Signalling ballet in space and time

Abstract

Although we have amassed extensive catalogues of signalling network components, our understanding of the spatiotemporal control of emergent network structures has lagged behind. Dynamic behaviour is starting to be explored throughout the genome, but analysis of spatial behaviours is still confined to individual proteins. The challenge is to reveal how cells integrate temporal and spatial information to determine specific biological functions. Key findings are the discovery of molecular signalling machines such as Ras nanoclusters, spatial activity gradients and flexible network circuitries that involve transcriptional feedback. They reveal design principles of spatiotemporal organization that are crucial for network function and cell fate decisions.

Figure 1. Versatile MAPK dynamics

Each panel schematically displays the RAF–MEK–ERK (RAF–MAPK/ERK kinase–extracellular signal-regulated kinase) cascade; the feedback from ERK to RAF, which is the initial mitogen-activated protein kinase (MAPK) activated by Ras-GTP, is indicated (when present). The different temporal responses of active, dually phosphorylated ERK (ppERK) to a constant Ras-GTP stimulus are obtained by changing the parameters of ERK-mediated feedback. A kinetic description of MAPK cascade reactions (rate equations) is given in Supplementary information S1 (table), in which parameter values are relative to the graph in part a. Parameters F and K_f describe the feedback regulation (F = 1, for no feedback, F < 1 for negative feedback, F > 1 for positive feedback; K_f equals ppERK concentration at which activation or inhibition is half-maximal), and indicates the maximal rate of a phosphatase reaction. a | No ERK–RAF feedback, F = 1. b | Negative ERK–RAF feedback, F=0.34, K_f = 25 nM. c | Negative ERK–RAF feedback, F=0.01, K_f = 1 nM, V₇^max = 0.175 nM s⁻¹. d | Negative ERK–RAF feedback, F=0.27, K_f=25 nM. e | Negative ERK–RAF feedback, F=0.01, K_f=25 nM. f | Positive ERK–RAF feedback, F=5, K_f=100 nM, k₁^cat= 0.025 s⁻¹, V₁₁^max =0.025 nMs⁻¹. Depending on the initial conditions (pre-existing activity level of the cascade), ppERK either descends to the low activity state (blue curves) or approaches the high-affinity state (red curves); the dashed line indicates a threshold.

Figure 2. Intrinsic transcriptional feedback inhibition of NF-κB

In resting cells nuclear factor-κB (NF-κB) is inactive because inhibitor of NF-κB (IκB) retains it in the cytosol. On activation, for example by tumour necrosis factor (TNF), TNF receptor 1 (TNFR1) forms a complex with the adaptor protein TNFR-associated DEATH domain (TRADD), which recruits different proteins to initiate dual signalling pathways. TRADD recruits FAS-associated death domain (FADD) to promote apoptosis by stimulating caspase 8 activation. By contrast, TRADD recruits the adaptor receptor interacting protein (RIP) to counteract apoptosis by activating NF-κB. NF-κB activation is enabled as a result of the stimulus-induced degradation of IκBs following their phosphorylation by IκB kinases (IKKs), which releases NF-κB from its cytosolic anchor proteins so that it can translocate to the nucleus. However, nuclear NF-κB also induces the transcription of its own inhibitors. IκBs can bind to nuclear NF-κB and export it back to the cytosol, and A20 can interrupt receptor-mediated NF-κB activation by inducing the degradation of RIP.

Figure 3. Scaffolds and spatial organization

a | G-protein coupled receptors (GPCRs) activate extracellular signal-regulated kinase (ERK) through two spatially and temporally separated pathways. Mechanistic details are omitted for the sake of clarity and were reviewed previously. Rapid ERK activation emanating from the plasma membrane through protein kinase C (PKC), Src, and receptor Tyr kinase stimulation is transient and β-arrestin independent, and allows ERK translocation to the nucleus. Sustained ERK activation is triggered from an endosomal β-arrestin-dependent RAF–MEK–ERK (RAF–MAPK/ERK kinase–extracellular signal-regulated kinase) module and restrains ERK signalling to the cytosol. The integrated dually phosphorylated ERK (ppERK) results from combining the nuclear and cytosolic ppERK levels. b | Ras activated at different subcellular compartments uses different scaffolding proteins to target ERK substrates. IQGAP1 (on the cytoskeleton) mediates negative ERK feedback phosphorylation of epidermal growth factor receptor (EGFR), and kinase suppressor of Ras (KSR) (at the plasma membrane) and interleukin-17 receptor D (IL-17RD; also known as SEF1) (in the Golgi) facilitate phosphorylation of cytoplasmic phospholipase A2 (CPLA2) by ERK activated at the plasma membrane or intracellular membranes, respectively. Ras activated at the endoplasmic reticulum (ER) might stimulate IL-17RD-bound ERK at the Golgi. However, Ras can also be activated directly at the Golgi. In either case, ERK phosphorylation activates CPLA2 to generate arachidonic acid, which is a precursor to signalling molecules such as leukotrienes and prostaglandins.

Figure 4. Ras nanoclusters digitize transmembrane signal transmission

a | Activation of epidermal growth factor receptors (EGFRs) generates KRAS-GTP on the plasma membrane. A fixed proportion of these KRAS-GTP molecules (the clustered fraction) assemble into signalling nanoclusters. Each cluster has a radius of ~9 nm and contains ~7 KRAS-GTP molecules. At higher EGF concentrations more nanoclusters form. b | Because KRAS-GTP levels are directly proportional to non-saturating EGF doses, and the KRAS-GTP clustered fraction remains constant as KRAS-GTP levels increase, the number of KRAS-GTP nanoclusters depends linearly on stimulating EGF concentration. c | After the recruitment of RAF and KSR–MEK–ERK (kinase suppressor of Ras–MAPK/ERK kinase–extracellular signal-regulated kinase) complexes from the cytosol, each nanocluster outputs a digital pulse of dually phosphorylated ERK (ppERK). The ppERK output is insensitive to RAF kinase input and is limited by disassembly of the nanocluster; a two-log range of relative RAF inputs is shown. d | As a result of b and c, the total system response to EGF, which is the aggregated digital outputs from all of the transiently active nanoclusters, is analogue (blue line). The gain of the response is increased if the KRAS clustered fraction increases from 40% to higher values (orange and purple lines).