COMMENTARY

Caught in the act – protein adaptation and the expanding roles ofthe PACS proteins in tissue homeostasis and diseaseGary Thomas1,2,*, Joseph E. Aslan3, Laurel Thomas1, Pushkar Shinde4, Ujwal Shinde4 and Thomas Simmen5

ABSTRACTVertebrate proteins that fulfill multiple and seemingly disparatefunctions are increasingly recognized as vital solutions tomaintaining homeostasis in the face of the complex cell and tissuephysiology of higher metazoans. However, the molecular adaptationsthat underpin this increased functionality remain elusive. In thisCommentary, we review the PACS proteins – which first appearedin lower metazoans as protein traffic modulators and evolved invertebrates to integrate cytoplasmic protein traffic and interorganellarcommunication with nuclear gene expression – as examples ofprotein adaptation ‘caught in the act’. Vertebrate PACS-1 and PACS-2increased their functional density and roles as metabolic switches byacquiring phosphorylation sites and nuclear trafficking signals withindisordered regions of the proteins. These findings illustrate onemechanism by which vertebrates accommodate their complex cellphysiology with a limited set of proteins. We will also highlight howpathogenic viruses exploit the PACS sorting pathways as well asrecent studies on PACS genes with mutations or altered expressionthat result in diverse diseases. These discoveries suggest thatinvestigation of the evolving PACS protein family provides a richopportunity for insight into vertebrate cell and organ homeostasis.

IntroductionAt the turn of the 20th century, Archibald Garrod,Walter Sutton andThomas HuntMorgan ushered in a new era of biological research byreplacing observational studies with mechanistic analyses (Kelvesand Hood, 1992). Their discoveries that chromosomes encodeheritable traits set the foundation for understanding geneticinheritance. More than 30 years later, through inactivation andmutational studies of the common bread mold Neurospora, GeorgeBeadle and Edward Tatum ultimately determined that genes encodeenzymes and laid a cornerstone of modern molecular biologythrough the ‘one gene – one protein’ hypothesis (Beadle and Tatum,1941). This provocative model inevitably collapsed under theweight of subsequent discoveries, which revealed that genesfrequently encode multiple proteins or collections of peptides thatdrive complex physiology and contribute to molecular promiscuitythrough mechanisms ranging from alternative RNA splicing to theproteolytic processing of polyproteins (‘one gene –many proteins’;Yang et al., 2016). The essence of these studies led to the present-

day ‘one protein –multiple functions’mantra, which recognizes thatindividual proteins often have multiple and seemingly disparatefunctions – a phenomenon known as ‘moonlighting’ (Jeffery, 1999;Henderson and Martin, 2014). The glycolytic enzymesglyceraldehyde-3-phosphate dehydrogenase (GAPDH) andenolase are widely cited examples of moonlighting proteins.Human GAPDH participates in as many as 20 different cellularfunctions ranging from the regulation of membrane traffic to themaintenance of genome integrity (Sirover, 2011). Human enolasecan be present on the cell surface of activated monocytes where itserves as a plasminogen receptor that promotes recruitment ofinflammatory cells to sites of tissue injury (Wygrecka et al., 2009).

Protein moonlighting, which by definition is limited to proteinsthat use a single primary sequence in the absence ofposttranslational modifications to mediate multiple functions(Jeffery, 1999), is inherently limited for solving evolutionarychallenges. Eventually, moonlighting gives way to geneduplication, which enables the resulting paralogs to acquiremutations that expand the molecular functions initiated by theancestral protein. Inevitably, such a hit-and-miss approach tomutational diversification will hit a roadblock in the form of rigidlyfolded domains, such as SH2 or PDZ domains found in manyproteins (Tompa et al., 2014). The dedicated structure of these∼100-amino-acid protein domains, which are typically involved inhigh-affinity binding to discrete ligands, limits amino acidsubstitutions, thereby reducing their ability to increase thefunctional density of associated cellular proteins. Therefore,protein adaptation represents an additional and vital solution toincrease the array of protein functions to an extent that is notpossible by moonlighting and gene duplication alone (Tompa et al.,2014). Rather than coaxing rigid protein domains to accept newroles, such proteins – notably vertebrate proteins – have acquiredintrinsically disordered regions (IDRs) to complement the limitedutility of folded domains (Dunker et al., 2005; Pancsa and Tompa,2016). IDRs do not fold into an autonomous structure, but containflexible 5–15-amino-acid small linear motifs (SLiMs) that canassume an ensemble of ‘structures’, which in turn interact with apanoply of binding partners through low-affinity but high-specificity interactions (Davey et al., 2015). SLiMs are frequentlysites of posttranslational modifications and undergo rapid evolution,greatly increasing the functional density of IDR-containing proteins(Tompa et al., 2014; Pancsa and Tompa, 2016). This plasticityenabled SLiMs from disparate proteins across a large taxonomicrange to acquire similar amino acid mutations by convergentevolution so that they may bind to a common globular proteindomain (Schlessinger et al., 2011; Davey et al., 2012, 2015; VanRoey et al., 2012; Jonas and Izaurralde, 2013). However, examplestracing how a single protein adapted to evolutionary pressure byacquiring specific SLiM mutations in order to fulfill new functionsthat are critical for vertebrate homeostasis are surprisingly limited.Studies on the phosphofurin acidic cluster sorting (PACS) proteins

1Department of Microbiology and Molecular Genetics, University of PittsburghSchool of Medicine, Pittsburgh, PA 15239, USA. 2University of Pittsburgh CancerInstitute, Pittsburgh, PA 15239, USA. 3Knight Cardiovascular Institute, OregonHealth & Science University, Portland, OR 97239, USA. 4Department ofBiochemistry andMolecular Biology, OregonHealth & Science University, Portland,OR 97239, USA. 5Department of Cell Biology, University of Alberta, Edmonton,Alberta, Canada T6G2H7.

*Author for correspondence (thomasg@pitt.edu)

G.T., 0000-0003-1976-7183

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provide a powerful illustration of such evolutionary proteinadaptations ‘caught in the act’ of connecting the increasinglycomplex organellar landscape that underpins vertebrate cellfunction.Here, we discuss how PACS proteins first appeared in metazoans

as membrane traffic regulators, and then expanded their functionalrepertoire in vertebrates by acquiring molecular switches, notablyphosphorylation sites and nuclear localization signals (NLSs)within their SLiMs. These gain-of-function adaptations enabledthe vertebrate PACS proteins to integrate cytoplasmic functionswith nuclear gene expression. In particular, we focus on theacquisition of an Akt phosphorylation site and NLS in vertebratePACS-2, which enable this protein to function as a metabolic switchthat integrates cytoplasmic membrane traffic and interorganellarcommunication with nuclear gene expression in response toanabolic or catabolic cues.

PACS proteins direct membrane trafficking in worms tohumansThe PACS proteins were discovered in a genetic screen forregulators of the secretory pathway proteinase furin (Wan et al.,1998; Thomas, 2002). Human PACS1 is located at 11q13.1-q13.2and contains 24 exons and at least 12 alternatively spliced variants.Human PACS-2, which is located near the telomere at 14q32.33,also contains 24 exons and at least 11 alternatively spliced variants.Genome-wide association studies (GWAS) identified the humanPACS1 locus as a susceptibility gene in severe early-onset obesity(Wheeler et al., 2013) and developmental delay (DecipheringDevelopmental Disorders Study, 2017), and mutations in PACS1underlie ‘PACS-1 syndrome’ (Schuurs-Hoeijmakers et al., 2012,2016; Stern et al., 2017), characterized by epileptic seizures, heartdefects, hypotonia and craniofacial malformations. The PACS2locus is highly susceptible to loss of heterozygosity in colorectalcancer, leading to a reduction or loss of PACS-2 protein expression(Anderson et al., 2001; Aslan et al., 2009). Similar to PACS1,mutations in PACS2 are associated with neonatal onset epilepsy,global developmental delay and intellectual disability (C. Thauvinand H. Olson, personal communication).The canonical 963-amino-acid PACS-1 and 889-amino-acid

PACS-2 proteins share 54% sequence identity. Bioinformaticsanalyses suggest both proteins lack folded globular domains and areinstead nearly 50% disordered (Fig. 1). The ∼150-amino-acid cargo(furin)-binding regions (FBRs) in PACS-1 and PACS-2, which arepredicted to be structured (Fig. 1), share nearly 80% sequenceidentity and bind client proteins, as well as the cytoplasmicmembrane trafficking machinery as described below. PACS-1 andPACS-2 also share a disordered middle region (MR), whichcontains an autoregulatory domain as well as NLSs and, specificallyin PACS-2, a critical Akt phosphorylation site that binds to 14-3-3proteins. The function of the shared C-terminal region (CTR) isunknown. PACS-1 additionally contains an N-terminal extensioncalled the atrophin-1-related region (ARR), which has homology tothis nuclear transcriptional co-repressor (Zhang et al., 2006).The PACS genes are a recent addition to the eukaryotic genome,

appearing first in lower metazoans (Fig. 2). Invertebrates, includingnematodes, arthropods and echinoderms, possess a single PACSlocus that is apparently dedicated to membrane trafficking. Notably,Caernorhabditis elegans PACS (cePACS, T18H9.7a) localizes toearly endosomes at the presynaptic terminus of the neuromuscularjunction where it mediates synaptic transmission (Sieburth et al.,2005). Lower chordates, including Amphioxus and tunicates alsopossess only a single gene. The PACS gene was duplicated with the

appearance of the vertebrates, resulting in the PACS-1 and PACS-2genes.

The regulation of membrane traffic remains a role that isconserved among the vertebrate PACS proteins. The PACS-1 FBRbinds to acidic clusters that can be phosphorylated by casein kinase2 (CK2), as well as α-helices on a large number of client proteins(Wan et al., 1998; Youker et al., 2009; Dikeakos et al., 2012). Thus,PACS-1, which interacts with the clathrin adaptors AP-1 and AP-3,as well as the monomeric adaptor GGA3, mediates localization offurin and other client proteins to the trans-Golgi network (TGN) andalso targets a subset of client proteins to the primary cilium,including the adaptor protein nephrocystin (also known as NPHP1)and the olfactory cyclic-nucleotide-gated ion channel (CNG), thelatter by binding to the β1 subunit (CNGB1) (Fig. 3A) (Wan et al.,1998; Schermer et al., 2005; Jenkins et al., 2009; Youker et al.,2009). PACS-1 acquired a CK2-phosphorylated acidic cluster of itsown, which is located in the disordered MR (see Fig. 1). CK2phosphorylation of Ser278 in the PACS-1 autoregulatory domaincontrols intramolecular binding to the FBR, which regulates theinteraction with client proteins (Scott et al., 2003). PACS-2, whichinteracts with the coatomer COPI, mediates the localization of cargoto the endoplasmic reticulum (ER) and, similar to cePACS, directstrafficking from early endosomes (Youker et al., 2009) (Fig. 3A, seealso Fig. 4A). PACS-2 stabilizes a pool of the metalloproteinaseADAM17 (also known as TACE) on early endosomes, from wherethe enzyme is trafficked to the cell surface to shed ErbB ligands,including those that ligate the epidermal growth factor receptor(EGFR) (Dombernowsky et al., 2015). In the absence of PACS-2,ADAM17 is diverted to lysosomes, which reduces ErbB shedding.Correspondingly, EGFR signaling is reduced in the intestinalepithelium of PACS2−/− mice (Dombernowsky et al., 2015). Therole of the candidate autoregulatory domain in the PACS-2 MR hasnot been established.

Several pathogenic viruses acquired furin-like acidic clusters toexploit the endosomal sorting steps mediated by PACS-1 andPACS-2. This viral mimicry (see Davey et al., 2011) enables theviruses to assemble progeny, escape immune surveillance andprevent apoptosis. For example, PACS-1 binds to acidic clusters inthe human cytomegalovirus (HCMV) envelope glycoprotein gBand the Epstein–Barr virus (EBV) tegument protein BBLF1 andlocalizes them to the TGN to support virus assembly (Crump et al.,2003; Chiu et al., 2012) (see Fig. 3A). Furthermore, PACS-2interacts with a pair of small acidic clusters in the ubiquitin ligaseK5 of Kaposi sarcoma herpesvirus (KSHV) at the ER. Thisinteraction enables KSHV to downregulate the cell adhesionmolecule CD31 (also known as PECAM1), which may contributeto KSHV-induced cancer (Mansouri et al., 2006). The HIV-1accessory protein Nef uses a bipartite motif composed of a shortacidic cluster and the αB helix to interact with both PACS-1 andPACS-2 (Piguet et al., 2000; Atkins et al., 2008; Dikeakos et al.,2012). This bipartite binding enables HIV-1 Nef to downregulatemajor histocompatibility complex class I (MHC-I) from the cellsurface, which allows the virus to escape immune detection (Pawlakand Dikeakos, 2015) (Fig. 3B). To this end, Nef interacts withPACS-2 on early endosomes, which enables the HIV-1 protein toassemble a multi-kinase complex consisting of a Src family kinase(SFK), ζ-chain-associated protein kinase 70 (ZAP-70) and a class Iphosphoinositide-3 kinase (PI3K) (Blagoveshchenskaya et al.,2002; Hung et al., 2007; Atkins et al., 2008). The activated multi-kinase complex increases the amount of phosphatidylinositol(3,4,5)-trisphosphate (PIP3) underneath the plasma membrane,which recruits an ARF6 GEF to activate ARF6 and accelerate

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endocytosis of cell-surface MHC-I (Blagoveshchenskaya et al.,2002). Nef then connects the internalized MHC-I molecules toPACS-1 and AP-1 on an endosomal compartment to prevent theirrecycling to the cell surface and instead sequester them in the TGN,thereby protecting the virus from immune surveillance(Blagoveshchenskaya et al., 2002; Chaudhry et al., 2008;Noviello et al., 2008; Dikeakos et al., 2010; Dirk et al., 2016).Unlike KSHV and many other viruses, HIV-1 Nef does not inducedegradation of downregulated MHC-I (Blagoveshchenskaya et al.,2002; Dikeakos et al., 2010; Dirk et al., 2016). This chink in thearmor of HIV-1 provides an alternative approach to combat the virusby reversing the Nef-mediated immune evasion pathway. In supportof this possibility, treatment of HIV-1-infected primary CD4+

T-cells with small-molecule inhibitors of the multi-kinase complexrestores cell surface expression of MHC-I and sensitizes them tokilling by CD8+ T-cells (Hung et al., 2007; Dikeakos et al., 2010;and M. Ostrowski, personal communication).

Links between mammalian PACS proteins and TRAIL-induced apoptosisSurprisingly, while PACS-2 mediates trafficking of many pro-survival signaling molecules, it is also tasked with mediating death-ligand-induced apoptosis induced by TNF-related apoptosis-inducing ligand (TRAIL, also known as TNFSF10). Thisclinically important death ligand is an in vivo metastasis inhibitorthat selectively kills diseased cells, including cancer cells and virallyinfected cells (Johnstone et al., 2008). TRAIL-induced cell death

requires the coordinated permeabilization of multiple organelles,including mitochondria and lysosomes (Aslan and Thomas, 2009).TRAIL triggers mitochondrial outer membrane permeabilization(MOMP) by inducing dephosphorylation and proteolytic cleavageof the pro-apoptotic Bcl-2 protein Bid to form truncated Bid (tBid),which drives Bax-dependent MOMP and the consequent activationof executioner caspases (Aslan et al., 2009).

PACS-2 has essential roles in TRAIL-inducedMOMP (Fig. 4B). Inresponse to TRAIL, PACS-2 binds full-length dephosphorylated Bidand traffics it to mitochondria where it can be converted into tBid(Simmen et al., 2005). In parallel, TRAIL triggers assembly of acomplex that contains PACS-2, Bim (also known as BCL2L11) andBax, called the PIXosome, on lysosomal membranes (Werneburget al., 2012). The PIXosome drives lysosomal membranepermeabilization (LMP), which releases cathepsin B and otherhydrolases that are required for MOMP into the cytosol (Boya andKroemer, 2008). Several mechanisms interfere with theseproapoptotic functions of PACS-2. In hepatocellular carcinomacells, inhibitors of apoptosis (IAPs) target PACS-2 for proteasomaldegradation, thereby protecting the cancer cells from being killed byTRAIL (Guicciardi et al., 2014). In cardiomyocytes, miR499 preventsBid-dependent apoptosis by downregulating PACS-2 (Wang et al.,2014). Not surprisingly, herpesviruses also exploit the ability ofPACS-2 to traffic proteins to mitochondria. The HCMV protein viralinhibitor of apoptosis (vMIA) prevents cell death by trapping pro-apoptotic Bax on mitochondria (Arnoult et al., 2004). vMIA interactswith PACS-2 but not PACS-1 (our unpublished data), and the vMIA–

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Fig. 1. Disorder prediction for PACS-1 and PACS-2. The PrDOS server (http://prdos.hgc.jp/cgi-bin/top.cgi; Ishida and Kinoshita, 2007) was used to predictnatively disordered regions from the amino acid sequences of the human PACS-1 (UniProt Q6VY07) and PACS-2 (UniProt Q86VP3) proteins [false discovery rate(FDR)=2%]. The disorder probabilities for each residue were plotted as a function of length and the graphical profiles were juxtaposed with the predictedsecondary structures, which were obtained using an improved self-optimized prediction method (SOPMA) on a set of aligned members of the PACS-1 or PACS-2protein families (lower plots). The PACS-2 nuclear localization signal (NLS) and Akt site, which binds 14-3-3 proteins, together with the corresponding sequencesin PACS-1 are shown and predicted to reside in disordered regions. ARR, atrophin-1-related region; FBR, furin (cargo)-binding region; MR,middle region; CTR, C-terminal region. Red dots, phosphorylation sites [as predicted by PhosphoSitePlus (http://www.phosphosite.org/; Hornbeck et al., 2015)].

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http://prdos.hgc.jp/cgi-bin/top.cgihttp://prdos.hgc.jp/cgi-bin/top.cgihttp://www.phosphosite.org/http://www.phosphosite.org/

PACS-2 interaction is required for efficient translocation of vMIA tomitochondria (Salka et al., 2017). Similar to the fate of Bax, vMIAtraps PACS-2 onmitochondria. Notably, this sequestration of PACS-2is coupled with a blunted translocation of Bid to mitochondria inresponse to death ligands, suggesting vMIA may trap both inactivatedBax and PACS-2 on mitochondria to inhibit apoptosis in HCMV-infected cells (Fig. 5A; L.T., T.S., J.A. and G.T., unpublished results).The PACS-2-dependent recruitment of Bid to mitochondria prior toformation of tBid suggests this translocation step may be a checkpointfor apoptotic regulation. In support of this possibility, PACS-1 also

binds dephosphorylated Bid, but prevents translocation of Bid tomitochondria (T.S., L.T., J.A. and G.T., unpublished results).

PACS-2 regulates MAM-localized Ca2+ signaling, lipidmetabolism and autophagyMitochondria-associated membranes (MAMs) are ER–mitochondriacontacts, which were discovered as lipogeneic platforms in liver thatare responsive to feeding and starvation (Bernhard et al., 1952;Vance, 1990). Subsequent studies further revealed that MAMs are adynamic communication center that regulates Ca2+ signaling, lipid

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Fig. 2. Phylogenetic analysis of the PACS genes. Non-redundant protein sequences of the PACS family members were obtained from the UniProt andNCBI databases. The protein sequences were aligned using Muscle and were manually examined/modified for their accuracy within the non-conserveddomains that flank conserved domains. The program Gblocks was used to curate and eliminate poorly aligned positions and divergent regions with theprotein alignment prior to the phylogenetic analysis (Castresana, 2000). The program PhyML was used to estimate the maximum likelihood phylogenies fromalignments of amino acid sequences (Guindon et al., 2005). The tree (A) andmultiple alignments of the NLS and Akt sites (B) were visualized usingMega7. Blackdiamonds indicate invertebrate PACS proteins expressed from a single gene. Gray diamonds indicate cyclostome PACS proteins, which may be precursors toPACS-1 and PACS-2 and expressed from duplicated genes. Purple and magenta diamonds represent subfunctionalized PACS-1 and PACS-2 paralogs,respectively, that are expressed from duplicated genes in jawed vertebrates. A black background to the amino acid residue indicates identical residues, a redbackground to the amino acid residue indicates similar residues, and a yellow background divergent residues.

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metabolism and autophagy (Raturi and Simmen, 2013; Sood et al.,2014; Theurey et al., 2016). Mammalian MAMs are formed byprotein tethers, similar to the tetrameric tethering complexes in fungiknown as ER–mitochondria encounter structures (ERMES)(Kornmann et al., 2009). However, of the more than 30 proteinsinvolved in vertebrate MAM function, only two of them,GRAMD1A, which corresponds to yeast Lam6p, and MIRO (alsoknown as RHOT1), which corresponds to yeast Gem1p, areconserved in ERMES (Kornmann et al., 2011; Elbaz-Alon et al.,2015). This increased complexity of animal MAMs suggests theyacquired new roles beyond those controlled by ERMES (Herrera-Cruz and Simmen, 2017). This increased function correlates with theemerging realization that disturbances in MAM integrity areassociated with diseases ranging from obesity to neurodegenerativedisorders (Arruda and Hotamisligil, 2015; Paillusson et al., 2016).Knockdown or knockout of PACS-2 detaches the ER from

mitochondria and interferes with a number of key MAM functions

(Simmen et al., 2005) (see Fig. 4C). For example, PACS-2 is requiredfor starvation-induced autophagy because it promotes the recruitmentof the early autophagy marker Atg14 to MAMs (Hamasaki et al.,2013). PACS-2 knockdown also blocks Ca2+-mediated apoptosisprogression, which suggests that PACS-2 is required for efficient Ca2+

transfer between the ER andmitochondria (Simmen et al., 2005). ThisPACS-2 function may be coupled to its reported dynamic roles in thetrafficking of calnexin and perhaps other ER Ca2+ regulatory proteinsthat modulate ER–mitochondria Ca2+ flux (Myhill et al., 2008).Mechanistically, PACS-2 may modulate MAM formation bypreventing cleavage of the ER–mitochondria tethering proteinBAP31 (also known as BCAP31) (Iwasawa et al., 2011).Disturbances in these PACS-2 functions could contribute to disease.For example, in obese mice challenged by a high-fat diet, PACS-2 isresponsible for a chronic increase in MAM formation (Arruda et al.,2014), leading to toxic mitochondrial Ca2+ overload that consequentlyimpairs mitochondrial oxidative capacity, exacerbates insulin

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A BFig. 3. Protein traffic steps mediated byPACS-1 and PACS-2. (A) PACS-1 mediatesthe sorting of client proteins from lateendosomes (LE) to the TGN, from earlyendosomes (EE) to the plasma membrane(PM), as well as delivery to the primarycilium. PACS-2 mediates the localization ofcargo proteins to the ER, from earlyendosomes to the TGN or plasmamembrane, and also promotes MAMintegrity. (B) HIV-1 Nef usurps the sortingsteps mediated by PACS-2 and PACS-1 todownregulate the levels of cell surface MHC-I in CD4+ T-cells. Nef binds to Akt-phosphorylated PACS-2 on earlyendosomes (Atkins et al., 2008; Dikeakoset al., 2012, and L.T. and G.T., unpublishedresults). This allows Nef to traffic to the TGNregion where it binds and activates a Srcfamily kinase (SFK; Hck, Src or Lyn). TheNef–SFK complex then recruits ZAP-70 (inT-cells) or Syk (in monocytes and other celltypes) and a class I PI3K, which increasesthe level of PIP3 (maroon circles) at theplasma membrane. This recruits an ARF6GEF that accelerates MHC-I internalizationthrough an ARF6-regulated endocyticpathway. Nef diverts the internalized MHC-Imolecules from their local recyclingcompartment (dashed line) and combineswith AP-1 and PACS-1 to transport MHC-Ithrough early and late endosomes andsequester it in the TGN. The identity of theprecise compartment containing Nef, MHC-I,AP-1 and PACS-1 is under investigation.This MHC-I downregulation pathwayprotects HIV-1 from CD8+ T-cell killingthereby allowing the virus to evade immunesurveillance. Thus, small-molecule inhibitionof the multi-kinase complex re-exposesMHC-I on the cells surface and sensitizesHIV-1-infected cells to CD8+ T-cell killing.The steps shown here depict the ‘signalingmode’ of HIV-1 Nef-induced immuneevasion, which HIV-1 implements during thefirst 48 h post infection. A detailed discussionof Nef-induced immune evasion is presentedelsewhere (Dikeakos et al., 2010; Pawlakand Dikeakos 2015).

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resistance and disrupts glucose homeostasis (Arruda et al., 2014).Conversely, PACS-2 knockdown protects liver from overnutrition-induced steatosis and optimizes mitochondrial respiration and insulinsensitivity (Arruda et al., 2014). Aberrantly elevated PACS-2 andMAM formation are also found in neurons from Alzheimer’s patientsand Alzheimer’s mouse models, suggesting that the deleteriousconsequences from abnormally strongMAM formation in this diseasemay also result from dysregulated PACS-2 expression (Area-Gomezet al., 2012; Hedskog et al., 2013).

Vertebrate PACS-2 acquired an Akt phosphorylation site toswitch between its anabolic and catabolic rolesThe seemingly incongruous roles for PACS-2 in mediating pro-survival protein traffic and MAM function, as well as TRAIL-induced cell death, arose with the acquisition of an Akt

phosphorylation site that switches the function of PACS-2between these divergent roles. Akt phosphorylates PACS-2 onSer437, which promotes high-affinity binding to 14-3-3 proteins(Aslan et al., 2009). In response to insulin, MAM-localizedmTORC2 activates Akt, which phosphorylates PACS-2 tomodulate MAM integrity (Betz et al., 2013) (Fig. 4C, top). Akt-mediated phosphorylation of Ser437 is also required for PACS-2-dependent membrane trafficking steps (Fig. 4A) (Aslan et al.,2009). By contrast, dephosphorylation of PACS-2 Ser437, whichprevents 14-3-3 binding, blocks pro-survival membrane traffic anddisrupts MAMs, but is required for apoptotic trafficking tomitochondria and lysosomes and may support autophagyinduction (Fig. 4B,C, bottom) (Aslan et al., 2009; Werneburget al., 2012; Betz et al., 2013; Hamasaki et al., 2013; and L.T. andG.T., unpublished data).

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Fig. 4. The PACS-2 Akt site and NLS together modulate membrane traffic, TRAIL-induced apoptosis, MAM integrity and the response to DNA damage.(A) Protein trafficking. Akt-phosphorylated pSer437-PACS-2 (pPACS-2) interacts with ADAM17 on early endosomes (EE) and mediates delivery of theprotease to the cell surface where it sheds EGF ligands to stimulate EGFR signaling. In the absence of PACS-2, ADAM17 is degraded in lysosomes (Lys.).(B) TRAIL-induced MOMP. TRAIL triggers dephosphorylation of PACS-2 Ser437, which mediates two trafficking steps required for MOMP. In one trafficking step,PACS-2 binds full-length Bid and translocates Bid to mitochondria. In the other trafficking step, PACS-2 forms a complex with Bim and Bax on lysosomes calledthe PIXosome, which is required for lysosomemembrane permeabilization to release cathepsin B (cath. B). (C) MAMS. Top panel: insulin or growth factors triggeractivation of mTORC2 on mitochondria-associated membranes (MAMs; green shading at the ER–mitochondria contact site), which activates Akt tophosphorylate PACS-2. In turn, pPACS-2 increases MAM contacts, which may modulate ER–mitochondria exchange and support increased lipogenesis. The? denotes signaling pathways that may lead to Akt-dependent phosphorylation of PACS-2 independent of MAM-localized TORC2. Bottom panel: in starved cellsor cells treated with TRAIL, Akt is inhibited and PACS-2 Ser437 is dephosphorylated by a protein phosphatase (PPase). Dephosphorylated PACS-2 in turnremodels MAMs (red shading at the ER–mitochondria contact site), which may reduce lipogenesis but increase ER–mitochondrial Ca2+ exchange as well asinduction of autophagy. (D) DNA damage response. Top panel: to support induction of the NF-κB and Bcl-xL anti-apoptotic pathway, cytoplasmic PACS-2interacts with a pool of ATM released from the nucleus and maintains the DNA damage kinase in the cytoplasm. The cytoplasmic ATM then triggers activation ofthe canonical IκBα–NF-κB pathway that leads to induction of anti-apoptotic Bcl-xL. Bottom panel: to support induction of the p53–p21 cell cycle arrest pathway,pPACS-2 traffics to the nucleus where it binds and inhibits SIRT1 to protect acetylation of p53 bound to the p21 promoter, promoting p21 induction and cell cyclearrest. Green arrows, pro-survival-anabolic pathways mediated by pPACS-2. Red arrows, apoptotic or catabolic pathways mediated by dephosphorylatedPACS-2. Ac, acetylation; DDR, DNA-damage response.

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Akt and 14-3-3 proteins not only repress the apoptotic roles ofPACS-2 but also regulate other proapoptotic proteins, including theBcl-2 protein Bad and FOXO transcription factors (Datta et al.,1997, 2002; Brunet et al., 1999; Singh et al., 2010; Feehan andShantz, 2016). The regulation of Bad and FOXO proteins by Aktand 14-3-3 is akin to a simple molecular ‘on or off’ switch (VanRoey et al., 2012). However, the regulation of the anabolic (pro-survival) roles of PACS-2 versus the catabolic (apoptotic) rolesexerted by Akt and 14-3-3 appears to be more complex. We suggestthat the Akt site in PACS-2 resembles a ‘bifurcation’ switch suchthat both the phosphorylated and dephosphorylated states of PACS-2 are active, but in opposing directions (anabolic versus catabolicpathways, respectively). This model is supported by the ability of14-3-3 proteins to induce conformational changes in their partnersthat may enable PACS-2 to selectively bind to client proteins that areinvolved in anabolic (including pro-survival trafficking) pathways,which would be mediated by binding of 14-3-3 to phosphorylated

(p)Ser437, versus catabolic (including autophagic and apoptotictrafficking) pathways, which would be mediated by PACS-2 that isdephosphorylated on Ser437 (Yaffe, 2002).

PACS-2 acquired nuclear trafficking signals to modulategene expressionThe extrinsic and intrinsic apoptotic pathways induced by TRAIL orDNA damage, respectively, use different molecular steps to triggerMOMP. Nonetheless, these pathways are intimately coupled, as p53(also known as TP53) induces expression of the TRAIL receptorDR5 (also known as TNFRSF10B), and chemotherapeutics andTRAIL synergize to kill cancer cells (Sheikh et al., 1998; Ifeadiand Garnett-Benson, 2012). Surprisingly, PACS-2 has markedlydifferent roles in its response to TRAIL compared with DNAdamage; PACS2−/−mice are impaired in TRAIL-induced apoptosis,but are sensitized to DNA-damage-induced apoptosis (Aslan et al.,2009; Barroso-Gonzalez et al., 2016). These opposing roles for

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Fig. 5. Possible models for the regulation of Bid translocation to mitochondria by PACS proteins and vMIA. (A) Example of an experiment showing thatvMIA prevents translocation of Bid–GFP to mitochondria (L.T., T.S., J.A. and G.T., unpublished results). MCF-7 cells expressing Bid–GFP were left untreated(control, top) or transfected with a vector expressing vMIA (blue) followed by treatment with anti-Fas antibody (1 µg/ml) plus cycloheximide (CHX, 20 µg/ml) for3 h (bottom) to induce Bid translocation. Mitochondria were then labeled with Mitotracker Red. Image analysis showed that anti-Fas antibody concentrated Bid–GFP staining onmitochondria in untransfected cells (yellow asterisk) but not in cells expressing vMIA (white asterisks). Scale bar: 20 µm. (B) Conventional modelof Bid regulation. Dephosphorylation of full-length Bid exposes a cleavage site for caspase-8. Caspase-8-mediated cleavage generates tBid, which is thenmyristoylated and traffics to mitochondria to promote MOMP. (C) Alternative model of Bid regulation. Dephosphorylation of full-length Bid exposes a binding sitefor PACS-2 or PACS-1. Binding to PACS-2 promotes Bid translocation to mitochondria (solid lines), whereas binding of Bid to PACS-1 interrupts its translocationto mitochondria (upper dashed lines). In HCMV-infected cells, vMIA sequesters PACS-2 to mitochondria (lower dashed lines), thereby preventing Bid recruitmentand, ultimately, MOMP. TRAIL-R, TRAIL receptor.

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PACS-2 are partly explained by the phosphorylation state of Ser437,which is reduced by TRAIL, but increased upon DNA damage(Barroso-Gonzalez et al., 2016). Whereas TRAIL usesdephosphorylated PACS-2 to induce MOMP and LMP, the DNAdamage response uses phosphorylated PACS-2 to support cytostasisby coordinating NF-κB and Bcl-xL-dependent anti-apoptosis withp53- and p21 (CDKN1A)-dependent cell cycle arrest (Aslan et al.,2009; Barroso-Gonzalez et al., 2016).Clues to understanding the roles of PACS-2 in mediating the

p53–p21 and NF-κB–Bcl-xL pathways were provided by the DNArepair kinase ATM (Shiloh and Ziv, 2013). In response to DNAdamage, nuclear ATM phosphorylates p53, which stabilizes themulti-functional transcription factor to induce its target genes, thusfavoring cell cycle arrest and senescence over apoptosis (Xu andBaltimore, 1996; Sperka et al., 2012). Concurrently, a small pool ofactivated ATM translocates from the nucleus into the cytoplasmwhere it initiates a novel ‘nucleus-to-cytoplasm’ signaling pathwaythat promotes the NF-κB-dependent induction of anti-apoptoticBcl-xL through an as-yet-unresolved mechanism (Miyamoto,2011).PACS-2 intersects both ATMpathways to coordinate induction of

the NF-κB–Bcl-xL and p53–p21 pathways. Following DNAdamage, cytoplasmic PACS-2 interacts with ATM, therebysequestering the kinase in the cytoplasm where it induces TGF-β-activating kinase 1 (TAK1; also known as MAP3K7) to triggeractivation of IκB kinase (IκK) (Wu et al., 2010; Barroso-Gonzalezet al., 2016) (Fig. 4D, top). ATM-activated IκK then stimulates thecanonical IκBα–NF-κB induction of Bcl-xL. In the absence ofPACS-2, ATM fails to accumulate in the cytoplasm, which reducesthe induction of Bcl-xL. Consequently, induction of the apoptoticp53–Puma (Puma is also known as BBC3) pathway remains,thereby increasing MOMP and cell death (Barroso-Gonzalez et al.,2016).To promote p53–p21-dependent cell cycle arrest over p53–Puma-

dependent cell death, PACS-2 traffics to the nucleus where itincreases the transactivation of p53 bound to the p21 promoter(Atkins et al., 2014) (Fig. 4D, bottom). Notably, in addition to theATM-mediated phosphorylation of p53 within its N-terminalregion, acetylation of critical lysine residues near the C-terminusof p53 is also required for it to promote maximal transcriptionalactivity of its target genes (Gu and Roeder, 1997). The class IIIhistone deacetylase (HDAC) SIRT1, in turn, represses p53-mediated transcriptional activation by deacetylating p53 followingDNA damage (Kruse and Gu, 2009). In response to DNA damage,Akt-phosphorylated PACS-2 enters the nucleus where it binds toand inhibits SIRT1, which protects acetylated p53 bound to the p21promoter and, consequently, increases transcriptional output of p21(Atkins et al., 2014; and L.T. and G.T., unpublished results). In theabsence of PACS-2, the excessive SIRT1 activity reduces the p53-dependent induction of p21, which impedes cell cycle arrest andsensitizes cells to p53–Puma-dependent apoptosis. To access thenucleus, vertebrate PACS-2 acquired a polybasic nuclearlocalization signal (NLS) and a Crm1-dependent nuclear exportsignal (Atkins et al., 2014). Similar to the Ser437 Akt site of PACS-2,its NLS is located within a predicted IDR and is present only invertebrate PACS proteins (Figs 1 and 2).

PACS proteins as models of evolutionary protein adaptationThe acquisition of the Ser437 Akt site enabled cytoplasmic PACS-2to switch between its homeostatic (pSer437) and apoptotic(dephosphorylated Ser437) roles (see Fig. 4). PACS-2 proteins injawed vertebrates share an identical Akt motif nestled in a

disordered region that apparently became fixed by positiveselection (Fig. 2). Phylogenetic studies suggest this SLiM evolvedrapidly, albeit along a circuitous path (Figs 1 and 2). An Aktconsensus motif first appeared in lamprey PACS-1-related whichcoincided with an apparent gene duplication (Fig. 2). Thisconsensus phosphorylation site was disrupted in fish PACS-1 bythe acquisition of a proline residue at the +2 position. Surprisingly,in birds, the Akt motif reappeared in both PACS-1 and PACS-2.However, in mammals, the PACS-1 site again acquired a prolineresidue, this time in place of the phosphorylatable serine residue(Fig. 2). These findings suggest that PACS-2 acquired the Akt site toact as a vital ‘bifurcation’ switch between its anabolic versuscatabolic pathways and that evolutionary pressure assigned thisswitch to mammalian PACS-2 by negatively selecting against thephosphorylation site in mammalian PACS-1 (Figs 2 and 4) (Daveyet al., 2015).

Like the Akt site, the PACS-2 NLS, which is also located within adisordered region, is highly conserved across all jawed vertebrates(Figs 1 and 2). Phylogenetic studies suggest that a polybasic sitewith limited similarity to the vertebrate PACS NLS first appeared inamphioxus and highly homologous NLS sequences were firstacquired in fish (Fig. 2). Acquisition of the NLS by jawed vertebratePACS-2 parallels the evolutionary expansion of roles of p53 indirecting apoptosis versus cell cycle arrest. In worms and flies, p53is dedicated to driving apoptosis (Schumacher et al., 2001).However, vertebrate p53 additionally induces p21-dependent cellcycle arrest (Lu et al., 2009). Consistent with this finding, vertebratePACS-2, but not C. elegans PACS, can localize in the nucleus(Atkins et al., 2014). Thus, the ability of PACS-2 to traffic to thenucleus, where it modulates SIRT1 to promote p53-dependentinduction of p21, appears to have resulted from an evolutionaryadaptation required to support the more complex demands ofvertebrate p53 in responding to mild DNA damage by supportingp21-dependent cell cycle arrest. The predicted PACS-1 NLS is notfixed across all vertebrates. Mammals, birds and fish each express acharacteristic NLS in their PACS-1 sequences. The significance ofthis variation, as well as validation of the PACS-1 polybasic site as abona fide NLS, remains to be determined.

Conclusions and perspectivesThe PACS proteins first appeared in lower metazoans as membranetrafficking regulators and then adapted to evolutionary pressure byacquiring motifs to support the increasingly complex interorganellarcommunication pathways required for vertebrate cellularhomeostasis (Figs 1 and 2). The association of mutations in thePACS1 and PACS2 genes with diseases ranging from obesity toepilepsy and cancer underscores the broad and important roles thisgene family plays in vertebrate biology. As membrane traffickingregulators, the PACS proteins interact with sorting adaptors as wellas a variety of trafficking motifs on client proteins, ranging fromCK2-phosphorylatable acidic clusters to α-helices, in order tomodulate endomembrane protein traffic (Figs 3 and 4). The PACS-1and PACS-2 sorting pathways are also hijacked by pathogenicviruses to support production of progeny, immune evasion andprotection from apoptosis.

The acquisition of the Ser437 Akt site enabled cytoplasmic PACS-2 to act as a bifurcation switch that separates its roles betweenendomembrane trafficking (phosphorylated PACS-2) and apoptoticsignaling (unphosphorylated PACS-2) (see Fig. 4). The essentialrole for PACS-2 in mediating TRAIL-induced translocation of Bidto mitochondria was surprising as it expanded the prevailing modelof Bid action. This conventional model posits that death receptors

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trigger dephosphorylation of Bid, which permits caspase-8-dependent proteolytic cleavage and myristoylation of human Bidat Asp↓-Gly61 to form tBid (Li et al., 1998; Desagher et al., 2001;Kaufmann et al., 2012). However, and congruent with live-cellimaging analyses demonstrating that full-length Bid can translocateto mitochondria prior to the generation of tBid, we found thatPACS-2 binds to and traffics full-length dephosphorylated Bid tomitochondria (Simmen et al., 2005; Ward et al., 2006). Thesefindings suggest that myristoylation of tBid at Gly61 may not beabsolutely required for Bid action. Indeed, granzyme B, whichcleaves human Bid at Asp↓-Ser76 to generate a non-myristoylatedBid species, requires PACS-2 to recruit Bid to mitochondria (Liet al., 1998; Brasacchio et al., 2014). These findings suggest thatfollowing dephosphorylation, full-length Bid follows one of twodistinct pathways leading to MOMP. One pathway is irreversibleand relies on cleavage by caspase-8 to generate tBid, whichobligates cleaved Bid to trigger MOMP (Fig. 5B). The otherpathway is reversible and relies on the PACS-2-dependenttranslocation of full-length dephosphorylated Bid to mitochondria(Fig. 5C). Following translocation, Bid can be cleaved by multipleproteases, including caspase-8 and granzyme B, to trigger MOMP.Conversely, PACS-1 can bind de-phosphorylated Bid and blocktranslocation to mitochondria, suggesting a mechanism to impedethe apoptotic program. In HCMV-infected cells, vMIA traps PACS-2 and Bax on mitochondria, suppressing Bid recruitment andMOMP (Arnoult et al., 2004; Salka et al., 2017; Fig. 5A,C).The PACS-2 Ser437 Akt site may also act as a bifurcation switch

between anabolic and catabolic roles of the MAM. In response toinsulin, MAM-localized mTORC2 activates Akt proteins tophosphorylate PACS-2 Ser437, which increases ER–mitochondriacontacts (Betz et al., 2013). Accordingly, by increasing MAMintegrity, Akt-phosphorylated PACS-2 may increase lipogenesis,modulate ER–mitochondria Ca2+ transfer and repress autophagyinduction (Bernhard et al., 1952; Simmen et al., 2005; Betz et al.,2013). By contrast, starvation triggers PACS-2 Ser437

dephosphorylation, which would remodel MAMs to reducelipogenesis but may increase ER–mitochondria Ca2+ transfer andautophagy (Fig. 4). Future work will investigate to what the extentthe phosphorylation state of PACS-2 modulates MAM ‘thickness’or changes in contacts between mitochondria with smooth or roughendoplasmic reticulum, which may in turn regulate the switchbetween anabolic versus catabolic functions (Giacomello andPellegrini, 2016). It will also be interesting to determine whetherPACS-2 (as well PACS-1) has roles at additional intermembranecontact sites in other contexts (Schrader et al., 2015). Finally, it willbe important to determine to what extent PACS-2 Ser437

phosphorylation is regulated by mTORC2 and Akt localized toMAMs versus other compartments, and whether other signalingpathways might converge on Akt to phosphorylate PACS-2 (Herset al., 2011; Betz and Hall, 2013). It will also be important toidentify the PACS-2 Ser437 phosphatase.Since autophagy is generally viewed as a survival mechanism, it

may at first appear surprising that dephosphorylation of PACS-2Ser437 is also required for TRAIL to induce Bid translocation tomitochondria and cell death (Simmen et al., 2005; Levine andKroemer, 2008; Aslan et al., 2009; Rubinstein and Kimchi, 2012).Recent studies show, however, that autophagy is intimately coupledwith TRAIL-induced cell death and can influence whether TRAILkills cells by apoptosis (a death pathway in which cells are removedwithout membrane disruption) or necroptosis (an inflammatorynecrotic-like death pathway that involves cell breakage) (Goodallet al., 2016). In response to TRAIL, induction of early stage

autophagic machinery can promote assembly of the necrosome,which diverts the TRAIL-induced mode of killing from apoptosis tonecroptosis. It will therefore be interesting to determine towhat extentdephosphorylated PACS-2 modulates common or separate steps inthe decision between autophagy and autophagy-modulated cell death.

The evolutionally recent acquisition of nuclear trafficking signalsby PACS-1 and PACS-2 expanded their roles to also modulatenuclear gene expression (Atkins et al., 2014). The role of the PACSproteins in the nucleus is just beginning to be understood. Notably,PACS-2 is the most recent addition to a small collection of proteinsthat regulate SIRT1, which include deleted in breast cancer 1(DBC1; also known as CCAR2), active regulator of SIRT1 (AROS;also known as RPS19BP1) and the moonlighting protein GAPDH(Kim et al., 2007, 2008; Atkins et al., 2014; Chang et al., 2015). Itwill be important to determine whether PACS-2 acts in the same ordifferent pathways to the other SIRT1 regulators and whetherPACS-2 or PACS-1 regulates HDACs in addition to SIRT1.Interestingly, the description of PACS-2 as an in vivo modulator ofSIRT1 in response to DNA damage suggests that it may be involvedin additional pathways controlled by SIRT1 (Brooks and Gu, 2009).For example, in response to fasting, liver SIRT1 increases theexpression of genes that encode proteins involved in fatty acidoxidation and ketogenesis – pathways that protect from diet-inducedobesity (Purushotham et al., 2009). Thus, the recent report thatPACS-2 knockdown in liver protects mice from diet-inducedobesity (Arruda et al., 2014), raises the possibility that the acquiredNLS and Akt sites enable PACS-2 to coordinate SIRT1-dependentnuclear gene expression with MAM-dependent changes inmitochondrial respiration to modulate liver metabolism.

In summary, studies of the PACS proteins illustrate the vital roleof protein adaptation in coordinating the seemingly autonomousactions of the nucleus, mitochondria and endomembrane systems inresponse to the complex challenges faced by vertebrate organsystems. Moreover, the findings that mutation or altered expressionof PACS proteins are associated with pathologies ranging fromcancer, obesity and viral pathogenesis to epilepsy andneurodegeneration points to a rich opportunity for insight into themolecular basis of disease through analysis of this adaptable genefamily.

AcknowledgementsThe authors thank R. Chaillet and J. Dacks for critically reading the manuscript,J. D. Dikeakos for helpful suggestions and C. H. Hung and C. Crump forcontributions to studies described here. We also thankM. Ostrowski, C. Thauvin andH. Olson for communication of unpublished results.

Competing interestsThe authors declare no competing or financial interests.

FundingThis work in our laboratories was supported by the National Institutes of Health (NIH)(grants CA151564 and DK112844 to G.T.), Natural Sciences and EngineeringResearch Council of Canada (NSERC) (grant RGPIN-2015-04105 to T.S.) and theAmerican Heart Association (AHA) (grant 17SDG33350075 to J.E.A.). Deposited inPMC for release after 12 months.

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