Negative Regulation of T Cell Antigen Receptor Signal Transduction by Hematopoietic Tyrosine Phosphatase (HePTP)

The hematopoietic tyrosine phosphatase (HePTP) is predominantly expressed in thymocytes and T lymphocytes and at lower levels in other hematopoietic cells. Expression of the gene is enhanced by the T cell growth factor interleukin-2, suggesting a role for HePTP in T cell proliferation or differentiation. We report that HePTP blocks T cell antigen receptor (TCR)-induced transcriptional activation of a reporter gene driven by a nuclear factor of activated T cells(NFAT)/AP-1 element taken from the interleukin-2 gene promoter. This effect was specific to HePTP and was abolished by a mutation (C270S) that impaired its phosphatase activity. Co-expression of HePTP also reduced TCR-induced activation of the mitogen-activated protein kinase Erk2 and the TCR-induced appearance of phosphorylated Erk. In contrast, HePTP did not affect the activation of the N-terminal c-Jun kinase, Jnk. Together these findings suggest that HePTP plays an active negative role in TCR signaling by dephosphorylating one or several signaling molecules between the receptor and the mitogen-activated protein kinase pathway. The hematopoietic tyrosine phosphatase (HePTP) is predominantly expressed in thymocytes and T lymphocytes and at lower levels in other hematopoietic cells. Expression of the gene is enhanced by the T cell growth factor interleukin-2, suggesting a role for HePTP in T cell proliferation or differentiation. We report that HePTP blocks T cell antigen receptor (TCR)-induced transcriptional activation of a reporter gene driven by a nuclear factor of activated T cells(NFAT)/AP-1 element taken from the interleukin-2 gene promoter. This effect was specific to HePTP and was abolished by a mutation (C270S) that impaired its phosphatase activity. Co-expression of HePTP also reduced TCR-induced activation of the mitogen-activated protein kinase Erk2 and the TCR-induced appearance of phosphorylated Erk. In contrast, HePTP did not affect the activation of the N-terminal c-Jun kinase, Jnk. Together these findings suggest that HePTP plays an active negative role in TCR signaling by dephosphorylating one or several signaling molecules between the receptor and the mitogen-activated protein kinase pathway. One of the earliest biochemical events seen in T lymphocytes triggered through the T cell antigen-receptor (TCR) 1The abbreviations used are: TCR, T cell antigen receptor; Erk2, extracellular signal-regulated protein kinase 2; HePTP, hematopoietic cell PTPase; MAPK, mitogen-activated protein kinase; NFAT, nuclear factor of activated T cells; PTK, protein tyrosine kinase; PTPase, protein tyrosine phosphatase; LMPTP, low molecular weight PTPase; VHR, vaccinia H1-related; TCPTP, T cell PTPase. 1The abbreviations used are: TCR, T cell antigen receptor; Erk2, extracellular signal-regulated protein kinase 2; HePTP, hematopoietic cell PTPase; MAPK, mitogen-activated protein kinase; NFAT, nuclear factor of activated T cells; PTK, protein tyrosine kinase; PTPase, protein tyrosine phosphatase; LMPTP, low molecular weight PTPase; VHR, vaccinia H1-related; TCPTP, T cell PTPase.is an enhanced, but transient, phosphorylation of a number of cellular proteins on tyrosine residues. Inhibition of this event by pharmacological agents prevents T cell activation as measured by both functional read-outs and biochemical assays (1Mustelin T. Coggeshall K.M. Isakov N. Altman A. Science. 1990; 247: 1584-1587Crossref PubMed Scopus (369) Google Scholar, 2June C. Fletcher M.C. Ledbetter J.A. Schieven G.L. Siegel J.N. Phillips A.F. Samelson L.E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7722-7727Crossref PubMed Scopus (432) Google Scholar). It has become evident that several protein tyrosine kinases (PTKs) and the CD45 protein tyrosine phosphatase (PTPase) play crucial roles (reviewed in Ref. 3Mustelin T. Immunity. 1994; 1: 351-356Abstract Full Text PDF PubMed Scopus (82) Google Scholar) and that the TCR-induced cascade of transient tyrosine phosphorylation events depends on a dynamic interplay between these and, presumably, many additional PTKs and PTPases. In addition to CD45 (4Pingel J.T. Thomas M.L. Cell. 1989; 58: 1055-1065Abstract Full Text PDF PubMed Scopus (437) Google Scholar, 5Koretzky G.A. Picus J. Thomas M.L. Weiss A. Nature. 1990; 346: 66-68Crossref PubMed Scopus (394) Google Scholar, 6Mustelin T. Coggeshall K.M. Altman A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6306-6502Crossref Scopus (408) Google Scholar, 7Mustelin T. Altman A. Oncogene. 1990; 5: 809-813PubMed Google Scholar), only three other PTPases have been implicated in T cell activation, namely SHP1 (8Schultz L.D. Schweitzer P.A. Rajan T.V. Yi T. Ihle J.N. Matthews R.J. Thomas M.L. Beier D.R. Cell. 1993; 73: 1445-1454Abstract Full Text PDF PubMed Scopus (686) Google Scholar, 9Plas D.R. Johnson R. Pingel J.T. Matthews R.J. Dalton M. Roy G. Chan A.C. Thomas M.L. Science. 1996; 272: 1173-1176Crossref PubMed Scopus (330) Google Scholar), SHP2 (10Tailor P. Jascur T. Williams S. von Willebrand M. Couture C. Mustelin T. Eur. J. Biochem. 1996; 237: 736-742Crossref PubMed Scopus (20) Google Scholar), and the low molecular weight PTPase, LMPTP-B (11Tailor P. Gilman J. Williams S. Couture C. Mustelin T. J. Biol. Chem. 1997; 272: 5371-5374Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). The hematopoietic protein tyrosine phosphatase (HePTP) was originally cloned from human T lymphocytes (12Zanke B. Suzuki H. Kishihara K. Mizzen L. Minden M. Pawson A. Mak T.W. Eur. J. Immunol. 1992; 22: 235-239Crossref PubMed Scopus (85) Google Scholar, 13Zanke B. Squire J. Greisser H. Henry M. Suzuki H. Patterson B. Minden M. Mak T.W. Leukemia. 1994; 8: 236-244PubMed Google Scholar, 14Adaichi M. Sekiya M. Isobe M. Kumura Y. Ogita Z.-I. Hinoda Y. Imai K. Yachi A. Biochem. Biophys. Res. Commun. 1992; 186: 1607-1615Crossref PubMed Scopus (47) Google Scholar). The gene is expressed in thymus and at lower levels in spleen, but not in nonhematopoietic tissues. The exon/intron structure of the HePTP gene (15Adaichi M. Miyachi T. Sekiya M. Hinoda Y. Yachi A. Imai K. Oncogene. 1994; 9: 3031-3035PubMed Google Scholar) is quite similar to that of the phosphatase domains of human CD45. In contrast to CD45, however, the 38-kDa HePTP consists of only a single PTPase domain, which occupies the C-terminal three-fourths of the enzyme and is preceded by a ∼70-amino acid noncatalytic N terminus. Presently, very little is known about the physiological function of HePTP, but some indications may be derived from the findings that its gene maps to chromosome 1q32.1 (13Zanke B. Squire J. Greisser H. Henry M. Suzuki H. Patterson B. Minden M. Mak T.W. Leukemia. 1994; 8: 236-244PubMed Google Scholar), which is a site for frequent chromosomal abnormalities in preleukemic myeloproliferative disease (16Fonatsch C. Haase D. Freund M. Bartels H. Tesch H. Cancer Genet. Cytogenet. 1991; 56: 243-253Abstract Full Text PDF PubMed Scopus (44) Google Scholar, 17Mamacv N. Mamaeva S.E. Pavlova V. Patterson D. Cancer Genet. Cytogenet. 1988; 35: 21-25Abstract Full Text PDF PubMed Scopus (10) Google Scholar), and that gene amplification and overexpression of HePTP have been reported for leukemic cells (13Zanke B. Squire J. Greisser H. Henry M. Suzuki H. Patterson B. Minden M. Mak T.W. Leukemia. 1994; 8: 236-244PubMed Google Scholar). In addition, expression of HePTP in NIH3T3 cells resulted in altered cell morphology and decreased contact inhibition (13Zanke B. Squire J. Greisser H. Henry M. Suzuki H. Patterson B. Minden M. Mak T.W. Leukemia. 1994; 8: 236-244PubMed Google Scholar). Together, these findings suggest a role for HePTP in the regulation of cell proliferation, survival, or differentiation. This possibility is supported by the up-regulation of HePTP mRNA in T cells in response to interleukin-2 (IL-2) (18Adaichi M. Sekiya M. Ishino M. Sasaki H. Hinoda Y. Imai K. Yachi A FEBS Lett. 1994; 338: 47-52Crossref PubMed Scopus (17) Google Scholar), and by the rapid phosphorylation of HePTP on tyrosine in RBL-2H3 mast cells stimulated through their FcεRI (19Swieter M. Berenstein E.H. Swaim W.D. Siraganian R.P. J. Biol. Chem. 1995; 270: 21902-21906Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Since HePTP is preferentially expressed in T cells and may be involved in the regulation of cell proliferation, we decided to investigate the involvement of this enzyme in TCR-induced T cell activation. We report that HePTP had a strong inhibitory effect on the transcriptional activation of a reporter gene driven by three tandem nuclear factor of activated T cells (NFAT)/AP-1 elements derived from the 5′ IL-2 gene promotor, while a catalytically inactive C270S mutant HePTP did not. HePTP also reduced TCR-induced activation of the mitogen-activated protein kinase (MAPK) Erk2, but not of the N-terminal c-Jun kinase (Jnk). Based on these findings we suggest that HePTP plays a negative role in TCR signaling by acting on signaling molecules upstream of MAPK and transcriptional activation of the IL-2 gene. The anti-Tyr(P) mAb 4G10 was from Upstate Biotechnology Inc. (Lake Placid, NY). The 12CA5 anti-hemagglutinin mAb was from Boehringer Mannheim and was used for immunoprecipitation, and the 16B12 anti-hemagglutinin was from Babco (Richmond, CA) was used for immunoblotting. The 9E10 hybridoma producing the mAb that recognizes the c-Myc epitope tag and the OKT3 hybridoma that produces the anti-CD3ε mAb were from American Type Culture Collection (Bethesda, MD). Both mAbs were used as ascites. The polyclonal anti-extracellular signal-regulated kinase 2 (Erk2) was from Santa Cruz Biotechnology Inc., and the polyclonal anti-phospho-Erk was from New England Biolabs. The cDNA for HePTP was obtained from Dr. Brent Zanke, and the coding region was subcloned into the pEF/HA vector (20von Willebrand M. Jascur T. Bonnefoy-Berard N. Yano H. Altman A. Matsuda Y. Mustelin T. Eur. J. Biochem. 1996; 235: 828-835Crossref PubMed Scopus (64) Google Scholar), which adds a hemagglutinin (HA) tag to the N terminus of the insert. HePTP was also subcloned into the pGEX-2T prokaryotic expression vector with a glycine kinker. The cDNAs for LMPTP (11Tailor P. Gilman J. Williams S. Couture C. Mustelin T. J. Biol. Chem. 1997; 272: 5371-5374Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) and VHR were generated using the polymerase chain reaction and a Jurkat cell cDNA library and were cloned into pEF/HA and verified by sequencing. The cDNAs for SHP1 (from M. Thomas), TCPTP (from Deborah Cool), and Syk (21Couture C. Baier G. Altman A. Mustelin T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5301-5305Crossref PubMed Scopus (115) Google Scholar) were also cloned into pEF/HA, while PTP36 (from M. Ogata) and Lck (22Couture C. Songyang Z. Jascur T. Williams S. Tailor P. Cantley L.C. Mustelin T. J. Biol. Chem. 1996; 271: 24880-24884Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) were in pEF-neo (driven by the same promotor, but without N-terminal tag) and c-Cbl (from A. Veillette) in pMX139. The c-Myc-tagged Erk2 (from C. Marshall) was in pEF-neo. The NFAT/AP-1-luc construct (from G. Crabtree) contains three tandem NFAT/AP-1 sites driving the expression of a luciferase gene. To generate a catalytically inactive mutant of HePTP, the codon for Cys-270 was changed into a codon for serine in the pEF/HA-HePTP plasmid using the transformerTM site-directed mutagenesis kit as recommended by the manufacturer (CLONTECH). The resulting C270S mutation was verified by sequencing. The C216S mutant of TCPTP was generated and verified by the same procedure. Jurkat T leukemia cells and two variants of this cell line: J-TAg (from Dr. M. Karin), which is stably transfected with simian virus 40 large T antigen, and JCaM1.6 (from Dr. A. Weiss), which lacks Lck (23Straus D.B. Weiss A. Cell. 1992; 70: 585-593Abstract Full Text PDF PubMed Scopus (928) Google Scholar), were kept at logarithmic growth in RPMI 1640 medium with 5% fetal calf serum, l-glutamine, and antibiotics. These cells were transiently transfected with a total of 5–10 μg of DNA by electroporation at 950 microfarads and 240 V. Empty vector was added to control samples to make a constant amount of DNA in each sample. Cells were used for experiments 48 h after transfection. COS-1 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. These cells were transfected by lipofection with a total of 5–10 μg of DNA and grown for 48 h prior to the experiments. These assays were performed as described previously (24Jascur T. Gilman J. Mustelin T. J. Biol. Chem. 1997; 272: 14483-14488Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Briefly, cells were transiently transfected with 2 μg of NFAT/AP-1-luc together with empty vector or PTK and/or PTPase plasmids. Two days later, the cells were either stimulated with the anti-CD3ε mAb OKT3 (5 or 10 μg/ml) or left untreated. After the cells were with and in of were by at for The of of in of The was measured in an The total protein in each cell was by the protein and was used to the luciferase activity. the in protein between samples was Cells were in 10 and and and by at for The were on protein and with for 2 by protein were three in in with in and either in or used kinase These were performed as described previously (20von Willebrand M. Jascur T. Bonnefoy-Berard N. Yano H. Altman A. Matsuda Y. Mustelin T. Eur. J. Biochem. 1996; 235: 828-835Crossref PubMed Scopus (64) Google Scholar, C. Songyang Z. Jascur T. Williams S. Tailor P. Cantley L.C. Mustelin T. J. Biol. Chem. 1996; 271: 24880-24884Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Briefly, cells were transfected with μg of c-Myc-tagged Erk2 μg of HePTP and μg of Lck Empty vector was added to control samples to make constant amount of DNA in each sample. Cells were 2 days after into two and either stimulated with OKT3 (5 μg/ml) for at or left untreated. Cells were as described and the Erk2 with 2 μg of the 9E10 mAb by of protein The kinase reaction was performed for at in of kinase 10 μg of 10 of The were by 2 and to for 2 The samples were on and the proteins by The of of the Erk2 was verified by using the at and the by the enhanced to the The of HePTP from Jurkat T cells was measured as described (11Tailor P. Gilman J. Williams S. Couture C. Mustelin T. J. Biol. Chem. 1997; 272: 5371-5374Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). The reaction was for at in of with as a The of was measured as at The coding region of HePTP was by the polymerase chain reaction using to both of the and with human HePTP from B. as the The amplification was cloned into the vector and The obtained was to the (12Zanke B. Suzuki H. Kishihara K. Mizzen L. Minden M. Pawson A. Mak T.W. Eur. J. Immunol. 1992; 22: 235-239Crossref PubMed Scopus (85) Google Scholar). expression of the pEF/HA-HePTP construct in or J-TAg cells resulted in the appearance of a tag adds protein that was both and with the tag mAbs 12CA5 and The expression plasmid was also expressed in and in J-TAg cells. similar expression levels were seen with the catalytically inactive C270S mutant of a PTPase of obtained from cells transfected with empty pEF/HA vector or the HePTP or PTPase TCPTP and its inactive the had while the two did not. The of and proteins were similar The of human HePTP was also subcloned into the prokaryotic expression vector pGEX-2T a glycine and the protein was by by and used for of two The resulting were a 38-kDa protein in Jurkat T cells and a protein in T cells or cells transfected with the pEF/HA-HePTP construct The polyclonal also active HePTP from Jurkat T the assays an A at of and with and The for cells were and and of T cells through the TCR molecules to the transcriptional activation of the IL-2 by and of this to specific on activated T cells and the cells through the cell resulting in the proliferation of T cells. TCR-induced activation of the IL-2 gene is measured by T cells with a luciferase reporter construct driven by elements from the IL-2 gene 5′ promoter. we transfected J-TAg cells with the NFAT/AP-1-luc reporter together with HePTP or empty pEF/HA Two days after the cells were with OKT3 for and the of the luciferase be seen in the of of HePTP reduced the of the to This was obtained in three experiments in to the large T antigen, these cells levels of transiently transfected the to we used in which expressed at levels to the of signaling proteins S. Couture C. Gilman J. Jascur T. M. Altman A. Mustelin T. Eur. J. Biochem. 1997; PubMed Scopus Google Scholar). These cells also Lck (23Straus D.B. Weiss A. Cell. 1992; 70: 585-593Abstract Full Text PDF PubMed Scopus (928) Google Scholar) and have very low levels of Syk J. M. von Weiss A. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar) and not to mAbs either kinase is we transfected these cells with Lck or in with HePTP or catalytically inactive together with the NFAT/AP-1-luc Two days after the cells were with OKT3 for and the luciferase in of expression of Lck the of the to In of active HePTP reduced the activation of the reporter construct to while the C270S mutant did not have This was obtained in experiments in and control that HePTP and Lck were expressed in lower To the effect of HePTP was specific for the with we that expression of Syk in cells also the TCR to a activation of the NFAT/AP-1 reporter gene S. Couture C. Gilman J. Jascur T. M. Altman A. Mustelin T. Eur. J. Biochem. 1997; PubMed Scopus Google Scholar). HePTP was with Syk and was a very in the activation by OKT3 with cells transfected with Syk and The from was with a total in many experiments. In contrast, the catalytically inactive the activation of the reporter but this was and was not seen in experiments. we that was to these of and HePTP with a number of other PTPases. be seen in TCPTP, VHR, LMPTP in the same and the c-Cbl had a effect on the NFAT/AP-1 activity. the strong inhibition by HePTP is to this PTPase. We also used a control in these namely which we have to with Syk in TCR-induced NFAT/AP-1 activation M. S. Couture C. Mustelin T. Altman A. Immunity. 1996; 5: Full Text PDF PubMed Scopus Google Scholar). Co-expression of with Syk the activation of the reporter construct about This that the effect of the control and c-Cbl that Syk is expressed in samples and that the PTPases expressed at and that and HePTP at the IL-2 activation experiments suggest that HePTP may play a role in TCR-induced T cell activation between the TCR and the IL-2 we a for TCR namely the activation of the MAPK pathway. cells were transiently with a Erk2 MAPK together with HePTP or Two days after each was in was left while the other was with mAbs for at After cell the Erk2 was with the 9E10 and after these were to in kinase assays with protein as a and the phosphorylation of the was by be seen in the Erk2 was not activated in cells with empty pEF/HA vector and Co-expression of Lck the mAb to activation of HePTP had a inhibitory effect on MAPK activation the The amount of Erk2 was to be similar in the samples and HePTP and were expressed The expression of Lck was also similar in This was obtained in several experiments. To that the effect of HePTP on Erk2 an inhibitory effect on the of MAPK activation a effect in the kinase assays or some other event to the transfected we from cells transfected with Lck with or without HePTP, TCPTP, or by with specific for the phosphorylated and activated of Erk. These that was only by OKT3 in cells Lck and that catalytically active HePTP reduced this In contrast, the C270S mutant of HePTP, as as the TCPTP did not. that samples similar of MAPK and that HePTP and TCPTP were and expressed as was Lck This was obtained in three experiments. To the negative effect of HePTP on NFAT/AP-1 activation, we decided to also the effect of this PTPase on the kinase which is involved in the activation of a of the Jurkat cells were transfected with empty or together with Two days after samples were with OKT3 and or with the later, the cells were and the with the 12CA5 mAb and to in kinase assays using as a These experiments to effect of HePTP activation of and expression of both and the PTPase We that the for HePTP involved in MAPK but not in activation. tyrosine phosphorylation is a for the regulation of cell and These the of both PTKs and which also each other in a In T the of PTKs involved in the earliest signaling events by of the TCR and its is In contrast, the PTPases that these PTKs and that their phosphorylation of signaling molecules The PTPase CD45 was the PTPase in T cell activation T. Immunity. 1994; 1: 351-356Abstract Full Text PDF PubMed Scopus (82) Google Scholar, J.T. Thomas M.L. Cell. 1989; 58: 1055-1065Abstract Full Text PDF PubMed Scopus (437) Google Scholar, 5Koretzky G.A. Picus J. Thomas M.L. Weiss A. Nature. 1990; 346: 66-68Crossref PubMed Scopus (394) Google Scholar, 6Mustelin T. Coggeshall K.M. Altman A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6306-6502Crossref Scopus (408) Google Scholar, 7Mustelin T. Altman A. Oncogene. 1990; 5: 809-813PubMed Google Scholar). This enzyme the C-terminal negative tyrosine in the PTKs Lck T. Coggeshall K.M. Altman A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6306-6502Crossref Scopus (408) Google Scholar, 7Mustelin T. Altman A. Oncogene. 1990; 5: 809-813PubMed Google Scholar) and T. T. M. M. L.C. P. Eur. J. Immunol. 1992; 22: PubMed Scopus Google Scholar). This function of CD45 is not by TCR but in a and is for T cell for activation, by a large of Lck and in a active T. Immunity. 1994; 1: 351-356Abstract Full Text PDF PubMed Scopus (82) Google Scholar) and by a of tyrosine phosphorylation of the activation in the and PTPase, the PTPase has been as a negative of signaling from the TCR as as many other hematopoietic The for its in TCR however, is not and may be that SHP1 is involved in signaling by inhibitory as and G.A. Biol. 1995; 5: Full Text Full Text PDF PubMed Scopus Google Scholar). We have previously added the PTPase, SHP2 (10Tailor P. Jascur T. Williams S. von Willebrand M. Couture C. Mustelin T. Eur. J. Biochem. 1996; 237: 736-742Crossref PubMed Scopus (20) Google Scholar), and the low molecular weight PTPase, LMPTP-B (11Tailor P. Gilman J. Williams S. Couture C. Mustelin T. J. Biol. Chem. 1997; 272: 5371-5374Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), to the of PTPases that may in TCR SHP2 is while LMPTP-B is TCR but is for functional role of these PTPases in T cell activation. the PTPases that the activation sites in Lck or or the phosphorylation sites in and or the of tyrosine phosphorylation sites in the for these In this we that T PTPase, HePTP, has a strong negative effect in TCR-induced T cell activation. We that expression of HePTP blocks the of and the phosphorylation and activation of Erk. This effect of HePTP did not overexpression of the enzyme and was to HePTP in with several other PTPases LMPTP, and The effect also the of In some experiments the catalytically inactive C270S mutant of HePTP had a to with It is also that expression of HePTP did not affect the of cellular tyrosine phosphorylation Together, these experiments that HePTP of the TCR signaling at one of which is upstream of the MAPK In contrast, experiments with TCPTP not of this PTPase in TCR It is that HePTP NFAT/AP-1 to a reduced MAPK activation This be to the of additional signaling molecules by HePTP to inhibition of other for NFAT/AP-1 activation. the of MAPK activation seen at is to the for NFAT/AP-1 activation at h after receptor a It is that HePTP also the of the MAPK In HePTP as a negative of T cell activation and that at one site of HePTP is between the TCR and the phosphorylation and activation of experiments be to in the signaling cascade that HePTP and to its role and in T cell activation. We to Dr. Brent for the of the HePTP