ABSTRACT
In developing brains, axons exhibit remarkable precision in selecting synaptic partners among many non-partner cells. Evolutionarily conserved teneurins are transmembrane proteins that instruct synaptic partner matching. However, how intracellular signaling pathways execute teneurins' functions is unclear. Here, we use in situ proximity labeling to obtain the intracellular interactome of a teneurin (Ten-m) in the Drosophila brain. Genetic interaction studies using quantitative partner matching assays in both olfactory receptor neurons (ORNs) and projection neurons (PNs) reveal a common pathway: Ten-m binds to and negatively regulates a RhoGAP, thus activating the Rac1 small GTPases to promote synaptic partner matching. Developmental analyses with single-axon resolution identify the cellular mechanism of synaptic partner matching: Ten-m signaling promotes local F-actin levels and stabilizes ORN axon branches that contact partner PN dendrites. Combining spatial proteomics and high-resolution phenotypic analyses, this study advanced our understanding of both cellular and molecular mechanisms of synaptic partner matching.
ABSTRACT
Molecular interactions at the cellular interface mediate organized assembly of single cells into tissues and, thus, govern the development and physiology of multicellular organisms. Here, we developed a cell-type-specific, spatiotemporally resolved approach to profile cell-surface proteomes in intact tissues. Quantitative profiling of cell-surface proteomes of Drosophila olfactory projection neurons (PNs) in pupae and adults revealed global downregulation of wiring molecules and upregulation of synaptic molecules in the transition from developing to mature PNs. A proteome-instructed in vivo screen identified 20 cell-surface molecules regulating neural circuit assembly, many of which belong to evolutionarily conserved protein families not previously linked to neural development. Genetic analysis further revealed that the lipoprotein receptor LRP1 cell-autonomously controls PN dendrite targeting, contributing to the formation of a precise olfactory map. These findings highlight the power of temporally resolved in situ cell-surface proteomic profiling in discovering regulators of brain wiring.
Subject(s)
Olfactory Pathways/metabolism , Olfactory Receptor Neurons/metabolism , Proteomics/methods , Animals , Axons/metabolism , Brain/metabolism , Dendrites/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , Gene Expression Profiling/methods , Gene Expression Regulation, Developmental/genetics , Membrane Proteins/metabolism , Neurogenesis/physiology , Olfactory Nerve/metabolism , Olfactory Pathways/cytology , Olfactory Pathways/physiology , Receptors, Lipoprotein/metabolism , Smell/physiologyABSTRACT
Cellular compartments that cannot be biochemically isolated are challenging to characterize. Here we demonstrate the proteomic characterization of the synaptic clefts that exist at both excitatory and inhibitory synapses. Normal brain function relies on the careful balance of these opposing neural connections, and understanding how this balance is achieved relies on knowledge of their protein compositions. Using a spatially restricted enzymatic tagging strategy, we mapped the proteomes of two of the most common excitatory and inhibitory synaptic clefts in living neurons. These proteomes reveal dozens of synaptic candidates and assign numerous known synaptic proteins to a specific cleft type. The molecular differentiation of each cleft allowed us to identify Mdga2 as a potential specificity factor influencing Neuroligin-2's recruitment of presynaptic neurotransmitters at inhibitory synapses.
Subject(s)
Cell Adhesion Molecules, Neuronal/metabolism , GABAergic Neurons/metabolism , Immunoglobulins/metabolism , Membrane Glycoproteins/metabolism , Nerve Tissue Proteins/metabolism , Proteome/metabolism , Synaptic Membranes/metabolism , Animals , Antigens, CD/metabolism , Glutamic Acid/metabolism , HEK293 Cells , Humans , Mice , Neural Cell Adhesion Molecules/metabolism , Peroxidase/genetics , Peroxidase/metabolism , Proteomics , Rats , Receptors, GABA/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Thalamus/metabolismABSTRACT
While a mutation in C9ORF72 is the most common genetic contributor to amyotrophic lateral sclerosis (ALS), much remains to be learned concerning the function of the protein normally encoded at this locus. To elaborate further on functions for C9ORF72, we used quantitative mass spectrometry-based proteomics to identify interacting proteins in motor neurons and found that its long isoform complexes with and stabilizes SMCR8, which further enables interaction with WDR41. To study the organismal and cellular functions for this tripartite complex, we generated Smcr8 loss-of-function mutant mice and found that they developed phenotypes also observed in C9orf72 loss-of-function animals, including autoimmunity. Along with a loss of tolerance for many nervous system autoantigens, we found increased lysosomal exocytosis in Smcr8 mutant macrophages. In addition to elevated surface Lamp1 (lysosome-associated membrane protein 1) expression, we also observed enhanced secretion of lysosomal components-phenotypes that we subsequently observed in C9orf72 loss-of-function macrophages. Overall, our findings demonstrate that C9ORF72 and SMCR8 have interdependent functions in suppressing autoimmunity as well as negatively regulating lysosomal exocytosis-processes of potential importance to ALS.
Subject(s)
Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/physiopathology , Autoimmunity/genetics , Carrier Proteins/metabolism , Exocytosis/genetics , Lysosomes/metabolism , Animals , C9orf72 Protein/genetics , C9orf72 Protein/metabolism , Carrier Proteins/genetics , Gene Expression Regulation/genetics , Humans , Lymph Nodes/pathology , Lysosomal-Associated Membrane Protein 1/genetics , Macrophages/pathology , Mice , Mice, Knockout , Mutation , Protein Isoforms , Protein Stability , Splenomegaly/geneticsABSTRACT
Planar cell polarity (PCP) signaling polarizes epithelial cells within the plane of an epithelium. Core PCP signaling components adopt asymmetric subcellular localizations within cells to both polarize and coordinate polarity between cells. Achieving subcellular asymmetry requires additional effectors, including some mediating post-translational modifications of core components. Identification of such proteins is challenging due to pleiotropy. We used mass spectrometry-based proximity labeling proteomics to identify such regulators in the Drosophila wing. We identified the catalytic subunit of protein phosphatase1, Pp1-87B, and show that it regulates core protein polarization. Pp1-87B interacts with the core protein Van Gogh and at least one serine/threonine kinase, Dco/CKIε, that is known to regulate PCP. Pp1-87B modulates Van Gogh subcellular localization and directs its dephosphorylation in vivo. PNUTS, a Pp1 regulatory subunit, also modulates PCP. While the direct substrate(s) of Pp1-87B in control of PCP is not known, our data support the model that cycling between phosphorylated and unphosphorylated forms of one or more core PCP components may regulate acquisition of asymmetry. Finally, our screen serves as a resource for identifying additional regulators of PCP signaling.
Subject(s)
Drosophila Proteins , Membrane Proteins , Animals , Cell Polarity/physiology , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Membrane Proteins/metabolism , Protein Phosphatase 1/genetics , Protein Phosphatase 1/metabolism , Protein Processing, Post-Translational , Protein Serine-Threonine Kinases/metabolism , Signal TransductionABSTRACT
Casitas B-lineage lymphoma (CBL) encodes an E3 ubiquitin ligase and signaling adaptor that regulates receptor and nonreceptor tyrosine kinases. Recurrent CBL mutations occur in myeloid neoplasms, including 10% to 20% of chronic myelomonocytic leukemia (CMML) cases, and selectively disrupt the protein's E3 ubiquitin ligase activity. CBL mutations have been associated with poor prognosis, but the oncogenic mechanisms and therapeutic implications of CBL mutations remain incompletely understood. We combined functional assays and global mass spectrometry to define the phosphoproteome, CBL interactome, and mechanism of signaling activation in a panel of cell lines expressing an allelic series of CBL mutations. Our analyses revealed that increased LYN activation and interaction with mutant CBL are key drivers of enhanced CBL phosphorylation, phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) recruitment, and downstream phosphatidylinositol 3-kinase (PI3K)/AKT signaling in CBL-mutant cells. Signaling adaptor domains of CBL, including the tyrosine kinase-binding domain, proline-rich region, and C-terminal phosphotyrosine sites, were all required for the oncogenic function of CBL mutants. Genetic ablation or dasatinib-mediated inhibition of LYN reduced CBL phosphorylation, CBL-PIK3R1 interaction, and PI3K/AKT signaling. Furthermore, we demonstrated in vitro and in vivo antiproliferative efficacy of dasatinib in CBL-mutant cell lines and primary CMML. Overall, these mechanistic insights into the molecular function of CBL mutations provide rationale to explore the therapeutic potential of LYN inhibition in CBL-mutant myeloid malignancies.
Subject(s)
Class Ia Phosphatidylinositol 3-Kinase/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins c-cbl/genetics , src-Family Kinases/metabolism , Hematologic Neoplasms/genetics , Hematologic Neoplasms/metabolism , Humans , Mutation , Protein Interaction Maps , Proto-Oncogene Proteins c-cbl/metabolism , Signal TransductionABSTRACT
Proximity labeling catalyzed by promiscuous enzymes, such as TurboID, have enabled the proteomic analysis of subcellular regions difficult or impossible to access by conventional fractionation-based approaches. Yet some cellular regions, such as organelle contact sites, remain out of reach for current PL methods. To address this limitation, we split the enzyme TurboID into two inactive fragments that recombine when driven together by a protein-protein interaction or membrane-membrane apposition. At endoplasmic reticulum-mitochondria contact sites, reconstituted TurboID catalyzed spatially restricted biotinylation, enabling the enrichment and identification of >100 endogenous proteins, including many not previously linked to endoplasmic reticulum-mitochondria contacts. We validated eight candidates by biochemical fractionation and overexpression imaging. Overall, split-TurboID is a versatile tool for conditional and spatially specific proximity labeling in cells.
Subject(s)
Endoplasmic Reticulum/metabolism , Membrane Proteins/metabolism , Mitochondria/metabolism , Mitochondrial Membranes/metabolism , Proteome/analysis , Biotinylation , HEK293 Cells , Humans , Proteome/metabolism , Staining and LabelingABSTRACT
Obtaining complete protein inventories for subcellular regions is a challenge that often limits our understanding of cellular function, especially for regions that are impossible to purify and are therefore inaccessible to traditional proteomic analysis. We recently developed a method to map proteomes in living cells with an engineered peroxidase (APEX) that bypasses the need for organellar purification when applied to membrane-bound compartments; however, it was insufficiently specific when applied to unbounded regions that allow APEX-generated radicals to escape. Here, we combine APEX technology with a SILAC-based ratiometric tagging strategy to substantially reduce unwanted background and achieve nanometer spatial resolution. This is applied to map the proteome of the mitochondrial intermembrane space (IMS), which can freely exchange small molecules with the cytosol. Our IMS proteome of 127 proteins has >94% specificity and includes nine newly discovered mitochondrial proteins. This approach will enable scientists to map proteomes of cellular regions that were previously inaccessible.
Subject(s)
Mitochondrial Proteins/metabolism , Proteome/metabolism , Blotting, Western , Cell Fractionation , HEK293 Cells , Humans , Isotope Labeling , Mitochondrial Membranes/metabolismABSTRACT
Lenalidomide is a highly effective treatment for myelodysplastic syndrome (MDS) with deletion of chromosome 5q (del(5q)). Here, we demonstrate that lenalidomide induces the ubiquitination of casein kinase 1A1 (CK1α) by the E3 ubiquitin ligase CUL4-RBX1-DDB1-CRBN (known as CRL4(CRBN)), resulting in CK1α degradation. CK1α is encoded by a gene within the common deleted region for del(5q) MDS and haploinsufficient expression sensitizes cells to lenalidomide therapy, providing a mechanistic basis for the therapeutic window of lenalidomide in del(5q) MDS. We found that mouse cells are resistant to lenalidomide but that changing a single amino acid in mouse Crbn to the corresponding human residue enables lenalidomide-dependent degradation of CK1α. We further demonstrate that minor side chain modifications in thalidomide and a novel analogue, CC-122, can modulate the spectrum of substrates targeted by CRL4(CRBN). These findings have implications for the clinical activity of lenalidomide and related compounds, and demonstrate the therapeutic potential of novel modulators of E3 ubiquitin ligases.
Subject(s)
Casein Kinase I/metabolism , Myelodysplastic Syndromes/genetics , Myelodysplastic Syndromes/physiopathology , Thalidomide/analogs & derivatives , Ubiquitination/drug effects , Amino Acid Sequence , Animals , Casein Kinase I/genetics , Cell Line , Gene Expression Regulation/drug effects , HEK293 Cells , Humans , Immunologic Factors/pharmacology , Jurkat Cells , K562 Cells , Lenalidomide , Mice , Molecular Sequence Data , Peptide Hydrolases/chemistry , Proteolysis/drug effects , Sequence Alignment , Sequence Deletion , Species Specificity , Thalidomide/pharmacology , Ubiquitin-Protein Ligases/metabolismABSTRACT
Although purification of biotinylated molecules is highly efficient, identifying specific sites of biotinylation remains challenging. We show that anti-biotin antibodies enable unprecedented enrichment of biotinylated peptides from complex peptide mixtures. Live-cell proximity labeling using APEX peroxidase followed by anti-biotin enrichment and mass spectrometry yielded over 1,600 biotinylation sites on hundreds of proteins, an increase of more than 30-fold in the number of biotinylation sites identified compared to streptavidin-based enrichment of proteins.
Subject(s)
Antibodies/metabolism , Biotin/metabolism , Peptides/chemistry , Proteins/chemistry , Biotechnology/methods , Biotinylation , Chromatography, Liquid , HEK293 Cells , Humans , Jurkat Cells , Proteins/isolation & purification , Staining and Labeling , Streptavidin/metabolism , Tandem Mass SpectrometryABSTRACT
Thalidomide and its derivatives, lenalidomide and pomalidomide, are clinically effective treatments for multiple myeloma and myelodysplastic syndrome with del(5q). These molecules lack activity in murine models, limiting investigation of their therapeutic activity or toxicity in vivo. Here, we report the development of a mouse model that is sensitive to thalidomide derivatives because of a single amino acid change in the direct target of thalidomide derivatives, cereblon (Crbn). In human cells, thalidomide and its analogs bind CRBN and recruit protein targets to the CRL4CRBN E3 ubiquitin ligase, resulting in their ubiquitination and subsequent degradation by the proteasome. We show that mice with a single I391V amino acid change in Crbn exhibit thalidomide-induced degradation of drug targets previously identified in human cells, including Ikaros (Ikzf1), Aiolos (Ikzf3), Zfp91, and casein kinase 1a1 (Ck1α), both in vitro and in vivo. We use the Crbn I391V model to demonstrate that the in vivo therapeutic activity of lenalidomide in del(5q) myelodysplastic syndrome can be explained by heterozygous expression of Ck1α in del(5q) cells. We found that lenalidomide acts on hematopoietic stem cells with heterozygous expression of Ck1α and inactivation of Trp53 causes lenalidomide resistance. We further demonstrate that Crbn I391V is sufficient to confer thalidomide-induced fetal loss in mice, capturing a major toxicity of this class of drugs. Further study of the Crbn I391V model will provide valuable insights into the in vivo efficacy and toxicity of this class of drugs.
Subject(s)
Antineoplastic Agents/pharmacology , Lenalidomide/pharmacology , Myelodysplastic Syndromes/drug therapy , Nerve Tissue Proteins/genetics , Point Mutation , Thalidomide/pharmacology , Adaptor Proteins, Signal Transducing , Animals , Antineoplastic Agents/chemistry , Casein Kinase I/metabolism , Disease Models, Animal , Female , Hematopoiesis/drug effects , Lenalidomide/chemistry , Male , Mice , Mice, Inbred C57BL , Myelodysplastic Syndromes/genetics , Myelodysplastic Syndromes/metabolism , Nerve Tissue Proteins/metabolism , Thalidomide/analogs & derivativesABSTRACT
Introduction of antibodies specific for acetylated lysine has significantly improved the detection of endogenous acetylation sites by mass spectrometry. Here, we describe a new, commercially available mixture of anti-lysine acetylation (Kac) antibodies and show its utility for in-depth profiling of the acetylome. Specifically, seven complementary monoclones with high specificity for Kac were combined into a final anti-Kac reagent which results in at least a twofold increase in identification of Kac peptides over a commonly used Kac antibody. We outline optimal antibody usage conditions, effective offline basic reversed phase separation, and use of state-of-the-art LC-MS technology for achieving unprecedented coverage of the acetylome. The methods were applied to quantify acetylation sites in suberoylanilide hydroxamic acid-treated Jurkat cells. Over 10,000 Kac peptides from over 3000 Kac proteins were quantified from a single stable isotope labeling by amino acids in cell culture labeled sample using 7.5 mg of peptide input per state. This constitutes the deepest coverage of acetylation sites in quantitative experiments obtained to-date. The approach was also applied to breast tumor xenograft samples using isobaric mass tag labeling of peptides (iTRAQ4, TMT6 and TMT10-plex reagents) for quantification. Greater than 6700 Kac peptides from over 2300 Kac proteins were quantified using 1 mg of tumor protein per iTRAQ 4-plex channel. The novel reagents and methods we describe here enable quantitative, global acetylome analyses with depth and sensitivity approaching that obtained for other well-studied post-translational modifications such as phosphorylation and ubiquitylation, and should have widespread application in biological and clinical studies employing mass spectrometry-based proteomics.
Subject(s)
Antibodies, Monoclonal/metabolism , Liver/metabolism , Lysine/metabolism , Mammary Neoplasms, Experimental/metabolism , Proteomics/methods , Acetylation , Animals , Female , Humans , Jurkat Cells , Lysine/immunology , Mass Spectrometry/methods , Mice , Protein Processing, Post-Translational , WorkflowABSTRACT
Detection of endogenous ubiquitination sites by mass spectrometry has dramatically improved with the commercialization of anti-di-glycine remnant (K-ε-GG) antibodies. Here, we describe a number of improvements to the K-ε-GG enrichment workflow, including optimized antibody and peptide input requirements, antibody cross-linking, and improved off-line fractionation prior to enrichment. This refined and practical workflow enables routine identification and quantification of â¼20,000 distinct endogenous ubiquitination sites in a single SILAC experiment using moderate amounts of protein input.
Subject(s)
Proteome/analysis , Proteomics/methods , Ubiquitination , Amino Acids/metabolism , Antibodies/chemistry , Antibodies/immunology , Binding Sites , Chromatography, Liquid/methods , Cross-Linking Reagents/chemistry , Cysteine Proteinase Inhibitors/pharmacology , Glycylglycine/immunology , Humans , Isotope Labeling/methods , Jurkat Cells , Leupeptins/pharmacology , Proteasome Endopeptidase Complex/metabolism , Proteome/chemistry , Proteome/metabolism , Reproducibility of Results , Tandem Mass Spectrometry/methods , Ubiquitinated Proteins/analysis , Ubiquitinated Proteins/chemistry , Ubiquitinated Proteins/metabolismABSTRACT
IRE1α is an endoplasmic reticulum (ER) sensor that recognizes misfolded proteins to induce the unfolded protein response (UPR). We studied cholera toxin (CTx), which invades the ER and activates IRE1α in host cells, to understand how unfolded proteins are recognized. Proximity labeling colocalized the enzymatic and metastable A1 segment of CTx (CTxA1) with IRE1α in live cells, where we also found that CTx-induced IRE1α activation enhanced toxicity. In vitro, CTxA1 bound the IRE1α lumenal domain (IRE1αLD), but global unfolding was not required. Rather, the IRE1αLD recognized a seven-residue motif within an edge ß-strand of CTxA1 that must locally unfold for binding. Binding mapped to a pocket on IRE1αLD normally occupied by a segment of the IRE1α C-terminal flexible loop implicated in IRE1α oligomerization. Mutation of the CTxA1 recognition motif blocked CTx-induced IRE1α activation in live cells, thus linking the binding event with IRE1α signal transduction and induction of the UPR.
Subject(s)
Cholera Toxin , Endoribonucleases , Protein Serine-Threonine Kinases , Unfolded Protein Response , Cholera Toxin/genetics , Cholera Toxin/metabolism , Endoplasmic Reticulum Stress , Endoribonucleases/genetics , Endoribonucleases/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Signal Transduction , Humans , Animals , Mice , Cell LineABSTRACT
PCP signaling polarizes epithelial cells within the plane of an epithelium. Core PCP signaling components adopt asymmetric subcellular localizations within cells to both polarize and coordinate polarity between cells. Achieving subcellular asymmetry requires additional effectors, including some mediating post-translational modifications of core components. Identification of such proteins is challenging due to pleiotropy. We used mass spectrometry-based proximity labeling proteomics to identify such regulators in the Drosophila wing. We identified the catalytic subunit of Protein Phosphatase1, Pp1-87B, and show that it regulates core protein polarization. Pp1-87B interacts with the core protein Van Gogh and at least one Serine/Threonine kinase, Dco/CKIε, that is known to regulate PCP. Pp1-87B modulates Van Gogh subcellular localization and directs its dephosphorylation in vivo. PNUTS, a Pp1 regulatory subunit, also modulates PCP. While the direct substrate(s) of Pp1-87B in control of PCP is not known, our data support the model that cycling between phosphorylated and unphosphorylated forms of one or more core PCP components may regulate acquisition of asymmetry. Finally, our screen serves as a resource for identifying additional regulators of PCP signaling.
ABSTRACT
Transcription factors specify the fate and connectivity of developing neurons. We investigate how a lineage-specific transcription factor, Acj6, controls the precise dendrite targeting of Drosophila olfactory projection neurons (PNs) by regulating the expression of cell-surface proteins. Quantitative cell-surface proteomic profiling of wild-type and acj6 mutant PNs in intact developing brains, and a proteome-informed genetic screen identified PN surface proteins that execute Acj6-regulated wiring decisions. These include canonical cell adhesion molecules and proteins previously not associated with wiring, such as Piezo, whose mechanosensitive ion channel activity is dispensable for its function in PN dendrite targeting. Comprehensive genetic analyses revealed that Acj6 employs unique sets of cell-surface proteins in different PN types for dendrite targeting. Combined expression of Acj6 wiring executors rescued acj6 mutant phenotypes with higher efficacy and breadth than expression of individual executors. Thus, Acj6 controls wiring specificity of different neuron types by specifying distinct combinatorial expression of cell-surface executors.
Subject(s)
Drosophila Proteins , Olfactory Receptor Neurons , Animals , Dendrites/physiology , Drosophila/metabolism , Drosophila Proteins/metabolism , Ion Channels/metabolism , Membrane Proteins/metabolism , Nerve Tissue Proteins/metabolism , Olfactory Pathways/physiology , Olfactory Receptor Neurons/metabolism , POU Domain Factors/metabolism , Proteomics , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
Conventional approaches to identify secreted factors that regulate homeostasis are limited in their abilities to identify the tissues/cells of origin and destination. We established a platform to identify secreted protein trafficking between organs using an engineered biotin ligase (BirA*G3) that biotinylates, promiscuously, proteins in a subcellular compartment of one tissue. Subsequently, biotinylated proteins are affinity-enriched and identified from distal organs using quantitative mass spectrometry. Applying this approach in Drosophila, we identify 51 muscle-secreted proteins from heads and 269 fat body-secreted proteins from legs/muscles, including CG2145 (human ortholog ENDOU) that binds directly to muscles and promotes activity. In addition, in mice, we identify 291 serum proteins secreted from conditional BirA*G3 embryo stem cell-derived teratomas, including low-abundance proteins with hormonal properties. Our findings indicate that the communication network of secreted proteins is vast. This approach has broad potential across different model systems to identify cell-specific secretomes and mediators of interorgan communication in health or disease.
Subject(s)
Carbon-Nitrogen Ligases/metabolism , Escherichia coli Proteins/metabolism , Proteomics/methods , Repressor Proteins/metabolism , Staining and Labeling/methods , Animals , Animals, Genetically Modified , Biotin/metabolism , Biotinylation , Carbon-Nitrogen Ligases/genetics , Cell Line , Disease Models, Animal , Drosophila , Embryonic Stem Cells , Escherichia coli Proteins/genetics , Female , Humans , Male , Mice , Protein Engineering , Protein Transport , Repressor Proteins/genetics , Tandem Mass Spectrometry/methods , Teratoma/diagnosis , Teratoma/pathologyABSTRACT
Driver genes with a mutually exclusive mutation pattern across tumor genomes are thought to have overlapping roles in tumorigenesis. In contrast, we show here that mutually exclusive prostate cancer driver alterations involving the ERG transcription factor and the ubiquitin ligase adaptor SPOP are synthetic sick. At the molecular level, the incompatible cancer pathways are driven by opposing functions in SPOP. ERG upregulates wild type SPOP to dampen androgen receptor (AR) signaling and sustain ERG activity through degradation of the bromodomain histone reader ZMYND11. Conversely, SPOP-mutant tumors stabilize ZMYND11 to repress ERG-function and enable oncogenic androgen receptor signaling. This dichotomy regulates the response to therapeutic interventions in the AR pathway. While mutant SPOP renders tumor cells susceptible to androgen deprivation therapies, ERG promotes sensitivity to high-dose androgen therapy and pharmacological inhibition of wild type SPOP. More generally, these results define a distinct class of antagonistic cancer drivers and a blueprint toward their therapeutic exploitation.
Subject(s)
Nuclear Proteins/metabolism , Oncogene Proteins/metabolism , Prostatic Neoplasms/metabolism , Repressor Proteins/metabolism , Transcriptional Regulator ERG/metabolism , Ubiquitin-Protein Ligase Complexes/metabolism , Animals , Biomarkers, Tumor/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Co-Repressor Proteins/genetics , Co-Repressor Proteins/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , HEK293 Cells , Humans , Immunohistochemistry , Immunoprecipitation , Male , Mice , Mice, Nude , Mutation/genetics , Nuclear Proteins/genetics , Oncogene Proteins/genetics , Prostatic Neoplasms/genetics , Protein Binding , Proteomics , Receptors, Androgen/metabolism , Repressor Proteins/genetics , Signal Transduction/physiology , Transcriptional Regulator ERG/genetics , Ubiquitin-Protein Ligase Complexes/geneticsABSTRACT
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
ABSTRACT
Protein ubiquitylation is involved in a plethora of cellular processes. While antibodies directed at ubiquitin remnants (K-É-GG) have improved the ability to monitor ubiquitylation using mass spectrometry, methods for highly multiplexed measurement of ubiquitylation in tissues and primary cells using sub-milligram amounts of sample remains a challenge. Here, we present a highly sensitive, rapid and multiplexed protocol termed UbiFast for quantifying ~10,000 ubiquitylation sites from as little as 500 µg peptide per sample from cells or tissue in a TMT10plex in ca. 5 h. High-field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) is used to improve quantitative accuracy for posttranslational modification analysis. We use the approach to rediscover substrates of the E3 ligase targeting drug lenalidomide and to identify proteins modulated by ubiquitylation in models of basal and luminal human breast cancer. The sensitivity and speed of the UbiFast method makes it suitable for large-scale studies in primary tissue samples.