Quantitative Phosphoproteome Profiling of Wnt3a-mediated Signaling Network INDICATING THE INVOLVEMENT OF RIBONUCLEOSIDE-DIPHOSPHATE REDUCTASE M2 SUBUNIT

The complexity of canonical Wnt signaling comes not only from the numerous components but also from multiple post-translational modifications. Protein phosphorylation is one of the most common modifications that propagates signals from extracellular stimuli to downstream effec-tors. To investigate the global phosphorylation regulation and uncover novel phosphoproteins at the early stages of canonical Wnt signaling, HEK293 cells were metabolically labeled with two stable isotopic forms of lysine and were stimulated for 0, 1, or 30 min with purified Wnt3a. After phosphoprotein enrichment and LC-MS/MS analysis, 1057 proteins were identified in all three time points. In total 287 proteins showed a 1.5-fold or greater change in at least one time point. In addition to many known Wnt signaling transducers, other phosphoproteins were iden-tified and quantitated, implicating their involvement in ca-nonical Wnt signaling. k-Means clustering analysis showed dynamic patterns for the differential phosphopro-teins. Profile pattern and interaction network analysis of the differential phosphoproteins implicated the possible roles for those unreported components in Wnt signaling. Moreover 100 unique phosphorylation sites were identi-fied, and 54 of them were quantitated in the three time points. Site-specific phosphopeptide quantitation re-vealed that Ser-20 phosphorylation on RRM2 increased upon 30-min Wnt3a stimulation. Further studies with mu-tagenesis, the Wnt reporter gene assay, and RNA inter-ference indicated that RRM2 functioned downstream of -catenin as an inhibitor of Wnt signaling and that Ser-20 phosphorylation of RRM2 counteracted its inhibition ef-fect. Our systematic profiling of dynamic phosphorylation changes responding to Wnt3a stimulation not only pre-sented a comprehensive phosphorylation network regu-lated by canonical Wnt signaling but also found novel molecules and phosphorylation involved in Wnt signaling