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Phosphorylation-Dependent Mechanism in a T-Cell Line (T Lymphocyte/Signal Transduction/Phosphatase) BRUCE C

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Phosphorylation-Dependent Mechanism in a T-Cell Line (T Lymphocyte/Signal Transduction/Phosphatase) BRUCE C Proc. Natl. Acad. Sci. USA Vol. 90, pp. 5544-5548, June 1993 Immunology Interleukin 2 regulates Raf-1 kinase activity through a tyrosine phosphorylation-dependent mechanism in a T-cell line (T lymphocyte/signal transduction/phosphatase) BRUCE C. TURNER*t, NICHOLAS K. TONKS*, ULF R. RAPP§, AND JOHN C. REED*$ *Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6082; §Laboratory of Viral Carcinogenesis, National Cancer Institute, Frederick, MD 21701; and *Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724-2208 Communicated by Peter C. Nowell, March 15, 1993 ABSTRACT Previously we found that interleukin 2 (IL-2) Ultimately, however, it is believed that growth factor- induces tyrosine phosphorylation and activation of the ser- activated PTKs pass their information on to serine/ ine/threonine-specific kinase encoded by the raf-l protoonco- threonine-specific kinases in a signal transduction cascade gene in a T-cell line, CTLL-2. Here we extended these rmdings that eventually reaches the nucleus, resulting in the changes by exploring the effects of selective removal of phosphate from in gene expression that lead to cellular proliferation, sup- tyrosines in p72-74-Raf-i kinase that had been immunopre- pression of apoptosis, and other responses. cipitated from IL-2-stimulated CTLL-2 cells. Treatment in The 72- to 74-kDa kinase encoded by the raf-J protoonco- vito of IL-2-activated Raf-i with the tyrosine-specific phos- gene is a good candidate for a serine/threonine-specific phatases CD45 and TCPTP (formerly called T-cell protein kinase that receives its activation signals directly from up- tyrosine phosphatase) reduced Raf kinase activity to nearly stream PTKs. Although somewhat controversial, Raf-1 was baseline levels. This effect was completely inhibited by the originally reported to become phosphorylated on tyrosines in phosphatase inhibitor sodium orthovanadate. In contrast, platelet-derived growth factor-stimulated fibroblasts and was treatment of Raf-i with a serine/threonine-specific phospha- shown to physically associate with platelet-derived growth tase, protein phosphatase 1 (PP-1), resulted in a more modest factor receptors (13). Subsequently, the lymphokines IL-2, decrease in Raf in vitro kinase activity, and this effect was IL-3, and granulocyte-macrophage colony-stimulating factor prevented by okadaic acid. Two-dimensional phosphoamino were found to induce tyrosine phosphorylation and activation acid analysis confirmed the selective removal of phosphate of Raf-1 in hematolymphoid cells (14-16). The tyrosine from tyrosine by CD45 and from serine and threonine by PP-1. phosphorylation of Raf-1 occurs with high stoichiometry in The immunoreactivity of p72-74-Raf-i with anti-phosphoty- some lymphokine-dependent cells, including IL-2-stimulated rosine antibodies was also completely abolished by treatment CTLL-2 cells where 20-50% of the total incorporated 32P04 with CD45 in the absence but not in the presence of sodium was found on tyrosines (14). Furthermore, a physical asso- orthovanadate. These findings provide evidence that the IL- ciation between Raf-1 and the p75 13 chain ofthe IL-2 receptor 2-stimulated phosphorylation of Raf-i on tyrosines plays an has been described (17), suggesting that it may be a proximal important role in upregulating the activity of this ser- player in an IL-2-regulated signal transduction cascade. An ine/threonine-specific kinase in CTLL-2 cells and, as such, important gap in these scenarios that have placed Raf-1 in a provides a model system for studying the transfer of growth likely position to relay information from PTKs to down- factor-initiated signals from protein tyrosine kinases to ser- stream serine/threonine-specific kinases concerns the func- ine/threonine-specific kinases. tional significance of the tyrosine phosphorylation that has been observed in Raf-1 after stimulation of cells with lym- Interleukin 2 (IL-2) regulates the growth, survival, and cy- phokines and other types ofgrowth factors. Here we describe tolytic killing activity of T lymphocytes (1). Although this the results of experiments in which tyrosine-specific protein lymphokine has shown some efficacy in enhancing immune phosphatases were used to remove phosphates selectively responses to tumors in patients, the severe side effects of from tyrosine residues in Raf-1 and the effects on Raf-1 in systemic IL-2 administration have drastically limited its vitro kinase activity were then determined. clinical uses (2). An improved knowledge of the molecular events that occur in T cells upon binding of IL-2 thus could potentially contribute to more effective manipulation of im- MATERIALS AND METHODS mune-cell responses against cancer. Cell Cultures. CTLL-2 cells were adapted to growth in CTLL-2 was originally described as a tumor-specific cy- RPMI 1640 medium containing 1 mM L-glutamine, 10% tolytic T cell that was isolated from mice and that could be (vol/vol) heat-inactivated fetal bovine serum, and 10% (vol/ maintained long-term in culture with IL-2 (3). This T-cell line vol) conditioned medium from the IL-2-producing cell line has been used extensively as a model for investigations ofthe MLA-144 (18) and were then maintained in this medium as mechanisms of IL-2 signal transduction. Unlike many other described (14). All experiments were performed 3 days after growth factor receptors (4), the known subunits of the IL-2 plating CTLL-2 cells, when the cells had reached their receptor complex lack homology with protein-tyrosine ki- plateau phase of growth and had consumed most of the IL-2 nases (PTKs) (5-7). Nevertheless, tyrosine phosphorylation in the cultures. Cells were washed three times with RPMI of numerous intracellular proteins occurs rapidly in CTLL-2 1640 medium to remove residual lymphokine and then re- and other T cells when stimulated with IL-2 (8-10), and pharmacological inhibitors of PTKs prevent most of the Abbreviations: IL-2, interleukin 2; PTK, protein-tyrosine kinase; biochemical and cellular events that normally follow from TCPTP, T-cell protein tyrosine phosphatase; PP-1, protein phospha- exposure ofT cells to IL-2 (see refs. 11 and 12 for examples). tase 1. tPresent address: Department of Neurological Surgery, University of Florida/Shands Hospital, Gainesville, FL 32606. The publication costs of this article were defrayed in part by page charge $To whom reprint requests should be addressed at: La Jolla Cancer payment. This article must therefore be hereby marked "advertisement" Research Foundation, 10901 North Torrey Pines Road, La Jolla, CA in accordance with 18 U.S.C. §1734 solely to indicate this fact. 92037. 5544 Downloaded by guest on September 24, 2021 Immunology: Tumer et al. Proc. Natl. Acad. Sci. USA 90 (1993) 5545 turned to culture for 7-10 h in medium lacking IL-2. In some dithiothreitol, 5 mM EDTA, 0.05% Triton X-100, 5% (vol/ cases, the cells were resuspended in phosphate-free medium vol) glycerol, 5% 2-mercaptoethanol, and 1 mg of bovine and 32P04 (0.2 mCi/ml; 1 Ci = 37 GBq) was added to the serum albumin per ml (22-24). For some samples, either 1 cultures during this 7- to 10-h period to metabolically label the mM sodium orthovanadate or 1 ,uM okadaic acid was added cells. Quiescent CTLL-2 cells were then restimulated with before the phosphatases, but all reactions were stopped by 100 units of recombinant human IL-2 per ml (generous gift addition of these phosphate inhibitors. For 32P04-labeled from Cetus/Chiron) (19) for 5 min. Raf-1, the immunoprecipitates were washed five times in Antibodies, Immunoprecipitations, and Immunoblotting. modified RIPA lysis buffer (14) before analysis by SDS/ The Raf-l-specific monoclonal antibody URP3OS3 was used PAGE using 7.5% gels. In some cases, the bands correspond- for most experiments (20). In some cases, rabbit antisera ing to 32P04-labeled Raf-1 were excised from gels, acid generated against the SP63 peptide corresponding to the C hydrolyzed, and analyzed by two-dimensional electrophore- terminus of Raf kinases were used (21). The anti- sis with 0.1-mm plastic-backed thin-layer cellulose plates phosphotyrosine monoclonal antibody 4G10 was purchased (EM Science) as described (14). For unlabeled Raf-1, immu- from Upstate Biotechnology (Lake Placid, NY). Immuno- noprecipitates were washed twice with a buffer containing precipitations and immunoblot analysis of Raf-1 were per- 1% Triton X-100 (14), once with 0.5 M LiC12/50 mM formed exactly as described except that 4G10 was used at a Tris HCl, pH 7.4, and once with water before in vitro kinase dilution of 1:50,000 (vol/vol) and an emission chemilumines- assays using a synthetic peptide as substrate exactly as cence detection system was used (Amersham). Exposure described (14, 25, 26). times with Kodak XRP film were typically 15-30 s. Phosphatase Treatments and in Vitro Raf Kinase Assays. RESULTS Raf-l-containing immunoprecipitates were prepared from either unlabeled or 32P04-labeled CTLL-2 cells and treated To determine the functional significance of tyrosine phos- for 30-60 min at 30°C with either 100 units of CD45 per ml, phorylation in regulation of Raf-1 kinase activity in IL-2- 100 units of TCPTP (T-cell protein tyrosine phosphatase) per stimulated CTLL-2 cells, we used purified tyrosine-specific ml, or 76 units of protein phosphatase 1 (PP-1) per ml in 50 phosphatases CD45 and TCP to specifically dephosphorylate p1 of a solution containing 25 mM Hepes (pH 7.2), 2 mM tyrosines in p72-74-Raf-1 and then determined the effect of Table 1. Effect of phosphatase treatments on in vitro kinase activity of Raf-1 Phosphatase Stimulation Phosphatase inhibitor Raf kinase activity with IL-2 CD45 TCP PPI Na3VO4 Oka Total cpm Corr cpm Rel.
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