found in AKT substrates, such as glycogen synthase kinase 3β (GSK3β) and BRF1 (Fig. 1, A and B), suggesting that NF90 is most likely to be a substrate of AKT kinase.


Figure 1
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  		FIGURE 1. Phosphorylation at Ser647 regulates nuclear export of NF90. A, Structural schematic of NF90; B, the alignment of conserved sequences of NF90 and other AKT substrates BRF1, GSK3β; C, subcellular distribution of GFP-NF90 and GFP-NF90-Ser647A in Jurkat cells. Cells were treated or untreated with anti-CD3 and anti-CD28 after transfection. Four hours later, the cells were fixed and subcellular distribution of GFP fusion proteins was determined by fluorescence microscopy. Nuclei were counterstained with 4',6'-diamidino-2-phenylindole; D, immunoblotting analysis of subcellular distribution of Myc-NF90 and Myc-NF90-Ser647A in Jurkat cells. Transiently transfected cells were stimulated with anti-CD3 and anti-CD28. Two hours after stimulation, the cells were collected and nuclear and cytoplasmic extracts were prepared. The relative amounts of Myc-NF90 and Myc-NF90-Ser647A proteins were determined by immunoblotting. Actin and histone 3 were used as the cytoplasmic and nuclear markers, respectively.

 
Nuclear export is sufficient for IL-2 mRNA stabilization by NF90. To examine whether NF90-Ser647 is required for nuclear export, we constructed NF90-Ser647A. If Ser647 indeed could be phosphorylated upon CD28 costimulation, the mutant would be unable to be phosphorylated. We determined the subcellular distribution of wild-type and mutant NF90 after stimulation (Fig. 1D). In nonstimulated Jurkat cells, more Myc-NF90 was detected in the nuclear fraction than in the cytoplasm. After anti-CD28 and anti-CD3 Ab treatment, Myc-NF90 was much more abundant in the cytoplasmic fraction. Nuclear translocation was not detected in cells transfected with Myc-NF90-Ser647A. This is consistent with results from microscopy analysis (Fig. 1C). Jurkat cells were transiently transfected with either wild-type or mutant NF90, as indicated. A major portion of GFP-NF90 migrated into the cytoplasm after treatment with anti-CD28 and anti-CD3 Abs, which was not found in cells transfected with GFP-NF90-Ser647A. These results supported our hypothesis that nuclear export of NF90 was regulated through phosphorylation.

NF90 and AKT protein interaction

To confirm the possible enzyme-substrate relation between NF90 and AKT, their interaction was first examined in mammalian cells. We constructed HA-AKT and Myc-NF90, and transfected these plasmids into 293T. Forty-eight hours after transient transfection, cell lysates were prepared and incubated with Myc or HA mAb and agarose beads. Following IP, HA or Myc mAb was used for Western blot analysis. The results are shown in Fig. 2A. When Myc-NF90 and HA-AKT were cotransfected, but not control vectors, AKT was detected in the fraction precipitated with the Myc mAb, and Myc-NF90 was detected in the fraction precipitated with the HA mAb. This indicates that the exogenously expressed Myc-NF90 and HA-AKT could interact with each other.


Figure 2
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  		FIGURE 2. NF90 and AKT interact with each other and colocalize in the nucleus. A, IP results with Myc-NF90 and HA-AKT in 293T cells. Lys: whole cell lysate. B, IP results with endogenous NF90 and AKT in Jurkat cells; C, colocalization of NF90 and AKT in Hela cells.

 
To further verify whether NF90 and AKT interact under physiological conditions in T cells, Jurkat cell lysates were incubated with AKT mAb. As shown in Fig. 2B (left panel), NF90 was detected in the endogenous IP fraction by AKT Ab, but not by the control mouse IgG. The reciprocal experiment in Jurkat cells demonstrated the same results (Fig. 2B, right panel). Therefore, our results indicate that AKT and NF90 also interact in vivo.

AKT locates in the cytoplasm before cell activation and translocates into nucleus following activation via phosphorylation. Activated AKT phosphorylates and activates other transcriptional factors. Because NF90 contains a conserved nuclear localization signal sequence, we examined whether activated AKT colocalized with NF90. Twenty-four hours after cotransfection with pEGFP-NF90 and pDsRed-AKT constructs into HeLa cells, we incubated the cells in serum-free medium overnight. The cells were then cultured in medium with 10% serum for 45 min and fixed for cofocal microscopy. Indeed, we found that AKT was in nucleus and colocalized with NF90 (Fig. 2C). We conclude from the above experiments that NF90 and AKT interact in vivo and colocalize in the nucleus.

AKT phosphorylates NF90 at Ser647

To further confirm NF90 as a substrate of AKT, we performed an in vitro kinase assay. First, we expressed wild-type NF90500–702 and NF90-Ser647A500–702 with His tag in Escherichia coli, and purified the proteins with Ni-NTA His affinity Resin (Novagen). Purified NF90500–702 or NF90-Ser647A500–702 proteins were incubated with AKT kinase and {gamma}-32P. In this experiment, we used GST and GSK3β proteins as negative and positive controls, respectively. As shown in Fig. 3, A and B, AKT-phosphorylated wild-type NF90 and the degree of phosphorylation were dependent on the protein levels of AKT or NF90. However, AKT did not phosphorylate NF90-Ser647A, although the amount of AKT protein was 5 times that of wild-type NF90.


Figure 3
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  		FIGURE 3. AKT phosphorylates NF90 in vivo and in vitro. A and B, E. coli-expressed NF90500–702 (different doses), NF90-Ser647A500–702, GST (negative control), and/or GSK3β (positive control) were incubated with AKT kinase at different doses, 1 µl of {gamma}-32P-labeled ATP (10 mCi/ml), and kinase buffer for 30 min. The reaction mixture was separated on SDS-PAGE and subjected to autoradiography. The amount of corresponding proteins was indicated with Coomassie blue staining. C, Phosphorylation of NF90 in CHO cells. CHO cells were transfected with expression constructs, as shown in the figure. Thirty-six hours after transfection, Myc Ab was used to pull down tagged protein in the cell lysates. Left panel, Shows the amount of NF90 precipitated. Right panel, Phosphorylation of NF90 in the pull-down fractions was detected with specific phosphorylation Ab for AKT substrates.

 
An in vivo phosphorylation experiment was performed with Chinese hamster ovary (CHO) cells because it had higher transfection efficiency than 293T cells. First, constitutively active AKT (AKT-CA) or inactive AKT were cotransfected with Myc-NF90 or Myc-NF90-Ser647A into CHO cells. After pulling down NF90 protein with Myc Ab, we detected phosphorylation of NF90 at Ser647 with Ab specific for AKT substrate phosphorylation. This Ab specifically recognizes phosphorylated serine within RXRXXS, the consensus sequence in AKT substrates. As shown in Fig. 3C, phosphorylation at Ser647 can only be detected in the precipitated fraction when wild-type NF90 and AKT-CA were cotransfected. In precipitated fractions with NF90 and inactive AKT cotransfection or NF90-Ser647A and AKT-CA cotransfection, we did not detect the phosphorylation. Thus, the above experiments confirmed that AKT phosphorylates NF90 in vitro and in vivo, and NF90 indeed is a substrate of AKT.

Phosphorylation at Ser647 by AKT is critical for NF90 to stabilize IL-2 mRNA

Previous studies showed that NF90 could bind to the AREs located within IL-2 mRNA 3' UTR, and attenuate the decay of IL-2 mRNA (16). We have found that Ser647 is important for NF90 nuclear export and could be phosphorylated by AKT. Therefore, we hypothesized that the phosphorylation of Ser647 may be critical for NF90 to stabilize IL-2 mRNA. Myc-NF90, Myc-NF90-Ser647A (defective for phosphorylation), or the empty Myc vector was transfected into Jurkat T cells. Eighteen hours after transfection, PMA and Ca2+ ionophore A23187 were added to the culture for 5 h to increase IL-2 transcription. We used CsA to specifically block IL-2 transcription. RNA was collected from the cells every 30 min after CsA addition and was quantified with real-time RT-PCR. The result showed that IL-2 mRNA was degraded rapidly in cells transfected with empty Myc vector and its t1/2 is ~30 min (Fig. 4A). However, introduction of Myc-NF90, but not Myc-NF90-Ser647A, increased the t1/2 of IL-2 mRNA to ~60 min. To further confirm the effect of Ser647 phosphorylation, we mutated serine at the 647 position to glutamate (NF90-Ser647E) to mimic the phosphorylation state. We found that NF90-Ser647E stabilized IL-2 mRNA with the same potential as the wild-type NF90, possibly due to constitutive AKT activation in Jurkat cells. Therefore, the phosphorylation at Ser647 is critical for NF90 to stabilize IL-2 mRNA.


Figure 4
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  		FIGURE 4. AKT phosphorylates NF90 to stabilize IL-2 mRNA. A, Myc-NF90, Myc-NF90-Ser647A (defective for phosphorylation), or empty Myc vector was transfected into Jurkat cells. Eighteen hours after transfection, PMA and Ca2+ ionophore A23187 were added and cultured for 5 h to increase IL-2 transcription. Following CsA addition, RNA was collected every 30 min and quantified using real-time RT-PCR. B, RNA stability was measured as in A, except that the cells were transfected with the plasmids Myc-NF90 or Myc-NF90-Ser647A together with AKT. C, Transfection and cell stimulation were performed same as in A. LY294002 (5 and 10 µM) was added at the same time with CsA. Results shown were the average of three independent experiments (n = 6).

 
We have shown that AKT phosphorylated NF90 at Ser647, and this phosphorylation was closely related with its ability to stabilize IL-2 mRNA. The question was then whether AKT modulates IL-2 mRNA stability through phosphorylating NF90 at Ser647. First, we examined IL-2 mRNA stability in Jurkat cells cotransfected with AKT and NF90 or NF90-Ser647A, and found that IL-2 mRNA was much more stable in cells cotransfected with AKT and NF90 than cells cotransfected with AKT and NF90-Ser647A, or transfected with NF90 alone (Fig. 4B). Second, we monitored the ability of NF90 or NF90-Ser647A to stabilize IL-2 mRNA when AKT activity was inhibited. Similar to the previous experiment, Jurkat cells were first transfected with NF90 or NF90-Ser647A. Following stimulation with PMA and A23187 for 5 h, we added CsA to end transcription. At the same time, LY294002, an AKT inhibitor, was also added to the culture. IL-2 mRNA was quantified, as previously described. As shown in Fig. 4C, the stabilization of IL-2 mRNA by NF90 is sensitive to LY294002 treatment, and in the presence of a larger dose of the inhibitor, IL-2 mRNA was more unstable. As predicted, LY294002 did not alter the effect of NF90-Ser647A on IL-2 mRNA stability. Taken together, our experiments indicate that expressing AKT by cotransfection promoted the stabilization of IL-2 mRNA mediated by NF90, and inhibiting AKT activity abrogated the ability of NF90, but not NF90-Ser647A, to stabilize IL-2 mRNA. Therefore, we conclude that AKT modulates IL-2 mRNA stability through phosphorylation of NF90-Ser647.

CD28 costimulation induces NF90-Ser647 phosphorylation by AKT

IL-2 expression level has been reported to be rapidly elevated upon CD28 costimulation due to increased transcription and posttranscriptional stabilization. In Jurkat cells, AKT activated by CD28 signaling promotes IL-2 transcription, whereas PMA did not activate AKT kinase (44). Based on our previous results, we hypothesized that CD28 costimulation-activated AKT could phosphorylate NF90 to stabilize IL-2 mRNA. To test this hypothesis, we stimulated Jurkat T cells with anti-CD28 and anti-CD3 or together with LY294002 for 1 h. After using NF90 mAb to IP endogenous NF90, we examined phosphorylation of NF90-Ser647 with AKT substrate phosphorylation Ab. At the same time, the phosphorylation state of AKT was also determined using Ab to phosphorylated AKT. As shown in Fig. 5A, CD28 costimulation induced phosphorylation of both AKT and NF90-Ser647. If AKT were inhibited with LY294002 following the stimulation, phosphorylation of NF90 at Ser647 would also diminish. The physiological relevance of these results was confirmed with human primary T cells (Fig. 5B). Therefore, by activating AKT, CD28 costimulation induced NF90-Ser647 phosphorylation.


Figure 5
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  		FIGURE 5. AKT and NF90 phosphorylation is induced in response to CD28 costimulation. A, Jurkat cells were incubated with different concentrations of CD28 and CD3 Abs with or without LY294002 (5 µM) for 1 h. Cell lysate was immunoprecipitated with NF90 Ab. Phosphorylation of NF90 was detected with specific phosphorylation Ab for AKT substrates, and the amount of total NF90 pulled down was examined with NF90 Ab. B, Phosphorylation of AKT and total AKT in the cell lysate was determined with corresponding Abs. Human T cells were isolated from PBMC by CD3 MACS MicroBeads, stimulated, and assayed, as previously described.

 
NF90-Ser647 phosphorylation regulates IL-2 production in response to CD28 costimulation

To further study the effect of NF90-Ser647 phosphorylation in CD28 costimulation-induced T cell activation, we transfected Jurkat cells with NF90 or NF90-Ser647A, stimulated the cells with anti-CD3 and anti-CD28, and examined the production of IL-2 protein by a radioimmunoactivity assay. Empty vector was used as a negative control. In the experiment, anti-CD3 and anti-CD28 were added to the cell culture 18 h after transfection, and the stimulation lasted for 48 h. The culture medium was collected every several hours for quantifying IL-2 protein (Fig. 6A). In Fig. 6B, CsA was added to block IL-2 transcription after 24 h of the stimulation. As shown in Fig. 6B, during the time period before CsA addition, the amount of IL-2 in cell medium was almost the same for cells transfected with NF90 or NF90-Ser647A, which was higher than that transfected with empty vector (n = 6). This indicates that NF90 increases IL-2 transcription and is consistent with previous reports (35, 36). Moreover, this promotion does not depend on phosphorylation at Ser647. Furthermore, 3 h after transcription ended by CsA, there was an obvious difference in levels of IL-2 in culture medium of cells transfected with NF90 or NF90-Ser647A. The amount of IL-2 was much higher in culture medium of cells transfected with NF90 than in those transfected with NF90-Ser647A or empty vector. This indicates that phosphorylation of NF90 at Ser647 plays an important role in the posttranscriptional regulation of IL-2 mRNA induced by CD28 costimulation and is consistent with the data presented above. Of interest is the result that cells transfected with NF90-Ser647A secreted less IL-2 than those transfected with the empty vector. NF90-Ser647A may mediate blocking of endogenous NF90 export to stabilize IL-2 mRNA. In conclusion, we show that phosphorylation of NF90 at Ser647 is a key step in IL-2 production in response to CD28 costimulation.


Figure 6
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  		FIGURE 6. Phosphorylation of NF90 at Ser647 modulates IL-2 secretion induced by CD28 costimulation. A, Jurkat cells were transfected with NF90, NF90-Ser647A, or empty vector. Eighteen hours after transfection, anti-CD3 and anti-CD28 were added to stimulate IL-2 production for 24 h, and IL-2 secretion was measured at every point, as shown. B, Transfection and stimulation were performed, as previously described, except that CsA was added to block IL-2 transcription after 24 h of stimulation. C, The model of NF90 phosphorylation modulates IL-2 mRNA stabilization by CD28 costimulation.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 Disclosures
 References
 
Our data demonstrated that NF90 is involved in the up-regulation of IL-2 expression by CD28 costimulation. CD28 promotes AKT phosphorylation of NF90 at Ser647, which results in nuclear export of NF90 and IL-2 mRNA stabilization. AKT is an important component of the PI3K signaling pathway and is activated in T cells stimulated by CD28 Ab (45, 46). Phosphorylation of AKT activates downstream substrates, which efficiently stabilize ARE-containing mRNAs. A consensus sequence for AKT substrates is found in the NF90 coding sequence, with Ser647 being the conserved phosphorylation site. The in vitro and in vivo kinase assay experiments proved that AKT indeed could phosphorylate NF90 at Ser647 (Fig. 3).

The stability of IL-2 mRNA is controlled by the presence of ARE in the 3' UTR. The current model is that constitutive ARE-binding proteins, such as tristetraprolin, recruit a multicomponent exosome that degrades the associated mRNA molecule. The signals that interfere with exosome recruitment and induce mRNA stabilization are not well understood. Phosphorylation or competition with nonexosome-recruiting ARE-binding proteins is linked to the stabilization of some transcripts. Based on the demonstration that NF90 could bind and stabilize IL-2 mRNA and that phosphorylation of AKT substrates also stabilizes some ARE-containing mRNAs, we hypothesized that phosphorylation of NF90-Ser647 by AKT might affect its function on IL-2 mRNA stabilization. The difference in stability of IL-2 mRNA between the wild-type and the mutant NF90 depended on phosphorylation of NF90-Ser647, indicating that phosphorylation of NF90-Ser647 is indeed crucial for its ability to stabilize IL-2 mRNA. However, the t1/2 of IL-2 mRNA in cells transfected with NF90-Ser647E was similar to or even higher than cells transfected with wild-type NF90, which could be explained by Jurkat cells lacking PTEN, an inhibitor of PI3K. In this study, we used PMA to activate the Jurkat cells and induce IL-2 production because PMA does not influence AKT phosphorylation in Jurkat cells (data not shown). This result is consistent with previously reported mechanisms of regulation for many other AUBPs. For example, TTP is phosphorylated at Ser52 and Ser178 by MAPPK2 and p38. This phosphorylation results in its binding with 14-3-3. TTP loses its mRNA degradation capacity once it binds to 14-3-3 (47, 48, 49, 50). After T cell stimulation by CD28, TTP is phosphorylated by kinases and binds to 14-3-3 (51). This stabilizes the ARE-containing mRNAs such as IL-2 mRNA. BRF1, which contains the same zinc finger protein binding domain as TTP, can be phosphorylated at Ser92 by AKT and stabilizes IL-3 mRNA (46). We also found that phosphorylation of Ser647 was required for NF90 nuclear export, which may explain how phosphorylation of NF90-Ser647 may affect IL-2 mRNA stability (Fig. 1, C and D). Phosphorylation has been shown to regulate nuclear export of some proteins, including NF-AT (41, 42). The protein conformation might be affected upon phosphorylation, and nuclear export of NF90 mediated through the NES sequence located in its N terminus could be facilitated by the conformation change.

In T cells, the expression of IL-2 increases rapidly upon CD28 costimulation due to increased transcription and mRNA stabilization. Even though PMA is another activator of IL-2 production, PMA did not change AKT activity in Jurkat cells (44). We detected significant increase of NF90 phosphorylation in response to CD28 costimulation, which was accompanied by increased AKT phosphorylation. Once AKT was inhibited, phosphorylation of NF90-Ser647 decreased (Fig. 5). This demonstrates the existence of a phosphorylation cascade from CD28 costimulation to AKT and then to NF90. Moreover, we found that both NF90 and NF90-Ser647A also increased IL-2 protein levels 24 h after stimulation with anti-CD3 and anti-CD28. This phenomenon lasted until the addition of CsA to stop IL-2 transcription. The above observation indicates that NF90 itself could increase IL-2 transcription, which was not dependent on Ser647 phosphorylation. NF90 was originally purified as a protein binding to the same regulatory sequence of IL-2 gene as with other NF-AT family members. However, NF90 does not contain the specific sequences for DNA binding. When Jurkat cell lysate was blocked with NF90 Ab in the in vitro transcription assay, the transcription of the reporter gene containing the IL-2 transcription-regulatory elements showed significant down-regulation (35, 36). NF90, NF45, Ku protein, and DNA-dependent protein kinase have been found in a large complex associated with IL-2 gene regulatory region in human bronchial endothelial cells (52). This complex activated IL-2 transcription in response to immune-stimulating signals. We also observed that NF90 and NF90-Ser647A affected CD28 costimulation-induced IL-2 expression differently after CsA addition. This is consistent with our results shown in Fig. 4. Because NF90 could stabilize IL-2 mRNA, IL-2 protein levels continued to increase and remained high even after transcription was interrupted by CsA. Meanwhile, NF90-Ser647A cells secreted less IL-2 than cells transfected with the vector, indicating the inability of NF90-Ser647A to stabilize IL-2 mRNA. This might be attributed to the inhibition of nuclear export of endogenous wild-type NF90 by exogenous NF90-Ser647A, thus hampering the ability of endogenous NF90 to stabilize IL-2 mRNA.

Based on our results, we propose a mechanism of IL-2 mRNA stabilization. CD28 signaling activates AKT to phosphorylate NF90 at Ser647. This phosphorylation might change the protein conformation and facilitate the nuclear export of NF90 mediated through the NES sequence located in its N terminus. Phosphorylated NF90 relocates to and accumulates in the cytoplasm and stabilizes IL-2 mRNA. Nevertheless, further studies are also needed to examine whether there are other sites on NF90 that can also be regulated by CD28 signaling and to better understand how the phosphorylation of NF90-Ser647 stabilizes IL-2 mRNA.


   Acknowledgments
 
We thank Alison Woo for editorial assistance in the preparation of this manuscript. We thank Dr. Glen Barber (University of Miami) for supplying NF90 cDNA and Dr. Brian A. Hemmings from Friedrich Miescher Institute for AKT wild-type plasmid and AKT mutant version. Dr. Zhongzhou Yang from Dr. Hemming’s group also gives us help.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
Disclosures
References
 
The authors have no financial conflict of interest.


   Acknowledgments
 
We thank Alison Woo for editorial assistance in the preparation of this manuscript. We thank Dr. Glen Barber (University of Miami) for supplying NF90 cDNA and Dr. Brian A. Hemmings from Friedrich Miescher Institute for AKT wild-type plasmid and AKT mutant version. Dr. Zhongzhou Yang from Dr. Hemming’s group also gives us help.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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.

1 This work was supported by the National 973 Program of China (2004CB518605), 863 Projects of China (2006AA020501), the Project of the Shanghai Municipal Science and Technology Commission (03dz14086), and the National Natural Science Foundation of China (30024001). Back

2 Address correspondence and reprint requests to Dr. Long Yu, Institute of Genetics, Fudan University, 220 Handan Road, Shanghai 200433, China. E-mail address: longyu{at}fudan.edu.cn Back

3 Abbreviations used in this paper: ARE, AU-rich element; AKT-CA, constitutively active AKT; AUBP, AU-binding protein; CHO, Chinese hamster ovary; CsA, cyclosporin A; CT, threshold cycle; HA, hemagglutinin; IP, immunoprecipitation; UTR, untranslated region; TTP, tristetraprolin; BRF1, butyrate response factor; NES, nuclear export signal; GSK3β, glycogen synthase kinase 3β. Back

Received for publication April 30, 2007. Accepted for publication September 28, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 Disclosures
 References
 

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