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  Director : Oreste Acuto (oacuto@pasteur.fr)


  abstract

 

Our Unit studies the molecular mechanisms driving T cell activation, a process that contributes to the development of the adaptive immune response. This includes T cell antigen and ancillary (co-receptors/co-stimulators) receptor engagement, signal triggering and propagation leading to gene activation and cell differentiation. Signal transduction is governed by protein and lipid kinases, phosphatases, lipases, small GTPases and adaptor proteins. We study the regulation of protein tyrosine kinase (PTK) Lck/Fyn, Syk/Zap-70 and Tec family and of their substrates which connect the TCR and co-stimulatory receptors to various intracellular signaling pathways.



  report

cale

Fine regulation of Zap-70 protein tyrosine kinase (Vincenzo Di Bartolo)

Zap-70 (Zeta chain-associated protein of 70 kDaltons) is a PTK critical for triggering T cell activation. Zap-70 binds to the TCR-associated ITAM (Immune-receptor tyrosine-based activation motif) following engagement with the antigen/MHC. The interdomain B of Zap-70 (a short region connecting the two SH2 domains to the kinase domain) contains regulatory tyrosines which become phosphorylated upon TCR triggering. In previous work, we described the TCR-directed phosphorylation of tyrosines 315 and 319 in the interdomain B and their relative importance in regulating T cell activation in vitro. We have carried out a collaborative study with the group of B. and M. Malissen in Marseille to clarify their role in vivo. Recombinant mice carrying a mutation of Tyr315 show a slight defect in T cell development and activation. We have found that T cells harboring the Tyr315 mutation display a defect in the binding of Zap-70 to phospho-ITAM binding suggesting that this tyrosine plays a role in the conformation of ZAP-70. In another collaborative study (with Antonella Isacchi, Pharmacia-Upjohn, Milan), we have established that the SH2 domains of Zap-70 inhibit its kinase activity but that their binding to the ITAM results in an initial change in kinase activity. Thus, Zap-70 seems to be regulated by an initial allosteric effect due to ITAM binding, which depends on Tyr315, followed by tyrosine phosphorylation events, likely to be required for its full activation.


The protein tyrosine phosphatase CD45: its role in the initiation and maintenance of T cell activation signal (Claudine Irles and Frédérique Michel).

The membrane PTPase CD45 is known to have a primary function as a positive regulator of T cell activation through a direct action on Lck, although recent data strongly suggest that CD45 exerts, at the same time, a negative regulatory role. CD45 is therefore essential in setting the activation threshold of T cells by contributing to maintaining the basal steady-state tyrosine phosphorylation level in the cell. Moreover, it has been suggested that during activation CD45 may be sequestered away from the TCR in order to sustain the signal. However, it is unclear how CD45 function is regulated in unstimulated cells and during activation. To begin to clarify the underlying mechanism, we have carried out a complementation analysis using CD45 negative T cell lines and chimeric constructs of CD45 whose extracellular region has been replaced by the homologous regions of Thy-1, CD2 and CD43. The specific question to be addressed during this investigation was whether the size and/or the intrinsic structure of the CD45 extracellular region has a role in regulating its activity and therefore T cell activation. The extracellular regions of these chimeric proteins vary considerably in size and glycosylation. Our results indicate that the capacity to reconstitute the activation signal is a function of the size of the extracellular region of the chimeras, but the peculiar structure of CD45 must also be important. This is true both for activation induced by anti-TCR antibodies and Ag/APC. Indeed, CD45 is, by far, the most effective in reconstituting T cell activation, whereas CD43/CD45 (the " longest " protein) has an intermediate capacity and Thy-1/CD45 or CD2/CD45 (the " shortest " ones) reconstitute very poorly. Our data suggest, therefore, that the extracellular region of CD45 is required for regulating its activity. One interesting possibility is that the size and/or glycosylation of the CD45 extracellular region is a key factor in regulating the accessibility of this enzyme to its substrates within the cells (e.g. perhaps by limiting the contacts with Lck localized in the lipid rafts).


The mechanism of CD28 co-stimulation revisited (F. Michel, G. Mangino, S. Mise).

CD28 is largely responsible for delivering a second signal required for T cell activation. This signal can be exclusively induced by "professional" APCs, that is, cells expressing on their surface high levels of CD28 ligands, B7.1 and/or B7.2. Previous work suggested that CD28 controls a unique signaling pathway capable of cooperating at the transcriptional and translational levels with signals generated by the TCR. However, this model has been questioned and the mechanism through which CD28 costimulation potentiates T cell antigen receptor (TCR)-driven gene expression remains unclear. Vav-1, an exchange factor for Rho GTPases thought to regulate various signaling components leading to cytokine gene expression, is tyrosine phosphorylated upon CD28 engagement. We have provided evidence for a key role of Vav-1 in CD28-mediated signaling. Overexpression of Vav-1 in Jurkat cells in combination with CD28 ligation strongly reduced the concentration of SEE/MHC required for TCR-induced Nuclear factor of activated T cells (NF-AT) activation. Surprisingly, upon Vav-1 overexpression CD28 ligation sufficed to activate NF-AT in the absence of TCR engagement. This effect was not mediated by overexpression of ZAP-70 nor of SLP-76, but required the intracellular tail of CD28, the intactness of the TCR-proximal signaling cascade, the Src-homology domain 2 (SH2) domain of Vav-1 and SLP-76 phosphorylation, an event which was favoured by Vav-1 itself. Cells overexpressing Vav-1 formed lamellipodia and microspikes reminiscent of Rac-1 and Cdc42 activation, respectively, for which the SH2 domain of Vav-1 was dispensable. Together, these data suggest that CD28 engagement activates Vav-1 to boost TCR signals through a synergistic mechanism implicating the adapter SLP-76.



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
 

Wendy HOUSSIN, IP, whoussin@pasteur.fr

Oreste ACUTO, IP, oacuto@pasteur.fr

Frédérique MICHEL, IP, fmichel@pasteur.fr

Vincenzo DI BARTOLO, IP, vbartolo@pasteur.fr

Giorgio MANGINO, post-doc, gmangino@pasteur.fr

Setsuko MISE, post-doc, smise@pasteur.fr

Claudine IRLES, PhD student, cirles@pasteur.fr

Maria Elena MARQUEZ CAMPOS, PhD student, trujillo@pasteur.fr

Fabien BLANCHET, PhD student, fablanc@pasteur.fr

Evelyne DUFOUR, IP, technician, edufour@pasteur.fr

Géraldine ATTAL-BONNEFOY, IP, technician, gattal@pasteur.fr

Emmanuel SECHET, IP, technician, esechet@pasteur.fr


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