B and T lymphocyte attenuator (BTLA) functions as a negative regulator of T cell activation and proliferation. Although the role of BTLA in regulating T cell responses has been characterized, a thorough investigation into the precise molecular mechanisms involved in BTLA-mediated lymphocyte attenuation and, more specifically, its role in regulating B cell activation has not been presented. In this study, we have begun to elucidate the biochemical mechanisms by which BTLA functions to inhibit B cell activation. We describe the cell surface expression of BTLA on various human B cell subsets and confirm its ability to attenuate B cell proliferation upon associating with its known ligand, herpesvirus entry mediator (HVEM). BTLA associates with the BCR and, upon binding to HVEM, recruits the tyrosine phosphatase Src homology 2 domain-containing phosphatase 1 and reduces activation of signaling molecules downstream of the BCR. This is exemplified by a quantifiable decrease in tyrosine phosphorylation of the protein tyrosine kinase Syk, as measured by absolute quantification mass spectrometry. T he B and T lymphocyte attenuator (BTLA) 2 is an Ig superfamily coinhibitory receptor with structural and functional similarities to programmed cell death 1 (PD-1) and CTLA-4 (1-3). However, unlike PD-1 and CTLA-4, which bind members of the B7 Ig superfamily receptors, the established ligand for BTLA is the TNFR family member herpesvirus entry mediator (HVEM) (TNFRSF14) (3-5). HVEM binding leads to tyrosine phosphorylation of the cytoplasmic tail of BTLA, which contains a proposed Grb2/Grb2-related adaptor protein (Grap) docking site and two ITIMs (5-11). Biochemical analysis has revealed that phosphorylation at multiple tyrosines within the cytoplasmic tail of BTLA is necessary and required for efficient recruitment of protein tyrosine phosphatases and BTLA-mediated attenuation of T cell effector response and cell proliferation (5,8,10,11).The role of BTLA appears to be limited to lymphocyte activation as shown in BTLA-deficient mice, which present normal lymphoid organ development and near wild-type lymphocyte numbers. However, these mice display hyperproliferative T and B cell responses to TCR-and BCR-mediated activation, respectively (9, 11). Furthermore, loss of BTLA function leads to increased susceptibility to experimental autoimmune encephalomyelitis and MHC-mismatched allograft rejection, supporting the role of BTLA in modulating T cell activation and effector responses (11,12).Surface expression analysis of BTLA indicates that it is expressed on a wide number of lymphocytes in mice. It is most highly expressed on B cells, followed by CD4 ϩ T cells, and lower expression on CD8 ϩ T cells, macrophages, dendritic cells, and NK cells (9, 13). During murine B cell development, BTLA expression first appears in pre-B cells and shows increased expression through B cell maturation, with the highest expression on mature B cells (9). In this study, we show a detailed cell surface expression profile of BTLA during human B cell deve...
Ca2ϩ channel  subunits regulate cell-surface expression and gating of voltage-dependent Ca 2ϩ channel ␣1 subunits. Based on primary sequence comparisons,  subunits are predicted to be modular structures composed of five domains (A-E) that are related to the large family of membrane-associated guanylate kinase proteins. The crystal structure of the  subunit core B-D domains has been reported recently; however, little is known about the structures of the A and E domains. The N-terminal A domain differs among the four subtypes of Ca 2ϩ channel  subunits ( 1 - 4 ) primarily as the result of two duplications of an ancestral gene containing multiple alternatively spliced exons. At least nine A domain sequences can be generated by alternative splicing. In this report, we focus on one A domain sequence, the highly conserved  4a A domain. We solved its three-dimensional structure and show that it is expressed in punctate structures throughout the molecular layer of the cerebellar cortex. We also demonstrate that it does not participate directly in Ca v 2.1 Ca 2ϩ channel gating but serves as a binding site in protein-protein interactions with synaptotagmin I and the LC2 domain of microtubule-associated protein 1A. With respect to  4 subunits, the interactions are specific for the  4a splice variant, because they do not occur with the  4b A domain. These results have strong bearing on our current understanding of the structure of alternatively spliced Ca 2ϩ channel  subunits and the cell-specific roles they play in the CNS.
HIV-1 Tat is required for the expression of the viral genome. Tat binds to an RNA stem-loop and mediates the recruitment of human coactivators to facilitate HIV-1 transcription. The coactivator and acetyltransferase CREB binding protein (CBP), and the paralog p300, are recruited to the HIV-1 promoter by Tat. Here we identify the interacting domains of Tat and CBP. Circular dichroism and pulldown assays show that full-length Tat binds to the KIX domain of CBP, but not to the C/H1 or CR2 domains. Circular dichroism and NMR studies of Tat deletion mutants localize the KIX-binding domain of Tat to the N-terminal 24 residues of Tat. Transient cotransfections demonstrate that exogenous KIX behaves as a dominant negative to Tat-mediated transcription in human T-cells, suggesting that Tat and KIX interact in vivo. These findings indicate that Tat targets the KIX domain of CBP and provide insight into the molecular interactions involved in regulating HIV-1 gene expression.
The HTLV-1 transcriptional activator Tax is required for viral replication and pathogenesis. In concert with human CREB, Tax recruits the human transcriptional coactivator and histone acetyltransferase p300/CBP to the HTLV-1 promoter. Here we investigate the structural features of the interaction between Tax and the KIX domain of p300/CBP. Circular dichroism spectroscopy, nuclear magnetic resonance chemical shift perturbation mapping, and sedimentation equilibrium analysis show that KIX binds a Tax subdomain corresponding to residues 59-98 of Tax (called Tax(59-98)). Circular dichroism spectroscopy suggests that Tax(59-98) is intrinsically disordered (natively unfolded) in isolation and adopts an ordered conformation upon binding KIX. The interaction is disrupted by a single amino acid variation of Tax(59-98) in which leucine 68 is substituted with proline. Chemical shift perturbation mapping reveals that the Tax-binding surface of KIX is distinct from that utilized by CREB, and corresponds to the site of KIX that interacts with the human transcription factors c-Jun and mixed lineage leukemia protein (MLL). Sedimentation equilibrium analysis shows that Tax and the phosphorylated KID domain of CREB can simultaneously bind KIX to form a ternary 1:1:1 complex. The results provide a molecular description of the concerted recruitment of p300/CBP via the KIX domain by Tax and phosphorylated CREB during Tax-mediated gene expression.
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