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.

Genome-wide kinase-MAM interactome screening reveals the role of CK2A1 in MAM Ca2+ dynamics linked to DEE66.

The endoplasmic reticulum (ER) and mitochondria form a unique subcellular compartment called mitochondria-associated ER membranes (MAMs). Disruption of MAMs impairs Ca2+ homeostasis, triggering pleiotropic effects in the neuronal system. Genome-wide kinase-MAM interactome screening identifies casein kinase 2 alpha 1 (CK2A1) as a regulator of composition and Ca2+ transport of MAMs. CK2A1-mediated phosphorylation of PACS2 at Ser207/208/213 facilitates MAM localization of the CK2A1-PACS2-PKD2 complex, regulating PKD2-dependent mitochondrial Ca2+ influx. We further reveal that mutations of PACS2 (E209K and E211K) associated with developmental and epileptic encephalopathy-66 (DEE66) impair MAM integrity through the disturbance of PACS2 phosphorylation at Ser207/208/213. This, in turn, causes the reduction of mitochondrial Ca2+ uptake and the dramatic increase of the cytosolic Ca2+ level, thereby, inducing neurotransmitter release at the axon boutons of glutamatergic neurons. In conclusion, our findings suggest a molecular mechanism that MAM alterations induced by pathological PACS2 mutations modulate Ca2+-dependent neurotransmitter release.

Do PACS1 variants impeding adaptor protein binding predispose to syndromic intellectual disability?

To date, PACS1-neurodevelopmental disorder (PACS1-NDD) has been associated with recurrent variation of Arg203 and is considered diagnostic of PACS1-NDD, an autosomal dominant syndromic intellectual disability disorder. Although incompletely defined, the proposed disease mechanism for this variant is altered PACS1 affinity for its client proteins. Given this proposed mechanism, we hypothesized that PACS1 variants that interfere with binding of adaptor proteins might also give rise to syndromic intellectual disability. Herein, we report a proposita and her mother with phenotypic features overlapping PACS1-NDD and a novel PACS1 variant (NM_018026.3:c.[755C > T];[=], p.(Ser252Phe)) that impedes binding of the adaptor protein GGA3 (Golgi-associated, gamma-adaptin ear-containing, ARF-binding protein 3). We hypothesize that attenuating PACS1 binding of GGA3 also gives rise to a disorder with features overlapping those of PACS1-NDD. This observation better delineates the mechanism by which PACS1 variation predisposes to syndromic intellectual disability.

The Path to Therapeutic Furin Inhibitors: From Yeast Pheromones to SARS-CoV-2.

The spurious acquisition and optimization of a furin cleavage site in the SARS-CoV-2 spike protein is associated with increased viral transmission and disease, and has generated intense interest in the development and application of therapeutic furin inhibitors to thwart the COVID-19 pandemic. This review summarizes the seminal studies that informed current efforts to inhibit furin. These include the convergent efforts of endocrinologists, virologists, and yeast geneticists that, together, culminated in the discovery of furin. We describe the pioneering biochemical studies which led to the first furin inhibitors that were able to block the disease pathways which are broadly critical for pathogen virulence, tumor invasiveness, and atherosclerosis. We then summarize how these studies subsequently informed current strategies leading to the development of small-molecule furin inhibitors as potential therapies to combat SARS-CoV-2 and other diseases that rely on furin for their pathogenicity and progression.

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.

A Phase 2 Study of PCI-27483, a Factor VIIa Inhibitor in Combination with Gemcitabine for Advanced Pancreatic Cancer.

OBJECTIVES: Tissue factor overexpression is associated with tumor progression, venous thromboembolism, and worsened survival in patients with cancer. Tissue factor and activated factor VII (FVIIa) complex may contribute to tumor invasiveness by promoting cell migration and angiogenesis. The study objective was to evaluate safety, pharmacokinetics, and efficacy of PCI-27483, a selective FVIIa inhibitor. METHODS: This was an open-label, multicenter phase 2 trial of patients with advanced pancreatic cancer. Part A of the study was an intrapatient dose escalation lead-in portion in patients concurrently receiving gemcitabine, and in part B, patients were randomized 1: 1 to the recommended phase 2 dose combination PCI-27483-gemcitabine versus gemcitabine alone. RESULTS: Target international normalized ratio (between 2.0-3.0) was achieved following PCI-27483 treatment. Overall safety of PCI-27483-gemcitabine (n = 26) was similar to gemcitabine alone (n = 16), with a higher incidence of mostly low-grade bleeding events (65% vs. 19%). Progression-free survival (PFS) and overall survival (OS) were not significantly different between patients treated with PCI-27483-gemcitabine (PFS: 3.7 months, OS: 5.7 months) and those treated with gemcitabine alone (PFS: 1.9 months, OS: 5.6 months). CONCLUSIONS: Targeted inhibition of the coagulation cascade was achieved by administering PCI-27483. PCI-27483-gemcitabine was well tolerated, but superiority to single agent gemcitabine was not demonstrated.

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.

The mechanism by which a propeptide-encoded pH sensor regulates spatiotemporal activation of furin.

The proprotein convertase furin requires the pH gradient of the secretory pathway to regulate its multistep, compartment-specific autocatalytic activation. Although His-69 within the furin prodomain serves as the pH sensor that detects transport of the propeptide-enzyme complex to the trans-Golgi network, where it promotes cleavage and release of the inhibitory propeptide, a mechanistic understanding of how His-69 protonation mediates furin activation remains unclear. Here we employ biophysical, biochemical, and computational approaches to elucidate the mechanism underlying the pH-dependent activation of furin. Structural analyses and binding experiments comparing the wild-type furin propeptide with a nonprotonatable His-69 → Leu mutant that blocks furin activation in vivo revealed protonation of His-69 reduces both the thermodynamic stability of the propeptide as well as its affinity for furin at pH 6.0. Structural modeling combined with mathematical modeling and molecular dynamic simulations suggested that His-69 does not directly contribute to the propeptide-enzyme interface but, rather, triggers movement of a loop region in the propeptide that modulates access to the cleavage site and, thus, allows for the tight pH regulation of furin activation. Our work establishes a mechanism by which His-69 functions as a pH sensor that regulates compartment-specific furin activation and provides insights into how other convertases and proteases may regulate their precise spatiotemporal activation.

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.

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.

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.

Small molecule inhibitors of the HIV-1 virulence factor, Nef.

Although antiretroviral therapy has revolutionized the clinical management of AIDS, life-long treatment is required because these drugs do not eradicate HIV- infected cells. Chronic antiretroviral therapy may not protect AIDS patients from cognitive impairment, raising important quality of life issues. Because of the rise of HIV strains resistant to current drugs and uncertain vaccine prospects, an urgent need exists for the discovery and development of new therapeutic approaches. This review is focused on one such approach, which involves targeting HIV-1 Nef, a viral accessory protein essential for AIDS pathogenesis.

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.

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.

Low-dose naltrexone reduced anxiety in persons with multiple sclerosis during the COVID-19 pandemic.

Persons with multiple sclerosis (PwMS) have been considered at high risk for vaccination and/or acquisition of COVID-19 related to their reduced immune systems and daily regimen of immune suppressing therapy. Substantiated and unsubstantiated reports on these unknown circumstances increased anxiety and depression. Low-dose naltrexone (LDN) is a potentially effective off-label therapy shown to be effective at controlling fatigue for several autoimmune disorders including MS. This study utilized a small population of PwMS from central Pennsylvania in order to determine whether LDN therapy altered their perceived anxiety or depression during the early months of COVID-19. Utilizing mailed surveys, self-reported anxiety and depression scores were found to be significantly lower for PwMS who were prescribed LDN either alone or as an adjuvant to a standard disease modifying therapy (DMT) in comparison to those on oral disease-modifying therapies (DMTs). The data suggest that the non-toxic, inexpensive biotherapeutic may be beneficial in lessening anxiety.

Death by committee: organellar trafficking and communication in apoptosis.

Apoptosis proceeds through a set of evolutionarily conserved processes that co-ordinate the elimination of damaged or unneeded cells. This program of cell death is carried out by organelle-directed regulators, including the Bcl-2 proteins, and ultimately executed by proteases of the caspase family. Although the biochemical mechanisms of apoptosis are increasingly understood, the underlying cell biology orchestrating programmed cell death remains enigmatic. In this review, we summarize the current understanding of Bcl-2 protein regulation and caspase activation while examining cell biological mechanisms and consequences of apoptotic induction. Organellar contributions to apoptotic induction include death receptor endocytosis, mitochondrial and lysosomal permeabilization, endoplasmic reticulum calcium release and fragmentation of the Golgi apparatus. These early apoptotic events are accompanied by stabilization of the microtubule cytoskeleton and translocation of organelles to the microtubule organizing center. Together, these phenomena establish a model of apoptotic induction whereby a cytoskeletal-dependent coalescence and 'scrambling' of organelles in the paranuclear region co-ordinates apoptotic communication, caspase activation and cell death.

Oxidative protein folding in the endoplasmic reticulum: tight links to the mitochondria-associated membrane (MAM).

The production of secretory proteins at the ER (endoplasmic reticulum) depends on a ready supply of energy and metabolites as well as the close monitoring of the chemical conditions that favor oxidative protein folding. ER oxidoreductases and chaperones fold nascent proteins into their export-competent three-dimensional structure. Interference with these protein folding enzymes leads to the accumulation of unfolded proteins within the ER lumen, causing an acute organellar stress that triggers the UPR (unfolded protein response). The UPR increases the transcription of ER chaperones commensurate with the load of newly synthesized proteins and can protect the cell from ER stress. Persistant stress, however, can force the UPR to commit cells to undergo apoptotic cell death, which requires the emptying of ER calcium stores. Conversely, a continuous ebb and flow of calcium occurs between the ER and mitochondria during resting conditions on a domain of the ER that forms close contacts with mitochondria, the MAM (mitochondria-associated membrane). On the MAM, ER folding chaperones such as calnexin and calreticulin and oxidoreductases such as ERp44, ERp57 and Ero1alpha regulate calcium flux from the ER through reversible, calcium and redox-dependent interactions with IP3Rs (inositol 1,4,5-trisphophate receptors) and with SERCAs (sarcoplasmic/endoplasmic reticulum calcium ATPases). During apoptosis progression and depending on the identity of the ER chaperone and oxidoreductase, these interactions increase or decrease, suggesting that the extent of MAM targeting of ER chaperones and oxidoreductases could shift the readout of ER-mitochondria calcium exchange from housekeeping to apoptotic. However, little is known about the cytosolic factors that mediate the on/off interactions between ER chaperones and oxidoreductases with ER calcium channels and pumps. One candidate regulator is the multi-functional molecule PACS-2 (phosphofurin acidic cluster sorting protein-2). Recent studies suggest that PACS-2 mediates localization of a mobile pool of calnexin to the MAM in addition to regulating homeostatic ER calcium signaling as well as MAM integrity. Together, these findings suggest that cytosolic, membrane and lumenal proteins combine to form a two-way switch that determines the rate of protein secretion by providing ions and metabolites and that appears to participate in the pro-apoptotic ER-mitochondria calcium transfer.

ß-endorphin and opioid growth factor as biomarkers of physical ability in multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is an autoimmune-mediated degenerative disorder with increased peripheral inflammation disrupting the blood brain barrier. With increasing MS-related healthcare costs, the requirement to validate minimally invasive biomarkers has become imperative. METHODS: Relapsing-remitting MS patients on disease modifying therapies were consented at the Penn State Health MS Clinic to provide blood samples for analyses of serum cytokines and endogenous opioid peptides, as well as to complete the MSQOL-54 survey. RESULTS: Serum OGF levels in MS patients on glatiramer acetate (mean = 326 pg/ml), dimethyl fumarate (mean = 193.3 pg/ml) and natalizumab (mean = 393.4 pg/ml) were significantly elevated (p < 0.01) compared to healthy controls (mean = 98.46 pg/ml). Individuals with elevated OGF levels also had increased levels of TNFα (r = 0.78) and IL-17A (r = 0.81). Only patients treated with glatiramer acetate had significant (p < 0.01) elevations in serum ß-endorphin levels. Analyses of MS-QoL 54 data showed no significant differences in physical or mental composite scores between treatment groups. However, serum levels of ß-endorphin had a direct correlation with physical health composite score (r = 0.70) in all treatments. Serum vitamin D levels had an indirect relationship with 25-foot walk test times (r = 0.47). CONCLUSION: Both regression and cohort data suggest that serum levels of OGF, ß-endorphin, and vitamin D are potential biomarkers for physical disease status in MS.

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".

PACS-1 mediates phosphorylation-dependent ciliary trafficking of the cyclic-nucleotide-gated channel in olfactory sensory neurons.

Impaired ciliary protein transport in olfactory sensory neurons (OSNs) leads to anosmia, and is a newly recognized clinical manifestation of a class of human disorders called ciliopathies. Surprisingly little is known regarding the mechanisms controlling trafficking to this unique neuronal compartment. Here, we show a novel role for phosphofurin acidic cluster-sorting protein 1 (PACS-1) in the ciliary trafficking of the olfactory cyclic-nucleotide-gated (CNG) channel. PACS-1 is an intracellular sorting protein that mediates its effects through the binding of acidic clusters on cargo protein. This interaction is dependent on CK2 phosphorylation of both PACS-1 and its cargo. We show that CNGB1b contains two putative PACS-1 binding sites, which are phosphorylated by the serine/threonine protein kinase, CK2. Additionally, we show that PACS-1 is expressed in OSNs and interacts in complex with the CNG channel. CK2 inhibition in native OSNs causes a loss of CNG channel from cilia and subsequent olfactory dysfunction, while adenoviral expression of mutant PACS-1 causes similar mislocalization. These results provide a mechanism for the subunit-dependent ciliary trafficking of the CNG channel and offer insight into the mechanisms of ciliary transport.