Control of Glucocorticoid Receptor Levels by PTEN Establishes a Failsafe Mechanism for Tumor Suppression.

The PTEN tumor suppressor controls cell death and survival by regulating functions of various molecular targets. While the role of PTEN lipid-phosphatase activity on PtdIns(3,4,5)P3 and inhibition of PI3K pathway is well characterized, the biological relevance of PTEN protein-phosphatase activity remains undefined. Here, using knockin (KI) mice harboring cancer-associated and functionally relevant missense mutations, we show that although loss of PTEN lipid-phosphatase function cooperates with oncogenic PI3K to promote rapid mammary tumorigenesis, the additional loss of PTEN protein-phosphatase activity triggered an extensive cell death response evident in early and advanced mammary tumors. Omics and drug-targeting studies revealed that PI3Ks act to reduce glucocorticoid receptor (GR) levels, which are rescued by loss of PTEN protein-phosphatase activity to restrain cell survival. Thus, we find that the dual regulation of GR by PI3K and PTEN functions as a rheostat that can be exploited for the treatment of PTEN loss-driven cancers.

Structural insights into the multifunctionality of rabies virus P3 protein.

Viruses form extensive interfaces with host proteins to modulate the biology of the infected cell, frequently via multifunctional viral proteins. These proteins are conventionally considered as assemblies of independent functional modules, where the presence or absence of modules determines the overall composite phenotype. However, this model cannot account for functions observed in specific viral proteins. For example, rabies virus (RABV) P3 protein is a truncated form of the pathogenicity factor P protein, but displays a unique phenotype with functions not seen in longer isoforms, indicating that changes beyond the simple complement of functional modules define the functions of P3. Here, we report structural and cellular analyses of P3 derived from the pathogenic RABV strain Nishigahara (Nish) and an attenuated derivative strain (Ni-CE). We identify a network of intraprotomer interactions involving the globular C-terminal domain and intrinsically disordered regions (IDRs) of the N-terminal region that characterize the fully functional Nish P3 to fluctuate between open and closed states, whereas the defective Ni-CE P3 is predominantly open. This conformational difference appears to be due to the single mutation N226H in Ni-CE P3. We find that Nish P3, but not Ni-CE or N226H P3, undergoes liquid-liquid phase separation and this property correlates with the capacity of P3 to interact with different cellular membrane-less organelles, including those associated with immune evasion and pathogenesis. Our analyses propose that discrete functions of a critical multifunctional viral protein depend on the conformational arrangements of distant individual domains and IDRs, in addition to their independent functions.

Human Tim8a, Tim8b and Tim13 are auxiliary assembly factors of mature Complex IV.

Human Tim8a and Tim8b are paralogous intermembrane space proteins of the small TIM chaperone family. Yeast small TIMs function in the trafficking of proteins to the outer and inner mitochondrial membranes. This putative import function for hTim8a and hTim8b has been challenged in human models, but their precise molecular function(s) remains undefined. Likewise, the necessity for human cells to encode two Tim8 proteins and whether any potential redundancy exists is unclear. We demonstrate that hTim8a and hTim8b function in the assembly of cytochrome c oxidase (Complex IV). Using affinity enrichment mass spectrometry, we define the interaction network of hTim8a, hTim8b and hTim13, identifying subunits and assembly factors of the Complex IV COX2 module. hTim8-deficient cells have a COX2 and COX3 module defect and exhibit an accumulation of the Complex IV S2 subcomplex. These data suggest that hTim8a and hTim8b function in assembly of Complex IV via interactions with intermediate-assembly subcomplexes. We propose that hTim8-hTim13 complexes are auxiliary assembly factors involved in the formation of the Complex IV S3 subcomplex during assembly of mature Complex IV.

Novel CDKL5 targets identified in human iPSC-derived neurons.

CDKL5 Deficiency Disorder (CDD) is a debilitating epileptic encephalopathy disorder affecting young children with no effective treatments. CDD is caused by pathogenic variants in Cyclin-Dependent Kinase-Like 5 (CDKL5), a protein kinase that regulates key phosphorylation events in neurons. For therapeutic intervention, it is essential to understand molecular pathways and phosphorylation targets of CDKL5. Using an unbiased phosphoproteomic approach we identified novel targets of CDKL5, including GTF2I, PPP1R35, GATAD2A and ZNF219 in human iPSC-derived neuronal cells. The phosphoserine residue in the target proteins lies in the CDKL5 consensus motif. We validated direct phosphorylation of GTF2I and PPP1R35 by CDKL5 using complementary approaches. GTF2I controls axon guidance, cell cycle and neurodevelopment by regulating expression of neuronal genes. PPP1R35 is critical for centriole elongation and cilia morphology, processes that are impaired in CDD. PPP1R35 interacts with CEP131, a known CDKL5 phospho-target. GATAD2A and ZNF219 belong to the Nucleosome Remodelling Deacetylase (NuRD) complex, which regulates neuronal activity-dependent genes and synaptic connectivity. In-depth knowledge of molecular pathways regulated by CDKL5 will allow a better understanding of druggable disease pathways to fast-track therapeutic development.

PI3Kα Translocation Mediates Nuclear PtdIns(3,4,5)P3 Effector Signaling in Colorectal Cancer.

The canonical view of PI3Kα signaling describes phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3) generation and activation of downstream effectors at the plasma membrane or at microtubule-bound endosomes. Here, we show that colorectal cancer (CRC) cell lines exhibit a diverse plasma membrane-nuclear distribution of PI3Kα, controlling corresponding levels of subcellular PtdIns(3,4,5)P3 pools. PI3Kα nuclear translocation was mediated by the importin ß-dependent nuclear import pathway. By PtdIns(3,4,5)P3 affinity capture mass spectrometry done in the presence of SDS on CRC cell lines with PI3Kα nuclear localization, we identified 867 potential nuclear PtdIns(3,4,5)P3 effector proteins. Nuclear PtdIns(3,4,5)P3 interactome proteins were characterized by noncanonical PtdIns(3,4,5)P3-binding domains and showed overrepresentation for nuclear membrane, nucleolus, and nuclear speckles. The nuclear PtdIns(3,4,5)P3 interactome was enriched for proteins related to RNA metabolism, with splicing reporter assays and SC-35 foci staining suggesting a role of epidermal growth factor-stimulated nuclear PI3Kα signaling in modulating pre-mRNA splicing. In patient tumors, nuclear p110α staining was associated with lower T stage and mucinous histology. These results indicate that PI3Kα translocation mediates nuclear PtdIns(3,4,5)P3 effector signaling in human CRC, modulating signaling responses.

N-Terminomic Changes in Neurons During Excitotoxicity Reveal Proteolytic Events Associated With Synaptic Dysfunctions and Potential Targets for Neuroprotection.

Excitotoxicity, a neuronal death process in neurological disorders such as stroke, is initiated by the overstimulation of ionotropic glutamate receptors. Although dysregulation of proteolytic signaling networks is critical for excitotoxicity, the identity of affected proteins and mechanisms by which they induce neuronal cell death remain unclear. To address this, we used quantitative N-terminomics to identify proteins modified by proteolysis in neurons undergoing excitotoxic cell death. We found that most proteolytically processed proteins in excitotoxic neurons are likely substrates of calpains, including key synaptic regulatory proteins such as CRMP2, doublecortin-like kinase I, Src tyrosine kinase and calmodulin-dependent protein kinase IIß (CaMKIIß). Critically, calpain-catalyzed proteolytic processing of these proteins generates stable truncated fragments with altered activities that potentially contribute to neuronal death by perturbing synaptic organization and function. Blocking calpain-mediated proteolysis of one of these proteins, Src, protected against neuronal loss in a rat model of neurotoxicity. Extrapolation of our N-terminomic results led to the discovery that CaMKIIα, an isoform of CaMKIIß, undergoes differential processing in mouse brains under physiological conditions and during ischemic stroke. In summary, by identifying the neuronal proteins undergoing proteolysis during excitotoxicity, our findings offer new insights into excitotoxic neuronal death mechanisms and reveal potential neuroprotective targets for neurological disorders.

Inhibition of P21-activated kinases 1 and 4 synergistically suppresses the growth of pancreatic cancer by stimulating anti-tumour immunity.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal types of cancer, and KRAS oncogene occurs in over 90% of cases. P21-activated kinases (PAK), containing six members (PAK1 to 6), function downstream of KRAS. PAK1 and PAK4 play important roles in carcinogenesis, but their combinational effect remains unknown. In this study, we have determined the effect of dual inhibition of PAK1 and PAK4 in PDA progression using knockout (KO) cancer cell lines. METHODS: Murine wild-type (WT) and PAK1KO pancreatic cancer cell lines were isolated from PAK1+/+ and PAK1-/- KPC (LSL-KrasG12D/+; LSL-Trp53 R172H/+; Pdx-1-Cre) mice. KPC PAK4KO and KPC PAK1&4 KO cell lines were generated from KPC WT and KPC PAK1KO cell lines respectively using the CRISPR-CAS9 gene knockout technique. PAK WT and KO cell lines were used in mouse models of pancreatic tumours. Cells and tumour tissue were also used in flow cytometry and proteomic studies. A human PDA tissue microarray was stained by immunohistochemistry. RESULTS: Double knock out of PAK1 and PAK4 caused complete regression of tumour in a syngeneic mouse model. PAK4KO inhibited tumour growth by stimulating a rapid increase of cytotoxic CD8+ T cell infiltration. PAK1KO synergistically with PAK4KO increased cytotoxic CD8+ T cell infiltration and stimulated a sustained infiltration of CD8+ T cells at a later phase to overcome the immune evasion in the PAK4KO tumour. The human PDA tissue microarray study showed the important role of PAK1 and PAK4 in intra-tumoral T-cell function. CONCLUSION: Our results demonstrated that dual inhibition of PAK1 and PAK4 synergistically suppressed PDA progression by stimulating cytotoxic CD8 + T cell response.

Structure activity relationship and target prediction for ABX464 analogues in Caenorhabditis elegans.

Global challenges with treatment failures and/or widespread resistance in parasitic worms against commercially available anthelmintics lend impetus to the development of new anthelmintics with novel mechanism(s) of action. The free-living nematode Caenorhabditis elegans is an important model organism used for drug discovery, including the screening and structure-activity investigation of new compounds, and target deconvolution. Previously, we conducted a whole-organism phenotypic screen of the 'Pandemic Response Box' (from Medicines for Malaria Venture, MMV) and identified a hit compound, called ABX464, with activity against C. elegans and a related, parasitic nematode, Haemonchus contortus. Here, we tested a series of 44 synthesized analogues to explore the pharmacophore of activity on C. elegans and revealed five compounds whose potency was similar or greater than that of ABX464, but which were not toxic to human hepatoma (HepG2) cells. Subsequently, we employed thermal proteome profiling (TPP), protein structure prediction and an in silico-docking algorithm to predict ABX464-target candidates. Taken together, the findings from this study contribute significantly to the early-stage drug discovery of a new nematocide based on ABX464. Future work is aimed at validating the ABX464-protein interactions identified here, and at assessing ABX464 and associated analogues against a panel of parasitic nematodes, towards developing a new anthelmintic with a mechanism of action that is distinct from any of the compounds currently-available commercially.

Bovine milk-derived extracellular vesicles enhance doxorubicin sensitivity in triple negative breast cancer cells by targeting metabolism and STAT signalling.

Metastatic triple-negative breast cancer (TNBC) has a low 5-year survival rate of below 30% with systemic chemotherapy being the most widely used treatment. Bovine milk-derived extracellular vesicles (MEVs) have been previously demonstrated to have anti-cancer attributes. In this study, we isolated bovine MEVs from commercial milk and characterised them according to MISEV guidelines. Bovine MEVs sensitised TNBC cells to doxorubicin, resulting in reduced metabolic potential and cell-viability. Label-free quantitative proteomics of cells treated with MEVs and/or doxorubicin suggested that combinatorial treatment depleted various pro-tumorigenic interferon-inducible gene products and proteins with metabolic function, previously identified as therapeutic targets in TNBC. Combinatorial treatment also led to reduced abundance of various STAT proteins and their downstream oncogenic targets with roles in cell-cycle and apoptosis. Taken together, this study highlights the ability of bovine MEVs to sensitise TNBC cells to standard-of-care therapeutic drug doxorubicin, paving the way for novel treatment regimens.

Proteomics analysis of C2C12 myotubes treated with atrophy inducing cancer cell-derived factors.

Cancer-associated cachexia is a wasting syndrome that results in dramatic loss of whole-body weight, predominantly due to loss of skeletal muscle mass. It has been established that cachexia inducing cancer cells secrete proteins and extracellular vesicles (EVs) that can induce muscle atrophy. Though several studies examined these cancer-cell derived factors, targeting some of these components have shown little or no clinical benefit. To develop new therapies, understanding of the dysregulated proteins and signaling pathways that regulate catabolic gene expression during muscle wasting is essential. Here, we sought to examine the effect of conditioned media (CM) that contain secreted factors and EVs from cachexia inducing C26 colon cancer cells on C2C12 myotubes using mass spectrometry-based label-free quantitative proteomics. We identified significant changes in the protein profile of C2C12 cells upon exposure to C26-derived CM. Functional enrichment analysis revealed enrichment of proteins associated with inflammation, mitochondrial dysfunction, muscle catabolism, ROS production, and ER stress in CM treated myotubes. Furthermore, strong downregulation in muscle structural integrity and development and/or regenerative pathways were observed. Together, these enriched proteins in atrophied muscle could be utilized as potential muscle wasting markers and the dysregulated biological processes could be employed for therapeutic benefit in cancer-induced muscle wasting.

Proteomic analysis of the small extracellular vesicles and soluble secretory proteins from cachexia inducing and non-inducing cancer cells.

Cancer cachexia is a wasting syndrome characterised by the loss of fat and/or muscle mass in advanced cancer patients. It has been well-established that cancer cells themselves can induce cachexia via the release of several pro-cachectic and pro-inflammatory factors. However, it is unclear how this process is regulated and the key cachexins that are involved. In this study, we validated C26 and EL4 as cachexic and non-cachexic cell models, respectively. Treatment of adipocytes and myotubes with C26 conditioned medium induced lipolysis and atrophy, respectively. We profiled soluble secreted proteins (secretome) as well as small extracellular vesicles (sEVs) released from cachexia-inducing (C26) and non-inducing (EL4) cancer cells by label-free quantitative proteomics. A total of 1268 and 1022 proteins were identified in the secretome of C26 and EL4, respectively. Furthermore, proteomic analysis of sEVs derived from C26 and EL4 cancer cells revealed a distinct difference in the protein cargo. Functional enrichment analysis using FunRich highlighted the enrichment of proteins that are implicated in biological processes such as muscle atrophy, lipolysis, and inflammation in both the secretome and sEVs derived from C26 cancer cells. Overall, our characterisation of the proteomic profiles of the secretory factors and sEVs from cachexia-inducing and non-inducing cancer cells provides insights into tumour factors that promote weight loss by mediating protein and lipid loss in various organs and tissues. Further investigation of these proteins may assist in highlighting potential therapeutic targets and biomarkers of cancer cachexia.

The Proteome and Lipidome of Extracellular Vesicles from Haemonchus contortus to Underpin Explorations of Host-Parasite Cross-Talk.

Many parasitic worms have a major adverse impact on human and animal populations worldwide due to the chronicity of their infections. There is a growing body of evidence indicating that extracellular vesicles (EVs) are intimately involved in modulating (suppressing) inflammatory/immune host responses and parasitism. As one of the most pathogenic nematodes of livestock animals, Haemonchus contortus is an ideal model system for EV exploration. Here, employing a multi-step enrichment process (in vitro culture, followed by ultracentrifugation, size exclusion and filtration), we enriched EVs from H. contortus and undertook the first comprehensive (qualitative and quantitative) multi-omic investigation of EV proteins and lipids using advanced liquid chromatography-mass spectrometry and informatics methods. We identified and quantified 561 proteins and 446 lipids in EVs and compared these molecules with those of adult worms. We identified unique molecules in EVs, such as proteins linked to lipid transportation and lipid species (i.e., sphingolipids) associated with signalling, indicating the involvement of these molecules in parasite-host cross-talk. This work provides a solid starting point to explore the functional roles of EV-specific proteins and lipids in modulating parasite-host cross-talk, and the prospect of finding ways of disrupting or interrupting this relationship to suppress or eliminate parasite infection.

Pep2Graph: A standalone tool to analyse proteolytic cleavages by proteases from gel-based mass spectrometry data.

Proteases are enzymes that regulate substrates via proteolytic activation and coordinate essential cellular functions including DNA replication, DNA transcription, cell proliferation, differentiation, migration and apoptosis. However, techniques to identify proteolytic events in a high-throughput manner is limited. PROtein TOpography and Migration Analysis Platform (PROTOMAP) is a technique that relies on mass spectrometry-based proteomics to globally identify the shifts in the in-gel migration of proteins and their corresponding fragments that are obtained by proteolysis. However, user-friendly software tool to analyse the proteomic data to identify proteolytic events is needed. Here, we report Pep2Graph, a user-friendly standalone tool that integrates peptide sequence information from in-gel proteomics and presents the data as two-dimensional peptographs with in-gel migration, sequence coverage and MS/MS spectra counts. Pep2Graph (http://www.mathivananlab.org/Pep2Graph) allows users to utilize in-gel proteomics data to study proteolytic events that may play a significant role in normal physiology and pathology.

Phosphomatics: interactive interrogation of substrate-kinase networks in global phosphoproteomics datasets.

MOTIVATION: Mass spectrometry-based phosphoproteomics can routinely identify and quantify thousands of phosphorylated peptides from a single experiment. However interrogating possible upstream kinases and identifying key literature for phosphorylation sites is laborious and time-consuming. RESULTS: Here, we present Phosphomatics-a publicly available web resource for interrogating phosphoproteomics data. Phosphomatics allows researchers to upload phosphoproteomics data and interrogate possible relationships from a substrate-, kinase- or pathway-centric viewpoint. AVAILABILITY AND IMPLEMENTATION: Phosphomatics is freely available via the internet at: https://phosphomatics.com. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Arginine in C9ORF72 Dipolypeptides Mediates Promiscuous Proteome Binding and Multiple Modes of Toxicity.

C9ORF72-associated Motor Neuron Disease patients feature abnormal expression of 5 dipeptide repeat (DPR) polymers. Here we used quantitative proteomics in a mouse neuronal-like cell line (Neuro2a) to demonstrate that the Arg residues in the most toxic DPRS, PR and GR, leads to a promiscuous binding to the proteome compared with a relative sparse binding of the more inert AP and GA. Notable targets included ribosomal proteins, translation initiation factors and translation elongation factors. PR and GR comprising more than 10 repeats appeared to robustly stall on ribosomes during translation suggesting Arg-rich peptide domains can electrostatically jam the ribosome exit tunnel during synthesis. Poly-GR also recruited arginine methylases, induced hypomethylation of endogenous proteins, and induced a profound destabilization of the actin cytoskeleton. Our findings point to arginine in GR and PR polymers as multivalent toxins to translation as well as arginine methylation that may explain the dysfunction of biological processes including ribosome biogenesis, mRNA splicing and cytoskeleton assembly.

HIGD2A is Required for Assembly of the COX3 Module of Human Mitochondrial Complex IV.

Assembly factors play a critical role in the biogenesis of mitochondrial respiratory chain complexes I-IV where they assist in the membrane insertion of subunits, attachment of co-factors, and stabilization of assembly intermediates. The major fraction of complexes I, III and IV are present together in large molecular structures known as respiratory chain supercomplexes. Several assembly factors have been proposed as required for supercomplex assembly, including the hypoxia inducible gene 1 domain family member HIGD2A. Using gene-edited human cell lines and extensive steady state, translation and affinity enrichment proteomics techniques we show that loss of HIGD2A leads to defects in the de novo biogenesis of mtDNA-encoded COX3, subsequent accumulation of complex IV intermediates and turnover of COX3 partner proteins. Deletion of HIGD2A also leads to defective complex IV activity. The impact of HIGD2A loss on complex IV was not altered by growth under hypoxic conditions, consistent with its role being in basal complex IV assembly. Although in the absence of HIGD2A we show that mitochondria do contain an altered supercomplex assembly, we demonstrate it to harbor a crippled complex IV lacking COX3. Our results redefine HIGD2A as a classical assembly factor required for building the COX3 module of complex IV.

Pannexin-1 channel regulates nuclear content packaging into apoptotic bodies and their size.

Apoptotic bodies (ApoBDs), which are large extracellular vesicles exclusively released by apoptotic cells, possess therapeutically exploitable properties including biomolecule loadability and transferability. However, current limited understanding of ApoBD biology has hindered its exploration for clinical use. Particularly, as ApoBD-accompanying cargoes (e.g., nucleic acids and proteins) have major influence on their functionality, further insights into the mechanism of biomolecule sorting into ApoBDs are critical to unleash their therapeutic potential. Previous studies suggested pannexin 1 (PANX1) channel, a negative regulator of ApoBD biogenesis, can modify synaptic vesicle contents. We also reported that trovafloxacin (a PANX1 inhibitor) increases proportion of ApoBDs containing DNA. Therefore, we sought to define the role of PANX1 in regulating the sorting of nuclear content into ApoBDs. Here, using flow cytometry and label-free quantitative proteomic analyses, we showed that targeting PANX1 activity during apoptosis, via either pharmacological inhibition or genetic disruption, resulted in enrichment of both DNA and nuclear proteins in ApoBDs that were unexpectedly smaller in size. Our data suggest that PANX1, besides being a key regulator of ApoBD formation, also functions as a negative regulator of nuclear content packaging and modulator of ApoBD size. Together, our findings provide further insights into ApoBD biology and form a novel conceptual framework for ApoBD-based therapies through pharmacologically manipulating ApoBD contents.

What Are We Missing by Using Hydrophilic Enrichment? Improving Bacterial Glycoproteome Coverage Using Total Proteome and FAIMS Analyses.

Hydrophilic interaction liquid chromatography (HILIC) glycopeptide enrichment is an indispensable tool for the high-throughput characterization of glycoproteomes. Despite its utility, HILIC enrichment is associated with a number of shortcomings, including requiring large amounts of starting materials, potentially introducing chemical artifacts such as formylation when high concentrations of formic acid are used, and biasing/undersampling specific classes of glycopeptides. Here, we investigate HILIC enrichment-independent approaches for the study of bacterial glycoproteomes. Using three Burkholderia species (Burkholderia cenocepacia, Burkholderia Dolosa, and Burkholderia ubonensis), we demonstrate that short aliphatic O-linked glycopeptides are typically absent from HILIC enrichments, yet are readily identified in whole proteome samples. Using high-field asymmetric waveform ion mobility spectrometry (FAIMS) fractionation, we show that at high compensation voltages (CVs), short aliphatic glycopeptides can be enriched from complex samples, providing an alternative means to identify glycopeptide recalcitrant to hydrophilic-based enrichment. Combining whole proteome and FAIMS analyses, we show that the observable glycoproteome of these Burkholderia species is at least 25% larger than what was initially thought. Excitingly, the ability to enrich glycopeptides using FAIMS appears generally applicable, with the N-linked glycopeptides of Campylobacter fetus subsp. fetus also being enrichable at high FAIMS CVs. Taken together, these results demonstrate that FAIMS provides an alternative means to access glycopeptides and is a valuable tool for glycoproteomic analysis.

Dafachronic acid promotes larval development in Haemonchus contortus by modulating dauer signalling and lipid metabolism.

Here, we discovered an endogenous dafachronic acid (DA) in the socioeconomically important parasitic nematode Haemonchus contortus. We demonstrate that DA promotes larval exsheathment and development in this nematode via a relatively conserved nuclear hormone receptor (DAF-12). This stimulatory effect is dose- and time-dependent, and relates to a modulation of dauer-like signalling, and glycerolipid and glycerophospholipid metabolism, likely via a negative feedback loop. Specific chemical inhibition of DAF-9 (cytochrome P450) was shown to significantly reduce the amount of endogenous DA in H. contortus; compromise both larval exsheathment and development in vitro; and modulate lipid metabolism. Taken together, this evidence shows that DA plays a key functional role in the developmental transition from the free-living to the parasitic stage of H. contortus by modulating the dauer-like signalling pathway and lipid metabolism. Understanding the intricacies of the DA-DAF-12 system and associated networks in H. contortus and related parasitic nematodes could pave the way to new, nematode-specific treatments.

Oestrogen Activates the MAP3K1 Cascade and ß-Catenin to Promote Granulosa-like Cell Fate in a Human Testis-Derived Cell Line.

Sex determination triggers the differentiation of the bi-potential gonad into either an ovary or testis. In non-mammalian vertebrates, the presence or absence of oestrogen dictates gonad differentiation, while in mammals, this mechanism has been supplanted by the testis-determining gene SRY. Exogenous oestrogen can override this genetic trigger to shift somatic cell fate in the gonad towards ovarian developmental pathways by limiting the bioavailability of the key testis factor SOX9 within somatic cells. Our previous work has implicated the MAPK pathway in mediating the rapid cellular response to oestrogen. We performed proteomic and phosphoproteomic analyses to investigate the precise mechanism through which oestrogen impacts these pathways to activate ß-catenin-a factor essential for ovarian development. We show that oestrogen can activate ß-catenin within 30 min, concomitant with the cytoplasmic retention of SOX9. This occurs through changes to the MAP3K1 cascade, suggesting this pathway is a mechanism through which oestrogen influences gonad somatic cell fate. We demonstrate that oestrogen can promote the shift from SOX9 pro-testis activity to ß-catenin pro-ovary activity through activation of MAP3K1. Our findings define a previously unknown mechanism through which oestrogen can promote a switch in gonad somatic cell fate and provided novel insights into the impacts of exogenous oestrogen exposure on the testis.