The Second Domain of the CD45 Protein Tyrosine Phosphatase Is Critical for Interleukin-2 Secretion and Substrate Recruitment of TCR-ζ in Vivo

The CD45 protein tyrosine phosphatase (PTPase) has been shown to regulate the activity of Lck and Fyn protein tyrosine kinases in T cells. However, it is not clear that these constitute the only CD45 substrates. Moreover, the manner by which PTPase activity and substrate recruitment are regulated, is poorly understood. Previousin vitro studies suggest that the first cytoplasmic PTPase domain (D1) of CD45 is the active PTPase, which may be regulated by an enzymatically inactive second PTPase domain (D2). However, the function of CD45 D2 in vivo is unknown. In this study, reconstitution of CD45− T cells with specific CD45 PTPase mutants allowed demonstration of a critical role for D2 in TCR-mediated interleukin (IL)-2 production. Specifically, replacement of CD45 D2 with that of the LAR PTPase to form a CD45/LAR:D2 chimera, abrogates CD45-dependent IL-2 production. This effect cannot be accounted for by loss of PTPase activity per se. The expression of D1 substrate-trapping mutants reveals an in vivo interaction between CD45 and TCR-ζ that is dependent on CD45 D2. Thus, cells expressing CD45 lacking D2 exhibit abnormal TCR-mediated signaling characterized by hyperphosphorylation of ζ and deficient ZAP-70 phosphorylation. These data suggest an essential role for CD45 D2 in TCR-regulated IL-2 production through substrate recruitment of the ζ chain. The CD45 protein tyrosine phosphatase (PTPase) has been shown to regulate the activity of Lck and Fyn protein tyrosine kinases in T cells. However, it is not clear that these constitute the only CD45 substrates. Moreover, the manner by which PTPase activity and substrate recruitment are regulated, is poorly understood. Previousin vitro studies suggest that the first cytoplasmic PTPase domain (D1) of CD45 is the active PTPase, which may be regulated by an enzymatically inactive second PTPase domain (D2). However, the function of CD45 D2 in vivo is unknown. In this study, reconstitution of CD45− T cells with specific CD45 PTPase mutants allowed demonstration of a critical role for D2 in TCR-mediated interleukin (IL)-2 production. Specifically, replacement of CD45 D2 with that of the LAR PTPase to form a CD45/LAR:D2 chimera, abrogates CD45-dependent IL-2 production. This effect cannot be accounted for by loss of PTPase activity per se. The expression of D1 substrate-trapping mutants reveals an in vivo interaction between CD45 and TCR-ζ that is dependent on CD45 D2. Thus, cells expressing CD45 lacking D2 exhibit abnormal TCR-mediated signaling characterized by hyperphosphorylation of ζ and deficient ZAP-70 phosphorylation. These data suggest an essential role for CD45 D2 in TCR-regulated IL-2 production through substrate recruitment of the ζ chain. CD45 is a family of transmembrane PTPases 1The abbreviations used are: PTPase, protein tyrosine phosphatase; CS, Cys-to-Ser (replacement); D1, first cytoplasmic phosphatase domain; D2, second cytoplasmic phosphatase domain; PTK, protein tyrosine kinase; wt, wild-type; PAGE, polyacrylamide gel electrophoresis; IL-2, interleukin-2; mAb, monoclonal antibody; AEBSF, aminoethylbenzenesulfonyl fluoride; ITAM, immunoreceptor tyrosine-based activation motif. critically involved in lymphocyte activation. Multiple alternatively spliced isoforms differ in the length and glycosylation of their extracellular domains but share identical cytoplasmic PTPase domains (1Ralph S.J. Thomas M.L. Morton C.C. Trowbridge I.S. EMBO J. 1987; 6: 1251-1257Crossref PubMed Scopus (179) Google Scholar, 2Streuli M. Hall L.R. Saga Y. Schlossman S.F. Saito H. J. Exp. Med. 1987; 166: 1548-1566Crossref PubMed Scopus (276) Google Scholar). CD45− mutant T cell lines fail to normally phosphorylate cellular proteins or produce IL-2 after TCR ligation (3Pingel J.T. Thomas M.L. Cell. 1989; 58: 1055-1065Abstract Full Text PDF PubMed Scopus (438) Google Scholar, 4Koretzky G.A. Picus J. Thomas M.L. Weiss A. Nature. 1990; 346: 66-68Crossref PubMed Scopus (395) Google Scholar). Initial studies comparing CD45− mutants to wild-type or revertant cells indicated that CD45 up-regulates Lck and Fyn activity by dephosphorylating their negative regulatory (COOH-terminal) tyrosine sites (5Ostergaard H.L. Shackelford D.A. Hurley T.R. Johnson P. Hyman R. Sefton B.M. Trowbridge I.S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8959-8963Crossref PubMed Scopus (413) Google Scholar, 6Mustelin T. Coggeshall K.M. Altman A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6302-6306Crossref PubMed Scopus (407) Google Scholar, 7Hurley T.R. Hyman R. Sefton B. Mol. Cell Biol. 1993; 13: 1651-1656Crossref PubMed Scopus (166) Google Scholar, 8McFarland E.D. Hurley T.R. Pingel J.T. Sefton B.M. Shaw A. Thomas M.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1402-1406Crossref PubMed Scopus (192) Google Scholar). However, subsequent analysis demonstrated that CD45 can also down-regulate Fyn and Lck activity by dephosphorylating their positive regulatory autophosphorylation sites (9Burns C.M. Sakaguchi K. Appella E. Ashwell J.D. J. Biol. Chem. 1994; 269: 13594-13600Abstract Full Text PDF PubMed Google Scholar, 10D'Oro U. Sakaguchi K. Appella E. Ashwell J. Mol. Cell. Biol. 1996; 16: 4996-5003Crossref PubMed Scopus (86) Google Scholar). It is also not clear that these PTKs constitute the only CD45 substrates. Cytoplasmic PTPases appear to be targeted to their appropriate substrates by protein interaction and cellular localization motifs (11Mauro L.J. Dixon J.E. Trends Biochem. Sci. 1994; 19: 151-155Abstract Full Text PDF PubMed Scopus (181) Google Scholar). Such interactions with target proteins may also enhance the activity of these PTPases (12Leichleider R. Sugimoto S. Bennett A. Kashishian A. Cooper JA Shoelson S. Walsh C. Neel B. J. Biol. Chem. 1993; 268: 21478-21481PubMed Google Scholar, 13Pei D. Lorenz U. Klingmuller U. Neel B. CT W. Biochemistry. 1994; 33: 15483-15493Crossref PubMed Scopus (186) Google Scholar). However, for transmembrane PTPases, very little is known about how substrate specificity and activity are regulated. Recent evidence indicates that the extracellular domain is able to regulate signaling through the cytoplasmic PTPase domains (14Novak T. Farber D.L. Leitenberg D. Hong S.-C. Johnson J. Bottomly K. Immunity. 1994; 1: 109-119Abstract Full Text PDF PubMed Scopus (110) Google Scholar, 15McKenney D.W. Onodera H. Gorman L. Mimura T. Rothstein D.M. J. Biol. Chem. 1995; 270: 24949-24954Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 16Onodera H. Motto D.G. Koretzky G.A. Rothstein D.M. J. Biol. Chem. 1996; 271: 22225-22230Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 17Majeti R. Bilwes A. Noel J. Hunter T. Weiss A. Science. 1998; 279: 88-91Crossref PubMed Scopus (219) Google Scholar). In this regard, most transmembrane PTPases, including CD45, share tandem PTPase domains (18Streuli M. Krueger N.X. Hall L. Schlossman S. Saito H. J. Exp. Med. 1988; 168: 1553-1562Crossref PubMed Scopus (185) Google Scholar, 19Tonks N.K. Neel B.G. Cell. 1996; 87: 365-368Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar). Curiously, the second PTPase domain (D2) of most of these molecules appears to have little or no PTPase activity against various in vitro substrates. Furthermore, PTPase consensus sequences in D2 are less well conserved than in domain 1 (D1), and in some cases, key residues required for enzymatic activity (for example, the catalytic center Cys residue), are absent (20Krueger N. Streuli M. Saito H. EMBO J. 1990; 9: 3241-3252Crossref PubMed Scopus (370) Google Scholar, 21Krueger N. Saito H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7417-7421Crossref PubMed Scopus (201) Google Scholar). Such findings suggest an alternative role for D2 in transmembrane PTPases. In the case of CD45, mutation of the D1 catalytic center Cys to Ser (position 828) completely abrogates in vitro PTPase activity (22Streuli M. Krueger N.X. Thai T. Tang M. Saito H. EMBO J. 1990; 9: 2399-2407Crossref PubMed Scopus (268) Google Scholar, 23Johnson P. Ostergaard H.L. Wasden C. Trowbridge I.S. J. Biol. Chem. 1992; 267: 8035-8041Abstract Full Text PDF PubMed Google Scholar), whereas, mutation of the analogous Cys to Ser in D2 (position 1144), has no effect. While, deletion of D2 in its entirety destabilizes D1 rendering it inactive (22Streuli M. Krueger N.X. Thai T. Tang M. Saito H. EMBO J. 1990; 9: 2399-2407Crossref PubMed Scopus (268) Google Scholar, 23Johnson P. Ostergaard H.L. Wasden C. Trowbridge I.S. J. Biol. Chem. 1992; 267: 8035-8041Abstract Full Text PDF PubMed Google Scholar), small deletions within D2 strikingly alter the relative activity of CD45 against various substrates in vitro. These results suggest that D2 is not an active PTPase but may play a regulatory role for D1 (22Streuli M. Krueger N.X. Thai T. Tang M. Saito H. EMBO J. 1990; 9: 2399-2407Crossref PubMed Scopus (268) Google Scholar). Nevertheless, it has been reported that deletion of large portions of D1 can activate D2 in vitro (24Tan X. Stover D.R. Walsh K.A. J. Biol. Chem. 1993; 268: 6835-6838Abstract Full Text PDF PubMed Google Scholar), at least raising the possibility that D2 could have cryptic PTPase activity in vivo. Although Cys to Ser (CS) mutation of the catalytic center inactivates PTPase activity, substrate binding is preserved and substrate trapping within the enzyme active site is actually promoted (19Tonks N.K. Neel B.G. Cell. 1996; 87: 365-368Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar, 25Black D. Bliska J. EMBO J. 1997; 16: 2730-2744Crossref PubMed Scopus (288) Google Scholar). Glutathione S-transferase-CD45 fusion proteins containing CS mutation of D1 were able to co-precipitate phospho-ζ from lysates obtained from activated Jurkat cells (26Furukawa T. Itoh M. Krueger N.X. Streuli M. Saito H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10928-10932Crossref PubMed Scopus (150) Google Scholar). When D1 was active, phospho-ζ was no longer co-precipitated, suggesting that ζ might be a direct substrate for CD45 D1. Swapping either CD45 D1 or D2 with the corresponding domain from the LAR PTPase prevented this in vitro association. These findings suggested that CD45 D2 may influence D1 by playing a role in substrate recruitment. To establish a regulatory role for D2 among transmembrane PTPases and identify in vivo substrates critical to our understanding of CD45 function, we now address the physiologic role of CD45 D2 and substrate recruitment of ζ in TCR-mediated signal transduction. We have transfected a CD45− version of the Jurkat human leukemic T cell line with either wild-type CD45 or PTPase mutants. Direct comparison of these cell lines demonstrates that IL-2 production is dependent on an intact CD45 D1 PTPase. Importantly, we now show that CD45 D2 is also critical for IL-2 production. However, this is not related to loss of D2 PTPase activity per se, nor by a requirement for D2 in the interaction between CD45 and Lck. While enzymatically inactive substrate-trapping mutants of CD45 D1 coprecipitate phospho-ζ, this interaction is dependent on the presence of CD45 D2. Despite active CD45 D1, replacement of CD45 D2 with that of LAR resulted in hyperphosphorylation of TCR-ζ and deficient activation-induced phosphorylation of ZAP-70. These data support a mechanism whereby CD45 D2 regulates IL-2 production through the recruitment of TCR-ζ as a CD45 substrate. The CD45(0) construct encodes wild-type (wt) CD45 PTPase domains in the context of the smallest CD45 extracellular domain under control of the SRα promoter (15McKenney D.W. Onodera H. Gorman L. Mimura T. Rothstein D.M. J. Biol. Chem. 1995; 270: 24949-24954Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Plasmids encoding the isolated CD45 cytoplasmic domains containing CS point mutations of: D1 (position 828), D2 (position 1144), or wt CD45 D1 (amino acids 584–895) fused to wt LAR D2 (amino acids 1590–1881) (26Furukawa T. Itoh M. Krueger N.X. Streuli M. Saito H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10928-10932Crossref PubMed Scopus (150) Google Scholar, 27Itoh M. Streuli M. Krueger N. Saito H. J. Biol. Chem. 1992; 267: 12356-12363Abstract Full Text PDF PubMed Google Scholar) were kindly provided by Dr. H. Saito (Dana-Farber Cancer Institute, Boston, MA). Using convenient restriction sites, segments of these constructs were used to replace the analogous wt region of CD45(0) generating full-length (transmembrane) CD45 PTPase mutants. A double mutant, containing CS mutations in both D1 and D2, was generated usingPstI sites. Using polymerase chain reaction, a CS mutation was inserted into position 828 of D2:LAR to generate D1:CS/D2:LAR. DNA sequencing (Keck Biotechnology Center, Yale University) confirmed the expected mutation. The CD4+CD45− J-AS Jurkat clone lacks endogenous CD45 expression by virtue of a stably integrated antisense gene targeting the 5′-untranslated region of CD45 (15McKenney D.W. Onodera H. Gorman L. Mimura T. Rothstein D.M. J. Biol. Chem. 1995; 270: 24949-24954Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 16Onodera H. Motto D.G. Koretzky G.A. Rothstein D.M. J. Biol. Chem. 1996; 271: 22225-22230Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). J-AS was transfected with CD45(0) constructs encoding either wt or mutant PTPase domains along with the PGK-hyg vector by electroporation, as described (15McKenney D.W. Onodera H. Gorman L. Mimura T. Rothstein D.M. J. Biol. Chem. 1995; 270: 24949-24954Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Hygromycin-resistant clones were screened for CD45 expression by immunofluorescence and Western blotting. Two previously described clones expressing the wt CD45R0 isoform, J(0)-2 and J(0)-3, were also used in these studies (16Onodera H. Motto D.G. Koretzky G.A. Rothstein D.M. J. Biol. Chem. 1996; 271: 22225-22230Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). All cells were grown in RPMI 1640 media supplemented with 10% iron-fortified calf serum,l-glutamine, and gentamycin. Transfectants were removed from G418 and hygromycin for 7–10 days before use in assays. Cell phenotype was routinely monitored for CD3, CD4, and CD45 expression using commercially available mAbs and analyzed on a FACSTAR IV (Becton Dickinson, Mountain View, CA) (10,000 cells/sample), as described (15McKenney D.W. Onodera H. Gorman L. Mimura T. Rothstein D.M. J. Biol. Chem. 1995; 270: 24949-24954Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Anti-CD45 (9.4) and anti-CD3 (OKT3) hybridomas were from the ATCC. mAbs were purified from culture supernatants using Protein-A-Sepharose according to standard methods. The following were kindly provided as gifts: anti-phosphotyrosine (anti-Tyr(P)) mAb 4G10 (from Dr. B. Drucker, University of Oregon, Portland, OR), anti-ζ mAb 6B10.2 (from Dr. A. Weiss, University of California at San Francisco, San Francisco, CA), polyclonal rabbit anti-ζ (from Dr. K. Bottomly, Yale University, New Haven, CT), and polyclonal rabbit anti-Lck (from Dr. C. Rudd, Dana-Farber Cancer Institute, Boston, MA). 105 cells/well in triplicate flat bottom 96-well tissue culture plates were stimulated with anti-CD3 (OKT3) (from 0.1 to 0.01 μg/ml) plus goat anti-mouse cross-linking (at a 1:1 ratio with OKT3). Phorbol 12-myristate 13-acetate (1 ng/ml) was added to all wells, as described (15McKenney D.W. Onodera H. Gorman L. Mimura T. Rothstein D.M. J. Biol. Chem. 1995; 270: 24949-24954Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). IL-2 secretion in 20-h cell culture supernatants was determined by enzyme-linked immunosorbent assay (Genzyme Corp.). In each experiment, the data were normalized to the response of transfectants expressing (wt) CD45(0) to 0.1 μg/ml anti-CD3 (15McKenney D.W. Onodera H. Gorman L. Mimura T. Rothstein D.M. J. Biol. Chem. 1995; 270: 24949-24954Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). IL-2 secretion after stimulation with 1 ng/ml phorbol 12-myristate 13-acetate and 1 μm ionomycin, was used as a positive control. As described previously (15McKenney D.W. Onodera H. Gorman L. Mimura T. Rothstein D.M. J. Biol. Chem. 1995; 270: 24949-24954Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar,16Onodera H. Motto D.G. Koretzky G.A. Rothstein D.M. J. Biol. Chem. 1996; 271: 22225-22230Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar), cells (5 × 107/ml) were stimulated at 37 °C with either anti-CD3 (17 μg/ml) or pervanadate (3 mmH2O2, 100 mmNa3VO4) plus 10 mm phenylarsine oxide, which mimic the effects of TCR ligation (28Garcia-Morales P. Minami Y. Luong E. Klausner R. Samelson L.E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9255-9259Crossref PubMed Scopus (235) Google Scholar, 29Secrist J.P. Burns L.A. Karnitz L. Koretzky G.A. Abraham R.T. J. Biol. Chem. 1993; 268: 5886-5893Abstract Full Text PDF PubMed Google Scholar). At the indicated times, ice-cold stop solution (phosphate-buffered saline with phosphatase inhibitors) was added, cells were pelleted and lysed in ice-cold 1% Brij-97 lysis buffer (CD45 immunoprecipitation) or 1% Nonidet P-40 (ζ immunoprecipitation) containing 50 mmTris-HCl (pH 8.0), 150 mm NaCl, 1 mmaminoethylbenzenesulfonyl fluoride (AEBSF), 10 mg/ml aprotinin, 10 mg/ml leupeptin, 10 mm iodoacetamide, 10 mmsodium fluoride, and 10 mm sodium pyrophosphate, as described (15McKenney D.W. Onodera H. Gorman L. Mimura T. Rothstein D.M. J. Biol. Chem. 1995; 270: 24949-24954Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 16Onodera H. Motto D.G. Koretzky G.A. Rothstein D.M. J. Biol. Chem. 1996; 271: 22225-22230Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Immunoprecipitations were performed as described (15McKenney D.W. Onodera H. Gorman L. Mimura T. Rothstein D.M. J. Biol. Chem. 1995; 270: 24949-24954Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 16Onodera H. Motto D.G. Koretzky G.A. Rothstein D.M. J. Biol. Chem. 1996; 271: 22225-22230Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Briefly, after preclearing, postnuclear supernatants containing equivalent amounts of protein (DC protein assay; Bio-Rad) were incubated with antibody, followed by immunoprecipitation with Protein A-Sepharose (UBI). Immunoprecipitates or postnuclear supernatants (whole cell lysates) were subjected to SDS-PAGE and transferred to nitrocellulose. Membranes were blocked (5% nonfat milk in phosphate-buffered saline), probed with primary antibody followed by horseradish peroxidase-conjugated secondary antibody, and developed with enhanced chemiluminescence. Cells expressing either CD45(0) or D2:LAR were lysed in 1% digitonin, 50 mm Tris-HCl (pH 8.0), 150 mm NaCl, AEBSF, aprotinin, and leupeptin (as above), followed by immunoprecipitation with anti-CD45, anti-Lck, or control rabbit anti-mouse Ab. After washing 4 times, immunoprecipitates were resuspended in 40 μl of kinase buffer (25 mm HEPES (pH 7.6), 10 mm MnCl2 plus 10 μCi of [γ-32P]ATP), for 15 min, as described (30Ross S. Schraven B. Goldman F.D. Crabtree J. Koretzky G.A. Biochem. Biophys. Res. Commun. 1994; 198: 88-96Crossref PubMed Scopus (16) Google Scholar). Sepharose beads were then washed twice in lysis buffer, boiled in Laemmli sample buffer, run on 12% SDS-PAGE, and subjected to autoradiography. After lysis in 0.5% Triton X-100 containing 20 mm Tris (pH 7.5), 150 mm NaCl, 2 mm EDTA, and AEBSF, aprotinin, and leupeptin (as above), CD45 immunoprecipitates from each cell line were extensively washed and resuspended in 25 mm imidazole (pH 7.2), 50 mmNaCl, 1 mm EDTA with 5 mm dithiothreitol. Equal amounts of each CD45 protein, as confirmed by Western analysis, were analyzed for PTPase activity using the tyrosine phosphatase assay (Promega Corp., Madison WI) which determines the concentration of free phosphate released from a tyrosyl-phosphorylated peptide substrate by the absorbance of a molybdate-malachite green-phosphate complex. Both phosphopeptide substrates, END(pY)INASL and DADE(pY)LIPQQG, supplied with the assay gave equivalent results. Because of the potential regulatory role of the CD45 extracellular domain (15McKenney D.W. Onodera H. Gorman L. Mimura T. Rothstein D.M. J. Biol. Chem. 1995; 270: 24949-24954Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 16Onodera H. Motto D.G. Koretzky G.A. Rothstein D.M. J. Biol. Chem. 1996; 271: 22225-22230Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) and association of LPAP with the CD45 transmembrane domain (31Schraven B. Schoenhaut D. Bruyns E. Koretzky G. Eckerskorn C. Wallich R. Kirchgessner H. Sakorafas P. Labkovsky B. Ratnofsky S. Meuer S. J. Biol. Chem. 1994; 269: 29102-29111Abstract Full Text PDF PubMed Google Scholar, 32McFarland E. Thomas M. J. Biol. Chem. 1995; 270: 28103-28107Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), the physiologic function of the CD45 PTPase domains was addressed using intact transmembrane CD45 molecules. To this end, CD45− J-AS Jurkat cells were stably transfected to express either wild-type (wt) CD45, or PTPase mutations in the context of the smallest (CD45RO or CD45(0)) extracellular domain. The J-AS parental cell line was initially generated by means of antisense gene targeting of endogenous CD45, as we described (15McKenney D.W. Onodera H. Gorman L. Mimura T. Rothstein D.M. J. Biol. Chem. 1995; 270: 24949-24954Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). These cells combine specific targeting of CD45 with expression levels that are undetectable by Western analysis. J-AS cells were stably transfected with (wt) CD45(0), inactivating CS point mutation of the catalytic center of CD45 domain 1 (D1:CS), domain 2 (D2:CS), or domains 1 and 2 (D1:CS/D2:CS) (see Fig.1). In addition, chimeric proteins containing D2 of the LAR PTPase, either in the context of active CD45 D1 (D2:LAR) or inactive CD45 D1 (D1:CS/D2:LAR) were expressed. The transfectants expressed similar levels of CD45, CD3, and CD4 (See Fig.2). Expression of only a single CD45 isoform of 180 kDa was confirmed by Western blotting (as seen in Fig.6 C). Two independent transfected clones expressing each CD45 construct were selected for these studies.Figure 2Phenotypic analysis of surface expression of transfectants. Representative immunofluorescence analysis of CD45, CD4, and CD3 expression of J-AS and (J-AS derived) transfectants expressing various CD45 constructs as noted. Isotype-matched negative controls are depicted in the first column, as dotted lines. The x and y axes represent log fluorescence and cell number, respectively.View Large Image Figure ViewerDownload (PPT)Figure 6Association of phospho-ζ with CD45 PTPase mutants in vivoin an activation and phosphorylation dependent fashion. Transfectants before and after stimulation with phenyl arsine oxide/pervanadate were lysed in Brij-97. Equivalent amounts of each lysate were subjected to immunoprecipitation with anti-CD45 followed by resolution on 12.5% SDS-PAGE (reducing conditions) and immunoblotting with polyclonal α-Zeta (Panel A). The membrane was stripped and reprobed using α-Tyr(P) (Panel B). 4 × 106 cell equivalents of each lysate used for the immunoprecipitates above were run on 6% SDS-PAGE (non-reducing) and immunoblotted with anti-CD45 (Panel C). L.C. denotes light chain.View Large Image Figure ViewerDownload (PPT) Before examining the effects of these CD45 mutations on cellular function, the PTPase activity of each was examined in vitro(see Fig. 3). Consistent with previous analysis of isolated cytoplasmic domains, CS mutation of D1 (D1:CS), or D1 and D2 (D1:CS/D2:CS), abrogates the PTPase activity of intact CD45 molecules immunoprecipitated from each transfectant. Likewise, CS mutation of CD45 D2 alone (D2:CS) was without detectable effect (22Streuli M. Krueger N.X. Thai T. Tang M. Saito H. EMBO J. 1990; 9: 2399-2407Crossref PubMed Scopus (268) Google Scholar,23Johnson P. Ostergaard H.L. Wasden C. Trowbridge I.S. J. Biol. Chem. 1992; 267: 8035-8041Abstract Full Text PDF PubMed Google Scholar). Importantly, comparison of the D2:LAR chimera to wild-type CD45 (CD45(0) shows that the chimeric protein retains full catalytic activity (Fig. 3). This is not surprising, since D2 of both LAR and CD45 lack activity in vitro and are believed to share similar structure based on conservation of grouped residues at key positions and ∼40% overall amino acid identity (22Streuli M. Krueger N.X. Thai T. Tang M. Saito H. EMBO J. 1990; 9: 2399-2407Crossref PubMed Scopus (268) Google Scholar, 33Streuli M. Krueger N.X. Tsai A.M. Saito H. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8689-8702Crossref Scopus (246) Google Scholar, 34Desai D. Sap J. Silvennoinen O. Schlessinger J. Weiss A. EMBO. 1994; 13: 4002-4010Crossref PubMed Scopus (91) Google Scholar, 35Barford D. Flint A.J. Tonks N.K. Science. 1994; : 397-1404PubMed Google Scholar). To examine the in vivo role of CD45 D2, TCR-mediated IL-2 production was assessed (See Fig. 4). In agreement with our previous results, CD45− J-AS cells produce only small amounts of IL-2 in response to anti-CD3 stimulation and IL-2 production is reconstituted by expression of the CD45(0) isoform (15McKenney D.W. Onodera H. Gorman L. Mimura T. Rothstein D.M. J. Biol. Chem. 1995; 270: 24949-24954Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Expression of CD45 containing CS mutations of both D1 and D2 eliminates the restoration of IL-2 production, confirming the principal role of PTPase activity in CD45 function. Consistent with results suggesting that CD45 D1 contains most if not all of the detectable PTPase activity (Fig. 3) (22Streuli M. Krueger N.X. Thai T. Tang M. Saito H. EMBO J. 1990; 9: 2399-2407Crossref PubMed Scopus (268) Google Scholar, 23Johnson P. Ostergaard H.L. Wasden C. Trowbridge I.S. J. Biol. Chem. 1992; 267: 8035-8041Abstract Full Text PDF PubMed Google Scholar), mutation of CD45 D1 alone (D1:CS) also prevented IL-2 secretion, whereas, mutation of the D2 catalytic center (D2:CS) resulted in only a small decrease in IL-2 production that was not statistically different from wt CD45(0). Similar results were obtained using concentrations of anti-CD3 ranging from 0.1 to .01 mg/ml (Fig. 4). Each cell line displayed a similar inherent capacity to produce IL-2 when the proximal signaling apparatus was bypassed using phorbol 12-myristate 13-acetate and ionomycin (data not shown). These data indicate that if D2 does exhibit cryptic PTPase activity in vivo, it does not play a significant role in TCR-induced IL-2 production. Concordantly, expression of chimeric proteins containing active CD45 D1 (in the presence of wt or inactive D2) in CD45− cells was necessary and sufficient to reconstitute TCR-mediated tyrosine phosphorylation of cellular proteins and dephosphorylate Lck at its carboxyl-terminal regulatory site (34Desai D. Sap J. Silvennoinen O. Schlessinger J. Weiss A. EMBO. 1994; 13: 4002-4010Crossref PubMed Scopus (91) Google Scholar,36Niklinska B.B. Hou D. June C.H. Weissman A.M. Ashwell J.D. Mol. Cell Biol. 1994; 14: 8078-8084Crossref PubMed Scopus (14) Google Scholar). Taken together, these results cast doubt on physiologically significant PTPase activity of CD45 D2 in vivo. Lack of PTPase activity does not preclude important function. Therefore, to further examine the role of CD45 D2 in IL-2 production, D2 of CD45 was replaced with that of the LAR PTPase, generating a catalytically active chimeric protein containing CD45 D1 and LAR D2 (D2:LAR). In marked contrast to the results obtained with point mutation of the D2 catalytic center (D2:CS), replacement of CD45 D2 with that of LAR (D2:LAR) abrogated the ability of CD45 to promote IL-2 production (Fig. 4). Similar results were obtained using various anti-CD3 concentrations. These results suggest that CD45 D2 plays an important regulatory role in CD45 function and this is not explained by loss of D2 PTPase activity per se. One explanation for these findings is that CD45 D2 plays an important role in substrate recruitment. In this regard, Lck acts as a CD45 substrate and co-precipitates with CD45 in non-disruptive detergents (30Ross S. Schraven B. Goldman F.D. Crabtree J. Koretzky G.A. Biochem. Biophys. Res. Commun. 1994; 198: 88-96Crossref PubMed Scopus (16) Google Scholar, 37Schraven B. Schirren A. Kirchgessner H. Siebert B. Meuer S.C. Eur. J. Immunol. 1992; 22: 1857-1863Crossref PubMed Scopus (48) Google Scholar, 38Koretzky G.A. Kohmetscher M. Ross S. J. Biol. Chem. 1993; 268: 8958-8964Abstract Full Text PDF PubMed Google Scholar). Although Lck interacts with the CD45 cytoplasmic domain (39Autero M. Saharinen J. Pessa-Morihawa T. Soula-Rothnut M. Oetken C. Gassmann M. Alitalo U. Burn P. Gahmberg C.G. Mustelin T. Mol. Cell. Biol. 1994; 14: 1398-1421Crossref Scopus (118) Google Scholar, 40Ng D.H. Watts J.D. Aebersold R. Johnson P. J. Biol. Chem. 1996; 271: 1295-1300Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), it is unknown whether CD45 D2 is involved. To determine whether D2 is required for this interaction, we compared overall and CD45-associated Lck activity in cells expressing either CD45(0) or D2:LAR using immune complex kinase assays. As seen in Fig.5 A, equivalent Lck activity, as measured by Lck autophosphorylation (56–60 kDa band), is precipitated from both cell lines. Thus, loss of CD45 D2 does not appear to affect overall Lck activity. CD45 immunoprecipitates from both transfectants reveal autophosphorylated Lck, as well as associated LPAP (32–34 kDa) which undergoes in vitro phosphorylation in this assay, as described (30Ross S. Schraven B. Goldman F.D. Crabtree J. Koretzky G.A.