Forskolin-inducible cAMP Pathway Negatively Regulates T-cell Proliferation by Uncoupling the Interleukin-2 Receptor Complex*

Background: Within activated T-cells, the binding of IL-2 to its receptor initiates the Jak3/Stat5 cascade culminating in proliferation. Results: Elevated levels of cAMPi within activated T-cells suppresses proliferation by targeting multiple levels of the IL-2R cascade. Conclusion: Cross-talk occurs between cAMP/PKA and the IL-2R/Jak3/Stat5 cascade in human T-cells. Significance: Therapeutic potential exists in manipulating the described cross-talk for the treatment of various immune diseases. Cytokine-mediated regulation of T-cell activity involves a complex interplay between key signal transduction pathways. Determining how these signaling pathways cross-talk is essential to understanding T-cell function and dysfunction. In this work, we provide evidence that cross-talk exists between at least two signaling pathways: the Jak3/Stat5 and cAMP-mediated cascades. The adenylate cyclase activator forskolin (Fsk) significantly increased intracellular cAMP levels and reduced proliferation of the human T-cells via inhibition of cell cycle regulatory genes but did not induce apoptosis. To determine this inhibitory mechanism, effects of Fsk on IL-2 signaling was investigated. Fsk treatment of MT-2 and Kit 225 T-cells inhibited IL-2-induced Stat5a/b tyrosine and serine phosphorylation, nuclear translocation, and DNA binding activity. Fsk treatment also uncoupled IL-2 induced association of the IL-2Rβ and γc chain, consequently blocking Jak3 activation. Interestingly, phosphoamino acid analysis revealed that Fsk-treated cells resulted in elevated serine phosphorylation of Jak3 but not Stat5, suggesting that Fsk can negatively regulate Jak3 activity possibly mediated through PKA. Indeed, in vitro kinase assays and small molecule inhibition studies indicated that PKA can directly serine phosphorylate and functionally inactivate Jak3. Taken together, these findings suggest that Fsk activation of adenylate cyclase and PKA can negatively regulate IL-2 signaling at multiple levels that include IL-2R complex formation and Jak3/Stat5 activation.

Interleukin-2 receptor (IL-2R) 2 signaling in T-cells provides a necessary third signal for driving cellular proliferation during an immune response. The transmission of a proliferative signal by IL-2 requires a heterotrimeric receptor complex comprised of ␣, ␤, and common ␥ (␥ c ) chains. Subsequently, Janus tyrosine kinase 1 (Jak1) and Jak3 are recruited to the ␤ and ␥ c chains, respectively, allowing for autoactivation of both proteins. Activated Jaks then phosphorylate tyrosine residues on the IL-2R␤ chain, creating docking sites for downstream effectors, including signal transducer and activator of transcription 5a/b (Stat5a/b), which associate via their Src homology 2 domains (1). Stat5a/b become phosphorylated on Tyr 694 /Ser 726 /Ser 780 and Ser 193 /Tyr 699 /Tyr 725 /Ser 731 /Tyr 740 /Tyr 743 , respectively, dissociate from the receptor, form hetero-or homodimers, and translocate to the nucleus where they mediate transcription of genes important for cell growth and differentiation (2)(3)(4)(5)(6)(7)(8). Conversely, this signaling pathway is negatively regulated by three families of proteins, suppressor of cytokine signaling, protein tyrosine phosphatases, and protein inhibitors of activated Stats (PIAS), which target different levels within the Jak/Stat signaling cascade (reviewed in Ref. 9).
T-cell activity is also tightly controlled by other effectors, including Mapk and PI3K pathways, as well as non-protein signaling molecules such as cAMP (10). cAMP is produced by adenylate cyclase (AC), a 12-transmembrane-spanning enzyme that catalyzes the conversion of ATP to 3Ј,5Ј-cAMP and pyrophosphate (11). Cellular phosphodiesterases then degrade newly synthesized cAMP by hydrolyzing the phosphodiester bonds to produce 5ЈAMP (12). While cAMP has multiple functions including activation of cyclic nucleotide-gated (CNG) ion channels (13) and exchange protein activated by cAMP (EPAC) (14), in T-cells it predominately exerts its effects through Protein kinase A (PKA) (15). PKA is a ubiquitous serine/threonine kinase comprised of two catalytic and two regulatory subunits. Interestingly, cAMPi levels can be elevated via addition of forskolin (Fsk), phosphodiesterase inhibitors, prostaglandin E2, or adenosine, which results in suppression of T-cell proliferation (16 -18). Although the antiproliferative effects of cAMP within cells of the immune system are well documented (19 -23), the mechanism of action is less clear. It is tempting to speculate that cAMP-mediated effectors can suppress a T-cell proliferative signaling pathway such as Jaks and Stats. Nearly a decade earlier, Zhang et al. (16) demonstrated that adenosine, a cAMP activator, can suppress T-cell proliferation that may involve Stat5 but not Jak3.
Similarly, previous studies have reported that high intensity activation of the TCR can inhibit IL-2, IL-4, IL-6, and IFN-␣ signaling in murine T-cells by uncoupling Erk and Mek (24). Ivashkiv and co-workers (25) have also shown that inhibition of cytokine signaling was independent of new protein synthesis not dependent on suppressor of cytokine signaling, but implicated Stat5, Jak3, and Akt as possible targets. His work suggests that PKC/Mek/Erk signaling may uncouple this pathway (25). However, a role for PKA was not investigated or linked to negative regulation of these cytokine-dependent pathways.
The objective of this study was to determine the role cAMP-PKA plays in regulating IL-2R signaling, how it affects T-cell activity, and consequently immune function. Evidence is provided that Fsk activation of AC/cAMP/PKA disrupts IL-2R complex formation, Jak3 catalytic activity, Stat5 signaling, and expression of cell cycle-dependent genes. This work suggests that a novel cross-talk pathway dependent on cAMPi can negatively affect T-cell proliferation and Jak/Stat signaling.

EXPERIMENTAL PROCEDURES
Cell Culture and Treatment-The human leukemic cell line MT-2 (26) and human embryonic kidney cells 293 (Hek293) were maintained as described previously (27). Human IL-2 dependent leukemic Kit 225 cells (28) were similarly maintained but supplemented with 100 IU/ml human recombinant IL-2 (NCI Preclinical Repository). Prior to cell treatment with Fsk or 3-isobutyl-1-methylxanthine (IBMX), MT-2 cells were made quiescent by growing them to exhaustion or culturing them in 1% FBS overnight at 37°C. Kit 225 cells were made quiescent by CO 2 washes and subsequently culturing in 10% FBS overnight at 37°C. Quiescent cells were subsequently treated with the indicated concentrations of Fsk (Calbiochem), IBMX (Sigma-Aldrich), or anti-CD3 monoclonal antibody (Santa Cruz Biotechnology) and cultured at 37°C for different amounts of time as indicated. Control cells were treated with 1% dimethyl sulfoxide (DMSO, vehicle) and cultured as treated cells. Following pre-treatment, cells were stimulated with 1 ϫ 10 4 IU IL-2 at 37°C for the times shown. MT-2 cells exposed to ultraviolet (UV) light were subjected to 120,000 mJ/cm 2 /min for 1 min prior to the final 5 h of incubation.
Proliferation Assays-Quiescent Kit 225 or MT-2 cells were seeded into 96-well plates at 5 ϫ 10 4 cells per well. Cells were then pretreated for 1 h with 1% DMSO (vehicle) or Fsk at 1, 5, 10, 25, 50, and 100 M concentrations. The cells were stimulated with IL-2 as above and cultured for an additional 20 h at 37°C. Control cells were treated with 1% DMSO for 20 h. During the final 4 h of incubation, the cells were pulsed with [ 3 H]thymidine (PerkinElmer Life Sciences) at a concentration of 0.5 Ci/200 l. Cells were harvested onto fiberglass filters and analyzed using liquid scintillation counting.
cAMP Production Assay-Kit 225 or MT-2 cells were treated with 1, 5, 10, 20, 50, or 100 M Fsk for 20 min at 37°C. Cells were lysed and clarified by centrifugation, and concentration of cAMP was detected by direct cAMP ELISA (Assay Designs). Optical density was measured at 405 nM, and the concentration of intracellular cAMP was calculated using a weighted four parameter logistic curve according to the manufactures instructions.
In Vitro Kinase Assays-For Jak3 kinase assays, Fsk-treated MT-2 cells were lysed, clarified, and immunoprecipitated using Jak3 antibody as described above. Kinase reactions were carried out as described previously (31) at 30°C for 20 min. For PKA kinase assays, untreated MT-2 cells were lysed, and Jak3 was immunoprecipitated and bound to PAS beads as described previously (31). Immunoprecipitated Jak3 was washed with kinase buffer (50 mM Hepes-NaOH (pH 7.4), 10 mM MgCl 2 , 0.5 mM EGTA, 0.5 mM DTT, 20 g/ml aprotinin, 10 g/ml leupeptin, 1 g/ml pepstatin A) and incubated with 200 M ATP and purified protein kinase A catalytic subunit (PKAc) (Invitrogen) as indicated in the figure legends. Kinase reactions were carried out at 32°C for 30 min followed by vigorous washing of the beads with cold kinase wash buffer as described previously (31). For [␥-32 P]ATP radiolabeled kinase assays using recombinant Jak3, Hek293 cells were transfected with wild type (WT) Jak3 or kinase-dead Jak3 K855A (31) using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen). Cells were lysed and immunoprecipitated with Jak3 antibody (described above). Jak3-bound PAS beads were washed three times in cold lysis buffer (described above) followed by kinase buffer. Kinase reactions were initiated by adding 10 Ci [␥-32 P]ATP, 10 M unlabeled ATP, and 1 g of purified PKAc (Invitrogen) to Jak3bound PAS bead reaction mixtures. Kinase reactions were performed at 32°C for 30 min. Jak3-bound PAS beads were washed three times in radioimmunoassay buffer (10 mM Tris-HCl, pH 7.4, 75 mM NaCl, 20 mM EDTA, 10 mM EGTA, 20 mM Na 4 P 2 O 7 , 50 mM NaF, 20 mM 2-glycerolphosphate, 1 mM p-nitrophenylphosphate, 0.1% Triton X-100) and one time in kinase wash buffer (described above). The reactions were stopped by adding 2ϫ SDS-PAGE sample buffer followed by SDS-PAGE. Coomassie stainable Jak3 bands were excised from the PVDF membrane and subjected to phosphoamino acid analysis.
[ 32 P]Orthophosphate Labeling and Phosphoamino Acid Analysis-Jak3 and Stat5b obtained from MT-2, Kit 225, or Hek293 (transfected with Jak3) were metabolically labeled, separated by SDS-PAGE, transferred to PVDF membrane, and visualized using autoradiography. Coomassie blue staining or Western blotting was used to determine total protein; bands corresponding to Jak3 and Stat5b were excised from the membranes and subjected to two-dimensional phosphoamino acid analysis as described previously (33).
Nuclear Extract and Electrophoretic Mobility Shift Assays-Nuclear extracts were prepared from MT-2 cell pellets as reported previously (34). Oligonucleotide sequences corresponding to the ␤-casein promoter (5Ј-AGATTTCTAG-GAATTCAATCC-3Ј) were purchased from Santa Cruz Biotechnology.
RT 2 Profiler PCR Array-Kit 225 cells were treated with 1% DMSO (vehicle) or 100 M Fsk for 1 h prior to stimulation with IL-2 as above for 30 h at 37°C. Total RNA was isolated from ϳ4 ϫ 10 6 cells using the RNeasy kit (Qiagen) and then were DNase-treated (Qiagen RNase-free DNase set). Complementary DNA was synthesized using RT 2 first strand kit (SA Biosciences) as recommended by the manufacturer from 2 g of total RNA/each sample/plate. SA Biosciences' human cell cycle PCR array (PAHS-020) was used according to the manufacturer's instructions. Quantification based on real-time monitoring of amplification was determined using a Bio-Rad iQ5 machine and 2ϫ SYBR Green Mastermix (SA Biosciences). Samples were run in 25-l reaction volumes. Treatments were performed in duplicate, and data were analyzed using the ⌬⌬C t method normalized to the average of four housekeeping genes (B2M, RPL13A, GAPDH, and ACTB) using SA Biosciences' data analysis web tool. Genes with C t Ͼ 32 were ignored in the analysis.

Forskolin-induced cAMPi Inhibits T-cell Proliferation and Disrupts Expression of Cell Cycle Progression Genes-Published
reports have extensively documented the inhibitory effects of adenylate cyclase linked G protein-coupled receptor agonists on lymphocyte proliferation (16,19,21,35). To identify the molecular mechanism responsible for this effect, we first sought to determine whether Fsk, a potent adenylate cyclase activator, affects the proliferation of the human T-cell lines such as Kit 225 ( To further support the notion that Fsk-mediated inhibition of MT-2 and Kit 225 cell proliferation was due to an increase in cAMPi, cAMPi generation was measured using an ELISA. Indeed, Fsk treatment (10 -100 M) increased cAMPi levels ϳ5to 20-fold above basal levels, which reached maximum levels between 50 -100 M Fsk (Fig. 1D).
Several reports have indicated that cAMPi can block expression of cell cycle genes (37,38). To determine whether the reduction in T-cell proliferation was due to Fsk similarly affecting cell cycle regulators, these genes were analyzed (Fig. 1E) by quantitative RT-PCR. Indeed, cell cycle-dependent genes, CCNB1 and CCNB2, as well as the cell cycle checkpoint genes CDKN2A, CDKN2B, and CDKN3, were found to be down-regulated following Fsk treatment of Kit 225 cells. Expression of the G 2 /M transition cell cycle regulatory genes GTSE1 and BIRC5 were similarly down-regulated. Interestingly, the p53 tumor suppressor gene, TP53, was shown to be up-regulated. Similar results were obtained for MT-2 cells (data not shown). These data suggest that cAMP can inhibit cell cycle gene expression, which may serve as a possible inhibitory mechanism of cell proliferation.
Forskolin-cAMPi Inhibits IL-2-induced Stat5 Activation, Translocation, and DNA Binding-One possibility for the reduced expression of these critical cell cycle genes could be due to uncoupled cytokine signals. Stat5 has been shown to be critical for T-cell expansion and survival in this signaling cascade by targeting G 1 /S transition and anti-apoptotic genes (34,39,40). To determine whether Fsk-cAMPi-activated pathways disrupted IL-2 activation of Stat5, its tyrosine phosphorylation status was examined by Western blot. For this analysis, MT-2 cells were treated with increasing concentrations of Fsk and Stat5b tyrosine phosphorylation monitored ( Fig. 2A, upper  panel). Indeed, Fsk inhibited Stat5b tyrosine phosphorylation with an IC 50 ϳ10 M Fsk without effecting Stat5b expression ( Fig. 2A). This experiment was repeated in triplicate, and the densitometry of tyrosine phosphorylated Stat5 normalized to total Stat5 was plotted ( Fig. 2A, lower panel). Similar results were observed for Stat5a (data not shown). To determine the kinetics of Fsk-mediated Stat5b inactivation, MT-2 cells were pretreated with 100 M Fsk for various time points prior to IL-2 stimulation (10 min). As shown in Fig. 2B, 80% inhibition of Stat5b tyrosine phosphorylation occurred by 10 min, whereas maximal inhibition occurred after 60 min (Fig. 2B, lane h). Tyrosine phosphorylation of Stat5 is required for optimal dimerization, nuclear translocation, and DNA binding (3,4,41). To determine whether Fsk-cAMPi activated pathways disrupt this event, Western blot analysis of cytoplasmic and nuclear protein fractions from MT-2 cells before and after IL-2 stimulation were investigated (Fig. 2C). Both nuclear and cytoplasmic tyrosine phosphorylated Stat5 and nuclear Stat5 protein levels were reduced following Fsk treatment compared with vehicle-treated cells (Fig. 2C, lanes d and h). Similar results were confirmed by Stat5 EMSA analysis (Fig. 2D), where 100 M Fsk inhibited IL-2 induced Stat5 DNA binding to a radiolabeled ␤-casein promoter by ϳ60% (Fig. 2D, lane d). These data support the notion that Fsk-induced cAMPi can disrupt IL-2mediated Stat5 tyrosine phosphorylation, nuclear translocation, and DNA binding activity.
Forskolin-cAMPi Inhibits IL-2 Activation of Jak3-Following IL-2 binding to its cognate receptor, Jak3 is activated and com- MARCH 8, 2013 • VOLUME 288 • NUMBER 10 JOURNAL OF BIOLOGICAL CHEMISTRY 7139 petent to tyrosine phosphorylate substrates such as the IL-2R␤, ␥ c , and Stat5. Because Jak3 is critical for Stat5 activation, cAMPi may exert its effects by blocking Jak3 activity. To test this model, MT-2 cells were pretreated with Fsk and Jak3 tyrosine phosphorylation status examined. Anti-phosphotyrosine Western blot revealed that Jak3 tyrosine phosphorylation was inhibited by 80% after Fsk pretreatment (Fig. 3A, lane c) when replicated, and data were normalized from three separate experiments (Fig. 3A, graph). Phosphorylation of Tyr 980 , Tyr 981 , and Tyr 939 have been shown to regulate Jak3 enzymatic activity (31,42). Given the importance of Tyr 939 for Stat5 association, we examined Fsk treatment on Jak3 Tyr 939 phosphorylation (Fig. 3B). Indeed, Fsk treatment completely abrogated Jak3 Tyr 939 phosphorylation (Fig. 3B, lane c) as compared with control (Fig. 3B, lane b). Because Tyr 939 phosphorylation is also critical for Jak3 activity, its enzymatic status was tested using the GST-tagged cytoplasmic portion of ␥ c as a substrate (31). As shown in Fig. 3C, Fsk treatment inhibited Jak3 tyrosine phosphorylation of ␥ c in a dose-dependent manner with an IC 50 between 1 and 10 M. These results closely parallel those Fsk concentrations required to reduce T-cell proliferation (Fig. 1B).

Forskolin/cAMP Uncouples Interleukin-2 Receptor Signaling
cAMPi Elevators Induce Jak3 Serine Phosphorylation but Disrupt IL-2-inducible Stat5 Serine Phosphorylation-The positive regulatory role of tyrosine phosphorylation of IL-2R signaling proteins has been well documented (3,42,43), and many also undergo serine phosphorylation to positively or negatively regulate their activity (8, 44 -46). The role of serine phosphorylation within IL-2R signaling, however, is not fully established. Because cAMPi most notably activates the serine-threonine protein kinase PKA (10), we sought to determine the effects of   and c) at 37°C. Immunoprecipitated Jak3 protein was Western blotted using anti-phosphotyrosine (pY) antibody (upper panel) or total Jak3 (lower panel). Densitometric analyses were carried out (n ϭ 3), and tyrosine phosphorylation levels of Jak3 were normalized to total Jak3 protein and plotted (lower panel). B, cells were treated as described in A and Western blotted with anti-phosphotyrosine 939 (pY939). C, MT-2 cells were treated with increasing concentrations of Fsk for 40 min prior to stimulation with 100 nM IL-2 for 10 min. Immunocaptured Jak3 was used for in vitro kinase assays with GST-␥ c as a substrate and Western blotted with anti-phosphotyrosine or anti-GST antibodies. Densitometric analysis was performed, and tyrosine phosphorylation of GST-␥ c was normalized to total GST expression. The results are presented as percentage of IL-2 activated Jak3 activity in the absence or presence of Fsk.
Fsk treatment on Jak3 and Stat5b serine phosphorylation by phosphoamino acid analysis (Fig. 4). Parallel mass spectrometry analysis was used to confirm the purity of Jak3 and Stat5b proteins (data not shown). Indeed, Fsk-cAMPi induced Jak3 serine phosphorylation (Fig. 4A, panel c) as compared with control (Fig. 4A, panel b). In contrast, IL-2-inducible Stat5b serine phosphorylation was inhibited by Fsk treatment (Fig. 4B, panel  c) but not untreated Stat5b (Fig. 4B, panel b). It should be noted that under the hydrolysis conditions used to maximize the detection of phosphoserine, the more labile tyrosine and threonine phosphorylation is lost. Nevertheless, one possible mech-anism by which cAMPi disrupts Jak3 catalytic activity may occur via serine phosphorylation. To determine whether Fskinduced Jak3 serine phosphorylation was confined to the human lymphotropic virus-1 transformed MT-2 cells, we next examined non-virally transformed T-cells, Kit 225. As was the case in MT-2 cells, Fsk induced Jak3 serine phosphorylation (Fig. 4C, upper panel, panel b), which could be uncoupled with the PKA inhibitor KT5720 prior to Fsk treatment (Fig. 4C,  upper panel, panel c). These data suggest that PKA is an important mediator within the Fsk-cAMPi cascade to promote Jak3 serine phosphorylation. To further substantiate this notion, cells were treated with the nonspecific phosphodiesterase inhibitor, IBMX, which can elevate cAMPi by preventing its degradation. As shown in Fig. 4C (upper panel, panel d) IBMXpretreated MT-2 cells in the absence of Fsk also induced Jak3 serine phosphorylation (representative data from two independent experiments). Interestingly, treatment of Kit 225 cells with anti-CD3 monoclonal antibody also increased Jak3 serine phosphorylation (Fig. 4C, upper panel, panel e).
To determine whether PKA is capable of directly phosphorylating serine residues on Jak3, a radiolabeled in vitro kinase assay using purified PKAc and wild type (WT) Jak3 or catalytically inactive Jak3 (K855A) was performed in conjunction with phosphoamino acid analysis. Under these conditions, WT Jak3 showed an increase in total phosphorylation in the presence of PKAc (Fig. 5A, lower panel, lane d). Subsequent phosphoamino acid analysis revealed a 5.8-fold increase in serine and a 2-fold decrease in tyrosine autophosphorylation in the presence of PKAc (Fig. 5A, upper panel, panel b). Interestingly, threonine phosphorylation was not detected on WT Jak3 (lane c). To confirm that Jak3 tyrosine and serine phosphorylation were due to WT Jak3 and PKAc, respectively, catalytically inactive Jak3, K855A, was tested. For this analysis, autoradiography revealed that PKAc increased serine phosphorylation of catalytically inactive Jak3 5-fold (Fig. 5A, lower panel, lane f), whereas threonine and tyrosine phosphorylation were not detected (Fig. 5A,  upper panel, lane d). These data suggest that Jak3 can be inhibited by a Fsk-cAMPi-PKA pathway via serine phosphorylation. For this analysis, GST-␥ c was used as a substrate, and Jak3 inactivation by increasing concentrations of PKAc was monitored. As shown in Fig. 5B, as little as 0.1 g of PKAc disrupted the ability of Jak3 to tyrosine phosphorylate the GST-␥ c substrate.
Multiple lots of PKAc were tested and revealed a similar dose response in disrupting Jak3 catalytic activity. Normalized data from three separate experiments are shown in Fig. 5B, lower panel. These results suggest that PKA can phosphorylate Jak3 serine residues and negatively regulate its catalytic activity.
Fsk-cAMPi Disrupt IL-2 Receptor Subunit Association-IL-2 binds to the IL-2R␣ (55 kDa, K d of 10 Ϫ8 M), which allows for IL-2R␤ (75 kDa, K d of 10 Ϫ9 M) to bind and finally for the recruitment of the ␥ c (65 kDa, K d of 10 Ϫ11 M) (4,47). Cytoplasmically localized Jak1 and Jak3 bind to the IL-2R␤ and ␥ c , respectively, allowing for Jak autophosphorylation (9) and subsequent tyrosine phosphorylation of the receptor subunits. This in turn promotes Stat5 binding and subsequent tyrosine phosphorylation. To determine whether the loss of Stat5b tyrosine phosphorylation mediated by Fsk-cAMPi (Fig. 2) was due to inactivated IL-2R complex, the following experiment was performed. MT-2 cells were either untreated or treated with Fsk (100 M for 1 h) prior to stimulation with IL-2, and receptor association was analyzed. The IL-2R␤ chain was immunoprecipitated and subjected to SDS-PAGE and Western blotted with antibodies directed to IL-2R␤ or ␥ c (Fig. 6). Western blot analysis revealed that Fsk-cAMPi blocked IL-2R␤ and ␥ c association by ϳ60% (Fig. 6, lane d), which was confirmed in triplicate with densitometry (Fig. 6, lower panel). Reverse co-immunoprecipitation by first capturing the ␥ c subunit to detect IL-2R␤ by Western blot yielded similar results (data not shown). These data indicate Fsk-cAMPi can disrupt IL-2R␤/␥ c association in the presence of IL-2 as another mechanism to uncouple cytokine activation of Jak/Stat signaling.

DISCUSSION
Immune system function is dependent on positive and negative regulation of T-cell signaling pathways, including the Jak3/Stat5 cascade. These key signaling proteins have been shown to be hyperactivated in various hematopoietic cancers, including certain lymphomas and leukemias (48 -50), and therefore represent therapeutic targets for treating various diseases. In this study, IL-2 activation of Stat5 (Fig. 2) and Jak3 (Fig.  3) was shown to be inhibited by elevated cAMP levels induced by Fsk in a dose-dependent manner, which resulted in a loss of T-cell proliferation (Fig. 1). Multiple cell cycle genes were identified as possible targets (Fig. 1E), including the gene for KD-67, a protein present during all phases of the cell cycle except G 0 (51). It is likely that cAMPi accumulation does not allow T-cells to exit the G 0 phase. This hypothesis is supported by a similarly identified and reported case where cAMP induced an anergiclike state in Th1 cells (52).
The reason for the loss of Jak3/Stat5 activity appears to be the result of uncoupling of the IL-2R complex and disruption of IL-2-mediated association of the IL-2R␤ and ␥ c (Fig. 6). Although the mechanism by which this occurs is currently under investigation by our lab, it is tempting to speculate that cAMP activates a kinase that phosphorylates the ␤ or ␥ c chains, which disrupts their functional association. This may represent the first level of IL-2R uncoupling (Fig. 7a). Within this same level, cAMP results in Jak3 serine phosphorylation that suppresses its catalytic activity (Fig. 7b) and ability to phosphorylate substrates such as Stat5 (Fig. 7c) and GST-␥ c (Fig. 3C). It is important to note that Jak3 becomes stabilized as part of the IL-2R complex over time (53), and thus, a loss of Jak3-mediated stabilization of the receptor complex may account for the reduced IL-2R␤/␥ c association following Fsk treatment in this study. PKA may represent a key kinase in this cascade as it was  -f). Jak3-bound beads were then washed free of PKAc and used in a second in vitro kinase assay with GST-␥ c substrate. The upper panel shows Western blots of the latter in vitro kinase reaction using anti-phosphotyrosine antibody to assess ␥ c tyrosine phosphorylation and reblotted for total GST (lower panel). Each error bar represents the mean Ϯ S.D. of three independent experiments. ␥ c tyrosine phosphorylation normalized to GST is plotted below.  c and d). Cells were lysed, immunoprecipitated for IL-2R␤, separated by SDS-PAGE, and Western blotted using antibodies against ␥ c or IL-2R␤ (upper panel). IL-2R␥ band intensities were normalized to IL-2R␤ levels using densitometric analysis and presented as percentage of IL-2R association (lower panel). Representative data from three independent experiments are shown. activated by Fsk or IBMX and the event inhibited by KT5720 (Fig. 4). Indeed, PKA could directly serine phosphorylate Jak3 and disrupt its catalytic activity (Fig. 5). The identification and validation of putative PKA consensus sites within Jak3 are currently under investigation. As such, PKA or a PKA-dependent pathway may act to negatively regulate the IL-2R complex (Fig.  6), Jak3 catalytic activity (Fig. 5), and Stat5 (Figs. 2 and 4). Attempts to deplete PKA via shRNA and other approaches did not reveal significant protein knockdown, making confirmation difficult.
Previous studies have identified a mechanism by which AC activity modulates T-cell responses. Activation of the TCR complex and Ca 2ϩ mobilization are known to activate AC (54). Early events in T-cell activation require the intracellular accumulation of calcium to activate key signaling pathways. Human T-cells and lymphoid cell lines, including MT-2 and Kit 225 cells, express AC III 3 known to be activated by calcium (55). Similarly, other groups have shown that high avidity TCR engagement inhibits T-cell proliferation by blocking activation of Stat5, Jak1, and Jak3 mediated by IL-2 or IL-4 (25). Similar to our results, TCR ligation blocked activation of cell cycle regulatory proteins. Inhibition of IL-4 signaling following TCR ligation was shown to be dependent on PKC, Erk, and Ca 2ϩ signaling through calcineurin in murine T-cells (24,56). We found that activation of the TCR complex by anti-CD3 cross-linking induced serine phosphorylation of Jak3 (Fig. 4C) perhaps mimicking the Fsk effect. In addition to Mek/Erk and Ca 2ϩ signaling, PKA may represent a possible cross-talk between these pathways and the Jak/Stat cascade within T-cells.
Physiologically, inhibition of T-cell proliferation is needed at the culmination of a successful immune response. Because Jak3 is not expressed in naïve or resting T-cells, the inhibitory effects of cAMP on IL-2R signaling may be a mechanism for downregulating T-cell activity. Indeed, for primed T-cells, TCR signaling/Ca 2ϩ mobilization (signal 1 and 2) would inhibit IL-2R signaling (signal 3) and possibly initiate activation-induced cell death via Ca 2ϩ mobilization/activated AC/PKA. This is possible because the Jak3/Stat5 pathway is critical for delivering T-cell survival signals. The finding that TCR activation induces cAMP production in T-cells (57) may support this hypothesis. A second possible physiological mechanism is that T regulatory cells would suppress autoreactive T-cells by inducing activation-induced cell death or anergy. Indeed, it has been reported that T regulatory cells maintain high levels of intracellular cAMP and can transfer it through gap junctions into CD4 ϩ T-cells (58), whereas others have reported that T-cells enter an anergic state in response to cAMP (52).
The findings presented herein provide new insights into the mechanism underlying immunosuppression in disease, including human immunodeficiency virus (HIV) infection and severe combined immunodeficiency (SCID) disorder. Indeed, it has been reported that replication of HIV requires cAMP and increases intracellular cAMP concentrations (59,60). Thus, uncoupling the Jak3/Stat5 pathway may be one possible mechanism by which HIV may inhibit T-cell function. Although SCID can result from mutations in Jak3 or the ␥ c subunit, this phenotype can also be manifested in individuals with an adenosine deaminase deficiency. Because adenosine binds an AClinked GPCR that can prevent IL-2 induced phosphorylation of Stat5 (16) and lymphocyte proliferation (61), it is tempting to speculate that cAMP mediates this process. Further identification and characterization of putative serine residues targeted by PKA in the IL-2R complex remain to be identified and possibly hold new strategies to control T-cell-mediated diseases. In summary, this work demonstrates that elevated cAMPi can disrupt IL-2R complex formation, Jak3 catalytic activity, and its ability to phosphorylate Stat5 and transcriptionally responsive Stat5 genes (Fig. 7), resulting in a loss of T-cell proliferation.