Identification of CMTM7 as a Transmembrane Linker of BLNK and the B-Cell Receptor

BLNK is a pivotal adaptor protein in the signal transduction pathway from the IgM class B-cell receptor. BLNK is phosphorylated by Syk and binds various signaling intermediates, leading to cellular events including MAP-kinase activation, culminating in cellular activation. It remains unclear how BLNK is initially recruited to the surface IgM (sIgM) complex to which Syk is also recruited. Here we show that CMTM7, a tetra-spanning membrane protein of unknown function, co-localized with clathrin and sIgM at the plasma membrane. RNA-interference-mediated knockdown of CMTM7 expression in B cells resulted in an impairment of sIgM-ligation-induced tyrosine phosphorylation of BLNK, which was due to an impaired interaction of BLNK and Syk, and in a failure to activate JNK and ERK, but not upstream kinases such as Src-family kinases and Syk. CMTM7 was bound to BLNK in a membrane fraction, and their association was augmented after sIgM ligation. Exogenous CMTM7 or a mutant with an N-terminal deletion (ΔN), but not one with a C-terminal deletion (ΔC) that is defective in membrane localization, were able to restore BLNK-Syk binding, BLNK phosphorylation and ERK activation in the CMTM7-knockdown B cells. In addition, CMTM7 and the ΔN, but not the ΔC, were constitutively associated with sIgM, and this binding was required for BLNK recruitment to sIgM. From these data, we conclude that CMTM7 functions to link sIgM and BLNK in the plasma membrane, to recruit BLNK to the vicinity of Syk, and to initiate the BLNK-mediated signal transduction.


Introduction
Upon ligation with antigen, B-cell antigen receptors (BCR) cluster on the cell surface, rapidly transduce signals into the cytoplasm, and are eventually internalized with bound antigen, primarily through a clathrin-mediated endocytosis pathway [1,2]. The BCR on the membrane of naïve B cells is a complex composed of surface immunoglobulin M (sIgM) and the signal transducing subunits, Iga and Igb. Signal transduction is initiated with the phosphorylation by Src-family kinases such as Lyn of tyrosine residues in an immunoreceptor tyrosine-based activation motif (ITAM) contained within the cytoplasmic domains of both Iga and Igb [3]. Syk is then recruited to the ITAM phosphotyrosines, activated and subsequently phosphorylates the adaptor protein BLNK to which signaling factors such as Btk, phospholipase Cc2 (PLCc2), Vav and Grb2 are recruited through their SH2 or SH3 domains. Syk then phosphorylates and activates Btk and Vav, which activate PLCc2 and Rac, respectively. The activated PLCc2 hydrolyzes phosphatidylinositol 4,5-bisphosphate into inositol trisphosphate (IP 3 ) and diacyl glycerol (DAG). IP 3 triggers Ca 2+ mobilization, while DAG activates Ras through RasGRP [4,5]. Ras and Rac trigger signaling cascades eventually activating MAP-kinases such as ERK and JNK. Intracellular calcium and DAG also activate enzymes such as PKC, which initiates signaling cascades including those activating NF-kB. These biochemical events culminate in the activation of transcription factors that induce activation, proliferation and/or differentiation of B cells [6].
BLNK (also known as SLP65 or BASH) plays a crucial role in signal transduction from the BCR, especially of the IgM class, and the pre-BCR. In BLNK-deficient mice, B-cell development is markedly affected at both pre-B-cell and immature B-cell stages. The spleen contains fewer mature B cells than normal, and the B cells present respond poorly to BCR ligation-induced proliferation in vitro and the mice also have a defective antibody response to Tindependent type-2 antigens in vivo [7][8][9][10][11]. BLNK-deficient B cells also have defects in BCR-triggered Ca 2+ flux and activation of PLCc2, ERK, JNK, p38, and NF-kB [7,8,12,13]. Thus, BLNK functions as a multivalent adaptor molecule that gathers signaling intermediates to form a 'signalosome' beneath the sIgM complex.
In order to exert its adaptor function, BLNK must first be recruited to the BCR to be phosphorylated by Syk, which is bound there to Iga/Igb. However, the mechanism for BLNK recruitment to the BCR remains unclear. It has been shown that, upon phosphorylation, a non-ITAM tyrosine (Y 204 in mice) of Iga binds the C-terminal src-homology 2 (SH2) domain of BLNK, and that this contributes to BLNK phosphorylation upon BCR-or Igacrosslinking to various extents depending on the experimental setting [14][15][16][17][18]. It has also been proposed that BLNK directly binds to Syk [19] through the BLNK SH2 domain and the Cterminal region of Syk [20,21]. However, deletion of the SH2 domain resulted only in a modest reduction of tyrosine phosphorylation of BLNK upon BCR ligation [20], implicating an SH2 domain-independent mechanism for the interaction of BLNK with Syk. An N-terminal basic region of BLNK, and most likely a leucine zipper motif in this region, was shown to be necessary and sufficient for constitutive association of BLNK with the plasma membrane [22], although a BLNK variant lacking the leucine zipper motif could still be phosphorylated by Syk in a Tcell reconstitution system [20].
We have previously identified CMTM3 (formerly termed BNAS2) as a binding partner of BLNK [23]. CMTM3 belongs to a nine-member protein family (CKLF and CMTM1,8) of unknown function, which have homology to a tetra-spanning transmembrane domain called MARVEL (MAL and related protein for vesicle trafficking and membrane linking) [24]. Some of the MARVEL domain-containing proteins are involved in cholesterol-rich membrane apposition events, such as biogenesis of vesicular transport carriers or tight junction regulation, but the functions of others remain unknown [25]. Genes encoding this family, except for CMTM5, cluster at two loci on different chromosomes; CKLF and CMTM1,4 on chromosome 8 in mice and 16 in humans, and CMTM6,8 on chromosome 9 in mice and 3 in humans. The expression profile of each member shows a distinctive pattern, with CMTM3 and 7 being expressed selectively in hematopoietic cells. In the course of our study of CMTM3 function, we noted that CMTM7 is also bound with BLNK. Thus far, no reports have been published about CMTM7 except for one describing its genomic configuration [24]. Here we report that CMTM7 is crucial for BCR-induced BLNK interaction with and phosphorylation by Syk, and for activation of downstream signaling pathways to ERK and JNK. We propose that CMTM7 functions to connect sIgM and BLNK, thus facilitating formation of the BLNK-nucleated sIgM signalosome.

CMTM7 is localized at the plasma membrane in association with clathrin and surface IgM
To clarify the localization of CMTM7 in mammalian cells, we transfected HeLa cells with a T7 epitope-tagged human CMTM7 expression construct and stained the cells with anti-T7 antibody together with antibodies against various organelle markers. Confocal microscopy revealed that CMTM7 is co-localized with clathrin accumulating near the plasma membrane, presumably representing clathrin-coated pits and vesicles, in a perinuclear region partly overlapping with the Golgi apparatus, and also with some early endosomes, whereas there was no apparent colocalization with the endoplasmic reticulum (ER) marker calnexin ( Figure 1A). To clarify the membrane topology of CMTM7, HEK293T cells were transfected with expression constructs encoding mouse CMTM7 tagged with HA or FLAG epitopes at N-or C-termini, respectively. Both epitopes could be detected with specific antibodies by flow cytometry on non-permeabilized cells ( Figure 1B), indicating that both the N-and C-terminal moieties of CMTM7 are exposed outside of cells.
To confirm the plasma membrane localization of CMTM7 in B cells and examine possible changes in localization dynamics after ligation of surface IgM (sIgM), we made a mouse B-cell lymphoma cell line, BAL17, stably transfected with an expression construct encoding T7-tagged mouse CMTM7 (BAL17/T7-CMTM7). The intact BAL17/T7-CMTM7 cells were stained with anti-T7 and anti-IgM antibodies and cultured at 37uC to allow the ligation-mediated reorganization of sIgM and its subsequent signaling. Before the culture, CMTM7 was clearly detected at plasma membrane, co-localizing almost exactly with sIgM. Some of the CMTM7 were internalized into cytoplasm by 5 minutes after the initiation of the culture, but others remaining on the plasma membrane were concomitantly segregated with sIgM into clusters. By 15 minutes, sIgM was internalized with the surface CMTM7 and mostly co-localized with CMTM7 at vesicular structures in the cytoplasm ( Figure 1C). The observed tight association of sIgM and CMTM7 during the active sIgM movement strongly suggests a physical interaction between the two molecules.

CMTM7 is required for BLNK phosphorylation by Syk and for signal transduction upon sIgM ligation
To investigate the function of CMTM7 in B cells, we generated BAL17 transductants in which CMTM7 gene expression was suppressed by shRNAs targeting two independent sequences (kd1 and kd2), as well as cells transduced with a control shRNA vector (mock) (Figure 2A). We found that BCR-ligation-induced tyrosine-phosphorylation of several proteins was attenuated in the CMTM7-knockdown cells as compared with the mocktransduced cells ( Figure 2B and Figure S1A). Activationassociated phosphorylation of ERK and JNK was greatly attenuated and that of PLCc2 was moderately, whereas that of Src-family kinases (SFK) and Syk were unaffected by CMTM7 knockdown ( Figure 2C and Figure S1B). In addition, BCRinduced tyrosine-phosphorylation of BLNK was greatly attenuated in the CMTM7-knockdown cells ( Figure 2D and Figure S1C), which was attributable to impaired association of BLNK with Syk ( Figure 2E and Figure S1D). Moreover, reciprocal immunoprecipitation experiments demonstrated association of CMTM7 and BLNK, which was augmented after sIgM-ligation ( Figure 2F and 2G). These data strongly suggest that, upon sIgM-ligation, CMTM7 functions to recruit BLNK to the plasma membrane in the vicinity of BCR-bound Syk and thus facilitate BLNK phosphorylation by Syk.

The C-terminal region of CMTM7 is necessary for its membrane localization and for BLNK phosphorylation
To further investigate the mode of CMTM7 interaction, we made expression vectors that encode T7-tagged full length (full) CMTM7, and mutants lacking the N-terminal (DN) or the Cterminal (DC) putative extracellular regions. A silent mutation at the shRNA recognition site was introduced into the full and DN constructs to prevent their recognition and silencing by the shRNA ( Figure 3A). When transiently expressed in HeLa cells, the full and DN versions of CMTM7 were localized to the plasma membrane and perinuclear regions as described above, whereas the DC version was diffusely localized in the cytosol and the nucleus ( Figure 3B). We transfected these constructs into CMTM7-knockdown BAL17 (kd1) cells and made stable clones expressing the full, DN, or DC versions of CMTM7 proteins. These clones as well as the original BAL17 and parental kd1 cells expressed equivalent levels of BLNK and IgM H chain proteins ( Figure 3C). A subcellular fractionation experiment confirmed that most of the full and DN CMTM7 proteins were present in the membrane fraction, whereas the DC was exclusively in a cytosolic fraction ( Figure 3D). In the membrane fraction, the full and the DN CMTM7 were bound with BLNK but not Lyn ( Figure 3E). BCR-induced BLNK association with Syk was restored in the kd1 cells reconstituted with the full or the DN, but not the DC, CMTM7 ( Figure 3F), indicating that the C-terminal domain of CMTM7, or membrane localization of CMTM7, is critical for the binding of both proteins. Accordingly, BCR-induced tyrosinephosphorylation of BLNK and ERK activation were fully restored in the kd1 cells reconstituted with the full or the DN, but not the DC, CMTM7 ( Figure 3G and 3H).

CMTM7 is associated with sIgM and recruits BLNK
It is well known that, upon ligation of IgM, Syk is recruited to the Iga/Igb subunits of the IgM BCR complex via binding to phosphorylated ITAMs in their cytoplasmic tails [3]. Therefore, the observed CMTM7-dependent interaction of BLNK with Syk led us to hypothesize that CMTM7 is associated with the IgM complex and thus mediates interaction of BLNK and Syk. Indeed, mH chain was co-precipitated with CMTM7 from BAL17/T7-CMTM7 cell lysates ( Figure 4A), and CMTM7 was present in the surface IgM complex in the same cells irrespective of sIgM ligation ( Figure 4B). Although both full and DN CMTM7 was coprecipitated with sIgM in the reconstituted kd1 cells, DC CMTM7 was not ( Figure 4C). Furthermore, BLNK was co-precipitated with the sIgM in the BAL17/T7-CMTM7 cells ( Figure 4B) and the kd1 cells reconstituted with the full or DN, but not DC, CMTM7 ( Figure 4C). Therefore, BLNK recruitment to the sIgM complex is dependent on CMTM7 integrated in the plasma membrane.

Discussion
BLNK has been characterized as a multivalent adaptor protein that plays a pivotal role in signal transduction from BCR of the IgM class. It has been demonstrated that Syk is the dominant tyrosine kinase that phosphorylates BLNK and that this phosphorylation is mandatory for the BLNK adaptor function [26]. However, less is known how BLNK is initially recruited to the sIgM complex, to which Syk is already recruited and thereby activated, after ligation of the BCR. Here we have identified a previously unknown transmembrane protein CMTM7 as the missing link between BLNK and the BCR. We demonstrated that CMTM7 binds both BLNK and sIgM, and is necessary for Syk interaction with and phosphorylation of BLNK, and ultimately for activation of downstream MAP-kinases after sIgM ligation ( Figure 5). Confocal microscopy data indicated a tight association of the two proteins in the plasma membrane as well as in the membrane of vesicles trafficking through the cytoplasm. Although the diffuse cytoplasmic localization of BLNK hampered confocal microscopic visualization of its plasma membrane recruitment, biochemical analyses clearly demonstrated that BLNK is bound with CMTM7 in the membrane fraction, and also that BLNK is associated with the sIgM complex only when CMTM7 is normally integrated in the plasma membrane. These data strongly suggest that CMTM7 is a part of sIgM complex that binds BLNK for its phosphorylation by Syk and to nucleate the 'signalosome'.
BLNK has been shown to directly bind to Iga [14][15][16] or to Syk [20,21] via its SH2 domain. While these interactions appear to be biologically significant, they do not appear to be essential for BLNK phosphorylation since BLNK lacking the SH2 domain could still be phosphorylated in B cells upon BCR ligation [20]. Likewise, the N-terminal leucine zipper motif of BLNK, a requisite for its plasma-membrane association [22], is not essential for BLNK phosphorylation [20]. Nevertheless, given their considerable contributions to BLNK phosphorylation, these interactions are likely involved in the process whereby BLNK is recruited to the sIgM complex and interacts with Syk, in addition to the CMTM7-BLNK interaction demonstrated in the present study. Whether these interactions represent different steps of the process leading to full BLNK phosphorylation, or are redundant mechanisms for this process, remains to be examined. Our data demonstrated that BLNK is co-precipitated with CMTM7 before sIgM ligation but that the amount of co-precipitate increased after the ligation. It appears that crosslinking of sIgM causes multi-merization of the associated CMTM7, which may stabilize the binding of BLNK. BLNK SH2 domain-mediated binding to Iga and/or Syk may further stabilize the association of BLNK with the sIgM complex.  Close colocalization of CMTM7 and sIgM in B cells before and after sIgM ligation suggests a direct interaction of the two proteins, although we have not yet identified the region(s) in these proteins that are involved in this interaction. It was previously reported that the membrane-proximal C m 4 domain of the m H chain constant region is critical for the assembly of immobile sIgM oligomers that are competent for signaling when bound by monovalent, membrane-tethered antigens [27]. Although the mechanism for the C m 4 domain-mediated sIgM oligomerization remains unknown, it may be mediated by integral membrane proteins that bind to this domain. It is tempting to speculate that CMTM7 binds to the C m 4 domain of sIgM through its extracellular regions and thereby mediates oligomerization of the sIgM. We demonstrated that the CMTM7 DC mutant lacking the C-terminal extracellular region failed to associate with sIgM ( Figure 4C), but the interpretation of this data is complicated by the fact that the DC variant is also not integrated into the plasma membrane. This might indicate that the C-terminal region of CMTM7 is necessary for the association with sIgM and this association is required for initial membrane integration of nascent CMTM7 at ER. This explanation would be unlikely, however, without a supposition that the C-terminal region can also be associated with other membrane receptor(s) expressed in HeLa cells (see Figure 3B). Alternatively, the C-terminal region may be intrinsically required for the membrane integration of CMTM7, although there has been no evidence that a C-terminal region neighboring the last transmembrane domain is necessary for the membrane integration of multi-spanning proteins. If the latter is the case, the role of the C-terminal region of CMTM7 in sIgM association remains obscure. Thus, further study is necessary to determine the molecular basis for the interaction of CMTM7 and sIgM.
Our confocal microscopy analysis indicated that CMTM7 is also associated with clathrin ( Figure 1A) and co-internalized with sIgM after its ligation, although the majority of the sIgM-CMTM7 complex appears to remain on the cell surface at least for ten minutes after sIgM ligation before it is internalized ( Figure 1C). Therefore, CMTM7 may also play some role in the process of clathrin-mediated sIgM internalization, but well after the early phase of BCR-mediated signal transduction. Concerning its role in ligation-induced sIgM internalization, we observed that this process was delayed in BAL17 cells overexpressing CMTM7 and, conversely, was accelerated in the CMTM7-knockdown BAL17 cells. Normal kinetics could by restored by reconstitution of the knockdown cells with full or DN, but not DC, forms of CMTM7 (our unpublished data). Thus, it appears that CMTM7, perhaps with associated BLNK, negatively regulates sIgM internalization to retain the BLNK-nucleated signalosome at the plasma membrane for the requisite time period required for optimal signal transduction. After that point, sIgM is presumably dissociated from Iga/b, which remains at the plasma membrane [28,29], CMTM7 dissociates from BLNK, which is now bound to Iga, and then the sIgM, accompanied by CMTM7, may be endocytosed via clathrin-coated pits and vesicles and delivered to early endosomes.
We previously identified CMTM3, another member of the CMTM protein family, as a binding partner of BLNK, and showed that it binds to an N-terminal part of BLNK [23]. We observed that the expression level of CMTM3 mRNA is relatively low compared to that of CMTM7 in BAL17 cells, while it is relatively high in the DC2.4 dendritic cell line (our unpublished data). Indeed, according to the expression profiles in the RefDIC public database (http://refdic.rcai.riken.jp/welcome.cgi), CMTM7 is highly expressed in almost all hematopoietic lineage cells, but high expression of CMTM3 is restricted to dendritic cells and macrophages. Therefore, CMTM7 appears to play a more dominant role in B cells than CMTM3. On the other hand, CMTM3 may be more important in dendritic cells and macrophages, in which BLNK as well as an analogous adaptor protein SLP76 are expressed. In this regard, we found that CMTM3, but not CMTM7, binds also to SLP76 in DC2.4 cells and Raw263.7 macrophage cells (our unpublished data). We have observed distinct functions of BLNK and SLP76 in endocytosis and signaling through a cell-surface receptor in dendritic cells (manuscript in preparation), a finding that might be related to the different binding preferences of these adaptor molecules for CMTM3 and CMTM7.

Real time RT-PCR
cDNA was synthesized with ReverTra Ace (Toyobo). Real-time PCR was performed with an Applied Biosystems 7500Fast. To amplify CMTM7 cDNA, the following primers were used: 59agatggtcaccctgctgatt39 and 59caggtgagcacacggtagaa39.

Plasmid constructions
The human CMTM7 (hCMTM7), mouse CMTM7 (mCMTM7) and its variants (DN; 38-168 aa, DC; 1-152 aa) were cloned into the pCAT7 vector [23]. The mCMTM7 and those appended with an N-terminal HA-tag or a C-terminal 36FLAG-tag via a glycine/serine linker (GGGGS) were also cloned into a pcDNA3.1 vector. To generate CMTM7-knockdown cells (kd1 and kd2), short hairpin (shRNA)-expressing retroviral vectors were constructed using the pSIREN-RetroQ vector (Clontech) and the following RNA interference sequences: for mCMTM7, 59acccagtcttcagatgcatct39 (shRNA-1 for kd1) and 59gccttcatctgtgtacgaagc39 (shRNA-2 for kd2); and for an irrelevant sequence (luciferase) used for mock-transduction, 59gtgcgttgctagtac-caac39. To reconstitute the kd1 cells, the mCMTM7 sequence was mutated at the shRNA-1 targeting site by PCR using two complementary primers, 59cctgtataacccagtcgtcggatgcatctgcctg39 and 59caggcagatgcatccgacgactgggttatacagg39 (mutated nucleotides are indicated by bold letters), pCAT7-mCMTM7 and pCAT7-mCMTM7 DN as templates, and KOD polymerase (Toyobo). PCR products were treated with Dpn1 to digest the template DNA and used for bacterial transformation. The CMTM7 sequences of the resultant clones were verified by nucleotide sequencing.

Immunoprecipitation and Western blot analysis
Immunoprecipitations were performed as described [19]. The immunoprecipitated proteins and/or cell lysates were resolved by SDS-PAGE, transferred onto polyvinylidene fluoride membranes, probed with the indicated horseradish peroxidase (HRP)-labeled or unlabeled antibodies, and with the secondary HRP antibodies for the latter, and the signals were visualized with Western Lightning Chemiluminescence Reagent Plus (PerkinElmer). The blot was stripped off and reprobed with other antibodies as indicated. The chemiluminescence of the protein bands was detected and imaged by LAS-3000 (Fujifilm), the intensities of which were quantified using the Science Lab 2001 Image Gauge software (Fujifilm). The values of the intensities of phosphorylated proteins were normalized as relative to those of the corresponding total proteins, and expressed as relative values in each data.

Immunofluorescence microscopy
HeLa cell transfectants cultured on glass-bottomed dishes were fixed with 4% paraformaldehyde, permeabilized with 0.1% TrironX-100, then stained with rabbit anti-T7 and the other indicated antibodies, and then with TRITC-anti-rabbit IgG antibody and appropriate FITC-labeled secondary antibodies. BAL17 cell transfectants were stained with biotin-goat F(ab9) 2 antimouse IgM and rabbit anti-T7 antibodies on ice, incubated at 37uC for the indicated time periods, or kept on ice (for 0 min), then fixed with 4% paraformaldehyde and permeabilized with 0.1% Tween-100, and stained with anti-FccRII/III blocking Ab (2.4G2). The cells were then incubated with blocking buffer (5% skim milk in PBS), stained with TRITC-anti-rabbit IgG antibody and FITC-streptavidin, extensively washed, and finally placed on slide glasses and coverslipped. The samples were analyzed with a Leica TCS SP2 confocal laser-scanning microscope (CLSM) using a 636 objective.

Subcellular fractionation
For preparation of subcellular fractions, cells were suspended in hypotonic solution (20 mM Tris, 5 mM EDTA, 5 mM EGTA) for 10 min, passed through a 26-gauge needle for 15 strokes on ice, and centrifuged at 10,0006 g for 5 min at 4uC. The supernatant fraction was further centrifuged at 110,0006 g for 15 min at 4uC. The pellet and supernatant were used as membrane and cytoplasmic fractions, respectively. The membrane fraction was solubilized with 1% TNE buffer containing 1% NP-40 [19].
All the data shown are representative of three or more independent experiments

Supporting Information
Figure S1 CMTM7 is required for BLNK association with Syk, its phosphorylation and signal transduction from BCR. (A, B, C, D) The CMTM7-knockdown (kd2) or mock-transduced BAL17 cells were stimulated with anti-IgM antibody for the indicated time periods and then analyzed as in Figure 2 (B, C, D, E). (TIF)