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Home , BCAP31, FBRS, FIS1, PACS1, Protein moonlighting, Thomas Hunt Morgan

© 2017. Published by The Company of Biologists Ltd | Journal of Cell (2017) 130, 1865-1876 doi:10.1242/jcs.199463

COMMENTARY Caught in the act – adaptation and the expanding roles of the PACS in tissue homeostasis and disease Gary Thomas1,2,*, Joseph E. Aslan3, Laurel Thomas1, Pushkar Shinde4, Ujwal Shinde4 and Thomas Simmen5

ABSTRACT day ‘one protein – multiple functions’ mantra, which recognizes that Vertebrate proteins that fulfill multiple and seemingly disparate individual proteins often have multiple and seemingly disparate – ‘ ’ functions are increasingly recognized as vital solutions to functions a phenomenon known as moonlighting (Jeffery, 1999; maintaining homeostasis in the face of the complex cell and tissue Henderson and Martin, 2014). The glycolytic of higher metazoans. However, the molecular adaptations glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and that underpin this increased functionality remain elusive. In this are widely cited examples of moonlighting proteins. Commentary, we review the PACS proteins – which first appeared GAPDH participates in as many as 20 different cellular in lower metazoans as protein traffic modulators and evolved in functions ranging from the regulation of membrane traffic to the vertebrates to integrate cytoplasmic protein traffic and interorganellar maintenance of genome integrity (Sirover, 2011). Human enolase communication with nuclear expression – as examples of can be present on the cell surface of activated monocytes where it protein adaptation ‘caught in the act’. Vertebrate PACS-1 and PACS-2 serves as a plasminogen that promotes recruitment of increased their functional density and roles as metabolic switches by inflammatory cells to sites of tissue injury (Wygrecka et al., 2009). acquiring sites and nuclear trafficking signals within Protein moonlighting, which by definition is limited to proteins disordered regions of the proteins. These findings illustrate one that use a single primary sequence in the absence of mechanism by which vertebrates accommodate their complex cell posttranslational modifications to mediate multiple functions physiology with a limited set of proteins. We will also highlight how (Jeffery, 1999), is inherently limited for solving evolutionary pathogenic viruses exploit the PACS sorting pathways as well as challenges. Eventually, moonlighting gives way to gene recent studies on PACS with or altered expression duplication, which enables the resulting paralogs to acquire that result in diverse diseases. These discoveries suggest that mutations that expand the molecular functions initiated by the investigation of the evolving PACS protein family provides a rich ancestral protein. Inevitably, such a hit-and-miss approach to opportunity for insight into vertebrate cell and organ homeostasis. mutational diversification will hit a roadblock in the form of rigidly folded domains, such as SH2 or PDZ domains found in many proteins (Tompa et al., 2014). The dedicated structure of these Introduction ∼100-amino-acid protein domains, which are typically involved in At the turn of the 20th century, Archibald Garrod, and high-affinity binding to discrete ligands, limits amino acid Thomas Hunt Morgan ushered in a new era of biological research by substitutions, thereby reducing their ability to increase the replacing observational studies with mechanistic analyses (Kelves functional density of associated cellular proteins. Therefore, and Hood, 1992). Their discoveries that encode protein adaptation represents an additional and vital solution to heritable traits set the foundation for understanding genetic increase the array of protein functions to an extent that is not inheritance. More than 30 years later, through inactivation and possible by moonlighting and alone (Tompa et al., mutational studies of the common bread mold Neurospora, George 2014). Rather than coaxing rigid protein domains to accept new Beadle and ultimately determined that genes encode roles, such proteins – notably vertebrate proteins – have acquired enzymes and laid a cornerstone of modern molecular intrinsically disordered regions (IDRs) to complement the limited through the ‘one gene – one protein’ hypothesis (Beadle and Tatum, utility of folded domains (Dunker et al., 2005; Pancsa and Tompa, 1941). This provocative model inevitably collapsed under the 2016). IDRs do not fold into an autonomous structure, but contain weight of subsequent discoveries, which revealed that genes flexible 5–15-amino-acid small linear motifs (SLiMs) that can frequently encode multiple proteins or collections of peptides that assume an ensemble of ‘structures’, which in turn interact with a drive complex physiology and contribute to molecular promiscuity panoply of binding partners through low-affinity but high- through mechanisms ranging from alternative RNA splicing to the specificity interactions (Davey et al., 2015). SLiMs are frequently proteolytic processing of polyproteins (‘one gene – many proteins’; sites of posttranslational modifications and undergo rapid , Yang et al., 2016). The essence of these studies led to the present- greatly increasing the functional density of IDR-containing proteins (Tompa et al., 2014; Pancsa and Tompa, 2016). This plasticity enabled SLiMs from disparate proteins across a large taxonomic 1Department of Microbiology and Molecular , University of Pittsburgh range to acquire similar amino acid mutations by convergent School of Medicine, Pittsburgh, PA 15239, USA. 2University of Pittsburgh Institute, Pittsburgh, PA 15239, USA. 3Knight Cardiovascular Institute, Oregon evolution so that they may bind to a common globular protein Health & Science University, Portland, OR 97239, USA. 4Department of domain (Schlessinger et al., 2011; Davey et al., 2012, 2015; Van Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, OR 97239, USA. 5Department of Cell Biology, University of Alberta, Edmonton, Roey et al., 2012; Jonas and Izaurralde, 2013). However, examples Alberta, Canada T6G2H7. tracing how a single protein adapted to evolutionary pressure by acquiring specific SLiM mutations in order to fulfill new functions *Author for correspondence ([email protected]) that are critical for vertebrate homeostasis are surprisingly limited.

G.T., 0000-0003-1976-7183 Studies on the phosphofurin acidic cluster sorting (PACS) proteins Journal of Cell Science

1865 COMMENTARY Journal of Cell Science (2017) 130, 1865-1876 doi:10.1242/jcs.199463 provide a powerful illustration of such evolutionary protein appearance of the vertebrates, resulting in the PACS-1 and PACS-2 adaptations ‘caught in the act’ of connecting the increasingly genes. complex organellar landscape that underpins vertebrate cell The regulation of membrane traffic remains a role that is function. conserved among the vertebrate PACS proteins. The PACS-1 FBR Here, we discuss how PACS proteins first appeared in metazoans binds to acidic clusters that can be phosphorylated by casein kinase as membrane traffic regulators, and then expanded their functional 2 (CK2), as well as α-helices on a large number of client proteins repertoire in vertebrates by acquiring molecular switches, notably (Wan et al., 1998; Youker et al., 2009; Dikeakos et al., 2012). Thus, phosphorylation sites and nuclear localization signals (NLSs) PACS-1, which interacts with the adaptors AP-1 and AP-3, within their SLiMs. These gain-of-function adaptations enabled as well as the monomeric adaptor GGA3, mediates localization of the vertebrate PACS proteins to integrate cytoplasmic functions and other client proteins to the trans-Golgi network (TGN) and with nuclear gene expression. In particular, we focus on the also targets a subset of client proteins to the primary cilium, acquisition of an Akt phosphorylation site and NLS in vertebrate including the adaptor protein nephrocystin (also known as NPHP1) PACS-2, which enable this protein to function as a metabolic switch and the olfactory cyclic-nucleotide-gated (CNG), the that integrates cytoplasmic membrane traffic and interorganellar latter by binding to the β1 subunit (CNGB1) (Fig. 3A) (Wan et al., communication with nuclear gene expression in response to 1998; Schermer et al., 2005; Jenkins et al., 2009; Youker et al., anabolic or catabolic cues. 2009). PACS-1 acquired a CK2-phosphorylated acidic cluster of its own, which is located in the disordered MR (see Fig. 1). CK2 PACS proteins direct membrane trafficking in worms to phosphorylation of Ser278 in the PACS-1 autoregulatory domain controls intramolecular binding to the FBR, which regulates the The PACS proteins were discovered in a genetic screen for interaction with client proteins (Scott et al., 2003). PACS-2, which regulators of the secretory pathway proteinase furin (Wan et al., interacts with the coatomer COPI, mediates the localization of cargo 1998; Thomas, 2002). Human PACS1 is located at 11q13.1-q13.2 to the (ER) and, similar to cePACS, directs and contains 24 exons and at least 12 alternatively spliced variants. trafficking from early (Youker et al., 2009) (Fig. 3A, see Human PACS-2, which is located near the telomere at 14q32.33, also Fig. 4A). PACS-2 stabilizes a pool of the metalloproteinase also contains 24 exons and at least 11 alternatively spliced variants. ADAM17 (also known as TACE) on early endosomes, from where Genome-wide association studies (GWAS) identified the human the is trafficked to the cell surface to shed ErbB ligands, PACS1 locus as a susceptibility gene in severe early-onset obesity including those that ligate the epidermal growth factor receptor (Wheeler et al., 2013) and developmental delay (Deciphering (EGFR) (Dombernowsky et al., 2015). In the absence of PACS-2, Developmental Disorders Study, 2017), and mutations in PACS1 ADAM17 is diverted to , which reduces ErbB shedding. underlie ‘PACS-1 syndrome’ (Schuurs-Hoeijmakers et al., 2012, Correspondingly, EGFR signaling is reduced in the intestinal 2016; Stern et al., 2017), characterized by epileptic seizures, heart epithelium of PACS2−/− mice (Dombernowsky et al., 2015). The defects, and craniofacial malformations. The PACS2 role of the candidate autoregulatory domain in the PACS-2 MR has locus is highly susceptible to loss of heterozygosity in colorectal not been established. cancer, leading to a reduction or loss of PACS-2 protein expression Several pathogenic viruses acquired furin-like acidic clusters to (Anderson et al., 2001; Aslan et al., 2009). Similar to PACS1, exploit the endosomal sorting steps mediated by PACS-1 and mutations in PACS2 are associated with neonatal onset epilepsy, PACS-2. This viral mimicry (see Davey et al., 2011) enables the global developmental delay and intellectual disability (C. Thauvin viruses to assemble progeny, escape immune surveillance and and H. Olson, personal communication). prevent . For example, PACS-1 binds to acidic clusters in The canonical 963-amino-acid PACS-1 and 889-amino-acid the human cytomegalovirus (HCMV) envelope glycoprotein gB PACS-2 proteins share 54% sequence identity. Bioinformatics and the Epstein–Barr virus (EBV) tegument protein BBLF1 and analyses suggest both proteins lack folded globular domains and are localizes them to the TGN to support virus assembly (Crump et al., instead nearly 50% disordered (Fig. 1). The ∼150-amino-acid cargo 2003; Chiu et al., 2012) (see Fig. 3A). Furthermore, PACS-2 (furin)-binding regions (FBRs) in PACS-1 and PACS-2, which are interacts with a pair of small acidic clusters in the ubiquitin ligase predicted to be structured (Fig. 1), share nearly 80% sequence K5 of Kaposi sarcoma herpesvirus (KSHV) at the ER. This identity and bind client proteins, as well as the cytoplasmic interaction enables KSHV to downregulate the cell adhesion membrane trafficking machinery as described below. PACS-1 and molecule CD31 (also known as PECAM1), which may contribute PACS-2 also share a disordered middle region (MR), which to KSHV-induced cancer (Mansouri et al., 2006). The HIV-1 contains an autoregulatory domain as well as NLSs and, specifically accessory protein Nef uses a bipartite motif composed of a short in PACS-2, a critical Akt phosphorylation site that binds to 14-3-3 acidic cluster and the αB helix to interact with both PACS-1 and proteins. The function of the shared C-terminal region (CTR) is PACS-2 (Piguet et al., 2000; Atkins et al., 2008; Dikeakos et al., unknown. PACS-1 additionally contains an N-terminal extension 2012). This bipartite binding enables HIV-1 Nef to downregulate called the atrophin-1-related region (ARR), which has homology to major histocompatibility complex class I (MHC-I) from the cell this nuclear transcriptional co-repressor (Zhang et al., 2006). surface, which allows the virus to escape immune detection (Pawlak The PACS genes are a recent addition to the eukaryotic genome, and Dikeakos, 2015) (Fig. 3B). To this end, Nef interacts with appearing first in lower metazoans (Fig. 2). Invertebrates, including PACS-2 on early endosomes, which enables the HIV-1 protein to nematodes, and echinoderms, possess a single PACS assemble a multi-kinase complex consisting of a Src family kinase locus that is apparently dedicated to membrane trafficking. Notably, (SFK), ζ-chain-associated protein kinase 70 (ZAP-70) and a class I Caernorhabditis elegans PACS (cePACS, T18H9.7a) localizes to phosphoinositide-3 kinase (PI3K) (Blagoveshchenskaya et al., early endosomes at the presynaptic terminus of the neuromuscular 2002; Hung et al., 2007; Atkins et al., 2008). The activated multi- junction where it mediates synaptic transmission (Sieburth et al., kinase complex increases the amount of phosphatidylinositol 2005). Lower chordates, including Amphioxus and tunicates also (3,4,5)-trisphosphate (PIP3) underneath the plasma membrane, possess only a single gene. The PACS gene was duplicated with the which recruits an ARF6 GEF to activate ARF6 and accelerate Journal of Cell Science

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PACS-2 NLS Akt/14-3-3 PKKQRRSIV RSTpSLKERQ FBR MR CTR 1.0

0.5 Disorder probability 0

33.3% α-Helix 15.5% β-Strand 5.3% β-Turn 45.6% Random 0 200 400 600 800

PACS-1 RSTPLKERQ ARR FBR VKKTRRKLT MR CTR 1.0

0.5 Disorder probability 0 30.6% α-Helix 15.8% β-Strand 5.3% β-Turn 48.3% Random 0 200 400 600 800 Amino acid

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 predict natively disordered regions from the amino acid sequences of the human PACS-1 (UniProt Q6VY07) and PACS-2 (UniProt Q86VP3) proteins [false discoveryrate (FDR)=2%]. The disorder probabilities for each residue were plotted as a function of length and the graphical profiles were juxtaposed with the predicted secondary structures, which were obtained using an improved self-optimized prediction method (SOPMA) on a set of aligned members of the PACS-1 or PACS-2 protein families (lower plots). The PACS-2 nuclear localization signal (NLS) and Akt site, which binds 14-3-3 proteins, together with the corresponding sequences in 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)]. endocytosis of cell-surface MHC-I (Blagoveshchenskaya et al., requires the coordinated permeabilization of multiple organelles, 2002). Nef then connects the internalized MHC-I molecules to including mitochondria and lysosomes (Aslan and Thomas, 2009). PACS-1 and AP-1 on an endosomal compartment to prevent their TRAIL triggers mitochondrial outer membrane permeabilization recycling to the cell surface and instead sequester them in the TGN, (MOMP) by inducing dephosphorylation and proteolytic cleavage thereby protecting the virus from immune surveillance of the pro-apoptotic Bcl-2 protein Bid to form truncated Bid (tBid), (Blagoveshchenskaya et al., 2002; Chaudhry et al., 2008; which drives Bax-dependent MOMP and the consequent activation Noviello et al., 2008; Dikeakos et al., 2010; Dirk et al., 2016). of executioner caspases (Aslan et al., 2009). Unlike KSHV and many other viruses, HIV-1 Nef does not induce PACS-2 has essential roles in TRAIL-induced MOMP (Fig. 4B). In degradation of downregulated MHC-I (Blagoveshchenskaya et al., response to TRAIL, PACS-2 binds full-length dephosphorylated Bid 2002; Dikeakos et al., 2010; Dirk et al., 2016). This chink in the and traffics it to mitochondria where it can be converted into tBid armor of HIV-1 provides an alternative approach to combat the virus (Simmen et al., 2005). In parallel, TRAIL triggers assembly of a by reversing the Nef-mediated immune evasion pathway. In support complex that contains PACS-2, Bim (also known as BCL2L11) and of this possibility, treatment of HIV-1-infected primary CD4+ Bax, called the PIXosome, on lysosomal membranes (Werneburg T-cells with small-molecule inhibitors of the multi-kinase complex et al., 2012). The PIXosome drives lysosomal membrane restores cell surface expression of MHC-I and sensitizes them to permeabilization (LMP), which releases cathepsin B and other killing by CD8+ T-cells (Hung et al., 2007; Dikeakos et al., 2010; hydrolases that are required for MOMP into the (Boya and and M. Ostrowski, personal communication). Kroemer, 2008). Several mechanisms interfere with these proapoptotic functions of PACS-2. In hepatocellular carcinoma Links between mammalian PACS proteins and TRAIL- cells, inhibitors of apoptosis (IAPs) target PACS-2 for proteasomal induced apoptosis degradation, thereby protecting the cancer cells from being killed by Surprisingly, while PACS-2 mediates trafficking of many pro- TRAIL (Guicciardi et al., 2014). In cardiomyocytes, miR499 prevents survival signaling molecules, it is also tasked with mediating death- Bid-dependent apoptosis by downregulating PACS-2 (Wang et al., ligand-induced apoptosis induced by TNF-related apoptosis- 2014). Not surprisingly, herpesviruses also exploit the ability of inducing ligand (TRAIL, also known as TNFSF10). This PACS-2 to traffic proteins to mitochondria. The HCMV protein viral clinically important death ligand is an in vivo metastasis inhibitor inhibitor of apoptosis (vMIA) prevents cell death by trapping pro- that selectively kills diseased cells, including cancer cells and virally apoptotic Bax on mitochondria (Arnoult et al., 2004). vMIA interacts infected cells (Johnstone et al., 2008). TRAIL-induced cell death with PACS-2 but not PACS-1 (our unpublished data), and the vMIA– Journal of Cell Science

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NLS Akt site Mammals P 240 Human P-KKQRRSI-V RS-TSLKERQ442 201 403 Gorilla P-KKQRRSI-V RS-TSLKERQ 201 403 Orangutan P-KKQRRSI-V RS-TSLKERQ 240 442 Chimpanzee P-KKQRRSV-V RS-TSLKERQ 240 443 Mouse P-KKQRRSI-V RS-TSLKERQ Mammals 240 Rat P-KKQRRSI-V RS-TSLKERQ442 P1 P2 201 403 Dog P-KKQRRSI-V RS-TSLKERQ 173 375 Cat P-KKQRRSI-V RS-TSLKERQ 201 403 Panda P-KKQRRSI-V RS-TSLKERQ 103 305 Marmoset P-KKQRRSI-V RS-TSLKERQ Birds Birds P1 P2 255 458 Finch P-KKQRRSI-V RS-TSLKERQ 286 489

Chicken P-KKQRRSI-V RS-TSLKERQ PACS-2 203 406 Turkey P-KKQRRSI-V RS-TSLKERQ Fish Fish P-KKQRRSI-V239 RS-TSLKERQ433 Zebrafish 251 445 P1 P2 Stickleback P-KKQRRSI-V RS-TSLKERQ 204 406 J. rice fish P-KKQRRSI-V RS-TSLKERQ 321 504 Pufferfish P-KKQRRSI-V RS-TSLKERQ 240 Cyclostomes Tilapia P-KKQRRSI-V RS-TSLKERQ430 189 383 S. platyfish P-KKQRRSI-V RS-TSLKERQ Mammals 319 517 Human V-KKTRRKL-T RS-TPLKERQ Gorilla V-KKTRRKL-T257 RS-TPLKERQ455 318 516 Orangutan V-KKTRRKL-T RS-TPLKERQ 319 517 Chimpanzee V-KKTRRKL-T RS-TPLKERQ 317 515 Tunicates Mouse V-KKTRRKL-T RS-TPLKERQ 317 515 Rat V-KKTRRKL-T RS-TPLKERQ 318 516 Dog V-KKTRRKL-T RS-TPLKERQ 127 325 Cat V-KKTRRKL-T RS-TPLKERQ Cephalochordates 316 514 Panda V-KKTRRKL-T RS-TPLKERQ 204 405 Marmoset V-KKTRRKL-T RS-TPLKERQ Birds 220 Finch T-KKARRKM-N RS-TSVKDRQ417 Chicken T-KKARRKM-I213 RS-TSVKDRQ409 PACS-1 Echinoderms Turkey T-KKARRKM-I207 RS-TSVKDRQ409 Fish Zebrafish --KKPRRKL-P253 RG-TPMKERQ452 Stickleback AVNVSELKL-E218 RI-TPAKERQ418 J. rice fish --KKPRRKL-E199 NSSTPMKERQ403 Pufferfish --KKPRRKL-P203 RVSTPMKERQ400 Arthropods Tilapia --KKPRRKL-P250 RISTPMKERQ451 S. platyfish --KKPRRKL-P203 SSNTPMKERQ404 Lower chordates 206 401 Lamprey SKKSRHRKV-S RS-SSLRERS Nematodes 196 Lamprey P1 rel PKPRRKNQP -S-TSLRERP390 497 Amphioxious K-KSGRRKV--331 RS-ISMRERK

Fig. 2. Phylogenetic analysis of the PACS genes. Non-redundant protein sequences of the PACS family members were obtained from the UniProt and NCBI databases. The protein sequences were aligned using Muscle and were manually examined/modified for their accuracy within the non-conserved domains that flank conserved domains. The program Gblocks was used to curate and eliminate poorly aligned positions and divergent regions with the protein alignment prior to the phylogenetic analysis (Castresana, 2000). The program PhyML was used to estimate the maximum likelihood phylogenies from alignments of amino acid sequences (Guindon et al., 2005). The tree (A) and multiple alignments of the NLS and Akt sites (B) were visualized using Mega7. Black diamonds indicate invertebrate PACS proteins expressed from a single gene. Gray diamonds indicate cyclostome PACS proteins, which may be precursorsto PACS-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 red background to the amino acid residue indicates similar residues, and a yellow background divergent residues.

PACS-2 interaction is required for efficient translocation of vMIA to binds dephosphorylated Bid, but prevents translocation of Bid to mitochondria (Salka et al., 2017). Similar to the fate of Bax, vMIA mitochondria (T.S., L.T., J.A. and G.T., unpublished results). traps PACS-2 on mitochondria. Notably, this sequestration of PACS-2 is coupled with a blunted translocation of Bid to mitochondria in PACS-2 regulates MAM-localized Ca2+ signaling, lipid response to death ligands, suggesting vMIA may trap both inactivated metabolism and autophagy Bax and PACS-2 on mitochondria to inhibit apoptosis in HCMV- Mitochondria-associated membranes (MAMs) are ER–mitochondria infected cells (Fig. 5A; L.T., T.S., J.A. and G.T., unpublished results). contacts, which were discovered as lipogeneic platforms in liver that The PACS-2-dependent recruitment of Bid to mitochondria prior to are responsive to feeding and starvation (Bernhard et al., 1952; formation of tBid suggests this translocation step may be a checkpoint Vance, 1990). Subsequent studies further revealed that MAMs are a 2+ for apoptotic regulation. In support of this possibility, PACS-1 also dynamic communication center that regulates Ca signaling, lipid Journal of Cell Science

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Fig. 3. Protein traffic steps mediated by A B PACS-1 and PACS-2. (A) PACS-1 mediates the sorting of client proteins from late ARF6 MHC-I PIP3 GEF endosomes (LE) to the TGN, from early Primary endosomes (EE) to the plasma membrane ARF6 cilium (PM), as well as delivery to the primary Nephrocystin cilium. PACS-2 mediates the localization of CNGB1b PM cargo proteins to the ER, from early ADAM17 endosomes to the TGN or plasma membrane, and also promotes MAM Furin PACS-1 HIV-1 RE replication integrity. (B) HIV-1 Nef usurps the sorting PM steps mediated by PACS-2 and PACS-1 to downregulate the levels of cell surface MHC- PACS-2 I in CD4+ T-cells. Nef binds to Akt- phosphorylated PACS-2 on early Nef endosomes (Atkins et al., 2008; Dikeakos et al., 2012, and L.T. and G.T., unpublished EE results). This allows Nef to traffic to the TGN ZAP-70 region where it binds and activates a Src EE LE Syk family kinase (SFK; Hck, Src or Lyn). The P – PACS-2 SFK Nef SFK complex then recruits ZAP-70 (in PI3K T-cells) or Syk (in monocytes and other cell Nef types) and a class I PI3K, which increases

PACS-1 the level of PIP3 (maroon circles) at the HIV-1 Nef plasma membrane. This recruits an ARF6 GEF that accelerates MHC-I internalization Furin through an ARF6-regulated endocytic 14-3-3 CI-MPR P pathway. Nef diverts the internalized MHC-I SorLA EE molecules from their local recycling HCMV gB compartment (dashed line) and combines EBV BBLF1 LE with AP-1 and PACS-1 to transport MHC-I Golgi PACS-2 through early and late endosomes and Nef AP-1 sequester it in the TGN. The identity of the Nef precise compartment containing Nef, MHC-I, PACS-2 PACS-1 AP-1 and PACS-1 is under investigation. This MHC-I downregulation pathway protects HIV-1 from CD8+ T-cell killing Calnexin thereby allowing the virus to evade immune Polycystin-2 surveillance. Thus, small-molecule inhibition Profurin SFK of the multi-kinase complex re-exposes Nef MHC-I on the cells surface and sensitizes HIV-1-infected cells to CD8+ T-cell killing. Nef The steps shown here depict the ‘signaling ’ MAM mode of HIV-1 Nef-induced immune PACS-2 evasion, which HIV-1 implements during the first 48 h post infection. A detailed discussion of Nef-induced immune evasion is presented Golgi ER elsewhere (Dikeakos et al., 2010; Pawlak and Dikeakos 2015).

metabolism and autophagy (Raturi and Simmen, 2013; Sood et al., (Simmen et al., 2005) (see Fig. 4C). For example, PACS-2 is required 2014; Theurey et al., 2016). Mammalian MAMs are formed by for starvation-induced autophagy because it promotes the recruitment protein tethers, similar to the tetrameric tethering complexes in fungi of the early autophagy marker Atg14 to MAMs (Hamasaki et al., known as ER–mitochondria encounter structures (ERMES) 2013). PACS-2 knockdown also blocks Ca2+-mediated apoptosis (Kornmann et al., 2009). However, of the more than 30 proteins progression, which suggests that PACS-2 is required for efficient Ca2+ involved in vertebrate MAM function, only two of them, transfer between the ER and mitochondria (Simmen et al., 2005). This GRAMD1A, which corresponds to yeast Lam6p, and MIRO (also PACS-2 function may be coupled to its reported dynamic roles in the known as RHOT1), which corresponds to yeast Gem1p, are trafficking of calnexin and perhaps other ER Ca2+ regulatory proteins conserved in ERMES (Kornmann et al., 2011; Elbaz-Alon et al., that modulate ER–mitochondria Ca2+ flux (Myhill et al., 2008). 2015). This increased complexity of MAMs suggests they Mechanistically, PACS-2 may modulate MAM formation by acquired new roles beyond those controlled by ERMES (Herrera- preventing cleavage of the ER–mitochondria tethering protein Cruz and Simmen, 2017). This increased function correlates with the BAP31 (also known as BCAP31) (Iwasawa et al., 2011). emerging realization that disturbances in MAM integrity are Disturbances in these PACS-2 functions could contribute to disease. associated with diseases ranging from obesity to neurodegenerative For example, in obese mice challenged by a high-fat diet, PACS-2 is disorders (Arruda and Hotamisligil, 2015; Paillusson et al., 2016). responsible for a chronic increase in MAM formation (Arruda et al., Knockdown or knockout of PACS-2 detaches the ER from 2014), leading to toxic mitochondrial Ca2+ overload that consequently mitochondria and interferes with a number of key MAM functions impairs mitochondrial oxidative capacity, exacerbates insulin Journal of Cell Science

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A C EGF Insulin D 14-3-3 P ADAM17 DNA mTORC2 damage PM MAM PACS-2 ATM MAM ? 14-3-3 X P Lys. 14-3-3 κ P NF- B Akt ATM PACS-2 ADAM17 PACS-2 DDR EE Lipogenesis Autophagy NF-κB 2+ (Anti-apoptosis) Bcl-xL B Anabolic signaling Ca transfer

Catabolic signaling 14-3-3 Bid PACS-2 P

TRAIL PACS-2 SIRT1 mTORC2 Lys. Ac Bax MAM MAM Ac p53 P PACS-2 Bim ? tBid ( arrest) p21 Cath. B Akt PPase PACS-2 X Lipogenesis Nucleus 14-3-3 Autophagy P Cytoplasm Ca2+ transfer

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 the 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 called the PIXosome, which is required for membrane permeabilization to release cathepsin B (cath. B). (C) MAMS. Top panel: insulin or growth factors trigger activation of mTORC2 on mitochondria-associated membranes (MAMs; green shading at the ER–mitochondria contact site), which activates Akt to phosphorylate 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 cells or cells treated with TRAIL, Akt is inhibited and PACS-2 Ser437 is dephosphorylated by a protein phosphatase (PPase). Dephosphorylated PACS-2 in turn remodels MAMs (red shading at the ER–mitochondria contact site), which may reduce lipogenesis but increase ER–mitochondrial Ca2+ exchange as well as induction of autophagy. (D) DNA damage response. Top panel: to support induction of the NF-κB and Bcl-xL anti-apoptotic pathway, cytoplasmic PACS-2 interacts with a pool of ATM released from the nucleus and maintains the DNA damage kinase in the cytoplasm. The cytoplasmic ATM then triggers activation of the 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 cycle arrest. Green arrows, pro-survival-anabolic pathways mediated by pPACS-2. Red arrows, apoptotic or catabolic pathways mediated by dephosphorylated PACS-2. Ac, acetylation; DDR, DNA-damage response. resistance and disrupts glucose homeostasis (Arruda et al., 2014). phosphorylation site that switches the function of PACS-2 Conversely, PACS-2 knockdown protects liver from overnutrition- between these divergent roles. Akt phosphorylates PACS-2 on induced steatosis and optimizes mitochondrial respiration and insulin Ser437, which promotes high-affinity binding to 14-3-3 proteins sensitivity (Arruda et al., 2014). Aberrantly elevated PACS-2 and (Aslan et al., 2009). In response to insulin, MAM-localized MAMformationarealsofoundinneuronsfromAlzheimer’s patients mTORC2 activates Akt, which phosphorylates PACS-2 to and Alzheimer’s mouse models, suggesting that the deleterious modulate MAM integrity (Betz et al., 2013) (Fig. 4C, top). Akt- consequences from abnormally strong MAM formation in this disease mediated phosphorylation of Ser437 is also required for PACS-2- mayalsoresultfromdysregulatedPACS-2expression(Area-Gomez dependent membrane trafficking steps (Fig. 4A) (Aslan et al., et al., 2012; Hedskog et al., 2013). 2009). By contrast, dephosphorylation of PACS-2 Ser437, which prevents 14-3-3 binding, blocks pro-survival membrane traffic and Vertebrate PACS-2 acquired an Akt phosphorylation site to disrupts MAMs, but is required for apoptotic trafficking to switch between its anabolic and catabolic roles mitochondria and lysosomes and may support autophagy The seemingly incongruous roles for PACS-2 in mediating pro- induction (Fig. 4B,C, bottom) (Aslan et al., 2009; Werneburg survival protein traffic and MAM function, as well as TRAIL- et al., 2012; Betz et al., 2013; Hamasaki et al., 2013; and L.T. and induced cell death, arose with the acquisition of an Akt G.T., unpublished data). Journal of Cell Science

1870 COMMENTARY Journal of Cell Science (2017) 130, 1865-1876 doi:10.1242/jcs.199463

A B TRAILC TRAIL

PM TRAIL-RPM TRAIL-R

P Bid translocation P aborted Bid Bid ?

Control P PACS-1 Bid P

Bid-GFP Mito. Bid Bid * Caspase-8 PACS-2 * Apoptosis vMIA blocked PACS-2 * vMIA + vMIA tBid + Fas Ab/CHX * * * * * * PACS-2 Bid Bid-GFP* Mito.* vMIA* tBid translocates to mitochondria tBid Caspase-8 and tBid other

c Cytochrome Cytochrome c

Fig. 5. Possible models for the regulation of Bid translocation to mitochondria by PACS proteins and vMIA. (A) Example of an experiment showing that vMIA 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) for 3 h (bottom) to induce Bid translocation. Mitochondria were then labeled with Mitotracker Red. Image analysis showed that anti-Fas antibody concentrated Bid– GFP staining on mitochondria in untransfected cells (yellow asterisk) but not in cells expressing vMIA ( asterisks). Scale bar: 20 µm. (B) Conventional model of Bid regulation. Dephosphorylation of full-length Bid exposes a cleavage site for caspase-8. Caspase-8-mediated cleavage generates tBid, which is then myristoylated and traffics to mitochondria to promote MOMP. (C) Alternative model of Bid regulation. Dephosphorylation of full-length Bid exposes a binding site for 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 translocation to mitochondria (upper dashed lines). In HCMV-infected cells, vMIA sequesters PACS-2 to mitochondria (lower dashed lines), thereby preventing Bid recruitment and, ultimately, MOMP. TRAIL-R, TRAIL receptor.

Akt and 14-3-3 proteins not only repress the apoptotic roles of (p)Ser437, versus catabolic (including autophagic and apoptotic PACS-2 but also regulate other proapoptotic proteins, including the trafficking) pathways, which would be mediated by PACS-2 that is Bcl-2 protein Bad and FOXO transcription factors (Datta et al., dephosphorylated on Ser437 (Yaffe, 2002). 1997, 2002; Brunet et al., 1999; Singh et al., 2010; Feehan and Shantz, 2016). The regulation of Bad and FOXO proteins by Akt PACS-2 acquired nuclear trafficking signals to modulate and 14-3-3 is akin to a simple molecular ‘on or off’ switch (Van gene expression Roey et al., 2012). However, the regulation of the anabolic (pro- The extrinsic and intrinsic apoptotic pathways induced by TRAIL or survival) roles of PACS-2 versus the catabolic (apoptotic) roles DNA damage, respectively, use different molecular steps to trigger exerted by Akt and 14-3-3 appears to be more complex. We suggest MOMP. Nonetheless, these pathways are intimately coupled, as p53 that the Akt site in PACS-2 resembles a ‘bifurcation’ switch such (also known as TP53) induces expression of the TRAIL receptor that both the phosphorylated and dephosphorylated states of PACS- DR5 (also known as TNFRSF10B), and chemotherapeutics and 2 are active, but in opposing directions (anabolic versus catabolic TRAIL synergize to kill cancer cells (Sheikh et al., 1998; Ifeadi pathways, respectively). This model is supported by the ability of and Garnett-Benson, 2012). Surprisingly, PACS-2 has markedly 14-3-3 proteins to induce conformational changes in their partners different roles in its response to TRAIL compared with DNA that may enable PACS-2 to selectively bind to client proteins that are damage; PACS2−/− mice are impaired in TRAIL-induced apoptosis, involved in anabolic (including pro-survival trafficking) pathways, but are sensitized to DNA-damage-induced apoptosis (Aslan et al., which would be mediated by binding of 14-3-3 to phosphorylated 2009; Barroso-Gonzalez et al., 2016). These opposing roles for Journal of Cell Science

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PACS-2 are partly explained by the phosphorylation state of Ser437, disordered region that apparently became fixed by positive which is reduced by TRAIL, but increased upon DNA damage selection (Fig. 2). Phylogenetic studies suggest this SLiM evolved (Barroso-Gonzalez et al., 2016). Whereas TRAIL uses rapidly, albeit along a circuitous path (Figs 1 and 2). An Akt dephosphorylated PACS-2 to induce MOMP and LMP, the DNA consensus motif first appeared in lamprey PACS-1-related which damage response uses phosphorylated PACS-2 to support cytostasis coincided with an apparent gene duplication (Fig. 2). This by coordinating NF-κB and Bcl-xL-dependent anti-apoptosis with consensus phosphorylation site was disrupted in fish PACS-1 by p53- and p21 (CDKN1A)-dependent cell cycle arrest (Aslan et al., the acquisition of a proline residue at the +2 position. Surprisingly, 2009; Barroso-Gonzalez et al., 2016). in birds, the Akt motif reappeared in both PACS-1 and PACS-2. Clues to understanding the roles of PACS-2 in mediating the However, in mammals, the PACS-1 site again acquired a proline p53–p21 and NF-κB–Bcl-xL pathways were provided by the DNA residue, this time in place of the phosphorylatable serine residue repair kinase ATM (Shiloh and Ziv, 2013). In response to DNA (Fig. 2). These findings suggest that PACS-2 acquired the Akt site to damage, nuclear ATM phosphorylates p53, which stabilizes the act as a vital ‘bifurcation’ switch between its anabolic versus multi-functional to induce its target genes, thus catabolic pathways and that evolutionary pressure assigned this favoring cell cycle arrest and over apoptosis (Xu and switch to mammalian PACS-2 by negatively selecting against the Baltimore, 1996; Sperka et al., 2012). Concurrently, a small pool of phosphorylation site in mammalian PACS-1 (Figs 2 and 4) (Davey activated ATM translocates from the nucleus into the cytoplasm et al., 2015). where it initiates a novel ‘nucleus-to-cytoplasm’ signaling pathway Like the Akt site, the PACS-2 NLS, which is also located within a that promotes the NF-κB-dependent induction of anti-apoptotic disordered region, is highly conserved across all jawed vertebrates Bcl-xL through an as-yet-unresolved mechanism (Miyamoto, (Figs 1 and 2). Phylogenetic studies suggest that a polybasic site 2011). with limited similarity to the vertebrate PACS NLS first appeared in PACS-2 intersects both ATM pathways to coordinate induction of amphioxus and highly homologous NLS sequences were first the NF-κB–Bcl-xL and p53–p21 pathways. Following DNA acquired in fish (Fig. 2). Acquisition of the NLS by jawed vertebrate damage, cytoplasmic PACS-2 interacts with ATM, thereby PACS-2 parallels the evolutionary expansion of roles of p53 in sequestering the kinase in the cytoplasm where it induces TGF-β- directing apoptosis versus cell cycle arrest. In worms and flies, p53 activating kinase 1 (TAK1; also known as MAP3K7) to trigger is dedicated to driving apoptosis (Schumacher et al., 2001). activation of IκB kinase (IκK) (Wu et al., 2010; Barroso-Gonzalez However, vertebrate p53 additionally induces p21-dependent cell et al., 2016) (Fig. 4D, top). ATM-activated IκK then stimulates the cycle arrest (Lu et al., 2009). Consistent with this finding, vertebrate canonical IκBα–NF-κB induction of Bcl-xL. In the absence of PACS-2, but not C. elegans PACS, can localize in the nucleus PACS-2, ATM fails to accumulate in the cytoplasm, which reduces (Atkins et al., 2014). Thus, the ability of PACS-2 to traffic to the the induction of Bcl-xL. Consequently, induction of the apoptotic nucleus, where it modulates SIRT1 to promote p53-dependent p53–Puma (Puma is also known as BBC3) pathway remains, induction of p21, appears to have resulted from an evolutionary thereby increasing MOMP and cell death (Barroso-Gonzalez et al., adaptation required to support the more complex demands of 2016). vertebrate p53 in responding to mild DNA damage by supporting To promote p53–p21-dependent cell cycle arrest over p53–Puma- p21-dependent cell cycle arrest. The predicted PACS-1 NLS is not dependent cell death, PACS-2 traffics to the nucleus where it fixed across all vertebrates. Mammals, birds and fish each express a increases the transactivation of p53 bound to the p21 promoter characteristic NLS in their PACS-1 sequences. The significance of (Atkins et al., 2014) (Fig. 4D, bottom). Notably, in addition to the this variation, as well as validation of the PACS-1 polybasic site as a ATM-mediated phosphorylation of p53 within its N-terminal bona fide NLS, remains to be determined. region, acetylation of critical lysine residues near the C-terminus of p53 is also required for it to promote maximal transcriptional Conclusions and perspectives activity of its target genes (Gu and Roeder, 1997). The class III The PACS proteins first appeared in lower metazoans as membrane histone deacetylase (HDAC) SIRT1, in turn, represses p53- trafficking regulators and then adapted to evolutionary pressure by mediated transcriptional activation by deacetylating p53 following acquiring motifs to support the increasingly complex interorganellar DNA damage (Kruse and Gu, 2009). In response to DNA damage, communication pathways required for vertebrate cellular Akt-phosphorylated PACS-2 enters the nucleus where it binds to homeostasis (Figs 1 and 2). The association of mutations in the and inhibits SIRT1, which protects acetylated p53 bound to the p21 PACS1 and PACS2 genes with diseases ranging from obesity to promoter and, consequently, increases transcriptional output of p21 epilepsy and cancer underscores the broad and important roles this (Atkins et al., 2014; and L.T. and G.T., unpublished results). In the gene family plays in vertebrate biology. As membrane trafficking absence of PACS-2, the excessive SIRT1 activity reduces the p53- regulators, the PACS proteins interact with sorting adaptors as well dependent induction of p21, which impedes cell cycle arrest and as a variety of trafficking motifs on client proteins, ranging from sensitizes cells to p53–Puma-dependent apoptosis. To access the CK2-phosphorylatable acidic clusters to α-helices, in order to nucleus, vertebrate PACS-2 acquired a polybasic nuclear modulate endomembrane protein traffic (Figs 3 and 4). The PACS-1 localization signal (NLS) and a Crm1-dependent nuclear export and PACS-2 sorting pathways are also hijacked by pathogenic signal (Atkins et al., 2014). Similar to the Ser437 Akt site of PACS-2, viruses to support production of progeny, immune evasion and its NLS is located within a predicted IDR and is present only in protection from apoptosis. vertebrate PACS proteins (Figs 1 and 2). The acquisition of the Ser437 Akt site enabled cytoplasmic PACS- 2 to act as a bifurcation switch that separates its roles between PACS proteins as models of evolutionary protein adaptation endomembrane trafficking (phosphorylated PACS-2) and apoptotic The acquisition of the Ser437 Akt site enabled cytoplasmic PACS-2 signaling (unphosphorylated PACS-2) (see Fig. 4). The essential to switch between its homeostatic (pSer437) and apoptotic role for PACS-2 in mediating TRAIL-induced translocation of Bid (dephosphorylated Ser437) roles (see Fig. 4). PACS-2 proteins in to mitochondria was surprising as it expanded the prevailing model jawed vertebrates share an identical Akt motif nestled in a of Bid action. This conventional model posits that death receptors Journal of Cell Science

1872 COMMENTARY Journal of Cell Science (2017) 130, 1865-1876 doi:10.1242/jcs.199463 trigger dephosphorylation of Bid, which permits caspase-8- autophagic machinery can promote assembly of the necrosome, dependent proteolytic cleavage and myristoylation of human Bid which diverts the TRAIL-induced mode of killing from apoptosis to at Asp↓-Gly61 to form tBid (Li et al., 1998; Desagher et al., 2001; necroptosis. It will therefore be interesting to determine to what extent Kaufmann et al., 2012). However, and congruent with live-cell dephosphorylated PACS-2 modulates common or separate steps in imaging analyses demonstrating that full-length Bid can translocate the decision between autophagy and autophagy-modulated cell death. to mitochondria prior to the generation of tBid, we found that The evolutionally recent acquisition of nuclear trafficking signals PACS-2 binds to and traffics full-length dephosphorylated Bid to by PACS-1 and PACS-2 expanded their roles to also modulate mitochondria (Simmen et al., 2005; Ward et al., 2006). These nuclear gene expression (Atkins et al., 2014). The role of the PACS findings suggest that myristoylation of tBid at Gly61 may not be proteins in the nucleus is just beginning to be understood. Notably, absolutely required for Bid action. Indeed, granzyme B, which PACS-2 is the most recent addition to a small collection of proteins cleaves human Bid at Asp↓-Ser76 to generate a non-myristoylated that regulate SIRT1, which include deleted in breast cancer 1 Bid species, requires PACS-2 to recruit Bid to mitochondria (Li (DBC1; also known as CCAR2), active regulator of SIRT1 (AROS; et al., 1998; Brasacchio et al., 2014). These findings suggest that also known as RPS19BP1) and the moonlighting protein GAPDH following dephosphorylation, full-length Bid follows one of two (Kim et al., 2007, 2008; Atkins et al., 2014; Chang et al., 2015). It distinct pathways leading to MOMP. One pathway is irreversible will be important to determine whether PACS-2 acts in the same or and relies on cleavage by caspase-8 to generate tBid, which different pathways to the other SIRT1 regulators and whether obligates cleaved Bid to trigger MOMP (Fig. 5B). The other PACS-2 or PACS-1 regulates HDACs in addition to SIRT1. pathway is reversible and relies on the PACS-2-dependent Interestingly, the description of PACS-2 as an in vivo modulator of translocation of full-length dephosphorylated Bid to mitochondria SIRT1 in response to DNA damage suggests that it may be involved (Fig. 5C). Following translocation, Bid can be cleaved by multiple in additional pathways controlled by SIRT1 (Brooks and Gu, 2009). proteases, including caspase-8 and granzyme B, to trigger MOMP. For example, in response to fasting, liver SIRT1 increases the Conversely, PACS-1 can bind de-phosphorylated Bid and block expression of genes that encode proteins involved in fatty acid translocation to mitochondria, suggesting a mechanism to impede oxidation and ketogenesis – pathways that protect from diet-induced the apoptotic program. In HCMV-infected cells, vMIA traps PACS- obesity (Purushotham et al., 2009). Thus, the recent report that 2 and Bax on mitochondria, suppressing Bid recruitment and PACS-2 knockdown in liver protects mice from diet-induced MOMP (Arnoult et al., 2004; Salka et al., 2017; Fig. 5A,C). obesity (Arruda et al., 2014), raises the possibility that the acquired The PACS-2 Ser437 Akt site may also act as a bifurcation switch NLS and Akt sites enable PACS-2 to coordinate SIRT1-dependent between anabolic and catabolic roles of the MAM. In response to nuclear gene expression with MAM-dependent changes in insulin, MAM-localized mTORC2 activates Akt proteins to mitochondrial respiration to modulate liver metabolism. phosphorylate PACS-2 Ser437, which increases ER–mitochondria In summary, studies of the PACS proteins illustrate the vital role contacts (Betz et al., 2013). Accordingly, by increasing MAM of protein adaptation in coordinating the seemingly autonomous integrity, Akt-phosphorylated PACS-2 may increase lipogenesis, actions of the nucleus, mitochondria and endomembrane systems in modulate ER–mitochondria Ca2+ transfer and repress autophagy response to the complex challenges faced by vertebrate organ induction (Bernhard et al., 1952; Simmen et al., 2005; Betz et al., systems. Moreover, the findings that or altered expression 2013). By contrast, starvation triggers PACS-2 Ser437 of PACS proteins are associated with pathologies ranging from dephosphorylation, which would remodel MAMs to reduce cancer, obesity and viral pathogenesis to epilepsy and lipogenesis but may increase ER–mitochondria Ca2+ transfer and points to a rich opportunity for insight into the autophagy (Fig. 4). Future work will investigate to what the extent molecular basis of disease through analysis of this adaptable gene the phosphorylation state of PACS-2 modulates MAM ‘thickness’ family. or changes in contacts between mitochondria with smooth or rough endoplasmic reticulum, which may in turn regulate the switch Acknowledgements between anabolic versus catabolic functions (Giacomello and The 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 for Pellegrini, 2016). It will also be interesting to determine whether contributions to studies described here. We also thank M. Ostrowski, C. Thauvin and PACS-2 (as well PACS-1) has roles at additional intermembrane H. Olson for communication of unpublished results. contact sites in other contexts (Schrader et al., 2015). Finally, it will be important to determine to what extent PACS-2 Ser437 Competing interests phosphorylation is regulated by mTORC2 and Akt localized to The authors declare no competing or financial interests. MAMs versus other compartments, and whether other signaling pathways might converge on Akt to phosphorylate PACS-2 (Hers Funding This work in our laboratories was supported by the National Institutes of Health (NIH) et al., 2011; Betz and Hall, 2013). It will also be important to (grants CA151564 and DK112844 to G.T.), Natural Sciences and Engineering 437 identify the PACS-2 Ser phosphatase. Research Council of Canada (NSERC) (grant RGPIN-2015-04105 to T.S.) and the Since autophagy is generally viewed as a survival mechanism, it American Heart Association (AHA) (grant 17SDG33350075 to J.E.A.). Deposited in may at first appear surprising that dephosphorylation of PACS-2 PMC for release after 12 months. 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