The IL-2 Receptor Promotes Lymphocyte Proliferation and Induction of the c-myc, bcl-2, and bcl-x Genes Through the trans-Activation Domain of Stat51 James D. Lord,*†‡ Bryan C. McIntosh,* Philip D. Greenberg,†‡§ and Brad H. Nelson2*‡ Studies assessing the role of Stat5 in the IL-2 proliferative signal have produced contradictory, and thus inconclusive, results. One factor confounding many of these studies is the ability of IL-2R to deliver redundant mitogenic signals from different cytoplasmic tyrosines on the IL-2R ␤-chain (IL-2R␤). Therefore, to assess the role of Stat5 in mitogenic signaling independent of any redun- dant signals, all cytoplasmic tyrosines were deleted from IL-2R␤ except for Tyr510, the most potent Stat5-activating site. This deletion mutant retained the ability to induce Stat5 activation and proliferation in the T cell line CTLL-2 and the pro-B cell line BA/F3. A set of point mutations at or near Tyr510 that variably compromised Stat5 activation also compromised the proliferative signal and revealed a quantitative correlation between the magnitude of Stat5 activation and proliferation. Proliferative signaling by a receptor mutant with a weak Stat5 activating site could be rescued by overexpression of wt Stat5a or b. Additionally, the ability of this receptor mutant to induce c-myc, bcl-x, and bcl-2 was enhanced by overexpression of wt Stat5. By contrast, overexpression of a version of Stat5a lacking the C-terminal trans-activation domain inhibited the induction of these genes and cell proliferation. Thus, Stat5 is a critical component of the proliferative signal from Tyr510 of the IL-2R and regulates expression of both mitogenic and survival genes through its trans-activation domain. The Journal of Immunology, 2000, 164: 2533–2541. he T cell mitogen IL-2 binds to a heterotrimeric receptor Stat5 refers to either of two highly homologous members of the complex consisting of IL-2R␣, IL-2R␤, and ␥c chains (1), larger Stat family of signal-transducing activators of transcription, T with the consequent heterodimerization of IL-2R␤ and ␥c Stat5a and Stat5b (16–18). Stat activation conventionally com- inducing the proliferative signal (2, 3). ␥c contributes to prolifer- mences with selective binding of a Stat SH2 domain to a phos- ative signaling by recruiting the tyrosine kinase Jak3(4–8), the phorylated tyrosine motif on a ligated receptor (19–22), approxi- activation of which also requires a membrane-proximal S region of mating the Stat molecule with a receptor-associated, activated IL-2R␤ (9, 10). Additionally, at least one of three cytoplasmic Janus kinase which phosphorylates a key tyrosine residue on the tyrosine phosphorylation sites (Tyr338, Tyr393,orTyr510) distal to Stat molecule (23, 24). These Stat phosphotyrosines preferentially the S region on IL-2R␤ must be present for proliferation, suggest- bind to Stat SH2 domains (21), causing Stat molecules to dimerize, ing that obligate mitogenic signaling molecules interact with these release from receptors, enter the nucleus, and bind palindromic phosphotyrosines (11, 12). Indeed, the adapter molecule Shc in- DNA sequences (22) in certain promoters (25–27), inducing gene teracts with Tyr338 (11, 13) and can deliver a proliferative signal transcription via a C-terminal trans-activation domain (TAD)3 (14). The other two tyrosines, Tyr510 and Tyr393, provide docking (28–31). Some members of the Stat family also demonstrate less sites for the transcription factor Stat5 (11), which has led to the conventional activities. Stat3, for example, serves as an adapter hypothesis that Stat5 mediates a proliferative signal parallel to the molecule between PI3 kinase and type I IFN receptors (32). Fur- one involving Shc. However, Shc and Stat5 have very different thermore, Stat1 and, in cooperation with the glucocorticoid recep- biochemical properties, which raises the question of whether they tor, Stat5 mediate the expression of certain genes through a TAD- could indeed generate redundant proliferative signals from IL- independent mechanism (33–35). Thus, not all signaling functions 2R␤. In addition, the fact that a single phosphotyrosine on a re- of Stat molecules involve the conventional trans-activation ceptor chain can interact with multiple signaling molecules, as domain. exemplified by studies of the platelet-derived growth factor recep- A critical role for Stat5 in IL-2-mediated mitogenesis has been tor (15), raises the possibility that a factor other than Stat5 medi- suggested by the failure of TCR-stimulated T cells from mice lack- ates the proliferative signal from Tyr393 and/or Tyr510. ing both the Stat5a and Stat5b genes to proliferate in response to IL-2 (36). However, the IL-2R can mediate proliferation in the absence of detectable Stat5 activation through alternative mito- *Virginia Mason Research Center, Seattle, WA 98101; †Fred Hutchinson Cancer Research Center, Seattle, WA 98104; and Departments of ‡Immunology and §Med- genic signaling molecules, such as Shc (11, 14). Indeed, IL-2R␤ icine, University of Washington, Seattle, WA 98195 mutants that fail to activate Stat5 mediate proliferation in cultured Received for publication September 3, 1999. Accepted for publication December cell lines (11, 37, 38), and primary T cells (39). Thus, the inability 10, 1999. of Stat5-null T cells to proliferate may reflect a recently described The costs of publication of this article were defrayed in part by the payment of page essential role for Stat5 in TCR signaling (40) rather than a requisite charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. role in IL-2 signaling. 1 This work was supported by Grant GM57931 from the National Institutes of Health. This report assesses the role of Stat5 in signals derived from the J.D.L. was supported by a National Defense Science and Engineering Graduate fel- IL-2R in cell lines that do not require TCR stimulation for prolif- lowship from the U.S. Department of Defense and by a grant from the Poncin Schol- eration. A receptor mutagenesis and rescue strategy was used to arship Fund. 2 Address correspondence and reprint requests to Dr. Brad Nelson, Virginia Mason Research Center, 1201 9th Avenue, Seattle, WA 98101-2795. 3 Abbreviations used in this paper: TAD, trans-activation domain; wt, wild type. Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00 2534 IL-2 MEDIATES PROLIFERATION THROUGH THE TAD OF Stat5 study signaling by Stat5 in the absence of any redundant signals resed on acrylamide gels and transferred to nitrocellulose. Nitrocellulose from Shc or other molecules potentially associating with IL-2R␤. blots were blocked with 0.1 M Tris base (pH 7.5), 0.9% sodium chloride, We demonstrate that Tyr510 of IL-2R␤ mediates proliferation 0.05% Tween 20 (TTBS) containing 1% BSA (for antiphosphotyrosine probes) or 5% powdered skim milk (Carnation, Glendale, CA), and probed through the activation of Stat5 and that either the Stat5a or the with rabbit antisera recognizing Jak1 (Santa Cruz Biotechnology) or Jak3 Stat5b isoform can transduce the mitogenic signal. Furthermore, (Upstate Biotechnology, Lake Placid, NY) or murine Abs recognizing proliferative signaling by Stat5 is critically dependent on its TAD, phosphotyrosine (4G10; Upstate Biotechnology) or Stat5 (Transduction indicating that Stat5a mediates proliferation through its conven- Laboratories, Lexington, KY). Blots were then washed with TTBS, probed with peroxidase-conjugated goat anti-rabbit or anti-mouse Abs (Life Tech- tional role as a transcription factor. Indeed, IL-2-induced transcrip- nologies), and washed again with TTBS. Bound Abs were detected by tion of the proliferative and survival genes c-myc, bcl-2, and bcl-x enhanced chemiluminescence (DuPont NEN, Boston, MA). Blots were is mediated by Stat5a through the TAD. stripped between probings with a 30-min, 50°C incubation in 62.6 mM Tris-HCl (pH 6.7), 0.1 M ␤-mercaptoethanol, and 2% SDS. Materials and Methods Plasmid construction Electrophoretic mobility shift assays Expression vectors encoding the chimeric ␣␥- and ␤␤-chains (formerly Cells stimulated as indicated were washed once with buffer H (20 mM denoted GM␣/2␥ and GM␤/2␤) under the control of the human ␤-actin HEPES (pH 7.9), 1 mM EDTA, 0.1 mM EGTA, 2 mM magnesium chlo- promoter have been described previously (2, 9, 14, 41). Mutants of ␤␤ ride, 1 mM sodium o-vanadate, 20 mM sodium fluoride, 1 mM DTT, 0.1 were generated by annealing sense and antisense oligonucleotides encod- mM 4-(2-aminoethyl)benzenesulfonyl fluoride, and 1 mg/ml leupeptin) ing novel C-terminal sequences for ␤␤ and/or premature stop codons, and and lysed in buffer H plus 0.2% Nonidet P-40 at 0°C. Nuclei were pelleted cloning overhanging ends of these annealed primers between a unique AflII by centrifugation and extracted with buffer K (buffer H plus 0.42 M sodium site in the cytoplasmic domain of ␤␤ and a unique XbaI site immediately chloride and 20% v/v glycerol). To generate a probe for Stat activity, 3Ј to the stop codon of ␤␤. For analyses in BA/FG cells, the full length, incompletely overlapping oligonucleotides corresponding to the sense and nonchimeric human IL-2R␤ cDNA was cloned into the expression vector antisense strands of the Stat-responsive DNA element from the Fc␥R1 used above, and the C terminus of this chain, between the AflII site and a promoter were annealed, radiolabeled with [␣-32P]dCTP by an end-filling unique 3Ј ScaI site, was then replaced with the respective mutated se- T4 polymerase reaction, and purified with a MicroSpin G-25 column (Phar- quences. The ⌬713 mutant of Stat5a was generated by PCR with an anti- macia, Piscataway, NJ). The probe was added to nuclear extracts in 50 mM sense oligonucleotide encoding residues 708 to 712 of Stat5a followed by potassium chloride, 10 mM HEPES (pH 7.9), 10% glycerol, 1 mM DTT, the flag epitope, a stop codon, and an EcoRV site.