- Email: [email protected]
CD148 induces tyrosine-phosphorylation and modulates CD3 signalling on human lymphocytes
Monday, 23 June 1997 - Oral presentations
phorylation of the associated CD+&- and c-chain cytoplasmlc tails. These events lead to the induction of the intracellular signalling pathways with concomitant receptor down-regulation. Although the TCR is down-regulated from the cell surface by the activation of PKC and subsequent sedne phospholylation of the CD3ychain. whether the TCR tails are involved in this process is not kllOWll.
Matertetr and Methods: Wildtype and C-terminal truncated TCR-chains were generated using PCR-based techniques. These mutant TCRs were intro duced into the TCRu-,9- T cell hybtfcbma, 58, using a retroviral system. The effect of PKC-induced TCR and CD4 down-regulation were measured, using standard FACS techniques. Raw& We report here that the TCRu-chain cytoplasmic tail is necessary for PKC-mediated internalization of the TCR complex. The requirement for the TCRachain cytoplasmic tail is specific for internalization of the TCR complex, since down-regulation of CD4 is still intact in hybrtdoma cells expressing a tall-less TCRa-chaln. The absence of TCR internalization directly correlates with defective PKC-mediated phosphorylation of the CD3y-chain. Despite deficient PKC-mediated TCR down-regulation, the tail-less a,9 TCR still transduces antigenic signals resulting in the production of IL-2. Condu&n: Although the TCR tails are not obviously required for signal transduction, the TCR&ail may serve as a targeting domain for PKC-mediated down-regulation of the TCR complex.
0.1.02.3
The She adaptor protein mediates TCR dependent IL-2 gene expression by associating with the orotein tvrosine kinase ZAP-70
E. Milia ‘, MM. Di Somma 1.2,C. Ulivieri ‘, L. Lanfrancone 3, P.G. Pelicci 3, J.L. Telford 2, C.T. Baldari ’ ’ Depament of EwMonary Bio/w Siena University, ItaQ 2IRIS-Biocine, Siena, ltalx 3European Institute of oncology, Milan. Ma/v T-cell antigen receptor (TCR) triggering results both in recruitment of the protein tyrosine kinase ZAP-70 to the TCR zeta chain and in phosphorylation of the She adaptor, which transduces activating signals to Ras. Both ZAP-70 and Ras are essential for the activation of IL-2 gene expression in response to TCR triggering. We have analyzed the functional relationship between She and ZAP-70 in TCR signal transduction. Our results show that She associates with ZAP-70 in response to TCR triggering. This association is dependent on the presence of the She aminotenninal domain (PTB), which was recently identified as a phosphotyrosine binding domain with a different specificity as compared to SH2 domains. In vitro binding experiments using a phosphopepttde spanning a putative PTB binding domain on ZAP-70 showed a direct binding between the She PTB domain and ZAP-70. The potential role of She in TCR signaling was tested by measuring the effect of a She mutant containing the isolated PTB domain on the activation of the IL-2 gene promoter. We hypothized that the isolated PTB domain could non productively compete with endogenous She for binding to tyrosine phosphorylated proteins and thus interfere with Ras activation. NF-AT activation in response to TCR triggering was signiftcantty inhibited by the F’TB-Shemutant but not by a similar mutant encoding the isolated SH2 domain of She. Expression of a ZAP-70 substitution mutant lacking the site of interaction whh She also transdominantly inhibited TCR dependent NF-AT activation. These results shows that She is-essential for TCR signaling to Ras and suggests an important role for ZAP-70 association with the She PTB domain in TCR signal transduction.
10.1.02.4
1 TCR slgnalling in SHP-1 deficient Tcells
K.G. Johnson ‘, F.G. LeRoy I, D. Kioussis 2, R.J. Matthews I. 1Dept.of Medicine, Tenovus Building, UWCM, Canlie UK, 2Laboratory of Molecular Immunolog)! National Institute for Medical Research, Mill Hill, London, UK
Signal transduction in lymphocytes
3
with a nucleopmtein peptide and irradiated splenocytes +/- IL-2 and pulsed overnight with trftiated thymidine after 2-4 days. Results: Following CD3 stimulation, primary thymocytes and thymocyte blasts from motheaten animals when compared to littermate controls, showed no significant dferences in the tyrosine phosphorylatton state of the zeta chain, the level of recruitment of ZAP-70 to the CD3 complex or the kfnase activity of ZAP-70. In addition, in time-course experfments comparfno motheaten and control thymocyte blasts after CD3 stimulation, we have’been-unable to detect significant differences in the tymsine phorphorylation states of PLCy and Vav and their associated phospho-proteins p36 and SLP-76 respectively. Furthermore, motheaten and control thymocytes protiferate with identical kinetics in response to either CD3 or ConA stimulation in the presence of 11-2. However, motheaten thymocytes showed a significantly increased and more sustained response to CD3 stimulation in the absence of exogenous IL-2. As with CD3 stimulation, a difference in the responsiveness of F5 positive motheaten and littermate control thymocytes to a stimulatoty nucleopmtein peptide is only detected in the absence of IL-2. Conclusion: Our failure, when comparing motheaten and litttennate thymocvtes, to detect differences in a) the twosine phosphorvlation state of the CD3complex and b) proliferation in the p-&sence.of exogenous IL-2 following anti-CD3 stimulation indicates that SHP-1 is not involved in regulating the early events in TCR signalling that eventually lead to the up-regulation of the high-affinity IL-2 Receptor. However, the more sustained responses of motheaten thymocytes to TCR triggering in the absence of IL-2 suggests that SHP-1 may regulate pathways that influence the survival of T-cells.
) 0.1.02.5 1 Molecular cloning of a novel protein, SKAP55, that associates with the protein tyroslne kinase P59v” in human T lymphocytes
specifically
A. Marie-Cardine ‘. E. Bruvns'. C. Eckerskom 2. H. Kirchaessner ‘. S. Meuer ‘, 0. Schraven ’ ..’ R&cht-Karls LJni&sity of l?eidelbe& Institute of Immunology Heidelberg, Germany, 2Max-Plan& institute for Biochemistry Martinsried, Germany Introduction: Stimulation of T lymphocytes by antigen or anti T-cell receptor (TCR) antibodies causes rapid phosphorylation of several cellular proteins on tyrosine residues. Two src-family protein tyrosine kinases (PTKs), ~56~ and p59m, have been implicated in the lnittation of the TCR-induced signaling cascade. Whereas the requirement of Lck in TCWCD3 mediated actfvatfon pathways is well established, the role of Fyn is less well defined. To gain insights into the mechanisms by which Fyn is involved in T-cell actiiation, we puriffed and cloned a novel Fyn-associated protein, SKAP55 (Src Kinase Associated Phosphopmtein). Materlals end Methods: SKAP55 was purified from lysates of vanadatetreated Jurkat cells using a GST-SHP domain of Fyn. Following 2Dgelelectmphoresis Coomassie stained SKAP55 was excised, digested and subjected to microsequencing. Molecular cloning was performed using standard pmcedures. ReeuL: SKAP55 represents a novel phosphoprotein. that binds to the Fyn-SH2 domain in T-cells. It is preferentially expressed in lymphoid tissues and encompasses a pleckstnn homology (PH) domain, a C-terminal SH3 domain, and several tyrosine residues that could potentially be phosphorylated by src PTKs. SKAP55 is constiiutively phosphorylated on tyrosine residue(s) in resting T lymphocytes and in Jurkat cells. However, despite identical expression and phosphorylation, SKAP55 only coprecipitates with Fyn in resting T-lymphocytes but not in continuously proliferating T-cell lines. In addition, the interaction between Fyn and SKAP55 is lost in long term proliferating T-lymphocytes. Conotuslon: The molecular organisation of SKAP55 suggests a signaling function during T-cell activation. The loss of association with fyn in transformed T-cell lines and long term proliferating human T-lymphocytes could further indicate that SKAP55 is involved in the reaulation of human T-cell omliferation.
introduction:The spontaneous mouse mutant motheaten is genetically deficient for the intracellular protein tymsine phosphatase, SHP-1. which has been implicated as a pleiotropic negative regulator of signalling through a number of receptors found on haematopoietfc cells. However, a conclusive role for SHP-1 in T cells has not been established. We have utfllsed the motheaten mouse to examine signalling triggered by an anti-CD3 antibody on T-cells that are deficient for SHP-1. In addition, in order to examine T-Cell Receptor (TCR) signalling in a more phvsioloaical manner the transaenic TCR, F5, that recoonises a oeotide from the influenza virus nucleopmtein-has been introduced into the motheaten genetic background. Materlet and Yetttodez Primary thvmccvtes or thvmocvte blasts venerated using ConA and IL-2 were activated 4th a&CD3 Ab, tys& and pmbins punfied by immunoprecipitatfon with specific Abs and subjected to SDS-PAGE and Western blotting with an antiphosphotyrosine containing antibody. For pmltferation assays, thymocytes from motheaten and ltttermate control animals were incubated with anti-CD3 coated plates +/- IL-2 and pulsed overnight with tritiated thymidine after 2-7 days. Alternatively, thymocytes from motheaten and control animals possessing a copy of the F5 TCR transgene were incubated
0.1.02.6
CD149 induces tyrosine-phosphorylation and modulates CD3 signalling on human lymphocytes
E. Palou ‘, M.A. de la Fuente-Garcfa ‘, J.M. Nicolas 2, J. Vives’, A. Gaya l. ’ Servei d’lmmunologia, Hospital Clfnic, Barcelona, Spain, 2Medicina hterna. Hospital Clinic, Barcelona, Spain Infroduotlon: CD148 is a new Cluster of Differentiation defined in the VI Intemattonal Workshop on Leucocyte Diiensntation Antigens (WLDA). Although this molecule was officiaflv recoonized at VI WLDA it reomsents the hematoooietic form of a previously~desc&sd membrane protein tymsine phosphatase HPTPq/DEP-1. In the present work we were interested to know whether CD143 could influence the signal transduction through the antigen receptor in a similar way to CD45 the main protein tyrosine phosphatase expressed on the membrane of lymphocytes. Matertals and h4elWds:Cells previously coated wtth CD3, CD143 or both were stimulated with rabbit anti-mouse antisera. Ca2+ mobilization after cell
4
Monday 23 June 1997 - Oral presentations
Genetics of MHC
stimulation was analyzed by computer-aided fluorescence imaging using lymphocytes loaded with fura2/AM. Tyrosine-phosphorytated protein patterns were detected by immunobiotting. Reeutts: When CD148 was co-cross-linked with CD3 a clear inhibition of the subsequent calcium mobilization was observed. Furthermore, the pattern of protein tyroslne phosphorytation after CD3 cross-linking was also modified when it was co-cross-linked with CD148. In addition, we observed that the stimulation of lymphocytes with CD148 alone was able to induce an increase in [Ca2+]iwhich was abolished in the presence of genistein and by co-cross-linking with CD45 These data, together with the induction of tyrosine phosphorylation after CD148 cross-linking, suggest the involvement of tyrosine kinases based signalling pathways in this process. Conclusion:Our data show that CD148, a recently described membrane protein tyrosine phosphatase, induces tyrosine phosphorylation probably by the activation of protein tyrosine kinase(s) and could be involved in CD3 signalling in T lymphocytes.
0.1.02.7
Role of the proto-oncogene product Cbl in B cell receptor signalllng
L. Smit, G. van der Horst, J. Borst. Division of Cellular Biochemistr)! The Netherlands Cancer Institute, Amsterdam, The Netherlands Previously, we have shown that the B cell antigen receptor (BCR) induces formation of a Shc!Grb2 complex, which most likely couples it to the Ras signalling cascade. In this adaptor protein complex we observed a 110 kDa protein, which was strongly phosphorylated on tyrosine upon BCR triggering. lmmunoblotting identified this major kinase substrate as CM. A truncated form of Cbl. v-Cbl. is the moduct of the Cas-NSl murine mtrovirus which induces B cell’lymphbmas and myeloid leukemias. Cbl has been shown to acquire transforming capacity upon specific deletion mutations involving a 17 amino acid region. We want to establish how wild type Cbl participates in antigen receptor signalling and elucidate the mechanism underlying _ - the transforming caoacltv of?bl d&stlon mutants. ‘We found that Cbl is constitutively associated with the SH3 domain of Grt12. In addition. Cbl associates with Crk and CtkL uoon BCR stimulation. suwestlna an interaction of Cbl with the SH2 domains of these two related adaptors. In analogy with Grb2, the Crk proteins were also found to associate with guanine nucleotide exchange factors. Crk inducibly interacts with Vav upon BCR triggering, while CrkL is constkutlvely associated with C3G, an exchange factor for the Ras-related GTPase Raol. In summarv. the BCR couples to three nucleotide exchange factors via three different a&ptor molecules. Tymsine phosphorylated Cbl is found in all three complexes upon stimulation of the BCR. Based on these findings, we postulate that Cbl might affect the signalling pathways involving these three nucleotide exchangers. To test this hypothesis, we have transfsed wild type Cbl and two transf&ning mutants into Cos and NIH3T3 cells to investigate the deregulation of these different signalling pathways. The transforming mutants are still phospholylated on tymsine upon stimulation of the EGF receptor and can still bind to the receptor in Cos cells suggesting an EGF receptor binding at the n-terminal part of Cbl. At the moment we are looking whether there is a differential interaction of intracellulair signalling proteins with the wild type and transfomting mutants of Cbl. In addition, we are generating a transgenic mouse line expressing oncogenic Cbl to determine the effects on lymphoid development and transformation, as well as on antigen receptor-induced signalling.
10.1.02.8
) CD28 can promote T-cell survival through both Pl3Kdependent and -Independent mechanisms
Y. Collette, M. Ghiotto, D. Olive. INSERM U119, Sd Lel Rowe, 27, 13009 Marseille, France Apoptosis is believed to be of central importance for the precise regulation of cell numbers and a major mechanism to remove unwanted cells. Many signals may act to suppress or promote this cell death process. Following the apop totic signal, a cascade of signals leads to the induction of cysteine pmteases and finally to chromatln condensation and DNA fragmentation. Several survival factors might prevent the induction of apoptosis by reducing the quantity or quality of death effector molecules, or by enhancing the activity of anti-apoptotic proteins such as Bcl2 and Bcl-XL.Activated T-cells may undergo apoptosis due to growth-factors deprivation or activation-induced cell death. Upon mitogenic or antigenic stimulation, T cells become sensitive to anti-CD3-triggered apoptosis. Recent studies have demonstrated that CD28 is a major costimulatory molecule for T-cell activation. CD28 triggering markedly enhances the production of cytoktnes and cytokine receptors, and can also promote T cell survival by enhancing the expression of Bcl-XL. Functional studies have revealed that beyond Its ability to generate signals similar to CD3 (PLCy 1 phosphorylation, Grb2 recruitment,...), CD28 can also use distinct mechanisms, including tyrosine phosphorytatton of specific substrates, recruitment of Tee family tymsine kinases as well as PI3 kinase, and together wtth CD3, can also activate the MAP
kinase JNK. The recently identified role of Pl3kinase in prevention of apoptosis of PC12 neuronal cells upon growth factor deprivation led us to examine the role of PI3 kinase recruitment in the prevention by CD28 of CDMriggered apoptosis. In a previously described huCD28 stably transfected murtne T cell hybrldoma. triggering of CD3 rapidly led to cell death characterized by the cleavage of the specific cystein protease substrate PARP, DNA fragmentation and cellular destruction. Costimulation by CD28 prevented the apoptotlc process and neither treatment with wortmannin nor LY 294002 PI3 kinase inhibitors was found to affect this function. lmmunoblotttng experiments evidenced an increase in the atready high basal level of Bcl-x~ expression, following CD28 costimulatlon, and which was prevented by wortmannin treatment. Cultivation under low serum concentration significantly sensttized the cells to apoptosis biggered by wortmannin, as previously described in neumnal cells. Under this low serum culture conditions, costtmulation by CD28 efficlentty recruited PI3 kinase, prevented CDB-triggered apoptosis and Bcl-XLexpression was signifiintly induced. Strlkingly, prevention of apoptosis by CD28 was markedly impaired by wortmannin treatment under these exoenmental conditions. Together, these results identify two mechanisms by which CD28 can prevent CDI-triggered apoptosis. One pathway, dominant upon high serum culture, is independent of PI3 ktnase activation by CD28. The second pathway, revealed only upon low serum culture, depends on PI3 kinase activity. Our data also indicate a strong correlation between PI3 kinase recruitment by CD28 and Bcl-xL induction.
14:0&16:00
0.1.03 F[0 1 03 1
0.1.03.2
Room M
Genetics of MHC No abstract available
Molecular analysls of a recomblnatlonal hot spot In the class Ill region of the mouse NlHC, lmpllcatlons for disease susceMlbllltv . I
M. Snoek I, C. Teuscher*, H. van Vugt ’ . ’ Division of Molecular Genetics, the Netherlands Cancer institute, Plesmanlaan 121, Amsterdam, The Nethedands, ’ Deparhent of Veterinary Pathobiology, the University of Illinois, Ubana, USA
Introduction:The large number of available intra /f2 recombinant haptotypes and the increasing knowledge on the molecular structure of the MHC provide an interesting genetic system to study meiottc recombination. A remarkable fact is that within the mouse MHC recombination is not random, but occurs more often at specific sites- hot spots. At least seven areas harboring a recombinational hot spot have been described between the K and D region genes. We previously described a hot spot of recombination in the Hsp70-G7 interval in the class Ill region. Loci controlling the susceptibility to experimentally induced allergic orchitis, to cortlcosteroid induced cleft palate, vitamin A sensittvity to this cleft palate, as well as tumor susceptibility genes for chemically induced lung tumors all map to this region. Materials and Methods:Cosmid derived DNA from the relevant area was sequenced. We designed primers flanking the simple sequence repeats in this intewal and searched for polymorphisms in the parental haplotypes of a variety of recombinant haplotype combinations. In two recombinants this approach led to the defining of a crossover intewal of 2.4 kb. We assumed that recombinattons in other haplotypes occurred in the near vicinity, and using PCR amplllcation and cycle sequencing we analysed this region in a number of parentahaplotypes in order to define haplotype specific nucteotide substitutions. Reeutts: Sequence analysis of the entire region, the generation of new microsatellite markers and the implication of additional haplotypes has revealed a recombinational hot spot telomerfc of G7a. We found a 3 kb fragment where the crossover breakpoints of 22 recombinants of a variety of haplotype combinations are clustered. However not atl recombinants defining the disease susceptibilities are using this 3 kb hot spot area. Conclusions: At present, only the Eb and Lmp2 hot spots were characterized in detail, comparisons with the here presented hot spot indicates that the suggested recombinational signals (MT-family of repetitive sequence, retmvtral elements) are no prerequisite for meiotic recombinatton. The localization of the breakpoints of phenotypicaky differing recombinants is needed for the positional cloning of candidate disease susceptibility genes.
phorylation of the associated CD+&- and c-chain cytoplasmlc tails. These events lead to the induction of the intracellular signalling pathways with concomitant receptor down-regulation. Although the TCR is down-regulated from the cell surface by the activation of PKC and subsequent sedne phospholylation of the CD3ychain. whether the TCR tails are involved in this process is not kllOWll.
Matertetr and Methods: Wildtype and C-terminal truncated TCR-chains were generated using PCR-based techniques. These mutant TCRs were intro duced into the TCRu-,9- T cell hybtfcbma, 58, using a retroviral system. The effect of PKC-induced TCR and CD4 down-regulation were measured, using standard FACS techniques. Raw& We report here that the TCRu-chain cytoplasmic tail is necessary for PKC-mediated internalization of the TCR complex. The requirement for the TCRachain cytoplasmic tail is specific for internalization of the TCR complex, since down-regulation of CD4 is still intact in hybrtdoma cells expressing a tall-less TCRa-chaln. The absence of TCR internalization directly correlates with defective PKC-mediated phosphorylation of the CD3y-chain. Despite deficient PKC-mediated TCR down-regulation, the tail-less a,9 TCR still transduces antigenic signals resulting in the production of IL-2. Condu&n: Although the TCR tails are not obviously required for signal transduction, the TCR&ail may serve as a targeting domain for PKC-mediated down-regulation of the TCR complex.
0.1.02.3
The She adaptor protein mediates TCR dependent IL-2 gene expression by associating with the orotein tvrosine kinase ZAP-70
E. Milia ‘, MM. Di Somma 1.2,C. Ulivieri ‘, L. Lanfrancone 3, P.G. Pelicci 3, J.L. Telford 2, C.T. Baldari ’ ’ Depament of EwMonary Bio/w Siena University, ItaQ 2IRIS-Biocine, Siena, ltalx 3European Institute of oncology, Milan. Ma/v T-cell antigen receptor (TCR) triggering results both in recruitment of the protein tyrosine kinase ZAP-70 to the TCR zeta chain and in phosphorylation of the She adaptor, which transduces activating signals to Ras. Both ZAP-70 and Ras are essential for the activation of IL-2 gene expression in response to TCR triggering. We have analyzed the functional relationship between She and ZAP-70 in TCR signal transduction. Our results show that She associates with ZAP-70 in response to TCR triggering. This association is dependent on the presence of the She aminotenninal domain (PTB), which was recently identified as a phosphotyrosine binding domain with a different specificity as compared to SH2 domains. In vitro binding experiments using a phosphopepttde spanning a putative PTB binding domain on ZAP-70 showed a direct binding between the She PTB domain and ZAP-70. The potential role of She in TCR signaling was tested by measuring the effect of a She mutant containing the isolated PTB domain on the activation of the IL-2 gene promoter. We hypothized that the isolated PTB domain could non productively compete with endogenous She for binding to tyrosine phosphorylated proteins and thus interfere with Ras activation. NF-AT activation in response to TCR triggering was signiftcantty inhibited by the F’TB-Shemutant but not by a similar mutant encoding the isolated SH2 domain of She. Expression of a ZAP-70 substitution mutant lacking the site of interaction whh She also transdominantly inhibited TCR dependent NF-AT activation. These results shows that She is-essential for TCR signaling to Ras and suggests an important role for ZAP-70 association with the She PTB domain in TCR signal transduction.
10.1.02.4
1 TCR slgnalling in SHP-1 deficient Tcells
K.G. Johnson ‘, F.G. LeRoy I, D. Kioussis 2, R.J. Matthews I. 1Dept.of Medicine, Tenovus Building, UWCM, Canlie UK, 2Laboratory of Molecular Immunolog)! National Institute for Medical Research, Mill Hill, London, UK
Signal transduction in lymphocytes
3
with a nucleopmtein peptide and irradiated splenocytes +/- IL-2 and pulsed overnight with trftiated thymidine after 2-4 days. Results: Following CD3 stimulation, primary thymocytes and thymocyte blasts from motheaten animals when compared to littermate controls, showed no significant dferences in the tyrosine phosphorylatton state of the zeta chain, the level of recruitment of ZAP-70 to the CD3 complex or the kfnase activity of ZAP-70. In addition, in time-course experfments comparfno motheaten and control thymocyte blasts after CD3 stimulation, we have’been-unable to detect significant differences in the tymsine phorphorylation states of PLCy and Vav and their associated phospho-proteins p36 and SLP-76 respectively. Furthermore, motheaten and control thymocytes protiferate with identical kinetics in response to either CD3 or ConA stimulation in the presence of 11-2. However, motheaten thymocytes showed a significantly increased and more sustained response to CD3 stimulation in the absence of exogenous IL-2. As with CD3 stimulation, a difference in the responsiveness of F5 positive motheaten and littermate control thymocytes to a stimulatoty nucleopmtein peptide is only detected in the absence of IL-2. Conclusion: Our failure, when comparing motheaten and litttennate thymocvtes, to detect differences in a) the twosine phosphorvlation state of the CD3complex and b) proliferation in the p-&sence.of exogenous IL-2 following anti-CD3 stimulation indicates that SHP-1 is not involved in regulating the early events in TCR signalling that eventually lead to the up-regulation of the high-affinity IL-2 Receptor. However, the more sustained responses of motheaten thymocytes to TCR triggering in the absence of IL-2 suggests that SHP-1 may regulate pathways that influence the survival of T-cells.
) 0.1.02.5 1 Molecular cloning of a novel protein, SKAP55, that associates with the protein tyroslne kinase P59v” in human T lymphocytes
specifically
A. Marie-Cardine ‘. E. Bruvns'. C. Eckerskom 2. H. Kirchaessner ‘. S. Meuer ‘, 0. Schraven ’ ..’ R&cht-Karls LJni&sity of l?eidelbe& Institute of Immunology Heidelberg, Germany, 2Max-Plan& institute for Biochemistry Martinsried, Germany Introduction: Stimulation of T lymphocytes by antigen or anti T-cell receptor (TCR) antibodies causes rapid phosphorylation of several cellular proteins on tyrosine residues. Two src-family protein tyrosine kinases (PTKs), ~56~ and p59m, have been implicated in the lnittation of the TCR-induced signaling cascade. Whereas the requirement of Lck in TCWCD3 mediated actfvatfon pathways is well established, the role of Fyn is less well defined. To gain insights into the mechanisms by which Fyn is involved in T-cell actiiation, we puriffed and cloned a novel Fyn-associated protein, SKAP55 (Src Kinase Associated Phosphopmtein). Materlals end Methods: SKAP55 was purified from lysates of vanadatetreated Jurkat cells using a GST-SHP domain of Fyn. Following 2Dgelelectmphoresis Coomassie stained SKAP55 was excised, digested and subjected to microsequencing. Molecular cloning was performed using standard pmcedures. ReeuL: SKAP55 represents a novel phosphoprotein. that binds to the Fyn-SH2 domain in T-cells. It is preferentially expressed in lymphoid tissues and encompasses a pleckstnn homology (PH) domain, a C-terminal SH3 domain, and several tyrosine residues that could potentially be phosphorylated by src PTKs. SKAP55 is constiiutively phosphorylated on tyrosine residue(s) in resting T lymphocytes and in Jurkat cells. However, despite identical expression and phosphorylation, SKAP55 only coprecipitates with Fyn in resting T-lymphocytes but not in continuously proliferating T-cell lines. In addition, the interaction between Fyn and SKAP55 is lost in long term proliferating T-lymphocytes. Conotuslon: The molecular organisation of SKAP55 suggests a signaling function during T-cell activation. The loss of association with fyn in transformed T-cell lines and long term proliferating human T-lymphocytes could further indicate that SKAP55 is involved in the reaulation of human T-cell omliferation.
introduction:The spontaneous mouse mutant motheaten is genetically deficient for the intracellular protein tymsine phosphatase, SHP-1. which has been implicated as a pleiotropic negative regulator of signalling through a number of receptors found on haematopoietfc cells. However, a conclusive role for SHP-1 in T cells has not been established. We have utfllsed the motheaten mouse to examine signalling triggered by an anti-CD3 antibody on T-cells that are deficient for SHP-1. In addition, in order to examine T-Cell Receptor (TCR) signalling in a more phvsioloaical manner the transaenic TCR, F5, that recoonises a oeotide from the influenza virus nucleopmtein-has been introduced into the motheaten genetic background. Materlet and Yetttodez Primary thvmccvtes or thvmocvte blasts venerated using ConA and IL-2 were activated 4th a&CD3 Ab, tys& and pmbins punfied by immunoprecipitatfon with specific Abs and subjected to SDS-PAGE and Western blotting with an antiphosphotyrosine containing antibody. For pmltferation assays, thymocytes from motheaten and ltttermate control animals were incubated with anti-CD3 coated plates +/- IL-2 and pulsed overnight with tritiated thymidine after 2-7 days. Alternatively, thymocytes from motheaten and control animals possessing a copy of the F5 TCR transgene were incubated
0.1.02.6
CD149 induces tyrosine-phosphorylation and modulates CD3 signalling on human lymphocytes
E. Palou ‘, M.A. de la Fuente-Garcfa ‘, J.M. Nicolas 2, J. Vives’, A. Gaya l. ’ Servei d’lmmunologia, Hospital Clfnic, Barcelona, Spain, 2Medicina hterna. Hospital Clinic, Barcelona, Spain Infroduotlon: CD148 is a new Cluster of Differentiation defined in the VI Intemattonal Workshop on Leucocyte Diiensntation Antigens (WLDA). Although this molecule was officiaflv recoonized at VI WLDA it reomsents the hematoooietic form of a previously~desc&sd membrane protein tymsine phosphatase HPTPq/DEP-1. In the present work we were interested to know whether CD143 could influence the signal transduction through the antigen receptor in a similar way to CD45 the main protein tyrosine phosphatase expressed on the membrane of lymphocytes. Matertals and h4elWds:Cells previously coated wtth CD3, CD143 or both were stimulated with rabbit anti-mouse antisera. Ca2+ mobilization after cell
4
Monday 23 June 1997 - Oral presentations
Genetics of MHC
stimulation was analyzed by computer-aided fluorescence imaging using lymphocytes loaded with fura2/AM. Tyrosine-phosphorytated protein patterns were detected by immunobiotting. Reeutts: When CD148 was co-cross-linked with CD3 a clear inhibition of the subsequent calcium mobilization was observed. Furthermore, the pattern of protein tyroslne phosphorytation after CD3 cross-linking was also modified when it was co-cross-linked with CD148. In addition, we observed that the stimulation of lymphocytes with CD148 alone was able to induce an increase in [Ca2+]iwhich was abolished in the presence of genistein and by co-cross-linking with CD45 These data, together with the induction of tyrosine phosphorylation after CD148 cross-linking, suggest the involvement of tyrosine kinases based signalling pathways in this process. Conclusion:Our data show that CD148, a recently described membrane protein tyrosine phosphatase, induces tyrosine phosphorylation probably by the activation of protein tyrosine kinase(s) and could be involved in CD3 signalling in T lymphocytes.
0.1.02.7
Role of the proto-oncogene product Cbl in B cell receptor signalllng
L. Smit, G. van der Horst, J. Borst. Division of Cellular Biochemistr)! The Netherlands Cancer Institute, Amsterdam, The Netherlands Previously, we have shown that the B cell antigen receptor (BCR) induces formation of a Shc!Grb2 complex, which most likely couples it to the Ras signalling cascade. In this adaptor protein complex we observed a 110 kDa protein, which was strongly phosphorylated on tyrosine upon BCR triggering. lmmunoblotting identified this major kinase substrate as CM. A truncated form of Cbl. v-Cbl. is the moduct of the Cas-NSl murine mtrovirus which induces B cell’lymphbmas and myeloid leukemias. Cbl has been shown to acquire transforming capacity upon specific deletion mutations involving a 17 amino acid region. We want to establish how wild type Cbl participates in antigen receptor signalling and elucidate the mechanism underlying _ - the transforming caoacltv of?bl d&stlon mutants. ‘We found that Cbl is constitutively associated with the SH3 domain of Grt12. In addition. Cbl associates with Crk and CtkL uoon BCR stimulation. suwestlna an interaction of Cbl with the SH2 domains of these two related adaptors. In analogy with Grb2, the Crk proteins were also found to associate with guanine nucleotide exchange factors. Crk inducibly interacts with Vav upon BCR triggering, while CrkL is constkutlvely associated with C3G, an exchange factor for the Ras-related GTPase Raol. In summarv. the BCR couples to three nucleotide exchange factors via three different a&ptor molecules. Tymsine phosphorylated Cbl is found in all three complexes upon stimulation of the BCR. Based on these findings, we postulate that Cbl might affect the signalling pathways involving these three nucleotide exchangers. To test this hypothesis, we have transfsed wild type Cbl and two transf&ning mutants into Cos and NIH3T3 cells to investigate the deregulation of these different signalling pathways. The transforming mutants are still phospholylated on tymsine upon stimulation of the EGF receptor and can still bind to the receptor in Cos cells suggesting an EGF receptor binding at the n-terminal part of Cbl. At the moment we are looking whether there is a differential interaction of intracellulair signalling proteins with the wild type and transfomting mutants of Cbl. In addition, we are generating a transgenic mouse line expressing oncogenic Cbl to determine the effects on lymphoid development and transformation, as well as on antigen receptor-induced signalling.
10.1.02.8
) CD28 can promote T-cell survival through both Pl3Kdependent and -Independent mechanisms
Y. Collette, M. Ghiotto, D. Olive. INSERM U119, Sd Lel Rowe, 27, 13009 Marseille, France Apoptosis is believed to be of central importance for the precise regulation of cell numbers and a major mechanism to remove unwanted cells. Many signals may act to suppress or promote this cell death process. Following the apop totic signal, a cascade of signals leads to the induction of cysteine pmteases and finally to chromatln condensation and DNA fragmentation. Several survival factors might prevent the induction of apoptosis by reducing the quantity or quality of death effector molecules, or by enhancing the activity of anti-apoptotic proteins such as Bcl2 and Bcl-XL.Activated T-cells may undergo apoptosis due to growth-factors deprivation or activation-induced cell death. Upon mitogenic or antigenic stimulation, T cells become sensitive to anti-CD3-triggered apoptosis. Recent studies have demonstrated that CD28 is a major costimulatory molecule for T-cell activation. CD28 triggering markedly enhances the production of cytoktnes and cytokine receptors, and can also promote T cell survival by enhancing the expression of Bcl-XL. Functional studies have revealed that beyond Its ability to generate signals similar to CD3 (PLCy 1 phosphorylation, Grb2 recruitment,...), CD28 can also use distinct mechanisms, including tyrosine phosphorytatton of specific substrates, recruitment of Tee family tymsine kinases as well as PI3 kinase, and together wtth CD3, can also activate the MAP
kinase JNK. The recently identified role of Pl3kinase in prevention of apoptosis of PC12 neuronal cells upon growth factor deprivation led us to examine the role of PI3 kinase recruitment in the prevention by CD28 of CDMriggered apoptosis. In a previously described huCD28 stably transfected murtne T cell hybrldoma. triggering of CD3 rapidly led to cell death characterized by the cleavage of the specific cystein protease substrate PARP, DNA fragmentation and cellular destruction. Costimulation by CD28 prevented the apoptotlc process and neither treatment with wortmannin nor LY 294002 PI3 kinase inhibitors was found to affect this function. lmmunoblotttng experiments evidenced an increase in the atready high basal level of Bcl-x~ expression, following CD28 costimulatlon, and which was prevented by wortmannin treatment. Cultivation under low serum concentration significantly sensttized the cells to apoptosis biggered by wortmannin, as previously described in neumnal cells. Under this low serum culture conditions, costtmulation by CD28 efficlentty recruited PI3 kinase, prevented CDB-triggered apoptosis and Bcl-XLexpression was signifiintly induced. Strlkingly, prevention of apoptosis by CD28 was markedly impaired by wortmannin treatment under these exoenmental conditions. Together, these results identify two mechanisms by which CD28 can prevent CDI-triggered apoptosis. One pathway, dominant upon high serum culture, is independent of PI3 ktnase activation by CD28. The second pathway, revealed only upon low serum culture, depends on PI3 kinase activity. Our data also indicate a strong correlation between PI3 kinase recruitment by CD28 and Bcl-xL induction.
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Genetics of MHC No abstract available
Molecular analysls of a recomblnatlonal hot spot In the class Ill region of the mouse NlHC, lmpllcatlons for disease susceMlbllltv . I
M. Snoek I, C. Teuscher*, H. van Vugt ’ . ’ Division of Molecular Genetics, the Netherlands Cancer institute, Plesmanlaan 121, Amsterdam, The Nethedands, ’ Deparhent of Veterinary Pathobiology, the University of Illinois, Ubana, USA
Introduction:The large number of available intra /f2 recombinant haptotypes and the increasing knowledge on the molecular structure of the MHC provide an interesting genetic system to study meiottc recombination. A remarkable fact is that within the mouse MHC recombination is not random, but occurs more often at specific sites- hot spots. At least seven areas harboring a recombinational hot spot have been described between the K and D region genes. We previously described a hot spot of recombination in the Hsp70-G7 interval in the class Ill region. Loci controlling the susceptibility to experimentally induced allergic orchitis, to cortlcosteroid induced cleft palate, vitamin A sensittvity to this cleft palate, as well as tumor susceptibility genes for chemically induced lung tumors all map to this region. Materials and Methods:Cosmid derived DNA from the relevant area was sequenced. We designed primers flanking the simple sequence repeats in this intewal and searched for polymorphisms in the parental haplotypes of a variety of recombinant haplotype combinations. In two recombinants this approach led to the defining of a crossover intewal of 2.4 kb. We assumed that recombinattons in other haplotypes occurred in the near vicinity, and using PCR amplllcation and cycle sequencing we analysed this region in a number of parentahaplotypes in order to define haplotype specific nucteotide substitutions. Reeutts: Sequence analysis of the entire region, the generation of new microsatellite markers and the implication of additional haplotypes has revealed a recombinational hot spot telomerfc of G7a. We found a 3 kb fragment where the crossover breakpoints of 22 recombinants of a variety of haplotype combinations are clustered. However not atl recombinants defining the disease susceptibilities are using this 3 kb hot spot area. Conclusions: At present, only the Eb and Lmp2 hot spots were characterized in detail, comparisons with the here presented hot spot indicates that the suggested recombinational signals (MT-family of repetitive sequence, retmvtral elements) are no prerequisite for meiotic recombinatton. The localization of the breakpoints of phenotypicaky differing recombinants is needed for the positional cloning of candidate disease susceptibility genes.