Distinct NF-kB activation pathways engaged by T-cell receptor and co-receptor CD28 on T-cells

The transcription factor nuclear factor-kB (NF-kB) is critical for the induction of inflammatory responses in T-cells, survival and differentiation. Antigen receptor (TCR) and co-receptor CD28 are the central regulators of NF-kB activation in T-cells. Progress in understanding NF-kB activation in T-cells has occurred over the years with the identification of individual adapters such as ADAP and GRB-2 and enzymes such as PKC-θ that regulate NF-kB. However, little is known whether the engagement of distinct modules by the TCR and CD28 account for the cooperative effects of the two receptors in activating NF-kB. In this review, we discuss recent advances in our understanding of NF-kB regulation by TCR and CD28.This work was supported by Wellcome Trust Program Grant (PG) PKAG/504 to Principal Research Fellow (PRF) C.E. Rudd.This is the final version of the article. It first appeared from Smart Science & Technology via http://dx.doi.org/10.14800/ics.61

Distinct NF-kB activation pathways engaged by T-cell receptor and co-receptor CD28 on T-cells

The transcription factor nuclear factor-kB (NF-kB) is critical for the induction of inflammatory responses in T-cells, survival and differentiation. Antigen receptor (TCR) and co-receptor CD28 are the central regulators of NF-kB activation in T-cells. Progress in understanding NF-kB activation in T-cells has occurred over the years with the identification of individual adapters such as ADAP and GRB-2 and enzymes such as PKC-θ that regulate NF-kB. However, little is known whether the engagement of distinct modules by the TCR and CD28 account for the cooperative effects of the two receptors in activating NF-kB. In this review, we discuss recent advances in our understanding of NF-kB regulation by TCR and CD28

TCR and CD28 activate the transcription factor NF-κB in T-cells via distinct adaptor signaling complexes.

The transcription factor NF-κB is needed for the induction of inflammatory responses in T-cells. Whether its activation by the antigen-receptor and CD28 is mediated by the same or different intracellular signaling pathways has been unclear. Here, using T-cells from various knock-out (Cd28(-/-), adap(-/-)) and knock-in (i.e. Cd28 Y-170F) mice in conjunction with transfected Jurkat T-cells, we show that the TCR and CD28 use distinct pathways to activate NF-κB in T-cells. Anti-CD28 ligation alone activated NF-κB in primary and Jurkat T-cells as measured by NF-κB reporter and EMSA assays. Anti-CD28 also activated NF-κB normally in primary T-cells from adap(-/-) mice, while anti-CD3 stimulation required the adaptor ADAP. Over-expression of ADAP or its binding partner SKAP1 failed to enhance anti-CD28 activation of NF-κB, while ADAP greatly increased anti-CD3 induced NF-κB activity. By contrast, CD28 activation of NF-κB depended on GRB-2 binding to CD28 as seen in CD28 deficient Jurkat T-cells reconstituted with the CD28 YMN-FM mutant, and in primary T-cells from CD28 Y170F mutant knock-in mice. CD28 associated with GRB-2, and GRB-2 siRNA impaired CD28 NF-κB activation. GRB-2 binding partner and guanine nucleotide exchange factor, VAV1, greatly enhanced anti-CD28 driven activation of NF-κB. Further, unlike in the case of anti-CD28, NF-κB activation by anti-CD3 and its cooperation with ADAP was strictly dependent on LAT expression. Overall, we provide evidence that CD28 and the TCR complex regulate NF-κB via different signaling modules of GRB-2/VAV1 and LAT/ADAP pathways respectively.This work was supported by Wellcome Trust Progam Grant (PG) PKAG/504 to Principal Research Fellow (PRF) C.E. Rudd. We are grateful to Oreste Ocuto and Enzo Cerundolo from University of Oxford for providing CHC17 and 1G4 (Cd28−/−) Jurkat cells.This paper was originally published in Immunology Letters (Thaker YR, Schneider H, Rudd CE, Immunology Letters 2015, 163, 1, 113–119, doi:10.1016/j.imlet.2014.10.020)

GTPase-activating protein Rasal1 associates with ZAP-70 of the TCR and negatively regulates T-cell tumor immunity.

Immunotherapy involving checkpoint blockades of inhibitory co-receptors is effective in combating cancer. Despite this, the full range of mediators that inhibit T-cell activation and influence anti-tumor immunity is unclear. Here, we identify the GTPase-activating protein (GAP) Rasal1 as a novel TCR-ZAP-70 binding protein that negatively regulates T-cell activation and tumor immunity. Rasal1 inhibits via two pathways, the binding and inhibition of the kinase domain of ZAP-70, and GAP inhibition of the p21ras-ERK pathway. It is expressed in activated CD4 + and CD8 + T-cells, and inhibits CD4 + T-cell responses to antigenic peptides presented by dendritic cells as well as CD4 + T-cell responses to peptide antigens in vivo. Furthermore, siRNA reduction of Rasal1 expression in T-cells shrinks B16 melanoma and EL-4 lymphoma tumors, concurrent with an increase in CD8 + tumor-infiltrating T-cells expressing granzyme B and interferon γ-1. Our findings identify ZAP-70-associated Rasal1 as a new negative regulator of T-cell activation and tumor immunity

Specific motifs of the V-ATPase a2-subunit isoform interact with catalytic and regulatory domains of ARNO

AbstractWe have previously shown that the V-ATPase a2-subunit isoform interacts specifically, and in an intra-endosomal acidification-dependent manner, with the Arf-GEF ARNO. In the present study, we examined the molecular mechanism of this interaction using synthetic peptides and purified recombinant proteins in protein-association assays. In these experiments, we revealed the involvement of multiple sites on the N-terminus of the V-ATPase a2-subunit (a2N) in the association with ARNO. While six a2N-derived peptides interact with wild-type ARNO, only two of them (named a2N-01 and a2N-03) bind to its catalytic Sec7-domain. However, of these, only the a2N-01 peptide (MGSLFRSESMCLAQLFL) showed specificity towards the Sec7-domain compared to other domains of the ARNO protein. Surface plasmon resonance kinetic analysis revealed a very strong binding affinity between this a2N-01 peptide and the Sec7-domain of ARNO, with dissociation constant KD=3.44×10−7M, similar to the KD=3.13×10−7M binding affinity between wild-type a2N and the full-length ARNO protein. In further pull-down experiments, we also revealed the involvement of multiple sites on ARNO itself in the association with a2N. However, while its catalytic Sec7-domain has the strongest interaction, the PH-, and PB-domains show much weaker binding to a2N. Interestingly, an interaction of the a2N to a peptide corresponding to ARNO's PB-domain was abolished by phosphorylation of ARNO residue Ser392. The 3D-structures of the non-phosphorylated and phosphorylated peptides were resolved by NMR spectroscopy, and we have identified rearrangements resulting from Ser392 phosphorylation. Homology modeling suggests that these alterations may modulate the access of the a2N to its interaction pocket on ARNO that is formed by the Sec7 and PB-domains. Overall, our data indicate that the interaction between the a2-subunit of V-ATPase and ARNO is a complex process involving various binding sites on both proteins. Importantly, the binding affinity between the a2-subunit and ARNO is in the same range as those previously reported for the intramolecular association of subunits within V-ATPase complex itself, indicating an important cell biological role for the interaction between the V-ATPase and small GTPase regulatory proteins

Caspase interaction of anti-apoptotic Livin as well as the Vacuolar ATPase (V-ATPase) and structural insights into the subunit d and a of the yeast V-ATPase

Apoptosis is a critical process to remove the non-functional and redundant cells regulated by pro- and anti-apoptotic factors. Perturbation of balance between pro- and anti-apoptotic components is the leading cause of several physiopathological conditions such as neurodegenerative and cancer malignancies. Here, the pro-apoptotic cellular protein, ARTS as well as an anti-apoptotic protein, Livin, a family member of inhibitor of apoptosis have been studied. Results showed that ARTS is not the target of Livin E3 ligase activity in apoptotic cells co-expressing ARTS and Livin. In turn, Livin was found to undergo cleavage in ARTS promoted apoptosis which was independent of its self-ubiquitination activity, normally observed in healthy cells. The exhaustion of Livin during ARTS-promoted apoptosis could partially be suppressed by the caspase inhibitors, implying a possible role of caspases concomitant with high active caspase 7 levels found in ARTS-promoted staurosporine-induced apoptosis. Not only Livin, caspase do cleave several important cellular components during apoptosis and here, I have identified subunit d of V-ATPase as a new target of caspase 3. V ATPases do play critical role in health and disease by maintaining proper acid/base balance pH. Additionally, homogenous protein preparation of yeast subunit d protein was used to determine its first low resolution shape by small angle X-ray spectroscopy (SAXS), revealing two distinct domains of 6.5 nm and 3.5 nm widths forming a “boxing glove” shape. Using previously solved low resolution structure of VO domain as a template, subunit d could be assigned inside the VO, allowing its clear localization on the top of VO domain of V-ATPase. Moreover, biochemical approaches of fluorophore labeling, tryptic digestion and MALDI-TOF analysis led to the identification of a cysteine bridge between Cys36 and Cys329.DOCTOR OF PHILOSOPHY (SBS