Evelyne Dufour

Tyrosine 319, a Newly Identified Phosphorylation Site of ZAP-70, Plays a Critical Role in T Cell Antigen Receptor Signaling

Following T cell antigen receptor (TCR) engagement, the protein tyrosine kinase (PTK) ZAP-70 is rapidly phosphorylated on several tyrosine residues, presumably by two mechanisms: an autophosphorylation and a trans-phosphorylation by the Src-family PTK Lck. These events have been implicated in both positive and negative regulation of ZAP-70 activity and in coupling this PTK to downstream signaling pathways in T cells. We show here that Tyr315 and Tyr319 in the interdomain B of ZAP-70 are autophosphorylated in vitro and become phosphorylated in vivo upon TCR triggering. Moreover, by mutational analysis, we demonstrate that phosphorylation of Tyr319 is required for the positive regulation of ZAP-70 function. Indeed, overexpression in Jurkat cells and in a murine T cell hybridoma of a ZAP-70 mutant in which Tyr319 was replaced by phenylalanine (ZAP-70-Y319F) dramatically impaired anti-TCR-induced activation of the nuclear factor of activated T cells and interleukin-2 production, respectively. Surprisingly, an analogous mutation of Tyr315 had little or no effect. The inhibitory effect of ZAP-70-Y319F correlated with a substantial loss of its activation-induced tyrosine phosphorylation and up-regulation of catalytic activity, as well as with a decreased in vivocapacity to phosphorylate known ZAP-70 substrates, such as SLP-76 and LAT. Collectively, our data reveal the pivotal role of Tyr319phosphorylation in the positive regulation of ZAP-70 and in TCR-mediated signaling.

Human Opiorphin, a natural antinociceptive modulator of opioid-dependent pathways

Mammalian zinc ectopeptidases play important roles in turning off neural and hormonal peptide signals at the cell surface, notably those processing sensory information. We report here the discovery of a previously uncharacterized physiological inhibitor of enkephalin-inactivating zinc ectopeptidases in humans, which we have named Opiorphin. It is a QRFSR peptide that inhibits two enkephalin-catabolizing ectoenzymes, human neutral ecto-endopeptidase, hNEP (EC 3.4.24.11), and human ecto-aminopeptidase, hAP-N (EC 3.4.11.2). Opiorphin displays potent analgesic activity in chemical and mechanical pain models by activating endogenous opioid-dependent transmission. Its function is closely related to the rat sialorphin peptide, which is an inhibitor of pain perception and acts by potentiating endogenous μ- and δ-opioid receptor-dependent enkephalinergic pathways. Here we demonstrate the functional specificity in vivo of human Opiorphin. The pain-suppressive potency of Opiorphin is as effective as morphine in the behavioral rat model of acute mechanical pain, the pin-pain test. Thus, our discovery of Opiorphin is extremely exciting from a physiological point of view in the context of endogenous opioidergic pathways, notably in modulating mood-related states and pain sensation. Furthermore, because of its in vivo properties, Opiorphin may have therapeutic implications.

Tyrosine 319 in the Interdomain B of ZAP-70 Is a Binding Site for the Src Homology 2 Domain of Lck

T-cell antigen receptor-induced signaling requires both ZAP-70 and Lck protein-tyrosine kinases. One essential function of Lck in this process is to phosphorylate ZAP-70 and up-regulate its catalytic activity. We have previously shown that after T-cell antigen receptor stimulation, Lck binds to ZAP-70 via its Src homology 2 (SH2) domain (LckSH2) and, more recently, that Tyr319 of ZAP-70 is phosphorylated in vivo and plays a positive regulatory role. Here, we investigated the possibility that Tyr319 mediates the SH2-dependent interaction between Lck and ZAP-70. We show that a phosphopeptide encompassing the motif harboring Tyr319, YSDP, interacted with LckSH2, although with a lower affinity compared with a phosphopeptide containing the optimal binding motif, YEEI. Moreover, mutation of Tyr319 to phenylalanine prevented the interaction of ZAP-70 with LckSH2. Based on these results, a gain-of-function mutant of ZAP-70 was generated by changing the sequence Y319SDP into Y319EEI. As a result of its increased ability to bind LckSH2, this mutant induced a dramatic increase in NFAT activity in Jurkat T-cells, was hyperphosphorylated, and displayed a higher catalytic activity compared with wild-type ZAP-70. Collectively, our findings indicate that Tyr319-mediated binding of the SH2 domain of Lck is crucial for ZAP-70 activation and consequently for the propagation of the signaling cascade leading to T-cell activation.

T cell receptor for antigen induces linker for activation of T cell–dependent activation of a negative signaling complex involving Dok-2, SHIP-1, and Grb-2

Adaptor proteins positively or negatively regulate the T cell receptor for antigen (TCR) signaling cascade. We report that after TCR stimulation, the inhibitory adaptor downstream of kinase (Dok)-2 and its homologue Dok-1 are involved in a multimolecular complex including the lipid phosphatase Src homology 2 domain–containing inositol polyphosphate 5′-phosphatase (SHIP)-1 and Grb-2 which interacts with the membrane signaling scaffold linker for activation of T cells (LAT). Knockdown of LAT and SHIP-1 expression indicated that SHIP-1 favored recruitment of Dok-2 to LAT. Knockdown of Dok-2 and Dok-1 revealed their negative control on Akt and, unexpectedly, on Zap-70 activation. Our findings support the view that Dok-1 and -2 are critical elements of a LAT-dependent negative feedback loop that attenuates early TCR signal. Dok-1 and -2 may therefore exert a critical role in shaping the immune response and as gatekeepers for T cell tolerance.

TCR/CD3 Down-Modulation and ζ Degradation Are Regulated by ZAP-70

TCR down-modulation following binding to MHC/peptide complexes is considered to be instrumental for T cell activation because it allows serial triggering of receptors and the desensitization of stimulated cells. We studied CD3/TCR down-modulation and ζ degradation in T cells from two ZAP-70-immunodeficient patients. We show that, at high occupancy of the TCR, down-modulation of the CD3/TCR is comparable whether T cells express or do not express ZAP-70. However, if TCR occupancy was low, we found that CD3/TCR was down-regulated to a lesser extent in ZAP-70-negative than in ZAP-70-positive T cells. We studied CD3/TCR down-modulation in P116 (a ZAP-70-negative Jurkat cell-derived clone) and in P116 transfected with genes encoding the wild-type or a kinase-dead form of ZAP-70. Down-modulation of the TCR at high occupancy did not require ZAP-70, whereas at low TCR occupancy down-modulation was markedly reduced in the absence of ZAP-70 and in cells expressing a dead kinase mutant of ZAP-70. Thus, the presence of ZAP-70 alone is not sufficient for down-modulation; the kinase activity of this molecule is also required. The degradation of ζ induced by TCR triggering is also severely impaired in T cells from ZAP-70-deficient patients, P116 cells, and P116 cells expressing a kinase-dead form of ZAP-70. This defect in TCR-induced ζ degradation is observed at low and high levels of TCR occupancy. Our results identify ZAP-70, a tyrosine kinase known to be crucial for T cell activation, as a key player in TCR down-modulation and ζ degradation.

Tyrosine 315 determines optimal recruitment of ZAP-70 to the T cell antigen receptor.

Recruitment of ZAP‐70 protein tyrosine kinase to the T cell antigen receptor (TCR) is mediated by the binding of the SH2 domains of this enzyme to phosphorylated ITAM motifs in the CD3 and TCRζ subunits. We have previously shown that the efficiency of both positive and negative thymocyte selection was decreased in knock‐in mice expressing ZAP‐70 mutated at Tyr315 (ZAP‐70‐Y315F), a residue laying in the interdomain B of this protein. Surprisingly, in these cells the amount of phosphorylated TCRζ chain co‐precipitating with ZAP‐70‐Y315F was significantly reduced compared to control mice. We report now that the binding affinity of ZAP‐70‐Y315F to phosphorylated ITAM is reduced as compared to the wild‐type protein, whereas the intrinsic catalytic activity is untouched. Consequently, phosphorylated ITAM appear to be more accessible to protein tyrosine phosphatases (PTP) and can be readily dephosphorylated. We provide evidence suggesting that the defective ITAM binding induced by Tyr315 mutation is independent of the putative role of this residue as a binding site for Vav‐1. Finally, we found that the extracellular signal‐regulated kinase pathway is impaired in ZAP‐70‐Y315F‐expressing mice. Collectively, these results demonstrate that Tyr315 has an unsuspected structural role in ZAP‐70 and may allosterically regulate the function of the nearby SH2 domains.

Structure- Activity Relationship Study and Function-Based Petidomimetic Design of Human Opiorphin with Improved Bioavailability Property and Unaltered Analgesic Activity

Human opiorphin inhibits enkephalin-inactivating ectopeptidases to produce analgesic and antidepressant-like effects in standard murine models via activation of μ and/or δ opioid pathways. It is an endogenous peptide regulator of enkephalin bioavailability. Opiorphin molecule, a QRFSR-peptide, is thus a promising prototype for the design of an improved class of analgesics. The major limitation on the clinical use of peptide drugs is their rapid degradation by circulating peptidases. Our goal was, therefore, to search for functional derivatives of opiorphin with improved metabolic stability. In order to identify the functional amino acid groups required for opiorphin inhibitory potency toward both AP-N and NEP human ectopeptidases, we used the Structure-Activity Relationship (SAR) method. From this data, a series of opiorphin derivatives was designed and selected. The best performing compound then underwent a complete metabolic profile using in vitro kinetic models. Finally, its safety profile relative to the native peptide as well as its efficacy in an in vivo rat model was evaluated. We demonstrated a tight structural selectivity in the functional interaction of opiorphin with both human NEP and AP-N targets by SAR studies. Nevertheless, we found that the addition of an N-terminal Zn-chelating group, a Cys-thiol group and the replacement of the first labile peptide bond by a polyethylene surrogate, a [CH2]6 linker,and, finally, the substitution of Ser4 by Ser-O-[CH2]8, results in a high performing C-[(CH2)6]-QRF[S-O-[CH2]8]-R peptidomimetic product. This designed opiorphin analog shows reinforced inhibitory potency toward human AP-N activity (more than 10-fold increase) and NEP activities (more than 40-fold increase) relative to the QRFSR native peptide. It also has increased metabolic stability in human plasma and yet retains full analgesic activity in the behavioral formalin-induced rat pain model. C-[(CH2)6]-QRF[S-O-[CH2]8]-R thus represents a very attractive and promising analgesic drug-candidate.