Journal
NATURE COMMUNICATIONS
Volume 13, Issue 1, Pages -Publisher
NATURE PORTFOLIO
DOI: 10.1038/s41467-022-34980-5
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Funding
- Yale School of Medicine startup fund
- National Institutes of Health [GM124296, F32CA224946, GM125951]
- Yale School of Medicine
- Yale West Campus
- Systems Biology Institute
- Cancer Biology Institute
- Jeff Sampson Technology
- Agilent Technology
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Protein phosphorylation is a crucial post-translational modification that regulates cellular processes and protein-protein interactions. Traditional strategies for functional assignment of phosphorylation events have become inadequate in keeping up with the rapid identification pace through proteomic surveillance. However, the development of phospho-amino-acid-specific orthogonal translation systems offers a kinase-independent method to assess the physiological function of phosphorylation.
Protein phosphorylation is a ubiquitous post-translational modification used to regulate cellular processes and proteome architecture by modulating protein-protein interactions. The identification of phosphorylation events through proteomic surveillance has dramatically outpaced our capacity for functional assignment using traditional strategies, which often require knowledge of the upstream kinase a priori. The development of phospho-amino-acid-specific orthogonal translation systems, evolutionarily divergent aminoacyl-tRNA synthetase and tRNA pairs that enable co-translational insertion of a phospho-amino acids, has rapidly improved our ability to assess the physiological function of phosphorylation by providing kinase-independent methods of phosphoprotein production. Despite this utility, broad deployment has been hindered by technical limitations and an inability to reconstruct complex phopho-regulatory networks. Here, we address these challenges by optimizing genetically encoded phosphothreonine translation to characterize phospho-dependent kinase activation mechanisms and, subsequently, develop a multi-level protein interaction platform to directly assess the overlap of kinase and phospho-binding protein substrate networks with phosphosite-level resolution. Protein phosphorylation is a ubiquitous post-translational modification used to regulate cellular processes and proteome architecture by modulating protein-protein interactions. Here the authors optimize genetically encoded phosphothreonine to study the regulation of CHK2 kinase using large-scale DNA arrays that enable phosphoproteome expression techniques to identify sitespecific overlap between CHK2 substrates and 14-3-3 interactions.
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