Phosphoproteome Profiling Revealed the Importance of mTOR Inhibition on CDK1 Activation to Further Regulate Cell Cycle Progression

The
mammalian target of rapamycin (mTOR) functions as a critical
regulator of cell cycle progression. However, the underlying mechanism
by which mTOR regulates cell cycle progression remains elusive. In
this study, we used stable isotope labeling of amino acids in cell
culture with a two-step strategy for phosphopeptide enrichment and
high-throughput quantitative mass spectrometry to perform a global
phosphoproteome analysis of mTOR inhibition by rapamycin. By monitoring
the phosphoproteome alterations upon rapamycin treatment, downregulation
of mTOR signaling pathway was detected and enriched. Further functional
analysis of phosphoproteome revealed the involvement of cell cycle
events. Specifically, the elevated profile of cell cycle-related substrates
was observed, and the activation of CDK1, MAPK1, and MAPK3 kinases
was determined. Second, pathway interrogation using kinase inhibitor
treatment confirmed that CDK1 activation operated downstream from
mTOR inhibition to further regulate cell cycle progression. Third,
we found that the activation of CDK1 following 4–12 h of mTOR
inhibition was accompanied by the activation of the Greatwall–endosulfine
complex. In conclusion, we presented a high-confidence phosphoproteome
map inside the cells upon mTOR inhibition by rapamycin. Our data implied
that mTOR inhibition could contribute to CDK1 activation for further
regulating cell cycle progression, which was mediated by the Greatwall–endosulfine
complex.