Ca2+ influx and protein scaffolding via TRPC3 sustain PKCβ and ERK activation in B cells

Ca2+ signaling mediated by phospholipase C that produces inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and diacylglycerol (DAG) controls lymphocyte activation. In contrast to store-operated Ca2+ entry activated by Ins(1,4,5)P3-induced Ca2+ release from endoplasmic reticulum, the importance of DAG-activated Ca2+ entry remains elusive. Here, we describe the physiological role of DAG-activated Ca2+ entry channels in B-cell receptor (BCR) signaling. In avian DT40 B cells, deficiency of transient receptor potential TRPC3 at the plasma membrane (PM) impaired DAG-activated cation currents and, upon BCR stimulation, the sustained translocation to the PM of protein kinase Cβ (PKCβ) that activated extracellular signal-regulated kinase (ERK). Notably, TRPC3 showed direct association with PKCβ that maintained localization of PKCβ at the PM. Thus, TRPC3 functions as both a Ca2+-permeable channel and a protein scaffold at the PM for downstream PKCβ activation in B cells.


Introduction
the physiological importance of these DAG-activated cation channels (DACCs), previous studies have demonstrated their function as nonselective cation channels inducing membrane depolarization, which in turn activates voltage-dependent channels to induce action potentials (Lucas et al., 2003) and/or depolarizationinduced Ca 2+ influx, which is responsible for Ca 2+ -dependent cellular responses such as muscle contraction (Inoue et al., 2001;Welsh et al., 2002) and activation of transcription factor NFAT (Thebault et al., 2006;Onohara et al., 2006). However, in contrast to the depolarizing function in excitable cells, the physiological significance of Ca 2+ entry occurring directly through DACCs and subsequent Ca 2+ signals is largely unknown.
DAG is recognized classically as the potent activator of protein kinase C (PKC), a family of serine/threonine kinases that play crucial roles in a plethora of biological functions, such as proliferation, differentiation, development and more specialized cellular functions (Nishizuka, 1995). The 'so-called' conventional PKCs (cPKCs) are activated by recruitment of the protein to membranes via the Ca 2+ -dependent binding of C2 domains to phospholipids, which is potentiated by the binding of C1 domains to DAG. Spatial and temporal targeting critical for the enzymatic activation of cPKC is mostly driven by the spatial and temporal properties of the Ca 2+ signaling machinery (Oancea and Meyer, 1998;Maasch et al., 2000;Pinton et al., 2002;Mogami et al., 2003;Reither et al., 2006). Specifically, local changes in intracellular Ca 2+ concentration ([Ca 2+ ] i ) control membrane translocation of cPKCs, and different modes of Ca 2+ influx and release target cPKCs to distinct areas in the cell (Maasch et al., 2000;Pinton et al., 2002). In B cells, PKC isoforms are the major Ca 2+ and DAG-regulated cPKCs (Mischak et al., 1991), and their important roles in BCR signaling and cell survival have been demonstrated using PKCknockout mice with impaired humoral immune responses and reduced cellular responses of B cells (Leitges et al., 1996). However, despite the physiological importance of PKC established in the context of B-cell biology, specific subtypes of Ca 2+ -permeable channels responsible for PKC translocation and activation have not been elucidated in B cells.
Previous studies have suggested that activation of PKC and the duration of activation of a mitogen-activated protein (MAP) kinase, extracellular signal-regulated kinase (ERK), play important roles in development of B cells (King and Monroe, 2000;Koncz et al., 2002). Immature B cells undergo apoptosis upon BCR stimulation to eliminate self-antigen reactive cells, whereas mature B cells proliferate and differentiate by BCR stimulation. It has been demonstrated that this differential functional response of immature and mature B cells is partly attributable to the activation of PKC and differences in the duration of ERK activation. In immature B cells, ligation of BCR is uncoupled from the activation of PKC (King and Monroe, 2000), and transient phosphorylation of ERK and activation of ERKdependent transcription factors are involved in triggering apoptosis. In mature B cells, sustained ERK activation induces survival and cell activation (Koncz et al., 2002). Furthermore, we previously demonstrated that Ca 2+ entry is coupled to translocation and secondary activation of PLC2, which amplifies Ins(1,4,5)P 3 production and Ca 2+ signaling upon BCR stimulation in the avian B cell line DT40 (Nishida et al., 2003). The same Ca 2+ entry mechanism also induced sustained MAP kinase signaling that was sensitive to blockade by PKC inhibitors. This, together with the above-stated importance of temporal patterns in controlling BCR responses, has prompted us to now investigate the Ca 2+ entry mechanism linked to DAG and PKC signaling at the 'sustained' phase.
Here, we describe an investigation of DACCs and their physiological significance in the context of BCR signaling. In DT40 cells, genetic disruption of translocation of TRPC3 proteins to the PM has revealed that native TRPC3 forms DACCs but not SOCs. Upon BCR activation, the DAG-activated Ca 2+ influx via TRPC3 amplifies Ca 2+ signals and downstream NFAT activation by controlling PM translocation of PLC2 and sustains PKC translocation and activation. Furthermore, direct physical interaction between TRPC3 and PKC also regulates the stable retention of PKC at the PM, leading to the sustained BCR-induced MAP kinase activation. These results suggest that TRPC3 has a dual function in BCR-induced signaling: it is a DACC, which elicits PM translocation of PLC2 and PKC, and a scaffolding platform at the PM for PKC.

Disruption of PM expression of endogenous TRPC3 channels in DT40 B cells
The expression of DAG-activated TRPC3 and TRPC7 channels was previously demonstrated in DT40 cells (Nishida et al., 2003). Furthermore, recombinant expression studies suggested that TRPC3 channels are in part responsible for Ca 2+ entry associated with translocation and sustained activation of PLC2 (Nishida et al., 2003). Therefore, to study the possible physiological significance of DAG-activated Ca 2+ -permeable TRPC3 channels in PLC2mediated BCR signaling, the TRPC3 gene locus was disrupted by deletion of the exon encoding amino acid residues (a.a.) 681-750, containing the well conserved TRP domain (Okada et al., 1999), through homologous recombination in DT40 B cells (Fig. 1A,B). RT-PCR revealed that TRPC3-mutant (MUT) DT40 cells expressed truncated TRPC3 transcripts in which the targeted exon was deleted (Fig. 1C), in accordance with immunoblotting detecting a slightly smaller band in MUT cells (Fig. 1D). Evaluation of channel function of mouse TRPC3 (mC3) with the corresponding deletion [mC3(667-736): a.a. 667-736 in mC3 corresponds to a.a. 681-750 in chicken TRPC3] revealed that it lacks Ca 2+ influx channel activity upon stimulation by ATP, carbachol (CCh), and the membrane permeable DAG analogue, 1-oleolyl-2-acetyl-sn-glycerol (OAG), when expressed in the HEK293 cell system (supplementary material Fig. S1A). Confocal images revealed an intracellular localization of a mC3(667-736)-EGFP fusion construct, in contrast to WT mC3-EGFP which was localized in the PM (supplementary material Fig. S1B). These results clearly indicate that deletion of the TRP domain ablates targeting of TRPC3 proteins to the PM. Consistent with these results, immunofluorescence staining using anti-TRPC3 antibody revealed intracellular localization of TRPC3 proteins in MUT cells but PM localization in WT cells, suggesting that the endogenous truncated mutant of TRPC3 in MUT cells has a defect in PM expression (supplementary material Fig. S1C). The level of cell surface expression of BCR examined by staining with FITCconjugated anti-chicken IgM antibody on MUT clones was indistinguishable from that of parental DT40 cells (supplementary material Fig. S1D).

TRPC3 constitutes a DACC but not SOC in DT40 B cells
DAG-induced ionic currents in WT and MUT DT40 cells were analyzed using the whole-cell patch-clamp technique ( Fig. 1E-G). In WT cells, bath application of 10 M OAG induced relatively sustained inward currents (1.74±0.45 pA/pF, n7) at a holding potential of -60 mV (Fig. 1G). The current-voltage (I-V) relationship showed a slight outward rectification similar to the recombinant TRPC3 current (Lintschinger et al., 2000) with reversal potential of 0 mV (Fig. 1F). The time between OAG challenge and current activation varied among WT cells: the time to maximum current amplitude after OAG application was from 78 to 301 seconds and the average time to maximum was 196±31 seconds. In contrast to WT cells, MUT cells showed significantly reduced OAG-induced currents (0.51±0.27 pA/pF, n8; Fig. 1E-G). BCR ligation induced rapid release of Ca 2+ from internal stores and depletion of Ca 2+ stores subsequently activating store-operated Ca 2+ entry (SOCE), a major Ca 2+ entry pathway in B cells (Mori et al., 2002). We compared Ca 2+ release-activated Ca 2+ currents (I CRAC ) that correspond to SOCE in WT and MUT cells. Intracellular dialysis with 10 M Ins(1,4,5)P 3 via the patch pipette elicited inward currents that showed the salient features of I CRAC : a positive reversal potential and inward rectification over the voltage range from -150 to 50 mV (Fig. 1H). Peak current densities and activation kinetics of I CRAC recorded in MUT cells were comparable to those in WT cells (Fig. 1I S2). These results clearly indicate that DACCs are ablated, but SOCs are unaffected by the expression defect of functional TRPC3 channels at the PM in DT40 B cells.

TRPC3 plays a critical role in BCR-induced Ca 2+ signaling in DT40 B cells
We next analyzed [Ca 2+ ] i mobilization in response to physiological stimuli via BCR in WT and MUT DT40 cells (Fig. 2). In the presence of 2 mM extracellular Ca 2+ , [Ca 2+ ] i increases, induced by BCR ligation, were significantly suppressed in MUT cells ( Fig. 2A,C). BCR-induced Ca 2+ mobilization in MUT cells was indistinguishable from that in WT cells in the absence of extracellular Ca 2+ (Fig. 2B,C), indicating that the Ca 2+ response defect in MUT cells is attributable to Ca 2+ influx defect. In support of this observation, the compromised Ca 2+ influx in MUT cells was restored in MUT cells stably transfected with mC3 cDNA ( Fig. 2A-C). The stable MUT transfectant exhibited immunolocalization of TRPC3 at the PM and intracellular TRPC3, which may represent exogenous mC3 and an endogenous TRPC3 truncation mutant, respectively (supplementary material Fig. S1C;   Fig. S3B). Thus, disruption of functional TRPC3 expression at the PM, which mediates DAG-activated currents, elicits a Ca 2+ influx defect in DT40 B cells.
We have previously shown that BCR stimulation induces the initial Ca 2+ responses followed by Ca 2+ -entry-dependent sustained and/or oscillatory responses (Nishida et al., 2003). In MUT cells, the amplitudes of Ca 2+ oscillations were reduced compared with those in WT cells ( Fig. 2D-F) when the same concentration of anti-IgM (1 g/ml) as Fig. 2A-C was employed for BCR stimulation. By contrast, the frequency of Ca 2+ oscillation was indistinguishable between WT and MUT cells. This suggests that Ca 2+ influx via DAG-activated TRPC3 channels is crucial for the modulation of Ca 2+ oscillations.
A previous study revealed that Ca 2+ -influx-dependent membrane translocation, secondary activation of PLC2 and secondary production of Ins(1,4,5)P 3 are required for the generation of Ca 2+ Journal of Cell Science 123 (6) oscillations (Nishida et al., 2003). TRPC3 has been considered as a candidate for the unidentified molecular entity of that Ca 2+ entry pathway, based on the observation that PLC2 was shown to be functionally and physically coupled to TRPC3 in a HEK293 heterologous expression system (Nishida et al., 2003). Thus, we next examined coupling of native TRPC3 channels with PLC2 translocation in DT40 cells by observing EYFP-tagged PLC2 (PLC2-EYFP) with time-lapse confocal laser microscopy. BCR ligation induced PM translocation of PLC2-EYFP in WT cells, whereas this was nearly abolished in MUT cells (Fig. 2G,H). Thus, the suppression of BCR-induced Ca 2+ oscillations in MUT cells may be attributable to the defect in BCR-induced PLC2 translocation.
For PM translocation of PLC2, Ca 2+ influx through TRPC3 is particularly important, because expression of red fluorescent protein (mStrawberry)-tagged mC3 (mC3-mStrawberry), but not that of mStrawberry-tagged mutant mC3 with a defect in the pore-forming region (mC3PD-mStrawberry), restored BCR-induced PLC2 translocation in MUT cells (supplementary material Fig. S4A). In addition, [Ca 2+ ] i elevation by ionomycin also failed to translocate PLC2 to the PM (supplementary material Fig. S4B). Interestingly, overexpression of an mStrawberry-tagged mC3 subfragment containing a.a. 23-73 [mStrawberry-mC3(23-73)], which interacts with PLC (van Rossum et al., 2005) and suppressed PLC2 translocation in WT cells, failed to recover the translocation defect of PLC2 in MUT cells (supplementary material Fig. S4C,D). This excludes the possibility that the PLC2 translocation defect in MUT cells endogenously expressing the truncated mutant TRPC3 is solely attributable to its dominant-negative effect anticipated from the coimmunoprecipitation of PLC2 with the counterpart mC3(667-736) mutant in HEK293 cells (supplementary material Fig. S4E), and supports the importance of functional TRPC3 localized at the PM.

TRPC3-mediated Ca 2+ influx is required for PM translocation of PKC
The secondary PLC2 activation should enhance production of DAG as well as Ins(1,4,5)P 3 , since equimolar DAG and Ins(1,4,5)P 3 are generated through phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ] hydrolysis by PLC. Therefore, we next examined the impact of TRPC3 deficiency on BCR-induced DAG production and its downstream PKC activation (Fig. 3). Among nine PKC subtypes, our study focused on cPKCs, particularly PKC isoforms, as potential target molecules of Ca 2+ influx via TRPC3 channels, because of the requirement of Ca 2+ and DAG for their activation as well as the fact that PKC isoforms are the major PKC isozymes expressed in B cells (Mischak et al., 1991). PM translocation of endogenous PKC was assessed using the membrane fractionation method, since PM translocation of PKC has been considered as the most critical step in its activation and has been frequently used to assess PKC activation (Liu et al., 1998). BCR stimulation caused sustained membrane translocation of PKC for at least 30 minutes in the presence of extracellular Ca 2+ in WT cells (Fig. 3A). As observed for WT cells in the absence of extracellular Ca 2+ , MUT cells exhibited transient PKC PM translocation in the presence of extracellular Ca 2+ . The translocation defect was rescued by the recombinant expression of mC3 (Fig. 3A). Levels of BCRdependent PKC activation were compared between WT and MUT cells by analyzing PM translocation of the EGFP fusion protein for PKCII (PKCII-EGFP), the most abundant PKC isoform in DT40 cells (T. N., unpublished data), at the single cell level using confocal microscopy. PKCII-EGFP was diffusively distributed throughout the cytosol in resting cells, and rapidly translocated, within 1 minute, from cytosol to the PM upon BCR stimulation. In WT cells, PKCII-EGFP translocation persisted in the presence of extracellular Ca 2+ but became transient in the absence of extracellular Ca 2+ (Fig. 3B,D). By contrast, in the presence of extracellular Ca 2+ , PKCII-EGFP translocation was transient in MUT cells. Although BCR-induced sustained translocation of PKCII-EGFP to the PM was recovered by the expression of mC3-mStrawberry, mC3PD-mStrawberry failed to restore the defect in sustained PKCII PM translocation in response to BCR stimulation in MUT cells (Fig. 3C,D). These studies suggest that TRPC3-mediated Ca 2+ entry is required for BCR-induced PKCII PM translocation.
Since translocation of PKC is affected by DAG via the C1 domain (Oancea and Meyer, 1998), we next analyzed the OAGinduced PM translocation of PKCII-EGFP. Application of OAG to WT cells caused a clear translocation of PKCII-EGFP to the PM in the presence of extracellular Ca 2+ , whereas in the absence of extracellular Ca 2+ , the OAG-induced translocation was nearly abolished (Fig. 3E,G). Interestingly, in the presence of extracellular Ca 2+ , OAG failed to translocate PKCII-EGFP to the PM in MUT cells. Although OAG-induced sustained translocation of PKCII-EGFP to the PM was recovered by the expression of mC3-mStrawberry, mC3PD-mStrawberry again failed to restore the defect in sustained PKCII PM translocation in response to OAG stimulation in MUT cells (Fig. 3F,G). These results suggest that OAG-activated Ca 2+ influx mediated by TRPC3 is required for the OAG-induced PM translocation of PKCII.
PKC carries the C2 domain that interacts with Ca 2+ and anionic phospholipids, and cPKC is known to translocate to the PM in response to a rapid, generalized increase in [Ca 2+ ] i induced by ionomycin (Maasch et al., 2000) through passive leakage from Ca 2+ stores and Ca 2+ influx (supplementary material Fig. S2A). In fact, ionomycin evoked the accumulation of PKCII-EGFP at the PM, which persisted for 10 minutes in WT cells. By contrast, PKCII-EGFP failed to show significant accumulation at the PM in MUT cells (see data in the presence of U73343, an inactive analogue for the PLC inhibitor U73122 in supplementary material Fig. S5A). These results suggest that TRPC3 mediates localization of PKC at the PM induced by a generalized [Ca 2+ ] i increase. Interestingly, after PKCII-EGFP transiently accumulated at the PM, it showed cytosolic localization at 10 minutes in WT cells pretreated with the PLC inhibitor U73122 (supplementary material Fig. S5A). Thus, activation of PLC2, production of DAG, and Ca 2+ influx via DAGactivated TRPC3 channels may be involved in PM translocation of PKCII-EGFP in response to a generalized [Ca 2+ ] i increase.

TRPC3 functions as a scaffold for BCR-induced sustained translocation of PKC to the PM in DT40 B cells
We next tested the possibility that PKC might colocalize with TRPC3 at the PM, given PM translocation of PKC upon BCR stimulation. PKC was co-immunoprecipitated with TRPC3 before BCR stimulation in the presence or absence of extracellular Ca 2+ in WT and MUT DT40 cells (Fig. 4A). The association between TRPC3 and PKC increased after 15 minutes BCR stimulation in an extracellular-Ca 2+ -dependent manner in WT cells. By contrast, no such increase of interaction was observed after 15 minutes BCR stimulation in MUT cells. Interestingly, the increased phase of the interaction between TRPC3 and PKC coincided with the sustained phase of PKC PM translocation in which TRPC3 plays a critical role (Fig. 3A,B,D). We next assessed in vitro binding of the mC3 constructs with PKC in DT40 cell extracts or with the PKC Journal of Cell Science 123 (6)  purified preparations in a glutathione S-transferase (GST) pull-down assay (Fig. 4B,C). The TRPC3 C-terminal residues Asn659-Glu836 and Asn659-Phe753 bound to PKC, whereas residues of Asn754-Glu836 only showed faint interaction. Furthermore, deletion of Asp667-Arg736 at the C-terminus, which corresponds to deletion of the TRPC3 mutant expressed in MUT cells, failed to affect the interaction of TRPC3 with PKC in vitro (Fig. 4B,C). These results suggest that Asn659-Glu666 and/or Leu737-Phe753 (supplementary material Fig. S5B) are essential for the interaction of TRPC3 with PKC.
To investigate the functional relevance of the direct interaction between TRPC3 and PKC in PKC translocation, the effects of the transiently expressed PKC-interacting mC3 fragment (Leu726-Phe753) fused to red fluorescent protein mCherry [mCherry-mC3(726-753)] were tested on BCR-induced translocation of PKCII-EGFP towards the PM in WT DT40 cells. mCherry alone and mCherry-mC3(726-753) were indistinguishable in their subcellular localization in WT cells (Fig. 4D). However, expression of mCherry-mC3(726-753) significantly suppressed the sustained phase of the BCR-induced PKC translocation compared with mCherry alone (Fig. 4D). Thus, TRPC3 residues Leu726-Phe753 are sufficient for disruption of PKC translocation to the PM, suggesting that TRPC3 is important in anchoring PKC to the PM through physical interaction as well as through eliciting Ca 2+ influx.

TRPC3 is required for sustained localization of PKC at the PM in HeLa cells
PM translocation and activation of PKCII have been previously analyzed using real-time imaging in HeLa cells (Violin et al., 2003).
To examine the precise spatiotemporal patterns of the functional coupling between TRPC3 and PKC, we analyzed histamineinduced PM translocation of PKCII-EGFP in HeLa cells using confocal microscopy. HeLa cells were transfected with small interfering RNAs (siRNAs) specific for human TRPC3 to knockdown endogenous TRPC3 levels (supplementary material Fig.  S6). Histamine at a concentration of 100 M evoked gradual concentration of the PKCII-EGFP fluorescence at the PM after rapid accumulation in the initial phase, and the PM concentration of PKCII-EGFP showed oscillations in control HeLa cells (Fig.  4E,F). The removal of extracellular Ca 2+ reduced both oscillatory translocation and persistent PM localization of PKCII-EGFP. In TRPC3-deficient cells, although oscillatory movement of PKCII-EGFP was still observed, the localization of PKCII-EGFP at the PM was gradually suppressed. The membrane fractionation experiment also showed that both the removal of extracellular Ca 2+ and the siRNAs against TRPC3 abolished PM accumulation of PKCII-EGFP after 15 minutes of histamine stimulation (Fig. 4G). These results suggest that TRPC3 channels stabilize PKC at the PM also in HeLa cells, raising the possibility that this mechanism is shared by different types of cells.

Sustained PM translocation of PKC is important for BCRinduced sustained activation of ERK in DT40 B cells
Previous studies have suggested that PKC is required for the BCRinduced activation of ERK, a MAP kinase (Sakata et al., 1999;Cao et al., 2001;Teixeira et al., 2003;Nishida et al., 2003;Aiba et al., 2004). We examined the roles played by TRPC3 channels in BCRinduced ERK activation via PKC in DT40 B cells (Fig. 5). In WT cells, BCR stimulation maintained the increase in ERK phosphorylation over 45 minutes in the presence of extracellular Ca 2+ . Strikingly, removal of extracellular Ca 2+ resulted in transient ERK activation in WT cells. MUT cells exhibited suppression of ERK activation prominently at the sustained phase and significantly but moderately at the initial phase in the presence of extracellular Ca 2+ . The sustained ERK activation was restored by the heterologous expression of mC3. Hence, in DT40 B cells, Ca 2+ influx via TRPC3 channels is required for full ERK activation, in which TRPC3 protein also plays an additional role as a platform for signal transduction at the PM.

Discussion
The results of the present study establish TRPC3 as a DACC that plays an important role in B-cell signaling. TRPC3 channels are responsible for Ca 2+ influx that induces PM translocation of PLC2, and amplification of Ca 2+ signaling and NFAT activation downstream. Importantly, TRPC3 channels also elicit sustained PM translocation and activation of PKC by mediating Ca 2+ influx and acting as platforms at the PM for PKC. Sustained PM translocation of PKC has significant impact on downstream ERK activation (Fig.  6).
In MUT DT40 cells expressing an endogenous PM-expressiondeficient TRPC3 mutant, suppression of BCR-induced Ca 2+ influx was attributable to the defect in DAG-activated Ca 2+ influx (Fig.  1). MUT cells also exhibited impairments in BCR-induced Ca 2+ oscillations and PLC2 accumulation at the PM (Fig. 2), supporting the key role played by DACC TRPC3 in translocation and subsequent secondary activation of PLC2 that regulates Ca 2+ oscillations in the sustained phase of Ca 2+ signaling in B cells (Nishida et al., 2003). Our previous report demonstrated that the accumulation of PLC2 at the PM is completely abolished by the removal of extracellular Ca 2+ or by treatment with the TRPC3selective inhibitor Pyr3 (Nishida et al., 2003;Kiyonaka et al., 2009). Furthermore, overexpression of the PLC-interacting mC3(23-73) subfragment significantly suppressed PLC2 PM translocation in WT cells, but it failed to restore the defect of PLC2 PM translocation in MUT cells (supplementary material Fig. S4C,D). These observations suggest that the accumulation of PLC2 at the PM specifically requires both TRPC3-mediated Ca 2+ influx and the direct interaction between PLC2 and PM-localized TRPC3. The specific requirement of TRPC3-mediated Ca 2+ influx for PM translocation of PLC2 is also suggested by the additional finding that a generalized [Ca 2+ ] i increase by ionomycin and expression of the mC3PD mutant failed to restore BCR-induced PM translocation of PLC2 in MUT cells (supplementary material Fig. S4A,B,D).

Journal of Cell Science 123 (6)
It has been known that BLNK, also known as SLP65, is the critical scaffolding protein for PLC2 in BCR signaling (Fu et al., 1998;Kurosaki et al., 2000). We have not yet studied the exact molecular composition of the signal complex containing TRPC3 and PLC2 in DT40 cells to determine whether BLNK is a constituent of this complex. However, considering the fact that both TRPC3 and BLNK positively regulate PLC2 activation, it is more likely that TRPC3 and BLNK cooperate in the same complex to amplify BCR signaling than that they compete and antagonize each other. However, an alternative possibility cannot be excluded that BLNK and TRPC3 share the same binding site on PLC2 and regulate BCR signaling with different time dependencies.
In different types of cells, sustained Ca 2+ increase and/or oscillations are required for the activation of NFAT (Gwack et al., 2007). It has been suggested that SOCs form the only major Ca 2+ influx pathway responsible for NFAT activation in lymphocytes (Feske, 2007). Our previous work in fact demonstrated that disruption of store-operated TRPC1 channels suppresses the frequency of BCR-induced Ca 2+ oscillations and NFAT activation in DT40 cells (Mori et al., 2002). However, NFAT activity is also reduced in MUT cells (Fig. 2I). Therefore, our study provides evidence for the first time, that Ca 2+ influx via DAG-activated TRPC3 plays a role in Ca 2+ oscillations and subsequent NFAT activation in lymphocytes.
With regard to the activation mechanism for cPKC, the coordination of Ca 2+ and DAG signals is known to determine the kinetics of translocation and activation (Oancea and Meyer, 1998). Recently, Singh et al. suggested that a DAG-activated TRPC6 channel signals the membrane translocation and activation of PKC, and thereby induces RhoA activation and endothelial contraction (Singh et al., 2007). However, the mechanism underlying PKC-PM interaction caused by TRPC6-mediated Ca 2+ entry was not clarified. Our study describes a precise process of cPKC recruitment to the membrane. TRPC3 is capable of providing the sustained Ca 2+ influx required for sustained PM localization of PKC upon BCR stimulation, because DAG is continuously produced by PLC2, and TRPC3 activity is in turn sustained. Importantly, to recruit PKC to the PM, OAG per se failed but required Ca 2+ influx via OAG-activated TRPC3 channels (Fig. 3E-G). Furthermore, sustained PM translocation of PKC evoked by ionomycin became transient following inhibition of DAG production, and was abolished by disruption of TRPC3 expression at the PM (supplementary material Fig. S5A), suggesting that the sustained PM translocation of PKC requires TRPC3-mediated Ca 2+ influx and persistent production of DAG. Since PKC-mediated phosphorylation has been reported to negatively regulate TRPC3 (Trebak et al., 2003;Venkatachalam et al., 2003;Trebak et al., 2005), the sustained translocation of PKC towards PM is the consequence of the TRPC3-mediated Ca 2+ influx but is unlikely a requisite of TRPC3 activation. Interestingly, in HeLa cells, histamine stimulation evoked oscillatory translocation on top of gradual accumulation of PKCII-EGFP at the PM, while knockdown of TRPC3 only suppressed the latter (Fig. 4E,F). This suggests that DAG-dependent persistent localization of PKCII requires TRPC3-mediated Ca 2+ influx, whereas the oscillatory translocation of PKCII is induced by repetitive Ca 2+ spikes in the cytosol. Thus, the persistent localization of cPKCs mediated by DAG-activated Ca 2+ influx could be a common mechanism shared by many types of cells.
Previous in vitro studies predicted that binding of two Ca 2+ ions to the C2 domain of PKC causes slow and low-affinity membrane interaction and an additional third Ca 2+ ion subsequently binds to the C2 domain and stabilizes the C2 domain-membrane complex, which allows PKC to search for the C1 domain ligand DAG on the membrane (Nalefski and Newton, 2001;Kohout et al., 2002). Notably, it has been proposed that the binding affinity of the third Ca 2+ ion is too low to promote occupancy except when Ca 2+ levels reach the millimolar range, or additional groups for Ca 2+ coordination are provided, as in the presence of phospholipids (Nalefski and Newton, 2001). Since the local Ca 2+ concentration ([Ca 2+ ]) within nanometers of the mouths of Ca 2+ channels is orders of magnitude larger than bulk cytosolic [Ca 2+ ] (Marsault et al., 1997), TRPC3 may sufficiently augment [Ca 2+ ] to provide the third Ca 2+ ion to the C2 domain, once PKC directly interacts with TRPC3 ( Fig. 4A-D). In addition, since TRPC3 also directly interacts with PLC2 and regulates PM translocation and activation of PLC2, DAG also should be concentrated near TRPC3 and PKC. Thus, TRPC3 may increase local concentrations of Ca 2+ and DAG in organizing the nanodomain that supports sustained PM localization of PKC and stabilization of a ternary complex of PKC, Ca 2+ and lipid. Interestingly, it has recently been demonstrated that TRPC3 interacts with a receptor for activated C-kinase-1, which is known to be a scaffolding protein for PKC via its N-terminus (Bandyopadhyay et al., 2008). In the Drosophila phototransduction system, TRP functions both as a Ca 2+ -permeable channel and as a Fig. 6. Proposed model of DAG-activated Ca 2+ influx mediated by TRPC3 in BCR signaling. BCR stimulation induces PLC2 activation via phosphorylation by Syk kinases, and activated PLC2 hydrolyses PtdInsP 2 to produce Ins(1,4,5)P 3 (IP 3 ) and DAG. Ins(1,4,5)P 3 leads to Ca 2+ release from the ER followed by Ca 2+ influx via SOCs, whereas DAG induces Ca 2+ influx via TRPC3. TRPC3-mediated Ca 2+ influx promotes PLC2 translocation from the cytosol to the PM. The targeted PLC2 elicits secondary production of Ins(1,4,5)P 3 and DAG to amplify Ca 2+ signaling via Ins(1,4,5)P 3 receptormediated Ca 2+ release and TRPC3-mediated Ca 2+ influx, respectively. TRPC3mediated Ca 2+ influx is also required for sustained PKC membrane translocation and activation, resulting in persistent activation of ERK. TRPC3 protein acts as a platform for PKC through direct protein-protein interaction. molecular anchor for signalplexes (Li and Montell, 2000). Notably, Leu737-Phe753, the binding region of TRPC3 to PKC, is not highly conserved in TRPC6 and TRPC7 (supplementary material Fig. S5B), suggesting a specific function of TRPC3 in anchoring PKC at the PM. Thus, TRPC3 stabilizes PKC PM localization directly, as a platform, and also indirectly as an amplifier of Ca 2+ and DAG signals for organizing specific signal complex to achieve specificity and efficiency in BCR-induced signaling.
We can propose a mechanism underlying enhancement of TRPC3 interaction with PKC after 15 minutes of BCR stimulation (Fig. 4A). A recent study suggested a control of surface expression of TRPC3 by the interaction between PLC1 and TRPC3 (van Rossum et al., 2005). Our data demonstrate that TRPC3-mediated Ca 2+ influx elicits translocation of PLC2 to the PM (Fig. 2G,H; supplementary material Fig. S4) and that the TRP domain is required for proper targeting of TRPC3 proteins to the PM (supplementary material Fig. S1B,C). Therefore, surface expression of TRPC3 is likely to be positively regulated by PLC2 membrane translocation in the sustained phase of BCR stimulation, which may consequently augment the interaction between TRPC3 and PKC at the PM.
Our study also addresses the mechanisms that link Ca 2+ influx with BCR-induced ERK activation. In MUT cells, PKC translocation and ERK activation was suppressed particularly at later time points after BCR stimulation ( Fig. 3A-D, Fig. 5). This is similar to the effect of the cPKC selective inhibitor Gö6976 on ERK activation (Cao et al., 2001), suggesting that PKC activity is responsible for the activation of ERK in the sustained phase. Interestingly, it has been reported that TRPC3 regulates brain-derived neurotrophic factordependent ERK activation and calcium/cAMP-response elementbinding protein phosphorylation in cerebellar granule neurons (Jia et al., 2007). Thus, the regulation of sustained ERK activation by TRPC3 can be shared by various cell types.
Previous studies have correlated differential functional responses of immature and mature B cells with the activation of PKC and differences in the duration of ERK activation (King and Monroe, 2000;Koncz et al., 2002). It is possible that the developmentally regulated differential expression of TRPC3 may account for the differences in signaling processes such as the activation of PKC and the duration of ERK activation between immature and mature B cells. Alternatively, the production of DAG may be insufficient to activate TRPC3 and PKC in immature B cells, since it has been reported that an increase of intracellular Ca 2+ and hydrolysis of PtdIns(4,5)P 2 are induced in response to BCR cross-linking in mature B cells but an increase of intracellular Ca 2+ levels is induced in the relative absence of PtdIns(4,5)P 2 hydrolysis in immature B cells (King and Monroe, 2000). These aspects can be addressed using TRPC3 knockout mice, in which B-cell function has not been demonstrated yet (Hartmann et al., 2008;Kim et al., 2009). Interestingly, our preliminary results demonstrated that application of the TRPC3-selective inhibitor Pyr3 (Kiyonaka et al., 2009) suppressed BCR-induced Ca 2+ responses, sustained PKC PM translocation, and ERK activation in murine primary splenic B cells (T.N., unpublished data). Hence, a combination of genetic and pharmacological approaches can reveal the importance of the in vivo DAG-activated TRPC3 channel and its associated mechanism in the context of the developmental maturation of B cells.

Cell cultures and cDNA expression
EGFP-fused chicken PKCII cDNA (Aiba et al., 2004) was first established in the pEGFP-N1 vector (Clontech), and then transferred into pA-puro expression vector (Takata et al., 1994). mC3 cDNA was subcloned into pA-puro vector. DT40 cells were transfected with these constructs by electroporation (550 V, 25 F) and selected in the presence of 0.5 g/ml puromycin. WT and PD mutant (in which Leu609, Phe610, Trp611 were changed to alanines) of mC3 (mouse TRPC3) were fused at the C-terminus with mStrawberry and then transferred into pMX (Mori et al., 2002). PKC-interacting (Leu726-Phe753) or PLC2-interacting (Ser23-Glu73) regions of mC3 were amplified using PCR and fused at the N-terminus with mCherry or mStrawberry, respectively, and cloned into pMX. Cell cultures and cDNA expression in DT40 cells using the vesicular stomatitis virus glycotyped pseudotyped retrovirus were performed as described previously (Mori et al., 2002). An mC3 mutant with deletion of amino acids 667-736 [mC3(667-736)] was constructed using PCR and cloned into pCI-neo (Promega) or pEGFP-N1 to be tagged with EGFP C-terminally. HeLa and HEK293 cells, grown in DMEM supplemented with 10% FBS, were transfected using Superfect (Qiagen) according to the manufacturer's instructions.

Generation of TRPC3-deficient DT40 cells
The chicken genomic TRPC3 DNA was obtained by PCR using pairs of primers chTRPC3-P1 and chTRPC3-P14, chTRPC3-P5 and chTRPC3-P10, respectively (supplementary material Table S1). The targeting vector was constructed by replacing the genomic sequence, contains the exon corresponding to the sequence distal to the H8 transmembrane region containing TRP domain (EWKFAR) of chicken TRPC3, with a histidinol (hisD) or neomycin (neo) resistance gene cassette as shown in Fig.  1A (Takata et al., 1994). The upstream 2.1-kb and downstream 4-kb genomic sequences of TRPC3 were used as a targeting vector. The targeting vector transfection and isolation of several clones were performed as described previously (Mori et al., 2002). Clones were further screened by Southern blot analysis of XbaI-digested genomic DNA hybridized with a 3Ј-flanking probe using Gene Images random prime labeling and detection system (GE Healthcare) according to the manufacturer's instructions. 2+ ] i was performed, as we previously described (Mori et al., 2002). Images of the fura-2 fluorescence of the cells were recorded in a physiological salt solution (PSS; in mM): 150 NaCl, 8 KCl, 2 CaCl 2 , 1 MgCl 2 , 5 Hepes, 5.6 glucose (pH 7.4 adjusted with NaOH), and analyzed with a video image analysis system (Aqua Cosmos; Hamamatsu Photonics). The Ca 2+ -free solution contained 0.5 mM EGTA but no added CaCl 2 .

Immunofluorescence staining
DT40 cells were fixed with 4% paraformaldehyde for 5 minutes, immobilized on slides using cytospin centrifugation, and permeabilized with 0.2% Triton X-100 for 5 minutes. After blocking with 5% BSA, cells were incubated with anti-TRPC3 antibody for 2 hours (Nishida et al., 2003). The primary antibodies were detected using anti-rabbit secondary antibodies labeled with Alexa Fluor 488 (Invitrogen).

Confocal microscopy and image analysis
Fluorescent protein-expressing DT40 or HeLa cells were plated onto poly-L-lysinecoated glass coverslips. Fluorescence images were acquired with a confocal laserscanning microscope (FV500; Olympus) using the 488-nm line of an argon laser for excitation and a 505-nm to 525-nm band-pass filter for emission (EYFP or EGFP), or the 543-nm line of a HeNe laser for excitation and a 560-nm long-pass filter for emission (mStrawberry or mCherry). The specimens were viewed at high magnification using plan oil objectives (60ϫ, 1.40 NA; Olympus). DT40 cells were stimulated with 1 g/ml anti-IgM or 0.3 M OAG. HeLa cells were stimulated with 100 M histamine. To obtain a measure of the membrane translocation of PLC2 and PKCII, regions of interest (ROI) were defined over the outer border (typical thickness of regions, 0.5 m; Fig. 2G). Fluorescence intensities over the expected localization of the PM (F PM ) were divided by the fluorescence intensities over the whole cell (F total ) and expressed as percentages of total fluorescence. For experiments shown in Fig. 4E, confocal images were recorded for 15 minutes at 10-second intervals. Non-overlapping ROI of identical size were placed on the PM and cytosol of each PKCII-EGFP-positive HeLa cell (Fig. 4E). The average fluorescence intensity for each ROI were measured during the recording, and the ratio (F PM /F cytosol ) was calculated. These values were then plotted as a function of time and normalized to the fluorescence intensities recorded at time 0. To quantify changes of fluorescence increase in the PM after 15 minutes of histamine stimulation in HeLa cells (shown in Fig. 4E), the fluorescence intensities of the PM were averaged and normalized according to the following equation: R(R-R 0 )/R 0 , where R is the ratio of fluorescence intensity of the PM to that of the whole cell at 15 minutes after histamine stimulation, and R 0 is the ratio at time 0.

Separation of membrane and cytosolic fractions
DT40 or HeLa cells were stimulated with 10 g/ml anti-IgM or 100 M histamine in serum-free PSS, respectively. Membrane fractionation was performed as described previously (Krotova et al., 2003). Samples were resolved by SDS-PAGE and subjected to immunoblotting with anti-PKC monoclonal antibody (BD Transduction Laboratories). The bands were scanned and the density of each band was determined using ImageJ software.

GST pull-down assay
cDNAs for mC3 fragments and the GST were subcloned into the pET23 vector (Novagen). Purification of GST fusion proteins and pull-down assays were performed as described previously (Kiyonaka et al., 2007). DT40 cells were lysed in NP-40 lysis buffer. Purified human PKCII was obtained from Sigma. The samples were subjected to immunoblotting using anti-PKC antibody.

Expression analysis
The PCR protocol used for the expression analysis was described previously (Inoue et al., 2001). The PCR primers used are listed in supplementary material Table S1.

Analysis of ERK activity
DT40 cells were stimulated with 10 g/ml anti-IgM in serum-free PSS and then lysed as described previously (Nishida et al., 2003).

Flow cytometric analysis
Cell surface expression of BCR on WT and MUT cells was analyzed with Epics Altra (Beckman Coulter) using a FITC-conjugated anti-chicken IgM antibody (Bethyl) as previously described (Aiba et al., 2004).

Statistical analysis
All data are expressed as means ± s.e.m. The data represent at least three independent experiments for each condition. Statistical significance was evaluated using the Student's t-test for comparisons between two mean values. Multiple comparisons between more than three groups were carried out using an ANOVA followed by Tukey-Kramer test.