The increase in BIK expression following ERK1/2 pathway inhibition is a consequence of G1 cell-cycle arrest and not a direct effect on BIK protein stability

Abstract

BIK (BCL2-interacting killer) is a pro-apoptotic BH3 (BCL2 homology domain 3)-only protein and a member of the BCL2 protein family. It was proposed recently that BIK abundance is controlled by ERK1/2 (extracellular-signal-regulated kinase 1/2)-catalysed phosphorylation, which targets the protein for proteasome-dependent destruction. In the present study, we examined ERK1/2-dependent regulation of BIK, drawing comparisons with BIM(EL) (BCL2-interacting mediator of cell death; extra long), a well-known target of ERK1/2. In many ERK1/2-dependent tumour cell lines, inhibition of BRAF(V600E) (v-raf murine sarcoma viral oncogene homologue B1, V600E mutation) or MEK1/2 (mitogen-activated protein kinase/ERK kinase 1/2) had very little effect on BIK expression, whereas BIM(EL) was strongly up-regulated. In some cell lines we observed a modest increase in BIK expression; however, this was not apparent until ~16 h or later, whereas BIM(EL) expression increased rapidly within a few hours. Although BIK was degraded by the proteasome, we found no evidence that this was regulated by ERK1/2 signalling. Rather, the delayed increase in BIK expression was prevented by actinomycin D, and was accompanied by increases in BIK mRNA. Finally, the delayed increase in BIK expression following ERK1/2 inhibition was phenocopied by a highly selective CDK4/6 (cyclin-dependent kinases 4 and 6) inhibitor, which caused a strong G₁ cell-cycle arrest without inhibiting ERK1/2 signalling. In contrast, BIM(EL) expression was induced by ERK1/2 inhibition, but not by CDK4/6 inhibition. We conclude that BIK expression is not subject to direct regulation by the ERK1/2 pathway; rather, we propose that BIK expression is cell-cycle-dependent and increases as a consequence of the G₁ cell-cycle arrest which results from inhibition of ERK1/2 signalling.