Endoplasmic Reticulum-associated Degradation Controls Cell Surface Expression of γ-Aminobutyric Acid,Type B Receptors

Metabotropic GABA_B receptors are crucial for controlling the excitability of neurons by mediating slow inhibition in the CNS. The strength of receptor signaling depends on the number of cell surface receptors, which is thought to be regulated by trafficking and degradation mechanisms. Although the mechanisms of GABA_B receptor trafficking are studied to some extent, it is currently unclear whether receptor degradation actively controls the number of GABA_B receptors available for signaling. Here we tested the hypothesis that proteasomal degradation contributes to the regulation of GABA_B receptor expression levels. Blocking proteasomal activity in cultured cortical neurons considerably enhanced total and cell surface expression of GABA_B receptors, indicating the constitutive degradation of the receptors by proteasomes. Proteasomal degradation required Lys⁴⁸-linked polyubiquitination of lysines 767/771 in the C-terminal domain of the GABA_B2 subunit. Inactivation of these ubiquitination sites increased receptor levels and GABA_B receptor signaling in neurons. Proteasomal degradation was mediated by endoplasmic reticulum-associated degradation (ERAD) as shown by the accumulation of receptors in the endoplasmic reticulum upon inhibition of proteasomes, by the increase of receptor levels, as well as receptor signaling upon blocking ERAD function, and by the interaction of GABA_B receptors with the essential ERAD components Hrd1 and p97. In conclusion, the data support a model in which the fraction of GABA_B receptors available for plasma membrane trafficking is regulated by degradation via the ERAD machinery. Thus, modulation of ERAD activity by changes in physiological conditions may represent a mechanism to adjust receptor numbers and thereby signaling strength.     

Numerous Classes of General Anesthetics Inhibit Etomidate Binding to ��-Aminobutyric Acid Type A (GABAA) Receptors

Enhancement of γ-aminobutyric acid type A receptor (GABA_AR)-mediated inhibition is a property of most general anesthetics and a candidate for a molecular mechanism of anesthesia. Intravenous anesthetics, including etomidate, propofol, barbiturates, and neuroactive steroids, as well as volatile anesthetics and long-chain alcohols, all enhance GABA_AR function at anesthetic concentrations. The implied existence of a receptor site for anesthetics on the GABA_AR protein was supported by identification, using photoaffinity labeling, of a binding site for etomidate within the GABA_AR transmembrane domain at the β-α subunit interface; the etomidate analog [³H]azietomidate photolabeled in a pharmacologically specific manner two amino acids, α1Met-236 in the M1 helix and βMet-286 in the M3 helix (Li, G. D., Chiara, D. C., Sawyer, G. W., Husain, S. S., Olsen, R. W., and Cohen, J. B. (2006) J. Neurosci. 26, 11599–11605). Here, we use [³H]azietomidate photolabeling of bovine brain GABA_ARs to determine whether other structural classes of anesthetics interact with the etomidate binding site. Photolabeling was inhibited by anesthetic concentrations of propofol, barbiturates, and the volatile agent isoflurane, at low millimolar concentrations, but not by octanol or ethanol. Inhibition by barbiturates, which was pharmacologically specific and stereospecific, and by propofol was only partial, consistent with allosteric interactions, whereas isoflurane inhibition was nearly complete, apparently competitive. Protein sequencing showed that propofol inhibited to the same extent the photolabeling of α1Met-236 and βMet-286. These results indicate that several classes of general anesthetics modulate etomidate binding to the GABA_AR: isoflurane binds directly to the site with millimolar affinity, whereas propofol and barbiturates inhibit binding but do not bind in a mutually exclusive manner with etomidate.     

Neurosteroids Allosterically Modulate Binding of the Anesthetic Etomidate to ��-Aminobutyric Acid Type A Receptors

Photoaffinity labeling of γ-aminobutyric acid type A (GABA_A)-receptors (GABA_AR) with an etomidate analog and mutational analyses of direct activation of GABA_AR by neurosteroids have each led to the proposal that these structurally distinct general anesthetics bind to sites in GABA_ARs in the transmembrane domain at the interface between the β and α subunits. We tested whether the two ligand binding sites might overlap by examining whether neuroactive steroids inhibited etomidate analog photolabeling. We previously identified (Li, G. D., Chiara, D. C., Sawyer, G. W., Husain, S. S., Olsen, R. W., and Cohen, J. B. (2006) J. Neurosci. 26, 11599–11605) azietomidate photolabeling of GABA_AR α1Met-236 and βMet-286 (in αM1 and βM3). Positioning these two photolabeled amino acids in a single type of binding site at the interface of β and α subunits (two copies per pentamer) is consistent with a GABA_AR homology model based upon the structure of the nicotinic acetylcholine receptor and with recent αM1 to βM3 cross-linking data. Biologically active neurosteroids enhance rather than inhibit azietomidate photolabeling, as assayed at the level of GABA_AR subunits on analytical SDS-PAGE, and protein microsequencing establishes that the GABA_AR-modulating neurosteroids do not inhibit photolabeling of GABA_AR α1Met-236 or βMet-286 but enhance labeling of α1Met-236. Thus modulatory steroids do not bind at the same site as etomidate, and neither of the amino acids identified as neurosteroid activation determinants (Hosie, A. M., Wilkins, M. E., da Silva, H. M., and Smart, T. G. (2006) Nature 444, 486–489) are located at the subunit interface defined by our etomidate site model.GABA_A³ receptors (GABA_AR) are major mediators of brain inhibitory neurotransmission and participate in most circuits and behavioral pathways relevant to normal and pathological function (1). GABA_AR are subject to modulation by endogenous neurosteroids, as well as myriad clinically important central nervous system drugs including general anesthetics, benzodiazepines, and possibly ethanol (1, 2). The mechanism of GABA_AR modulation by these different classes of drugs is of major interest, including identification of the receptor amino acid residues involved in binding and action of the drugs.In the absence of high resolution crystal structures of drug-receptor complexes, the locations of anesthetic binding sites in GABA_ARs have been predicted based upon analyses of functional properties of point mutant receptors, which identified residues in the α and β subunit M1–M4 transmembrane helices important for modulation by volatile anesthetics (primarily α subunit) and by intravenous agents, including etomidate and propofol (β subunit) (3–5). Position βM2–15, numbered relative to the N terminus of the helix, functions as a major determinant of etomidate and propofol potency as GABA modulators in vitro and in vivo (6–8). By contrast, this residue is not implicated for modulation by the neurosteroids, potent endogenous modulators of GABA_AR (9).Photoaffinity labeling, which allows the identification of residues in proximity to drug binding sites (10, 11), has been used to identify two GABA_AR amino acids covalently modified by the etomidate analog [³H]azietomidate (12): α1Met-236 within αM1 and βMet-286 within βM3. Photolabeling of these residues was inhibited equally by nonradioactive etomidate and enhanced proportionately by GABA present in the assay, consistent with the presence of these two residues in a common drug binding pocket that would be located at the interface between the β and α subunits in the transmembrane domain (12). Mutational analyses identify these positions as etomidate and propofol sensitivity determinants (13–15).A recent mutagenesis study (16) identified two other residues in GABA_AR αM1 and βM3 as critical for direct activation by neurosteroids, αThr-236 (rat numbering, corresponding to α1Thr-237, bovine numbering used here and by Li et al. (12))⁴ and βTyr-284. These residues were also proposed to contribute to a neurosteroid binding pocket in the transmembrane domain at the interface between β and α subunits, based upon their location in an alternative GABA_AR structural model that positioned those amino acids, and not α1Met-236 or βMet-286, at the subunit interface. For GABA_ARs and other members of the Cys-loop superfamily of neurotransmitter-gated ion channels, the transmembrane domain of each subunit is made up of a loose bundle of four α helices (M1–M4), with M2 from each subunit contributing to the lumen of the ion channel and M4 positioned peripherally in greatest contact with lipid, as seen in the structures of the Torpedo nicotinic acetylcholine receptor (nAChR) (17) and in distantly related prokaryote homologs (18). However, uncertainties in the alignment of GABA_AR subunit sequences relative to those of the nAChR result in alternative GABA_AR homology models (12, 19, 20) that differ in the location of amino acids in the M3 and M4 membrane-spanning helices and in the M1 helix in some models (16, 21).If etomidate and neurosteroids both bind at the same β/α interface in the GABA_AR transmembrane domain, the limited space available for ligand binding suggests that their binding pockets might overlap and that ligand binding would be mutually exclusive. To address this question, we photolabeled purified bovine brain GABA_AR with [³H]azietomidate in the presence of different neuroactive steroids and determined by protein microsequencing whether active neurosteroids inhibited labeling of α1Met-236 and βMet-286, as expected for mutually exclusive binding, or resulted in [³H]azietomidate photolabeling of other amino acids, a possible consequence of allosteric interactions. Active steroids failed to inhibit labeling and enhanced labeling of α1Met-236, clearly indicating that the neurosteroid and the etomidate sites are distinct. Our GABA_AR homology model that positions α1Met-236 and βMet-286 at the β/α interface, but not that of Hosie et al. (16), is also consistent with cysteine substitution cross-linking studies (20, 22), which define the proximity relations between amino acids in the αM1, αM2, αM3, and βM3 helices, and these results support the interpretation that the two residues photolabeled by [³H]azietomidate are part of a single subunit interface binding pocket, whereas the steroid sensitivity determinants identified by mutagenesis neither are at the β/α subunit interface nor are contributors to a common binding pocket.     

Blocking L-type Voltage-gated Ca2+ Channels with Dihydropyridines Reduces ��-Aminobutyric Acid Type A Receptor Expression and Synaptic Inhibition

γ-Aminobutyric acid type A receptors (GABA_ARs) are the major sites of fast inhibitory neurotransmission in the brain, and the numbers of these receptors at the cell surface can determine the strength of GABAergic neurotransmission. Chronic changes in neuronal activity lead to an adaptive modulation in the efficacy of GABAergic synaptic inhibition, brought about in part by changes in the number of synaptic GABA_ARs, a mechanism known as homeostatic synaptic plasticity. Reduction in the number of GABA_ARs in response to prolonged neuronal activity blockade is dependent on the ubiquitin-proteasome system. The underlying biochemical pathways linking chronic activity blockade to proteasome-dependent degradation of GABA_ARs are unknown. Here, we show that chronic blockade of L-type voltage-gated calcium channels (VGCCs) with nifedipine decreases the number of GABA_ARs at synaptic sites but not the overall number of inhibitory synapses. In parallel, blockade of L-type VGCCs decreases the amplitude but not the frequency of miniature inhibitory postsynaptic currents or expression of the glutamic acid decarboxylase GAD65. We further reveal that the activation of L-type VGCCs regulates the turnover of newly translated GABA_AR subunits in a mechanism dependent upon the activity of the proteasome and thus regulates GABA_AR insertion into the plasma membrane. Together, these observations suggest that activation of L-type VGCCs can regulate the abundance of synaptic GABA_ARs and the efficacy of synaptic inhibition, revealing a potential mechanism underlying the homeostatic adaptation of fast GABAergic inhibition to prolonged changes in activity.     

Mutations at Beta N265 in γ-Aminobutyric Acid Type A Receptors Alter Both Binding Affinity and Efficacy of Potent Anesthetics

Etomidate and propofol are potent general anesthetics that act via GABAA receptor allosteric co-agonist sites located at transmembrane β+/α− inter-subunit interfaces. Early experiments in heteromeric receptors identified βN265 (M2-15′) on β2 and β3 subunits as an important determinant of sensitivity to these drugs. Mechanistic analyses suggest that substitution with serine, the β1 residue at this position, primarily reduces etomidate efficacy, while mutation to methionine eliminates etomidate sensitivity and might prevent drug binding. However, the βN265 residue has not been photolabeled with analogs of either etomidate or propofol. Furthermore, substituted cysteine modification studies find no propofol protection at this locus, while etomidate protection has not been tested. Thus, evidence of contact between βN265 and potent anesthetics is lacking and it remains uncertain how mutations alter drug sensitivity. In the current study, we first applied heterologous α1β2N265Cγ2L receptor expression in Xenopus oocytes, thiol-specific aqueous probe modification, and voltage-clamp electrophysiology to test whether etomidate inhibits probe reactions at the β-265 sidechain. Using up to 300 µM etomidate, we found both an absence of etomidate effects on α1β2N265Cγ2L receptor activity and no inhibition of thiol modification. To gain further insight into anesthetic insensitive βN265M mutants, we applied indirect structure-function strategies, exploiting second mutations in α1β2/3γ2L GABAA receptors. Using α1M236C as a modifiable and anesthetic-protectable site occupancy reporter in β+/α− interfaces, we found that βN265M reduced apparent anesthetic affinity for receptors in both resting and GABA-activated states. βN265M also impaired the transduction of gating effects associated with α1M236W, a mutation that mimics β+/α− anesthetic site occupancy. Our results show that βN265M mutations dramatically reduce the efficacy/transduction of anesthetics bound in β+/α− sites, and also significantly reduce anesthetic affinity for resting state receptors. These findings are consistent with a role for βN265 in anesthetic binding within the β+/α− transmembrane sites.     

Small GTPase Rab17 Regulates the Surface Expression of Kainate Receptors but Not α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors in Hippocampal Neurons via Dendritic Trafficking of Syntaxin-4 Protein

Glutamate receptors are fundamental for control synaptic transmission, synaptic plasticity, and neuronal excitability. However, many of the molecular mechanisms underlying their trafficking remain elusive. We previously demonstrated that the small GTPase Rab17 regulates dendritic trafficking in hippocampal neurons. Here, we investigated the role(s) of Rab17 in AMPA receptor (AMPAR) and kainate receptor (KAR) trafficking. Although Rab17 knockdown did not affect surface expression of the AMPAR subunit GluA1 under basal or chemically induced long term potentiation conditions, it significantly reduced surface expression of the KAR subunit GluK2. Rab17 co-localizes with Syntaxin-4 in the soma, dendritic shaft, the tips of developing hippocampal neurons, and in spines. Rab17 knockdown caused Syntaxin-4 redistribution away from dendrites and into axons in developing hippocampal neurons. Syntaxin-4 knockdown reduced GluK2 but had no effect on GluA1 surface expression. Moreover, overexpression of constitutively active Rab17 promoted dendritic surface expression of GluK2 by enhancing Syntaxin-4 translocation to dendrites. These data suggest that Rab17 mediates the dendritic trafficking of Syntaxin-4 to selectively regulate dendritic surface insertion of GluK2-containing KARs in rat hippocampal neurons.     

Dendritic Assembly of Heteromeric ��-Aminobutyric Acid Type B Receptor Subunits in Hippocampal Neurons

Understanding the mechanisms that control synaptic efficacy through the availability of neurotransmitter receptors depends on uncovering their specific intracellular trafficking routes. γ-Aminobutyric acid type B (GABA_B) receptors (GABA_BRs) are obligatory heteromers present at dendritic excitatory and inhibitory postsynaptic sites. It is unknown whether synthesis and assembly of GABA_BRs occur in the somatic endoplasmic reticulum (ER) followed by vesicular transport to dendrites or whether somatic synthesis is followed by independent transport of the subunits for assembly and ER export throughout the somatodendritic compartment. To discriminate between these possibilities we studied the association of GABA_BR subunits in dendrites of hippocampal neurons combining live fluorescence microscopy, biochemistry, quantitative colocalization, and bimolecular fluorescent complementation. We demonstrate that GABA_BR subunits are segregated and differentially mobile in dendritic intracellular compartments and that a high proportion of non-associated intracellular subunits exist in the brain. Assembled heteromers are preferentially located at the plasma membrane, but blockade of ER exit results in their intracellular accumulation in the cell body and dendrites. We propose that GABA_BR subunits assemble in the ER and are exported from the ER throughout the neuron prior to insertion at the plasma membrane. Our results are consistent with a bulk flow of segregated subunits through the ER and rule out a post-Golgi vesicular transport of preassembled GABA_BRs.The efficacy of synaptic transmission depends on the intracellular trafficking of neurotransmitter receptors (1, 2). The trafficking of glutamatergic and GABA_A⁶ receptors has been extensively studied, and their implications for synaptic plasticity have been well documented (3, 4). For example, differential trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors modifies synaptic strength and influences experience-dependent plasticity in vivo (5). The molecular mechanisms that govern the trafficking of metabotropic GABA_BRs and their consequences for synaptic inhibition remain less clear. In particular, limited information is available regarding the relationship between the trafficking of GABA_BRs and the topological complexity of the secretory pathway in neurons.GABA_BRs mediate the slow component of synaptic inhibition by acting on pre- and postsynaptic targets (6–8). They are implicated in epilepsy, anxiety, stress, sleep disorders, nociception, depression, and cognition (9). They also represent attractive targets for the treatment of withdrawal symptoms from drugs of addiction such as cocaine (10). They are obligatory heteromers composed of GABA_BR1 and GABA_BR2 subunits. GABA_BR1 contains an RXR-type sequence in the intracellular C-terminal domain that functions as an ER retention motif (11, 12). The ER retention sequence is masked upon assembly with GABA_BR2 resulting in the appearance of functional receptors at the plasma membrane. Only GABA_BR1 binds GABA with high affinity, whereas G protein signaling is exclusively mediated by the second and third intracellular loops of GABA_BR2 (13–15). GABA_BRs are located in dendrites and axons, but their distribution does not coincide with the active zone or the postsynaptic density. Rather, they are adjacent to both compartments constituting perisynaptic receptors (16, 17).If GABA_BR subunits are synthesized in the soma, at least two possibilities exist for their anterograde transport, assembly, and insertion in dendrites. First, the subunits may be synthesized in the cell body, assembled in the somatic ER, and targeted preassembled in post-Golgi vesicles to their site of insertion in dendrites. Alternatively, they may be synthesized in the soma and transported through the ER membrane as non-heteromeric subunits. In the latter scenario, newly assembled receptors may exit the ER throughout the somatodendritic compartment prior to insertion at the plasma membrane and diffuse laterally for retention at functional sites. No evidence exists to discriminate between these possibilities. We reasoned that a prevalence of associated subunits in post-Golgi vesicles in dendrites would favor the first alternative, whereas the existence of non-associated subunits in intracellular compartments would support a somatodendritic assembly mechanism. Here we explore the presence of associated GABA_BR subunits using fluorescence recovery after photobleaching (FRAP), biochemistry, and quantitative colocalization. In addition, we report for the first time the use of BiFC (18) to study GABA_BR assembly in neurons. Our results demonstrate that GABA_BR subunits are differentially mobile in dendrites and that a high proportion of non-associated subunits prevail in an intracellular fraction of the adult brain. They also show that GABA_BR subunits are heteromeric at the plasma membrane but segregated in intracellular compartments of dendrites of hippocampal neurons. Importantly, treatment with brefeldin A (BFA) or interference of the coatomer protein complex II impair ER export and result in the accumulation of assembled subunits in intracellular compartments throughout the somatodendritic arbor. We conclude that GABA_BR subunits are synthesized in the soma and remain segregated in intracellular compartments prior to somatodendritic assembly. Our observations rule out a post-Golgi vesicular transport of preassembled GABA_BRs and suggest an alternative mechanism of receptor targeting.     

Combining Valosin-containing Protein (VCP) Inhibition and Suberanilohydroxamic Acid (SAHA) Treatment Additively Enhances the Folding,Trafficking, and Function of Epilepsy-associated γ-Aminobutyric Acid,Type A (GABAA) Receptors

GABA_A receptors are the primary inhibitory ion channels in the mammalian central nervous system. The A322D mutation in the α1 subunit results in its excessive endoplasmic reticulum-associated degradation at the expense of plasma membrane trafficking, leading to autosomal dominant juvenile myoclonic epilepsy. Presumably, valosin-containing protein (VCP)/p97 extracts misfolded subunits from the endoplasmic reticulum membrane to the cytosolic proteasome for degradation. Here we showed that inhibiting VCP using Eeyarestatin I reduces the endoplasmic reticulum-associated degradation of the α1(A322D) subunit without an apparent effect on its dynamin-1 dependent endocytosis and that this treatment enhances its trafficking. Furthermore, coapplication of Eeyarestatin I and suberanilohydroxamic acid, a known small molecule that promotes chaperone-assisted folding, yields an additive restoration of surface expression of α1(A322D) subunits in HEK293 cells and neuronal SH-SY5Y cells. Consequently, this combination significantly increases GABA-induced chloride currents in whole-cell patch clamping experiments than either chemical compound alone in HEK293 cells. Our findings suggest that VCP inhibition without stress induction, together with folding enhancement, represents a new strategy to restore proteostasis of misfolding-prone GABA_A receptors and, therefore, a potential remedy for idiopathic epilepsy.     

Agonist-dependent Endocytosis of γ-Aminobutyric Acid Type A (GABAA) Receptors Revealed by a γ2(R43Q) Epilepsy Mutation

GABA-gated chloride channels (GABA_ARs) trafficking is involved in the regulation of fast inhibitory transmission. Here, we took advantage of a γ2(R43Q) subunit mutation linked to epilepsy in humans that considerably reduces the number of GABA_ARs on the cell surface to better understand the trafficking of GABA_ARs. Using recombinant expression in cultured rat hippocampal neurons and COS-7 cells, we showed that receptors containing γ2(R43Q) were addressed to the cell membrane but underwent clathrin-mediated dynamin-dependent endocytosis. The γ2(R43Q)-dependent endocytosis was reduced by GABA_AR antagonists. These data, in addition to a new homology model, suggested that a conformational change in the extracellular domain of γ2(R43Q)-containing GABA_ARs increased their internalization. This led us to show that endogenous and recombinant wild-type GABA_AR endocytosis in both cultured neurons and COS-7 cells can be amplified by their agonists. These findings revealed not only a direct relationship between endocytosis of GABA_ARs and a genetic neurological disorder but also that trafficking of these receptors can be modulated by their agonist.     

Cysteine Substitutions Define Etomidate Binding and Gating Linkages in the α-M1 Domain of γ-Aminobutyric Acid Type A (GABAA) Receptors

Etomidate is a potent general anesthetic that acts as an allosteric co-agonist at GABA_A receptors. Photoreactive etomidate derivatives labeled αMet-236 in transmembrane domain M1, which structural models locate in the β+/α- subunit interface. Other nearby residues may also contribute to etomidate binding and/or transduction through rearrangement of the site. In human α1β2γ2L GABA_A receptors, we applied the substituted cysteine accessibility method to α1-M1 domain residues extending from α1Gln-229 to α1Gln-242. We used electrophysiology to characterize each mutant''s sensitivity to GABA and etomidate. We also measured rates of sulfhydryl modification by p-chloromercuribenzenesulfonate (pCMBS) with and without GABA and tested if etomidate blocks modification of pCMBS-accessible cysteines. Cys substitutions in the outer α1-M1 domain impaired GABA activation and variably affected etomidate sensitivity. In seven of eight residues where pCMBS modification was evident, rates of modification were accelerated by GABA co-application, indicating that channel activation increases water and/or pCMBS access. Etomidate reduced the rate of modification for cysteine substitutions at α1Met-236, α1Leu-232 and α1Thr-237. We infer that these residues, predicted to face β2-M3 or M2 domains, contribute to etomidate binding. Thus, etomidate interacts with a short segment of the outer α1-M1 helix within a subdomain that undergoes significant structural rearrangement during channel gating. Our results are consistent with in silico docking calculations in a homology model that orient the long axis of etomidate approximately orthogonal to the transmembrane axis.     

A DNA Polymerase α Accessory Protein,Mcl1, Is Required for Propagation of Centromere Structures in Fission Yeast

Specialized chromatin exists at centromeres and must be precisely transmitted during DNA replication. The mechanisms involved in the propagation of these structures remain elusive. Fission yeast centromeres are composed of two chromatin domains: the central CENP-A^Cnp1 kinetochore domain and flanking heterochromatin domains. Here we show that fission yeast Mcl1, a DNA polymerase α (Polα) accessory protein, is critical for maintenance of centromeric chromatin. In a screen for mutants that alleviate both central domain and outer repeat silencing, we isolated several cos mutants, of which cos1 is allelic to mcl1. The mcl1-101 mutation causes reduced CENP-A^Cnp1 in the central domain and an aberrant increase in histone acetylation in both domains. These phenotypes are also observed in a mutant of swi7⁺, which encodes a catalytic subunit of Polα. Mcl1 forms S-phase-specific nuclear foci, which colocalize with those of PCNA and Polα. These results suggest that Mcl1 and Polα are required for propagation of centromere chromatin structures during DNA replication.     

Phosphorylated Ribosomal Protein S6 Is Required for Akt-Driven Hyperplasia and Malignant Transformation,but Not for Hypertrophy,Aneuploidy and Hyperfunction of Pancreatic β-Cells

Constitutive expression of active Akt (Akt^tg) drives hyperplasia and hypertrophy of pancreatic β-cells, concomitantly with increased insulin secretion and improved glucose tolerance, and at a later stage the development of insulinoma. To determine which functions of Akt are mediated by ribosomal protein S6 (rpS6), an Akt effector, we generated mice that express constitutive Akt in β-cells in the background of unphosphorylatable ribosomal protein S6 (rpS6^P-/-). rpS6 phosphorylation deficiency failed to block Akt^tg-induced hypertrophy and aneuploidy in β-cells, as well as the improved glucose homeostasis, indicating that Akt carries out these functions independently of rpS6 phosphorylation. In contrast, rpS6 phosphorylation deficiency efficiently restrained the reduction in nuclear localization of the cell cycle inhibitor p27, as well as the development of Akt^tg-driven hyperplasia and tumor formation in β-cells. In vitro experiments with Akt^tg and rpS6^P-/-;Akt^tg fibroblasts demonstrated that rpS6 phosphorylation deficiency leads to reduced translation fidelity, which might underlie its anti-tumorigenic effect in the pancreas. However, the role of translation infidelity in tumor suppression cannot simply be inferred from this heterologous experimental model, as rpS6 phosphorylation deficiency unexpectedly elevated the resistance of Akt^tg fibroblasts to proteotoxic, genotoxic as well as autophagic stresses. In contrast, rpS6^P-/- fibroblasts exhibited a higher sensitivity to these stresses upon constitutive expression of oncogenic Kras. The latter result provides a possible mechanistic explanation for the ability of rpS6 phosphorylation deficiency to enhance DNA damage and protect mice from Kras-induced neoplastic transformation in the exocrine pancreas. We propose that Akt1 and Kras exert their oncogenic properties through distinct mechanisms, even though both show addiction to rpS6 phosphorylation.