Fig 1: Virotrap interaction screen reveals novel BCAP-interaction partners. HEK293T cells were transfected with GAG-BCAP and a pMD2.G-pcDNA3-FLAG-VSV-G mix to generate FLAG-VSV-G–coated VLPs. After purification and tryptic digest, the VLP contents were analyzed by MS. A, volcano plot of BCAP VLP contents compared with the eDHFR control. False discovery rates (FDR) = 0.05 and S0 = 1. Proteins that are significantly enriched in either BCAP or eDHFR VLPs are highlighted in red. B, overview of significant BCAP virotrap hits sorted according to relative enrichment.
Fig 2: BCAP engages in SH3 domain interaction and tyrosine phosphorylation-dependent SH2 domain interactions. A, HEK293T cells were transfected with Myc-BCAP, Myc-BCAP Y374F, FLAG-GRB2, and FLAG-CRKL. At 24 h post-transfection, cells were lysed and subjected to immunoprecipitation with anti-FLAG antibody. Precipitates were split and immunostained for precipitation of FLAG-GRB2, FLAG-CRKL, and Myc-BCAP. B, purified GST-tagged GRB2, GRB2 SH2, CRKL; C, p85 N-SH2, p85 C-SH3, p85 SH3; D, PLC-γ2 N-SH2, PLC-γ2 C-SH2, and PLC-γ2 SH3 were immobilized on GST resin. Purified BCAP and dephosphorylated BCAP were subsequently applied to the resin and GST-tagged bait proteins were eluted from the resin and analyzed on SDS-PAGE. E, domain arrangement of BCAP-interacting proteins.
Fig 3: The BCAP DBB domain structure reveals striking similarities to TF TIG domains. (A) The structure is composed of a core TIG fold, followed by two C-terminal α helices. (B) Structural alignment of the DBB TIG fold (green) and TIG domains from various TF. Ebf1 (teal, PBD 3MLP), CAMTA1 (purple, PDB 2CXK), NFAT5 (gray, PDB 1IMH), and p50 NF-κB dimer (yellow, PDB1NFK). (C) Structural alignment of known TIG dimers from (A) with the BCAP DBB domain interface 1 putative dimer. The DBB domain TIG2α structure is shown (in green) with Ebf1 (teal), CAMTA1 (purple), NFAT5 (gray), and p50 NF-κB dimer (yellow).
Fig 4: The BCAP DBB domain is required for negative regulation of TLR signaling. (A) HEK293T cells were transiently transfected with FLAG-TIR, FLAG-TIR-TIG2α, FLAG-TIR-DBB, FLAG-TIR-DBB-ANK, FLAG-MyD88, and Myc-MAL. At 24 h posttransfection, cells were lysed and subjected to immunoprecipitation with anti-FLAG Ab. Precipitates were split and assayed for the presence of FLAG-tagged BCAP constructs or coprecipitation of Myc-tagged MAL. (B) As in (A), cells transfected with Myc-MyD88. (C) NF-κB reporter assay in HEK293T cells transiently transfected with MAL and BCAP constructs containing various domain boundaries. The results are presented as means ± SD based on quintuple samples from one of three independent experiments. Statistical significance was analyzed by using a two-tailed ANOVA test, in which p < 0.05 was deemed significant. (D) As in (C), cells transfected with MyD88. (E) HEK293T cells were transiently transfected with Myc-MAL, Myc-MyD88, and BCAP constructs containing various domain boundaries, as indicated. After lysis, samples were analyzed for NF-κB reporter activity and probed for the expression of Myc-MAL and Myc-MyD88 by Western blot. Myc-TIR-DBB is also detected in these experiments as it has a similar Mr to MAL and MyD88 (extra band in lanes 4 and 11). (F) Schematic depiction of domain boundaries for constructs used in this study.
Fig 5: Constitutive SH3 domain interactions facilitate rapid SH2 domain binding upon BCAP tyrosine phosphorylation. Stepwise binding model for the SH2 and SH3 domain-containing BCAP-interaction partners p85 and PLC-γ2. The PI3K p85 or PLC-γ2 SH3 domains constitutively interact with BCAP proline-rich regions (Pro). The preformed complex can then rapidly engage in N-SH2 domain interaction upon BCAP tyrosine phosphorylation. High-affinity N-SH2 interactions facilitate the binding of lower-affinity C-SH2 domain interaction resulting in full activation of PI3K and PLC-γ2.
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