Sustained activation of extracellular-signal-regulated kinase (ERK) has an important role in the decision regarding the cell fate of B-lymphocytes. Recently, we demonstrated that the diacylglycerol-activated non-selective cation channel canonical transient receptor potential 3 (TRPC3) is required for the sustained ERK activation induced by the B-cell receptor. However, the signalling mechanism underlying TRPC3-mediated ERK activation remains elusive. In the present study, we have shown that TRPC3 mediates Ca2+ influx to sustain activation of protein kinase D (PKD) in a protein kinase C-dependent manner in DT40 B-lymphocytes. The later phase of ERK activation depends on the small G-protein Rap1, known as a downstream target of PKD, whereas the earlier phase of ERK activation depends on the Ras protein. It is of interest that sustained ERK phosphorylation is required for the full induction of the immediate early gene Egr-1 (early growth response 1). These results suggest that TRPC3 reorganizes the BCR signalling complex by switching the subtype of small G-proteins to sustain ERK activation in B-lymphocytes.

INTRODUCTION

B-cell receptor (BCR) signalling is important for the development, maturation and activation of B-lymphocytes [1]. Antigen binding to the BCR activates a cascade of tyrosine kinases, leading to activation of multiple signalling pathways including phospholipase C (PLC)γ2-mediated Ca2+ signalling, Ras/extracellular-signal-regulated kinase (ERK) signalling and phosphoinositide 3-kinase (PI3K)–protein kinase B (Akt) signalling pathways [1].

ERK1/2 is one of the mitogen-activated protein kinases (MAPKs) and is important for cell survival, proliferation, differentiation and even apoptosis in a cell-context-dependent manner. In B-lymphocytes, pharmacological inhibition of ERK leads to the suppression of proliferation of pre-B-cells and mature splenic B-cells [2,3]. It was recently revealed that ERK is critical for pre-BCR-induced differentiation of pro-B-cells into pre-BII-cells and for the subsequent proliferation of pre-BII-cells [4]. However, it is unlikely that all ERK-mediated cellular responses are regulated by the simple on/off switching of ERK activation. In particular, the duration of active ERK signalling has been associated with specific biological outcomes [5,6]. In the PC12 phaeochromocytoma cell line, epidermal growth factor (EGF) and nerve growth factor (NGF) elicit transient and sustained ERK activation which results in proliferation and differentiation, respectively [7]. In B-lymphocytes, it has also been speculated that the duration of ERK activation determines BCR-induced apoptosis or proliferation in immature or mature B-cells, respectively [8]. Although the regulatory mechanism linking sustained ERK activation to functional outcome has been extensively studied [9,10], the molecular mechanism underlying sustained ERK activation remains largely elusive. Several signalling mechanisms have been identified as key regulators of sustained ERK activation such as intrinsic receptor property, activation mode of Ras, use of different Raf kinases, cAMP-activated kinase signalling and Ca2+ signalling [7,11,12].

It has been reported that BCR-induced ERK activation requires PLC-mediated Ca2+ signalling which originates from hydrolysis of phosphatidylinositol 4,5-bisphosphate into inositol 1,4,5-trisphosphate and diacylglycerol (DAG) [13]. Recently, we have demonstrated that DAG-activated TRPC3 also plays an important role for extracellular Ca2+-dependent sustained ERK phosphorylation on BCR activation in chicken DT40 B-lymphocytes [14]. BCR-induced sustained ERK activation is reflected by the sustained translocation of protein kinase C (PKC)β to the plasma membrane, for which canonical transient receptor potential 3 (TRPC3)-mediated Ca2+ entry is critical. However, the underlying signalling mechanism connecting sustained PKCβ membrane translocation to sustained ERK phosphorylation remains elusive. In the present study, we analysed the signalling mechanism leading to Ca2+ influx-dependent sustained ERK activation, and revealed that BCR-induced ERK activation can be divided into two phases: an initial Ras/Raf-1-dependent phase and a late and sustained Rap1/B-Raf-dependent phase. The downstream target of TRPC3-mediated PKCβ is phosphorylation and activation of protein kinase D (PKD) which presumably activates Rap1. Furthermore, sustained ERK phosphorylation is required for full induction of the immediate early gene Egr-1 (early growth response 1) in DT40 B-lymphocytes.

EXPERIMENTAL

Cell cultures and cDNA expression

DT40 wild-type (WT) and TRPC3 mutant (MUT) cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 1% chicken serum, 2 mM L-glutamine, 50 μM 2-mercaptoethanol, 30 units/ml penicillin and 30 μg/ml streptomycin. Enhanced green fluorescent protein (EGFP)-fused Ras guanine-nucleotide-releasing protein 3 (Ras GRP3) and EGFP-fused PKD1 WT or pleckstrin homology (PH)-domain-deleted mutant cDNAs were expressed using the vesicular stomatitis virus, a glycotyped pseudotyped retrovirus, as described previously [15]. DT40 cells were transfected with a mouse TRPC3 or Rap1 N17 construct by electroporation (550 V, 25 μF) and selected in the presence of 0.5 μg/ml of puromycin. A yellow fluorescent protein (YFP)-fused C3G construct was transfected into both DT40 cells using a neon transfection system (Life Technologies).

Analysis of ERK, MEK and PKD activity

Cells were stimulated with 10 μg/ml anti-immunoglobulin M (IgM) in serum-free PBS solution, and lysed as described previously [16]. The samples were resolved by SDS/10% PAGE and subjected to immunoblot analysis with anti-phospho-specific p44/p42 (ERK), anti-phospho-specific MAPK/ERK kinase 1/2 (MEK1/2) or anti-phospho-specific PKD antibodies (Cell Signaling Technology). The blot was stripped and re-probed with anti-ERK2 (Santa Cruz Biotechnology), anti-MEK1/2 (Cell Signaling Technology) or anti-PKCβ antibodies (BD Transduction Laboratories) to show loading equivalence. In Figure 1, PKCβ was used as the loading control because of the band shift and decrease in sensitivity of PKD1 through multiple phosphorylation on BCR stimulation. The blots were visualized using the ECL system (GE Healthcare). The bands were scanned and the density of each band was determined using ImageJ software (NIH).

Defect of TRPC3-mediated, Ca2+ influx-suppressed, BCR-induced PKD phosphorylation

Figure 1
Defect of TRPC3-mediated, Ca2+ influx-suppressed, BCR-induced PKD phosphorylation

BCR-induced PKD phosphorylation in WT and MUT DT40 cells. Cells were stimulated with 10 μg/ml anti-IgM for the indicated intervals, and cell lysates were analysed by Western blotting with anti-phospho-specific PKD antibody. The blot was re-probed with anti-PKD1 antibody and anti-PKCβ antibody, which indicated equal loading in each lane. Cells were treated with 10 μM Gö6976 10 min before BCR stimulation and kept treated during the experiments. The graph depicts the time course of PKD phosphorylation. **P<0.01 compared with WT cells stimulated in the presence of extracellular Ca2+ (Tukey–Kramer test).

Figure 1
Defect of TRPC3-mediated, Ca2+ influx-suppressed, BCR-induced PKD phosphorylation

BCR-induced PKD phosphorylation in WT and MUT DT40 cells. Cells were stimulated with 10 μg/ml anti-IgM for the indicated intervals, and cell lysates were analysed by Western blotting with anti-phospho-specific PKD antibody. The blot was re-probed with anti-PKD1 antibody and anti-PKCβ antibody, which indicated equal loading in each lane. Cells were treated with 10 μM Gö6976 10 min before BCR stimulation and kept treated during the experiments. The graph depicts the time course of PKD phosphorylation. **P<0.01 compared with WT cells stimulated in the presence of extracellular Ca2+ (Tukey–Kramer test).

Pull-down assays for the detection of activated Ras and Rap1

Pull-down assays were performed as described previously [17]. In brief, bacterially expressed GST-fusion proteins, prebound to glutathione–Sepharose beads, were prepared. GST–Raf-1-Ras-binding domain (RBD) and GST–Ral guanine nucleotide dissociation stimulator (RalGDS)-RBD fusion proteins were used to trap Ras-GTP and Rap1-GTP, respectively. Each anti-IgM-stimulated cell lysate in Mg2+-containing lysis buffer was incubated with the beads for 15 min at 4°C. Bound proteins were eluted with SDS/PAGE sample buffer, resolved by SDS/15% PAGE and subjected to Western blotting with anti-pan-Ras (Calbiochem) and anti-Rap1 antibodies (BD Transduction Laboratories).

Confocal microscopy and image analysis

RasGRP3–EGFP or TRPC3–EGFP-expressing DT40 cells were plated on to poly-L-lysine-coated glass coverslips. EGFP fluorescence images were acquired with a confocal laser-scanning microscope (FV500, Olympus) using the 488-nm line of an argon laser for excitation and a 505- to 525-nm band-pass filter for emission. The specimens were viewed at high magnification using plain oil objectives (×60, 1.40 numerical aperture; Olympus). DT40 cells were stimulated with 1 μg/ml anti-IgM. Measurement and analysis of the membrane and cytosolic region of EGFP fluorescence were carried out as described previously [14].

In vitro B-Raf kinase assay

In vitro kinase activity of B-Raf was measured as described previously [18]. In brief, B-Raf was immunoprecipitated and incubated with 20 μM ATP and GST–MEK1 fusion protein (Merck Millipore) at 30°C for 15 min in a kinase reaction buffer containing 20 mM Tris/HCl (pH 7.4), 20 mM NaCl, 1 mM DTT, 10 mM MgCl2 and 1 mM MnCl2. The samples were resolved using SDS/7.5% PAGE and subjected to immunoblot analysis with an anti-phospho-specific MEK1/2 antibody, followed by re-probing with an anti-B-Raf antibody (Santa Cruz Biotechnology).

Co-immunoprecipitation assay

YFP-fused C3G-expressing DT40 cells were lysed in 1% NP-40 lysis buffer [140 mM NaCl, 20 mM Tris/HCl, pH 7.8, 1% NP-40, 25 mM NaF, 0.04 mM EDTA, 5 mM sodium orthovanadate, protease inhibitor cocktail (Nacalai)]. Cell lysates were mixed with anti-GFP antibody (Abcam) overnight at 4°C, followed by incubation with protein A–Sepharose (GE Healthcare). Bound proteins were eluted using Laemli buffer and separated by SDS/15% PAGE. Immunoblots were probed with anti-Rap1 antibody (BD Transduction Laboratories).

Quantitative PCR

For reverse transcription (RT)–quantitative real-time PCR (qPCR), total RNAs were extracted from DT40 cells with ISOGEN (Nippon Gene Co., Ltd). The synthesis of cDNA was performed with an RNA LA PCR Kit (AMV), version 1.1 (TaKaRa), using 0.5 μg of total RNA, and RT–qPCR was performed using the LC-FastStart DNA master SYBR Green kit (Roche) and lightCycler system (Roche), using specific oligonucleotides (chicken Egr-1, forward 5′-CTTGACCACGCACATCCGC-3′, reverse 5′-GCTGAGACCGAAGCTGCCT-3′; GAPDH (glyceraldehyde-3-phosphate dehydrogenase), forward 5′-CGTGTTATCATCTCAGCTCCCT-3′, reverse 5′-CCAGCACCCGCATCAAAG-3′).

Statistical analysis

All data are expressed as means±S.E.M. The data were accumulated under each condition from at least three independent experiments. Statistical significance was evaluated using 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 a Tukey–Kramer test.

RESULTS

Sustained activation of PKD requires extracellular Ca2+ and TRPC3

To identify the signalling pathway linking TRPC3 and sustained ERK activity, we analysed the phosphorylation of PKD in either WT or MUT cells. MUT cells express TRPC3 with a deletion in its C-terminal cytoplasmic tail such that it cannot be expressed in the plasma membrane [14]. PKD has been known to be phosphorylated by PKC at Ser744 and/or Ser748. On BCR stimulation, PKD was rapidly and persistently phosphorylated over 45 min in WT DT40 cells (Figure 1A). In contrast, both MUT cells and WT cells, in the absence of extracellular Ca2+, showed transient PKD phosphorylation on BCR stimulation. Strikingly, the Ca2+- and TRPC3-dependent sustained phase of PKD phosphorylation was suppressed by the classic PKC inhibitor Gö6976, suggesting that PKD is one of the major downstream targeted molecules of the TRPC3–PKCβ signalling complex.

Rap1 mediates BCR-induced activation of ERK in the later phase

We next examined whether activation of the small GTPase Rap1, from the Ras family, mediates BCR-induced ERK activation, because Rap1 is responsible for the sustained phase of MAPK activation via B-Raf [19]. To analyse the contribution of Rap1 activity, BCR-induced ERK activation was analysed using DT40 cells expressing a dominant-negative mutant of Rap1, Rap1N17. Expression of Rap1N17 suppressed BCR-induced ERK activation, especially in the sustained phase (Figure 2A). The kinetics of suppressed ERK activation in Rap1N17-expressing DT40 WT cells were similar to those in MUT cells [14]. Next, BCR-induced Rap1 activation was analysed using a GST pull-down assay of active GTP-bound Rap1 (Figure 2B). Rap1 activation was observed from immediate early through later time periods after BCR stimulation in WT cells. Removal of extracellular Ca2+ and the TRPC3 deficiency (MUT expression) suppressed the sustained phase more significantly than the initial phase (Figure 2B). The activation kinetics of Rap1 are consistent with those of ERK, suggesting that the sustained phase of BCR-induced ERK activation is regulated by Rap1.

BCR-induced Rap1 activation is sustained, which is required for sustained ERK activation in DT40 cells

Figure 2
BCR-induced Rap1 activation is sustained, which is required for sustained ERK activation in DT40 cells

(A) BCR-induced ERK activation in WT and WT expressing Rap1N17 DT40 cells. Cells were stimulated with 10 μg/ml anti-IgM, and cell lysates were analysed using immunoblotting with anti-phospho-ERK1/2 antibody (upper panel). The blot was stripped and re-probed with anti-ERK2 antibody (bottom panel). The graph depicts the time course of an average fold increase over unstimulated cells, calculated from three independent experiments. **P<0.01, ***P<0.001; significant difference from WT cells stimulated in the presence of extracellular Ca2+ (Student's t-test). (B) BCR-induced Rap1 activation in WT and MUT DT40 cells. Cells were stimulated with 10 μg/ml anti-IgM for the indicated intervals, and cell lysates were mixed with beads containing the GST–RalGDS-RBD. Eluates of the beads (upper panel) or cell lysates (bottom panel) were subjected to immunoblotting with anti-Rap1 antibody. The graph depicts the time course of BCR-induced Rap1 activation. Results are presented as the average fold increases over unstimulated cells, calculated from three independent experiments. **P<0.01 compared with WT cells stimulated in the presence of extracellular Ca2+ (Tukey–Kramer test).

Figure 2
BCR-induced Rap1 activation is sustained, which is required for sustained ERK activation in DT40 cells

(A) BCR-induced ERK activation in WT and WT expressing Rap1N17 DT40 cells. Cells were stimulated with 10 μg/ml anti-IgM, and cell lysates were analysed using immunoblotting with anti-phospho-ERK1/2 antibody (upper panel). The blot was stripped and re-probed with anti-ERK2 antibody (bottom panel). The graph depicts the time course of an average fold increase over unstimulated cells, calculated from three independent experiments. **P<0.01, ***P<0.001; significant difference from WT cells stimulated in the presence of extracellular Ca2+ (Student's t-test). (B) BCR-induced Rap1 activation in WT and MUT DT40 cells. Cells were stimulated with 10 μg/ml anti-IgM for the indicated intervals, and cell lysates were mixed with beads containing the GST–RalGDS-RBD. Eluates of the beads (upper panel) or cell lysates (bottom panel) were subjected to immunoblotting with anti-Rap1 antibody. The graph depicts the time course of BCR-induced Rap1 activation. Results are presented as the average fold increases over unstimulated cells, calculated from three independent experiments. **P<0.01 compared with WT cells stimulated in the presence of extracellular Ca2+ (Tukey–Kramer test).

BCR-induced Ras activation is transient and not affected by extracellular Ca2+ and TRPC3

The best-characterized pathway leading to BCR-induced ERK activation is the Ras signalling pathway [17], which is regulated by PKCβ via phosphorylation of the Ras guanine-nucleotide-exchange factor (GEF) RasGRP3 [20,21]. Therefore, we next analysed the effect of TRPC3 deficiency on BCR-induced Ras activation. A GST pull-down assay of activated Ras, using a purified RBD of Raf-1, showed robust activation of Ras immediately after BCR stimulation (Figure 3A). However, Ras activation was not sustained but rapidly deactivated (Figure 3A). Furthermore, either DT40 WT cells, stimulated in the absence of extracellular Ca2+, or MUT cells showed levels of BCR-induced Ras activation comparable with those of WT cells stimulated in the presence of extracellular Ca2+ (Figure 3A). Consistent with these results, the translocation of RasGRP3 to the plasma membrane, which is a critical step for Ras activation, was also transient following BCR ligation, and was no different between WT and MUT cells (Figure 3B). These results indicate that Ras activation is not responsible for Ca2+-dependent sustained activation of the ERK pathway.

BCR-induced Ras activation is transient and independent of extracellular Ca2+ and TRPC3 channel in DT40 cells

Figure 3
BCR-induced Ras activation is transient and independent of extracellular Ca2+ and TRPC3 channel in DT40 cells

(A) BCR-mediated Ras activation in WT and MUT DT40 cells. Cells were stimulated with 10 μg/ml anti-IgM for the indicated intervals, and cell lysates were mixed with beads containing the Ras-binding domain of Raf-1. Eluates of the beads (upper panel) or cell lysates (bottom panel) were subjected to immunoblotting with anti-pan-Ras monoclonal antibody. The graph depicts the average time course of fold increases over unstimulated cells, calculated from three independent experiments. (B) Confocal fluorescence images indicating BCR-induced translocation of EGFP-tagged RasGRP3 to the plasma membrane in WT and MUT DT40 cells. The white scale bar indicates 2 μm. The graph depicts the time courses of average fluorescent changes of EGFP–RasGRP3 distributed in the plasma membrane regions (percentage total florescence) in WT and MUT DT40 cells.

Figure 3
BCR-induced Ras activation is transient and independent of extracellular Ca2+ and TRPC3 channel in DT40 cells

(A) BCR-mediated Ras activation in WT and MUT DT40 cells. Cells were stimulated with 10 μg/ml anti-IgM for the indicated intervals, and cell lysates were mixed with beads containing the Ras-binding domain of Raf-1. Eluates of the beads (upper panel) or cell lysates (bottom panel) were subjected to immunoblotting with anti-pan-Ras monoclonal antibody. The graph depicts the average time course of fold increases over unstimulated cells, calculated from three independent experiments. (B) Confocal fluorescence images indicating BCR-induced translocation of EGFP-tagged RasGRP3 to the plasma membrane in WT and MUT DT40 cells. The white scale bar indicates 2 μm. The graph depicts the time courses of average fluorescent changes of EGFP–RasGRP3 distributed in the plasma membrane regions (percentage total florescence) in WT and MUT DT40 cells.

Rap1 activation via PKD1 mediates BCR-induced sustained ERK activation

Rap1 has been reportedly activated by specific GEFs such as C3G (RapGEF1) and RasGRP3 in B-lymphocytes [19]. As RasGRP3 activation was transient and not affected by TRPC3 deficiency, we focused on the activation of Rap1 by C3G. To show the activation of Rap1 by C3G, the interaction between C3G and Rap1 on BCR stimulation was investigated (Figure 4A). A YFP-fusion construct of C3G was introduced into WT or MUT cells and immunoprecipitated with anti-GFP antibody after BCR stimulation. The interaction between C3G–YFP and Rap1 was rapidly increased in 5 min, and kept activated for 30 min after BCR stimulation in WT cells in the presence of extracellular Ca2+. In contrast, those interactions in either WT or MUT cells stimulated in the absence of extracellular Ca2+, or MUT cells stimulated in the presence of extracellular Ca2+, were increased only initially and returned to the basal level 30 min after BCR stimulation. These results indicate that the interaction of C3G and Rap1 depended on TRPC3-mediated Ca2+ influx, and sustained Rap1 activation was mainly mediated by C3G. A recent study has revealed that T-cell receptor activation induces Rap1 activation through PKD activation in its PH domain-dependent manner [22]. Therefore, we tested whether BCR-induced ERK activation requires the PH domain of PKD1-dependent Rap1 activation. BCR-induced ERK activation was analysed with DT40 WT cells expressing the vector PKD1(WT)–EGFP, or the PH-domain-deleted mutant of PKD1 [PKD1(ΔPH)]–EGFP (Figure 4B) in the presence of extracellular Ca2+. As shown in Figure 4B, expression of PKD1(ΔPH)–EGFP significantly suppressed BCR-induced ERK activation, indicating that PKD1 is a critical regulator of BCR-induced ERK activation in DT40 cells.

BCR-induced Rap1 activation is mediated by C3G and requires PKD1

Figure 4
BCR-induced Rap1 activation is mediated by C3G and requires PKD1

(A) Temporal changes of the interaction between Rap1 and C3G–YFP. Cells were stimulated with 1 μg/ml anti-IgM for the indicated times. Cell lysates were then immunoprecipitated (IP) with anti-GFP antibody, and the precipitates were immunoblotted (IB) with anti-Rap1 antibody. Within each set, panels show immunoprecipitated Rap1 (precipitate) and total Rap1 (input). Representative data from one of three independent experiments are shown. The graph depicts the average fold increases over unstimulated cells, calculated from three independent experiments. *P<0.05, **P<0.01; significant difference from WT cells stimulated in the presence of extracellular Ca2+ (Tukey–Kramer test). (B) BCR-induced ERK activation in DT40 WT cells expressing PKD1(WT)–EGFP or PKD1(ΔPH)–EGFP. The graph depicts the time courses of average fold increases over unstimulated cells, calculated from three independent experiments. **P<0.01, ***P<0.001 compared with WT cells stimulated in the presence of extracellular Ca2+ (Tukey–Kramer test).

Figure 4
BCR-induced Rap1 activation is mediated by C3G and requires PKD1

(A) Temporal changes of the interaction between Rap1 and C3G–YFP. Cells were stimulated with 1 μg/ml anti-IgM for the indicated times. Cell lysates were then immunoprecipitated (IP) with anti-GFP antibody, and the precipitates were immunoblotted (IB) with anti-Rap1 antibody. Within each set, panels show immunoprecipitated Rap1 (precipitate) and total Rap1 (input). Representative data from one of three independent experiments are shown. The graph depicts the average fold increases over unstimulated cells, calculated from three independent experiments. *P<0.05, **P<0.01; significant difference from WT cells stimulated in the presence of extracellular Ca2+ (Tukey–Kramer test). (B) BCR-induced ERK activation in DT40 WT cells expressing PKD1(WT)–EGFP or PKD1(ΔPH)–EGFP. The graph depicts the time courses of average fold increases over unstimulated cells, calculated from three independent experiments. **P<0.01, ***P<0.001 compared with WT cells stimulated in the presence of extracellular Ca2+ (Tukey–Kramer test).

B-Raf and MEK are responsible for BCR-induced sustained ERK activation in DT40 cells

Next, we analysed BCR-induced activation of MEK, an immediate upstream kinase of ERK, using an antibody specific for phosphorylated MEK. In WT cells stimulated in the presence of extracellular Ca2+, phosphorylation of MEK was sustained for up to 45 min, whereas BCR-induced MEK phosphorylation was significantly suppressed in the absence of extracellular Ca2+ (Figure 5A). MUT cells also exhibited suppressed MEK activation in the presence of extracellular Ca2+ (Figure 5A). Among Raf kinases located immediately downstream of Ras and upstream of MEK in the ERK cascade, two types of Raf kinases–Raf-1 and B-Raf–are expressed in DT40 cells, the latter being a dominant ERK activator in DT40 cells [23]. Furthermore, it has already been revealed that the single knockout of B-Raf suppressed BCR-mediated ERK activation, particularly in the immediate early and late phases in DT40 cells [23]. Therefore, we next analysed whether the removal of extracellular Ca2+ and TRPC3 deficiency affects BCR-induced activation of B-Raf by an in vitro B-Raf kinase assay, in which the immunoprecipitated B-Raf kinase was used to phosphorylate purified recombinant MEK protein in vitro (Figure 5B). In the presence of extracellular Ca2+, B-Raf activity increased gradually to a maximal level 15 min after BCR stimulation in WT cells, whereas B-Raf activity remained unchanged in WT cells in the absence of extracellular Ca2+, and in MUT cells in the presence of extracellular Ca2+ (Figure 5B). These results clearly demonstrate that BCR-induced B-Raf and MEK activation are dependent on Ca2+ influx mediated by TRPC3. The initial activation of MEK and B-Raf was also suppressed in WT cells activated in the absence of extracellular Ca2+. We analysed the localization of TRPC3 in the absence of extracellular Ca2+. It is interesting that removal of extracellular Ca2+ increased intracellular fluorescent vesicles compared with cells placed in the medium supplemented with 2 mM Ca2+ (Figure 5C). These results suggest that surface expression of TRPC3 requires the presence of extracellular Ca2+, and TRPC3 localized in the intracellular vesicles might have a dominant-negative effect on BCR-induced MEK and B-Raf activation.

B-Raf activation and MEK activation is suppressed by removal of extracellular Ca2+ and TRPC3 deficiency

Figure 5
B-Raf activation and MEK activation is suppressed by removal of extracellular Ca2+ and TRPC3 deficiency

(A) BCR-mediated MEK activation in WT and MUT DT40 cells. Cells were stimulated with 10 μg/ml anti-IgM, and cell lysates were analysed by immunoblotting with anti-phospho-MEK1/2 polyclonal antibody (upper panel). The blot was stripped and re-probed with anti-MEK1/2 polyclonal antibody (bottom panel). The graph depicts the time course of average fold increases over unstimulated cells, calculated from three independent experiments. (B) BCR-induced B-Raf activation in WT and MUT DT40 cells. Cells were stimulated with 10 μg/ml anti-IgM for the indicated intervals. B-Raf was immunoprecipitated from cell lysates, and in vitro phosphorylation of the B-Raf substrate GST–MEK1 was performed. Within each set, the upper panels show the phosphorylated GST–MEK1 and bottom panels show the total amount of the immunoprecipitated B-Raf. The graph depicts time courses of average fold increases over unstimulated cells, calculated from three independent experiments. *P<0.05, **P<0.01; compared with WT cells stimulated in the presence of extracellular Ca2+ (Tukey–Kramer test). (C) Localization of TRPC3 in DT40 cells in the presence or absence of extracellular Ca2+. The graph depicts intracellular fluorescence (percentage total fluorescence) in WT cells with (n=10) or without (n=10) extracellular Ca2+. The white scale bar indicates 2 μm. *P<0.05 (Student's t-test).

Figure 5
B-Raf activation and MEK activation is suppressed by removal of extracellular Ca2+ and TRPC3 deficiency

(A) BCR-mediated MEK activation in WT and MUT DT40 cells. Cells were stimulated with 10 μg/ml anti-IgM, and cell lysates were analysed by immunoblotting with anti-phospho-MEK1/2 polyclonal antibody (upper panel). The blot was stripped and re-probed with anti-MEK1/2 polyclonal antibody (bottom panel). The graph depicts the time course of average fold increases over unstimulated cells, calculated from three independent experiments. (B) BCR-induced B-Raf activation in WT and MUT DT40 cells. Cells were stimulated with 10 μg/ml anti-IgM for the indicated intervals. B-Raf was immunoprecipitated from cell lysates, and in vitro phosphorylation of the B-Raf substrate GST–MEK1 was performed. Within each set, the upper panels show the phosphorylated GST–MEK1 and bottom panels show the total amount of the immunoprecipitated B-Raf. The graph depicts time courses of average fold increases over unstimulated cells, calculated from three independent experiments. *P<0.05, **P<0.01; compared with WT cells stimulated in the presence of extracellular Ca2+ (Tukey–Kramer test). (C) Localization of TRPC3 in DT40 cells in the presence or absence of extracellular Ca2+. The graph depicts intracellular fluorescence (percentage total fluorescence) in WT cells with (n=10) or without (n=10) extracellular Ca2+. The white scale bar indicates 2 μm. *P<0.05 (Student's t-test).

Sustained ERK activation is important for BCR-induced expression of Egr-1

Previous studies have shown that the rapid induction of the immediate early gene Egr-1 in response to BCR activation is mediated through ERK1/2 in B-lymphocytes [2325]. Egr-1 expression is tightly correlated with B-lymphocyte activation [25]. We analysed whether TRPC3 deficiency affects BCR-mediated Egr-1 expression by using RT–qPCR analysis. Egr-1 expression after a 60-min BCR stimulation was significantly suppressed in MUT cells (Figure 6), suggesting that TRPC3-mediated Ca2+ influx is required for sustained ERK activation and subsequent expression of Egr-1. It is interesting that WT cells stimulated in the absence of extracellular Ca2+ showed more pronounced reduction of Egr-1 expression (Figure 6). This may indicate that a Ca2+ influx pathway other than the TRPC3 pathway is involved in BCR-induced Egr-1 expression.

BCR-induced expression of Egr-1 is suppressed by either the removal of extracellular Ca2+ or TRPC3 deficiency

Figure 6
BCR-induced expression of Egr-1 is suppressed by either the removal of extracellular Ca2+ or TRPC3 deficiency

Induction of Egr-1 in WT and MUT DT40 cells 30 and 60 min after stimulation by 10 μg/ml anti-IgM. The mRNA of Egr-1 was quantified by RT–qPCR. Egr-1 expression was normalized by GAPDH. *P<0.05, **P<0.01 compared with WT cells stimulated in the presence of extracellular Ca2+ (Tukey–Kramer test).

Figure 6
BCR-induced expression of Egr-1 is suppressed by either the removal of extracellular Ca2+ or TRPC3 deficiency

Induction of Egr-1 in WT and MUT DT40 cells 30 and 60 min after stimulation by 10 μg/ml anti-IgM. The mRNA of Egr-1 was quantified by RT–qPCR. Egr-1 expression was normalized by GAPDH. *P<0.05, **P<0.01 compared with WT cells stimulated in the presence of extracellular Ca2+ (Tukey–Kramer test).

DISCUSSION

In the present study, we addressed the mechanisms that link TRPC3-regulated PKCβ activation with sustained ERK activation, and demonstrated that BCR-induced ERK activation can be separated into two phases: one is a Ras-dependent early and transient phase and the other a Rap1-dependent late and sustained phase (Figure 7). TRPC3-mediated PKCβ activation leads to the phosphorylation of PKD, which then causes the activation of Rap1 and eventual activation of the B-Raf/MEK/ERK cascade (Figure 7).

Model of BCR-induced ERK activation

Figure 7
Model of BCR-induced ERK activation

BCR-induced ERK activation is separated into initial and sustained phases. Although the early phase of ERK activation is mainly dependent on the RasGRP3/Ras pathway, the sustained phase is predominantly mediated by the Rap1/B-Raf pathway. For activation of Rap1, PKD phosphorylation mediated by PKCβ is required. TRPC3 reorganizes a Rap1-containing signal complex on the plasma membrane for sustained ERK activation by recruiting initial regulators of PKCβ and PKD.

Figure 7
Model of BCR-induced ERK activation

BCR-induced ERK activation is separated into initial and sustained phases. Although the early phase of ERK activation is mainly dependent on the RasGRP3/Ras pathway, the sustained phase is predominantly mediated by the Rap1/B-Raf pathway. For activation of Rap1, PKD phosphorylation mediated by PKCβ is required. TRPC3 reorganizes a Rap1-containing signal complex on the plasma membrane for sustained ERK activation by recruiting initial regulators of PKCβ and PKD.

The importance of PKC in ERK activation has been described in previous reports of BCR-induced signalling [16,20,21,26,27]. The Ras signalling pathway, in which PKCβ modulates the Ras/ERK pathway via phosphorylation of RasGRP3 [20,21], is the best-characterized pathway leading to BCR-induced ERK activation [17]. Therefore, Ras was the initial candidate for a protein that links PKCβ to ERK in the present study. However, Ras activation was unaltered in MUT cells as well as in WT cells stimulated in the absence of extracellular Ca2+ (Figure 3A). Importantly, Oh-hora et al. [17] examined the kinetics of Ras and ERK activation in DT40 cells and found that ERK activation was sustained after the decay of Ras activation. Our previous study demonstrated that BCR-induced PKCβ translocation to the plasma membrane was suppressed, particularly at later time points after BCR stimulation, as observed for BCR-induced ERK activation in TRPC3-deficient cells [14]. In addition, the inhibitory effect of the Ca2+-dependent PKC-selective inhibitor Gö6976 on ERK activation was more pronounced later than earlier after BCR stimulation [26]. These results suggest that PKCβ activity is responsible for the activation of ERK in the sustained phase, in contrast to Ras which acts in the earlier phase. Therefore, we speculate that a Ras-independent pathway operates in sustained ERK activation. Activation of MEK, a kinase immediately upstream of ERK, was suppressed in MUT cells (Figure 4A), indicating that PKCβ regulates kinases upstream of MEK in the Ras-independent ERK activation pathway. DT40 B-lymphocytes express two isoforms of Raf kinases: Raf-1 and B-Raf, immediate upstream kinases for phosphorylation of MEK. It is of interest that the disruption of B-Raf but not that of Raf-1 strongly reduced BCR-mediated ERK activation, particularly in the immediate early and late phases [23]. This transient pattern of ERK activation in B-Raf-deficient DT40 cells is similar to that observed in MUT cells [14]. Indeed, BCR-induced B-Raf activation was suppressed in MUT cells (Figure 5B), suggesting that B-Raf mediates PKCβ-regulated sustained MEK/ERK activation. With regard to upstream regulators of Raf, it is known that Rap1 promotes a sustained activation of ERK in certain types of cells [19]. Although it has been previously demonstrated that ERK phosphorylation was not affected by the knockdown of Rap1b in B-lymphocytes, in that study the authors analysed only 2 min after BCR stimulation and provided no data for the sustained phase [28]. In the present study, as Rap1N17 expression more significantly suppressed the sustained phase of ERK phosphorylation in DT40 cells (Figure 2A), Rap1 would contribute more to the sustained rather than the early phase of ERK activation. Altogether, sustained ERK activation in response to BCR stimulation is mediated by a PKCβ/Rap1/B-Raf/MEK pathway in DT40 cells.

It has been reported that Rap1 can be activated by specific GEFs such as C3G (RapGEF1) and RasGRP3 in B-lymphocytes [19]. A recent study reported that PKC regulates Rap1 activation through PKD activation in TCR-induced β1-integrin activation [22]. The recruitment of Rap1 to the plasma membrane can promote the association of Rap1 with C3G and subsequent activation of Rap1. Indeed, BCR stimulation rapidly and sustainably induced Rap1 to interact with C3G, which is dependent on TRPC3-mediated Ca2+ entry (Figure 4A). Furthermore, our results demonstrate that PKD phosphorylation was attenuated by pre-treatment with the Ca2+-dependent PKC-specific inhibitor Gö6976, especially in the late phase of BCR stimulation in DT40 cells (Figure 1), as well as by the removal of extracellular Ca2+ and the loss of TRPC3 at the plasma membrane (Figure 1), supporting the idea that PKD is a downstream effector of PKC [29]. Importantly, kinase activity is not required for PKD-dependent Rap1 membrane translocation, but the PH domain of PKD is indispensable [22]. In addition, the function of the phosphorylation of Ser744/Ser748 in the PKD activation loop is to release autoinhibition by the PH domain for PKD activation [30]. Therefore we speculate that PKCβ facilitates the association of PKD and Rap1 by relieving the PH domain autoinhibition, which is supported by our observation that expression of the PH-domain-deleted mutant of PKD1 significantly suppressed BCR-induced ERK activation (Figure 4B). It was reported that Gq-coupled receptor-induced PKD activation could increase the duration of ERK activation in Swiss 3T3 cells [31]. In DT40 cells, the correlation of PKD activity and ERK activation has been reported [32,33]. Thus, TRPC3-mediated Ca2+ influx leads to the sustained activation of PKCβ to induce sustained activation of ERK via a PKD/Rap1/B-Raf/MEK pathway.

The importance of sustained ERK activation in B-lymphocytes remains elusive. We have demonstrated that the suppression of ERK activation in the sustained phase significantly reduced Egr-1 mRNA expression. This result is consistent with a previous report in which B-Raf knockdown suppressed sustained ERK activation and subsequent c-fos and Egr-1 expression in DT40 cells [23]. It has been shown that, similar to ERK, Egr-1 is critical for early B-cell development [4,34,35]. Furthermore, Rap1b, a dominant form of Rap1, is also important for early B-cell development [28]. These results suggest that TRPC3-regulated ERK activation may be critical for early B-cell development thorough the regulation of the Rap1/ERK/Egr-1 pathway in B-lymphocytes.

AUTHOR CONTRIBUTION

Takuro Numaga-Tomita and Motohiro Nishida carried out the acquisition, analysis and interpretation of data, and helped to draft the manuscript. James Putney analysed and interpreted the data, and helped to draft the manuscript. Yasuo Mori analysed and interpreted the data, and helped to draft and critically review the manuscript.

We thank M. Hikida and Y. Aiba for chicken RasGRP3 and for much technical advice and many helpful discussions; we also thank H. Kurose for Rap1N17.

FUNDING

This work was supported in part by the Intramural Research Program of the National Institutes of Health (NIH), National Institute of Environmental Health Sciences (to J.W.P.); Grants-in-Aid for Scientific Research [grant number 25670031] to M.N. from the Ministry of Education, Culture, Sports, Science and Technology of Japan; and grants from Naito Memorial Foundation (to M.N.).

Abbreviations

     
  • BCR

    B-cell receptor

  •  
  • DAG

    diacylglycerol

  •  
  • Egr-1

    early growth response 1

  •  
  • EGFP

    enhanced green fluorescent protein

  •  
  • ERK

    extracellular-signal-regulated kinase

  •  
  • GEF

    guanine-nucleotide-exchange factor

  •  
  • Ig

    immunoglobulin

  •  
  • MAPK

    mitogen-activated protein kinase

  •  
  • MEK

    MAPK/ERK kinase

  •  
  • MUT

    TRPC3 mutant

  •  
  • PH

    pleckstrin homology

  •  
  • PKC

    protein kinase C

  •  
  • PKD

    protein kinase D

  •  
  • PLC

    phospholipase C

  •  
  • qPCR

    quantitative real-time PCR

  •  
  • RalGDS

    Ral guanine nucleotide dissociation stimulator

  •  
  • RasGRP3

    Ras guanine-nucleotide-releasing protein 3

  •  
  • RBD

    Ras-binding domain

  •  
  • RT

    reverse transcription

  •  
  • TRPC3

    canonical transient receptor potential 3

  •  
  • YFP

    yellow fluorescent protein

  •  
  • WT

    wild-type

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