BANK1-PLCG2 complex formation is transient and induced by IgM stimulation.

<p>(A) Confocal images of Daudi B cells showing increase molecular proximity between endogenous BANK1 and PLCg2 proteins upon anti-IgM stimulation. The staining was done using <i>in situ</i> a PLA protocol with rabbit anti-BANK1 (ET-52) and mouse anti PLCg2 (Abcam). Nuclei were stained with DAPI in blue. The confocal images (PLA signals) were taken with a pinhole of 2.5 (Zeiss Plan-Apochromat 63× oil objective). Upper panel, non-stimulated cells. Low panel, cells stimulated for 1 minute with anti-human IgM-F(ab')2. To the right are shown digital magnifications. (B) Time variation of PLA BANK1-PLCg2 interaction upon stimulation with anti-IgM in two human B-cell lines, the Burkitt´s derived Daudi and the non-Hodgkin´s lymphoma derived RL. Results are shown as the mean from three independent experiments. Error bars represent the SD from the mean. *P<0,05 based on Student´s test comparing stimulated cells versus time 0 (non stimulated cells).(C) Merge confocal images (pinhole = 1) of Daudi cells showing the nuclei in green and the BANK1-PLCg2 PLA interaction in red to permit co-location analysis. PLA signals close to the nucleus appeared as yellow dots (arrowheads) and when localize in the periphery of the cell as red dots (arrows). (D) Time course upon anti-IgM stimulation of co-localization between the PLA signal and the nucleus. Quantification was done using the overlap coefficient after Manders because this coefficient is insensitive to differences in signal intensities between channels. After one minute of stimulation the co-localization decreases suggesting a translocation of the BANK1-PLCg2 complex. The graft shows two independent experiments, each point represents the analysis of at least 300 cells.**,P<0,01;***P<0,001 based on Student´s test comparing stimulated cells versus time 0 (non stimulated). (E) Immunoprecipitation of the BANK1-PLCg2 complex in Daudi cells. Anti-PLCg2 immunoprecipitates (above) and total cell lysate (below) were analyzed by immunoblotting with anti-BANK1 antibody (BANK1-ET-52). The position of the BANK1 protein is indicated by arrows.</p

BANK1 and BLK Act through Phospholipase C Gamma 2 in B-Cell Signaling

<div><p>The B cell adaptor protein with ankyrin repeats (BANK1) and the B lymphoid tyrosine kinase (BLK) have been genetically associated with autoimmunity. The proteins of these genes interact physically and work in concert during B-cell signaling. Little is know about their interactions with other B-cell signaling molecules or their role in the process. Using yeast two hybrid (Y2H) we sought for factors that interact with BANK1. We found that the molecular switch PLCg2 interacts with BANK1 and that the interaction is promoted by B-cell receptor (BCR) stimulation. We found further that the kinase activity of BLK enhanced BANK1- PLCg2 binding and that the interaction was suppressed upon BLK depletion. Immunoprecipitation and mutational analysis demonstrated that the interaction between BANK1 and PLCg2 was dependent on specific tyrosine and proline residues on the adaptor protein. Our results provide new information important to understand the role of these two genes in basic B-cell physiology and immune-related diseases.</p> </div

BANK1 Regulates IgG Production in a Lupus Model by Controlling TLR7-Dependent STAT1 Activation

<div><p>The purpose of our study was to investigate the effects of the adaptor Bank1 in TLR7 signaling using the B6.<i>Sle1</i>.<i>yaa</i> mouse, a lupus model that develops disease through exacerbated TLR7 expression. Crosses of B6.<i>Sle1</i>.<i>yaa</i> with <i>Bank1</i>^<i>-/-</i> mice maintained several B and myeloid cell phenotypes close to normal wild-type levels. Most striking was the reduction in total serum IgG antibodies, but not of IgM, and reduced serum levels of autoantibodies, IL-6, and BAFF. <i>Bank1</i> deficiency did modify numbers of MZ B cells and total B cell numbers, as well as expression of CXCR4 by follicular helper T cells. Other T cell changes were not observed. <i>Bank1</i> deficiency did not modify numbers of germinal center B cells or plasma cells or clinical disease outcomes. Purified B cells from <i>Bank1</i> deficient mice had strongly reduced <i>Ifnb</i>, <i>Ifna4</i>, <i>Irf7</i>, <i>Aicda</i> and <i>Stat1</i> gene expression following TLR7 agonist stimulation. Interestingly, phosphorylation of Tyr701, but not of Ser727 of STAT1, was impaired in splenic B cells from <i>B6</i>.<i>Sle1</i>.<i>yaa</i>.<i>Bank1-/-</i> mice, as was the nuclear translocation of IRF7 in response to TLR7 agonist stimulation. Further, <i>Bank1</i> deficiency in B6.<i>Sle1</i>.<i>yaa</i> mice reduced the production of IgG2c after <i>in vitro</i> TLR7 agonist stimulation. Our results demonstrate that <i>Bank1</i> controls TLR7-mediated type I interferon production. Combined with the control of the nuclear translocation of IRF7, the modulation of STAT1 transcription and phosphorylation, <i>Bank1</i> contributes to IgG production during development of autoimmune disease.</p></div

BANK1 and PLCG2 co-localize in cytoplasmic compartments.

<p>Confocal images of HEK293 cells co-expressing fluorescently tagged proteins. BANK1 is a cytoplasmic adaptor protein that shows a punctate and homogeneous pattern of distribution. PLCg2 shares the same sub-cellular compartments when co-expressed with BANK1. Upper row, co-expression of PLCg2 and BANK1 showing sub-cellular co-localization in punctate structures. Although a perfect co-localization between the two proteins is observed, there are dots (indicated by arrows, both in the merge image and in the amplification at the right upper corner) in which the ratio of the two proteins is reversed. Cross-talks between light channels were further ruled out by assessing the emission on cells expressing only one fluoresce protein and excitated sequencially with both lasers. Middle row, images showing co-localization between PLCg2 and BANK1 in a homogeneous cytoplasmic distribution. Lower row, images showing only partial co-localization between the cytoplasmic scavenger receptor, CD163 and BANK1. Scale bar: 10 µm.(-yfp; yellow fluorescence protein. –cfp; cyan fluorescence protein. –chr; cherry fluorescent protein. –gfp; green fluorescent protein).</p

Silencing of the BLK kinase leads to alteration of the association between BANK1 and PLCg2.

<p>(A) Immunoblot of extracts derived from human Daudi B-cells showing efficient silencing of endogenous BLK protein. The cell line shBLK was transduced with lentivirus coding for small hairpin RNAs targeting the BLK kinase while shControl lentivirus codes for unrelated sequences. Top, western blot analysis using antibody to BLK; bottom, western blot analysis using antibody to GAPDH as loading control. (B) The relative BLK mRNA is reduced to half in the silencing line (shBLK)(***P<0,001 based on Student´s test comparing mRNA expression between Daudi cell versus cBLK silencing cells and control silenced cells versus BLK silencing cells). (C) Immunoprecipitates (IPs) of stimulated silenced shBLK cells with anti-IgM, using anti-PLCg2 antibody and interrogated with anti-BANK1 to assay BANK1-PLCg2 association. Quantification of the immunoprecipitates normalized with BANK1 is displayed below the bands. Silencing of BLK leads to less association between BANK1-PLCg2. (D) Kinetics of the association between BANK1-PLCg2 assayed with <i>in situ</i> Proximity Ligation (PLA) in control and BLK-silenced cell lines. The association between BANK1-PLCg2 is reduced in silenced cells during resting conditions and after 15 minutes of IgM stimulation (***P≤0,001 based on Student´s test comparing control cells versus BLK silencing cells). The difference of BANK1-PLCg2 association between control and the silenced lines was not significant at 1 minute after stimulation.</p

Clones isolated in the yeast two-hybrid screen using the full-length BANK1.

<p>Clones isolated in the yeast two-hybrid screen using the full-length BANK1 (amino acids 1–785) as bait. The frame indicates if the coding sequences are in the same frame as Gal4-Activating Domain. In general polypeptides not having an in-frame (IF) position are not considered of biological interest. However, some of the proteins expressed from F1 or F2 can be translated in the correct frame, due to the existence of natural frame-shift events during translation in yeast. AA indicates the length of the clone in number of amino acids and (%) the percentage of frame corresponding to the annotation in GenBank.</p

The constitutive-active form of BLK enhances the binding between BANK1 and PLCG2.

<p>(A) Schematic representation of the constructs used to study the association between BANK1 and PLCg2 in transfected HEK293 cells. The constructs coding for wild-type forms of BLK, LYN, GFP and PLCg2 or the indicated mutated forms were fused to the epitope V5 at the C-termini. BANK1 was targeted with the Flag epitope at the N- terminus. The catalytic domains of BLK and PLCg2 are shown in red. The kinase dead form (BLK-KL-v5) has a substitution K (lysine) to L (leucine) at position 269 and the constitutively active form (BLK-YF-v5) has a Y501F substitution that prevents the phosphorylation of the inhibitory tyrosine. The lipidation in the amino terminal of BLK is indicated as back line. The myristoylation site was deleted by G2V substitution (glycine to valine) and the addition of an extra by palmitoylated site by L3C substitution (leucine to cysteine). The Src homology 3 domains (SH3) that bind to proline-rich motifs are drawn in orange and the SH2 domains in yellow. The Pleckstrin homology domain (PH) that binds to phosphatidylinositol lipids is shown in blue. In BANK1 are shown the Dof/BCAP/BANK (DBB) motif (amino acids 199–327), the double ankyrin repeat-like (ANK) motifs (amino acids 339-402) and the putative coiled coil (CC) region (amino acids 677–705). (B) HEK293 cells were transiently co-transfected with plasmids coding for the wild-type form of BLK, its functionally mutated forms (KL and YF), LYN or GFP in addition to plasmids expressing BANK1 and PLCg2. The lysates were immunoprecipitated using anti-PLCg2 antibody (above) and immunoblotted sequencially with anti-BANK1 antibody, anti-V5 to detect PLCg2, Srcs kinases and GFP and anti-phosphotyrosine antibody. (C). Mutation of lipidation sites of the kinases influence the formation of the BANK1-PLCg2 complex and the overall tyrosine phosphorylation on PLCg2. The blots were interrogated as in B.</p

<i>Bank1</i> deficiency reduces the nuclear translocation of IRF7.

<p>Levels of IRF7 protein in the (A) cytosol and the (B) nucleus following TLR7 agonist stimulation for the indicated times was detected by immunoblot on purified splenic B cells. Gapdh and Lamin B1 were used as control for the cytosolic and nuclear fractions, respectively. Statistical column plots present the relative band intensity relative to Gapdh (A) and Lamin B1 (B) of cytosolic and nuclear Irf7, respectively. NP: nuclear pellet; WC: whole cell lysate. Statistical plots are shown as mean with SD of 3 independent experiments. Mann-Whitney nonparametric test (<i>SY</i>.<i>Bank1</i>^<i>+/+</i> <i>vs</i>. <i>SY</i>.<i>Bank1</i>^<i>-/-</i>) (*p≤0.05, ns>0.05 [not shown]).</p

<i>Bank1</i> deficiency restores the cellular phenotypes of splenic lymphocytes from B6.<i>Sle1</i>.<i>yaa</i>.<i>Bank1</i>^<i>+/+</i> mice.

<p>(A) Splenocyte numbers (left) and spleen weight (right) in <i>SY</i>.<i>Bank1</i>^<i>-/-</i> and <i>SY</i>.<i>Bank1</i>^<i>+/+</i> mice. (B) <i>S</i>plenic B cells (left), follicular B cells (FO B; middle) and splenic B1a subset in <i>SY</i>.<i>Bank1</i>^<i>+/+</i> and <i>SY</i>.<i>Bank1</i>^<i>-/-</i> mice. Total mice analyzed: <i>SY</i>.<i>Bank1</i>^<i>+/+</i> (n = 14), <i>SY</i>.<i>Bank1</i>^<i>+/+</i> (n = 15), WT (n = 8). (C) CD23^-IgM^lo/- immature B cells in the spleen characterized as B220⁺CD93⁺CD23^-IgM^lo/-. The data are shown as percentage of B220⁺CD93⁺ immature B cells (IM B; left). Marginal zone B (MZ B) cells are characterized as B220⁺CD93^-CD23^loCD21^hi. Total mice analyzed: <i>SY</i>.<i>Bank1</i>^<i>+/+</i> (n = 11), <i>SY</i>.<i>Bank1</i>^<i>-/-</i> (n = 12 for immature B cells, n = 14 for MZ B), WT (n = 8). (D) Activation status of CD19⁺ splenic B cells and splenic T cells as determined by percentage of CD69⁺ cells out of CD19⁺B cells or TCRβ⁺T cells, and the frequency of splenic T cells in <i>SY</i>.<i>Bank1</i>^<i>+/+</i>, <i>SY</i>.<i>Bank1</i>^<i>-/-</i> and WT mice. Total mice analyzed: <i>SY</i>.<i>Bank1</i>^<i>+/+</i> (n = 10; n = 11 for splenic T), <i>SY</i>.<i>Bank1</i>^<i>-/-</i> (n = 11; n = 12 for splenic T), WT (n = 10). (E) The frequency of follicular helper T (T_fh) cells in splenic CD4⁺T and expression levels (MFI) of CXCR4 and CXCR5 in <i>SY</i>.<i>Bank1</i>^<i>-/-</i> and <i>SY</i>.<i>Bank1</i>^<i>+/+</i>mice, and germinal center B (GC B; CD19⁺IgD^-PNA^hiGL7⁺) cells and plasma cells (CD11c^-CD3^-B220^int/-CD138⁺) populations. (F) Immature B cells (B220⁺IgM⁺IgD^-) and mature recirculating B cells (B220⁺IgM⁺IgD^hi) from bone marrows. Total mice analyzed: <i>SY</i>.<i>Bank1</i>^<i>+/+</i> (n = 11), <i>SY</i>.<i>Bank1</i>^<i>-/-</i> (n = 11), WT (n = 10). Data pooled from 4 independent experimental cohorts of mice. Statistical plots are shown as mean with Mann-Whiney (<i>SY</i>.<i>Bank1</i>^<i>+/+</i> vs. <i>SY</i>.<i>Bank1</i>^<i>-/-</i>) nonparametric test (*p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001, ns>0.05).</p

Clones belonging to the phosphoinositide-specific phospholipase C and the family of Src kinases isolated in two yeast two-hybrid screens.

<p>(A) Representation of PLCg2 modular structure and the coding region of the clones identified in the Y2H. Clones named a- correspond to the first screen using as bait the full-length BANK1, clones starting with b- are the ones identified in the screen with the non autoactivating truncated protein BANK1 (331–785). The clone b-234 has a deletion of 25 aa between the cSH2 and SH3 domains. (B) Structure of the Src kinase FYN and the isolated clones belonging to this family of non- receptor kinase. PH: Pleckstrin homology domain, involved in the recruitment to membranes by binding to phosphatidylinositol containing lipids. X and Y are the two halves of the catalytic isomerase. SH2 (Src homology 2) conserved domain that typically binds to phosphorylated tyrosine residues. SH3 (Src homology 3) usually binds to proline-rich motifs. The C2 motif is present in many proteins that interact with membranes and are frequently involved in calcium dependent phospholipid binding and membrane targeting processes.</p