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 combina- tion 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; Barbera´n 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