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1. GRK2 kinases in the primary cilium initiate SMOOTHENED-PKA signaling in the Hedgehog cascade

   https://doi.org/10.1371/journal.pbio.3002685  

   Walker, Madison F; Zhang, Jingyi; Steiner, William; Ku, Pei-I; Zhu, Ju-Fen; Michaelson, Zachary; Yen, Yu-Chen; Lee, Annabel; Long, Alyssa B; Casey, Mattie J; et al (August 2024, PLOS Biology)

   Basler, Konrad (Ed.)

   During Hedgehog (Hh) signal transduction in development and disease, the atypical G protein-coupled receptor (GPCR) SMOOTHENED (SMO) communicates with GLI transcription factors by binding the protein kinase A catalytic subunit (PKA-C) and physically blocking its enzymatic activity. Here, we show that GPCR kinase 2 (GRK2) orchestrates this process during endogenous mouse and zebrafish Hh pathway activation in the primary cilium. Upon SMO activation, GRK2 rapidly relocalizes from the ciliary base to the shaft, triggering SMO phosphorylation and PKA-C interaction. Reconstitution studies reveal that GRK2 phosphorylation enables active SMO to bind PKA-C directly. Lastly, the SMO-GRK2-PKA pathway underlies Hh signal transduction in a range of cellular and in vivo models. Thus, GRK2 phosphorylation of ciliary SMO and the ensuing PKA-C binding and inactivation are critical initiating events for the intracellular steps in Hh signaling. More broadly, our study suggests an expanded role for GRKs in enabling direct GPCR interactions with diverse intracellular effectors. 

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2. Control of gastruloid patterning and morphogenesis by the Erk and Akt signaling pathways

   https://doi.org/10.1242/dev.201663  

   Underhill, Evan J.; Toettcher, Jared E. (August 2023, Development)

   ABSTRACT Many developmental processes rely on the localized activation of receptor tyrosine kinases and their canonical downstream effectors Erk and Akt, yet the specific roles played by each of these signals is still poorly understood. Gastruloids, 3D cell culture models of mammalian gastrulation and axial elongation, enable quantitative dissection of signaling patterns and cell responses in a simplified, experimentally accessible context. We find that mouse gastruloids contain posterior-to-anterior gradients of Erk and Akt phosphorylation induced by distinct receptor tyrosine kinases, with features of the Erk pattern and expression of its downstream target Snail exhibiting hallmarks of size-invariant scaling. Both Erk and Akt signaling contribute to cell proliferation, whereas Erk activation is also sufficient to induce Snail expression and precipitate profound tissue shape changes. We further uncover that Erk signaling is sufficient to convert the entire gastruloid to one of two mesodermal fates depending on position along the anteroposterior axis. In all, these data demonstrate functional roles for two core signaling gradients in mammalian development and suggest how these modules might be harnessed to engineer user-defined tissues with predictable shapes and cell fates. 

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3. E-cadherin mechanotransduction activates EGFR-ERK signaling in epithelial monolayers by inducing ADAM-mediated ligand shedding

   https://doi.org/10.1126/scisignal.adr7926  

   Houtekamer, Ronja M; van_der_Net, Mirjam C; Vliem, Marjolein J; Noordzij, Tomas_E_J C; van_Uden, Lisa; van_Es, Robert M; Sim, Joo Yong; Deguchi, Eriko; Terai, Kenta; Hopcroft, Matthew A; et al (May 2025, Science Signaling)

   The behavior of cells is governed by signals originating from their local environment, including mechanical forces exerted on the cells. Forces are transduced by mechanosensitive proteins, which can impinge on signaling cascades that are also activated by growth factors. We investigated the cross-talk between mechanical and biochemical signals in the regulation of intracellular signaling networks in epithelial monolayers. Phosphoproteomic and transcriptomic analyses on epithelial monolayers subjected to mechanical strain revealed the activation of extracellular signal–regulated kinase (ERK) downstream of the epidermal growth factor receptor (EGFR) as a predominant strain-induced signaling event. Strain-induced EGFR-ERK signaling depended on mechanosensitive E-cadherin adhesions. Proximity labeling showed that the metalloproteinase ADAM17, an enzyme that mediates shedding of soluble EGFR ligands, was closely associated with E-cadherin. A probe that we developed to monitor ADAM-mediated shedding demonstrated that mechanical strain induced ADAM activation. Mechanically induced ADAM activation was essential for mechanosensitive, E-cadherin–dependent EGFR-ERK signaling. Together, our data demonstrate that mechanical strain transduced by E-cadherin adhesion triggers the shedding of EGFR ligands that stimulate downstream ERK activity. Our findings illustrate how mechanical signals and biochemical ligands can operate within a linear signaling cascade. 

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4. Optimizing photoswitchable MEK

   https://doi.org/10.1073/pnas.1912320116  

   Patel, Aleena L.; Yeung, Eyan; McGuire, Sarah E.; Wu, Andrew Y.; Toettcher, Jared E.; Burdine, Rebecca D.; Shvartsman, Stanislav Y. (December 2019, Proceedings of the National Academy of Sciences)

   Optogenetic approaches are transforming quantitative studies of cell-signaling systems. A recently developed photoswitchable mitogen-activated protein kinase kinase 1 (MEK1) enzyme (psMEK) short-circuits the highly conserved Extracellular Signal-Regulated Kinase (ERK)-signaling cascade at the most proximal step of effector kinase activation. However, since this optogenetic tool relies on phosphorylation-mimicking substitutions in the activation loop of MEK, its catalytic activity is predicted to be substantially lower than that of wild-type MEK that has been phosphorylated at these residues. Here, we present evidence that psMEK indeed has suboptimal functionality in vivo and propose a strategy to circumvent this limitation by harnessing gain-of-function, destabilizing mutations in MEK. Specifically, we demonstrate that combining phosphomimetic mutations with additional mutations in MEK, chosen for their activating potential, restores maximal kinase activity in vitro. We establish that this modification can be tuned by the choice of the destabilizing mutation and does not interfere with reversible activation of psMEK in vivo in both Drosophila and zebrafish. To illustrate the types of perturbations enabled by optimized psMEK, we use it to deliver pulses of ERK activation during zebrafish embryogenesis, revealing rheostat-like responses of an ERK-dependent morphogenetic event. 

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5. Time-resolved live-cell spectroscopy reveals EphA2 multimeric assembly

   https://doi.org/10.1126/science.adg5314  

   Shi, Xiaojun; Lingerak, Ryan; Herting, Cameron J; Ge, Yifan; Kim, Soyeon; Toth, Paul; Wang, Wei; Brown, Benjamin P; Meiler, Jens; Sossey-Alaoui, Khalid; et al (December 2023, Science)

   Ephrin type-A receptor 2 (EphA2) is a receptor tyrosine kinase that initiates both ligand-dependent tumor-suppressive and ligand-independent oncogenic signaling. We used time-resolved, live-cell fluorescence spectroscopy to show that the ligand-free EphA2 assembles into multimers driven by two types of intermolecular interactions in the ectodomain. The first type entails extended symmetric interactions required for ligand-induced receptor clustering and tumor-suppressive signaling that inhibits activity of the oncogenic extracellular signal–regulated kinase (ERK) and protein kinase B (AKT) protein kinases and suppresses cell migration. The second type is an asymmetric interaction between the amino terminus and the membrane proximal domain of the neighboring receptors, which supports oncogenic signaling and promotes migration in vitro and tumor invasiveness in vivo. Our results identify the molecular interactions that drive the formation of the EphA2 multimeric signaling clusters and reveal the pivotal role of EphA2 assembly in dictating its opposing functions in oncogenesis. 

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