High-throughput phosphoproteomics: technologies and applications in systems biology
Abstract
Mass spectrometry-based proteomics has been used to study dynamic cell signalling for over a decade, and phosphoproteomics has become a burgeoning field in its own right. Developments in recent years have steadily increased the scope of phosphoproteomics studies, but major challenges remain. The ‘EasyPhos’ method is a robust and reproducible workflow aimed at resolving some of these. It simplifies phosphoproteomics experiments and allows the rapid measurement of large numbers of phosphoproteomes, accelerating dynamic and in vivo signalling studies in diverse areas ranging from insulin signalling in the liver to kappa opioid receptor signalling in the brain, as I will discuss in this talk.
Why is the yeast checkpoint kinase Rad53 so highly phosphorylated?
Abstract
As more and more phosphorylation sites, some occurring in clusters, have been identified in various proteins, one often asks what the functions of multiple phosphorylation sites are, and whether all phosphorylations are functionally relevant. Activation of the checkpoint kinase Rad53 upon S-phase DNA damage involves Rad9- or Mrc1-mediated phospho-priming by Mec1 kinase, followed by auto-activating T354-phosphorylation. However, specific mechanisms remain unclear. We report comprehensive and quantitative analyses of phosphorylations and interacting proteins of checkpoint kinase Rad53 under endogenous and untagged conditions, at different time points of the cell cycle, without and with DNA damages, for wildtype and several mutants. More than 40 phosphorylation sites have been characterized. Their variations, along with the variations in the binding partners, provide information on the functions of phosphorylation sites and the mechanism of kinase activation in vivo. For the phosphothreonine cluster at the N-terminus, we also show that the number of phosphorylations correlates with kinase activation and subsequent signaling events. In addition, we show that phospho-priming confers functionally relevant specificities for the autophosphorylation of Rad53 kinase.
References:
1. “Diphosphothreonine-specific interaction between SQ/TQ cluster and an FHA domain in the Rad53-Dun1 kinase cascade”. Hyun Lee, Chunhua Yuan, Andrew Hammet, Anjali Mahajan, Eric S.-W. Chen, Ming-Ru Wu, Mei-I Su, Jörg Heierhorst, Ming-Daw Tsai, Mol. Cell 30, 767-778 (2008).
2. “Use of quantitative mass spectrometric analysis to elucidate the mechanisms of phospho-priming and auto-activation of the checkpoint kinase Rad53 in vivo”. Eric S.-W. Chen, Nicolas C. Hoch, Shun-Chang Wang, Achille Pellicioli, Jörg Heierhorst, and Ming-Daw Tsai, Mol Cell Proteomics 13, 551-565 (2014).
Nrich and iNrich: N-terminomics tools for discovering various protein forms
Abstract
Various forms of protein (proteoforms) are generated by genetic variations, alternative splicing, alternative translation initiation, co- or post-translational modification and proteolysis. Different proteoforms are in part discovered by characterizing their N-terminal sequences using N-terminomics methods. Previously, we introduced an N-terminal-peptide-enrichment method, Nrich for this purpose. We identified 6,525 acetylated (or partially acetylated) and 6,570 free protein N-termini arising from 5,727 proteins in HEK293T human cells. The protein N-termini included translation initiation sites annotated in the UniProtKB database, putative alternative translational initiation sites, and N-terminal sites exposed after signal/transit/pro-peptide removal or unknown processing, revealing various proteoforms in cells. We next modified the method by performing multiple essential steps in a single-stage encapsulated solid phase extraction column. We named the new method as iNrich representing 'integrated Nrich'. iNrich could handle as little as 25 µg cell lysate, and was highly reproducible.
Fomryl-Methionine-Targeting Proteolytic System: the fMet/N-End Rule Pathway in the Yeast Saccharomyces cerevisiae
Abstract
Owing to the design of AUG genetic codon, protein biosynthesis begins with methionine (Met) in the cytosol of eukaryotes. In contrast, bacteria and eukaryotic organelles (mitochondria and chloroplasts), initiate the protein synthesis with formyl-methionine (fMet). Here we show, using the yeast Saccharomyces cerevisiae, that the formyltransferase Fmt1 of this eukaryote, while normally imported into mitochondria, can also mediate, in vivo, the synthesis of fMet-containing proteins by cytoplasmic ribosomes. The resulting fMet of eukaryotic proteins acts as as a specific protein degradation signal which can be targeted by the unprecedent polyubiquin-mediated and proteasome-dependent proteolytic system, termed the fMet/N-end rule pathway.
Rm#422, Industry-University Research Bldg., Yonsei University
50 Yonsei-ro, Seodeamoon-gu, Seoul 03722 Korea
Tel : +82-2-393-8328 Fax : +82-2-393-6589 E-mail : admin@khupo.org
COPYRIGHT ⓒ 2018 KHUPO. ALL RIGHTS RESERVED.
