Science Signaling

The c-Fes protein tyrosine kinase is a proto-oncogene that can also act as a tumor suppressor. We implemented an unbiased phosphoproteomics-based analysis that establishes cognate kinase-substrate associations, and revealed that c-Fes directly phosphorylates Dok1, Ptpn18 and Sts1, facilitating recruitment of the Src inhibitory kinase Csk to these substrates. These interactions resulted in modulation of Src signaling following B-cell receptor (BCR) stimulation and subsequent alteration of the protein levels of CD19, a membrane-localized BCR co-receptor and emerging key protein affecting the development of B- and plasma cell-lymphoma. Strikingly, manipulating c-Fes expression levels drove opposing biological outcomes. Low-level exogenous c-Fes expression led to a strong increase in CD19 protein levels while high c-Fes expression abolished CD19 protein levels. Thus, we propose that a balance of c-Fes and Src signaling can regulate receptor maintenance, which may influence cellular outcome such as tumorigenesis or tumor suppression.

Resistance to BRAF and MAPK inhibitors is a significant challenge in melanoma treatment, driven by adaptive and acquired mechanisms that allow tumour cells to evade therapy. Here, we examined early signalling responses to single and combined BRAF and MAPK inhibition in a BRAFV600E, drug-sensitive melanoma cell line and a drug-resistant ARID1A-knockout (KO) derivative. ARID1A, frequently mutated in melanoma, is associated with resistance and immune evasion. Using an innovative systems biology approach that integrates transcriptomics, proteomics, phosphoproteomics, and functional kinomics through matrix factorization and network analysis, we identified key signalling alterations and resistance mechanisms. We found that ARID1A-KO cells exhibited transcriptional rewiring, sustaining MAPK1/3 and JNK activity post-treatment, bypassing feedback sensitivity observed in parental cells. This rewiring suppressed PRKD1 activation, increased JUN activity--a central resistance network node--and disrupted PKC dynamics through elevated basal RTKs (e.g., EGFR, ROS1) and Ephrin receptor activity post-treatment. ARID1A mutations also reduced HLA-related protein expression and enriched extracellular matrix components, potentially limiting immune infiltration and reducing immunotherapy efficacy. Our graph-theoretical multi-omics approach uncovered novel resistance-associated signalling pathways, identifying PRKD1, JUN, and NCK1 as critical nodes. While receptor activation redundancies complicate single-target therapies, they also present opportunities for combination strategies. This study highlights ARID1As role in reshaping signalling and immune interactions, offering new insights into melanoma resistance mechanisms. By identifying actionable targets, including JUN and immune pathways, we provide a foundation for developing integrated therapeutic strategies to overcome resistance in BRAF/MAPK inhibitor-treated melanoma. One sentence summaryThis study reveals how ARID1A-mediated transcriptional rewiring drives resistance to MAPK inhibitors in melanoma by altering signalling pathways, immune interactions, and receptor dynamics, highlighting potential targets for combinatorial therapies.

The biophysical properties of ligand binding heavily influence the ability of receptors to specify cell fates. Understanding the rules by which ligand binding kinetics impact cell phenotype is challenging, however, because of the coupled information transfers that occur from receptors to downstream signaling effectors and from effectors to phenotypes. Here, we address that issue by developing an integrated mechanistic and data-driven computational modeling platform to predict cell responses to different ligands for the epidermal growth factor receptor (EGFR). Experimental data for model training and validation were generated using MCF7 human breast cancer cells treated with the high- and low-affinity ligands epidermal growth factor (EGF) and epiregulin (EREG), respectively. The integrated model captures the unintuitive, concentration-dependent abilities of EGF and EREG to drive signals and phenotypes differently, even at similar levels of receptor occupancy. For example, the model correctly predicts the dominance of EREG over EGF in driving a cell differentiation phenotype through AKT signaling at intermediate and saturating ligand concentrations and the ability of EGF and EREG to drive a broadly concentration-sensitive migration phenotype through cooperative ERK and AKT signaling. Parameter sensitivity analysis identifies EGFR endocytosis, which is differentially regulated by EGF and EREG, as one of the most important determinants of the alternative phenotypes driven by different ligands. The integrated model provides a new platform to predict how phenotypes are controlled by the earliest biophysical rate processes in signal transduction and may eventually be leveraged to understand receptor signaling system performance depends on cell context. One-sentence summaryIntegrated kinetic and data-driven EGFR signaling model identifies the specific signaling mechanisms that dictate cell responses to EGFR activation by different ligands.

Most antiviral agents are designed to target virus-specific proteins and mechanisms rather than the host cell proteins that are critically dysregulated following virus-mediated reprogramming of the host cell transcriptional state. To overcome these limitations, we propose that elucidation and pharmacologic targeting of host cell Master Regulator proteins--whose aberrant activities govern the reprogramed state of infected-coronavirus cells--presents unique opportunities to develop novel mechanism-based therapeutic approaches to antiviral therapy, either as monotherapy or as a complement to established treatments. Specifically, we propose that a small module of host cell Master Regulator proteins (ViroCheckpoint) is hijacked by the virus to support its efficient replication and release. Conventional methodologies are not well suited to elucidate these potentially targetable proteins. By using the VIPER network-based algorithm, we successfully interrogated 12h, 24h, and 48h signatures from Calu-3 lung adenocarcinoma cells infected with SARS-CoV, to elucidate the time-dependent reprogramming of host cells and associated Master Regulator proteins. We used the NYS CLIA-certified Darwin OncoTreat algorithm, with an existing database of RNASeq profiles following cell perturbation with 133 FDA-approved and 195 late-stage experimental compounds, to identify drugs capable of virtually abrogating the virus-induced Master Regulator signature. This approach to drug prioritization and repurposing can be trivially extended to other viral pathogens, including SARS-CoV-2, as soon as the relevant infection signature becomes available.

Pathological activation of the PI3K/AKT pathway is among the most frequent defects in human cancer and is also the cause of rare overgrowth disorders. Yet, there is currently no systematic understanding of the quantitative flow of information within PI3K/AKT signaling and how it is perturbed by disease-causing mutations. Here, we develop scalable, single-cell approaches for systematic analyses of signal processing within the PI3K pathway, enabling precise calculations of its information transfer for different growth factors. Using genetically-engineered human cell models with allele dose-dependent expression of PIK3CAH1047R, we show that this oncogene is not a simple, constitutive pathway activator but a context-dependent modulator of extracellular signal transfer. PIK3CAH1047Rreduces information transmission downstream of IGF1 while selectively enhancing EGF-induced signaling and transcriptional responses. This leads to a gross reduction in signaling specificity, akin to "blurred" signal perception. The associated increase in signaling heterogeneity promotes phenotypic diversity in a human cervical cancer cell line model and in human induced pluripotent stem cells. Collectively, these findings and the accompanying methodological advances lay the foundations for a systematic mapping of the quantitative mechanisms of PI3K/AKT-dependent signal processing and phenotypic control in health and disease. One-sentence summarySingle-cell signaling and information theoretic analyses reveal that oncogenic PI3K/AKT activation leads to a gross reduction in signaling specificity, context-dependent EGF response amplification as well as increased phenotypic heterogeneity.

Notch signaling relies on ligand-induced proteolysis to liberate a nuclear effector that drives cell fate decisions. The location and timing of individual steps required for proteolysis and movement of Notch from membrane to nucleus, however, remain unclear. Here, we use proximity labeling with quantitative multiplexed mass spectrometry to monitor the microenvironment of endogenous Notch2 after ligand stimulation in the presence of a gamma secretase inhibitor and then as a function of time after inhibitor removal. Our studies show that gamma secretase cleavage of Notch2 occurs in an intracellular compartment and that formation of nuclear complexes and recruitment of chromatin-modifying enzymes occurs within 45 minutes of inhibitor washout. This work provides a spatiotemporal map of unprecedented detail tracking the itinerary of Notch from membrane to nucleus after activation and identifies molecular events in signal transmission that are potential targets for modulating Notch signaling activity.

Genotoxic stress in mammalian cells, including that caused by anti-cancer chemotherapy, can induce temporary cell cycle arrest, DNA damage-induced senescence (DDIS) or apoptotic cell death. Despite obvious clinical importance, it is unclear how the signals emerging from DNA damage are integrated together with other cellular signaling pathways monitoring the cells environment and/or internal state to control these different cell fates. Here, using a combination of single cell-based signaling measurements and tensor PLSR/PCA computational approaches, we show that the JNK and Erk MAPK signaling pathways regulate the initiation of senescence through the transcription factor AP-1 at early times after extrinsic DNA damage, and the Senescence Associated Secretory Phenotype, a hallmark of DDIS, at late times after damage. These results identify a time-based separation of function for the same signaling pathways beyond the classic DNA damage response that control the cell senescence decision and modulate the tumor microenvironment following genotoxic stress, and reveal a fundamental similarity between signaling mechanisms responsible for oncogene-induced senescence and senescence caused by extrinsic DNA damaging agents.

The common {gamma}-chain receptor cytokines coordinate the proliferation and function of immune cell populations. One of these cytokines, interleukin (IL)-2, has potential as a therapy in autoimmune disease but is limited in effectiveness by its modest specificity toward regulatory T cells (Tregs). Engineering Treg-selective IL-2 has primarily focused on retaining binding to the high-affinity receptor, expressed more highly on Tregs, while reducing binding to the lower affinity receptor with broader expression. However, other parameters, such as the orientation and valency of Fc fusion, have signaling effects that have never been systematically explored. Here, we systematically profiled the signaling responses to a panel of wild type and mutein IL-2-Fc fusions across time, cell types, and concentrations. Exploring these responses, we found that dimeric muteins have unique specificity for Tregs through binding avidity. A mechanistic model of receptor interactions could capture these effects and directed the design of tetravalent IL-2-Fc fusions with greater Treg specificity than possible with current design strategies. Exploration of other surface targets on Tregs revealed that there are no other binding moieties that could be fused to IL-2 for greater selectivity. Instead, IL2R itself is a maximally unique surface target for Tregs, and so avidity is likely the only route to more selective Treg interaction. However, the binding model revealed that asymmetrical, multivalent IL-2 fusions can bias avidity effects toward IL2R for even further enhanced Treg selectivity. These findings present a comprehensive analysis of how ligand properties and their effects on surface receptor-ligand interactions translate to selective activation of immune cell populations, and consequently reveals two new routes toward therapeutic cytokines with superior Treg selectivity that can be exploited for designing selective therapies in many other contexts. Significance StatementSignaling in off-target immune cells has hindered the effectiveness of IL-2 as an immunotherapy. We show that IL-2-Fc fusions with higher valency can exhibit enhanced regulatory T cell selectivity. This altered selectivity is explained by the kinetics of surface receptor-ligand binding and can be quantitatively predicted using a multivalent binding model. Using these insights, we successfully develop two new strategies for IL-2 therapies with unprecedented selectivity. HighlightsO_LICurrent IL-2 therapies are limited by a selectivity/target potency tradeoff. C_LIO_LIMultivalency enhances selectivity for Tregs through IL2R avidity. C_LIO_LITreg selectivity cannot be enhanced by targeting other surface protein markers. C_LIO_LIMultivalency can decouple selectivity from signaling using asymmetric cytokine fusions. C_LI

Many G protein-coupled receptors (GPCRs) initiate a second phase of signaling after activation-induced endocytosis, but GPCRs vary considerably in their ability to internalize when activated. Here we show that this fundamental trafficking difference distinguishes the downstream signaling profiles of natively co-expressed GPCRs through the cAMP cascade. We focus on signaling to the nucleus stimulated by three different Gs-coupled GPCRs that are each endogenously co-expressed in human embryonic kidney cells but differ in their ability to internalize after activation: the adenosine-2B receptor that does not detectably internalize, the vasoactive intestinal peptide receptor-1 that internalizes very rapidly, and the {beta}2-adrenergic receptor that internalizes less rapidly. We show that each GPCR produces a distinct signaling profile differentiated by endocytosis. Our results support a model in which endocytosis compresses chemical information sensed by distinct GPCRs into a spatiotemporal cAMP code by setting receptor-specific differences in the amount and duration of cAMP production from endosomes relative to the plasma membrane and that this is decoded downstream in the pathway through sequential layers of processing by cytoplasmic and nuclear PKA activities. We propose that this biological information processing strategy has parallels to how computational encoder-decoder (autoencoder) networks denoise and recognize latent patterns in complex electrical signals.

Proteins of pathogens such as cardioviruses, kaposi sarcoma-associated herpes virus, varicella zoster virus and bacteria of the genus Yersinia were previously shown to use a common "DDVF" (D/E-D/E-V-F) short linear motif (SLiM) to hijack cellular kinases of the RSK (p90 ribosomal S6 kinases) family. Remarkable conservation of the SLiM docking site in RSKs suggested a physiological role for this site. Using SLiM prediction tools and AlphaFold docking, we screened the human proteome for proteins that would interact with RSKs through a DDVF-like SLiM. Using co-immunoprecipitation experiments, we show that two candidates previously known as RSK partners, FGFR1 and SPRED2, as well as two candidates identified as novel RSK partners, GAB3 and CNKSR2 do interact with RSKs through a similar interface as the one used by pathogens, as was recently documented for SPRED2. Moreover, we show that FGFR1 employs a DSVF motif to bind RSKs and that phosphorylation of the serine in this motif increases RSK binding. FGFR1, SPRED2, GAB3 and CNKSR2 as well as other candidate RSK binders act upstream of RSK in the RAS-ERK MAP kinase pathway. Analysis of ERK activation in cells expressing a mutated form of RSK lacking the DDVF-docking site suggests that RSK might interact with the DDVF-like SLiM of several partners to provide a negative feed-back to the ERK MAPK pathway. Thus, through SLiM mimicry, pathogens not only retarget RSKs toward unconventional substrates but also likely compete with human proteins to alter the regulation of the RAS-ERK MAP kinase pathway. Author SummaryShort linear motif (SLiM) are 3 to 10 amino acid-long protein sequences that can mediate the interaction with other proteins. We previously observed that highly unrelated pathogens, including viruses and bacteria, convergently evolved to hijack cellular enzymes of their host, through a common SLiM. In this work, we tested the hypothesis that the SLiM found in proteins of pathogens evolved to mimic a SLiM found in human proteins that regulate the cellular enzymes through the same interface. Protein-protein interactions mediated by SLiMs are often, low-affinity, transient interactions that are difficult to detect by conventional biochemical methods but that can nowadays be predicted with increasing confidence by artificial intelligence-based methods such as AlphaFold. Using such predictions, we identified several candidate human proteins and we confirmed experimentally that these proteins interact with the cellular enzymes the same way as pathogens proteins do. Identified proteins belong to the well-known RAS-ERK MAPK pathway which regulates important functions of the cell, suggesting that pathogens evolved to hijack this MAPK pathway by SLiM mimicry. By doing so, they can both dysregulate cellular physiology and hijack cellular enzymes to their own benefit.

Building mechanistic models of kinase-driven signaling pathways requires quantitative measurements of protein phosphorylation across physiologically relevant conditions, but this is rarely done because of the insensitivity of traditional technologies. By using a multiplexed deep phosphoproteome profiling workflow, we were able to generate a deep phosphoproteomics dataset of the EGFR-MAPK pathway in non-transformed MCF10A cells across physiological ligand concentrations with a time resolution of <12 min and in the presence and absence of multiple kinase inhibitors. An improved phosphosite mapping technique allowed us to reliably identify >46,000 phosphorylation sites on >6600 proteins, of which >4500 sites from 2110 proteins displayed a >2-fold increase in phosphorylation in response to EGF. This data was then placed into a cellular context by linking it to 15 previously published protein databases. We found that our results were consistent with much, but not all previously reported data regarding the activation and negative feedback phosphorylation of core EGFR-ERK pathway proteins. We also found that EGFR signaling is biphasic with substrates downstream of RAS/MAPK activation showing a maximum response at <3ng/ml EGF while direct substrates, such as HGS and STAT5B, showing no saturation. We found that RAS activation is mediated by at least 3 parallel pathways, two of which depend on PTPN11. There appears to be an approximately 4-minute delay in pathway activation at the step between RAS and RAF, but subsequent pathway phosphorylation was extremely rapid. Approximately 80 proteins showed a >2-fold increase in phosphorylation across all experiments and these proteins had a significantly higher median number of phosphorylation sites (~18) relative to total cellular phosphoproteins (~4). Over 60% of EGF-stimulated phosphoproteins were downstream of MAPK and included mediators of cellular processes such as gene transcription, transport, signal transduction and cytoskeletal arrangement. Their phosphorylation was either linear with respect to MAPK activation or biphasic, corresponding to the biphasic signaling seen at the level of the EGFR. This deep, integrated phosphoproteomics data resource should be useful in building mechanistic models of EGFR and MAPK signaling and for understanding how downstream responses are regulated.

Inflammasomes are intracellular protein complexes that control proteolytic maturation and secretion of inflammatory interleukin-1 (IL-1) family cytokines and are thus important in host defense. While some inflammasomes are activated simply by binding to pathogen-derived molecules, others, including those nucleated by NLRP3 and NLRP1, have more complex activation mechanisms that are not fully understood. We screened a library of small molecules to identify new inflammasome activators that might shed light on activation mechanisms. In addition to validating dipeptidyl peptidase (DPP) inhibitors as NLRP1 activators, we find that clinical tyrosine kinase inhibitors (TKIs) including imatinib and masitinib activate the NLRP3 inflammasome. Mechanistically, these TKIs cause lysosomal swelling and damage, leading to cathepsin-mediated destabilization of myeloid cell membranes and cell lysis. This is accompanied by potassium (K+) efflux, which activates NLRP3. Both lytic cell death and NLRP3 activation but not lysosomal damage induced by TKIs are prevented by the cytoprotectant high molecular weight polyethylene glycol (PEG). Our study establishes a screening method that can be expanded for inflammasome research and immunostimulatory drug development, and provides new insight into immunological off-targets that may contribute to efficacy or adverse effects of TKIs. One Sentence SummaryA functional small molecule screen identifies imatinib, masitinib and other tyrosine kinase inhibitors that destabilize myeloid cell lysosomes, leading to cell lysis and K+ efflux-dependent NLRP3 inflammasome activation.

Toll-like receptors (TLRs) are transmembrane proteins required for recognizing microbial components or cellular danger signals to activate intracellular signaling pathways leading to induction of anti-microbial and inflammatory genes. Inactive TLRs require ligand-induced activation to recruit adaptor proteins, e.g., MyD88, to trigger the synthesis of cytokines and interferons. TLR9 is an endosomal membrane-bound protein, which binds to CpG-containing microbial DNA or endogenous signals from dead cells or tissue damage. We showed that TLR9 activation requires EGFR, a tyrosine (Tyr) kinase, which interacts with and phosphorylates the cytoplasmic domain of TLR9. Blocking EGFR activity pharmacologically, or knocking out EGFR gene in myeloid cells, suppressed lethal TLR9-induced hepatotoxicity. Here, we reveal that TLR9 required two Src family of kinases, Syk and Lyn, which, together with EGFR, led to phosphorylation and activation of TLR9. Lack of either of these kinases inhibited TLR9-MyD88 interaction, thereby inhibiting TLR9-mediated gene induction. Unlike EGFR, which constitutively binds TLR9, activated Syk interacted with TLR9 in a CpG-dependent manner. Activated Syk interacted with TLR9 and was critical for activating TLR9-bound EGFR. Quantitative mass spectrometric analyses revealed TLR9 was phosphorylated, sequentially, on Tyr870 and Tyr980 by Syk and EGFR, respectively. Mutation of either of these tyrosines led to complete loss of TLR9-induced cytokine production. For activation, Syk was phosphorylated by Lyn, which was activated by CpG-mediated scavenger-receptor A, and surprisingly, independent of TLR9. In summary, our results uncovered the molecular details of TLR9 activation by its Tyr-phosphorylation, which is critical for TLR9-mediated intracellular signaling.

Epithelial-mesenchymal transition (EMT) occurs heterogeneously among malignant carcinoma cells to promote chemoresistance. Identifying the signaling pathways involved will nominate drug combinations to promote chemoresponse, but cell population-level studies are inherently fraught, and single-cell transcriptomics are limited to indirect ontology-based inferences. To understand EMT heterogeneity at a signaling protein level, we combined iterative indirect immunofluorescence imaging of pancreas cancer cells and tumors and mutual information (MI) modeling. Focusing first on MAP kinase pathways, MI predicted that cell-to-cell variation in ERK activity surprisingly dominated control of EMT heterogeneity in response to diverse growth factors and chemotherapeutics, but that JNK compensated when MEK was inhibited. Population-level models could not capture these experimentally validated MI predictions. The dominant role of ERK was predicted by MI even when analyzing seven potential EMT-regulating signaling nodes. More generally, this work provides an approach for studying highly multivariate signaling/phenotype relationships based on protein measurements in any setting.

AbstractOvarian cancer remains a major health threat with limited treatment options available. It is characterized by immunosuppressive tumor microenvironment (TME) maintained by tumor- associated macrophages (TAMs) hindering anti-tumor responses and immunotherapy efficacy. Here we show that targeting retinoblastoma protein (Rb) by disruption of its LxCxE cleft pocket, causes cell death in TAMs by induction of ER stress, p53 and mitochondria-related cell death pathways. A reduction of pro-tumor Rbhigh M2-type macrophages from TME in vivo enhanced T cell infiltration and inhibited cancer progression. We demonstrate an increased Rb expression in TAMs in women with ovarian cancer is associated with poorer prognosis. Ex vivo, we show analogous cell death induction by therapeutic Rb targeting in TAMs in post-surgery ascites from ovarian cancer patients. Overall, our data elucidates therapeutic targeting of the Rb LxCxE cleft pocket as a novel promising approach for ovarian cancer treatment through depletion of TAMs and re-shaping TME immune landscape. Statement of significanceCurrently, targeting immunosuppressive myeloid cells in ovarian cancer microenvironment is the first priority need to enable successful immunotherapy, but no effective solutions are clinically available. We show that targeting LxCxE cleft pocket of Retinoblastoma protein unexpectedly induces preferential cell death in M2 tumor-associated macrophages. Depletion of immunosuppressive M2 tumor-associated macrophages reshapes tumor microenvironment, enhances anti-tumor T cell responses, and inhibits ovarian cancer. Thus, we identify a novel paradoxical function of Retinoblastoma protein in regulating macrophage viability as well as a promising target to enhance immunotherapy efficacy in ovarian cancer.

Natural killer (NK) cells are cytotoxic lymphocytes of the innate immune system that eliminate virus-infected and transformed cells through the formation of a specialized immune synapse. Effective target cell killing requires coordinated plasma membrane remodeling and dynamic reorganization of the actin and microtubule cytoskeletons, enabling centrosome polarization and directed secretion of lytic granules. The scaffold protein CIP4 has emerged as an important regulator of cytoskeletal coordination in NK cells, yet how its subcellular localization is controlled during NK cell activation is unknown. CIP4 contains a unique protein kinase A (PKA) phosphorylation site (threonine 225, T225) within its F-BAR domain, a domain that mediates interactions with microtubules and the plasma membrane. We hypothesized that localized PKA signaling controls CIP4 redistribution during immune synapse assembly. To test this hypothesis, we analyzed CIP4 localization and phosphorylation in NK cells engaged with sensitive target cells using biochemical and imaging approaches. We show that NK-target cell interaction enhances PKA activity and promotes phosphorylation of CIP4, coinciding with its delocalization from microtubules and accumulation at the immune synapse. Importantly, this relocalization process requires the PKA-anchoring protein AKAP350, which positions PKA and CIP4 within the same protein complex, thereby facilitating CIP4 phosphorylation. Consistently, pharmacological inhibition of PKA prevented CIP4 delocalization from microtubules and reduced its accumulation at the immune synapse. The non-phosphorylatable CIP4 mutant T225A displayed increased association with microtubules compared with a phosphomimetic mutant, identifying phosphorylation at T225 as a key determinant of CIP4 spatial regulation. Together, these findings identify a signaling mechanism that links compartmentalized PKA activity to the spatial control of CIP4 during immune synapse formation, providing new insight into the molecular mechanisms governing immune synapse maturation.

G-protein-coupled receptors (GPCRs), the largest family of cell surface receptors, signal through the proximal effectors G proteins and {beta}-arrestins to influence nearly every biological process. Classically, the G protein and {beta}-arrestin signaling pathways have largely been considered separable. Recently, direct interactions between G protein and {beta}-arrestin have been described and suggest a distinct GPCR signaling pathway. Within these newly described G:{beta}-arrestin complexes, Gi/o, but not other G protein subtypes, have been appreciated to directly interact with {beta}-arrestin, regardless of canonical GPCR G protein subtype coupling. However it is unclear how biased agonists differentially regulate this newly described Gi:{beta}-arrestin interaction, if at all. Here we report that endogenous ligands (chemokines) of the GPCR CXCR3, CXCL9, CXCL10, and CXCL11, along with two small molecule biased CXCR3 agonists, differentially promote the formation of Gi:{beta}-arrestin complexes. The ability of CXCR3 agonists to form Gi:{beta}-arrestin complexes does not correlate well with either G protein signaling or {beta}-arrestin recruitment. Conformational biosensors demonstrate that ligands that promoted Gi:{beta}-arrestin complex formation generated similar {beta}-arrestin conformations. We find these Gi:{beta}-arrestin complexes can associate with CXCR3, but not with ERK. These findings further support that Gi:{beta}-arrestin complex formation is a distinct GPCR signaling pathway and enhance our understanding of biased agonism.

Cells integrate multiple, often concurrent signals through intertwined genetic circuits whose dynamics shape transcriptional programs and cell fate decisions. Among these, the tumor suppressor p53 and the inflammatory transcription factor NF-{kappa}B are central regulators of stress responses in normal and cancer cells, yet their dynamic crosstalk under co-activation remains poorly characterized. Here, we combine genetic approaches, live cell imaging, transcriptomic analysis and mathematical modeling to dissect their dynamic interplay. We find that co-activation of NF-{kappa}B by the inflammatory cytokines TNF- and IL-1{beta} significantly enhance p53 nuclear accumulation upon genotoxic stress or Nutlin3a, and this effect is absent in NF-{kappa}B-deficient cells. Mechanistically, we show that cytokines induce an NF-{kappa}B-mediated increase of TP53 transcription, and mathematical modeling indicates that it is sufficient to account for the observed increased p53 accumulation. Functionally, NF-{kappa}B co-activation rewires p53-dependent transcriptional programs and impairs p53-mediated DNA repair following genotoxic stress, due to a shift of p53 dynamics from oscillatory to more sustained accumulation; p53 oscillatory dynamics and DNA repair remain largely unaltered in absence of NF-{kappa}B. Our results uncover an amplification of p53 response in presence of inflammatory cues that is transcriptionally mediated by NF-{kappa}B and that results, counterintuitively, in functional antagonism. SIGNIFICANCE STATEMENTp53 dynamics have been shown to correlate with the cellular responses of cancer cells to genotoxic insults such as those delivered by chemo- and radiotherapies. However, these dynamics have been mostly studied in settings that do not account for the pro-inflammatory cues that cancer cells might receive from the microenvironment. By addressing this gap, our study uncovers a previously unappreciated mechanism by which inflammatory signals induce an increased p53 accumulation, leading to reduced DNA repair capacity. Our results provide mechanistic understanding on the origin of p53-NF-{kappa}B antagonism and show how dynamically interconnected signalling pathways can produce counterintuitive functional outcomes. Translationally, the contribution of inflammation to the sensitivity of normal and cancer cells to DNA damage might be exploited in the management of chemo- and radiotherapies.

Cytokines dimerize/oligomerize surface receptors to activate signaling. While cytokine receptors preferentially bind only one member of the JAK family, ancestral cytokine receptors, such as Gp130, promiscuously recruit different JAKs to elicit their activities. Here, we have explored how the identity of JAKs in Gp130 signaling complexes can regulate functional outcomes. Using a synthetic biology approach, we show that Gp130 bound to different JAKs propagates distinct STAT activation profiles. While Gp130-JAK1 complexes activated both, STAT1 and STAT3 very potently, Gp130-JAK2 complexes exhibited a clear preference for STAT3 activation. Gp130-TYK2 complexes triggered overall weaker signaling but with diminished STAT specificity. The three JAKs competed for binding to Gp130 and led to differential activation of phospho-Tyr in the Gp130 intracellular domain. JAK1, JAK2 and to a lower extent TYK2 bound with comparable affinities to Gp130, and in response to IL-6 stimulation efficiently drove Gp130 dimerization. However, the three JAKs differentially affected Gp130 surface expression, identifying JAK-dependent receptor trafficking as a critical determinant of signaling plasticity. Our results provide new mechanistic insights into how differential functional coupling in Gp130-JAK complexes translates into unique signaling signatures that likely contribute to its large functional diversity.

The cGAS-STING pathway is an evolutionarily conserved antimicrobial defense mechanism that senses cytosolic DNA to trigger innate immune responses. cGAS and STING play dual roles in tumorigenesis, promoting antitumor immunity and cell death while fueling tumor growth and metastasis. However, the mechanisms fine-tuning this pathway remain elusive. Using proteomic approaches, we report that Casein Kinase 1 alpha (CK1) operates as a bimodal regulator of the cGAS-STING pathway. CK1 supports optimal DNA sensing by preventing the proteasomal degradation of cGAS driven by the cullin-RING ubiquitin ligase 3 (CRL3). Conversely, CK1 facilitates STING degradation and signaling termination in response to STING agonists, tempering IRF3 activation. Exploiting these counterposing functions, we show that selective degradation of CK1 with molecular-glue degraders impaired the survival of a triple-negative breast cancer cell line with chronic cGAS-STING activation and synergized with a STING agonist to kill acute myeloid leukemia cells. Thus, CK1s dual regulatory role in the cGAS-STING pathway presents a promising target for therapeutic development. TEASERThis study unveils CK1 as a bimodal regulator of the cGAS-STING pathway.