Negative regulation of LRP6 function by casein kinase I epsilon phosphorylation.
ABSTRACT Wnt signaling acts in part through the low density lipoprotein receptor-related transmembrane proteins LRP5 and LRP6 to regulate embryonic development and stem cell proliferation. Up-regulated signaling is associated with many forms of cancer. Casein kinase I epsilon (CKIepsilon) is a known component of the Wnt-beta-catenin signaling pathway. We find that CKIepsilon binds to LRP5 and LRP6 in vitro and in vivo and identify three CKIepsilon-specific phosphorylation sites in LRP6. Two of the identified phosphorylation sites, Ser1420 and Ser1430, influence Wnt signaling in vivo, since LRP6 with mutation of these sites is a more potent activator of both beta-catenin accumulation and Lef-1 reporter activity. Whereas Wnt3a regulates CKIepsilon kinase activity, LRP6 does not, placing CKIepsilon upstream of LRP6. Mutation of LRP6 Ser1420 and Ser1430 to alanine strengthens its interaction with axin, suggesting a mechanism by which CKIepsilon may negatively regulate Wnt signaling. The role of CKIepsilon is therefore more complex than was previously appreciated. Generation of active CKIepsilon may induce a negative feedback loop by phosphorylation of sites on LRP5/6 that modulate axin binding and hence beta-catenin degradation.
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ABSTRACT: The Wnt and Hedgehog signaling pathways are essential for normal development and are misregulated in cancer. The casein kinase family of serine/threonine kinases regulates both pathways at multiple levels. However, it has been difficult to determine whether individual members of this family have distinct functions in vivo, due to their overlapping substrate specificities. In Drosophila melanogaster, photoreceptor differentiation is induced by Hedgehog and inhibited by Wingless, providing a sensitive system in which to identify regulators of each pathway. We used a mosaic genetic screen in the Drosophila eye to identify mutations in genes on the X chromosome required for signal transduction. We recovered mutations affecting the transcriptional regulator CREB binding protein, the small GTPase dynamin, the cytoskeletal regulator Actin-related protein 2, and the protein kinase Casein kinase 1α. Consistent with its reported function in the β-Catenin degradation complex, Casein Kinase 1α mutant cells accumulate β-Catenin and ectopically induce Wingless target genes. In contrast to previous studies based on RNA interference, we could not detect any effect of the same Casein Kinase 1α mutation on Hedgehog signaling. We thus propose that Casein kinase 1α is essential to allow β-Catenin degradation and prevent inappropriate Wingless signaling, but its effects on the Hedgehog pathway are redundant with other Casein kinase 1 family members.Genetics 11/2011; 190(2):601-16. DOI:10.1534/genetics.111.133827 · 4.87 Impact Factor
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ABSTRACT: Wnt proteins are secreted post-translationally modified proteins that signal locally to regulate development and proliferation. The production of bioactive Wnts requires a number of dedicated factors in the secreting cell whose coordinated functions are not fully understood. A screen for small molecules identified inhibitors of vacuolar acidification as potent inhibitors of Wnt secretion. Inhibition of the V-ATPase or disruption of vacuolar pH gradients by diverse drugs potently inhibited Wnt/β-catenin signaling both in cultured human cells and in vivo, and impaired Wnt-regulated convergent extension movements in Xenopus embryos. WNT secretion requires its binding to the carrier protein wntless (WLS); we find that WLS is ER-resident in human cells and WNT3A binding to WLS requires PORCN-dependent lipid modification of WNT3A at serine 209. Inhibition of vacuolar acidification results in accumulation of the WNT3A-WLS complex both in cells and at the plasma membrane. Modeling predictions suggest that WLS has a lipid-binding β-barrel that is similar to the lipocalin-family fold. We propose that WLS binds Wnts in part through a lipid-binding domain, and that vacuolar acidification is required to release palmitoylated WNT3A from WLS in secretory vesicles, possibly to facilitate transfer of WNT3A to a soluble carrier protein.Journal of Cell Science 10/2010; 123(Pt 19):3357-67. DOI:10.1242/jcs.072132 · 5.33 Impact Factor