RasGRP1 Sensitizes an Immature B Cell Line to Antigen Receptor-induced Apoptosis

Abstract

RasGRP1 is a guanine nucleotide exchange factor that activates Ras GTPases and is activated downstream of antigen receptors on both T and B lymphocytes. Ras-GRP1 provides signals to immature T cells that conferRasGRP1 is a guanine nucleotide exchange factor that transduces intracellular signaling by converting Ras GTPases to their active GTP-bound states (1-6). RasGRP1 is expressed most prominently in thymocytes and mature T cells but is also expressed in some immature B cell lines (2, 3, 5, 6). RasGRP1 is positively regulated by membrane translocation, which is driven by binding of its C1 domain to diacylglycerol (3, 4, 6 -9). This provides a mechanism for RasGRP1 activation by any receptor that couples to diacylglycerol-generating phospholipase Cs, including the T cell receptor (TCR) 1 (4) and the B cell receptor (BCR) (8).Recent work with genetically modified mice has demonstrated that RasGRP1 provides signals that enhance the survival and proliferation of T cells at several stage during their development and immunological selection. RasGRP1 is transcriptionally induced by pre-TCR signaling, and the resulting increase in RasGRP1 expression is sufficient to confer survival and proliferation in the absence of other pre-TCR-derived signals (10). RasGRP1 expression augments the effectiveness with which ␣␤TCR/major histocompatibility complex interactions confer survival and maturation through the process of positive selection (11) and increases the proliferative responses of thymic T cells to TCR ligation (10 -12). RasGRP1 deficiency does not noticeably alter the efficiency of TCR-dependent deletion (negative selection) of thymic T cells by a self-antigen (11) but impedes apoptosis induction in mature splenic T cells following TCR ligation (13). These experiments portray RasGRP1 as a promoter of survival and proliferation responses to pre-TCR or TCR during intrathymic T cell development but with the converse role of mediating TCR-induced deletion in mature peripheral T cells.B cell development in the bone marrow is also governed by a series of selections for immunologically competent but not selfreactive antigen receptors. To avoid auto-immunity, immature B cells carrying BCR that recognize self-antigen need to be suppressed either by anergization, by respecification through receptor editing, or by deletion through BCR-induced apoptosis (14 -16). Much of our knowledge of the signal transduction pathways that mediate BCR-induced deletion of immature B cells has been obtained from experiments with WEHI-231 cells, a B lymphoma-derived murine cell line that resembles immature B cells both phenotypically and functionally (17). WEHI-231 cells express surface IgM of unknown antigen specificity, but cross-linking with anti-IgM mimics the interaction of this BCR with a high affinity antigen. Anti-IgM-initiated signaling imposes cell cycle arrest on WEHI-231 cells within a few hours, and maintenance of BCR ligation beyond 1 day results in apoptosis. BCR-induced deletion of WEHI-231 cells shares many of the regulatory mechan...

IL-4 protects the B-cell lymphoma cell line CH31 from anti-IgM-induced growth arrest and apoptosis: contribution of the PI-3 kinase/AKT pathway

“…Banerji et al established that WEHI-231, another extensively documented, model B-lymphoma line, contained constitutively activated ERK, which was completely inactivated following BCR crosslinking [21]. Furthermore, Guilbault and Kay established that anti-IgM stimulated the loss of Ras effector signaling [36]. Therefore, we also examined the effect of IL-4 on Ras effector via monitoring activationspecific phosphorylation of the extensively characterized Ras/Raf target, MEK.…”

Regulation of RasGRP1 by B Cell Antigen Receptor Requires Cooperativity between Three Domains Controlling Translocation to the Plasma Membrane

“…The EF-hands of RasGRP1 (Figure 1) were the initial lead candidates for providing cooperativity with the C1 domain in maximizing BCR-induced plasma membrane localization, because they were the only other recognized regulatory domains within RasGRP1, and their deletion compromises RasGRP1-mediated apoptosis induction by BCR (Guilbault and Kay, 2004). However, a C-terminally deleted form of RasGRP1 that retains both the C1 domain and the EF-hands (RG1⌬C-term) failed to translocate to the plasma membrane in response to BCR ligation ( Figure 4A).…”

“…NIH 3T3 cells from American Type Culture Collection (Manassas, VA) were cultured in DMEM (Stem Cell Technologies) containing 10% bovine calf serum (Hyclone). The origin and culture of WEHI-231 cells are as described previously (Guilbault and Kay, 2004). DO11.10 cells (Morgan et al, 1999) were obtained from Barbara Osborne (University of Massachusetts), and they were cultured in DMEM/10% fetal bovine serum.…”

“…RasGRP1 is a guanine nucleotide exchange factor that couples antigen receptors to the activation of Ras GTPases (Dower et al, 2000;Ebinu et al, 2000;Priatel et al, 2002;Bivona et al, 2003;Caloca et al, 2003b;Layer et al, 2003;Norment et al, 2003;Oh-hora et al, 2003;Guilbault and Kay, 2004;Perez de Castro et al, 2004;Reynolds et al, 2004;Zugaza et al, 2004;Coughlin et al, 2005;Roose et al, 2005). Deletion of the RasGRP1 gene perturbs the immunological selection and activation of lymphocytes (Dower et al, 2000;Priatel et al, 2002Priatel et al, , 2006Layer et al, 2003) and mast cells (Liu et al, 2007), whereas aberrant expression of RasGRP1 initiates oncogenic transformation of lymphocytes (Li et al, 1999;Mikkers et al, 2002;Suzuki et al, 2002;Kim et al, 2003;Dupuy et al, 2005;Klinger et al, 2005), fibroblasts (Ebinu et al, 1998;Tognon et al, 1998) and keratinocytes (Oki-Idouchi and Lorenzo, 2007).…”

“…This mechanism has the potential to positively regulate RasGRP1 in response to any receptor that couples to a DAG-generating phospholipase C (PLC). In addition to the C1 domain, RasGRP1 has a pair of EF-hands that have been implicated as calcium-responsive positive regulators (Ebinu et al, 1998;Kawasaki et al, 1998;Guilbault and Kay, 2004), although no involvement of the EF-hands in regulating RasGRP1 localization has been established.In the DT40 B cell line, B cell antigen receptor (BCR) ligation induces translocation of RasGRP1 to the plasma membrane . Membrane translocation of RasGRP1 is also induced by T cell antigen receptor (TCR) ligation in the Jurkat T cell line, although this is variably reported to be directed to the plasma membrane (Carrasco Merida, 2004;Zugaza et al, 2004) or to Golgi membranes (Bivona et al, 2003;Perez de Castro et al, 2004).…”