Respective, Time‐dependent Phosphorylation Modules Shaping Phosphoproteome Abundance and Turnover

Abstract

Protein phosphorylation mediates protein-protein interactions and alters protein structure, stability, and subcellular localization—frequently in a modification site-specific manner. However, how phosphorylation impacts protein lifetime remains to be illustrated. We have devised a proteomic method, DeltaSILAC, to quantitatively assess the influence of site-specific phosphorylation on the turnover of thousands of proteins in live cells (1). We discovered many phosphosites prolonging protein lifetime, challenging the previous notion that phosphorylation mostly accelerates protein degradation. Extending this research analyzing steady-state cells, we herein aimed to characterize how the timing and duration of phosphorylation signaling dedicate cell phenotypes. In a classic signaling system, the differential stimulation of epidermal and nerve growth factors (EGF and NGF) can determine the cell fate. Whereas EGF “transiently” stimulates the mitogen-activated protein (MAP) kinases, ERK1 and ERK2, and provokes cellular proliferation, NGF stimulation induces “sustained” activation of MAPKs and cell differentiation. Herein, we applied both DeltaSILAC and label-free phosphoproteomics. We developed DeltaSILAC with a pulse labeling using stable isotope-labeled amino acids in cells (pSILAC) accompanying the EGF and NGF stimulations on PC12 cells. Our time-course design embraced both short- and long-term points: 0-sec, 15-sec, 30-sec, 1-min, 2-min, 5-min, 10-min, 30-min, 1-hour, 2 hour, 4-hour, 8-hour, 12-hour, 24-hour, 48-hour, and 72-hour. We also generated matching protein- and mRNA- profiles after 30 mins following stimulations, so that phosphorylation regulations can be dissected from gene expression dynamics. We utilized a novel peptide-level matching algorithm so that both abundance and lifetime can be measured and resolved for each phosphosite. By using a reproducible mass spectrometry method named data-independent acquisition (DIA-MS), collectively, we quantified 40,000 phosphopeptides and 9,000 proteins throughout the experiment, both globally recapitulated the “transient” and “sustained” patterns of EGF and NGF signaling. We discovered specific tyrosine phosphorylation as early as 15-30 seconds. Interesting EGFR phosphosites activated by NGF treatment were ascribed to P38 kinase activity. The modeling of our unique time-series datasets of long-term time points indicates that phosphosites involved in EGF/NGF signaling transduction and mRNA processing shortened the corresponding protein lifetime, whereas phosphosites in the apoptotic process stabilized protein expression. We conclude that while early phosphorylation events initiate signaling functions through phosphate-dependent transfer, the longer-lasting phosphorylation events could shape proteome stability and proteostasis in cell fate determination.1. C. Wu et al., Global and Site-Specific Effect of Phosphorylation on Protein Turnover . Developmental cell 10.1016/j.devcel.2020.10.025 (2020).