BCR/ABL activates Rap1 and B-Raf to stimulate the MEK/Erk signaling pathway in hematopoietic cells

BCR/ABL and IL-3 activate Rap1 to stimulate the B-Raf/MEK/Erk and Akt signaling pathways and to regulate proliferation, apoptosis, and adhesion

“…In accordance with our previous report (Mizuchi et al, 2005), IL-3 stimulation as well as the induction of BCR/ABL expression by Dox treatment activated B-Raf in Ton.B210 cells (Figure 1d). The SPA-1 expression in Ton.B210/SPA-1 cells partly inhibited activation of B-Raf by IL-3 and BCR/ABL in repeated experiments (Figure 1d and data not shown).…”

“…We first examined whether SPA-1 expressed in Ton.B210/SPA-1 cells inhibits Rap1 activation by IL-3 stimulation or by BCR/ABL expression. In accordance with our previous reports, IL-3 stimulation as well as the induction of BCR/ABL expression by Dox treatment activated Rap1 in Ton.B210 cells (Figure 1b) (Arai et al, 2001;Mizuchi et al, 2005). On the other hand, the SPA-1 expression in Ton.B210/SPA-1 cells not only reduced the basal level of Rap1-GTP significantly but also inhibited Rap1 activation by IL-3 and BCR/ABL almost completely (Figure 1b).…”

“…This is because SPA-1, unlike dominant-negative mutants of Rap1, very strongly inhibits Rap1 in cells through its GAP activity specific for Rap1 (Kurachi et al, 1997;van den Berghe et al, 1997;Mizuchi et al, 2005). As shown in Figure 1a, the expression of exogenous SPA-1 tagged with the Myc epitope in the selected clone Ton.B210/SPA-1 was confirmed by anti-Myc immunoblotting.…”

“…Kinase activities of B-Raf and Raf-1 were measured as described previously (Bondzi et al, 2000;Mizuchi et al, 2005). In brief, B-Raf or Raf-1 was immunoprecipitated and incubated with 20 mM ATP and GST-MEK-1 fusion protein (Upstate Biotechnology) at 301C for 15 min in a kinase reaction buffer containing 20 mM Tris (pH 7.4), 20 mM NaCl, 1 mM DTT, 10 mM MgCl 2 , and 1 mM MnCl 2 .…”

“…We previously found that hematopoietic cytokines, IL-3 and Epo, as well as BCR/ABL activate Rap1 in hematopoietic cells (Arai et al, 2001;Mizuchi et al, 2005). Rap1 is a small GTPase of the Ras family, which was initially identified as a homolog of Ras (Pizon et al, 1988) and was also identified independently by its ability to induce a flat revertant phenotype in K-Ras-transformed cells (Kitayama et al, 1989).…”