` 2013: Signaling Breakthroughs of the Year Jason D. Berndt and Nancy R. Gough (7 January 2014) Science Signaling 7 (307), eg1. [DOI: 10.1126/scisignal.2005013]

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Science Signaling (ISSN 1937-9145) is published weekly, except the last week in December, by the American Association for the Advancement of Science, 1200 New York Avenue, NW, Washington, DC 20005. Copyright 2008 by the American Association for the Advancement of Science; all rights reserved. EDITORIAL GUIDE

CELL BIOLOGY not only provides a molecular mechanism by which stress hormone signaling, and thus 2013: Signaling Breakthroughs of the Year mood and disease resistance, changes with the circadian cycle, it also is one of only a Jason D. Berndt1* and Nancy R. Gough2* few examples of neurotransmitter switching in adult neurons. The editorial staff and distinguished scientists in the fi eld of cell signaling nominat- It is well known that cells in the nervous ed diverse research as advances for 2013. Breakthroughs in understanding how system respond differently depending on the spatial and temporal signals control cellular behavior ranged from fi ltering high- precise spatial and temporal combinations of frequency stimuli to interpreting circadian inputs. This year’s nominations also inputs. Temporal information can be encoded highlight the importance of understanding cell signaling in the context of physiol- by biochemical events that take place across ogy and disease, such as links between the nervous system and cancer. Further- multiple systems or pathways. Chrousos rec- more, the application of new techniques to study cell signaling—such as optoge- ommended a study in which a cell receiving netics, DNA editing with CRISPR-Cas9, and sequencing untranslated regions of an initial signal responds by altering its abil- transcripts—continues to expand the realm and impact of signaling research. ity to respond to another signal through reg- ulation of alternative splicing. This work by A fter polling our Board of Reviewing Edi- Timing is everything in cells and in sig- Lal et al. (3) illustrates “cross-talk between tors and other prominent researchers to naling networks. Complex molecular cas- … two receptor systems that allows seamless Downloaded from bring you this 12th edition of “Signaling cades, feedback mechanisms, and crosstalk integration of extracellular and intracellular Breakthroughs of the Year,” we received are coordinated to enable cells to translate signaling pathways and genomic effects to several nominations with important implica- signals into physiological responses. We fi ne-tune biological responses.” The authors tions for human disease and focus on four begin our list of breakthroughs at the cellu- found that estrogen-mediated transcription main areas: (i) the increasing recognition lar level, focusing on the nervous system in regulates the splicing machinery, which ul- of the importance of temporal dynamics in cancer and mood disorders. Magnon et al. timately affects the function of G protein– signaling pathways; (ii) new understand- noted that neurons are commonly present in coupled receptors (GPCRs) that respond to stke.sciencemag.org ing of the mTOR pathway; (iii) previously tumors, leading the authors to ask whether other hormonal signals. Although chronic unknown roles for lipids, metabolites, and nervous system activation could play a role stress increases the risk of disease, and stress posttranslational modifi cations; and (iv) sev- in tumor progression (1). Using orthotopic hormones can contribute to disease pathol- eral techniques that provide unprecedented xenografts of human prostate cancer cells ogy, stress hormones are also essential in the insights into signaling biology. in mice, they found that tumors were infi l- response to injury and infl ammation and can We had an outstanding list of contribu- trated with both sympathetic and parasym- inhibit disease progression. CRH is a stress tors, including George P. Chrousos (Uni- pathetic nerve fi bers. Severing sympathetic hormone that can either increase the prolif- versity of Athens Medical School, Greece; nerves that normally innervate the prostate eration and invasion of breast cancer cells or on February 1, 2014 Georgetown University and National In- inhibited the growth of established tumors, inhibit estrogen-mediated cell proliferation stitute of Child Health and Development, as did inhibition of adrenergic or muscarinic in breast cancer. Estrogen inhibited the ex- USA); Henrik Dohlman (University of acetylcholine receptors. Analysis of pros- pression of the mRNA encoding serine- and North Carolina Chapel Hill, USA); James tate cancer samples from patients revealed arginine-rich splicing factor 55, ultimately Ferrell Jr. (Stanford University, USA); that the density of sympathetic and para- stimulating the expression of the gene en- Anne-Claude Gavin (European Molecular sympathetic nerve innervation correlated coding the CRH type-2 receptor and altering Biology Laboratory Heidelberg, Germany); with poor prognosis. the splicing of another gene encoding the Toby Gibson (European Molecular Biology The nervous system also contributes to CRH type-1 receptor. These changes were Laboratory Heidelberg, Germany); Tony circadian rhythm, a biological process in associated with decreased invasion of cul- Hunter (Salk Institute, USA); Stephen P. which cells respond to daily fl uctuations in tured breast cancer cells and with estrogen Jackson (Gurdon Institute University of light. Disruptions in circadian rhythm have receptor status in patients with breast cancer, Cambridge, UK); Norbert Perrimon (Har- profound effects on animal behavior and suggesting that this switch may contribute to vard Medical School, USA); Steve J. Smer- mood, as exemplifi ed by seasonal affective tumor progression. These results exemplify don (Medical Research Council National disorder, which is related to the decrease in a potentially new paradigm in the regulation Institute for Medical Research, UK); Solo- light during winter. Dulcis et al. found that of GPCRs through alternative splicing and mon Snyder (Johns Hopkins University, rodents exposed to periods of light mimick- show how the cell’s “history” and “previous USA); Arthur Weiss (University of Califor- ing long summer days and then switched experiences” set the responsiveness to sub- nia San Francisco, USA); and Michael B. to short periods of light mimicking winter sequent signals. Similar to CRH, transform- Yaffe (David H. Koch Institute for Integra- days exhibited a reversible switch in neu- ing growth factor–β (TGF-β) is both a tumor tive Cancer Research, The Broad Institute, rotransmitters in the hypothalamic neurons suppressor and tumor promoter. The study and Massachusetts Institute of Technology, that control the release of corticotropin- by Vizán et al. (4) revealed how differences USA). releasing hormone (CRH) (2). This study in the dynamics of the traffi cking of subunits of the TGF-β receptor altered the cellular re- 1 Associate Editor of Science Signaling, American Association for the Advancement of Science, 1200 sponse to subsequent exposure to TGF-β. New York Avenue, N.W., Washington, DC 20005, USA. 2Editor of Science Signaling, American Asso- ciation for the Advancement of Science, 1200 New York Avenue, N.W., Washington, DC 20005, USA. As with CRH, the effect of TGF-β on tumor *Corresponding author. E-mail: [email protected] (J.D.B.); [email protected] (N.R.G.) cells may depend on the cell’s history.

www.SCIENCESIGNALING.org 7 January 2014 Vol 7 Issue 307 eg1 1 EDITORIAL GUIDE

In addition to crosstalk between pathways, to those with proteins involved in terminating Temporal encoding in molecular signal- temporal information encoded in the signal the mitogenic signal and promoting cytoskel- ing is not limited to receptor tyrosine kinases itself can infl uence cellular responses (Fig. etal rearrangement, cell migration, and inva- and ERK. In fact, the sequential biochemical 1). Toettcher et al. showed that this principle sion. Thus, a single scaffolding protein can events that occur during the cell cycle have exists in signaling networks at the molecular serve as a both a network hub protein and a long been known to require exquisite tim- level by analyzing phosphorylation cascades central processing node to manage the tim- ing. Gibson and Ferrell recommended two downstream of receptor tyrosine kinases ing of diverse functional outputs. Weiss noted studies that refi ne our understanding of how through the use of optogenetic techniques that this study, “opened our eyes to more early multisite phosphorylation can direct this pro- to directly stimulate Ras in cells with light temporal regulation and feedback than we cess, and Gibson suggested that “signaling (5). High-frequency, short-duration bursts of previously appreciated took place.” researchers might want to take a fresh look at Ras activation failed to stimulate extracellu- Another mechanism by which hubs de- the distributions of serines and threonines in lar signal–regulated protein kinase (ERK), a code different inputs is exemplifi ed in a their favourite proteins.” Cyclin-dependent downstream target of Ras, indicating that the study by Kiel et al. (7). The authors showed kinases (CDKs) phosphorylate various sub- pathway from Ras to ERK suppresses noise that competition among proteins binding to a strates, and changes in the activity of CDKs in favor of more robust signals, thus effective- hub can govern how cells produce different toward different substrate proteins control ly acting as a low-pass fi lter. Both transient responses to the activation of the same recep- the cell cycle “clock.” Yeast Cdk1 is com- and sustained pulses of Ras activity stimu- tor. For proteins with a similar affi nity for a posed of (i) cyclins, (ii) catalytic subunits, lated ERK, but promoted phosphorylation common hub that bind in a mutually exclu- and (iii) the regulatory subunit Cks1p. The Downloaded from of different targets, suggesting that ERK can sive fashion, the relative abundance of these catalytic subunit recognizes two consensus decode not only amplitude but also temporal proteins determines which protein complex motifs, one with high affi nity and the other components of input signals and then convey forms. Therefore, changes in the abundance with low affi nity. In addition, the cyclin sub- that information to downstream targets. of competing binding partners could provide units dock onto motifs of certain substrates How might the temporal features of inputs a cellular memory of a previous stimulus and to increase CDK specifi city and activity. In a be decoded by signaling proteins like ERK? A a mechanism to change the fl ow of informa- pair of companion papers, Kõivomägi et al.

paper nominated by Weiss suggests that scaf- tion downstream of signaling hubs. (9) and McGrath et al. (10) showed that yeast stke.sciencemag.org folding proteins may integrate information Understanding the temporal aspects of Cks1p recognized a consensus sequence over time. In the work of Zheng et al. (6), the signaling could have clinical ramifi cations, containing residues that had been “primed” differential, temporally regulated phosphory- as noted by Gavin: “Some signaling hubs by CDK-mediated phosphorylation and thus lation of the scaffolding protein Shc1 (Src rely on stimulus-specifi c signal dynamics promoted processive multisite phosphoryla- homology 2 domain containing 1) coordi- to selectively activate downstream cellular tion by Cdk1. Through this molecular rec- nated the biochemical events after activation processes. Behar et al. (8) used a theoretical ognition mechanism, Cks1p lowered the of the EGF (epidermal growth factor) recep- approach to demonstrate that the dynam- threshold for complete phosphorylation and tor tyrosine kinase. Using quantitative mass ics features of signaling can be targeted contributed to the timing by which specifi c on February 1, 2014 spectrometry, the authors found that different pharmacologically to achieve therapeutic cell cycle regulatory substrates are activated sites in Shc1 were phosphorylated or dephos- specifi city.” Therapies that target individual or deactivated by CDK-mediated phosphory- phorylated at different times after stimulation proteins can have limited clinical use due lation. Gibson also nominated a related pa- with EGF, and this effect correlated with in- to the convergence of multiple signals on per by Lyons et al. (11), who found that the teractions between Shc1 and groups of pro- hubs, and thus the repression or activation degradation of the acetylase Eco1, which is teins with distinct cellular functions. Early of a hub protein’s function can result in un- required for cohesion between sister chro- waves of Shc1 phosphorylation promoted desirable side effects. A virtual screen of matids during S phase, required sequential its association with promitogenic and sur- perturbations to parameters in an idealized phosphorylation by three kinases. Similar vival pathway proteins, which was followed signaling module surrounding a theoretical to recognition of phosphorylation-primed by feedback phosphorylation events and the hub indicated that targeting nodes responsi- motifs by Cks1p, recognition of Eco1 by the subsequent recruitment of a phosphatase. ble for the “dynamic signaling code” could ubiquitin E3 ligase Cdc4 required precise Changes in the residues that were targeted for represent a novel strategy of developing spacing between phosphorylated residues. phosphorylation switched Shc1 interactions therapeutics for various human diseases. Gibson concludes, “Since clustered phos- phorylation sites are very common in native- Fig. 1. Temporal coding regu- ly disordered polypeptide[s], the take-home Input Response lates cell signaling and cellular message is that sequential priming is likely behavior. (A to C) The schematic to be an abundant cell regulatory mechanism A represents a theoretical outcome and there is no reason for it to be limited of cells exposed to different forms to protein destruction phosphodegron sig- of input. (A) Short-duration, high- nals....” Thus, understanding how the specifi c B amplitude spikes are fi ltered by

SCIENCE SIGNALING spacing and sequential phosphorylation of signaling molecules, resulting in no change in the cell’s behavior, substrates functions as an intrinsic molecular whereas (B) sustained activation clock is a breakthrough in our knowledge of C results in cell migration and (C) re- the molecular mechanisms that control the peated transient activation results timing and fl ow of biochemical information

CREDIT: V. ALTOUNIAN/ V. CREDIT: in cell division. through a signal transduction pathway.

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In other developments related to cell cy- only ‘effi cient’ substrates being phosphoryl- mLST8 structure with that of rapamycin cle, a study by Chang and Ferrell suggested ated when mTORC1 activity is low either bound to FKBP12 suggested that rapamycin how biochemical events that were initiated through starvation or rapamycin inhibition, inhibits mTOR by creating a cap across the on one side of a large frog egg cell undergo- [and] with serine being preferred over threo- catalytic cleft and that conserved residues in ing mitosis could be coordinated with those nine at effi cient target sites. A single protein the FRB domain formed a secondary sub- on the opposite side to enable rapid spatial can have both ‘good’ and ’poor’ mTORC1 strate recruitment motif, which may explain and temporal coordination of processes over sites illustrating that this is a property of the differences in substrate affi nity seen by “large” distances relative to the size of the the sequence.…A similar hierarchy may Kang et al. (13). molecules involved (12). Using a combina- well exist for other protein kinases dictating Plants also utilize TOR signaling to reg- tion of mathematical modeling and imaging which substrates are phosphorylated as ki- ulate growth. Xiong et al. (15) found that of mitotic events in a mitotic egg extract in nase activity increases over different thresh- light-activated glucose production in the vitro, the authors identifi ed a mechanism olds in response to a stimulus.” root meristem was required for root devel- that relied on a network of positive and neg- Yang et al. published the structure of opment at the point when plants transition ative feedback loops to produce a bistable mTOR (14), which according to Smerdon from heterotrophic (seed nutrient–depen- system that propagated through “trigger is a “structural tour-de-force and some- dent) to photoautotrophic (photosynthetic) waves.” The concept of trigger waves is the thing of a holy grail in kinase signaling … growth. The authors found that glucose basis for the propagation of action poten- [and] shed[s] new light on signaling by the promoted phosphorylation of the TOR sub- tials along an axon, and this study provides whole family of ‘giant’ PI3K-like kinases, strate S6-kinase and induced root growth in Downloaded from another molecular system in which trigger many of which are well-validated drug a TOR-dependent manner. Glucose-induced waves control biochemical processes that targets.” mTOR was cocrystalized with transcriptional responses were mediated in need to occur faster than the rate of molecu- mLST8 (mammalian lethal with SEC13 part by phosphorylation of the S phase–as- lar diffusion. protein 8), a subunit of both mTORC1 and sociated transcription factor E2Fa by TOR. Target of rapamycin (TOR in yeast or mTORC2. The structure revealed an in- Thus, this study expands the functions of mTOR in mammals) is a phosphoinositide trinsically active kinase domain formed by TOR signaling to include transcriptional

3-kinase (PI3K)–related kinase that once N- and C-terminal lobes separated by the regulation and shows that E2Fa is phos- stke.sciencemag.org again was featured in several nominations catalytic cleft. The positioning of the ATP- phorylated by TOR, perhaps bypassing the this year. This kinase is part of two pro- and substrate-binding residues was remark- need for CDK-retinoblastoma–mediated tein complexes, mTORC1 and mTORC2. ably similar to that of other protein kinases, phosphorylation. mTORC1 integrates information regarding such as CDK2. The active site was situated Protein kinases were not the only signal- the presence of growth factors with energy in a deep groove created by mLST8 and the ing components making the breakthrough and nutrient status to inhibit autophagy and FRB and LBE domains of mTOR, and por- list. New signaling roles for lipid kinases promote ribosome formation and protein tions of the kα9b helix hinder substrate ac- and their association with disease were also translation. mTORC2 responds to growth cess (Fig. 2). Many residues that when mu- revealed. Yaffe nominated research that on February 1, 2014 factors to enhance cellular metabolism, sur- tated result in increased activation of mTOR identifi ed a link between cancer and the vival, proliferation, and cytoskeletal rear- clustered near the distal end of the catalytic enzyme that phosphorylates a specifi c posi- rangements. mTOR is implicated in human cleft, which is blocked by the kα9b helix in tion of the inositol ring of phosphoinositide diseases and genetic disorders, such as can- the crystal. Superimposition of the mTOR- (PI)–type lipids. Although most therapeutic cer, diabetes, schizophrenia, and tuberous studies have focused on PI3K, which gen- sclerosis. Rapamycin is an mTOR inhibitor erates the lipid to which proteins such as with specifi city for mTORC1 that slows ag- Ras and Akt bind and are thus recruited to ing in yeast, fl ies, and mice, and rapamycin the plasma membrane and activated, Yaffe is used clinically as an immunosuppressant selected work by Emerling et al. (16) as a during kidney transplantation and a chemo- breakthrough because this study suggests therapeutic for Kaposi’s sarcoma. that enzymes in the initial steps of the Hunter nominated a paper that examined phosphoinositide polyphosphate pathway the substrate specifi city of mTORC1 (13). may also be important in cancer. Emer- Rapamycin binds to the 12-kD FK506- ling et al. found amplifi cation of PIP4K2B binding protein FKBP12 and acts as an al- and an increased abundance of PI5P4Ks in losteric inhibitor of mTORC1, but it only breast cancer cell lines and human epider- G

N inhibits the phosphorylation of some sub- mal growth factor receptor 2 (HER2)–posi- I

AL strates. In contrast, inhibitors that bind di- tive human breast tumors. Knockdown of N G I Fig. 2. The structure of mTOR bound to α β S rectly to the active site of mTOR affect the PI5P4K and PI5P4K in a p53-defi cient

CE mLST8 reveals a deep catalytic cleft. Rib-

N phosphorylation of all substrates equally. breast cancer cell line (genetically lacking E bon structure from PDB 4JSV shows the CI

S Kang et al. found that mTORC1 has a pre- TP53) enhanced the production of reac-

N/ N- and C-terminal lobes of mTOR fl anking A ferred consensus recognition motif and the active site containing an analog of ATP tive oxygen species and cell senescence, UNI O

T that substrate affi nity was inversely related (yellow). The FRB domain (red) of mTOR and inhibited glucose metabolism, and slowed L A

. to the inhibition of phosphorylation by ra- the growth of cells in culture or xenografts.

V the mTORC1 subunit mLST8 (green) extend :

T −/− −/− +/−

DI pamycin. As Hunter describes, “mTORC1 from the sides of the catalytic cleft to restrict TP53 PIP4K2A PIP4K2B mice had −/− CRE has a hierarchy of substrate affi nities, with substrate access. fewer tumors than did TP53 mice, sug-

www.SCIENCESIGNALING.org 7 January 2014 Vol 7 Issue 307 eg1 3 EDITORIAL GUIDE gesting that PI5P4Ks may be a good drug Complexity in signaling can arise through The interplay between protein meth- target in TP53-defi cient breast cancer. multiple types of posttranslational modifi - ylation and phosphorylation was selected The roles of lipids in cellular regulation cations of proteins. In addition to investiga- as a breakthrough in the understanding of can be technically diffi cult to assess be- tions of phosphorylation, studies looking signaling downstream of the bone morpho- cause of the complexity of the lipid species at other posttranslational modifi cations, genetic protein (BMP) receptors. BMP re- present in the cell and the lack of tools to including ubiquitylation and methylation, ceptor activation promotes phosphorylation selectively manipulate or visualize specifi c made this year’s list of signaling break- of Smads 1 and 5 (Smad1/5), which partner lipids. Montefusco et al. (17) devised an ap- throughs. Protein ubiquitylation is a per- with the cofactor Smad4 and translocate to proach to analyze the role of specifi c groups vasive posttranslational modifi cation that the nucleus to drive gene expression. Smad6 of ceramide species, which differ in N-acyl regulates virtually all aspects of cellular inhibits Smad1/5 by blocking their interac- chains and hydroxylations, to identify spe- function. Ubiquitin ligase complexes con- tion with the BMP receptor. Xu et al. (26) cifi c functions of these bioactive lipids in jugate ubiquitin moieties to lysines in tar- found that treating cultured keratinocytes yeast. Their work demonstrated that lipid get proteins and to one or more lysines in with BMP4 promoted an interaction between diversity has functional consequences in the ubiquitin itself to formed branched chains the arginine methyltransferase PRMT1 and cellular response to stress and in transcrip- with diverse linkage patterns. Lys48-linked Smad6. PRMT1 methylated Smad6, was re- tional regulation. polyubiquitylation targets proteins for quired for BMP-mediated phosphorylation In addition to proteins and lipids, many proteasomal degradation, whereas mono- of Smad1/5, and increased the expression of metabolites can serve regulatory functions. ubiquitylation or other patterns of polyubiq- target genes. Overexpression of the homo- Downloaded from Sutter et al. (18) found that the amino acid uitylation, such as Lys63-linkage, are gener- log of PRMT1 in fl ies prevented phenotypes methionine was suffi cient to inhibit non- ally associated with regulation of protein resulting from overexpression of Smad6. nitrogen starvation (NNS)–induced au- traffi cking and protein-protein interactions. Thus, methylation emerges as a key post- tophagy in yeast. Methionine increased the Deubiquitylating enzymes (DUBs) fall into translational modifi cation that is involved S-adenosyl methionine (SAM)–dependent two categories: (i) the ovarian tumor (OTU) in the response to metabolic signals, DNA methylation of the protein phosphatase class and (ii) ubiquitin-specifi c proteases damage, and morphogenetic stimuli.

PP2A. Methylated PP2A dephosphorylated (USPs). Jackson recommended the study by This year, we received several nomina- stke.sciencemag.org Npr2p (Nitrogen permease regulator 2p), a Mevissen et al. (23), which characterized 16 tions representing technical innovations that protein that is required for NNS-induced catalytically active OTUs and showed that, have yielded or have the potential to yield autophagy and is a negative regulator of unlike the nonspecifi c USPs, OTUs recog- important discoveries in cell signaling biol- TORC1. Laxman et al. (19) found that the nize only one or a few of the eight possible ogy. Optogenetics, the ability to genetically abundance of methionine and SAM also types of ubiquitin linkages. Structural anal- encode proteins that can be activated or in- controls tRNA thiolation, which enables ysis of related OTUs with different linkage hibited by light, was applied to understand tRNAs to read more than one codon and preferences revealed four distinct mecha- temporal encoding of the Ras-ERK pathway facilitates translation of genes associated nisms used to achieve linkage specifi city. by Toettcher et al. (5). Gibson nominated a on February 1, 2014 with rRNA processing, ribosome biogen- Understanding the specifi city of OTUs can study by Baarlink et al. (27) showing that esis, and translation. Thus, like glucose and be used to determine unknown linkage pat- optogenetic activation of the formin mDIA arginine, methionine plays a key regulatory terns on proteins of interest by performing induced nuclear actin fi lament formation. role in cellular metabolism. in vitro reactions with different OTUs. The results of this optogenetic study contin- Cyclic nucleotides, such as cyclic gua- Jackson and Yaffe both selected studies ue a thread from our 2011 Breakthroughs, nosine monophosphate (cGMP) and cyclic that identifi ed interplay between ubiquityl- highlighting Mouilleron et al. (in the Ar- adenosine monophosphate (cAMP), were ation and methylation in the DNA damage chives), which showed that monomeric nu- the fi rst identifi ed second messenger sig- response. Fradet-Turcotte et al. (24), recom- clear actin binds to the cofactor myocardin- naling metabolites. Snyder and colleagues mended by Jackson, found that the DNA related transcription factor A (MRTF-A, recommended a pair of papers that identi- repair protein 53BP1 recognized a bivalent also known as MAL) to inhibit activation fi ed cyclic guanosine monophosphate– epigenetic mark consisting of dimethylation of gene transcription in response to serum adenosine monophosphate (cGAMP) as a of histone-H4 on Lys20 and ubiquitylation of response factor. new second messenger that functions in the histone-2A on Lys15 through a previously un- Dohlman and Hunter both noted papers mammalian immune response to microbial characterized methyl-lysine binding Tudor that advanced antibody technologies. Dohl- pathogens, which includes interferon (IFN) domain and an extension on its C terminus. man nominated a study that demonstrated production triggered by the presence of for- Yaffe nominated work by Watanabe et al. the use of genetically encoded antibodies, eign DNA in the cytosol. Sun et al. (20), Wu (25) showing that the demethylase JMJD1C “nanobodies,” to visualize the subcellular et al. (21), and Gao et al. (22) found that associated with the E3 ubiquitin ligase RNF8 location of active GPCRs in living cells. cGAMP and the enzyme that produces it, at DNA double-strand breaks. JMJD1C Endocytosis can be associated with receptor cGAMP synthase, were critical mediators demethylated MDC1 (mediator of DNA- down-regulation or can contribute to signal- of the innate immune response to infection. damage checkpoint 1) and promoted its ubiq- ing from endosomal membranes. Irannejad Indeed, cGAMP produced in response to uitylation by RNF8, leading to the recruit- et al. (28) expressed green fl uorescent pro- the presence of DNA in the cytosol bound ment of the RAP80-BRCA1 E3 ligase com- tein (GFP) fused to a conformation-specifi c, to the adaptor protein STING (stimulator of plex, which further ubiquitylated MDC1. single-domain camelid antibody (Nb80- interferon genes), leading to the activation Thus, recognition and repair of DNA dam- GFP) that selectively binds to the agonist- β β of the transcription factor IRF3 and IFN age requires orchestration of multiple types bound 2-adrenergic receptor ( 2AR) and production. of posttranslational modifi cations. then tracked the location of the agonist-

www.SCIENCESIGNALING.org 7 January 2014 Vol 7 Issue 307 eg1 4 EDITORIAL GUIDE

bound receptors. In cells stimulated with the expression correlated with distinct cellular lation to enable tissue-specifi c protein pro- adrenergic agonist isoprenaline, Nb80-GFP maturation states or with the expression of duction. localized to both the plasma membrane master regulatory genes including Irf7 and Transitioning from RNA to genome edit- and early endosomes. Likewise, a similarly Stat2. Perrimon notes, “This approach is ing, CRISPR (clustered regularly interspaced constructed antibody that recognized the ‘game changing’ for investigating signaling short palindromic repeats)–based techniques α guanine nucleotide–free form of G s, rep- heterogeneity and temporal responses.” were used extensively in various cellular and resenting an intermediate state of G protein Yaffe nominated a study that investigated organismal model systems this year (32–35), β activation, localized to 2AR-containing the mechanism by which ubiquitously tran- as noted by Snyder and colleagues. CRISPR endosomes in stimulated cells. As Dohlman scribed genes show tissue-specifi c expres- sequences are fragments of viral or plasmid states, “the use of nanobodies represents sion. Lianoglou et al. (31) created a method DNA incorporated into the bacterial genome a novel approach for genetically-encoded to make libraries of the 3′-untranslated re- by the RNA-guided nuclease Cas9 as part of biosensor design. In the future, one can gions (3′-UTRs) of mRNAs from different the bacterial immune response. Researchers easily imagine that nanobodies could be di- tissues and isogenic cell lines and used deep have exploited the ability of Cas9 to cre- rected against other cellular targets, such as sequencing to quantify alternative cleavage ate site-specifi c DNA double-strand breaks metabolic control points, and thus it opens and polyadenylation. Tissue-specifi c tran- by creating synthetic guide RNAs that cor- up a new avenue to elucidate signaling scribed genes tended to produce transcripts respond to the target sequence. Because of mechanisms and the identifi cation of new with a single 3′-UTR, whereas ubiquitously the imprecise nature of nonhomologous end drug targets.” transcribed genes produced transcripts with joining, DNA repair often results in inser- Downloaded from In his nomination of the study by Kee et multiple 3′-UTRs (Fig. 3). The ratio of 3′- tions or deletions that ultimately produce al. (29), Hunter notes, “Histidine kinases UTR isoforms of ubiquitously transcribed truncated or nonfunctional proteins. In addi- and a pHis [phosphorylated histidine-spe- genes varied among tissues and between tion, providing a template with homologous cifi c] phosphatase have been reported, but different states of isogenic cell lines un- sequences to the targeted area can result a challenge in detecting phosphohistidine is dergoing transformation or differentiation. in recombination and enable site-specifi c the chemical instability of its phosphoarami- Both mechanisms of restricted gene expres- modifi cation or insertion of novel sequenc-

date linkage at low pH…. The availability sion contributed to genes involved in the es. Unlike similar technologies, such as zinc stke.sciencemag.org of antibodies that could detect pHis would same molecular pathways or cellular pro- fi nger nucleases (ZFNs) or transcription clearly facilitate the detection of histidine gram. Alteration in the ratio of the 3′-UTRs activator–like effector nucleases (TALENs), phosphorylation, just as pTyr antibodies contributed to differential gene expression the CRISPR-Cas9 system does not rely accelerated study of tyrosine phosphoryla- by changing the number of recognition sites on nucleotide recognition by amino acids. tion.” Kee et al. used a stable phosphohis- for microRNAs. Thus, differential 3′-UTR Therefore, construction of targeting vectors tidine mimetic to develop a pan-specifi c production represents another layer of regu- is both easy and inexpensive. phosphohistidine antibody Unlike RNAi or phar- and used it to explore chang- A B macological perturbation, on February 1, 2014 es in the phosphorylation of DNA DNA CRISPR-Cas9 enables com- metabolic enzymes in bac- plete genetic ablation in teria deprived of nitrogen. most cultured cells without Hunter cautions, “These the need to create a knock- antibodies will undoubtedly pre-mRNA 3’-UTR pre-mRNA out animal. In addition, be valuable, although their 5’ 3’ 3’-UTR CRISPR-Cas9 can be used cross-reactivity with pTyr 3’-UTR 5’ 3’ to create specifi c amino acid may limit their usefulness.” substitutions in endogenous Two nominations fo- Processing Processing proteins. Schwank et al. (36) cused on advances in the ap- used CRISPR-Cas9 and ho- plication of deep sequenc- mologous recombination in ing. Perrimon nominated the mRNA mRNA intestinal stem cells isolated study by Shalek et al. (30), Brain Brain from patients with cystic fi - which used RNA sequenc- brosis to correct the disease- ing to analyze gene expres- causing mutation in the gene sion in murine bone mar- encoding the cystic fi brosis row–derived dendritic cells transmembrane conductor stimulated with lipopoly- Kidney Kidney receptor (CFTR). Unlike the saccharide. By sequencing microRNA parental cells, CFTR-correct- mRNA from single cells, ed intestinal stem cells grown they found bimodal varia- as organoids in three-dimen-

SCIENCE SIGNALING tion in mRNA abundance sional culture swelled in re- and splicing in hundreds of Fig. 3. Alternative 3 ′-UTRs confer tissue specifi city to ubiquitously tran- sponse to cAMP, indicating immune-responsive genes, scribed genes.(A) Tissue-specifi c transcription results in the tissue-specifi c that the corrected allele was indicating extensive cellu- presence of proteins. (B) Ubiquitously transcribed genes undergo alternative functional and opening the lar transcriptional hetero- cleavage and polyadenylation of the 3′-UTR that enables differential targeting possibility for further studies

CREDIT: V. ALTOUNIAN/ V. CREDIT: geneity. Variation in gene by microRNAs to prevent protein translation in some tissues. in this system.

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Perspectives • D. A. Guertin, D. M. Sabatini, The phar- macology of mTOR inhibition. Sci. Sig- nal. 2, pe24 (2009). • S. Klumpp, J. Krieglstein, Reversible phosphorylation of histidine residues in proteins from vertebrates. Sci. Signal. 2, pe13 (2009). Editors’ Choice • J. D. Berndt, The lights on Ras avenue. Sci. Signal. 6, ec298 (2013).

• J. F. Foley, Arginine methylation dere- presses BMP signaling. Sci. Signal. 6, ec159 (2013).

• N. R. Gough, Transcriptional reprogram- ming by TOR. Sci. Signal. 6, ec86 (2013). Downloaded from

• N. R. Gough, Centered on SAM. Sci. Fig. 4. Human PSCs grown as organoids recapitulate disease and development. Image Signal. 6, ec170 (2013). of a cross section of a cerebral organoid stained for nuclei with Hoechst (blue), neural pro- genitor cells (red), or mature neurons (green). Organoids display regional specifi cation and laminar organization similar to the brain (37) • S. M. Hurtley, Nuclear actin in action. Sci.

Signal. 6, ec116 (2013). stke.sciencemag.org One particularly intriguing application systems that control cellular and organismal for CRISPR-Cas9 is in the study of develop- behavior—and whose disruption forms the • P. A. Kiberstis, Cancer hits a nerve. Sci. ment. CRISPR-Cas9 has been used to create etiology of human disease. Signal. 6, ec161 (2013). single-gene and even multigenic mutations in cultured pluripotent stem cells (PSCs) Related Resources • K. L. Mueller, DNA sensing is a (c)GAS. and to generate mutant mice and fi sh. In an- Editorial Guide Sci. Signal. 6, ec48 (2013). other recommendation by Snyder and col-

• E. M. Adler, 2011: Signaling break- on February 1, 2014 leagues, Lancaster et al. (37) demonstrated throughs of the year. Sci. Signal. 5, eg1 • K. L. Mueller, HIV detection is a (c)GAS. the ability to grow three-dimensional organ- (2012). Sci. Signal. 6, ec206 (2013). oids from PSCs that faithfully recapitulated many aspects of the development and archi- Research Articles • H. Pickersgill, Alarm bells. Sci. Signal. 6, tecture of the human brain (Fig. 4). Cerebral • K. A. Fujita, Y. Toyoshima, S. Uda, Y.-i. ec232 (2013). organoids from PSCs reprogrammed from Ozaki, H. Kubota, S. Kuroda, Decoupling the fi broblasts of a patient with severe mi- of receptor and downstream signals in the • L. B. Ray, Modifying deubiquitinases. crocephaly or from human embryonic PSCs Akt pathway by its low-pass fi lter charac- Sci. Signal. 6, ec35 (2013).

, 373–379, COPYRIGHT 2013 , 373–379, COPYRIGHT expressing short-hairpin RNAs targeting teristics. Sci. Signal. 3, ra56 (2010). 501 CDK5RAP2, the gene that was mutated in • L. B. Ray, Not mTORCing. Sci. Signal. 6, this patient, had epithelial hyperplasia and • S. Mouilleron, C. A. Langer, S. Guettler, ec178 (2013). NATURE reduced numbers of neural progenitor cells. N. Q. McDonald, R. Treisman, Structure Overexpressing CDK5RAP2 in patient- of a pentavalent G-actin•MRTF-A com- • P. R. Stern, Daylight determines dopa- derived cells rescued these defects. Thus, plex reveals how G-actin controls nucleo- mine. Sci. Signal. 6, ec95 (2013). the studies by Schwank et al. and Lancaster cytoplasmic shuttling of a transcriptional et al. suggest that combining advanced ge- coactivator. Sci. Signal. 4, ra40 (2011). • W. Wong, Remaining active in endo- nome-editing with sophisticated cell culture • T. Tomida, S. Oda, M. Takekawa, Y. Iino, somes. Sci. Signal. 6, ec78 (2013). techniques could yield important informa- H. Saito, The temporal pattern of stimu- tion about normal development and disease. lation determines the extent and duration References The nominations for breakthroughs in 1. C. Magnon, S. J. Hall, J. Lin, X. Xue, L. Gerber, S. of MAPK activation in a Caenorhabditis J. Freedland, P. S. Frenette, Autonomic nerve de- signaling this year uncover new regula- elegans sensory neuron. Sci. Signal. 5, velopment contributes to prostate cancer progres- tory molecules and mechanisms and new ra76 (2012). sion. Science 341, 1236361 (2013). interactions among physiological systems 2. D. Dulcis, P. Jamshidi, S. Leutgeb, N. C. Spitzer, Neurotransmitter switching in the adult brain regu- and highlight the development of a grow- Review lates behavior. Science 340, 449–453 (2013). ing repertoire of tools to dissect signaling • C. H. Emmerich, A. C. Schmukle, H. 3. S. Lal, A. Allan, D. Markovic, R. Walker, J. Ma- pathways. The science of signal transduc- Walczak, The emerging role of linear cartney, N. Europe-Finner, A. Tyson-Capper, D. K. Grammatopoulos, Estrogen alters the splicing of tion continues to expand into vast areas of ubiquitination in cell signaling. Sci. Sig- type 1 corticotropin-releasing hormone receptor in

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