A kinase translocation reporter reveals real-time dynamics of ERK activity in Drosophila

Extracellular Signal-Regulated Kinase (ERK) lies downstream of a core signalling cascade that controls all aspects of development and adult homeostasis. Recent developments have led to new tools to image and manipulate the pathway. However, visualising ERK activity in vivo with high temporal resolution remains a challenge in Drosophila. We adapted a kinase translocation reporter (KTR) for use in Drosophila, which shuttles out of the nucleus when phosphorylated by ERK. We show that ERK-KTR faithfully reports endogenous ERK signalling activity in developing and adult tissues, and that it responds to genetic perturbations upstream of ERK. Using ERK-KTR in time-lapse imaging, we made two novel observations: firstly, sustained hyperactivation of ERK by expression of dominant-active Epidermal Growth Factor Receptor raised the overall level but did not alter the kinetics of ERK activity; secondly, heterogeneity in ERK activity in retinal basal glia correlated with the direction of migration of individual cells. Our results show that KTR technology can be applied in Drosophila to monitor ERK activity in real-time and suggest that this modular tool can be further adapted to study other kinases.

Displacement of PKA catalytic subunit from AKAP signaling islands drives pathology in Cushing's syndrome

Mutations in the catalytic subunit of protein kinase A (PKAc) drive the stress hormone disorder adrenal Cushing's syndrome. Here we define mechanisms of action for the PKAc-L205R and W196R variants. Both Cushing's mutants are excluded from A kinase anchoring protein (AKAP) signaling islands and consequently diffuse throughout the cell. Kinase-dead experiments show that PKA activity is required for cortisol hypersecretion. However, kinase activation is not sufficient, as only cAMP analog drugs that displace native PKAc from AKAPs enhance cortisol release. Rescue experiments that incorporate mutant PKAc into AKAP signaling islands abolish cortisol overproduction, indicating that kinase anchoring restores normal endocrine function. Phosphoproteomics show that PKAc-L205R and W196R engage different mitogenic signaling pathways. ERK activity is elevated in adrenal-specific PKAc-W196R knock-in mice. Conversely, PKAc-L205R attenuates Hippo signaling, thereby upregulating the YAP/TAZ transcriptional co-activators. Thus, aberrant localization of each Cushing's variant promotes the transmission of a distinct downstream pathogenic signal.

Repression of MAPK/Erk signaling by Efnb2-Ephb4-Rasa1 is required for lymphatic valve formation

The lymphatic vascular system plays important roles in various physiological and pathological processes, and lack of lymphatic or lymphovenous valves always causes lymph or blood reflux, and can lead to lymphedema. However, the molecular mechanism underlying the valve formation is poorly understood. Here we report that the MAPK/Erk signaling needs to be repressed during the valve-forming lymphatic endothelial cells (LECs) fate determination, which differs from its positive role in the LECs specification. Up-regulation of MAPK/Erk signaling in ephb4b, efnb2a;efnb2b and rasa1a;rasa1b mutants leads to lymphatic valve defects, whereas simultaneous loss of Erk1 and Erk2 causes valve hyperplasia. Moreover, valve defects in ephb4b or rasa1a;rasa1b mutants are mitigated in the presence of MEK inhibitors, indicating a new function of Efnb2-Ephb4-Rasa1 cassette in lymphatic valve progenitor cells specification by repressing MAPK/Erk activity. Therefore, our findings provide a mechanistic understanding of the lymphatic valve formation and potential drug targets for related lymphatic diseases.

Quantifying ERK activity in response to inhibition of the BRAFV600E-MEK-ERK cascade using mathematical modelling

Background Simultaneous inhibition of multiple components of the BRAF-MEK-ERK cascade (vertical inhibition) has become a standard of care for treating BRAF-mutant melanoma. However, the molecular mechanism of how vertical inhibition synergistically suppresses intracellular ERK activity, and consequently cell proliferation, are yet to be fully elucidated. Methods We develop a mechanistic mathematical model that describes how the mutant BRAF inhibitor, dabrafenib, and the MEK inhibitor, trametinib, affect BRAFV600E-MEK-ERK signalling. The model is based on a system of chemical reactions that describes cascade signalling dynamics. Using mass action kinetics, the chemical reactions are re-expressed as ordinary differential equations that are parameterised by in vitro data and solved numerically to obtain the temporal evolution of cascade component concentrations. Results The model provides a quantitative method to compute how dabrafenib and trametinib can be used in combination to synergistically inhibit ERK activity in BRAFV600E-mutant melanoma cells. The model elucidates molecular mechanisms of vertical inhibition of the BRAFV600E-MEK-ERK cascade and delineates how elevated BRAF concentrations generate drug resistance to dabrafenib and trametinib. The computational simulations further suggest that elevated ATP levels could be a factor in drug resistance to dabrafenib. Conclusions The model can be used to systematically motivate which dabrafenib–trametinib dose combinations, for treating BRAFV600E-mutated melanoma, warrant experimental investigation.

The Downregulation of eIF3a Contributes to Vemurafenib Resistance in Melanoma by Activating ERK via PPP2R1B

Vemurafenib, a BRAF V600E inhibitor, provides therapeutic benefits for patients with melanoma, but the frequent emergence of drug resistance remains a challenge. An understanding of the mechanisms underlying vemurafenib resistance may generate novel therapeutic strategies for patients with melanoma. Here, we showed that eIF3a, a translational regulatory protein, was an important mediator involved in vemurafenib resistance. eIF3a was expressed at significantly lower levels in vemurafenib-resistant A375 melanoma cells (A375R) than in parental A375 cells. Overexpression of eIF3a enhanced the sensitivity to BRAF inhibitors by reducing p-ERK levels. Furthermore, eIF3a controlled ERK activity by regulating the expression of the phosphatase PPP2R1B via a translation mechanism, thus determining the sensitivity of melanoma cells to vemurafenib. In addition, a positive correlation between eIF3a and PPP2R1B expression was also observed in tumor samples from the Human Protein Atlas and TCGA databases. In conclusion, our studies reveal a previously unknown molecular mechanism of BRAF inhibitor resistance, which may provide a new strategy for predicting vemurafenib responses in clinical treatment.

Shedding light on developmental ERK signaling with genetically encoded biosensors

ABSTRACT The extracellular signal-regulated kinase (ERK) pathway governs cell proliferation, differentiation and migration, and therefore plays key roles in various developmental and regenerative processes. Recent advances in genetically encoded fluorescent biosensors have unveiled hitherto unrecognized ERK activation dynamics in space and time and their functional importance mainly in cultured cells. However, ERK dynamics during embryonic development have still only been visualized in limited numbers of model organisms, and we are far from a sufficient understanding of the roles played by developmental ERK dynamics. In this Review, we first provide an overview of the biosensors used for visualization of ERK activity in live cells. Second, we highlight the applications of the biosensors to developmental studies of model organisms and discuss the current understanding of how ERK dynamics are encoded and decoded for cell fate decision-making.

Optogenetic actuator/biosensor circuits for large-scale interrogation of ERK dynamics identify sources of MAPK signaling robustness.

Measurements of single-cell ERK activity dynamics provide unique insights in the MAPK network topology. We built genetic circuits consisting of optogenetic actuators activating ERK from different nodes within the MAPK network together with an ERK biosensor to measure single-cell ERK dynamics. Evaluating ERK dynamics induced by different temporal optogenetic inputs, in response to a large number of perturbations, shows that the MAPK network is robust to downregulation of most of its nodes. This robustness emerges in part because of the ERK-RSK2-SOS negative feedback. Bypassing this feedback, by direct activation of the RAS/RAF/MEK/ERK submodule, or by RSK2 perturbation, breaks MAPK network robustness. Targeting the RSK2-mediated feedback in a ErbB2-dependent oncogenic signaling model greatly sensitizes ERK to MEK inhibition, allowing efficient ERK activity shutdown within a cell population. Thus, the RSK2-mediated negative feedback is a weak node of the MAPK network whose perturbation enables potent inhibition of ERK.

ERK-mediated Curvature Feedback Regulates Branching Morphogenesis in Lung Epithelial Tissue

Intricate branching patterns emerge in internal organs because of the repetitive presence of simple deformations in epithelial tissues. During murine lung development, epithelial cells in distal tips of a single tube require fibroblast growth factor (FGF) signals generated by their surrounding mesenchyme to form repetitive tip bifurcations. However, it remains unknown how the cells employ FGF signaling to convert their behaviors to achieve the recursive branching processes. Here we show a self-sustained epithelial regulatory system during the murine lung branching morphogenesis, mediated by extracellular signal-regulated kinase (ERK), which acts as a downstream driver of FGF signaling. We found that tissue-scale curvature regulates ERK activity in the lung epithelium using two-photon live cell imaging and mechanical perturbations. ERK is activated specifically in epithelial tissues with a positive curvature, regardless of whether the change in curvature was attributable to morphogenesis or artificial perturbations. Moreover, we found that ERK activation accelerates actin polymerization specifically at the apical side of cells, and mechanically contributes to the extension of the apical membrane, leading to a decrease in epithelial tissue curvature. These results indicate the existence of a negative feedback loop between tissue curvature and ERK activity beyond scale. We confirmed that this regulation was sufficient to generate the recursive branching processes by a mathematical model. Taken together, we propose that ERK mediates the curvature feedback loop underlying the process of branching morphogenesis in developing lungs.

Disrupting the α7nAChR–NR2A protein complex exerts antidepressant-like effects

Major depressive disorder (MDD) is associated with significant morbidity and mortality. Most antidepressant medications target the serotonin and norepinephrine transporters, but a significant minority of patients do not respond to treatment and novel therapeutic targets are needed. We previously identified a protein complex composed of the α7 nicotinic acetylcholine receptor (nAChR) and NMDA glutamate receptors (NMDARs), through which α7nAChR upregulates NMDAR function. Disruption of the α7nAChR–NMDAR complex with an interfering peptide blocked α7nAChR-mediated upregulation of NMDAR function and cue-induced reinstatement of nicotine seeking in rat models of relapse. Here we report that disrupting the α7nAChR–NMDAR complex with the interfering peptide also has antidepressant-like effects in the forced swim test (FST), a common rat behaviour screening test for antidepressant effects. Furthermore, the interfering peptide significantly increases extracellular signal-regulated kinase (ERK) activity in the animals subjected to the FST. Our results provide a novel potential therapeutic target for the development of new antidepressant medications.