tyrosinephosphatase cd45 cd2-mediated 2038 biochemistry: koretzky et al. cells by using magnetic...

Tyrosinephosphatase CD45 CD2-mediated 2038 Biochemistry: Koretzky et al. cells by using magnetic beads
Tyrosinephosphatase CD45 CD2-mediated 2038 Biochemistry: Koretzky et al. cells by using magnetic beads
Tyrosinephosphatase CD45 CD2-mediated 2038 Biochemistry: Koretzky et al. cells by using magnetic beads
Tyrosinephosphatase CD45 CD2-mediated 2038 Biochemistry: Koretzky et al. cells by using magnetic beads
Tyrosinephosphatase CD45 CD2-mediated 2038 Biochemistry: Koretzky et al. cells by using magnetic beads
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  • Proc. Natl. Acad. Sci. USA Vol. 88, pp. 2037-2041, March 1991 Biochemistry

    Tyrosine phosphatase CD45 is required for T-cell antigen receptor and CD2-mediated activation of a protein tyrosine kinase and interleukin 2 production

    (signal transduction/phosphatidylinositol/second messengers/CD28)

    GARY A. KORETZKY*, JOEL Picust, TERRIE SCHULTZ*, AND ARTHUR WEISS* *Department of Medicine, Division of Rheumatology, Department of Microbiology and Immunology, Howard Hughes Medical Institute, and tCancer Research Institute, University of California, San Francisco, CA 94143

    Communicated by Marian E. Koshland, November 29, 1990 (received for review September 12, 1990)

    ABSTRACT CD45, a hematopoietic cell-specific surface antigen, has recently been shown to be a protein tyrosine phosphatase. Expression of CD45 is essential for the T-cell antigen receptor to couple with the phosphatidylinositol second messenger pathway and for antigen-mediated proliferation of T lymphocytes. In this report we describe a CD45-deficient mutant of the human T-cell leukemia line Jurkat. CD45 expression is required for the activation of a T-cell receptor- associated tyrosine kinase as well as the phosphatidylinositol pathway. Additionally, stimulation ofT lymphocytes by way of the accessory molecule CD2 requires the expression of CD45. The mutation in the CD45-deficient cell specifically impairs signal transduction by the T-cell receptor and CD2 because activation events by way of another accessory molecule, CD28, are unimpaired.

    Activation of T lymphocytes by way of the T-cell antigen receptor (TCR) results in the stimulation of at least two second messenger pathways. The TCR regulates the phos- phatidylinositol (PI) pathway characterized by activation of a phospholipase C specific for phosphatidylinositol 4,5- bisphosphate (PIP2) (1, 2). The hydrolysis products of PIP2 are inositol trisphosphate, which is responsible for the release of calcium from intracellular stores, and diacylglycerol, a cofactor in the activation of protein kinase C (PKC) (2, 3). Stimulation of the TCR also activates an as yet unidentified protein tyrosine kinase (PTK) (4-6). The physiologically relevant substrates of the PTK remain unknown; however, a component of the TCR itself, the chain, becomes phospho- rylated on tyrosine residues (6). Although there exist con- siderable data from studies using pharmacologic agents or various signal transduction mutants (2, 7, 8), the precise role of these two pathways in the initiation of subsequent biologic events remains unclear. T lymphocytes can also be activated when "accessory"

    receptors interact with their ligands. Thus, stimulation of the T-cell surface molecule CD2 results in the generation of PI-derived second messengers and the appearance of new phosphotyrosine-containing proteins (9-12). Another surface antigen, CD28, found on the majority of T lymphocytes can also deliver stimulatory signals (13, 14). Interestingly, acti- vation of T cells with monoclonal antibody (mAb) directed against CD28 cannot be explained by either of the known second messenger pathways stimulated by TCR activation (2, 6, 13), yet CD28 ligation does result in enhanced production of lymphokines such as interleukin 2 (IL-2). CD45 is expressed on all hematopoietic cells except those

    of erythrocyte lineage (15). Previous studies have shown that under various conditions T-cell activation can be either

    enhanced or inhibited in vitro by mAbs against CD45 (16, 17). Possible mechanisms for the influence of CD45 on T-cell activation were suggested when the cytoplasmic domain of CD45 was found to contain two tandem repeats that are homologous to a tyrosine phosphatase isolated from placenta (18). Moreover, immunoprecipitates of CD45 have intrinsic tyrosine phosphatase activity (19). We recently characterized a mutant derived from the

    human T-cell leukemia line HPB-ALL that lacks expression of CD45 (20). In contrast to CD45-positive clones, the TCR of the CD45-negative cells fails to couple to the PI second messenger pathway. Reconstitution of CD45 by transfection of cDNA for murine CD45 restored the ability of the TCR to stimulate the formation of PI-derived second messengers (20). These data, coupled with previous studies demonstrat- ing the importance of CD45 in the proliferative response of murine T-cell clones to specific antigen (21), suggested an important role for CD45 in controlling TCR-mediated signal transduction. Here we describe the isolation of a CD45-deficient mutant

    clone derived from the Jurkat human T-cell leukemia line. In contrast to HPB-ALL, Jurkat expresses CD2 and CD28 and is a potent producer of IL-2. We show that CD45 is essential for the TCR and CD2 to couple efficiently to the PI and PTK second messenger pathways and is necessary for TCR- and CD2-mediated production of IL-2. In contrast, CD45 is not necessary for signal transduction by way of CD28.

    MATERIALS AND METHODS mAbs. The anti-CD45, GAP 8.3, and anti-CD2, OKT11,

    hybridomas were obtained from the American Type Culture Collection. Purified anti-CD2 mAb, 9.6, anti-CD28 mAb, 9.3, and anti-CD45 mAb, 9.4, were generously supplied by On- cogen (Seattle). Purified anti-CD2 mAb, 9-1, was generously supplied by Bo Dupont (Sloan Kettering Institute for Cancer Research, New York). Anti-CD45, HLE-1, was purchased from Becton Dickinson. C305 (clonotypic anti-TCR) has been described (22).

    Cell Lines and the Derivation of a CD45-Deficient Jurkat Clone. Jurkat (clone E6-1), J.CaM1, and J.CaM2 have been described (22, 23). HBP.45.0 is a CD45-negative variant of HPB-ALL and HBP.45.1 is a CD45-positive variant of this cell line (20). J45.01, a CD45-deficient clone of Jurkat, was isolated after y irradiation (200 rads; 1 rad = 0.01 Gy) of Jurkat followed by complement-mediated lysis with the CD45 mAb GAP 8.3 and enrichment of surviving CD45-deficient

    Abbreviations: TCR, T-cell antigen receptor; PI, phosphatidylino- sitol; PIP2, phosphatidylinositol 4,5-bisphosphate; PTK, protein tyrosine kinase; IL-2, interleukin 2; mAb, monoclonal antibody; IP, inositol phosphate; MAP-2 kinase, microtubule-associated protein kinase 2; PMA, phorbol 12-myristate 13-acetate; PKC, protein kinase C.


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

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  • 2038 Biochemistry: Koretzky et al.

    cells by using magnetic beads coated with biotin (Miltenyl Biotec GmbH, Bergisch Gladbach, F.R.G.). CD45-deficient cells finally were isolated with a fluorescence-activated cell sorter and cloned by limiting dilution. Flow Cytometry and Transient Heterokaryon Assay. Cells

    were stained with mAbs and analyzed by flow cytometry as described (20). The heterokaryon assay for signal transduc- tion complementation analysis was performed as described (23). In Vitro Phosphatase Assay. Membranes were prepared

    from wild-type Jurkat and J45.01 by nitrogen cavitation and diluted to 5 mg of membrane protein per ml of buffer (20 mM Hepes, pH 8.0/2 mM MgCl2/1 mM EDTA/1 mM 2-mercap- toethanol/10% glycerol). Membranes were assayed for tyro- sine phosphatase activity by incubation with 10 mM o-phos- pho-L-tyrosine (Sigma) in assay buffer (100 mM sodium acetate, pH 6/1 mM EDTA) as described (24). Measurement of [3H]Inositol Phosphates ([3HJIPs). Cells

    were labeled with myo-[3H]inositol and stimulated with C305 (ascites, 1: 1000) or CD2 mAb (1 ,ug/ml each of 9-1 and 9.6). [3H]IPs were then extracted and quantified as described (25).

    Immunoprecipitation and Western Blotting. For analysis of phosphotyrosine-containing proteins, cells were stimulated with medium alone, anti-TCR, anti-CD2, or phorbol 12- myristate 13-acetate (PMA) for the indicated time. Lysates were assessed for the presence ofphosphotyrosine-containing proteins as described (26). For quantitation of CD45, lysates were immunoprecipitated with anti-CD45 mAb, 9.4. Samples were electrophoresed on 7% SDS/polyacrylamide gels that were then blotted with purified 9.4 mAb, 0.5 ,ug/ml.

    IL-2 Production and Biolgic Assay. Supernatants from cell cultures were assayed for IL-2 as described (22).

    RESULTS Characterization of a CD45-Deficient Jurkat Clone. To

    address the potential role of CD45 in coupling the TCR to the PTK second messenger pathway and the possible require- ment for CD45 in CD2 and CD28 signal transduction, we mutagenized the human T-cell leukemia line Jurkat and selected for CD45-deficient variants (see Materials and Methods). One of the clones obtained, J45.01, stains brightly (comparable to wild type) with mAb directed against the TCR, CD2, and CD28 but expresses only 8% as much CD45 on its surface when compared to the wild-type Jurkat parent, even when a panel of CD45 mAb was used (Table 1). To characterize J45.01 further, Northern blot analysis was

    performed on poly(A)+-selected mRNA obtained from Jurkat and J45.01, which revealed grossly equivalent levels of CD45 mRNA (data not shown). We therefore assessed the amount of CD45 protein expressed intracellularly and on the cell surface in the two clones by Western blotting ofwhole cell lysates with a mAb directed against all isoforms of CD45. In lysates, sub- stantial amounts of CD45 can be detected in Jurkat, whereas there is no detectable CD45 in J45.01 (Fig. LA). When immuno-

    Table 1. Staining characteristics of Jurkat and J45.01 Median


    Antigen Jurkat J45.01 TCR 68 66 CD2 58 58 CD28 39 25 CD45 99 8

    Median fluorescence was measured by flow cytometry (line