Ras homologue enriched in brain (Rheb) enhances apoptosis

The small GTPase Rheb has been described as a switch for mammalian target of rapamycin (mTOR) kinase-mediated cellular processes such as cell volume, growth, regeneration and autophagy^[1,2]. Here we show that overexpression of Rheb by transfection of primary cortical neurons enhances cell death after excitotoxic treatment with glutamate. Moreover, we demonstrate Rheb-enhanced apoptotic cell death after apoptotic stimulation by UV-light, TNFa or tunicamycin using Hela cells. Apoptotic stimuli such as UV treatment increased endogenous Rheb expression. Conversely, inhibition of endogenous or exogenous Rheb by either shRNA knockdown or rapamycin led to a partial protection. Furthermore, co-expression of a dominant active or dominant inactive cRaf mutant showed a protective effect indicating that the Raf kinase gene product is involved. Correspondingly, chemical shift assays derived from NMR backbone assignements ^[3,4] revealed a Ras effector-like binding of Rheb to cRaf-RBD. These results indicated an interaction between Rheb and Raf-RBD at low affinity and a Raf kinase activity-independent survival pathway. Correspondingly, small hairpin RNA-mediated knockdown of apoptosis signaling kinase (ASK-1) inhibited Rheb-enhanced apoptosis after application of UV light. We conclude that the Rheb-mTOR pathway is not only promoting cell growth and regeneration but potentiates apoptotic cell death by a Raf-kinase activity independent physiological mechanism mediated by ASK-1.