The allosteric landscape of the Src kinase
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ABSTRACT:
PROVIDER: PRJNA1040239 | ENA |
REPOSITORIES: ENA
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Project description:Enzymes catalyze the reactions of life and are the targets of nearly all small molecule drugs. Most drugs inhibit enzymes by binding to conserved active sites, causing problems of specificity and toxicity. Targeting regulatory allosteric sites can increase specificity, overcome drug resistance and tune or activate activity.However, the vast majority of enzymes have no known allosteric sites and methods do not exist to globally identify or predict them.Here we present a general and fast method to globally chart allosteric communication in enzymes and apply it to the Src protein kinase to produce the first comprehensive map of negative and positive allosteric control of enzymatic activity. Allosteric control of Src is pervasive, anisotropic and distance dependent, but fairly predictable from simple sequence and structural features. The comprehensive allosteric map enables unbiased identification of all the allosterically active surface pockets of the Src kinase for the development of activatory and inhibitory drugs.This general approach can be used to chart global allosteric maps of many kinases, enzymes, and other biochemical activities important for medicine and biotechnology.
2024-04-27 | GSE247740 | GEO
Project description:The allosteric mechanisms that control the different functional and conformational states of oncogenic kinases, and how these molecular switches can be targeted and therapeutically exploited remains largely unexplored. Here we show that c-terminal Tyr 530 is a de facto c- Src auto-phosphorylation site with slow time-resolution kinetics and strong intermolecular component. On the contrary, activation-loop Tyr 419 undergoes fast kinetics and a cis-to-trans phosphorylation-switch that controls c-terminal Tyr 530 phosphorylation, enzyme specificity and strikingly, c-Src non-catalytic function as a substrate. In line with this, we visualize by X-ray crystallography a snapshot of c-terminal Tyr 530 intermolecular phosphorylation between the enzyme and susbtrate acting kinases, and identify a functionally relevant c-terminal palindromic phospho-motif flanking Tyr 530 making important contacts at the interface. Perturbation of this phospho-motif accounts for c-Src disfunction in cancer, as indicated by viral and a colorectal cancer (CRC) associated c-terminal deleted variants. We show that c-terminal residues 531 to 536 are required for c-Src Tyr 530 auto-phosphorylation and overall phospho-tyrosine activity. However, c-terminal truncated forms display a minor delay in the capacity to phosphorylate intact c-Src substrates surrogates. On the contrary, when these c-terminal variants were used as intact substrate surrogates themselves, they were equally and efficiently phosphorylated, but strikingly they resulted in the allosteric inhibition of the active kinase. Our work reveals a bi-directional crosstalk between activation and c-terminal segments driven by auto-phosphorylation that controls the allosteric interplay between enzyme and susbtrate acting kinases. These findings have important implications for the drug-design and development of next generation c-Src inhibitors targeting non-catalytic and/or allosteric vulnerabilities.
2023-09-04 | PXD037287 | Pride
Project description:Here we describe a bead-based method capable of profiling tyrosine kinase phosphorylations in a multiplexed, high-throughput and low-cost manner. This approach allows for the discovery of tyrosine kinase-activating events, even when the DNA sequence is wild-type. In an effort to pilot the establishment of a tyrosine kinase activation catalog, we profiled tyrosine phosphorylation levels of 62 tyrosine kinases in 130 human cancer lines, and followed-up on the frequent SRC phosphorylation in glioblastoma. Keywords: quantitative measurements of tyrosine phosphorylation levels on tyrosine kinases Total protein lysates were collected from 130 cancer cell lines. Tyrosine phosphorylation levels on 62 tyrosine kinases were measured with the bead assay.
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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:Data-dependent nLC-MS/MS analysis of EYA3 phosphorylation by Src protein tyrosine kinase
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Project description:Data-dependent nLC-MS/MS analysis of EYA3 phosphorylation by Src protein tyrosine kinase
2019-12-17 | PXD016518 | Pride
Project description:Data-dependent nLC-MS/MS analysis of EYA3 phosphorylation by Src protein tyrosine kinase
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Project description:Data-dependent nLC-MS/MS analysis of EYA3 phosphorylation by Src protein tyrosine kinase
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Project description:Protein tyrosine kinases are involved in regulating growth and proliferation in cells and are often hyperactive in cancerous tissue. Tyrosine kinase inhibitors have been used to limit hyperactivity but become ineffective due to the appearance of resistance mutations. A common trait of hyperactive protein kinases is its strict client relationship with molecular chaperone Hsp90. However, the mechanism behind Hsp90 client kinase recognition is poorly understood. Here we measure the functional effect of thousands of single amino acid variants in the Src kinase domain to identify positions invovled in Hsp90 client recognition.
2023-05-22 | GSE218190 | GEO
Project description:Here we describe a bead-based method capable of profiling tyrosine kinase phosphorylations in a multiplexed, high-throughput and low-cost manner. This approach allows for the discovery of tyrosine kinase-activating events, even when the DNA sequence is wild-type. In an effort to pilot the establishment of a tyrosine kinase activation catalog, we profiled tyrosine phosphorylation levels of 62 tyrosine kinases in 130 human cancer lines, and followed-up on the frequent SRC phosphorylation in glioblastoma. Keywords: quantitative measurements of tyrosine phosphorylation levels on tyrosine kinases
2008-12-05 | GSE13808 | GEO