ABSTRACT
Epidermal growth factor receptor (EGFR) signalling is activated by ligand-induced receptor dimerization. Notably, ligand binding also induces EGFR oligomerization, but the structures and functions of the oligomers are poorly understood. Here, we use fluorophore localization imaging with photobleaching to probe the structure of EGFR oligomers. We find that at physiological epidermal growth factor (EGF) concentrations, EGFR assembles into oligomers, as indicated by pairwise distances of receptor-bound fluorophore-conjugated EGF ligands. The pairwise ligand distances correspond well with the predictions of our structural model of the oligomers constructed from molecular dynamics simulations. The model suggests that oligomerization is mediated extracellularly by unoccupied ligand-binding sites and that oligomerization organizes kinase-active dimers in ways optimal for auto-phosphorylation in trans between neighbouring dimers. We argue that ligand-induced oligomerization is essential to the regulation of EGFR signalling.
Subject(s)
ErbB Receptors/chemistry , ErbB Receptors/metabolism , Animals , Artifacts , Binding Sites , CHO Cells , Cricetinae , Cricetulus , Epidermal Growth Factor/metabolism , Fluorescence Resonance Energy Transfer , Ligands , Molecular Dynamics Simulation , Phosphorylation , Protein Domains , Protein Multimerization , Signal TransductionABSTRACT
Beyond penetrant germline and somatic mutations, there are substantial challenges in extrapolating phenotypes from linear DNA sequences and transcriptomics. This brings a molecular pathology emphasis to the properties of the main players responsible for executing actions, proteins. The proteomic attribute most frequently determined in pathology is (relative) content, but for many candidate biomarkers this is not the most important feature to understand. In keeping pace with the depth of knowledge of the mechanisms underlying pathologies, we need to ask more sophisticated questions about the state of proteins, for example, their oligomerization status, modification status, and location. This demands hitherto nonroutine approaches to proteomics, which we will discuss in this brief perspective.
Subject(s)
Biomarkers, Tumor/metabolism , Neoplasms/diagnosis , Neoplasms/metabolism , Proteomics/methods , Biomarkers, Tumor/analysis , Fluorescence Resonance Energy Transfer , Gene Expression Regulation, Neoplastic , Gene Regulatory Networks , Humans , Neoplasms/geneticsABSTRACT
Natural killer (NK) cells kill tumor cells and virally infected cells, and an effective NK cell response requires processes, such as motility, recognition, and directional secretion, that rely on cytoskeletal rearrangement. The Rho guanosine triphosphatase (GTPase) Cdc42 coordinates cytoskeletal reorganization downstream of many receptors. The Rho-related GTPase from plants 1 (ROP1) exhibits oscillatory activation behavior at the apical plasma membrane of growing pollen tubes; however, a similar oscillation in Rho GTPase activity has so far not been demonstrated in mammalian cells. We hypothesized that oscillations in Cdc42 activity might occur within NK cells as they interact with target cells. Through fluorescence lifetime imaging of a Cdc42 biosensor, we observed that in live NK cells forming immunological synapses with target cells, Cdc42 activity oscillated after exhibiting an initial increase. We used protein-protein interaction networks and structural databases to identify candidate proteins that controlled Cdc42 activity, leading to the design of a targeted short interfering RNA screen. The guanine nucleotide exchange factors RhoGEF6 and RhoGEF7 were necessary for Cdc42 activation within the NK cell immunological synapse. In addition, the kinase Akt and the p85α subunit of phosphoinositide 3-kinase (PI3K) were required for Cdc42 activation, the periodicity of the oscillation in Cdc42 activity, and the subsequent polarization of cytotoxic vesicles toward target cells. Given that PI3Ks are targets of tumor therapies, our findings suggest the need to monitor innate immune function during the course of targeted therapy against these enzymes.
Subject(s)
Immunological Synapses/immunology , Killer Cells, Natural/immunology , RNA, Small Interfering , cdc42 GTP-Binding Protein/immunology , Biological Clocks/genetics , Biological Clocks/immunology , Cell Line, Transformed , Cell- and Tissue-Based Therapy/methods , Class Ia Phosphatidylinositol 3-Kinase/genetics , Class Ia Phosphatidylinositol 3-Kinase/immunology , Class Ia Phosphatidylinositol 3-Kinase/metabolism , Cytoskeleton/genetics , Cytoskeleton/immunology , Cytoskeleton/metabolism , Enzyme Activation/genetics , Enzyme Activation/immunology , Guanine Nucleotide Exchange Factors/genetics , Guanine Nucleotide Exchange Factors/immunology , Guanine Nucleotide Exchange Factors/metabolism , Humans , Immunity, Cellular/genetics , Immunity, Innate/genetics , Immunological Synapses/enzymology , Immunological Synapses/genetics , Killer Cells, Natural/enzymology , Neoplasms/enzymology , Neoplasms/genetics , Neoplasms/immunology , Neoplasms/therapy , Proto-Oncogene Proteins c-akt , Rho Guanine Nucleotide Exchange Factors , cdc42 GTP-Binding Protein/genetics , cdc42 GTP-Binding Protein/metabolismABSTRACT
The presence of GH receptor (GHR) in the cell nucleus correlates with cell division, and targeting the GHR to the nucleus results in constitutive proliferation and transformation because of increased sensitivity to autocrine GH. Here we have sought additional mechanisms that might account for the enhanced proliferation seen with nuclear GHR, commencing with a yeast two-hybrid (Y2H) screen for interactors with the extracellular domain of the GHR [GH-binding protein (GHBP)]. We find that the GHBP is a transcriptional activator in yeast and mammalian cells, and this activity resides in the lower cytokine receptor module. Activity is dependent on S226, the conserved serine of the cytokine receptor consensus WSXWS box. By using parallel GHBP affinity columns and tandem mass spectrometry of tryptic digests of proteins bound to wild-type GHBP and S226A columns, we identified proteins that bind to the transcriptionally active GHBP. These include a nucleoporin and two transcriptional regulators, notably the coactivator activator (CoAA), which is also an RNA binding splicing protein. Binding of CoAA to the GHBP was confirmed by glutathione S-transferase pulldown and coimmunoprecipitation, and shown to be GH dependent in pro-B Ba/F3 cells. Importantly, stable expression of CoAA in Ba/F3 cells resulted in an increased maximum proliferation in response to GH, but not IL-3. Because CoAA overexpression has been identified in many cancers and its stable expression promotes cell proliferation and cell transformation in NIH-3T3 cells, we suggest CoAA contributes to the proliferative actions of nuclear GHR by the hormone-dependent recruitment of this powerful coactivator to the GHR.
Subject(s)
Receptors, Somatotropin/chemistry , Receptors, Somatotropin/metabolism , Transcription Factors/metabolism , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , CHO Cells , Cell Line , Cell Proliferation , Cricetinae , Cricetulus , DNA Primers/genetics , Growth Hormone/pharmacology , Humans , Interleukin-3/pharmacology , Mice , Models, Biological , Models, Molecular , Mutagenesis, Site-Directed , NIH 3T3 Cells , Protein Binding , Protein Structure, Tertiary , Receptors, Somatotropin/genetics , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Recombinant Proteins/pharmacology , Transcription Factors/genetics , Transcriptional Activation , Two-Hybrid System TechniquesABSTRACT
The proto-oncoprotein SYT is involved in the unique translocation t(X;18) found in synovial sarcoma SYT-SSX fusions. SYT has a conserved N-terminal domain (SNH domain) that interacts with the human paralog of Drosophila Brahma (hBRM) and Brahma-related gene 1 (BRG1) chromatin remodeling proteins and a C-terminal transactivating sequence rich in glutamine, proline, glycine, and tyrosine (QPGY domain). Here we reported the isolation of the ribonucleoprotein SYT-interacting protein/co-activator activator (SIP/CoAA), which specifically binds the QPGY domain of SYT and also the SYT-SSX2 translocation fusion. SIP/CoAA is a general nuclear co-activator and an RNA splicing modulator that contains two RNA recognition motifs and multiple hexapeptide repeats. We showed that the region consisting of the hexapeptide motif (YQ domain) is similar to the hexapeptide repeat domain found in EWS and in TLS/FUS family proteins. The YQ domain also resembles the QPGY region of SYT itself and like all these other domains acts as a transcriptional activator in reporter assays. Most interestingly, the last 84 amino acids adjacent to YQ down-modulate by 25-fold the YQ transactivation of the reporter gene, and both domains are important for SIP/CoAA binding to SYT. In addition, SYT acts together with SIP/CoAA in stimulating estrogen and glucocorticoid receptor-dependent transcriptional activation. Activation is hormone-dependent and requires functional hBRM and/or BRG1. The stimulation is strongly reduced if the N-terminal region of hBRM/BRG1 (amino acids 1-211) is deleted. This region encompasses the SNF11 binding domain (amino acids 156-211), which interacts specifically with SYT in vivo and in vitro.
Subject(s)
Cell Cycle Proteins/chemistry , Cell Nucleus/metabolism , Intracellular Signaling Peptides and Proteins/chemistry , Neoplasm Proteins/chemistry , Proto-Oncogene Proteins/metabolism , RNA-Binding Protein EWS/chemistry , RNA-Binding Protein FUS/chemistry , RNA-Binding Proteins/chemistry , Repressor Proteins/chemistry , Repressor Proteins/metabolism , Amino Acid Motifs , Amino Acid Sequence , Animals , COS Cells , Cell Line , Cell Line, Tumor , Chlorocebus aethiops , Chromatin/chemistry , Cloning, Molecular , Cytoplasm/metabolism , DNA, Complementary/metabolism , Down-Regulation , Drosophila , Gene Library , Glutamine/chemistry , Glutathione Transferase/metabolism , Glycine/chemistry , Hormones/metabolism , Humans , Immunoblotting , Immunoprecipitation , Intracellular Signaling Peptides and Proteins/metabolism , Ligands , Models, Biological , Molecular Sequence Data , Plasmids/metabolism , Proline/chemistry , Protein Binding , Protein Structure, Tertiary , Proto-Oncogene Proteins/chemistry , RNA Splicing , Recombinant Fusion Proteins/chemistry , Sarcoma, Synovial/metabolism , Sequence Homology, Amino Acid , Serine-Arginine Splicing Factors , Transcription, Genetic , Transcriptional Activation , Transfection , Translocation, Genetic , Two-Hybrid System Techniques , Tyrosine/chemistryABSTRACT
Many studies have now established that the SWI/SNF chromatin remodelling complexes are involved in activation and repression of a variety of genes. In mammalian cells, these complexes contain the BRM and BRG1 helicase-like proteins that are thought to be responsible for nucleosome remodelling. The proto-oncoprotein SYT, involved in the unique translocation t(X;18) found in synovial sarcoma, is known to interact with human BRM (hBRM), thus providing a link between chromatin remodelling factors and human cancer. In this work, we address how SYT interacts with hBRM and BRG1. We demonstrate that the conserved N-terminal SNH domain of SYT, which is also present in the oncoproteins SYT-SSX, binds to both hBRM and BRG1. We have also found that in vivo the C-terminus transactivation QPGY region of SYT can interact with itself. This results in an amplified interaction with hBRM and highlights a possible regulatory function of this domain in cells.