ABSTRACT
ß-Catenin-dependent WNT signal transduction governs development, tissue homeostasis, and a vast array of human diseases. Signal propagation through a WNT-Frizzled/LRP receptor complex requires proteins necessary for clathrin-mediated endocytosis (CME). Paradoxically, CME also negatively regulates WNT signaling through internalization and degradation of the receptor complex. Here, using a gain-of-function screen of the human kinome, we report that the AP2 associated kinase 1 (AAK1), a known CME enhancer, inhibits WNT signaling. Reciprocally, AAK1 genetic silencing or its pharmacological inhibition using a potent and selective inhibitor activates WNT signaling. Mechanistically, we show that AAK1 promotes clearance of LRP6 from the plasma membrane to suppress the WNT pathway. Time-course experiments support a transcription-uncoupled, WNT-driven negative feedback loop; prolonged WNT treatment drives AAK1-dependent phosphorylation of AP2M1, clathrin-coated pit maturation, and endocytosis of LRP6. We propose that, following WNT receptor activation, increased AAK1 function and CME limits WNT signaling longevity.
Subject(s)
Clathrin/metabolism , Endocytosis/physiology , Low Density Lipoprotein Receptor-Related Protein-6/metabolism , Protein Serine-Threonine Kinases/metabolism , Wnt Proteins/metabolism , Wnt Signaling Pathway/physiology , Animals , Feedback, Physiological , HEK293 Cells , Humans , Male , Mice , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitorsABSTRACT
The transcription factor FOXP1 (forkhead box protein P1) is a master regulator of stem and progenitor cell biology. In diffuse large B cell lymphoma (DLBCL), copy number amplifications and chromosomal translocations result in overexpression of FOXP1. Increased abundance of FOXP1 in DLBCL is a predictor of poor prognosis and resistance to therapy. We developed a genome-wide, mass spectrometry-coupled, gain-of-function genetic screen, which revealed that FOXP1 potentiates ß-catenin-dependent, Wnt-dependent gene expression. Gain- and loss-of-function studies in cell models and zebrafish confirmed that FOXP1 was a general and conserved enhancer of Wnt signaling. In a Wnt-dependent fashion, FOXP1 formed a complex with ß-catenin, TCF7L2 (transcription factor 7-like 2), and the acetyltransferase CBP [CREB (adenosine 3',5'-monophosphate response element-binding protein)-binding protein], and this complex bound the promoters of Wnt target genes. FOXP1 promoted the acetylation of ß-catenin by CBP, and acetylation was required for FOXP1-mediated potentiation of ß-catenin-dependent transcription. In DLBCL, we found that FOXP1 promoted sensitivity to Wnt pathway inhibitors, and knockdown of FOXP1 or blocking ß-catenin transcriptional activity slowed xenograft tumor growth. These data connect excessive FOXP1 with ß-catenin-dependent signal transduction and provide a molecular rationale for Wnt-directed therapy in DLBCL.
