In vitro tyrosine phosphorylation of ALIX
Ontology highlight
ABSTRACT:
INSTRUMENT(S): LTQ Orbitrap XL
ORGANISM(S): Homo Sapiens (ncbitaxon:9606)
SUBMITTER: 
Lalit Deshmukh
PROVIDER: MSV000088057 | MassIVE |
REPOSITORIES: MassIVE
Similar Datasets
Project description:Here we explore the interactome of the proline-rich motif regions of non-activated receptor tyrosine kinases by affinity purification mass spectrometry.
2024-10-01 | PXD041383 | Pride
Project description:Pyk2 is a multidomain non-receptor tyrosine kinase that undergoes a complex, multi-stage activation process. Ca2+-flux induces conformational rearrangements that relieve autoinhibitory FERM domain interactions. The kinase domain phosphorylates a key linker residue to recruit Src kinase. Pyk2 and Src mutually phosphorylate activation loop residues to confer full activation. While the mechanisms of autoinhibition are established, the conformational dynamics associated with autophosphorylation and Src recruitment remain unclear. Here, we employ hydrogen/deuterium exchange mass spectrometry (HDX-MS) to map the conformational changes associated with Src-mediated activation segment phosphorylation in a Pyk2 construct encompassing FERM and kinase domains (residues 20-692). Results reveal increased dynamics at regulatory interfaces spanning FERM and kinase domains. Phosphorylation of the activation segment stabilizes two antiparallel beta strands linking activation and catalytic loops. Increased dynamics of the C-terminal end of the activation loop propagate to the F-helix, explaining how phosphorylation prevents reversion to the autoinhibitory FERM interaction. HDX-guided site-directed mutagenesis and kinase activity profiling establish a mechanism for phosphorylation-induced active site sculpting to confer high activity.
2023-03-08 | PXD034028 | Pride
Project description:Src family kinases (SFKs), including Src, Fyn and Yes, play important roles in development and cancer. Despite being first discovered as the Yes-associated protein, the regulation of Yap by SFKs remains poorly understood. Here, through single-cell analysis and genetic lineage tracing, we show that the pan-epithelial ablation of C-terminal Src kinase (Csk) in the lacrimal gland unleashes broad Src signaling but causes cell competition-mediated extrusion and apoptosis of only acinar progenitors. Csk mutants can be phenocopied by constitutively active Yap and rescued by the genetic ablation of Yap or Taz, indicating remarkable functional equivalence between Src and Yap signaling. Although Csk inactivation induces tyrosine phosphorylation of Yap, mutating these tyrosine residues in both Yap and Taz fails to alleviate the Csk lacrimal gland phenotype. In contrast, Yap loses Hippo signaling-dependent serine phosphorylation and translocates into the nucleus in Csk mutants. Chemical genetics studies further demonstrate that acute inhibition of Csk enhances Crk/CrkL phosphorylation and Rac1 activity, whereas the deletion of either Crk/CrkL or Rac1/Rap1 rescues the Csk mutant phenotype. These results show that Csk controls Hippo-Yap signaling through the Crk/CrkL-Rac/Rap axis to regulate cell competition.
2025-01-19 | GSE253784 | GEO
Project description:we combined phosphoproteomics and several cellular and molecular biologic strategies to identify that Src specifically interacted with the members of Src-family kinases (SFKs), such as Fyn or Lyn to form heterodimers, and subsequently phosphorylate the tyrosine sites in SH2 and kinase domains, which critically contributed to the ESCC malignancy.
2022-03-15 | PXD032306 |
Project description:Alix is a ubiquitously expressed scaffold protein that participates in numerous cellular processes, relating to the remodeling/repair of membranes and the actin cytoskeleton. Alix exists in monomeric and dimeric/multimeric configurations, but how dimer formation occurs and what role the dimer has in Alix-mediated processes are questions still largely elusive. Here, we reveal a mechanism for Alix homodimerization mediated by disulfide bonds under physiological conditions and demonstrate that Alix dimer is enriched in exosome and F-actin cytoskeleton subcellular fractions. Proteomic analysis of exosomes derived from Alix−/− primary cells underlined Alix indispensable role in loading syntenin into exosomes, thereby regulating the cellular levels of this protein. Using a set of Alix deletion mutants, we could define the function of the Bro1 domain, which is solely required for Alix’ exosomal localization, and that of the V domain, which is needed for recruiting syntenin into exosomes. We reveal an essential role for Cys814 within the disordered proline rich domain (PRD) for Alix dimerization. By mutating this residue, Alix remains exclusively monomeric and, in this configuration, is more effective in loading syntenin into exosomes. In contrast, loss of dimerization affects Alix ability to associate with F-actin, thereby compromising Alix-mediated cytoskeleton remodeling. We propose that by 3 shifting the ratio between its dimeric and monomeric forms, Alix selectively executes two of its main functions, exosomal cargo loading or cytoskeleton remodeling.
2022-10-14 | PXD031450 | Pride
Project description:<p>The purpose of the original study was to search for somatic mutations in the tyrosine kinome of serous and clear cell endometrial carcinomas (human). The study was conducted in two phases.</p> <p>Phase 1: A mutation discovery screen, in which ~577 exons encoding the catalytic domains of 86 tyrosine kinases were PCR-amplified and bidirectionally Sanger sequenced from 24 serous, 11 clear cell, and 5 mixed histology endometrial tumors. This was followed by alignment of sequence reads to the human reference sequence and subsequent nucleotide variant calling to identify potential somatic (tumor-specific) mutations. Potential somatic mutations were confirmed by re-amplification and sequencing of the relevant tumor DNA as well as matched non-tumor ("normal") DNA from the same case.</p> <p>Phase 2: A mutation prevalence screen, in which the non-catalytic regions two tyrosine kinase genes, TNK2 and DDR1, were PCR-amplified and sequenced from the 40 discovery screen tumors, and all coding exons of TNK2 and DDR1 were PCR-amplified and sequenced from another 10 clear cell, 21 serous, and 41 endometrioid endometrial tumors, in an effort to identify additional somatic mutations in each gene. Exons encoding the exonuclease domain of POLE were also sequenced to document somatic mutations.</p>
| phs000841 | dbGaP
Project description:Tyrosine Kinase Inhibitor Cardiotoxicity
| PRJNA352077 | ENA
Project description:The ShcA adaptor possesses two phosphotyrosine binding motifs, which include an SH2 and a PTB domain. In the majority of cases, ShcA utilizes its PTB domain to engage activated receptor tyrosine kinases (RTKs). To establish the mportance of this domain during mammary tumorigenesis, we employed a ShcA mutant (R175Q) that no longer binds phospho-tyrosine residues via its PTB domain. We demonstrate that the ShcR175Q mutant delays mammary tumor onset n MMTV/MT transgenic animals. Paradoxically, we observe a robust increase in the growth and angiogenesis of emerging mammary tumors. ShcR175Q-expressing breast cancer cells increase fibronectin secretion and possess elevated levels f integrin α5/β1, the principle fibronectin receptor. Sustained integrin engagement activates Src, which in turn phosphorylates pro-angiogenic RTKs, including PDGFR, FGFR and Met, leading to increased VEGF secretion from ShcR175Q-xpressing breast cancer cells. Finally, we describe a ShcR175Q-dependent gene signature that stratifies breast cancer patients with a high microvessel density. This is the first study to demonstrate that intracellular signaling pathways ownstream of the ShcA PTB domain both positively and negatively regulate tumorigenesis during the various stages of breast cancer progression ShcA mutant (R175Q) vs NT/ShcA cells
2013-08-07 | E-GEOD-41718 | biostudies-arrayexpress
Project description:Increased fatty acid synthesis benefits glioblastoma malignancy. However, the coordinated regulation of cytosolic acetyl-CoA production, the exclusive substrate for fatty acid synthesis, remains unclear. Here, we show that proto-oncogene tyrosine kinase c-SRC is activated in glioblastoma and remodels cytosolic acetyl-CoA production for fatty acid synthesis. Firstly, acetate is an important substrate for fatty acid synthesis in glioblastoma. c-SRC phosphorylates acetyl-CoA synthetase ACSS2 at Tyr530 and Tyr562 to stimulate the conversion of acetate to acetyl-CoA in cytosol. Secondly, c-SRC inhibits citrate-derived acetyl-CoA synthesis by phosphorylating ATP-citrate lyase ACLY at Tyr682. ACLY phosphorylation shunts citrate to IDH1-catalyzed NADPH production to provide reducing equivalent for fatty acid synthesis. The c-SRC-unresponsive double-mutation of ACSS2 and ACLY significantly reduces fatty acid synthesis and hampers glioblastoma progression. In conclusion, this remodeling fulfills the dual needs of glioblastoma cells for both acetyl-CoA and NADPH in fatty acid synthesis and provides evidence for glioma treatment by c-SRC inhibition.
2024-07-29 | PXD054314 | Pride
Project description:The NLRP3 inflammasome is a multi-protein complex that triggers the activation of the inflammatory protein caspase-1 and the maturation of the cytokine IL-1 in response to microbes and other danger signals in host cells. Here, we sought a deeper understanding of how the NLRP3 inflammasome is regulated. We found that inflammasome activation induced the Src family kinase Lyn to phosphorylate NLRP3 at Tyr918, and that this phosphorylation of NLRP3 correlated with a subsequent increase in its ubiquitination, which facilitated its proteasome-mediated degradation. NLRP3 tyrosine phosphorylation and ubiquitination was abrogated in Lyn-deficient macrophages, which produced increased amounts of IL-1. Furthermore, mice lacking Lyn were highly susceptible to LPS-induced septic shock in an NLRP3-dependent manner. Our data demonstrate that Lyn-mediated tyrosine phosphorylation of NLRP3 is a prerequisite for its ubiquitination, thus dampening NLRP3 inflammasome activity.
2022-05-19 | PXD028582 | Pride