PROJECT SUMMARY
WNT signaling is crucial for embryonic development and adult tissue homeostasis, with aberrant signaling
resulting in developmental disorders and disease, including cancer. Although much is known, a deeper
mechanistic understanding of this signaling cascade will improve our understanding of cancer formation,
progression and metastasis, allowing for the development of more effective therapeutics. WNT/b-catenin
signaling is driven by the stabilization of the transcriptional co-activator, b-catenin. In the absence of WNT ligand,
a cytosolic destruction complex phosphorylates, ubiquitylates and degrades b-catenin. In the presence of WNT
ligand, the WNT receptors, Frizzled and LRP6, and intracellular proteins form an alternative complex called the
WNT signalosome. This results in b-catenin accumulation and activation of b-catenin target genes. Recent data
demonstrate that upon WNT ligand engagement, the signalosome is endocytosed. Although conflicting data
exist within the literature, a consensus is beginning to emerge that clathrin-dependent endocytosis of the
signalosome results in signalosome degradation. This training proposal and my thesis project is devoted to
elucidating the molecular events and dynamics of signalosome formation, stabilization and endocytosis in normal
cells and in cancer, with an emphasis on kinases. In the first half of my graduate training, I utilized a gain-of-
function screen of the kinome to identify AAK1 as a negative regulator of WNT signaling. I demonstrated that
AAK1 activates a transcription independent negative feedback loop to promote LRP6 internalization, resulting in
WNT signaling downregulation. In the course of these studies, we demonstrated that AAK1 promotes the
phosphorylation of a clathrin adapter protein, AP2M1, 8-10 hrs post-WNT3A and that AAK1 and AP2M1 interact
with the tumor suppressor, WTX. My lab previously discovered the WTX tumor suppressor as a component of
the signalosome and b-catenin destruction complex. Therefore, I will define comprehensive WNT3A and WTX-
dependent changes to the phosphoproteome by quantitative mass spectrometry and test whether WTX regulates
signalosome endocytosis via AAK1. Additionally, CSNK1g is known to regulate phosphorylation of LRP6, an
essential step for signalosome formation. CSNK1g has 3 isoforms, CSNK1g1/2/3, all identified as understudied
kinases. My preliminary data suggest each isoform functions differently to activate WNT signaling and promote
LRP6 internalization. A main focus for the remainder of my graduate work will be to functionally characterize the
role of each CSNK1g isoform in regulating WNT signaling, define comprehensive protein-protein interaction
networks and evaluate isoform specific changes to the WNT-driven phosphoproteome. Because this work is
descriptive in nature, I expect it to be submitted for publication in 14 months. To summarize, the precise role of
endocytosis in WNT signaling remains unclear, with numerous questions surrounding the mechanism(s) and
components of endocytosis and its effects on signaling. This work, and my future postdoctoral work, will provide
me training in and experience in the mechanisms of WNT signaling and feedback attenuation.