Contribution of RasGRP1 to BCR-induced deletion in the immature B cell line WEHI 231

RasGRP1 is a guanine nucleotide exchange factor (GEF) that activates Ras GTPases downstream of the B and T lymphocyte antigen receptors. RasGRP1 is a critical regulator of T cell homeostasis, and contributes to the maintenance of T cell-mediated tolerance in the host. Although Ras signalling is important during B cell development, relatively little is known about a role for RasGRP1 in B cells, or its contribution to B cell receptor (BCR) signalling. RasGRP1 expression is detected in the bone marrow and some B cell lines including the murine immature B cell line WEHI 231. The WEHI 231 cell line, which mimics immature B cell responses to self-antigen by undergoing cell cycle arrest and apoptosis in response to antigen stimulation, was used to determine whether RasGRP1 has the ability to modulate BCR-mediated responses in B cells. WEHI 231 cell populations with increased RasGRP1 expression (RasGRP1high cells) were generated by retroviral transduction. A two-fold increase in RasGRP1 protein levels correlated with increased Ras activity. A three-fold increase in the fraction of cells undergoing apoptosis was detected in RasGRP1high cells following BCR ligation, compared with control cells. Mutation of the GEF domain of RasGRP1, which is required for Ras activation, prevented the protein from sensitizing WEHI 231 cells to BCR-induced apoptosis. Expression of constitutively active Ras GTPases was sufficient to sensitize WEHI 231 cells to BCR-induced apoptosis. These results suggest that RasGRP1 acts as a positive regulator of BCR signalling, and has the ability to sensitize WEHI 231 cells to BCR-induced apoptosis via activation of Ras GTPases. Although increased RasGRP1 expression caused sustained activation of the Ras effectors ERK1/2, this effect was not required for the ability of RasGRP1 to sensitize WEHI 231 cells to BCR-induced apoptosis. Instead, increased RasGRP1 expression was found to inhibit the NF-KB pathway, a critical regulator of life and death decisions in WEHI 231 cells. While stimuli that activate NF-κB prevented increased BCR-induced apoptosis of RasGRP1high cells, inhibition of NF-κB was sufficient to sensitize WEHI 231 cells to BCR-induced apoptosis. These results suggest that RasGRP1 sensitizes WEHI 231 cells to BCR-induced apoptosis by causing down-regulation of the NF-κB pathway.Medicine, Faculty ofMedical Genetics, Department ofGraduat