Brown adipose receives extensive attention for dissipating stored chemical energy in heat via nonshivering thermogenesis. However, whether and how the dynamics of key transcription factors (TFs) phosphorylation contributes to initial activation of thermogenesis remains unknown. Here, we employed the stat-of-the-art data-independent acquisition (DIA) mass spectrometry to explore the global and temporal phosphorylation profiles of signaling networks to elucidate mechanism responsible for the TF activation for thermogenesis gene transcription. The nucleus was isolated before and after stimulation for 5, 10, 20, 30, 40, 45, 50, 55, 60, 70 and 80 min with NE. Consequently, 401 transcription regulators bearing 1007 phosphosites were identified with high reproducibility across 12 time points. Firstly, the UCP1 transcriptional level was measured, which began at 30min, then rose until 70min to the peakedness. The global phosphoproteome displayed dramatic changes early to 20-30min, which could be classified into two dynamic clusters, namely UCP1 pre-transcription phase and transcription phase. The former changed from 5min, and reached the extremum at 20-40min, which functioned before UCP1 action. Some well- characterized transcriptional regulators of UCP1 were founded to be phosphorylated, such as PRDM16 and RIP140. Alternatively, other phosphorylation events of TFs such as Jun and ATF2 began from 30min, and maintained until 70min, almost synchronizing with UCP1 transcription. Intriguingly, we found THRAP3 posed multiple phosphosites but involving in different regulation patterns. This might indicate that THRAP3 had distinct transcriptional regulation and functions upon thermogenesis. We, for the first time, uncovered temporal phosphoregulation profiles of TFs, which might be potentially required for initial activation of thermogenesis in brown fat. A new sight was provided to increase energy expenditure for treatment of obesity and other metabolic syndrome.
X. Gao: None.