[PMC free article] [PubMed] [Google Scholar]Li J, Liu J, Track J, Wang X, Weiss HL, Townsend CM, Jr, Gao T, Evers BM. general control nonderepressible 2 (GCN2), Protein kinase RNA-activated also known as protein kinase R (PKR), and The heme-regulated inhibitor (HRI) (Donnelly et al., 2013). There exists an inverse relationship between mTOR signaling and phospho-eIF2. Under pathological conditions, such as apoptosis, hypoxia, serum and nutrient deprivation, mTOR activity is usually Noopept downregulated, whereas phospho-eIF2is usually upregulated leading to diminished global protein synthesis (Deng et al., 2002; Hara et al., 1998; Kim et al., 2002; Koumenis et al., 2002; Liu et al., 2006; Schneider et al., 2008; Tee and Proud, 2001). In some instances mTOR and eIF2 may act in Noopept parallel. For example, deletion of TSC2, an inhibitor of mTOR signaling, augments signaling both by mTOR and PERK (Ozcan et al., 2008). Pharmacologic and genetic studies suggest that mTOR signaling does influence phospho-eIF2. For instance, the mTOR inhibitor, Noopept rapamycin, is known to upregulate phospho-eIF2 in some cells (Anand and Gruppuso, 2006; Kato et al., 2012; Kubota et al., 2003; Matsuo et al., 2005). Similarly, heat inactivation of mTOR potentiates phospho-eIF2 in yeast cells, whereas the genetic deletion of PTEN, a negative regulator of mTOR, reduces phospho-eIF2 in cancerous cells (Mounir et al., 2009; Valbuena et al., 2012). The GTPase Rheb is usually well established as an inducer of mTOR thereby augmenting protein synthesis. Here we demonstrate that Rheb plays a major role in inhibiting protein synthesis by enhancing PERK-mediated phospho-eIF2 levels. This action may underlie, in part, the reciprocal relationship of mTOR and eIF2 signaling. RESULTS GTPase Rheb inhibits protein synthesis The canonical mTOR pathway involves TSC1/2 binding to and inhibiting Rheb, preventing activation of mTOR signaling by Rheb (Inoki et al., 2003). Rheb, acting via mTOR, is generally Rabbit polyclonal to Nucleostemin regarded as a physiologic stimulant of protein synthesis (Hall et al., 2007; Wang et al., 2008). By contrast, in HEK293 cells, we observe that overexpression of Rheb is usually associated with diminished protein synthesis, as measured by incorporation of [35S]-Met (Physique. 1A). Tunicamycin, an ER stressor, elicits cell stress and inhibited protein synthesis (Physique 1A). We confirm that overexpressing Rheb augments phospho-S6 kinase, an index of mTOR signaling (Physique S1). The marked stimulation by tunicamycin of CHOP, an apoptotic protein, confirms its stressor actions (Physique S1). However, Rheb overexpression does not affect CHOP levels (Physique S1), indicating that diminished incorporation of [35S]-Met by Rheb (Physique 1A) may not be due to cellular stress. Inhibition of protein synthesis by Rheb is dependent upon its guanine-nucleotide binding, as it is usually absent with Rheb-D60K (Physique 1B), which cannot bind GTP or GDP (Aspuria and Tamanoi, 2004). Polysome profiles revealed the Rheb WT overexpressing cells showed reduced polysome/monosome ratio (Physique 1C). We next assessed whether Rheb can directly modulate protein synthesis (Physique 1D). Rheb WT, but not Rheb D60K, markedly decreases luciferase mRNA translation, an effect also elicited by active PERK kinase, a known inhibitor of protein synthesis (Harding et al., 1999). Rheb overexpression increased the viability of the HEK293 cells using MTS assay (which steps the mitochondrial activity) but it did not significantly alter the cell number (counted using hemocytometer) (Physique S2). In TSC2 depleted fibroblasts, which possess high RhebCmTOR activity (Inoki et al., 2003; Zhang et al., 2003), we observe diminished protein synthesis (Physique 1E), consistent with previous report compared to TSC2 intact cells (Auerbach et al., 2011). TSC2 depleted fibroblasts also exhibited reduced polysome/monosome ratio (Physique 1F) and diminished cell numbers, compared to TSC2 intact cells (Physique S3). Thus, GTPase.