Inhibitory protein-protein interactions of the SIRT1 deacetylase are choreographed by post-translational modification.

Regulation of SIRT1 activity is vital to energy homeostasis and plays important roles in many diseases. We previously showed that insulin triggers the epigenetic regulator DBC1 to prime SIRT1 for repression by the multifunctional trafficking protein PACS-2. Here, we show that liver DBC1/PACS-2 regulates the diurnal inhibition of SIRT1, which is critically important for insulin-dependent switch in fuel metabolism from fat to glucose oxidation. We present the x-ray structure of the DBC1 S1-like domain that binds SIRT1 and an NMR characterization of how the SIRT1 N-terminal region engages DBC1. This interaction is inhibited by acetylation of K112 of DBC1 and stimulated by the insulin-dependent phosphorylation of human SIRT1 at S162 and S172, catalyzed sequentially by CK2 and GSK3, resulting in the PACS-2-dependent inhibition of nuclear SIRT1 enzymatic activity and translocation of the deacetylase in the cytoplasm. Finally, we discuss how defects in the DBC1/PACS-2-controlled SIRT1 inhibitory pathway are associated with disease, including obesity and non-alcoholic fatty liver disease.

Neural deficits in a mouse model of PACS1 syndrome are corrected with PACS1- or HDAC6-targeting therapy.

PACS1 syndrome is a neurodevelopmental disorder (NDD) caused by a recurrent de novo missense mutation in PACS1 (p.Arg203Trp (PACS1R203W)). The mechanism by which PACS1R203W causes PACS1 syndrome is unknown, and no curative treatment is available. Here, we use patient cells and PACS1 syndrome mice to show that PACS1 (or PACS-1) is an HDAC6 effector and that the R203W substitution increases the PACS1/HDAC6 interaction, aberrantly potentiating deacetylase activity. Consequently, PACS1R203W reduces acetylation of α-tubulin and cortactin, causing the Golgi ribbon in hippocampal neurons and patient-derived neural progenitor cells (NPCs) to fragment and overpopulate dendrites, increasing their arborization. The dendrites, however, are beset with varicosities, diminished spine density, and fewer functional synapses, characteristic of NDDs. Treatment of PACS1 syndrome mice or patient NPCs with PACS1- or HDAC6-targeting antisense oligonucleotides, or HDAC6 inhibitors, restores neuronal structure and synaptic transmission in prefrontal cortex, suggesting that targeting PACS1R203W/HDAC6 may be an effective therapy for PACS1 syndrome.

RNA-targeted therapy corrects neuronal deficits in PACS1 syndrome mice.

Neurodevelopmental disorders (NDDs) are frequently associated with dendritic abnormalities in pyramidal neurons that affect arbor complexity, spine density, and synaptic communication 1,2. The underlying genetic causes are often complex, obscuring the molecular pathways that drive these disorders 3. Next-generation sequencing has identified recurrent de novo missense mutations in a handful of genes associated with NDDs, offering a unique opportunity to decipher the molecular pathways 4. One such gene is PACS1, which encodes the multi-functional trafficking protein PACS1 (or PACS-1); a single recurrent de novo missense mutation, c607C>T (PACS1R203W), causes developmental delay and intellectual disability (ID) 5,6. The processes by which PACS1R203W causes PACS1 syndrome are unknown, and there is no curative treatment. We show that PACS1R203W increases the interaction between PACS1 and the α-tubulin deacetylase HDAC6, elevating enzyme activity and appropriating control of its posttranscriptional regulation. Consequently, PACS1R203W reduces acetylation of α-tubulin and cortactin, causing the Golgi to fragment and enter developing neurites, leading to increased dendrite arborization. The dendrites, however, are beset with diminished spine density and fewer functional synapses, characteristic of ID pathology. Treatment of PACS1 syndrome mice with PACS1- or HDAC6-targeting antisense oligonucleotides restores neuronal structure and synaptic transmission, suggesting PACS1R203W/HDAC6 may be targeted for treating PACS1 syndrome neuropathology.

Rab32 uses its effector reticulon 3L to trigger autophagic degradation of mitochondria-associated membrane (MAM) proteins.

BACKGROUND: Rab32 is a small GTPase associated with multiple organelles but is particularly enriched at the endoplasmic reticulum (ER). Here, it controls targeting to mitochondria-ER contacts (MERCs), thus influencing composition of the mitochondria-associated membrane (MAM). Moreover, Rab32 regulates mitochondrial membrane dynamics via its effector dynamin-related protein 1 (Drp1). Rab32 has also been reported to induce autophagy, an essential pathway targeting intracellular components for their degradation. However, no autophagy-specific effectors have been identified for Rab32. Similarly, the identity of the intracellular membrane targeted by this small GTPase and the type of autophagy it induces are not known yet. RESULTS: To investigate the target of autophagic degradation mediated by Rab32, we tested a large panel of organellar proteins. We found that a subset of MERC proteins, including the thioredoxin-related transmembrane protein TMX1, are specifically targeted for degradation in a Rab32-dependent manner. We also identified the long isoform of reticulon-3 (RTN3L), a known ER-phagy receptor, as a Rab32 effector. CONCLUSIONS: Rab32 promotes degradation of mitochondrial-proximal ER membranes through autophagy with the help of RTN3L. We propose to call this type of selective autophagy "MAM-phagy".

Assessment of a mouse xenograft model of primary colorectal cancer with special reference to perfluorooctane sulfonate.

Colorectal cancer ranks third among the most commonly diagnosed cancers in the United States. Current therapies have a range of side effects, and the development of a reliable animal model to speed the discovery of safe effective preventative therapies would be of great value. A cross-sectional study in a large Appalachian population recently showed an association between low circulating levels of perfluorooctane sulfonate (PFOS) and a reduced prevalence of colorectal cancer. A study using APCmin (C57BL/6J-ApcMin/J) mice prone to familial adenomatous polyposis found PFOS was protective when exposure occurred during tumor development. To test the possible benefit of PFOS on spontaneous colorectal cancer, we developed a mouse model utilizing primary patient colorectal cancer implants into NSG (NOD.Cg-PrkdcscidIl2rgtm1Wjl /Sz) mice. Study goals included: (1) to assess potential factors supporting the successful use of colorectal cancer from heterogeneous tumors for PDX studies; and, (2) evaluate PFOS as a therapy in tumor matched pairs of mice randomized to receive PFOS or vehicle. The time in days for mice to grow primary tumors to 5 mm took almost 2 months (mean = 53.3, se = 5.7, range = 17-136). Age of mice at implantation, patient age, gender and race appeared to have no discernable effect on engraftment rates. Engraftment rates for low and high-grade patient tumors were similar. PFOS appeared to reduce tumor size dramatically in one group of tumors, those from the right ascending colon. That is, by 5 weeks of treatment in two mice, PFOS had eliminated their 52.4 mm3 and 124.6 mm3 masses completely, an effect that was sustained for 10 weeks of treatment; in contrast, their corresponding matched vehicle control mice had tumors that grew to 472.7 mm3 and 340.1 mm3 in size respectively during the same period. In a third xenograft mouse, the tumor growth was dramatically blunted although not eliminated, and compared favorably to their matched vehicle controls over the same period. These preliminary findings suggested that this mouse model may be advantageous for testing compounds of potential value in the treatment of colorectal cancer, and PFOS may have utility in selected cases.

An Insulin-Responsive Sensor in the SIRT1 Disordered Region Binds DBC1 and PACS-2 to Control Enzyme Activity.

Current models of SIRT1 enzymatic regulation primarily consider the effects of fluctuating levels of its co-substrate NAD+, which binds to the stably folded catalytic domain. By contrast, the roles of the sizeable disordered N- and C-terminal regions of SIRT1 are largely unexplored. Here we identify an insulin-responsive sensor in the SIRT1 N-terminal region (NTR), comprising an acidic cluster (AC) and a 3-helix bundle (3HB), controlling deacetylase activity. The allosteric assistor DBC1 removes a distal N-terminal shield from the 3-helix bundle, permitting PACS-2 to engage the acidic cluster and the transiently exposed helix 3 of the 3-helix bundle, disrupting its structure and inhibiting catalysis. The SIRT1 activator (STAC) SRT1720 binds and stabilizes the 3-helix bundle, protecting SIRT1 from inhibition by PACS-2. Identification of the SIRT1 insulin-responsive sensor and its engagement by the DBC1 and PACS-2 regulatory hub provides important insight into the roles of disordered regions in enzyme regulation and the mode by which STACs promote metabolic fitness.

A Recurrent De Novo PACS2 Heterozygous Missense Variant Causes Neonatal-Onset Developmental Epileptic Encephalopathy, Facial Dysmorphism, and Cerebellar Dysgenesis.

Developmental and epileptic encephalopathies (DEEs) represent a large clinical and genetic heterogeneous group of neurodevelopmental diseases. The identification of pathogenic genetic variants in DEEs remains crucial for deciphering this complex group and for accurately caring for affected individuals (clinical diagnosis, genetic counseling, impacting medical, precision therapy, clinical trials, etc.). Whole-exome sequencing and intensive data sharing identified a recurrent de novo PACS2 heterozygous missense variant in 14 unrelated individuals. Their phenotype was characterized by epilepsy, global developmental delay with or without autism, common cerebellar dysgenesis, and facial dysmorphism. Mixed focal and generalized epilepsy occurred in the neonatal period, controlled with difficulty in the first year, but many improved in early childhood. PACS2 is an important PACS1 paralog and encodes a multifunctional sorting protein involved in nuclear gene expression and pathway traffic regulation. Both proteins harbor cargo(furin)-binding regions (FBRs) that bind cargo proteins, sorting adaptors, and cellular kinase. Compared to the defined PACS1 recurrent variant series, individuals with PACS2 variant have more consistently neonatal/early-infantile-onset epilepsy that can be challenging to control. Cerebellar abnormalities may be similar but PACS2 individuals exhibit a pattern of clear dysgenesis ranging from mild to severe. Functional studies demonstrated that the PACS2 recurrent variant reduces the ability of the predicted autoregulatory domain to modulate the interaction between the PACS2 FBR and client proteins, which may disturb cellular function. These findings support the causality of this recurrent de novo PACS2 heterozygous missense in DEEs with facial dysmorphim and cerebellar dysgenesis.

Caught in the act - protein adaptation and the expanding roles of the PACS proteins in tissue homeostasis and disease.

Vertebrate proteins that fulfill multiple and seemingly disparate functions are increasingly recognized as vital solutions to maintaining homeostasis in the face of the complex cell and tissue physiology of higher metazoans. However, the molecular adaptations that underpin this increased functionality remain elusive. In this Commentary, we review the PACS proteins - which first appeared in lower metazoans as protein traffic modulators and evolved in vertebrates to integrate cytoplasmic protein traffic and interorganellar communication with nuclear gene expression - as examples of protein adaptation 'caught in the act'. Vertebrate PACS-1 and PACS-2 increased their functional density and roles as metabolic switches by acquiring phosphorylation sites and nuclear trafficking signals within disordered regions of the proteins. These findings illustrate one mechanism by which vertebrates accommodate their complex cell physiology with a limited set of proteins. We will also highlight how pathogenic viruses exploit the PACS sorting pathways as well as recent studies on PACS genes with mutations or altered expression that result in diverse diseases. These discoveries suggest that investigation of the evolving PACS protein family provides a rich opportunity for insight into vertebrate cell and organ homeostasis.

Suppression of calpain expression by NSAIDs is associated with inhibition of cell migration in rat duodenum.

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used for the alleviation of pain and inflammation, but these drugs are also associated with a suite of negative side effects. Gastrointestinal (GI) toxicity is particularly concerning since it affects an estimated 70% of individuals taking NSAIDs routinely, and evidence suggests the majority of toxicity is occurring in the small intestine. Traditionally, NSAID-induced GI toxicity has been associated with indiscriminate inhibition of cyclooxygenase isoforms, but other mechanisms, including inhibition of cell migration, intestinal restitution, and wound healing, are likely to contribute to toxicity. Previous efforts demonstrated that treatment of cultured intestinal epithelial cells (IEC) with NSAIDs inhibits expression and activity of calpain proteases, but the effects of specific inhibition of calpain expression in vitro or the effects of NSAIDs on intestinal cell migration in vivo remain to be determined. Accordingly, we examined the effect of suppression of calpain protease expression with siRNA on cell migration in cultured IECs and evaluated the effects of NSAID treatment on epithelial cell migration and calpain protease expression in rat duodenum. Our results show that calpain siRNA inhibits protease expression and slows migration in cultured IECs. Additionally, NSAID treatment of rats slowed migration up the villus axis and suppressed calpain expression in duodenal epithelial cells. Our results are supportive of the hypothesis that suppression of calpain expression leading to slowing of cell migration is a potential mechanism through which NSAIDs cause GI toxicity.

Oral perfluorooctane sulfonate (PFOS) lessens tumor development in the APCmin mouse model of spontaneous familial adenomatous polyposis.

BACKGROUND: Colorectal cancer is the second most common cause of cancer deaths for both men and women, and the third most common cause of cancer in the U.S. Toxicity of current chemotherapeutic agents for colorectal cancer, and emergence of drug resistance underscore the need to develop new, potentially less toxic alternatives. Our recent cross-sectional study in a large Appalachian population, showed a strong, inverse, dose-response association of serum perfluorooctane sulfonate (PFOS) levels to prevalent colorectal cancer, suggesting PFOS may have therapeutic potential in the prevention and/or treatment of colorectal cancer. In these preliminary studies using a mouse model of familial colorectal cancer, the APCmin mouse, and exposures comparable to those reported in human populations, we assess the efficacy of PFOS for reducing tumor burden, and evaluate potential dose-response effects. METHODS: At 5-6 weeks of age, APCmin mice were randomized to receive 0, 20, 250 mg PFOS/kg (females) or 0, 10, 50 and 200 mg PFOS/kg (males) via their drinking water. At 15 weeks of age, gastrointestinal tumors were counted and scored and blood PFOS levels measured. RESULTS: PFOS exposure was associated with a significant, dose-response reduction in total tumor number in both male and female mice. This inverse dose-response effect of PFOS exposure was particularly pronounced for larger tumors (r2 for linear trend = 0.44 for males, p's <0.001). CONCLUSIONS: The current study in a mouse model of familial adenomatous polyposis offers the first experimental evidence that chronic exposure to PFOS in drinking water can reduce formation of gastrointestinal tumors, and that these reductions are both significant and dose-dependent. If confirmed in further studies, these promising findings could lead to new therapeutic strategies for familial colorectal cancer, and suggest that PFOS testing in both preventive and therapeutic models for human colorectal cancer is warranted.

Inhibition of Kv channel expression by NSAIDs depolarizes membrane potential and inhibits cell migration by disrupting calpain signaling.

Clinical use of non-steroidal anti-inflammatory drugs (NSAIDs) is well known to cause gastrointestinal ulcer formation via several mechanisms that include inhibiting epithelial cell migration and mucosal restitution. The drug-affected signaling pathways that contribute to inhibition of migration by NSAIDs are poorly understood, though previous studies have shown that NSAIDs depolarize membrane potential and suppress expression of calpain proteases and voltage-gated potassium (Kv) channel subunits. Kv channels play significant roles in cell migration and are targets of NSAID activity in white blood cells, but the specific functional effects of NSAID-induced changes in Kv channel expression, particularly on cell migration, are unknown in intestinal epithelial cells. Accordingly, we investigated the effects of NSAIDs on expression of Kv1.3, 1.4, and 1.6 in vitro and/or in vivo and evaluated the functional significance of loss of Kv subunit expression. Indomethacin or NS-398 reduced total and plasma membrane protein expression of Kv1.3 in cultured intestinal epithelial cells (IEC-6). Additionally, depolarization of membrane potential with margatoxin (MgTx), 40mM K(+), or silencing of Kv channel expression with siRNA significantly reduced IEC-6 cell migration and disrupted calpain activity. Furthermore, in rat small intestinal epithelia, indomethacin and NS-398 had significant, yet distinct, effects on gene and protein expression of Kv1.3, 1.4, or 1.6, suggesting that these may be clinically relevant targets. Our results show that inhibition of epithelial cell migration by NSAIDs is associated with decreased expression of Kv channel subunits, and provide a mechanism through which NSAIDs inhibit cell migration and may contribute to NSAID-induced gastrointestinal (GI) toxicity.

The sorting protein PACS-2 promotes ErbB signalling by regulating recycling of the metalloproteinase ADAM17.

The metalloproteinase ADAM17 activates ErbB signalling by releasing ligands from the cell surface, a key step underlying epithelial development, growth and tumour progression. However, mechanisms acutely controlling ADAM17 cell-surface availability to modulate the extent of ErbB ligand release are poorly understood. Here, through a functional genome-wide siRNA screen, we identify the sorting protein PACS-2 as a regulator of ADAM17 trafficking and ErbB signalling. PACS-2 loss reduces ADAM17 cell-surface levels and ADAM17-dependent ErbB ligand shedding, without apparent effects on related proteases. PACS-2 co-localizes with ADAM17 on early endosomes and PACS-2 knockdown decreases the recycling and stability of internalized ADAM17. Hence, PACS-2 sustains ADAM17 cell-surface activity by diverting ADAM17 away from degradative pathways. Interestingly, Pacs2-deficient mice display significantly reduced levels of phosphorylated EGFR and intestinal proliferation. We suggest that this mechanism controlling ADAM17 cell-surface availability and EGFR signalling may play a role in intestinal homeostasis, with potential implications for cancer biology.

The multifunctional sorting protein PACS-2 regulates SIRT1-mediated deacetylation of p53 to modulate p21-dependent cell-cycle arrest.

SIRT1 regulates the DNA damage response by deacetylating p53, thereby repressing p53 transcriptional output. Here, we demonstrate that the sorting protein PACS-2 regulates SIRT1-mediated deacetylation of p53 to modulate the DNA damage response. PACS-2 knockdown cells failed to efficiently undergo p53-induced cell-cycle arrest in response to DNA damage. Accordingly, p53 acetylation was reduced both in PACS-2 knockdown cells and thymocytes from Pacs-2(-/-) mice, thereby blunting induction of the cyclin-dependent kinase inhibitor p21 (CDKN1A). The SIRT1 inhibitor EX-527 or SIRT1 knockdown restored p53 acetylation and p21 induction as well as p21-dependent cell-cycle arrest in PACS-2 knockdown cells. Trafficking studies revealed that cytoplasmic PACS-2 shuttled to the nucleus, where it interacted with SIRT1 and repressed SIRT1-mediated p53 deacetylation. Correspondingly, in vitro assays demonstrated that PACS-2 directly inhibited SIRT1-catalyzed p53 deacetylation. Together, these findings identify PACS-2 as an in vivo mediator of the SIRT1-p53-p21 axis that modulates the DNA damage response.

Discovery of a diaminoquinoxaline benzenesulfonamide antagonist of HIV-1 Nef function using a yeast-based phenotypic screen.

BACKGROUND: HIV-1 Nef is a viral accessory protein critical for AIDS progression. Nef lacks intrinsic catalytic activity and binds multiple host cell signaling proteins, including Hck and other Src-family tyrosine kinases. Nef binding induces constitutive Hck activation that may contribute to HIV pathogenesis by promoting viral infectivity, replication and downregulation of cell-surface MHC-I molecules. In this study, we developed a yeast-based phenotypic screen to identify small molecules that inhibit the Nef-Hck complex. RESULTS: Nef-Hck interaction was faithfully reconstituted in yeast cells, resulting in kinase activation and growth arrest. Yeast cells expressing the Nef-Hck complex were used to screen a library of small heterocyclic compounds for their ability to rescue growth inhibition. The screen identified a dihydrobenzo-1,4-dioxin-substituted analog of 2-quinoxalinyl-3-aminobenzene-sulfonamide (DQBS) as a potent inhibitor of Nef-dependent HIV-1 replication and MHC-I downregulation in T-cells. Docking studies predicted direct binding of DQBS to Nef which was confirmed in differential scanning fluorimetry assays with recombinant purified Nef protein. DQBS also potently inhibited the replication of HIV-1 NL4-3 chimeras expressing Nef alleles representative of all M-group HIV-1 clades. CONCLUSIONS: Our findings demonstrate the utility of a yeast-based growth reversion assay for the identification of small molecule Nef antagonists. Inhibitors of Nef function discovered with this assay, such as DQBS, may complement the activity of current antiretroviral therapies by enabling immune recognition of HIV-infected cells through the rescue of cell surface MHC-I.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein-induced lysosomal translocation of proapoptotic effectors is mediated by phosphofurin acidic cluster sorting protein-2 (PACS-2).

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis of liver cancer cell lines requires death receptor-5 (DR5)-dependent permeabilization of lysosomal membranes. Ligated DR5 triggers recruitment of the proapoptotic proteins Bim and Bax to lysosomes, releasing cathepsin B into the cytosol where it mediates mitochondria membrane permeabilization and activation of executioner caspases. Despite the requirement for lysosome membrane permeabilization during TRAIL-induced apoptosis, little is known about the mechanism that controls recruitment of Bim and Bax to lysosomal membranes. Here we report that TRAIL induces recruitment of the multifunctional sorting protein phosphofurin acidic cluster sorting protein-2 (PACS-2) to DR5-positive endosomes in Huh-7 cells where it forms an immunoprecipitatable complex with Bim and Bax on lysosomal membranes. shRNA-targeted knockdown of PACS-2 prevents recruitment of Bim or Bax to lysosomes, blunting the TRAIL-induced lysosome membrane permeabilization. Consistent with the reduced lysosome membrane permeabilization, shRNA knockdown of PACS-2 in Huh-7 cells reduced TRAIL-induced apoptosis and increased clonogenic cell survival. The determination that recombinant PACS-2 bound Bim but not Bax in vitro and that shRNA knockdown of Bim blocked Bax recruitment to lysosomes suggests that TRAIL/DR5 triggers endosomal PACS-2 to recruit Bim and Bax to lysosomes to release cathepsin B and induce apoptosis. Together, these findings provide insight into the lysosomal pathway of apoptosis.

An interdomain binding site on HIV-1 Nef interacts with PACS-1 and PACS-2 on endosomes to down-regulate MHC-I.

The human immunodeficiency virus type 1 (HIV-1) accessory protein Nef directs virus escape from immune surveillance by subverting host cell intracellular signaling and membrane traffic to down-regulate cell-surface major histocompatibility complex class I (MHC-I). The interaction of Nef with the sorting proteins PACS-1 and PACS-2 mediates key signaling and trafficking steps required for Nef-mediated MHC-I down-regulation. Little is known, however, about the molecular basis underlying the Nef-PACS interaction. Here we identify the sites on Nef and the PACS proteins required for their interaction and describe the consequences of disrupting this interaction for Nef action. A previously unidentified cargo subsite on PACS-1 and PACS-2 interacted with a bipartite site on Nef formed by the EEEE(65) acidic cluster on the N-terminal domain and W(113) in the core domain. Mutation of these sites prevented the interaction between Nef and the PACS proteins on Rab5 (PACS-2 and PACS-1)- or Rab7 (PACS-1)-positive endosomes as determined by bimolecular fluorescence complementation and caused a Nef mutant defective in PACS binding to localize to distorted endosomal compartments. Consequently, disruption of the Nef-PACS interaction repressed Nef-induced MHC-I down-regulation in peripheral blood mononuclear cells. Our results provide insight into the molecular basis of Nef action and suggest new strategies to combat HIV-1.

Small molecule inhibition of HIV-1-induced MHC-I down-regulation identifies a temporally regulated switch in Nef action.

HIV-1 Nef triggers down-regulation of cell-surface MHC-I by assembling a Src family kinase (SFK)-ZAP-70/Syk-PI3K cascade. Here, we report that chemical disruption of the Nef-SFK interaction with the small molecule inhibitor 2c blocks assembly of the multi-kinase complex and represses HIV-1-mediated MHC-I down-regulation in primary CD4(+) T-cells. 2c did not interfere with the PACS-2-dependent trafficking of Nef required for the Nef-SFK interaction or the AP-1 and PACS-1-dependent sequestering of internalized MHC-I, suggesting the inhibitor specifically interfered with the Nef-SFK interaction required for triggering MHC-I down-regulation. Transport studies revealed Nef directs a highly regulated program to down-regulate MHC-I in primary CD4(+) T-cells. During the first two days after infection, Nef assembles the 2c-sensitive multi-kinase complex to trigger down-regulation of cell-surface MHC-I. By three days postinfection Nef switches to a stoichiometric mode that prevents surface delivery of newly synthesized MHC-I. Pharmacologic inhibition of the multi-kinase cascade prevents the Nef-dependent block in MHC-I transport, suggesting the signaling and stoichiometric modes are causally linked. Together, these studies resolve the seemingly controversial models that describe Nef-induced MHC-I down-regulation and provide new insights into the mechanism of Nef action.

Akt and 14-3-3 control a PACS-2 homeostatic switch that integrates membrane traffic with TRAIL-induced apoptosis.

TRAIL selectively kills diseased cells in vivo, spurring interest in this death ligand as a potential therapeutic. However, many cancer cells are resistant to TRAIL, suggesting the mechanism mediating TRAIL-induced apoptosis is complex. Here we identify PACS-2 as an essential TRAIL effector, required for killing tumor cells in vitro and virally infected hepatocytes in vivo. PACS-2 is phosphorylated at Ser437 in vivo, and pharmacologic and genetic studies demonstrate Akt is an in vivo Ser437 kinase. Akt cooperates with 14-3-3 to regulate the homeostatic and apoptotic properties of PACS-2 that mediate TRAIL action. Phosphorylated Ser437 binds 14-3-3 with high affinity, which represses PACS-2 apoptotic activity and is required for PACS-2 to mediate trafficking of membrane cargo. TRAIL triggers dephosphorylation of Ser437, reprogramming PACS-2 to promote apoptosis. Together, these studies identify the phosphorylation state of PACS-2 Ser437 as a molecular switch that integrates cellular homeostasis with TRAIL-induced apoptosis.