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Sestrins represent a family of stress-inducible proteins that prevent the progression of many age- and obesity-associated disorders. Endogenous Sestrins maintain insulin-dependent AKT Ser/Thr kinase (AKT) activation during high-fat diet-induced obesity, and overexpressed Sestrins activate AKT in various cell types, including liver and skeletal muscle cells. Although Sestrin-mediated AKT activation improves metabolic parameters, the mechanistic details underlying such improvement remain elusive. Here, we investigated how Sestrin2, the Sestrin homolog highly expressed in liver, induces strong AKT activation. We found that two known targets of Sestrin2, mTOR complex (mTORC) 1 and AMP-activated protein kinase, are not required for Sestrin2-induced AKT activation. Rather, phosphoinositol 3-kinase and mTORC2, kinases upstream of AKT, were essential for Sestrin2-induced AKT activation. Among these kinases, mTORC2 catalytic activity was strongly up-regulated upon Sestrin2 overexpression in an in vitro kinase assay, indicating that mTORC2 may represent the major link between Sestrin2 and AKT. As reported previously, Sestrin2 interacted with mTORC2; however, we found here that this interaction occurs indirectly through GATOR2, a pentameric protein complex that directly interacts with Sestrin2. Deleting or silencing WDR24 (WD repeat domain 24), the GATOR2 component essential for the Sestrin2-GATOR2 interaction, or WDR59, the GATOR2 component essential for the GATOR2-mTORC2 interaction, completely ablated Sestrin2-induced AKT activation. We also noted that Sestrin2 also directly binds to the pleckstrin homology domain of AKT and induces AKT translocation to the plasma membrane. These results uncover a signaling mechanism whereby Sestrin2 activates AKT through GATOR2 and mTORC2. [ABSTRACT FROM AUTHOR]

p70 ribosomal S6 kinase (S6K1), a major substrate of the mammalian target of rapamycin (mTOR) kinase, regulates diverse cellular processes including protein synthesis, cell growth, and survival. Although it is well known that the activity of S6K1 is tightly coupled to its phosphorylation status, the regulation of S6K1 activity by other post-translational modifications such as acetylation has not been well understood. Here we show that the acetylation of the C-terminal region (CTR) of S6K1 blocks mTORC1-dependent Thr-389 phosphorylation, an essential phosphorylation site for S6K1 activity. The acetylation of the CTR of S6K1 is inhibited by the class III histone deacetylases, SIRT1 and SIRT2. An S6K1 mutant lacking acetylation sites in its CTR shows enhanced Thr-389 phosphorylation and kinase activity, whereas the acetylation-mimetic S6K1 mutant exhibits decreased Thr-389 phosphorylation and kinase activity. Interestingly, relative to the acetylation-mimetic S6K1 mutant, the acetylation-defective mutant displays higher affinity toward Raptor, an essential scaffolding component of mTORC1 that recruits mTORC1 substrates. These observations indicate that sirtuin-mediated regulation of S6K1 acetylation is an additional important regulatory modification that impinges on the mechanisms underlying mTORC1-dependent S6K1 activation. [ABSTRACT FROM AUTHOR]

The rapamycin-sensitive mTOR complex 1 (mTORC1) promotes protein synthesis, cell growth, and cell proliferation in response to growth factors and nutritional cues. To elucidate the poorly defined mechanisms underlying mTORC1 regulation, we have studied the phosphorylation of raptor, an mTOR-interacting partner. We have identified six raptor phosphorylation sites that lie in two centrally localized clusters (cluster 1, Ser696/Thr706 and cluster 2, Ser855/Ser859/Ser863/Ser877) using tandem mass spectrometry and generated phosphospecific antibodies for each of these sites. Here we focus primarily although not exclusively on raptor Ser863 phosphorylation. We report that insulin promotes mTORC1-associated phosphorylation of raptor Ser863 via the canonical PI3K/TSC/Rheb pathway in a rapamycin-sensitive manner. mTORC1 activation by other stimuli (e.g. amino acids, epidermal growth factor/MAPK signaling, and cellular energy) also promote raptor Ser863 phosphorylation. Rheb overexpression increases phosphorylation on raptor Ser863 as well as on the five other identified sites (e.g. Ser859, Ser855, Ser877, Ser696, and Thr706). Strikingly, raptor Ser863 phosphorylation is absolutely required for raptor Ser859 and Ser855 phosphorylation. These data suggest that mTORC1 activation leads to raptor multisite phosphorylation and that raptor Ser863 phosphorylation functions as a master biochemical switch that modulates hierarchical raptor phosphorylation (e.g. on Ser859 and Ser855). Importantly, mTORC1 containing phosphorylation site-defective raptor exhibits reduced in vitro kinase activity toward the substrate 4EBP1, with a multisite raptor 6A mutant more strongly defective that single-site raptor S863A. Taken together, these data suggest that complex raptor phosphorylation functions as a biochemical rheostat that modulates mTORC1 signaling in accordance with environmental cues. [ABSTRACT FROM AUTHOR]

We examined the role of glycogen synthase kinase-3β (GSK-3β) inhibition in airway smooth muscle hypertrophy, a structural change found in patients with severe asthma. LiCl, SB216763, and specific small interfering RNA (siRNA) against GSK-3β, each of which inhibit GSK-3β activity or expression, increased human bronchial smooth muscle cell size, protein synthesis, and expression of the contractile proteins α-smooth muscle actin, myosin light chain kinase, smooth muscle myosin heavy chain, and SM22. Similar results were obtained following treatment of cells with cardiotrophin (CT)-1, a member of the interleukin-6 superfamily, and transforming growth factor (TGF)-β, a proasthmatic cytokine. GSK-3β inhibition increased mRNA expression of α-actin and transactivation of nuclear factors of activated T cells and serum response factor. siRNA against eukaryotic translation initiation factor 2Bϵ (eIF2Bϵ) attenuated LiCl- and SB216763-induced protein synthesis and expression of β-actin and SM22, indicating that eIF2B is required for GSK-3β-mediated airway smooth muscle hypertrophy. eIF2Bϵ; siRNA also blocked CT-1- but not TGF-β-induced protein synthesis. Infection of human bronchial smooth muscle cells with pMSCV GSK-3β-A9, a retroviral vector encoding a constitutively active, nonphosphorylatable GSK-3β, blocked protein synthesis and α-actin expression induced by LiCl, SB216763, and CT-1 but not TGF-β. Finally, lungs from ovalbumin-sensitized and -challenged mice demonstrated increased β-actin and CT-1 mRNA expression, and airway myocytes isolated from ovalbumin-treated mice showed increased cell size and GSK-3β phosphorylation. These data suggest that inhibition of the GSK-3β/eIF2Bϵ translational control pathway contributes to airway smooth muscle hypertrophy in vitro and in vivo. On the other hand, TGF-β-induced hypertrophy does not depend on GSK-3β/eIF2B signaling. [ABSTRACT FROM AUTHOR]