Spatial Regulation and Generation of Diversity in Signaling Pathways

J Biosci (2021)46:30 Ó Indian Academy of Sciences

DOI: 10.1007/s12038-021-00150-w (0123456789().,-volV)(0123456789().,-volV) Review

Spatial regulation and generation of diversity in signaling pathways

1,2 1 NEETU SAINI and APURVA SARIN * 1Institute for Stem Cell Science and Regenerative Medicine, Bellary Road, Bengaluru 560 065, India 2Department of Biology, Manipal Academy of Higher Education, Manipal, India

*Corresponding author (Email, [email protected]) MS received 9 November 2020; accepted 19 February 2021

Signaling pathways orchestrate diverse cellular outcomes in the same tissue, spatially and temporally. These interactions, which are played out in micro-environments within cells and involve a relatively small number of core pathways, are the key to the development and function of multi-cellular organisms. How these outcomes are regulated has prompted interest in intracellular mechanisms that build diversity in signaling outcomes. This review speciﬁcally addresses spatial positioning of molecules as a means of enabling interactions and novel outcomes of signaling cascades. Using the Notch and Ras pathways as exemplars, we describe mechanisms that contribute to diverse signaling outcomes.

Keywords. Cross-talk; notch; signaling; spatial regulation

1. Introduction and Blair 1999; Baonza and Garcia-Bellido 2000; Becam et al. 2011), and in the lymph gland, the dif- Cell-to-cell communication is critical for the develop- ferentiation and survival of crystal cells (Duvic et al. ment and maintenance of multi-cellular organisms. 2002). In the PC12 neuronal cell line, activation of Development or repair following injury, requires receptor tyrosine kinase (RTK) in response to nerve interactions between many cell types for specific of cell growth factor (NGF) and epidermal growth factor fates, and breakdown or errors in these can lead to (EGF) regulates differentiation and proliferation various disorders or pathophysiological conditions respectively. Other highly conserved molecular cas- such as cancer, diabetes etc. (Giancotti 2014). Signal- cades such as wingless related (Wnt), transforming ing pathways – conserved modes of cell–cell and growth factor-b (TGF-b) and Hedgehog (Hh) (daSilva environment–cell communication – translate extracel- and Sommar 2003), regulate cellular processes ranging lular and intracellular cues into robust, precise cellular from cell growth, proliferation, differentiation to cell responses. The typical components of a signaling survival. How these signaling pathways generate dis- pathway are an input signal (ligand or growth factor), a tinct outcomes to control a wide range of cellular signal receiving molecule/receptor, transducer[s] and processes remains an area of active investigation effectors, which act in coordinated sequence[s] for involving multiple disciplines and approaches. reproducible cellular responses. However, final out- While referencing well studied examples, the review comes are tuned by cellular context, cross-talk, signal focuses on combinatorial associations of the Notch strength, and duration of signaling, amongst other pathway leading to differing outcomes, in particular factors. For example, Notch signaling in the developing those regulated by intracellular spatial cues, generating Drosophila wing regulates cell proliferation, pre- diversity in signaling outcomes. The mechanisms dis- sumptive vein cells differentiation and formation of the cussed here (figure 1) include (1) increased signaling dorsal/ventral boundary (Huppert et al. 1997; Micchelli intermediates, (2) differential kinetics of interactions, http://www.ias.ac.in/jbiosci 30 Page 2 of 17 Neetu Saini and Apurva Sarin

Figure 1. Mechanisms generating diversity in signaling outcomes: (A) Different combinations of ligand, receptor and effector molecules: EGF binds to EGFR or Trk receptor resulting in recruitment of Gab1 or GRB2 or CRK adaptor protein. Adaptor protein then activates PI3K or Ras/Phospholipase c or ERK pathways. (B) Differential activation kinetics: Binding of Dll1 or Dll4 ligand results in differential activation of Notch1 receptor culminating in the transcription of Hes1 or Hey1/ HeyL genes. (C) Cross-talk between signaling pathways: Activation of JAK/STAT pathway results in transcription of slbo gene. JAK/STAT pathway along with EGFR signaling results in transcription of pnt, blot, Mid, H15 genes. EGFR signaling along with BMP leads to mirr gene transcription. (D) Signaling activated from different sub-cellular location[s]: H-RAS activates PI3K or ERK or JNK signaling pathways when localized at Plasma membrane or Golgi or ER.

(3) cross-talk between signaling pathways and finally, leading to different outcomes, is another as is seen in (4) spatial regulation of signaling molecules. the development of the mammalian lung. A combination of Notch receptors and ligands regulate epithelial progenitor cells differentiation into secretory, ciliated 1.1 Increasing the number of signaling and neuroendocrine (NE) cells during lung develop- components ment. Notch2 signaling regulates differentiation of secretory and ciliated cells, whereas Notch1, Notch2 Increased representation of a family of signaling and Notch3 signal in an additive manner to tune the intermediates is a mechanism that leads to diverse differentiation of NE cells (Morimoto et al. 2012). phenotypic outcomes. Examples include the multiple Signaling activated by Jagged (Jagged1 and Jagged 2) ligands and receptors in the Notch family, G protein- ligands determines secretory vs ciliated cell fate of coupled receptor (GPCR), epidermal growth factor epithelial progenitors cells. On the other hand, Notch receptor (EGFR) and (receptor tyrosine kinase) RTK signaling when activated by its ligands Delta-like (Dll1 signaling pathways (Grassot et al. 2006; Mendoza and Dll4), regulates the number of the neuroendocrine et al. 2014; Barberań et al. 2016). Permutations and cells (NE) and NE associated secretory cells. Expres- combinations of different ligand-receptor interactions, sion of Jagged ligands is observed early during lung Spatial regulation and generation of diversity in signaling pathways Page 3 of 17 30 development in extrapulmonary airway progenitor cells are not yet elucidated, these culminate in very different at E12, whereas Dll ligands are expressed at E14.5 and outcomes. Notch1-Dll1 interaction promotes myogenic restricted to intrapulmonary airway epithelial progeni- differentiation, whereas DLL4-dependent signaling tor cells (Stupnikov et al. 2019) inhibits myogenic differentiation of chick somite cells An increase in signaling intermediates may underpin (Nandagopal et al. 2018). different outcomes. For example, in receptor tyrosine In the PC12 neuronal cell line, sustained activation kinase signaling, the kinase recruits one of various of the ERK pathway by nerve growth factor (NGF) downstream interacting proteins, epidermal growth promotes differentiation, whereas transient activation factor receptor substrate 15 (EPS15), CRK, Src of ERK by epidermal growth factor (EGF) triggers homology region 2 (SH2)-containing protein tyrosine proliferation (Traverse et al. 1992). Both the NGF and phosphatase 2 (SHP2), Casitas B-lineage lymphoma EGF receptors cause the formation of an activation (Cbl), Growth factor receptor-bound protein 2 (GRB2) complex comprising Crk, C3G, Rap1, and B-Raf, that or Src homology 2 domain containing (SHC) domains. activate the ERK pathway. However, EGF receptor These recognize different phosphorylation states of signaling causes formation of a short-lived activation RTK and thereby activate distinct signaling cascades complex, whereas activation of NGF receptor phos- (Schaeper et al. 2000; Wong et al. 1995). In another phorylates the scaffolding protein FRS2 resulting in a example, activated EGF receptor may recruit GRB2 stable complex and sustained activation of ERK (Kao protein leading to activation of Ras signaling et al. 2001). (Lowenstein et al. 1992) or Phospholipase Cc (Chat- In another example, p53 is activated in a series of topadhyay et al. 1999) or Gab1 protein culminating in repetitive pulses induced by c-irradiation induced DNA activation of PI3K-AKT signaling (Mattoon et al. damage observed in human (MCF-7, EB-1, H460, 2004). U2OS, UO31, UACC257 and UACC62) and mouse (NIH3T3, HEPA1C1C7, RAW264.3, and MAK) cell lines or a sustained signal in response to DNA damage 1.2 Modulating (oscillatory vs sustained) kinetics induced by UV radiation observed in MCF-7, A549 of activation and NIH3T3 cell lines (Speidel et al. 2006; Geva-Za- torsky et al. 2006; Batchelor et al. 2011; Stewart- Since signal propagation involves sequential activation Ornstein et al. 2017; Tsabar et al. 2020). Oscillatory of pathway components, tuning the kinetics of activa- activation of p53 following c-irradiation promotes tion of the same signaling step can culminate in novel DNA repair, whereas continues activation of p53 signaling outcomes. This is illustrated by examples of results in senescence in MCF7 cell line (Purvis et al. signaling by the Notch and extracellular signal-regu- 2012). The pulsatile or sustained activation of p53 is lated kinase (ERK) pathway. regulated by a feedback loop between ATM or ATR Notch receptors are activated following engagement kinase, p53-Mdm2 and wip1 phosphatase (Batchelor with one of multiple ligands by signaling which is et al. 2008, 2011). Oscillatory NF-kB activation in conserved across species (Artavanis-Tsakonas et al. response to tumor necrosis factor-alpha (TNF-a) 1999; Bray 2006; Kopan and Ilagan 2009). Differential induces transcription of cytokines such as IL-1a and kinetics of Notch1 activation by ligands Dll1 and Dll4 IL-1b, whereas continuous activation of NF-kB by has been shown to result in distinct signaling outcomes. lipopolysaccharide is a triggers for very different sig- In CHO-K1 cells Notch1 activity is pulsatile in nals including chemokine (C-C motif) ligand 5 response to DLL1 binding, whereas DLL4 activates (CCL5), CCL2 and chemokine (C-X-C motif) ligand Notch1 continuously. Notch activation in short pulses 10 (CXCL10) (Werner et al. 2005). The examples induces expression of HES1 protein, whereas sustained discussed above demonstrate that differences in acti- activation of Notch induces expression of HeyL/Hey1. vation dynamics of the same signaling molecule, result DLL1 binding to Notch1 receptor forms a cluster in distinct signaling outcomes. containing multiple receptors-ligands, which is endocytosed into the signaling cell, resulting in activation of multiple Notch1 receptors at the same time. In contrast, 1.3 Increasing cross-talk between signaling the DLL4-Notch1 receptor forms a small cluster or a cascades single Notch1-ligand pair, which is endocytosed into the signaling cell, ensuring continuous activation. Cross-talk between major signaling hubs is another While the mechanisms underpinning these two modes frequently encountered mechanism that generates 30 Page 4 of 17 Neetu Saini and Apurva Sarin diversity. Interactions between signaling pathways such body cells based on positional information. A gradient as TGF-b, Notch, Wnt, PI3K/Akt, MAPK, and of the JAK/STAT signaling pathway is generated by Hedgehog signaling has been reported (Mendoza et al. anterior and posterior polar cells, which produces 2011; Javelaud et al. 2012; Zhang et al. 2013a, b; Guo unpaired (upd) ligand for JAK-STAT signaling. Acti- and Wang 2009; Collu et al. 2014; Borggrefe et al. vation of the JAK/STAT pathway at the anterior pole 2016). Cross-talk between signaling pathways wherein induces expression of anterior specific gene-slbo, the activation of one pathway triggers the expression of whereas at the posterior pole JAK/STAT signaling activation or inhibition of another pathway has been along with EGFR signaling induces the expression of described in several reports (Galceran et al. 2004; posterior specific genes- pnt, blot, Mid and H15. EGFR Remy et al. 2004;Yiet al. 2005; Borday et al. 2012; signaling along with BMP in the main body cells, Borggrefe et al. 2016; Ammeux et al. 2016; Luo 2017; where JAK/STAT signaling is low, promotes the Regan et al. 2017) and is not covered here. expression of anterior-dorsal specific gene-mirr (Lo- Cross-talk between signaling pathways that triggers mas et al. 2016; Hombrıá and Reyes 2016). In these a distinct signaling output, may also occur. Droso- experiments, inhibition of Jax/STAT pathway resulted phila wing development provides a well-documented in ubiquitous expression of mirr and abrogated differ- example of cross-talk between Notch and Wnt sig- entiation of follicle cells in anterior or posterior cells. naling. Genetic mutant analysis of Notch and Wnt While this is one example, it demonstrates how sig- signaling showed that homozygous mutants of serrate naling pathways are integrated with developmental (Notch ligand) and wingless (Wnt pahway) lack outcomes. The examples discussed here elegantly wings, suggesting an interaction between the two demonstrate how cross-talk between essential signaling pathways (Sharma and Chopra 1976; Speicher et al. pathways is a means of generating diversity in cellular 1994). Consistent with this, Serrate-dependent Notch outcomes. and simultaneous Wingless signaling in leg discs are sufficient to trigger ectopic development of wing (Couso et al. 1995), requiring the expression of 1.4 Spatial regulation of signaling cascades vestigial, a key regulator of wing development (Klein and Arias 1998). This was shown to be regulated via Generally, the activation of a receptor triggers intra- enhancer elements, which tune the expression of cellular movement of effector molecules for signal vestigial and contain binding sites for Suppressor of propagation. For example, activation of the Wnt hairless and dTCF (Klein and Arias 1999). Cross-talk receptor triggers the translocation of b-catenin from between Notch and Wnt pathways is also reported in the cytoplasm to the nucleus to regulate downstream vertebrates. NICD and b-catenin are required for gene transcription (Behrens et al. 1996; Huber et al. differentiation of arterial endothelial cells (ECs) from 1996). Activation of the TGF-b receptor promotes vascular progenitor cells. Neither pathway alone is complexes of R-SMAD and c-SMAD and translo- sufficient to induce arterial endothelial cell fate. cation from the cytoplasm to the nucleus to regulate Further, a NICD-RBPj-k-b-catenin complex was downstream gene transcription (Inman et al. 2002) found to be associated with the induction of arterial Apart from intracellular movement for signal propa- marker genes (ephrinb2, neuropilin1 and cxcr4) in gation, distinct sub-cellular localization of signaling arterial ECs, which was not detected in venous ECs molecules can generate diversity as well as speci- (Yamamizu et al. 2010). ficity in signaling outcomes. An effector molecule Positional information is indispensable for the dif- can activate distinct signaling intermediates when ferentiation of specialized cell types during embryonic localized at different sub-cellular locations as dis- development. Integration of positional information into cussed in following sections. While the localization differentiation programmes can be orchestrated by of major signaling nodes in various subcellular cross-talk of signaling pathways, often mediated by locations promotes cross-talk between signaling building a gradient of morphogens. The establishment pathways, spatial restriction of molecular cascades of anterior–posterior and dorsal–ventral axes is a crit- also serves as a means of limiting the range of ical step in the maturation of the egg chamber in interactions and thereby signaling outcomes. In the Drosophila. Cross-talk between EGFR, JAK/STAT and sections that follow, we briefly summarize key BMP signaling pathways regulates follicle epithelial aspects of spatial control in the Ras and MEK/ERK cell fate controlling differentiation into specialized pathways and then turn to the current understanding polar (anterior, posterior, dorsal and ventral) or main of spatial control of Notch signaling. Spatial regulation and generation of diversity in signaling pathways Page 5 of 17 30 1.4.1 Ras signaling: RAS proteins are GTPases acti- M protein (M1), activated the JNK pathway more vated by GTP binding in response to EGFR or other efficiently than Akt or ERK. Conversely, H-Ras RKT signaling (Scolnick et al. 1979; Manne et al. restricted to the Golgi by fusing with cytosolic end of 1985; Rebollo and Martıńez 1999). Activated Ras the KDEL receptor, resulted in more efficient activation regulates various downstream signaling cascades of ERK and Akt than the JNK pathway (Chiu et al. including Raf/MEK/ERK, and P-I3Kinase/Akt path- 2002). Further, H-RAS signaling from plasma mem- ways etc. (Wood et al. 1992; Rodriguez-Viciana et al. brane induces proliferation and adipocytic differentia- 1994). While RAS signaling was known to be activated tion, whereas H-RAS signaling from the Golgi, induces at the plasma membrane, a body of work has demon- apoptosis in MCF-7 cells by Protein Tyrosine Phos- strated Ras signaling at endosomes, the ER and Golgi phatase receptor kappa (PTPRj)-mediated perturbation apparatus. As described in the section that follows, the in ERK activity (Herrero et al. 2006; Casar et al. 2018). spatial positioning of activated RAS results in distinct Together, these and other studies (Caloca et al. 2003; outcomes. Bivona et al. 2003; Onken et al. 2006; Matallanas et al. In one of the first such reports, the localization of 2006; Casar et al. 2009), provided compelling evidence H-RAS in MDCK cells analyzed using fluorescence that RAS activated at different cellular organelles and electron microscopy while confirming H-RAS at underpins pleiotropic outcomes associated with this the inner surface of the plasma membrane, observed pathway. protein localization in endocytic vesicles and the Golgi apparatus. Although the study reported RAS localiza- 1.4.2 MAPK/ERK signaling: ERK1/2 are extracellular tion at multiple organelles, functional activity at these signal-regulated serine/threonine protein kinases, acti- sites was not established (Willingham et al. 1980). The vated by phosphorylation at threonine and tyrosine first report of RAS activation from a non-plasma residues in response to growth factor-induced signal- membrane location, came from Michiyuki Matsuda’s ing. Activated ERKs either translocate to the nucleus to group, using the FRET (Fo¨rster resonance energy phosphorylate various transcription factors including transfer) based probe- Ras and interacting protein c-Myc, c-Foc, ELK1 or localize in the cytoplasm and chimaeric unit (Raichu) to monitor RAS activation. activate proteins such as SOS, RSK (Chen et al. 1996; Raichu consists of H-Ras and the Ras-binding domain Smith et al. 1999). Apart from the nucleus and cyto- of Raf (Raf RBD) tagged with FRET pair fluorescent plasm, ERK has also been shown to localize to the proteins, yellow fluorescent protein (YFP) and cyan endosome, mitochondria and Golgi (Cha and Shapiro fluorescent protein (CFP) respectively. Activation of 2001; Tamura et al. 2004; Nada et al. 2009). ERK Ras by binding of GTP promotes GTP–Ras binding to translocates from the cytoplasm to nucleus in response Raf RBD, which brings CFP and YFP in proximity, to growth factor stimulation to initiate cell cycle entry. resulting in increased FRET indicating the activated In the Chinese Hamster lung fibroblast cell line Ras in cells. With this approach they established that (CCL39), ERK activity in the nucleus, phosphorylates RAS activates Rap1 at the endosomal compartment and activates nuclear transcription factor ELK1 medi- (Mochizuki et al. 2001;Luet al. 2009). Apart from the ated transcription. Perturbing nuclear entry of ERK by endosomal compartment, activation of RAS signaling expressing a catalytically inactive form of cytoplasmic from other organelle membranes has also been descri- MAP kinase phosphatase (MKP-3/Pyst-1), and resul- bed. Mark R. Philips’s group demonstrated that dif- tant cytoplasmic sequestration of ERK in the cyto- ferential localization of RAS proteins activates distinct plasm, abrogated ELK1 mediated phosphorylation but downstream signaling pathways. Expression of a flu- not the phosphorylation of a cytoplasmic substrate orescent probe RBP-GFP, which binds to only acti- p90RSK1 (Brunet et al. 1999). In embryonic carci- vated RAS along with a constitutively active form of noma and stem cells, ERK activation in response to H-RAS (H-Ras61L) and N- RAS (N-Ras12D), showed retinoic acid treatment causes differentiation of these localization of the GFP (activated-Ras) protein at the cells into primitive endoderm lineage. ERK localiza- plasma membrane and Golgi. On the other hand, tion in the cytoplasm was established by cell fraction- expression of constitutively active K-RAS (K-Ras12V) ation and immunofluorescence approaches, and showed localization of the RBP-GFP to plasma mem- increased phosphorylation of its cytoplasmic substrate brane and not the Golgi. Constitutively active H-RAS RSK. ERK localization to the nucleus by perturbing the lacking localized at the ER and Golgi. Activated cytoskeleton using chemical inhibitors nocodazole, H-RAS restricted to the ER by fusion with the trans- colchicine, and cytochalasin D, inhibited differentiation membrane domain of avian infectious bronchitis virus and activated c-Fos, leading to cell proliferation (Smith 30 Page 6 of 17 Neetu Saini and Apurva Sarin et al. 2004). In the developing chick and mouse et al. 2002; Miyamoto et al. 2006; Schmidt et al. 2009; embryo, ERK signaling from cytoplasm promotes Yao et al. 2013; Zhang et al. 2013a, b; Nandhu et al. muscle differentiation of muscle progenitor/stem cells 2014; Brai et al. 2015). Apart from these reports, Notch in both head and trunk. Furthermore, restricting ERK to signaling also responds to the microenvironment, with the cytoplasm by inhibition of its nuclear localization pathway activation reported in response to shear stress, using Phospho-mimic (myristoylated) competitive hypoxia, hyperglycemia and temperature (Gustafsson peptide for binding importin 7, resulted in muscle et al. 2005; Diez et al. 2007; Wang et al. 2014; Masu- differentiation (Michailovici et al. 2014). ERK local- mura et al. 2009; Ishio et al. 2015). ization in the cytoplasm induced senescence in human In the canonical, conserved signaling pathway, and mouse fibroblast cells expressing oncogenic RAS. Notch receptors are activated by binding to one of its Conversely, forced nuclear localization of ERK inhibits membrane tethered ligands. Ligand binding triggers oncogenic RAS-induced senescence in cells (Gaumont- proteolytic cleavage (S2) by ADAM metalloprotease of Leclerc et al. 2004). The initial studies described above the extracellular domain, leaving the membrane-teth- and others have provided evidences that distinct sub- ered intracellular domain, which is referred to as the cellular localization of ERK dictates different cell fates Notch extracellular truncation (NEXT). Subsequent (Kumar et al. 2003; Vomastek et al. 2004; Whitehurst proteolytic cleavage (S3) of NEXT by c-secretase, et al. 2004; Casar et al. 2009). results in the release of the signaling active interme- The ERK protein does not contain a canonical diate, the Notch intracellular domain (NIC), from the nuclear localization signal sequence and its localization full-length receptor (Andersson et al. 2011). NIC sub- is regulated by a general nuclear translocation signal sequently translocates to the nucleus to interact with (NTS) encoding Serine-Proline-Serine in sequence. CBF1, Suppressor of Hairless, Lag-1 (CSL) and mas- Phosphorylation of NTS promotes its binding to termind-like (MAML) proteins, to regulate gene tran- importin7, which mediates translocation of ERK via scription (Kopan and Ilagan 2009). While the core nuclear pores (Chuderland et al. 2008). Cytoplasmic Notch pathway comprises these conserved steps, sev- localization of ERK is regulated by the actin and eral studies have described non-canonical Notch microtubule cytoskeleton and scaffolding proteins such activity, variously independent of ligand, c-secretase as sef-1, which tethers ERK to the Golgi membrane cleavage or RBPjk-mediated transcription (reviewed by and Paxillin, which sequesters ERK at focal adhesions Andersen et al. 2012; Ayaz et al. 2014). Spatial regu- (Reszka et al. 1995; Torii et al. 2004; Ishibe et al. lation of Notch activity is emerging as another mech- 2004). Nuclear export of ERK is regulated by binding anism underpinning diversity in signaling outputs and to PEA-15, an adaptor protein containing a nuclear is reviewed below (figure 2). export signal (Formstecher et al. 2001; Gaumont-Le- clerc et al. 2004). 2.1 Notch signaling from the plasma membrane

2. Notch signaling c-Secretase-mediated cleavage of the Notch receptor to release the Notch intracellular domain is central to the Notch receptors are single-pass transmembrane proteins highly conserved canonical Notch signaling pathway. comprising an extracellular domain containing EGF However, full-length Notch receptors also activate repeats, a transmembrane domain and an intracellular signaling from the plasma membrane. One example of domain containing RAM, ANK, TAD and PEST this is Notch mediated regulation of JNK signaling domains. Mammals have four Notch receptors (1–4) during dorsal closure in Drosophila. In Notch mutant and ﬁve ligands Delta-Like (DLL) 1, 3, 4 and Jagged1 embryos dorsal epidermal cells do not differentiate and 2 (Dumortier et al. 2005; D’Souza et al. 2008). normally, appearing to be more stretched as compared Notch signaling is not limited to inputs via ligand-re- to wild type embryos, phenocopying mutants with ceptor interactions but, also integrate signals from the hyperactivation of JNK signaling. In this background, extracellular milieu (LaFoya et al. 2016). Notch dorsal closure was rescued by Notch receptor inca- receptors are reported to directly or indirectly interact pacitated for ligand interactions. Thus, the expression with components of extracellular matrix- MAGP-2, of a modiﬁed full-length Notch receptor (FLND10–12), EGFL7, CCN3, Col IV, Col I, Reelin, Matrix Gla pro- which cannot bind the ligand Delta, or a chimeric tein (MGP) or Fibulin 3 which may positively or neg- Notch receptor with the ECD replaced by the extra- atively regulate Notch signaling outcomes (Sakamoto cellular domain of the Torso-receptor tyrosine kinase, Spatial regulation and generation of diversity in signaling pathways Page 7 of 17 30

Figure 2. Notch signaling from different sub-cellular locations. Notch signaling is activated at the cell surface or endosomes by full-length Notch receptor dependent or independent of ligand binding. NIC signals from the nucleus and also the cytoplasm, mitochondria or nucleolus. Notch signaling activated at these sub-cellular locations regulate cell proliferation, differentiation, cell survival or mitochondrial bioenergetics as summarized in the schematic (not to scale). rescued Notch dorsal closure phenotypes (Zecchini interacted with b-catenin analyzed by co-localization et al. 1999). While the mechanism by which JNK and and immuno-precipitation and promoted its sequestra- the Notch pathway interact is not elucidated, the data tion in endosomes, and targeted it for degradation in show that ligand-independent, full-length Notch mouse embryonic stem cells and Drosophila wing receptor signaling activated a novel cascade, by con- imaginal disc (Sanders et al. 2009; Kwon et al. 2011). verging on the JNK pathway. Ligand independent, full- Signaling by full-length Notch receptors is not length Notch1 receptor interacts with and regulates always independent of ligand. Ligand-induced, CSL- abundance of b-catenin protein in Drosophila, and in independent Notch1 signaling has been reported to mammalian embryonic stem and colon cancer cells inhibit differentiation of C2C12 myoblast into myo- (Sanders et al. 2009; Kwon et al. 2011). Inhibition of tubes (Nofziger et al. 1999; Shawber et al. 1996). Work NIC release from plasma membrane using a Val1744L from the Weinmaster group showed that Jagged1 ini- Notch1 form or mutant NEXT, rendered resistant to c- tiated full-length Notch signaling and suppressed secretase mediated cleavage, showed that receptor C2C12 differentiation. The result was recapitulated by processing and cleavage was not required for reducing chemical inhibition of a furin protease (AT-EK1) or b-catenin levels. Membrane tethered Notch receptor expressing a furin-resistant Notch1 receptor in C2C12 30 Page 8 of 17 Neetu Saini and Apurva Sarin cells. Inhibition of furin protease results in expression (Eps15). Further, NEXT was shown to undergo mono- of full-length Notch receptor, which cannot undergo ubiquitination, which is required for c-secretase medi- proteolytic cleavages, despite ligand binding, suggest- ated cleavage and release of NIC1. The study showed ing that ligand induced Notch1 signaling from the that c-secretase mediated activation of Notch was plasma membrane inhibits the muscle differentiation required for its localization to the endosome, however, programme (Bush et al. 2001). In other cells, Jagged1 direct evidence for ligand-mediated and Notch pro- binding to Notch1 in murine post-mitotic neurons, cessing by c-secretase was not assessed (Gupta-Rossi regulates expression of synaptic vesicle proteins et al. 2004). including synaptophysin-1 and VGLUT1, again inde- Inhibition of full-length Notch receptor entry into pendent of proteolytic cleavage of Notch. Since inhi- early endosomes using - dominant-negative shibire or bition of c-secretase using the chemical inhibitor N-[N- GTPase Rab5 or endocytic syntaxin Avl - attenuated (3,5-difluorophenacetyl)-L-alanyl]-(S)-phenylglycine Notch signaling (Vaccari et al 2008). Conversely, t-butyl ester (DAPT) did not modulate Notch signaling Notch signaling was elevated in ESCRT-I, -II, and - III vis-a-vis expression of synaptic vesicle proteins, it mutants, in which Notch targeting to the late endosome appeared that signaling activated from full-length or multi-vesicular body is blocked, resulting in its Notch receptor was sufficient for the observed outcome accumulation in early endosomes (Vaccari et al. 2008). (Hayashi et al. 2016). In dendritic cells, c-secretase Cilia and basal body protein, Bardet-Biedl syndrome independent, ligand-induced Notch signaling, in con- (BBS) and Alstrom syndrome1 (ALSM1) proteins are junction with the Toll-like receptor 4 signaling, regu- implicated in recycling of late endosomes. Loss of BBS lates cytokine production –inducing transcription of IL- or ALSM1 protein upregulated Notch signaling in 10 and IL-2 cytokines and inhibiting secretion of IL- Danio rerio embryos. Regulation of Notch signaling by 12. Notch along with Toll-like receptor 4 signaling cilia proteins was also observed in the human cell lines activates PI3K/AKT signaling downstream, required HEK293T and hTERT-RPE1. Furthermore, Notch1 for increased expression of IL-10 and IL-2 proteins receptor accumulated in the late endosome established (Gentle et al. 2012). using cell fractionation and co-localization with late These studies establish that full-length Notch endosomal markers following depletion of BBS pro- receptors can activate signaling in different cellular teins (Leitch et al. 2014). Vacuolar-ATPase involved in contexts. An important lesson from these studies is that acidification of late endosomes, is required for Notch perturbation of c-secretase mediated cleavage of Notch signaling activated from endosome in Drosophila, mice receptors may not necessarily abrogate Notch activity. and humans (Yan et al. 2009; Lange et al. 2011; Kobia, Hence, direct evidence, assessing receptor and/or et al. 2014; Faronato, et al. 2015). ligand involvement, is necessary to exclude Notch The aforementioned studies provided evidence that pathway activity. While studies described above pro- localization to the endosomes positively regulates vide compelling evidence of Notch signaling from canonical Notch signaling. This raises the question as plasma membrane, in many a role for canonical Notch to how Notch localization to the endosome regulated. signaling and its regulation in these contexts was not Some insights have been provided by the following assessed. studies. In Drosophila blood cells, Sima – the Droso- phila orthologue of Hif-a - and Deltex have been shown to regulates Notch localization in the endo- 2.2 Notch signaling from the endosomal somes, independent of their function in the regulation compartment of endosomal recycling. Notch signaling regulates lymph gland development and differentiation of Endosomal targeting has been reported to result in hemocytes in Drosophila. Sima stabilizes Notch both, the degradation and the activation of Notch receptor in the early endosomes and activates canonical receptors (Jehn et al. 2002; Shaye and Greenwald Notch signaling in a ligand-independent manner. Sima- 2002; Weber et al. 2003; Borgne et al. 2005). Initial mediated activation of Notch signaling controls sur- evidence of Notch activation at endosome came from vival of crystal cells during normal hematopoietic Christel Brou’s group, which showed that a truncated development and in conditions of hypoxia (Mukherjee form of Notch1 (N1DE/NEXT) lacking the extracel- et al. 2011). Deltex, an E3-ubiquitin ligase, interacts lular domain, is excluded from the nucleus if endocy- with and positively regulates Notch signaling. Deltex tosis is inhibited by overexpressing dominant-negative promotes Notch receptor internalization and activation, epidermal growth factor receptor pathway substrate 15 independent of ligand binding and induces expression Spatial regulation and generation of diversity in signaling pathways Page 9 of 17 30 of vestigial (vgBE) independent of Suppressor of 2009, 2010, 2012). This appeared to have physiologi- Hairless (Hori et al. 2004; Zheng and Conner 2018). In cal consequence, a ligand-dependent, non-nuclear another study, signaling activated from NIC seques- endogenous NIC1 activity was identified in murine tered in the endosome mediated by Asrij, promotes natural T-regulatory cells (nTregs), and shown to proliferation of lymph gland and differentiation of influence their function in the regulation of immune hemocytes (Kulkarni et al. 2011). Endosomal targeting response (Perumalsamy et al. 2012; Sakaguchi et al. of Notch receptor is observed in multiple organisms 2008). NIC1 is detected in immune-complexes, which including Drosophila, Danio rerio and mammals, include the lipid kinase PI3K, the nutrient sensor suggesting that activation of Notch signaling from mTORC2/Rictor and molecular intermediates of endosome may well be conserved. autophagy cascades. These pathways are required for NIC dependent survival in conditions of cytokine withdrawal and also modulate nTreg suppressor activ- 2.3 Notch signaling from the cytoplasm ity in vivo (Perumalsamy et al. 2012; Marcel and Sarin 2016). The processed Notch IntraCellular domain (NIC), has Suggesting a direct impact on regulation of cell been shown to signal from non-nuclear locations and metabolism, Jagged1-dependent NIC1 signaling has depends on integrating with other signaling hubs. The been shown to modulate mitochondrial bioenergetics. requirement for ligand-dependent Notch processing is Mitochondrial fractionation and co-localization analy- common to many reports thus far (Perumalsamy et al. sis showed that NIC1 localizes at the mitochondria in 2012; Shin et al. 2014; Sieiro et al. 2016; Hossain et al. Triple-Negative breast cancer cells (Hossain et al. 2018). Signaling activated by cytoplasmic NIC regu- 2018). Notch signaling mediated regulation of mito- lates NF-kB signaling in a T-cell leukaemia and breast chondrial morphology and function has also been cancer cells. NIC1 is reported to interact with PKC-h in reported in the proliferation of neuroblasts typeII cells immuno-precipitation analysis in CD4? T cells and in Drosophila larvae. In this context, Notch signaling T-cell lines and also promotes formation of the CBM synergizes with PTEN-induced kinase 1 (PINK) to complex (comprising CARMA1, BCL10, and MALT1) regulate mitochondrial function and morphology by downstream of the T-Cell Receptor (TCR), which activating the mTORC2-AKT pathway (Lee et al. activates NF-kB signaling during T-cell activation. 2013). While Notch1 has been reported in mitochon- Using NIC1 tagged with a nuclear export signal or drial fractions by biochemical analysis (Perumalsamy nuclear localization signal, the study showed that NIC- et al. 2010; Hossain et al. 2018), the mechanism con- NES regulates CBM complex formation (Shin et al. trolling localization and functional implications is not 2014). Isabella Screpanti’s group showed that in understood. malignant thymocytes, NIC3 from the cytoplasm acti- Despite the evidence of Notch1 activity from a non- vates NF-kB signaling, controlling proliferation and nuclear location in diverse cell types, evidence of this altered differentiation expressing high levels of Pre mode of signaling during developmental processes T-Cell receptor a (pTa) and CD25 markers usually remain sparse. Delta1 induced NIC1 signaling from the expressed at different stages of double negative thy- cytoplasm in the developing chick embryo inhibits mocyte maturation. Immuno-precipitation analysis GSK-3b beta activity, which stabilizes SNAI1 to revealed that NIC3 interacts with cytosolic IKKa, induces epithelial to mesenchymal transition and trig- required for activation of NF-kB pathway suggesting gers myogenesis. Using a membrane-tethered form of that NIC3 signaling from cytoplasm regulates NF-kB NIC (CD4-NIC), the authors showed that the effects in malignant thymocytes (Vacca et al. 2006). In breast could be recapitulated during myogenesis in the cancer cells, non-nuclear NIC1 signaling integrates developing chick embryo (Sieiro et al. 2016). That NIC with IKKa/IKKb to regulate the expression of IL-6 in sequestration in the cytoplasm may be a mechanism to an NF-kB-independent, but p53-dependent manner. tune canonical nuclear functions remains an intriguing, Expression of NIC1-ER (estrogen receptor fusion albeit largely unexplored possibility. Canonical NIC1 protein), which is localized in the cytoplasm in absence signaling is downregulated by ZnCl2 treatment, which of tamoxifen, continued to induce expression of IL-6, results in NIC1 sequestration in cytoplasm. Restriction confirming activity from this location (Jin et al. 2013). of NIC1 in cytoplasm was dependent on AKT signaling Work from our laboratory has shown that NIC1 leading to down regulation of NIC1 mediated tran- activity executed from the cytoplasm, inhibits apopto- scription of canonical targets (Baek et al. 2007; Song sis in mammalian cells (Perumalsamy et al. et al. 2008). These studies have been performed using 30 Page 10 of 17 Neetu Saini and Apurva Sarin ectopic expression of NIC1 in cell lines and any con- have been key to validating atypical signaling modal- firmation of this mechanism in primary cells and ities (Perumalsamy et al. 2012; Hossain et al. 2018; physiological role[s] remain to be discovered. Sieiro et al. 2016; Lee et al. 2013). The studies discussed above have provided evidence for non-nuclear Notch signaling in multiple organisms, although molecular control of this pathway remains 2.4 Notch signaling from the nucleolus poorly understood. NIC localization in the nucleus following release from plasma membrane is mediated Recent work from our laboratory has shown that Notch by three nuclear localization signal sequences (NLS) signaling is further compartmentalized in the nucleus. and a3, a4 and a7 importins (Huenniger et al. 2010). Nucleoli are the site for ribosome biogenesis and regulate Deacetylation of K-2157, K-2160, K-2164, K-2174 in process such as the DNA damage response, cell prolif- NIC1 by Sirtuin1, was shown to be necessary for eration and apoptosis (Tsai and McKay 2002; Boisvert cytoplasmic localization in T-regulatory cells (Marcel et al. 2007; Golstein 2017; Lindstro¨met al. 2018). Thus, et al. 2017). NIC1 sequestration in the cytoplasm was it is not surprising that nucleolar structure and function is dependent on PI3K/AKT as revealed cell fractionation altered in cancer (Ruggero 2013; Orsolic et al. 2016). A or imaging based approaches. Expression of myris- number of studies have associated Notch signaling in toylated Akt (myr-Akt), a constitutively active form of initiation and progression of cancer, and increased Notch AKT, resulted in cytoplasmic localization of NIC1 signaling has been correlated with treatment resistance (Baek et al. 2007; Song et al. 2008). In Cos-7 cells, (Simo˜eset al. 2015; Vermezovic et al. 2015; Aster et al. AKT-mediated phosphorylation of NIC4 at S1495, 2017). We recently reported that NIC4 is enriched in the S1847, S1865 and S1917 is reported to sequester NIC4 nucleolus and this was necessary and sufficient for pro- in the cytoplasm, with phosphorylated NIC4 found to tection of breast cancer cell lines from genotoxic stres- be enriched in the cytosolic fraction (Ramakrishnan sors. Further, activity from this location was independent et al. 2015). of, and uncoupled from, RBPj-k mediated canonical From the studies and examination of evolutionarily Notch signaling. NIC4 interactions with nucleolar pro- conserved Notch signaling the nucleus emerged as the teins – Nucleolin and Nucleophosphmin – lend credence predominant site for Notch signaling. However, evi- to the possibility of functional outcomes associated with dence that non-nuclear Notch signaling contributes to this localization (Saini and Sarin 2020). Whether, NIC4 pleiotropic signaling outcomes has grown substantially. activity regulates ribosome biogenesis remains to be Notably, a combination of microscopy, cell fractiona- tested. Localization of Notch4 was mediated by a tion approaches, coupled with functional read-outs Nucleolar Localization Sequence (NoLS) in NIC4 and

Table 1. Putative nucleolar signal sequence in Notch proteins from different organism predicted using NucleOlar localization sequence Detector (NoD) software http://www.compbio.dundee.ac.uk/www-nod/index.jsp

Species Protein Putative NucleOlar localization signal sequence Homo sapiens Notch1 1771-EGFKVSEASKKKRREPLGEDSVG-1793 Homo sapiens Notch4 1482-LPPGFTRRPRTQSAPHRRRPPLGE -1506 Mus musculus Notch1 1761-EGFKVSEASKKKRREPLGEDSVG-1783 Mus musculus Notch4 1481-PGFIRRPQTQQAPHRRRPPLG-1501 Rattus norvegicus Notch1 1761-EGFKVSEASKKKRREPLGEDSVG- 1783 Rattus norvegicus Notch4 1480-PPGFIRRPQTQQAPHRRRPPLG-1501 Danio rerio Notch1a 1763-GFKVNEPKKKRREPVGEDSV-1782 2152-KDSGKDIRTKKKKSGDGKNGG-2172 Danio rerio Notch1b 1749-GFKTSEPSKKKRREPVGEDSV-1769 2124-NMKPSVQSKKPRKPSTKGIGCKDG-2147 Danio rerio Notch2 2126-AKDLKEMKAKRRKKPTGGEGPS-2147 Gallus gallus Notch1 2179-GKDSKDLKARRKKSQDGKGCL-2199 Gallus gallus Notch2 2091-MPTNLTKDAKRRRKKSLSESKGQF-2114 Xenopous Levis Notch 1764-PDGFIPKEPSKKKRRDRLGEDSVG-1787 Xenopous Tropicalis Notch 1765-PEGFIPKEPSKKKRREPLGEDSVG-1788 Drosophila melanogaster Notch NIL Caenorhabditis elegans Lin-12 1312-HQPQPSRKVTRAPKKQTSRSKKESASNSR-1340 Spatial regulation and generation of diversity in signaling pathways Page 11 of 17 30 of signaling pathways during development is still limited and is an area of future investigation. Molecular mechanisms that regulate distinct localization patterns or the integration with canonical signaling pathways are not well delineated. This review largely focused on signaling by two molecules – Ras and Notch – to illustrate advances in the understanding of spatial control of signaling and also Figure 3. Multiple sequence alignment of putative NoLS of Notch1 protein in various organisms. NoLS amino acids highlight gaps in our understanding of these. Studies highlighted by red showing 100% conservation in Notch1 on the Ras proteins provide convincing demonstra- proteins in Human, Mouse and Rat and highlighted by green tion of outcomes of signaling from diverse locations amino acid showing *70% conservation other organisms. – plasma membrane, endosomes, ER and Golgi, but how localization to different sub-cellular compart- was impaired by the substitution of positively charged ments is controlled, remains unknown. Similarly, in residues with charge neutral amino acids the NIC4 the Notch pathway, Jagged1-dependent full-length NoLS. In mammals, a putative NoLS is present in Notch1 receptor signaling at plasma membrane sup- Notch1 (and functional in Notch4), whereas these are not pressed C2C12 differentiation (Bush et al. 2001), present in Notch2 and Notch3. However, immuno- whereas Jagged1 induced Notch processing also staining for endogenous Notch1 in breast cancer cell signals from the cytoplasm to modulate mitochon- lines, did not detect nucleolar localized Notch1, sug- drial bioenergetics (Hossain et al. 2018). Again, as to gesting that localization is under regulation. The putative how differential sub-cellular localization ensues in NoLS in Notch1 contains multiple lysine residues, which response to the same input (ligand) is not fully can be regulated by acetylation (Guarani et al. 2011; understood. A comprehensive understanding of spa- Popko-Scibor et al. 2011). Post-translation modification tial regulation of signal transduction cascades – their of Notch proteins regulates their subcellular distribution roles in developmental decisions and cues that drive (Ramakrishnan et al. 2015; Marcel et al. 2017) Thus, these – will be key to targeted manipulation[s] in the acetylation of Notch1 may be a possible mode of context of human diseases. inhibiting localization to nucleolus and tuning outcomes, remains to be investigated. Regardless, the possible functions arising from Notch family proteins localization Acknowledgements to the nucleolus, have implications for cell growth and differentiation in diverse contexts. Using NucleOlar Funding from DBT-inStem and the DBT programme localization sequence Detector (NoD) software (Scott grant BT/PR13446/COE/34/30/2015 is gratefully et al. 2011; http://www.compbio.dundee.ac.uk/www- acknowledged. We thank Drs. Arvind Ramanathan and nod/), Notch proteins in other organisms are observed Dasaradhi Palakodeti for their comments and feedback. to contain a putative nucleolar localization signal Schematics are created using BioRender. sequence. The analysis summarized in table 1 reveals that at least one Notch protein in all organisms has a putative NoLS. The Notch1 NoLS shows 100% con- References servation in mammals (Human, mouse and rat) and is Ammeux N, Housden BE, Georgiadis A, Hu Y and Perrimon *70% conserved in other organisms (figure 3). Notch N 2016 Mapping signaling pathway cross-talk in localization in the nucleolus and its functions therein is Drosophila cells. Proc. Natl. Acad. Sci. U. S. A. 113 an exciting avenue for investigation with implications 9940–9945 for physiology. Andersen P, Uosaki H, Shenje LT and Kwon C 2012 Non- canonical Notch signaling: emerging role and mechanism. Trends Cell Biol. 22 257–265 3. 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