Our previous work has pointed to a role for PLC-γ2 in Erk activation in response to BCR stimulation (37
). The data presented here demonstrate that RasGRP3 couples PLC-γ2 to Ras, accounting at least partly for the defective BCR-mediated Erk activation in PLC-γ2–deficient B cells. In addition, our results show that BCR and EGFR utilize distinct types of RasGEFs, the RasGRP family and the Sos family, respectively, to activate Ras in the same cellular context.
Because the two src homology (SH) 3 domains of Grb2 bind to proline-rich residues near the C terminus of Sos, Grb2 is thought to mediate the translocation of Sos to the plasma membrane, thereby allowing Sos to activate membrane-bound Ras (54
). In the case of EGFR, a requirement for Sos1 in EGFR-mediated Erk activation has been demonstrated already by using Sos1−/−
fibroblasts, but this Erk activation is only partially blocked in this mutant cell (59
), therefore, subsequently raising the question of whether the remaining Erk activation is accounted for by residual Sos2 or by other GEF families. Hence, an almost complete block of EGER-mediated Ras and subsequent Erk activation in Sos1/Sos2 double-deficient DT40 B cells ( B) supports the former possibility. Furthermore, this result, together with our previous evidence that Grb2 is required for EGFR-mediated Ras activation in DT40 B cells (31
), provides genetic evidence that the aforementioned mechanism indeed operates in B cells as well, at least in the case of transmembrane receptor tyrosine kinase signaling.
As in T cells, treatment of DAG analogues such as phorbol esters (PMAs) can induce the drastic activation of Ras in B cells (3
), suggesting the existence of other Ras activation modes, in addition to the Grb2–Sos mechanism. This PMA-dependent Ras activation was initially thought to be mediated by protein kinase C isoforms containing single or double C1 domains. However, recent identification of RasGRPs as Ras activators possessing the C1 domain has inspired the possibility that RasGRPs are additional potential candidates to connect between PMA and Ras (23
). Indeed, like a complete block of PMA-induced Ras activation in RasGRP1−/−
), RasGRP1/RasGRP3 double-deficient DT40 cells manifested almost complete block of PMA-induced Ras activation (unpublished data).
In fact, RasGRPs function in the BCR signaling context, because our data demonstrate the inhibition of BCR-mediated Ras activation in RasGRP1/RasGRP3 double-deficient DT40 B cells ( A). Despite a significant decrease in BCR-mediated Ras activation, this mutant cell line still exhibits Ras activation, which could be explained by the following possibilities. First, as there exists three Ras GEF families (RasGRF, RasGRP, and Sos family; reference 23
), the full BCR-mediated Ras activation might require other GEF families, Sos and/or RasGRF, in addition to RasGRP. Based on our data using Sos1/Sos2 double-deficient DT40 B cells, dominant involvement of the Sos family would be unlikely. Nevertheless, considering that a gene knockout approach sometimes causes functional up-regulation of other molecules in an attempt to cope with the deficit, it is still possible that the Sos family plays a role, albeit small, in BCR-mediated Ras activation. This possibility might explain the fact that Ras activation takes place, at least partly, independent of PLC-γ2 ( A), as Sos activation does not require PLC-γ. Second, because ablation of both RasGRP1 and RasGRP3 results in a slightly more severe defect in Ras activation than in single RasGRP3 deletion ( A), another RasGRP member (RasGRP2 or RasGRP4) could be a candidate for mediating the residual BCR-mediated Ras activation. Third, assuming that BCR activates Ras through operating two mechanisms concomitantly, by activating Ras GEFs and by inhibiting Ras GAPs, the residual Ras activation in RasGRP1/RasGRP3 double-deficient cells could be due to ongoing inhibition of Ras GAPs during the BCR signal (8
). Additional studies are underway to define which of the preceding possibilities is the most likely in DT40 B cells.
Because both Grb2–Sos and PLC-γ2–RasGRP pathways coexist in B cells, a question arises about how their selective engagement, depending on receptor types such as BCR or EGFR, takes place for Ras activation. Both receptors use Grb2 and PLC-γ2 in their signal transduction, because BCR-mediated Rac1 activation or EGFR-mediated calcium response is inhibited in Grb2- or PLC-γ2–deficient DT40 B cells, respectively (reference 61
; unpublished data). Thus, why would Grb2 or PLC-γ2 be used more preferentially for EGFR- or BCR-mediated Ras activation, respectively? As the NH2
-terminal Grb2 SH3 domain is able to bind to proline-rich residues in the B cell linker (BLNK) as well as Sos (62
), this Grb2-dependent association may be skewing more toward the BLNK when BCRs are stimulated. Indeed, this type of induced interaction between a proline-rich sequence in CD3
and an SH3 domain of adaptor molecule Nck has been reported in TCR-stimulated T cells (63
). Similarly, the association between Grb2 and the BLNK might be induced or stabilized upon BCR engagement, possibly sequestering Sos from Grb2. Hence, the selection of binding partners for Grb2, depending on receptor types, may underlie the basis for differential outcomes through Grb2.
In contrast to BCR, EGFR did not essentially require PLC-γ2 for Ras activation in DT40 B cells (unpublished data). This result is consistent with previous evidence that EGFR-mediated Erk activation occurs normally in PLC-γ1−/−
). These observations might simply suggest that DAG, a product of PLC-γ2 action, is necessary, but not sufficient, for activation of RasGRP3. If so, BCR, but not EGFR, could provide the second signal, in addition to generating DAG, both of which are required for activation of RasGRP3. Alternatively, given the relatively small activation of PLC-γ2 upon EGFR engagement, compared with the BCR (unpublished data), the differential requirement for PLC-γ2 might reflect the quantitative differences; low levels of PLC-γ2 activity are not sufficient for activation of RasGRP3.
Two lines of our evidence shown here support the proposed model that PLC-γ, after being activated upon antigen receptor engagement, generates DAG in the plasma membrane, thereby facilitating membrane recruitment of RasGRPs and its subsequent interaction with Ras (27
). First, the membrane recruitment of RasGRP3 was severely affected by loss of PLC-γ2, whereas absence of RasGRP3 did not affect PLC-γ2 activation. Moreover, this Ras GRP3 recruitment required its C1 domain. Second, the necessity of membrane localization of RasGRP3 for its activation was demonstrated by experiments using its membrane-attached construct ( C, ΔC1-pbRasGRP3). In line with this model, the defective membrane recruitment of RasGRP1 in Vav3-deficient DT40 cells is likely accounted for by the insufficient PLC-γ2 activation in this mutant line (65
). Recent results indicate that RasGRP1 may be involved not only in the stimulation of Ras at the plasma membrane but also at the Golgi apparatus in COS cells and Jurkat cells (66
). Indeed, consistent with a previous report (67
), in addition to the plasma membrane localization of RasGRP3 in DT40 cells, we also observed that RasGRP3 could localize at the perinuclear region, presumably Golgi in some DT40 cells after 10 min of BCR stimulation (Kashiwagi, K., and N. Saito, personal communication). Hence, it is possible that translocation of RasGRP3 to the perinuclear region might participate in the sustained Ras activation.
Constitutive localization of ΔC1-pbRasGRP3 enhanced activation status of Ras before BCR stimulation, but the Ras status was further activated upon BCR ligation, simply suggesting that additional BCR-mediated event might be required for optimal Ras activation. In this regard, consistent with the previous data (68
), we also detected the BCR-mediated electrophoretic mobility shift of RasGRP3 in DT40 and primary B cells (unpublished data), presumably reflecting phosphorylation status. Therefore, this phosphorylation-dependent modification could be a potential additional mechanism.
Although initiation of Ras activation in BCR signaling requires membrane localization of RasGRP3, the subsequent inactivation of Ras at 10 min after BCR stimulation ( A) appears not to be simply explained by dissociation of RasGRP3 from the membrane. Indeed, our biochemical and microscopic analyses indicated that RasGRP3 was still located in the membrane after 10 min of BCR stimulation. Hence, inactivation of RasGRP3, independently of membrane localization, might take place at this time point, or RasGAPs might be activated, thereby leading to a decrease in active Ras.
Despite the common ability of RasGRP and Sos to activate Ras, the pathways to which these GEFs are coupled seem not to be simply overlapping or redundant. Given that various receptors such as c-kit and cytokine receptors stimulate Ras and subsequent Erk, these pathways could provide the B cells with the ability to discriminate between these stimuli and possibly modulate the subsequent biological responses.