A live-cell kinase-inhibitor screen in primary mouse keratinocytes (Goglia et al., Cell Systems 2020) showed that inhibiting non-EGFR receptor tyrosine kinases (RTKs) such as Met and VEGFR unexpectedly raises Erk pulse frequency and baseline activity through an EGFR-dependent 'gain-enhancement' of transmission, and that high-frequency Erk pulses license cell-cycle entry only when coincident IGF1/insulin-PI3K-Akt activity is present. The paper left open the molecular mechanisms on both sides of this signaling-dynamics logic: which molecular intermediates and feedbacks implement antagonistic RTK-level control of EGFR-Ras-Erk transmission, and which endogenous decoding circuitry reads distinct Erk dynamic features (pulse frequency versus duration versus prominence, where prominence is the height of each pulse above baseline, quantified in the source screen as a per-pulse feature distinct from the overall Erk activity offset), together with permissive Akt activity, to decide cell-cycle entry. The molecular intermediates of the antagonistic RTK-level control of EGFR-Ras-Erk transmission and the endogenous effectors that decode pulse frequency versus duration versus prominence together with permissive Akt activity remain unidentified in the literature, leaving the mechanistic core open.

Background

The Ras-Erk (extracellular signal-regulated kinase) pathway is a conserved mitogen-activated protein kinase (MAPK) cascade that relays signals from cell-surface receptor tyrosine kinases (RTKs) - such as the EGF receptor (EGFR), Met (the hepatocyte growth factor receptor), and VEGFR (the vascular endothelial growth factor receptor) - to cell-cycle entry. Live-cell biosensors, in particular the Erk kinase translocation reporter (Erk-KTR, a fluorescent Erk substrate that shuttles between nucleus and cytoplasm in proportion to Erk activity), have revealed that Erk activity in single cells is not steady but pulsatile, and that these dynamics - pulse frequency, duration, prominence, and baseline - can carry information that plain activity levels do not. Goglia et al. (Cell Systems 2020) screened 429 kinase inhibitors in more than 80,000 primary mouse keratinocytes, an epithelial cell type exhibiting robust, growth-factor-independent 'idling motor' Erk pulses (self-sustained pulsatile signaling to which cells return even after strong perturbation), and quantified Erk dynamics per single cell. The screen revealed three drug classes: Class 1 (inhibitors of EGFR and of MEK, the kinase immediately upstream of Erk) suppressed Erk; Class 2 (paradox-activating B-Raf inhibitors, which stabilize dimers of Raf, the kinase between Ras and MEK) produced long, low-frequency pulses; and Class 3 (inhibitors of non-EGFR RTKs including Met, VEGFR, Kit, and the fibroblast growth factor receptors) increased Erk pulse frequency and baseline. Epistasis experiments - testing whether one drug's effect requires the target of another - showed, using the EGFR inhibitor lapatinib, that Class 2/3 drugs still require EGFR activity: inhibiting non-EGFR RTKs apparently increases the gain with which EGFR phosphorylation is transmitted to the Ras-Erk network rather than activating MAPK directly. Notably, EGFR phosphorylation itself was minimally increased by Class 3 drugs, while Erk phosphorylation rose strongly, consistent with strong amplification and nonlinearity between receptor and Erk. Functionally, the two dynamic regimes differ: optogenetic control of Ras (OptoSOS, a light-gated SOS/Ras activation system) and drug treatments showed that low-frequency, long Erk pulses trigger proliferation on their own, whereas short, high-frequency pulses (as produced by Class 3 drugs such as pazopanib, a clinically used VEGFR-directed kinase inhibitor) require coincident IGF1/insulin receptor signaling through PI3K-Akt (phosphoinositide 3-kinase-Akt, the pro-growth/pro-survival branch downstream of insulin receptors), since the PI3K inhibitor pictilisib blocks proliferation under pazopanib plus insulin. Immediate-early genes (IEGs, rapidly and transiently induced transcriptional targets of Erk such as FOS) are one established decoding layer, and mathematical work shows feedforward motifs can make IEG expression sensitive to Erk pulse duration; synthetic immediate-early genes have been engineered to read Erk duration and input combinations. What is missing is the endogenous molecular account on both ends: the crosstalk mechanism upstream (how non-EGFR RTKs normally restrain EGFR-driven Erk pulses, and which intermediates or feedbacks - for example adaptor competition, receptor trafficking, or paracrine ligand release - implement the gain change) and the decoding circuitry downstream (which molecular effectors distinguish pulse frequency from duration and how the PI3K-Akt arm makes high-frequency Erk pulses sufficient for cell-cycle entry).

Problem Statement

Define, at the molecular level, how pulsatile Erk dynamics are generated and decoded to control epithelial cell proliferation, as left open by Goglia et al. (Cell Systems 2020). The single overarching objective is a causal molecular account linking RTK-level inputs to cell-cycle entry through Erk dynamics, with two linked questions: (1) By what molecular mechanism does inhibition of non-EGFR receptor tyrosine kinases (such as Met and VEGFR) increase the frequency and baseline of EGFR-dependent Erk pulses - which molecular intermediates or feedbacks implement the apparent gain-enhancement of EGFR-to-Ras-to-Erk transmission (for example, receptor-level crosstalk, adaptor or feedback wiring, or ligand-release circuitry)? (2) What is the endogenous decoding circuitry that converts specific Erk dynamic features (pulse frequency, duration, and prominence), together with coincident inputs such as IGF1/insulin-PI3K-Akt activity, into robust cell-cycle entry - in particular, which downstream effectors read pulse frequency, pulse duration, and prominence, and by what biochemical mechanism does Akt activity license proliferation in the presence of high-frequency Erk pulses?

References

  1. Goglia AG, Wilson MZ, Jena SG, Silbert J, Basta LP, Devenport D, Toettcher JE (2020) A Live-Cell Screen for Altered Erk Dynamics Reveals Principles of Proliferative Control. Cell Systems 10:240-253.e6. DOI: 10.1016/j.cels.2020.02.005. https://doi.org/10.1016/j.cels.2020.02.005
  2. Ravindran PT, Wilson MZ, Jena SG, Toettcher JE (2020) Engineering combinatorial and dynamic decoders using synthetic immediate-early genes. Communications Biology 3:436. DOI: 10.1038/s42003-020-01171-1. https://doi.org/10.1038/s42003-020-01171-1
  3. Ravindran PT, McFann S, Thornton RH, Toettcher JE (2022) A synthetic gene circuit for imaging-free detection of signaling pulses. Cell Systems 13:131-142.e13. DOI: 10.1016/j.cels.2021.10.002. https://doi.org/10.1016/j.cels.2021.10.002
  4. Jena SG, Yu C, Toettcher JE (2021) Dynamics and heterogeneity of Erk-induced immediate-early gene expression. bioRxiv preprint. DOI: 10.1101/2021.04.30.442166. https://doi.org/10.1101/2021.04.30.442166
  5. Ram A, Murphy D, DeCuzzi N, Patankar M, Hu J, Pargett M, Albeck JG (2023) A guide to ERK dynamics, part 1: mechanisms and models. Biochemical Journal 480:1887-1907. DOI: 10.1042/BCJ20230276. https://doi.org/10.1042/BCJ20230276
  6. Ram A, Murphy D, DeCuzzi N, Patankar M, Hu J, Pargett M, Albeck JG (2023) A guide to ERK dynamics, part 2: downstream decoding. Biochemical Journal 480:1909-1928. DOI: 10.1042/BCJ20230277. https://doi.org/10.1042/BCJ20230277
  7. Stern AD, Smith GR, Santos LC, Sarmah D, Zhang X, Lu X, Iuricich F, Pandey G, Iyengar R, Birtwistle MR (2022) Relating individual cell division events to single-cell ERK and Akt activity time courses. Scientific Reports 12:18077. DOI: 10.1038/s41598-022-23071-6. https://doi.org/10.1038/s41598-022-23071-6
  8. Bennett JJR, Stern AD, Zhang X, Birtwistle MR, Pandey G (2024) Low-frequency ERK and Akt activity dynamics are predictive of stochastic cell division events. npj Systems Biology and Applications 10:65. DOI: 10.1038/s41540-024-00389-7. https://doi.org/10.1038/s41540-024-00389-7
  9. Nangia V, Ashraf H, Marikar N, Passanisi VJ, Ill CR, Spencer SL (2025) MAPK and mTORC1 signaling converge to drive cyclin D1 protein production to enable cell cycle reentry in melanoma persister cells. Science Signaling 18:eadw3231. DOI: 10.1126/scisignal.adw3231. https://doi.org/10.1126/scisignal.adw3231
  10. Ender P, Gagliardi PA, Dobrzynski M, Frismantiene A, Dessauges C, Hohener T, Jacques M-A, Cohen AR, Pertz O (2022) Spatiotemporal control of ERK pulse frequency coordinates fate decisions during mammary acinar morphogenesis. Developmental Cell 57:2153-2167.e6. DOI: 10.1016/j.devcel.2022.08.008. https://doi.org/10.1016/j.devcel.2022.08.008
  11. Hiratsuka T, Bordeu I, Pruessner G, Watt FM (2020) Regulation of ERK basal and pulsatile activity control proliferation and exit from the stem cell compartment in mammalian epidermis. Proceedings of the National Academy of Sciences 117:17796-17807. DOI: 10.1073/pnas.2006965117. https://doi.org/10.1073/pnas.2006965117
  12. Farahani PE, Lemke SB, Dine E, Uribe G, Toettcher JE, Nelson CM (2021) Substratum stiffness regulates Erk signaling dynamics through receptor-level control. Cell Reports 37:110181. DOI: 10.1016/j.celrep.2021.110181. https://doi.org/10.1016/j.celrep.2021.110181
  13. Okuda KS, Keyser MS, Gurevich DB, Sturtzel C, Mason EA, Paterson S, Chen H, Scott M, Condon ND, Martin P, Distel M, Hogan BM (2021) Live-imaging of endothelial Erk activity reveals dynamic and sequential signalling events during regenerative angiogenesis. eLife 10:e62196. DOI: 10.7554/eLife.62196. https://doi.org/10.7554/eLife.62196
  14. Raina D, Fabris F, Morelli LG, Schroter C (2022) Intermittent ERK oscillations downstream of FGF in mouse embryonic stem cells. Development 149:dev199710. DOI: 10.1242/dev.199710. https://doi.org/10.1242/dev.199710
  15. Toyooka Y, Aoki K, Usami FM, Oka S, Kato A, Fujimori T (2023) Generation of pulsatile ERK activity in mouse embryonic stem cells is regulated by Raf activity. Scientific Reports 13:9465. DOI: 10.1038/s41598-023-36424-6. https://doi.org/10.1038/s41598-023-36424-6
  16. Le Marois A, Bailey S, Hooper S, Kumar S, Sparks H, Alexandrov Y, Caswell D, Frohlich F, Schlegelmilch K, Valand K, Martin M, Narvaez A, Swanton C, Downward J, Dunsby C, French P, Sahai E (2024) Imaging of MAP kinase dynamics reveals endocytic regulation of pulsatile signalling and network re-wiring in response to targeted therapy in EGFR-mutant non-small cell lung cancer. bioRxiv preprint. DOI: 10.1101/2024.05.14.594112. https://doi.org/10.1101/2024.05.14.594112