Analysis of gamma c-Family Cytokine Target Genes

IDENTIFICATION OF DUAL-SPECIFICITY PHOSPHATASE 5 (DUSP5) AS A REGULATOR OF MITOGEN-ACTIVATED PROTEIN KINASE ACTIVITY IN INTERLEUKIN-2 SIGNALING*

Panu E. KovanenDagger §, Andreas Rosenwald§||**, Jacqueline FuDagger , Elaine M. Hurt||, Lloyd T. Lam||, Jena M. Giltnane||, George WrightDagger Dagger , Louis M. Staudt||§§, and Warren J. LeonardDagger §§¶¶

From the Dagger  Laboratory of Molecular Immunology, NHLBI, National Institutes of Health, the || Metabolism Branch, Center for Cancer Research, and the Dagger Dagger  Biometric Research Branch, National Cancer Institute, Bethesda, Maryland 20892

Received for publication, September 4, 2002, and in revised form, November 6, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Interleukin (IL)-2, IL-4, IL-7, IL-9, IL-15, and IL-21 form a family of cytokines based on their sharing the common cytokine receptor gamma  chain, gamma c, which is mutated in X-linked severe combined immunodeficiency (SCID). As a step toward further elucidating the mechanism of action of these cytokines in T-cell biology, we compared the gene expression profiles of IL-2, IL-4, IL-7, and IL-15 in T cells using cDNA microarrays. IL-2, IL-7, and IL-15 each induced a highly similar set of genes, whereas IL-4 induced distinct genes correlating with differential STAT protein activation by this cytokine. One gene induced by IL-2, IL-7, and IL-15 but not IL-4 was dual-specificity phosphatase 5 (DUSP5). In IL-2-dependent CTLL-2 cells, we show that IL-2-induced ERK-1/2 activity was inhibited by wild type DUSP5 but markedly increased by an inactive form of DUSP5, suggesting a negative feedback role for DUSP5 in IL-2 signaling. Our findings provide insights into the shared versus distinctive actions by different members of the gamma c family of cytokines. Moreover, we have identified a DUSP5-dependent negative regulatory pathway for MAPK activity in T cells.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The common cytokine receptor gamma c chain (gamma c)1 is essential for normal immune development and function. Mutations in gamma c result in X-linked severe combined immunodeficiency (XSCID) (1), a disease in which affected individuals are highly susceptible to infections resulting from the defective development of T and NK cells and nonfunctional B cells. gamma c is a component of the receptors for multiple cytokines, including IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 (2). These cytokines have both redundant and distinctive actions on lymphocytes. IL-2 is a growth and survival factor for activated T lymphocytes and is also essential for activation-induced cell death (AICD) and the prevention of T cell anergy, processes involved in the maintenance of peripheral tolerance (3). IL-4 is also a T-cell growth factor and is necessary for the development of Th2 cells (4), which regulate humoral immune responses, whereas IL-7 is required for the development and growth of T lymphocytes but can also support the growth of peripheral T lymphocytes (5-7). IL-15 has overlapping functions with IL-2, but, unlike IL-2, IL-15 is essential for NK-cell differentiation and is more important than IL-2 in the generation of CD8+ memory T cells (8). Although transgenic expression of IL-9 causes thymomas, suggesting that IL-9 can regulate lymphocyte growth (9), IL-9 knock-out mice do not manifest abnormalities in T-cell development or function but exhibit defective mast cell proliferation and mucus production (10). Based on in vitro studies, IL-21 has potential roles for T-cell, B-cell, and NK-cell biology (11).

Only limited information is available regarding the genes that are activated by gamma c-dependent cytokines. To investigate the basis for overlapping and distinctive actions of gamma c-dependent cytokines, we used the "Lymphochip" microarray, a specialized cDNA array enriched for genes expressed in lymphocytes (12), to identify genes regulated by IL-2, IL-4, IL-7, and IL-15 in peripheral blood T lymphocytes. IL-2, IL-7, and IL-15 regulated most genes in a similar manner, whereas the pattern seen with IL-4 was more distinctive. Although most gamma c-dependent genes are redundantly regulated by various stimuli, certain cytokine-specific patterns of gene expression were noted. One gene induced by IL-2, IL-7, and IL-15 but not IL-4 was dual-specificity phosphatase 5 (DUSP5) (13, 14). IL-2 is known to activate several signaling pathways, including Ras-MAP kinase, Jak-STAT, and phosphoinositol 3-kinase/Akt/p70 S6 kinase pathways (15), and we now provide evidence for a role for DUSP5 as part of a negative feedback loop that controls IL-2-induced MAPK activity in T cells. This is the first demonstration of a role for DUSP5 in T-cell biology and identifies a mechanism by which IL-2-mediated MAPK activation can be controlled.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell Cultures-- Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll (Amersham Biosciences) density gradient centrifugation, stimulated for 18 h with phytohemagglutinin (PHA, Roche Molecular Biochemicals, 2 µg/ml), and expanded for 2 weeks in RPMI 1640 medium containing 10% fetal bovine serum, 100 units/ml penicillin and streptomycin, and 2 mM glutamine (complete RPMI medium) supplemented with PHA (500 ng/ml) and IL-2 (50 units/ml). The cells were washed twice and resuspended in complete medium without PHA or IL-2 for 3 days. The purity of T cells was evaluated by flow cytometry, and only cultures with >95% CD3+ T cells were used for further studies. Cells were then not stimulated or stimulated with IL-2 (100 units/ml), IL-4 (100 units/ml), IL-7 (200 units/ml), or IL-15 (200 units/ml) for 4 h. We also separately performed more detailed time course experiments (0.5, 2, 4, 6, or 8 h) with IL-2 and IL-4. In some instances, PBMCs were stimulated for 1, 3, 6, or 24 h with phorbol 2-myristate 3-acetate (PMA) (50 ng/ml) and ionomycin (1.5 µM) (PI).

B lymphocytes (>98% pure by flow cytometry) were isolated from PBMCs by negative magnetic selection using StemCell Technologies human B-cell enrichment mixture and cultured for 0, 1, 3, 6, or 24 h at 5 × 106 cells/ml in complete RPMI medium containing 50 µg/ml anti-IgM (Jackson Laboratories).

Cell Lines and Chronic Lymphocytic Leukemia (CLL) Samples-- Cell lines (Jurkat, Ramos, SUDHL10, L428, MCF-7, and PC-3) were cultured in complete RPMI medium. OCI-Ly8 cells were grown in Iscove's modified Dulbecco's modified Eagle's medium in the presence of 20% human plasma and 1% penicillin/streptomycin. CLL cells were obtained from untreated patients, and CD19+ cells were purified by magnetic selection.

RNA Isolation-- For cDNA microarray analyses, mRNA was prepared using the Fast-Track kit (Invitrogen). Total RNA was isolated using the Trizol reagent (Invitrogen).

cDNA Microarray Analyses-- Microarray analysis was performed as described in the "Microarray Procedures" section under "Methods" in Ref. 16, using Lymphochips that contained either 7,296 or 12,672 array elements. RNA samples from cytokine-stimulated T cell cultures were reverse transcribed, labeled with Cy5, and these probes were hybridized to microarrays together with Cy3-labeled probes generated from nonstimulated control cultures. Cy5-labeled probes were also generated from T cells stimulated with IL-2 or IL-4 for various time periods, PBMCs activated with PI, B-cells stimulated with anti-IgM beads, and cell lines and CLL cells. These were hybridized together with Cy3-labeled cDNA generated from a common reference mRNA pool (16). This allowed us to compare the relative expression level of a given gene across all of our experiments. Microarrays were analyzed on a GenePix scanner (Axon Instruments), and data files were entered into a custom data base maintained at the National Institutes of Health (nciarray.nci.nih.gov). We used a standard global normalization approach for our expression data analogous to that previously used (16, 17). We extracted data for clustering analysis (programs "Cluster" and "TreeView") (19) that fulfilled the following requirements: spot size of at least 25 µm, minimum intensities of 100 relative fluorescent units (RFU) in the Cy3 and Cy5 channels or minimum intensity of at least 1000 RFU in one of the two channels. The 100 RFU criteria are as previously used for the Lymphochip and Axon 4000A scanner (17). Unless stated otherwise, the expression of a gene was considered induced or repressed if the median induction or repression was at least 2-fold with any one cytokine in at least two of three experiments (five experiments were done with IL-2, three with IL-4, four with IL-7, and 3 three with IL-15).

Northern Blotting and Real Time PCR-- Total RNA (15 µg/lane) was northern blotted using PCR-amplified inserts of the following IMAGE clones: 176940, 203132, 1336478, 714453, 342378, 1304437 for Mal, TRAIL, MAPKAPK3, IL-4Ralpha , DUSP5, and TSC-22R, respectively, as well as probes for IL-2Ralpha (X01075), and pHe7 (which corresponds to a housekeeping gene). Real time PCR was performed using a Light Cycler. 10 ng of total RNA was amplified for 40 cycles with either DUSP5 (sense 5'-AAAGGGGGATA TGAGACTTTC-3', antisense 5'-TTGGATGCATGGTAGGC ACTT-3') or beta -actin (Clontech)-specific primers. A standard curve was prepared by amplifying different amounts of total RNA prepared from IL-2-stimulated PBMCs. Results obtained with DUSP5-specific primers were normalized against beta -actin.

Transient and Stable Transfections-- 293T cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. For IL-2 receptor reconstitution experiments, cells were transfected in 10-cm plates with plasmids containing cDNAs for IL-2Rbeta (2 µg), gamma c (0.5 µg), Jak3 (0.25 µg), and with control vector pRV (3 µg), wild type DUSP5 (3 µg), or inactive mutant of DUSP5 (C263S mutant) (3 µg) or the constitutively active MEK1 (3 µg) using EffecteneTM transfection reagent (Qiagen). For stable transfections, CTLL-2 cells were grown in complete RPMI medium supplemented with 50 units/ml of IL-2 and electroporated (5 × 106 cells, 170 V, 960 µF) with Myc-epitope tagged DUSP5 wild type or mutant cDNAs (36 µg) as well as with pBabe-puro (4 µg) to confer puromycin resistance. Transfected cells were plated on 6-well plates, and puromycin (2.5 µg/ml) was added the next day. The cDNAs for Myc-epitope tagged wild type and mutant (C263S) DUSP5, human Jak3, and constitutively active MEK1 were kindly provided by Jack E. Dixon, John O'Shea, and Silvio Gutkind, respectively.

Western Blotting, Immunoprecipitation, and Kinase Assays-- For Western blotting, cells were harvested, washed with phosphate-buffered saline, and lysed in lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.5% Nonidet P-40, 1 mM Na3VO4, 5 mM NaF, 10 µg/ml each of leupeptin and aprotinin, and 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride and centrifuged at 14,000 × g at 4 °C for 15 min. 15 µg of protein per lane was Western blotted. For immunoprecipitations, 150 µg of protein lysate was used. ERK-1/2 kinase activity was determined using Elk-1 as a substrate and the p44/42 MAP kinase assay kit (Cell Signaling). We used antibodies to phosphorylated ERK-1/2, phosphorylated-MEK1, ERK-1/2, phosphorylated Stat5 (all from Cell Signaling), MEK1 (Transduction Laboratories), Myc-epitope (9E10), IL-2Rbeta phosphotyrosine antibody (PY99) (all from Santa Cruz Biotechnology), and anti-beta -actin (AC-15, Sigma). DUSP5 anti-serum was a gift from Dr. Jack E. Dixon.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IL-2, IL-7, and IL-15 Induce a Highly Similar Set of Genes, whereas IL-4 Induces Both Overlapping and Distinct Genes-- Using cDNA microarrays, we identified 137 genes that were induced at least 2-fold by IL-2, IL-4, IL-7, or IL-15 in preactivated T lymphocytes (genes induced or repressed at least 2-fold are in Figs. 1-3 and will be made available at llmpp.nih.gov/cytokines). A one-sample t test was performed on the log ratios to evaluate the significance of the observed fold changes. We observed that 99% of IL-2-regulated (five experiments), 100% of IL-7-regulated (four experiments), 74% of IL-4-regulated (three experiments), and 54% of IL-15-regulated (three experiments) genes were differentially expressed to a degree that achieved statistical significance (two-sided p < 0.05). We hypothesize that the lower significance for the IL-4 and IL-15 groups results in large part from the lower power associated with having only three experiments for each of these cytokines. The induced genes (Figs. 1 and 2) included many genes previously known to be regulated by IL-2, such as those encoding the IL-2Ralpha chain, cyclin D2 (15), SOCS1 (19), CIS1 (20), and Pim-1 (21), indicating the validity of our analysis. IL-4Ralpha was also induced by IL-4, as reported previously (22). We also identified 34 repressed genes (Fig. 3). Although the use of 2-fold induction criteria is common, smaller changes in gene expression levels can also be biologically significant, and certain known IL-2-induced genes were not identified by the 2-fold criteria. We therefore searched the microarray data for genes whose expression was induced or repressed by more than 40% (based on microarray analysis) in at least any 8 of the 15 experiments. 95 additional genes fulfilled these less stringent criteria (data not shown but will be made available at llmpp.nih.gov/cytokines). This group included c-myc (15) and Bcl-XL (23), genes whose expression levels are known to be regulated by IL-2. Thus, it is important to evaluate genes whose mRNA levels are changed less than 2-fold as well.


View larger version (59K):
[in this window]
[in a new window]
 
Fig. 1.   Hierarchical clustering of genes induced by IL-2, IL-4, IL-7, and IL-15. The color intensity reflects the magnitude of induction (red squares) or repression (green squares). Gray squares indicate missing or excluded data. Asterisks indicate genes whose identity was verified by re-sequencing. A, array dendrogram obtained by hierarchical clustering of expression data from 137 induced genes in a total of 15 array experiments comprising four different cytokines. Samples stimulated for 4 h by IL-2, IL-7, and IL-15 cluster together (black branches), indicating a similar gene expression response, whereas the samples induced by IL-4 form a separate group (blue branches). B, hierarchical clustering of genes similarly induced at least 2-fold by IL-2, IL-4, IL-7, and IL-15. Each column represents data from one experiment, and each row represents the measurements for a given gene across all experiments. C, hierarchical clustering of genes induced more potently by IL-2, IL-7, and IL-15 than by IL-4. Genes in this cluster were induced more than 2-fold by IL-2, IL-7, and IL-15, and their expression was at least two times higher in IL-2-, IL-7-, and IL-15-stimulated cultures than in IL-4-stimulated cultures. D, hierarchical clustering of genes preferentially induced by IL-4. Genes in this cluster were induced at least 2-fold by IL-4, and their expression was at least two times higher in IL-4 stimulated cultures than in cultures stimulated by IL-2, IL-7, or IL-15. E, kinetics of IL-2- and IL-4-induced gene expression. Gene expression data for highly induced genes after 4 h of stimulation with IL-2 (five experiments), IL-7 (four experiments), IL-15 (three experiments), and IL-4 (three experiments) and corresponding measurements in three time course experiments (two IL-2 experiments and one IL-4 experiment) at 0, 0.5, 2, 4, 6, and 8 h. The genes were ranked by their average up-regulation in response to IL-2, IL-7, and IL-15. Data from the 20 most induced genes are shown. F, genes induced more than 2-fold by IL-4 that, in addition, were at least 2-fold more highly induced by IL-4 than by IL-2, IL-7, or IL-15. In panels B-D the "gene tree" on the left indicates the degree of similarity in gene expression across all the experiments. The blue circles correspond to genes whose expression was also studied by Northern blotting in Fig. 3.


View larger version (45K):
[in this window]
[in a new window]
 
Fig. 2.   Analysis of expression patterns of cytokine-induced genes in PI-stimulated PBMCs (PBMC Act.), B cells after anti-IgM crosslinking (B-cell Act.), proliferating cell lines, and CLL cells. A, a set of genes induced by IL-2, IL-4, IL-7, or IL-15 that were 3-fold more expressed in proliferating cell lines than in CLL cells. B, cytokine-inducible genes that are induced >2-fold by PI stimulation of PBMCs and BCR stimulation of B cells but showed variable expression in the cell lines and CLL samples. C, cytokine-inducible genes that are induced >2-fold by PI treatment of PBMCs but not by BCR cross-linking of B cells. D, genes induced >2-fold with BCR cross-linking of B cells but not induced by PI. E, genes specifically induced by IL-2, IL-4, IL-7, or IL-15. Cytokine-inducible genes were assigned to this cluster based on lack of induction by PI stimulation of PBMCs or BCR cross-linking of B cells and variable expression cell lines and CLL samples. In panels A and B the blue circles correspond to genes whose expression was also studied by Northern blotting (Fig. 4). The time-course for PI stimulation and BCR cross-linking was 0, 1, 3, 6, and 24 h. The letters below the cell lines and CLL cells correspond to following: A, Jurkat; B, Ramos; C, OCI-Ly8; D, SUDHL10; E, L428; F, MCF-7; G, PC-3; H-L, CD19+ cells from five different CLL patients. Data are shown for one of three representative experiments. The data for IL-2, IL-7, and IL-15 were averaged.


View larger version (46K):
[in this window]
[in a new window]
 
Fig. 3.   Genes repressed by IL-2, IL-4, IL-7, and/or IL-15. Data are shown for 34 genes after 4 h stimulation with cytokines and with corresponding measurements in time-course experiments, PI stimulated PBMCs, B-cells after BCR cross-linking, proliferating cell lines, and CLL cells. The blue circles correspond to genes whose expression was also studied by Northern blotting in Fig. 4.

The gene expression data were analyzed using average linkage hierarchical clustering (the Pearson correlation coefficient was used as the distance metric) (18), yielding dendrograms that group experiments and genes based on the degree of similarity of their expression patterns (see Fig. 1). We separately analyzed genes that were induced by IL-2, IL-4, IL-7, or IL-15 (Fig. 1A), and the expression profiles seen with IL-2, IL-7, and IL-15 were very similar (see for example Fig. 1, B-D). Some genes were induced in a similar fashion by all four cytokines (Fig. 1B). A number of genes were strongly induced by IL-2, IL-7, and IL-15 but not induced or only weakly induced by IL-4 (Fig. 1C). A third set of genes was preferentially induced by IL-4 (Fig. 1D).

To determine whether the differences between the gene expression profiles for IL-2 and IL-4 represented differences in the kinetics of gene expression between the cytokines rather than induction of different genes, we studied gene expression 0.5, 2, 4, 6, and 8 h after cytokine treatment (Fig. 1, E and F). In the four panels on the left, multiple experiments are shown at the 4-h time point for IL-2, IL-7, IL-15, and IL-4, whereas in the three panels on the right data are shown for two time courses for IL-2 and one for IL-4. Most genes showed increased expression in a cytokine-restricted manner between 0.5 and 2 h, and this increase was sustained for at least 8 h. For example, leukemia inhibitory factor (LIF) and biliary glycoprotein were induced by IL-2 at 30 min but were not induced by IL-4 at any time point (second and third rows from the top in Fig. 1E).

gamma c-Dependent Cytokines Induce a Group of Genes That Are Highly Expressed in Proliferating Cell Lines but Are Expressed at a Low Level in CLL Cells-- Most of the genes we identified have not been previously linked to cytokine responses and many are functionally uncharacterized. One strategy for finding clues to the functions mediated by these genes is to define expression "signatures" characterizing cellular processes. One such expression signature is defined by a set of genes whose expression correlates with cell proliferation in that they are highly expressed in proliferating cell lines (Fig. 2A, third panel from the left, lanes A-G) but are expressed at a low level in CLL cells (lanes H-L) which are relatively quiescent in their growth properties (16). ~20% of the genes induced by IL-2, IL-4, IL-7, and/or IL-15 fulfilled these criteria.

Most Genes Induced by IL-2, IL-4, IL-7, or IL-15 Are Induced by Multiple Stimuli-- We sought to identify a set of genes that could distinguish a gamma c-dependent cytokine response from other activation events, and we compared genes induced or repressed by IL-2, IL-4, IL-7, and IL-15 with those regulated in PBMCs (>70% T cells) by PI and in B cells after antigen receptor cross-linking (Fig. 2). We found that 73% of the genes induced by the cytokines were also induced in PI-stimulated PBMCs (Fig. 2, A-C, first column of panels from the left). The induction typically occurred within 1 h, minimizing the possibility that PI-dependent cytokine production was responsible for the PI effect. BCR stimulation of B cells induced less overlapping (41%) gene expression profiles (Fig. 2, A and B versus C and D, compare first and second panels from the left), consistent with the use of nonshared signaling pathways or lineage-specific differences in expression. 23% of the gamma c-dependent genes showed a more restricted expression pattern, as they were not up-regulated by either PI treatment of PBMCs or BCR cross-linking of B cells.

gamma c-Dependent Cytokines Repress Certain Genes, Some of Which Are Highly Expressed in CLL Cells-- We also identified 34 genes that were consistently repressed by IL-2, IL-4, IL-7, and/or IL-15 (Fig. 3), most of which were also repressed in PBMCs treated with PI or B cells treated with anti-IgM. Analogous to many of the activated genes being poorly expressed in CLL cells, many of the repressed genes were more highly expressed in CLL cells than in highly proliferating cell lines (Fig. 3, third panel from the left, lanes H-L versus A-G), suggesting a correlation between the expression of these genes and establishing or maintaining a more quiescent state.

Confirmation of Microarray Results by Northern Blotting-- We confirmed the induction or repression of select genes (those encoding IL-2Ralpha , TRAIL, MAPKAPK3, DUSP5, Mal, IL-4Ralpha and TSC-22R; blue circles in Figs. 1-3) by Northern blot analysis (Fig. 4). IL-2Ralpha , TRAIL, and DUSP5 were more potently induced by IL-2, IL-7, and IL-15 than by IL-4. Mal and IL-4Ralpha were most strongly induced by IL-4. MAPKAPK3 was induced by all four cytokines, and TSC-22R was repressed by all of the cytokines.


View larger version (65K):
[in this window]
[in a new window]
 
Fig. 4.   Northern blot analysis of selected genes. PBMCs were either not stimulated (-) or stimulated (+) for 3 h with PI (lanes 1 and 2), and cultured T cells were either not stimulated (-) (lane 3) or stimulated with the indicated cytokines for 4 h (lanes 4-7). Data are shown for one of three representative experiments.

DUSP5 Regulates IL-2-Dependent Phosphorylation and Catalytic Activity of ERK-1/2-- One of the genes induced by IL-2, IL-7, and IL-15 but not by IL-4 was that encoding DUSP5, a dual-specificity phosphatase originally cloned from mammary epithelial (13) and liver (14) cell lines. DUSP5 is also known as hVH-3 (14) and is a dual-specificity phosphatase induced by serum stimulation and heat shock (13). DUSP5 can hydrolyze proteins at both phosphotyrosine and phosphoserine/threonine residues, and recombinant DUSP5 can decrease the catalytic activity of purified ERK-1 protein in vitro (13, 14); but its physiological role has not been evaluated, and it has not previously been shown to be expressed in T cells.

Because IL-2 can activate ERK-1/2 as well as induce DUSP5 expression, we hypothesized that DUSP5 induction might be part of a negative feedback loop controlling IL-2-induced MAPK activity. We first confirmed that IL-2 could induce ERK-1/2 phosphorylation in the T cells (Fig. 5A, top panel, lanes 1-3, see arrow). IL-15 also induced phosphorylation of ERK-1/2, but neither IL-4 nor IL-7 shared this property (Fig. 5A, top panel, lanes 10-12 versus 4-6 and 7-9). In the same experiments IL-2, IL-7, and IL-15 induced phosphorylation of Stat5 (Fig. 5A, middle panel, lanes 1-3, 7-9, and 10-12), and IL-4 induced phosphorylation of Stat6 (Fig. 5A, bottom panel, lanes 4-6), demonstrating the responsiveness of the cells to all of these cytokines. Using real time PCR, we confirmed the induction of DUSP5 mRNA in preactivated PBMCs within 30 min of stimulation with IL-2, with a subsequent decline (Fig. 5B). Similarly, an antiserum raised against a DUSP5·GST fusion protein Western blotted an IL-2-inducible band of a molecular weight appropriate for DUSP5 (Fig. 5C, upper panel), whereas a control anti-serum could not (data not shown), suggesting that IL-2 stimulation also increases DUSP5 protein levels.


View larger version (41K):
[in this window]
[in a new window]
 
Fig. 5.   IL-2-induced ERK-1/2 phosphorylation is inhibited by DUSP5. A, T cells were cultured as described under "Materials and Methods," rested for 3 days, and stimulated with IL-2, IL-4, IL-7, or IL-15 for 0, 10, or 30 min. 15 µg of protein lysate per lane was Western blotted using antibodies for phosphorylated-ERK-1/2 (top panel), phosphorylated Stat5 (middle panel), or phosphorylated Stat6 (bottom panel). B, PBMCs were activated with PHA for 48 h, rested for 2 days, and stimulated with IL-2 for the indicated times. DUSP5 mRNA expression was analyzed by real-time PCR, and the expression levels were normalized against beta -actin expression. Shown is the average of three independent experiments (± S.D.). C, 30 µg of total cellular protein lysates (TCL) from the experiment portrayed in panel B (lanes 1-6) were Western blotted using antibodies for DUSP5 (top panel) or beta -actin (bottom panel). Lanes 7 and 8 are lysates from parental CTLL-2 cells (-) or DUSP5-transfected CTLL cells (+), as controls. Shown is one of three representative experiments. D, 293T cells were transfected with IL-2 receptor components (see "Materials and Methods") and either the pRV control vector, wild type DUSP5, inactive mutant of DUSP5 (DUSP5mut), or constitutively active MEK1 (MEK1act). After 2 days, cells were not stimulated or stimulated with IL-2 (1000 units/ml), lysed, and 15 µg of protein lysates were Western blotted using an antibody specific for phosphorylated ERK-1/2. Data shown are from one of five experiments with similar results.

We next investigated whether DUSP5 could regulate IL-2-induced ERK-1/2 phosphorylation using an IL-2 receptor reconstitution system (24) in 293T cells. In this setting, ERK-1/2 phosphorylation was induced by IL-2 (Fig. 5D, lane 2 versus lane 1), and this phosphorylation was inhibited when the cells were also transfected with wild type DUSP5 (Fig. 5D, lanes 3 and 4). Transfection of an inactive (C263S) mutant of DUSP5 did not affect ERK-1/2 phosphorylation (Fig. 5D, compare lane 6 to lane 2), which is consistent with the fact that 293T cells express very low levels of endogenous DUSP5 (data not shown). As expected, 293T cells transfected with constitutively active MEK1 showed increased ERK-1/2 activity that was independent of IL-2 (Fig. 5D, lanes 7 and 8).

To further evaluate the possible role of DUSP5 in the regulation of IL-2-induced MAPK activity, we stably transfected IL-2-dependent CTLL-2 cells with wild type and inactive DUSP5. CTLL-2 is a murine T cell line that has been widely used to study IL-2 biology and signaling (see, for example, Refs. 25 and 26). We studied two clones that constitutively express wild type DUSP5 (WT1, WT2) and three clones that constitutively express an inactive mutant of DUSP5 (M1, M2, M3), as demonstrated by Western blotting with anti-Myc epitope antibody (Fig. 6A, top panel, lanes 1-20). The clones expressing the inactive form of DUSP5 showed markedly increased phosphorylation of ERK-1/2 (doublet band, Fig. 6A, second panel from the top, lanes 9-20) in response to IL-2 when compared with parental CTLL-2 cells (lanes 21-24) or clones expressing wild type DUSP5 (lanes 1-8). Similarly, as evaluated by an in vitro kinase assay, clones expressing inactive DUSP5 exhibited higher IL-2-induced ERK-1/2 kinase activity than did the parental cells (Fig. 6B, lanes 7-15 versus 16-19), whereas clones expressing wild type DUSP5 showed decreased activity (Fig. 6B, lanes 1-6). As ERK-1/2 protein levels were not affected by DUSP5 expression (Fig. 6A, third panel from the top, lanes 1-24), these results indicate that DUSP5 negatively regulates the activity of ERK-1 and ERK-2. We also studied the effect of DUSP5 on IL-2-induced phosphorylation of MEK1 and Stat5 (Fig. 6A, fourth and fifth panels from the top) and IL-2Rbeta (Fig. 6C), but no changes were observed. Thus, DUSP5 appears to selectively regulate ERK-1 and ERK-2 activity in IL-2 signaling.


View larger version (68K):
[in this window]
[in a new window]
 
Fig. 6.   DUSP5 regulates ERK-1/2 activity in CTLL-2 cells. IL-2 signaling events in clones of CTLL-2 cells expressing wild type DUSP5 (WT1 and WT2), three clones expressing an inactive DUSP5 mutant (M1-M3), or parental CTLL-2 cells. The cells were rested for 10 h in 5% fetal bovine serum in the absence of IL-2 and then either not stimulated or stimulated for 5, 15 or 30 min with 100 units/ml of IL-2. A, 15 µg of total cellular lysates were Western blotted using antibodies for Myc-epitope (to validate the expression levels of transfected DUSP5 proteins, top panel), phosphorylated ERK-1/2 (second panel from top), total ERK-1/2 (third panel from top), phosphorylated MEK1 (fourth panel from top), phosphorylated Stat5 (fifth panel from top), and beta -actin (bottom panel). B, ERK-1/2 kinase assay. Total cellular lysates (250 µg) were immunoprecipitated with antibodies to phosphorylated ERK-1/2 followed by kinase reaction in the presence of Elk1 substrate protein. The proteins were Western blotted using an antibody to phosphorylated Elk-1. C, total cellular lysates (250 µg) were immunoprecipitated with antibodies to IL-2Rbeta and Western blotted with anti-phosphotyrosine. Data are shown for one of four representative experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we investigated genes regulated by IL-2, IL-4, IL-7, and IL-15. Although some data regarding genes regulated by these cytokines have been previously generated, comparative data on the effects of these cytokines on large numbers of genes have not been available. Genes induced by IL-2 have been most extensively studied, and IL-2 is known to induce a number of genes including, for example, those encoding c-Myc (15), c-Fos (15), c-Jun (15), IL-2Ralpha chain (15), Pim-1 (21), Bcl-2 (15), and the SOCS family proteins SOCS1 (19) and CIS1 (20). Previous studies have sought to identify IL-2-regulated genes in a more systematic way (27-29) but have revealed largely nonoverlapping sets of IL-2-induced transcripts, suggesting that many other IL-2-regulated genes remained to be identified. In our microarray analysis137 genes appeared to be induced, and 34 genes appeared to repressed by IL-2, IL-4, IL-7, or IL-15. A significant number of these genes (20%) are related to cell proliferation based on their high level expression in proliferating cell lines but low expression in relatively quiescent CLL cells (16), in accord with the known mitogenic function of these cytokines on activated T cells.

Hierarchical clustering of these genes revealed that the induced genes fell into two major groups, i.e. those regulated preferentially by IL-2, IL-7, and IL-15, and those regulated by IL-4. It was noteworthy that IL-2, IL-7, and IL-15 induced almost identical gene expression patterns in T lymphocytes, at least at the doses we used. This supports the concept that multiple cytokines can similarly provide survival/growth signals and that the specificity of cytokine action is largely determined by cell type or developmental stage specific expression of cytokine receptor(s) and the availability of ligand(s) (30-32). However, it is possible that cell type-dependent differences also exist. For example, it has been suggested that IL-15 uses different receptor or signaling pathways depending on cell type (33, 34), which could also explain why IL-2 and IL-15 induce similar gene responses and proliferation in T cells, whereas only IL-15 is essential for NK cell differentiation.

Although IL-4 induced a set of genes overlapping those induced by IL-2, IL-7, and IL-15, there were also many differences. Most genes that were induced by IL-2, IL-7, or IL-15 were not induced by IL-4 or were induced at only a very low level. However, some genes (e.g. those endocing SOCS-1, CIS1, and Bcl-2) were induced in a similar fashion by all of the cytokines, whereas others (e.g. those encoding IL-4Ralpha and Mal) were more strongly induced by IL-4. The basis for this more distinctive pattern for IL-4 may be explained by the fact that IL-4 activates primarily Stat6, whereas the other cytokines preferentially activate Stat3, Stat5a, and Stat5b (35). For example, the promoter for IL-4Ralpha , which is regulated by IL-4, contains Stat6 binding sites (36), whereas the IL-2Ralpha promoter, which is regulated by IL-2, contains binding sites for Stat5a and Stat5b (37-39). It will be important to determine whether genes induced preferentially by IL-4, such as Mal, contribute to functions unique to IL-4 such as the induction of Th2 differentiation among T lymphocytes.

Most of the genes that we found to be induced by gamma c-dependent cytokines were also induced by the combination of PMA plus ionomycin (74%), and many were induced after BCR stimulation of B lymphocytes (57%). One of these genes, the IL-2Ralpha gene is regulated by at least five positive regulatory regions (PRRs) (15, 37-41). PRRI is presumably a T cell receptor response element as it contains an NF-kappa B binding site that is required for IL-2Ralpha promotor activity in response to PHA or PMA (15), whereas PRRIII and PRRIV (37-40) are both required for IL-2-induced IL-2Ralpha induction. A fifth element is a CD28 response element (41). Thus, in the IL-2Ralpha gene, different enhancer-like elements differentially respond to different stimuli. The coexistence of antigen and cytokine response elements in other genes as well might account for the highly overlapping gene expression profiles between gamma c-dependent cytokines and PI-stimulation. In this regard, IL-15 and T cell receptor stimulation were recently shown to induce many of the same genes in CD8+ memory T cells (42).

Only a few of the genes whose expression was regulated by gamma c-dependent cytokines were previously identified as functionally relevant target genes, such as those encoding IL-2Ralpha (43-45) and Bcl-2 (46), and most of the genes we identified have not been characterized as part of a cytokine response. One such gene is DUSP5, which we now show is induced by IL-2, IL-7, and IL-15, but not by IL-4. DUSP5 was previously shown in vitro to be capable of dephosphorylating ERK-1, but the physiological context was not investigated (13, 14). IL-2 signaling has been extensively studied, and along with the Jak-STAT and PI 3-kinase/Akt pathways, the MAP kinase pathway has been described as important (15). The activation of Stat5 proteins is mediated by phospho-tyrosine docking sites (Tyr-392 and Tyr-510) on the IL-2 receptor beta  chain (47, 48) and has been functionally linked to the regulation of proliferation and activation-induced cell death (48, 49). Activation of the PI 3-K/Akt pathway regulates IL-2-dependent cell survival (50) and may contribute to cell proliferation (50). Regarding the MAPK pathway, Tyr-338 on IL-2Rbeta directly binds the phospho-tyrosine binding (PTB) domain of Shc (48) and mediates the activation of ERK-1/2 by IL-2 (49, 51, 52). We provide evidence that DUSP5 induction by IL-2 may negatively regulate IL-2-dependent activation of ERK-1/2.

Studies in the 32D myeloid cell line revealed that IL-2Rbeta Tyr-338 is required for IL-2-dependent proliferation, suggesting that Shc-coupled MAPK activation is vital for proliferation (48). However, in primary T cells, simultaneous inactivation of Tyr-338 and Stat5 binding sites was required to reveal a decrease in proliferation, suggesting that in these cells either MAPK or Stat5-coupled pathways by themselves are sufficient for proliferation (49). This is consistent with our finding in CTLL-2 cells that DUSP5 alone did not decrease IL-2-mediated proliferation (data not shown). However, it is possible that MAPK and DUSP5 may have other effects in IL-2 biology.

It this study, we have shown that IL-2 potently activates ERK-1 and ERK-2 as well as DUSP5, which negatively regulates ERK-1 and ERK-2. In contrast, IL-4 does not potently activate either ERK-1 or ERK-2 nor does it induce DUSP5, thus providing an example of how differential gene regulation by gamma c-dependent cytokines can modulate cytokine-specific actions. As noted above in our microarray analysis, DUSP5 was induced in T cells either following treatment with cytokines or PMA plus ionomycin. This suggests that DUSP5-dependent negative feedback regulation of MAPK is not restricted to cytokine signaling. ERK-1/2 activation has been indicated in a range of important functions in T-cells and NK cells (53). Remarkably, in ERK-1-deficient mice the only observed defect is in thymic T-cell development at the double positive stage resulting in ~50% reduction in the numbers of single positive lymphocytes (54). Accordingly, we recently generated mice expressing DUSP5 transgene, which in preliminary experiments show an even more complete block in T lymphocyte development with ~70% reduction in numbers of single positive lymphocytes (data not shown). This is consistent with our observation that DUSP5 regulates both ERK-1 and ERK-2 in T cells and suggest that DUSP5 regulation of MAPK is a general theme in T lymphocyte activation and signaling.

    ACKNOWLEDGEMENTS

We thank Jack E. Dixon for kindly providing the DUSP5 cDNA constructs and antiserum and Drs. Keiji Zhao, Joost Oppenheim, Jian-Xin Lin, and John Kelly for critical comments.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ These authors should be considered as equal first authors.

Supported in part by grants from the Maud Kuistila Foundation, the Finnish Cultural Foundation, the Emil Aaltonen Foundation, and the Academy of Finland.

** Supported in part by a grant from the Deutsche Krebshilfe, Bonn, Germany.

§§ These authors should be considered as equal last authors.

¶¶ To whom correspondence should be addressed. Fax: 301-402-0971; E-mail: wjl@helix.nih.gov.

Published, JBC Papers in Press, November 14, 2002, DOI 10.1074/jbc.M209015200

    ABBREVIATIONS

The abbreviations used are: gamma c, cytokine receptor gamma  chain; IL, interleukin; DUSP5, dual-specificity phosphatase 5; MAP, mitogen-activated protein: MAPK, MAP kinase; ERK, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase; STAT, signal transducers and activators of transcription; PBMC, peripheral blood mononuclear cell; PHA, phytohemagglutinin; PMA, phorbol 2-myristate 3-acetate; PI, PMA plus ionomycin; PRR, positive regulatory region; CLL, chronic lymphocytic leukemia; BCR, B cell antigen receptor.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1. Noguchi, M., Yi, H., Rosenblatt, H. M., Filipovich, A. H., Adelstein, S., Modi, W. S., McBride, O. W., and Leonard, W. J. (1993) Cell 73, 147-157[Medline] [Order article via Infotrieve]
2. Leonard, W. J. (2001) Nature Rev. Immunol. 1, 200-208[CrossRef][Medline] [Order article via Infotrieve]
3. Van Parijs, L., and Abbas, A. K. (1998) Science 280, 243-248[Abstract/Free Full Text]
4. Nelms, K., Keegan, A. D., Zamorano, J., Ryan, J. J., and Paul, W. E. (1999) Annu. Rev. Immunol. 17, 701-738[CrossRef][Medline] [Order article via Infotrieve]
5. Chazen, G. D., Pereira, G. M., LeGros, G., Gillis, S., and Shevach, E. M. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 5923-5927[Abstract]
6. Peschon, J. J., Morrissey, P. J., Grabstein, K. H., Ramsdell, F. J., Maraskovsky, E., Gliniak, B. C., Park, L. S., Ziegler, S. F., Williams, D. E., and Ware, C. B. (1994) J. Exp. Med. 180, 1955-1960[Abstract]
7. von Freeden-Jeffry, U., Solvason, N., Howard, M., and Murray, R. (1997) Immunity 7, 147-154[Medline] [Order article via Infotrieve]
8. Waldmann, T. A., Dubois, S., and Tagaya, Y. (2001) Immunity 14, 105-110[CrossRef][Medline] [Order article via Infotrieve]
9. Renauld, J. C., van der Lugt, N., Vink, A., van Roon, M., Godfraind, C., Warnier, G., Merz, H., Feller, A., Berns, A., and Van Snick, J. (1994) Oncogene 9, 1327-1332[Medline] [Order article via Infotrieve]
10. Townsend, J. M., Fallon, G. P., Matthews, J. D., Smith, P., Jolin, E. H., and McKenzie, N. A. (2000) Immunity 13, 573-583[Medline] [Order article via Infotrieve]
11. Parrish-Novak, J., Dillon, S. R., Nelson, A., Hammond, A., Sprecher, C., Gross, J. A., Johnston, J., Madden, K., Xu, W., West, J., Schrader, S., Burkhead, S., Heipel, M., Brandt, C., Kuijper, J. L., Kramer, J., Conklin, D., Presnell, S. R., Berry, J., Shiota, F., Bort, S., Hambly, K., Mudri, S., Clegg, C., Moore, M., Grant, F. J., Lofton-Day, C., Gilbert, T., Rayond, F., Ching, A., Yao, L., Smith, D., Webster, P., Whitmore, T., Maurer, M., Kaushansky, K., Holly, R. D., and Foster, D. (2000) Nature 408, 57-63[CrossRef][Medline] [Order article via Infotrieve]
12. Alizadeh, A., Eisen, M., Davis, R. E., Ma, C., Sabet, H., Tran, T., Powell, J. I., Yang, L., Marti, G. E., Moore, D. T., Hudson, J. R., Jr., Chan, W. C., Greiner, T., Weisenburger, D., Armitage, J. O., Lossos, I., Levy, R., Botstein, D., Brown, P. O., and Staudt, L. M. (1999) Cold Spring Harbor Symp. Quant. Biol. 64, 71-78[Medline] [Order article via Infotrieve]
13. Ishibashi, T., Bottaro, D. P., Michieli, P., Kelley, C. A., and Aaronson, S. A. (1994) J. Biol. Chem. 269, 29897-29902[Abstract/Free Full Text]
14. Kwak, S. P., and Dixon, J. E. (1995) J. Biol. Chem. 270, 1156-1160[Abstract/Free Full Text]
15. Lin, J. X., and Leonard, W. J. (1997) Cytokine Growth Factor Rev. 8, 313-332[CrossRef][Medline] [Order article via Infotrieve]
16. Alizadeh, A. A., Eisen, M. B., Davis, R. E., Ma, C., Lossos, I. S., Rosenwald, A., Boldrick, J. C., Sabet, H., Tran, T., Yu, X., Powell, J. I., Yang, L., Marti, G. E., Moore, T., Hudson, J., Jr., Lu, L., Lewis, D. B., Tibshirani, R., Sherlock, G., Chan, W. C., Greiner, T. C., Weisenburger, D. D., Armitage, J. O., Warnke, R., Staudt, L. M., et al.. (2000) Nature 403, 503-511[CrossRef][Medline] [Order article via Infotrieve]
17. Rosenwald, A., Alizadeh, A. A., Widhopf, G., Simon, R., Davis, R. E., Yu, X., Yang, L., Pickeral, O. K., Rassenti, L. Z., Powell, J., Botstein, D., Byrd, J. C., Grever, M. R., Cheson, B. D., Chiorazzi, N., Wilson, W. H., Kipps, T. J., Brown, P. O., and Staudt, L. M. (2001) J. Exp. Med. 194, 1639-1647[Abstract/Free Full Text]
18. Eisen, M. B., Spellman, P. T., Brown, P. O., and Botstein, D. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 14863-14868[Abstract/Free Full Text]
19. Sporri, B., Kovanen, P. E., Sasaki, A., Yoshimura, A., and Leonard, W. J. (2001) Blood 97, 221-226[Abstract/Free Full Text]
20. Yoshimura, A., Ohkubo, T., Kiguchi, T., Jenkins, N. A., Gilbert, D. J., Copeland, N. G., Hara, T., and Miyajima, A. (1995) EMBO J. 14, 2816-2826[Abstract]
21. Dautry, F., Weil, D., Yu, J., and Dautry-Varsat, A. (1988) J. Biol. Chem. 263, 17615-17620[Abstract/Free Full Text]
22. Ohara, J., and Paul, W. E. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8221-8225[Abstract]
23. Akbar, A. N., Borthwick, N. J., Wickremasinghe, R. G., Panayoitidis, P., Pilling, D., Bofill, M., Krajewski, S., Reed, J. C., and Salmon, M. (1996) Eur. J. Immunol. 26, 294-299[Medline] [Order article via Infotrieve]
24. Lin, J. X., Mietz, J., Modi, W. S., John, S., and Leonard, W. J. (1996) J. Biol. Chem. 271, 10738-10744[Abstract/Free Full Text]
25. Turner, B., Rapp, U., App, H., Greene, M., Dobashi, K., and Reed, J. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 1227-1231[Abstract]
26. Burton, J. D., Bamford, R. N., Peters, C., Grant, A. J., Kurys, G., Goldman, C. K., Brennan, J., Roessler, E., and Waldmann, T. A. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 4935-4939[Abstract]
27. Beadling, C., Johnson, K. W., and Smith, K. A. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2719-2723[Abstract]
28. Herblot, S., Chastagner, P., Samady, L., Moreau, J. L., Demaison, C., Froussard, P., Liu, X., Bonnet, J., and Theze, J. (1999) J. Immunol. 162, 3280-3288[Abstract/Free Full Text]
29. Gonzalez, J., Harris, T., Childs, G., and Prystowsky, M. B. (2001) Blood Cells Mol. Dis. 27, 572-585[CrossRef][Medline] [Order article via Infotrieve]
30. Socolovsky, M., Dusanter-Fourt, I., and Lodish, H. F. (1997) J. Biol. Chem. 272, 14009-14012[Abstract/Free Full Text]
31. Goldsmith, M. A., Mikami, A., You, Y., Liu, K. D., Thomas, L., Pharr, P., and Longmore, G. D. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 7006-7011[Abstract/Free Full Text]
32. Stoffel, R., Ziegler, S., Ghilardi, N., Ledermann, B., de Sauvage, F. J., and Skoda, R. C. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 698-702[Abstract/Free Full Text]
33. Bulanova, E., Budagian, V., Pohl, T., Krause, H., Durkop, H., Paus, R., and Bulfone-Paus, S. (2001) J. Immunol. 167, 6292-6302[Abstract/Free Full Text]
34. Tagaya, Y., Burton, J. D., Miyamoto, Y., and Waldmann, T. A. (1996) EMBO J. 15, 4928-4939[Abstract]
35. Leonard, W. J., and O'Shea, J. J. (1998) Annu. Rev. Immunol. 16, 293-322[CrossRef][Medline] [Order article via Infotrieve]
36. Kotanides, H., and Reich, N. C. (1996) J. Biol. Chem. 271, 25555-25561[Abstract/Free Full Text]
37. Sperisen, P., Wang, S. M., Soldaini, E., Pla, M., Rusterholz, C., Bucher, P., Corthesy, P., Reichenbach, P., and Nabholz, M. (1995) J. Biol. Chem. 270, 10743-10753[Abstract/Free Full Text]
38. Lecine, P., Algarte, M., Rameil, P., Beadling, C., Bucher, P., Nabholz, M., and Imbert, J. (1996) Mol. Cell. Biol. 16, 6829-6840[Abstract]
39. John, S., Robbins, C. M., and Leonard, W. J. (1996) EMBO J. 15, 5627-5635[Abstract]
40. Kim, H. P., Kelly, J., and Leonard, W. J. (2001) Immunity 15, 159-172[CrossRef][Medline] [Order article via Infotrieve]
41. Yeh, J. H., Lecine, P., Nunes, J. A., Spicuglia, S., Ferrier, P., Olive, D., and Imbert, J. (2001) Mol. Cell. Biol. 21, 4515-4527[Abstract/Free Full Text]
42. Liu, K., Catalfamo, M., Li, Y., Henkart, P. A., and Weng, N. P. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 6192-6197[Abstract/Free Full Text]
43. Willerford, D. M., Chen, J., Ferry, J. A., Davidson, L., Ma, A., and Alt, F. W. (1995) Immunity 3, 521-530[Medline] [Order article via Infotrieve]
44. Nakajima, H., Liu, X. W., Wynshaw-Boris, A., Rosenthal, L. A., Imada, K., Finbloom, D. S., Hennighausen, L., and Leonard, W. J. (1997) Immunity 7, 691-701[Medline] [Order article via Infotrieve]
45. Sharfe, N., Dadi, H. K., Shahar, M., and Roifman, C. M. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 3168-3171[Abstract/Free Full Text]
46. Nakajima, H., Noguchi, M., and Leonard, W. J. (2000) Immunol. Today 21, 88-94[CrossRef][Medline] [Order article via Infotrieve]
47. Gaffen, S. L., Lai, S. Y., Ha, M., Liu, X., Hennighausen, L., Greene, W. C., and Goldsmith, M. A. (1996) J. Biol. Chem. 271, 21381-21390[Abstract/Free Full Text]
48. Friedmann, M. C., Migone, T. S., Russell, S. M., and Leonard, W. J. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 2077-2082[Abstract/Free Full Text]
49. Van Parijs, L., Refaeli, Y., Lord, J. D., Nelson, B. H., Abbas, A. K., and Baltimore, D. (1999) Immunity 11, 281-288[Medline] [Order article via Infotrieve]
50. Ahmed, N. N., Grimes, H. L., Bellacosa, A., Chan, T. O., and Tsichlis, P. N. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 3627-3632[Abstract/Free Full Text]
51. Gu, H., Maeda, H., Moon, J. J., Lord, J. D., Yoakim, M., Nelson, B. H., and Neel, B. G. (2000) Mol. Cell. Biol. 20, 7109-7120[Abstract/Free Full Text]
52. Moon, J. J., and Nelson, B. H. (2001) J. Immunol. 167, 2714-2723[Abstract/Free Full Text]
53. Rincon, M., Flavell, R. A., and Davis, R. J. (2001) Oncogene 20, 2490-2497[CrossRef][Medline] [Order article via Infotrieve]
54. Pages, G., Guerin, S., Grall, D., Bonino, F., Smith, A., Anjuere, F., Auberger, P., and Pouyssegur, J. (1999) Science 286, 1374-1377[Abstract/Free Full Text]


Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.