Global and Site-Specific Effect of Phosphorylation on Protein Turnover

To date, the effects of specific modification types and sites on protein lifetime have not been systematically illustrated. Here, we describe a proteomic method, DeltaSILAC, to quantitatively assess the impact of site-specific phosphorylation on the turnover of thousands of proteins in live cells. B...

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Bibliographic Details
Published in:Developmental cell 2021-01, Vol.56 (1), p.111-124.e6
Main Authors: Wu, Chongde, Ba, Qian, Lu, Dayun, Li, Wenxue, Salovska, Barbora, Hou, Pingfu, Mueller, Torsten, Rosenberger, George, Gao, Erli, Di, Yi, Zhou, Hu, Fornasiero, Eugenio F., Liu, Yansheng
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Cell Cycle - physiology
Cell Line, Tumor
Cyclin-Dependent Kinases - genetics
Cyclin-Dependent Kinases - metabolism
data-independent acquisition
DeltaSILAC
Glutamates - metabolism
Humans
Isotope Labeling - methods
mass spectrometry
Mass Spectrometry - methods
Peptides - metabolism
Peroxiredoxin VI - chemistry
Peroxiredoxin VI - metabolism
phosphomodiform
Phosphoproteins - chemistry
Phosphoproteins - metabolism
Phosphorylation
protein lifetime
protein turnover
Proteolysis
Proteome - genetics
Proteome - metabolism
proteomics
Proteomics - methods
pulse SILAC
RNA Splicing Factors - chemistry
RNA Splicing Factors - metabolism
Signal Transduction - genetics
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Summary:To date, the effects of specific modification types and sites on protein lifetime have not been systematically illustrated. Here, we describe a proteomic method, DeltaSILAC, to quantitatively assess the impact of site-specific phosphorylation on the turnover of thousands of proteins in live cells. Based on the accurate and reproducible mass spectrometry-based method, a pulse labeling approach using stable isotope-labeled amino acids in cells (pSILAC), phosphoproteomics, and a unique peptide-level matching strategy, our DeltaSILAC profiling revealed a global, unexpected delaying effect of many phosphosites on protein turnover. We further found that phosphorylated sites accelerating protein turnover are functionally selected for cell fitness, enriched in Cyclin-dependent kinase substrates, and evolutionarily conserved, whereas the glutamic acids surrounding phosphosites significantly delay protein turnover. Our method represents a generalizable approach and provides a rich resource for prioritizing the effects of phosphorylation sites on protein lifetime in the context of cell signaling and disease biology. [Display omitted] •A proteomic method reveals site-specific phosphorylation impacts protein degradation•Many phosphorylation sites delay protein turnover in growing cells•Phosphosites involved in cell cycle and cell fitness destabilize protein expression•Glutamic acids surrounding phosphosites significantly delay protein turnover By developing and applying a large-scale mass spectrometry-based method, Wu and Ba et al. quantitatively assessed the stabilizing and destabilizing effects of phosphorylation on protein expression. The findings revealed that phosphorylation impacts protein degradation in a site-specific manner, which is associated with surrounding amino acids, local structure, and phosphoprotein functions.
ISSN:1534-5807
1878-1551
DOI:10.1016/j.devcel.2020.10.025