Drug resistance profiling of thousands of Src kinase mutants uncovers a regulatory network that couples autoinhibition to catalytic domain dynamics
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ABSTRACT:
PROVIDER: PRJNA787284 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR17172500.fastq.gz | Fastqsanger.gz | |||
| SRR17172501.fastq.gz | Fastqsanger.gz | |||
| SRR17172502.fastq.gz | Fastqsanger.gz | |||
| SRR17172503.fastq.gz | Fastqsanger.gz | |||
| SRR17172504.fastq.gz | Fastqsanger.gz |
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Project description:Protein kinase inhibitors are effective cancer therapies, but acquired resistance often limits clinical efficacy. Despite the cataloguing of numerous resistance mutations with model studies and in the clinic, we still lack a comprehensive understanding of kinase inhibitor resistance. Here, we measured the resistance of thousands of Src tyrosine kinase mutants to a panel of ATP-competitive inhibitors. We found that ATP-competitive inhibitor resistance mutations are distributed throughout Src’s catalytic domain. In addition to inhibitor contact residues, residues that participate in regulating Src’s phosphotransferase activity were prone to the development of resistance. Unexpectedly, a resistance-prone cluster of residues that are on the top face of the N-terminal lobe of the catalytic domain contributes to Src autoinhibition by reducing the dynamics of the catalytic domain, and mutations in this cluster led to resistance by lowering inhibitor affinity and promoting kinase hyperactivation. Together, our studies demonstrate how comprehensive profiling of drug resistance can be used to understand potential resistance pathways and uncover new mechanisms of kinase regulation.
2021-12-17 | GSE190495 | GEO
Project description:MICAL proteins play a crucial role in cellular dynamics by binding and disassembling actin filaments, impacting processes like axon guidance, cytokinesis, and cell morphology. Their cellular activity is tightly controlled, as dysregulation can lead to detrimental effects on cellular morphology. Although previous studies have suggested that MICALs are autoinhibited, and require Rab proteins to become active, the detailed molecular mechanisms remained unclear. Here, we report the cryo-EM structure of human MICAL1 at a nominal resolution of 3.1 Å. Structural analyses, alongside biochemical and functional studies, show that MICAL1 autoinhibition is mediated by an intramolecular interaction between its N-terminal catalytic and C-terminal coiled-coil domains, blocking F-actin interaction. Moreover, we demonstrate that allosteric changes in the coiled-coil domain and the binding of the tripartite assembly of CH-L2α1-LIM domains to the coiled-coil domain are crucial for MICAL activation and autoinhibition. These mechanisms appear to be evolutionarily conserved, suggesting a potential universality across the MICAL family.
2024-10-30 | PXD057312 | Pride
Project description:The restoration of catalytic activity to mutant enzymes by small molecules is well-established for in vitro systems. Here we show that the protein tyrosine kinase Src R388A mutant can be rescued in live cells using the small molecule imidazole. Cellular rescue of a v-Src homolog was rapid and reversible and conferred predicted oncogenic properties. Using chemical rescue in combination with mass spectrometry, six known Src kinase substrates were confirmed, and several new protein targets identified. Chemical rescue data suggests that c-Src is active under basal conditions. Rescue of R388A c-Src also allowed contributions of Src to the MAP kinase pathway to be clarified. This chemical rescue approach is likely to be of broad utility in cell signaling. We were also interested in examining the impact of Src rescue on the kinetics of gene expression. Chronic gene expression changes in v-Src transformed colon cancer and NIH3T3 cells have been reported, but the chemical rescue method permits insights into rapid kinetic changes. We used gene microarray analysis of imidazole activated Src, 1 h after imidazole treatment of 8A7F cells as well as 6N7F control cells, a set of several genes show increases (>1.7-fold) at 1 h in the 8A7F cells and another set show decreases (>1.7-fold) at 1 h with minimal changes in 6N7F control cells. Thirteen of these genes were further analyzed using real-time RT-PCR and most of the genes tested showed similar changes using both techniques. These gene changes were not reported in cells chronically transformed with v-Src or rapidly stimulated with growth factors suggesting that rapid initiation of Src-mediated tyrosine phosphorylation may induce a specialized pattern of gene expression changes. However, these earlier experiments were done under different conditions which may also contribute to gene effects. Keywords: time course, cell type comparison
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Project description:Experimental aim: identification of SRC-2 and SRC-3-related genes in MCF-7 breast cancer cells. Experimental workflow: 1. shRNA transduction. MCF-7 cells were transduced with a mix of lentiviral particles containing five individual lentiviral pLKO.1-puro short hairpin (sh) RNA plasmids targeting different sequences on SRC-2 or SRC-3 mRNA and a pLKO.1-puro empty vector control. The puromycin selections were maintained for three weeks to obtain cells containing stably integrated shRNA. 2. Sample preparation. RNA was extracted from eight independent replicates expressing the pLKO.1-puro empty shRNA vector (shKTR), from eight replicates of SRC-2 shRNA expressing cells (shSRC-2), and from seven replicates of SRC-3 shRNA expressing cells (shSRC-3). 300 ng of total RNA from each cell sample was biotin-labelled and amplified.
2019-07-01 | E-MTAB-7018 | biostudies-arrayexpress
Project description:The RNA helicase UPF1 interacts with mRNAs, mRNA decay machinery, and the terminating ribosome to promote nonsense-mediated mRNA decay (NMD). Structural and biochemical data have revealed that UPF1 exists in an enzymatically autoinhibited “closed” state. Upon binding the NMD protein UPF2, UPF1 undergoes an extensive conformational change into a more enzymatically active “open” state, which exhibits enhanced ATPase and helicase activity. However, mechanically deficient UPF1 mutants can support efficient NMD, bringing into question the roles of UPF1 enzymatic autoinhibition and activation in NMD. Here, we identify two additional important features of the activated open state: slower nucleic acid binding kinetics and enhanced ATP-stimulated nucleic acid dissociation kinetics. Computational modeling based on empirical measurements of UPF1, UPF2, and RNA interaction kinetics predicts that the majority of UPF1-RNA binding and dissociation events in cells occur independently of UPF2 binding. We find that UPF1 mutants with either reduced or accelerated dissociation from RNA have NMD defects, whereas UPF1 mutants that are more dependent on UPF2 for catalytic activity remain active on well-established NMD targets. These findings support a model in which the kinetics of UPF1-mRNA interactions are important determinants of cellular NMD efficiency.
2024-05-29 | GSE246484 | GEO
Project description:The RNA helicase UPF1 interacts with mRNAs, mRNA decay machinery, and the terminating ribosome to promote nonsense-mediated mRNA decay (NMD). Structural and biochemical data have revealed that UPF1 exists in an enzymatically autoinhibited “closed” state. Upon binding the NMD protein UPF2, UPF1 undergoes an extensive conformational change into a more enzymatically active “open” state, which exhibits enhanced ATPase and helicase activity. However, mechanically deficient UPF1 mutants can support efficient NMD, bringing into question the roles of UPF1 enzymatic autoinhibition and activation in NMD. Here, we identify two additional important features of the activated open state: slower nucleic acid binding kinetics and enhanced ATP-stimulated nucleic acid dissociation kinetics. Computational modeling based on empirical measurements of UPF1, UPF2, and RNA interaction kinetics predicts that the majority of UPF1-RNA binding and dissociation events in cells occur independently of UPF2 binding. We find that UPF1 mutants with either reduced or accelerated dissociation from RNA have NMD defects, whereas UPF1 mutants that are more dependent on UPF2 for catalytic activity remain active on well-established NMD targets. These findings support a model in which the kinetics of UPF1-mRNA interactions are important determinants of cellular NMD efficiency.
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Project description:The non-receptor tyrosine kinase SRC is upregulated in various human cancers and plays crucial roles in cancer progression by promoting invasion and metastasis. We show that the transforming growth factor beta (TGF-β/SMAD pathway directly upregulates SRC during the epithelial-mesenchymal transition. In human epithelial MCF10A cells, TGF-β1 treatment markedly upregulated mRNA expression of SRC. Knockout of SMAD4 suppressed upregulation of SRC by TGF-β1. ChIP-sequencing analysis revealed that SRC was transcribed from the SRC1A promoter, which interacted with SMAD2 and SMAD4, in response to TGF-β1. These findings demonstrate that a direct interaction of the activated SMAD complex with the SRC1A promoter directly upregulates SRC and suggest that TGF-β contributes to SRC upregulation in the tumor microenvironment, where TGF-β-mediated tumor progression takes place.
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Project description:Cells responding to DNA damage implement complex adaptive programs that often culminate in two distinct outcomes: apoptosis or senescence. Here, to systematically identify factors driving each response, we analyzed human IMR-90 fibroblasts exposed to increasing doses of the genotoxin etoposide. We identified SRC as a key kinase contributing to this decision fork. SRC was activated by low (50 μM) etoposide but not by high (200 μM) etoposide levels. At low DNA damage, SRC-mediated activation of p38 critically promoted cell survival and senescence, and increased pro-survival BCL2L2 levels, while SRC-mediated repression of p53 prevented a rise in pro-apoptotic PUMA levels. At high DNA damage, SRC was not active, leading to elevation of p53, inhibition of p38, and apoptosis. In mice exposed to DNA damage, pharmacologic inhibition of SRC prevented the tissue accumulation of senescent cells. Inhibiting SRC could be exploited for inducing senescent-cell apoptosis in tissues to improve health outcomes.
2022-02-17 | GSE169037 | GEO
Project description:To address the molecular mechanisms underlying c-Src-mediated tumor progression, we previously developed a model system using Csk-deficient fibroblasts that can be transformed by wild-type c-Src. In this study, we applied this system for the analysis of the potential contribution of miRNA to c-Src-mediated transformation. Pair-wise significance analysis of the microarray indicated that seven miR genes were significantly upregulated and six miRNA genes were downregulated in c-Src-transformed cells with a P value below 0.01 and with a fold change over 2.0. Csk-/- mouse embryonic fibroblasts (Csk-/- MEFs) were transfected with empty vector, c-Src, Csk/c-Src, or Csk. Each sample was run in duplicate.
2011-02-17 | E-GEOD-23426 | biostudies-arrayexpress
Project description:The rapidly growing family of coregulators of nuclear receptors includes coactivators that promote transcription and corepressors that harbor the opposing function. In recent years, coregulators have begun to emerge as important regulators of metabolic homeostasis, including the p160 Steroid Receptor Coactivator (SRC) family. Members of the SRC family have been ascribed important roles in control of gluconeogenesis in liver and fatty acid oxidation in skeletal muscle. To provide a deeper and more granular understanding of the metabolic impact of SRC family members, we have performed targeted metabolomics analysis of key metabolic byproducts of glucose, fatty acid, and amino acid metabolism in mice with global knockout of SRC-1, SRC-2, or SRC-3. We measured amino acids, acyl carnitines, and organic acids in five tissues with key metabolic functions (liver, heart, skeletal muscle, brain, plasma) isolated from SRC-1, -2, or -3 knockout mice and their wild-type littermates in the fed and fasted conditions, thereby unveiling unique metabolic functions of each SRC coactivator. Overall, we observed entire groups or subgroups of metabolites belonging to discrete metabolic pathways that were influenced in different tissues and/or feeding states due to ablation of individual SRC isoforms. Surprisingly, we identified very few metabolites that changed universally across the three SRC knockout models. These findings demonstrate that coactivator function has very limited redundancy even within the homologous SRC coactivator family. Furthermore, this work also demonstrates the use of metabolomics as a means for identifying novel metabolic regulatory functions of transcriptional coregulators.
2013-04-10 | MTBLS30 | MetaboLights