Wnt/beta-catenin signaling controls a variety of cellular processes, including cell growth, oncogenesis, and development. Upon Wnt stimulation, the intracellular region of the coreceptor, LRP6 or 5, is phosphorylated by the membrane. recruited GSK3 beta and CK1. The cytoplasmic domain of LRP6/5 contains one Ser/Thr cluster and the PPPSP motifs, both of which are essential for propagation of the signal. While the phosphorylated PPPSP motifs are known to directly inhibit GSK3 beta, the biochemical role of the phosphorylated Ser/Thr cluster remains to be elucidated. Herein, we reveal that the Ser/Thr cluster plays an important role in the phosphorylation of the PPPSP motif. Interestingly, we observe that GSK3 beta activity oil the PPPSP motif requires a high ATP, concentration, close to that of the physiological condition. Taken together. these data suggest that the phosphorylated Ser/Thr Cluster Serves as a docking site for GSK3 beta to promote the phosphorylation of the PPPSP Motif. Our results provide insight into the molecular mechanism for the initial events of the Wnt/beta-catenin signaling. (C) 2009 Elsevier Inc. All rights reserved.