Extra-long Bim (BimEL) possesses a unique exon that encodes an ERK1/2 docking domain and three ERK1/2 phosphorylation find more sites [28, 29]. ERK1/2 phosphorylates Bim at Ser65, which downregulates Bim function by inducing either Bim degradation via the proteasomal pathway or Bim dissociation from Mcl-1 and Bcl-xL [30-32]. Since the MEK inhibitor diminished IL-15-mediated CD8αα+ iIEL survival (Fig. 1B), we examined the effect of IL-15 on Bim. BimEL is the predominant isoform expressed by CD8αα+ iIELs (Fig. 3A) as in other types of cells
. IL-15 treatment induced BimEL phosphorylation at Ser65 with similar kinetics as ERK1/2 phosphorylation (Fig. 3A). Withdrawal of IL-15 from cells that had been cultured in IL-15 for 40 h resulted in a simultaneous loss of BimEL and ERK1/2 phosphorylation (Fig. 3B). The similar kinetics between the change of Epigenetics inhibitor BimEL and ERK1/2 phosphorylation in response to IL-15 treatment or withdrawal implies a direct relationship between the two events. We examined this possibility using MEK and upstream PI3K inhibitors, and found that both inhibitors abolished IL-15-induced phosphorylation of ERK1/2 as well as BimEL (Fig. 3C). Moreover, neither IL-15 treatment (Fig. 3A and C) nor IL-15 withdrawal (Fig. 3B) affected the abundance of BimEL. Treatment with inhibitors to MEK or PI3K also did not alter BimEL abundance (Fig. 3C). Taken together, these results demonstrate
that IL-15 induces BimEL phosphorylation at Ser65 via activation of ERK1/2 without downregulating BimEL abundance in CD8αα+ iIELs. We then examined the from role of Bim in CD8αα+ iIEL survival. Bim−/− cells showed prolonged survival compared to WT cells in medium alone (Fig. 4A). IL-15 treatment enhanced the survival of both WT and Bim−/− cells to a similar level (Fig. 4A). Since Bim promotes cell death by binding to the prosurvival Bcl-2 members, we examined Bcl-2 expression in Bim−/− cells. The level of Bcl-2 in freshly isolated Bim−/− iIELs was slightly lower than that in the WT cells (Fig. 4B). IL-15 treatment upregulated Bcl-2 in Bim−/− iIELs to a similar level as in WT cells (Fig. 4B, line
graphs). Also similar to WT cells, ABT-737 reduced the survival of Bim−/− cells cultured in either medium alone or in IL-15 (Fig. 4C). The IC50 of ABT-737 followed the order of Bim−/−/IL-15 > Bim−/−/medium > WT/IL15 > WT/medium (Fig. 4C). Despite Bim−/− cells harboring slightly less Bcl-2 than WT cells, they required much more ABT-737 to diminish cell survival. As ABT-737 mimics the BH3-only protein in binding the prosurvival Bcl-2, the elevated IC50 suggests an increase of “free” Bcl-2 in Bim−/− cells that needed to be inhibited by ABT-737 and implies sequestering of Bcl-2 by Bim in WT CD8αα+ iIELs. This possibility is also in line with the elevation of ABT-737 IC50 for the IL-15-treated cells (Fig. 2D), as IL-15 upregulated Bcl-2 level (Fig. 2A).