Rax, the cellular pkr activator, potentiates ceramide-induced apoptosis in hematopoietic cells

Abstract

We previously identified a novel IL-3 signaling pathway that features regulation of protein synthesis via the double-stranded RNA kinase, PKR (Ito, T., Jaguas, R., and May, W. S. Proc. Natl. Acad. Sci. USA 91:7455–7459, 1994). Stress applications (e.g. IL-3 withdrawal) result in PKR activation and inhibition of protein synthesis. Recently, we discovered a novel murine cellular protein, RAX, which activates PKR (Ito, T., Yang, M., and May, W. S. J. Biol. Chem 274:15427–15432, 1999). RAX is ubiquitously expressed at low levels and exogenous expression augments cell death induced by IL-3 withdrawal suggesting that a stress stimulus can somehow up-regulate RAX. RAX and its human counterpart, PACT, represent the first cellular activators of PKR. RAX is phosphorylated upon IL-3 withdrawal not by PKR but by an as yet unidentified stress-activated protein kinase. Importantly, RAX activation is observed during diverse apoptotic stimuli. The second messenger molecule and potent apoptotic agent ceramide is (1) produced during diverse apoptotic stress conditions (including growth factor withdrawal) and (2) a potent agonist of stress-activated protein kinases. Therefore, we postulate that ceramide may regulate RAX. Consistent with this notion, we have now found that addition of C2-ceramide but not the inactive dihydro analog induces overexpression of human RAX in acute myeloid leukemia derived HL60 cells. Increases in human RAX expression with increasing ceramide concentrations correlate with the promotion of apoptosis in these cells. Moreover, the overexpression of exogenous RAX enhances ceramide-induced apoptosis in murine NSF/N1.H7 cells. These findings suggest a novel mechanism where ceramide may participate in stress-signaling pathways that activate RAX in hematopoietic cells. Since PKR has demonstrated potential as a tumor suppressor, it may be possible to exploit RAX activation to optimize this PKR tumor suppressor function and thus provide novel therapies for hematologic malignancies.