The Shc Family Protein Adaptor, Rai, Negatively Regulates T Cell Antigen Receptor Signaling by Inhibiting ZAP-70 Recruitment and Activation

Abstract

Rai/ShcC is a member of the Shc family of protein adaptors expressed with the highest abundance in the central nervous system, where it exerts a protective function by coupling neurotrophic receptors to the PI3K/Akt survival pathway. Rai is also expressed, albeit at lower levels, in other cell types, including T and B lymphocytes. We have previously reported that in these cells Rai attenuates antigen receptor signaling, thereby impairing not only cell proliferation but also, opposite to neurons, cell survival. Here we have addressed the mechanism underlying the inhibitory activity of Rai on TCR signaling. We show that Rai interferes with the TCR signaling cascade one of the earliest steps –recruitment of the initiating kinase ZAP-70 to the phosphorylated subunit of the TCR/CD3 complex, which results in a generalized dampening of the downstream signaling events. The inhibitory activity of Rai is associated to its inducible recruitment to phosphorylated CD3, which occurs in the physiological signaling context of the immune synapse. Rai is moreover found as a pre-assembled complex with ZAP-70 and also constitutively interacts with the regulatory p85 subunit of PI3K, similar to neuronal cells, notwithstanding the opposite biological outcome, i.e. impairment of PI-3K/Akt activation. The data highlight the ability of Rai to establish interactions with the TCR and key signaling mediators which, either directly (e.g. by inhibiting ZAP-70 recruitment to the TCR or sequestering ZAP-70/PI3K in the cytosol) or indirectly (e.g. by promoting the recruitment of effectors responsible for signal extinction) prevent full triggering of the TCR signaling cascade.

The Shc family protein adaptor, Rai, acts as a negative regulator of Th17 and Th1 cell development

“…We have reported previously that Rai fine tunes T cell activation and proliferation by attenuating TCR signaling, beginning from one of the key early steps downstream of CD3 phosphorylation-activation of ZAP-70 [28,29]. Here, we provide evidence that Rai is also implicated in the control of the balance among the Th1, Th2, and Th17 subsets, as revealed by the spontaneous Th1/Th17 skewing observed in Rai Ϫ/Ϫ mice.…”

“…Activations by TCR/CD3 cross-linking were performed as described [29]. Cells were resuspended at 2 ϫ 10 6 /mL in RPMI 7.5% FCS and activated using saturating amounts of anti-CD3 mAb (2c11), purified from hybridoma supernatants and plastic-immobilized secondary antibodies.…”

“…We have demonstrated recently that Rai is expressed in T and B lymphocytes, where it uncouples the antigen receptors from activation of the Ras/MAPK and Akt pathways, thereby acting as an attenuator of mitogenic and survival signaling [28]. The inhibitory effect of Rai on TCR and BCR signaling has been tracked to a key initiating step in the respective cascades, activation of the Syk family protein tyrosine kinases [28,29]. The requirement of the fine-tuning activity of Rai on antigen receptor signaling is witnessed by the development of systemic lupus-like autoimmunity in Rai Ϫ/Ϫ mice, characterized by the presence of circulating anti-dsDNA antibodies and glomerular deposition of autoimmune complexes, eventually leading to glomerulonephritis [28].…”

“…Single-cell suspensions were prepared from LNs and dispase-treated kidneys using cell strainer filters (BD Falcon; BD Biosciences Europe, Erembodegem, Belgium). Naïve CD4 ϩ T cells were enriched from spleen and LNs using the CD4 ϩ CD62L ϩ T cell isolation kit (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) or the CD4 ϩ CD62L ϩ CD44 low T cell column kit (R&D Systems, Minneapolis, MN, USA).Activations by TCR/CD3 cross-linking were performed as described [29]. Cells were resuspended at 2 ϫ 10 6 /mL in RPMI 7.5% FCS and activated using saturating amounts of anti-CD3 mAb (2c11), purified from hybridoma supernatants and plastic-immobilized secondary antibodies.…”

Cross-protective immunity against influenza A/H1N1 virus challenge in mice immunized with recombinant vaccine expressing HA gene of influenza A/H5N1 virus

“…De Groot et al [25] reported that the HA also possesses cross-conserved T-cell epitopes [26-28]. We had found that there was no correlation between the levels of antibody and HSI against influenza virus.…”