CMTM7 is required for BLNK association with Syk, its phosphorylation and signal transduction from BCR.

<p>(A) Expression levels of endogenous <i>CMTM7</i> mRNA in parental, mock-transduced, and in two independent CMTM7-knockdown BAL17 cells (kd1 and kd2) evaluated by real-time RT-PCR. (B, C, D, E) The kd1 or the mock-transduced BAL17 cells were stimulated with anti-IgM antibody for the indicated time periods. (B, C) The cell lysates were subjected to Western blot analysis with antibodies against the indicated molecules. pY: total phosphotyrosines detected by the PY20 antibody; SFK: src-family kinase. (D, E) The cell lysates were immunoprecipitated (IP) with anti-BLNK antibody or control rabbit IgG (D), or with anti-Syk antibody (E), and the precipitates and the lysates were subjected to Western blot analysis with the indicated antibodies. (F, G) BAL17/T7-CMTM7 cells were stimulated with anti-IgM for 10 min or untreated (0 min), and the cell lysates were immunoprecipitated (IP) with the indicated antibodies or species- and isotype-matched control antibodies and analyzed as in (E). (C, D) Numbers below each panel represent relative phosphorylation values of each protein (setting the value of the left-most sample in each panel as 1.0), normalized as relative to the corresponding total proteins as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031829#s4" target="_blank">Materials and Methods</a>, except for pSFK which contains the undefined number of Src-family proteins.</p

CMTM7 binds to sIgM and recruits BLNK to sIgM.

<p>(A) BAL17/T7-CMTM7 cells were stimulated with anti-IgM antibody for 10 min or untreated (0 min). Then the cell lysates were immunoprecipitated with mouse anti-T7 (IP: T7) or an isotype-matched control (IP: cont.) monoclonal antibodies. The precipitates and the lysate were analyzed by Western blot analysis. (B, C) BAL17/T7-CMTM7 cells (B) or the kd1 cells reconstituted with the indicated forms of CMTM7 (C) were treated with goat anti-IgM antibody (whole IgG) or untreated (-) and incubated at 37°C for the indicated time periods, or kept on ice (0 min). Then the cell lysates were incubated with protein G-coated beads. The bead-bound proteins (IP: IgM or -) and the lysates were analyzed by Western blot analysis using antibodies against μ H chain, T7 tag, and BLNK as indicated.</p

The C-terminal region of CMTM7 is necessary for its membrane localization and function.

<p>(A) Schematic representation of the mouse CMTM7 cDNA, and a T7-tagged (indicated by a blue globule) full-length version (full) or deletion mutants lacking the N-terminal (ΔN) or the C-terminal (ΔC) putative extracellular portions. Double point mutations (indicated in red) were introduced at the shRNA recognition site of the full and the ΔN CMTM7s. (B) HeLa cells transiently transfected with the full, ΔN, or ΔC CMTM7s were fixed, permeabilized, stained with anti-T7 antibody, and analyzed by confocal microscopy. (C) Western blot analysis of BAL17 cells, kd1 cells, and the latter reconstituted with the CMTM7 forms shown in (A). Antibodies against BLNK, IgM H chain (μ H), β-actin, and T7 tag (for the exogenous CMTM7s) were used for detection. (D) The reconstituted kd1 cells were fractionated into membrane and cytosol, and the lysates of both fractions were subjected to Western blot analysis using antibodies against Lyn and the T7-tag. (E) The same cells as in (D) were stimulated with anti-IgM for 1 min, and the lysates of the membrane fractions of these cells were immunoprecipitated with anti-T7 antibody [IP:T7(CMTM7)]. The precipitates and the lysates, as well as a cytosol fraction of the ‘full’ cells (the most right lane), were analyzed as in (D) with the addition of an anti-BLNK antibody. (F, G, H) BAL17 cells and the reconstituted kd1 cells were stimulated with anti-IgM antibody for the indicated time periods and the cell lysates were immunoprecipitated with the indicated antibodies (IP). The precipitates (F, G) and the lysates (F, H) were analyzed by Western blotting for the presence of BLNK and Syk (F), tyrosine-phosphorylated (pBLNK) and total BLNK (G), or activated (pERK) and total ERK (H). (G, H) Numbers below the panels represent relative phosphorylation values of each protein (setting the value of the left-most sample in each panel as 1.0), normalized as relative to the corresponding total proteins as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031829#s4" target="_blank">Materials and Methods</a>.</p

A model for the CMTM7 function in the B cell receptor complex.

<p>CMTM7 is associated with sIgM where it mediates interaction of BLNK and Syk, as well as BLNK phosphorylation by Syk, which is necessary for eventual activation of ERK and JNK, in part through PLCγ2 activation. Arrows with solid and dotted lines indicate direct and indirect interactions, respectively.</p

CMTM7 is localized at the plasma membrane in association with clathrin and sIgM.

<p>(A) HeLa cells stably expressing T7-hCMTM7 were fixed, permeabilized, and stained with antibodies for the indicated organelle makers (left panels) and T7 tag (middle panels) as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031829#s4" target="_blank">Materials and Methods</a> section. Right panels: merged images. (B) HEK293T cells transiently expressing mouse CMTM7 tagged with HA or FLAG epitopes at the N- or C-termini, respectively, or not (schematically represented at the top), were stained with anti-HA antibody (left) or anti-FLAG antibody (right) and analyzed by flow cytometry. The histograms indicate fluorescence intensities for non-tagged (red lines), and HA- (blue lines) or FLAG- (green lines) tagged CMTM7s. The predicted plasma membrane topology of CMTM7 is depicted at the bottom. (C) BAL17 cells stably expressing T7-tagged mouse CMTM7 (BAL17/T7-CMTM7) were stained with rabbit anti-T7 and biotin-anti-IgM antibodies, and incubated at 37°C for the indicated time periods. Then the cells were fixed, permeabilized, and stained with TRITC-anti-rabbit IgG and FITC-streptavidin. The fluorescence FITC (top), TRITC (middle), and their merged images (bottom) are shown.</p