Quantitative Phosphoproteome Profiling of Wnt3a-mediated Signaling Network INDICATING THE INVOLVEMENT OF RIBONUCLEOSIDE-DIPHOSPHATE REDUCTASE M2 SUBUNIT PHOSPHORYLATION AT RESIDUE SERINE 20 IN CANONICAL Wnt SIGNAL TRANSDUCTION*□S

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 effectors. 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 identified and quantitated, implicating their involvement in canonical Wnt signaling. k-Means clustering analysis showed dynamic patterns for the differential phosphoproteins. 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 identified, and 54 of them were quantitated in the three time points. Site-specific phosphopeptide quantitation revealed that Ser-20 phosphorylation on RRM2 increased upon 30-min Wnt3a stimulation. Further studies with mutagenesis, the Wnt reporter gene assay, and RNA interference indicated that RRM2 functioned downstream of -catenin as an inhibitor of Wnt signaling and that Ser-20 phosphorylation of RRM2 counteracted its inhibition effect. Our systematic profiling of dynamic phosphorylation changes responding to Wnt3a stimulation not only presented a comprehensive phosphorylation network regulated by canonical Wnt signaling but also found novel molecules and phosphorylation involved in Wnt signaling. Molecular & Cellular Proteomics 6:1952–1967, 2007. The Wnt family of secreted signaling molecules is highly conserved among animal species and has been implicated in three major pathways, the canonical pathway and the two noncanonical pathways including planar cell polarity and calcium pathway (1). The canonical Wnt signaling pathway plays an important role in developmental processes including cell adhesion (2), morphology (3), proliferation (4), and migration (5). Mutational deregulation of the Wnt cascade is closely associated with various tumors and other diseases (6, 7). Protein phosphorylation is one of the most important mechanisms for signaling propagation, and there is no exception for canonical Wnt signal transduction (8–13). Many regulators or downstream components in the canonical Wnt signaling pathway are known to be phosphorylated or dephosphorylated in the transduction of signals from extracellular Wnt stimulation to intracellular effectors. These include Wnt receptors low density lipoprotein receptor-related protein-6 (8) and Frizzled 3 (9) and signaling components glycogen synthase kinase-3 (GSK3 ) (10), adenomatous polyposis coli protein (APC) (11), Dishevelled (Dvl) (12), and -catenin (13). For example, as a key component in canonical Wnt signaling, -catenin is phosphorylated by serine/threonine kinases in the absence of Wnt stimulation, and the hyperphosphorylation leads to its ubiquitination and degradation by the proteasome. When Wnt is present, -catenin is free from phospho-