G Protein-coupled Receptor Kinases Phosphorylate LRP6 in the Wnt Pathway

Jiangbo Wang,Richard T Premont,Robert J Lefkowitz,Wei Chen,Tiffany R Garrison,Minyong Chen,Marc G Caron,Melanie Philipp

doi:10.1074/jbc.m109.047456

Jiangbo Wang, Richard T Premont + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m109.047456

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Abstract

Wnt ligands conduct their functions in canonical Wnt signaling by binding to two receptors, the single transmembrane low density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) and seven transmembrane (7TM) Frizzled receptors. Subsequently, phosphorylation of serine/threonine residues within five repeating signature PPPSP motifs on LRP6 is responsible for LRP6 activation. GSK3β, a cytosolic kinase for phosphorylation of a downstream effector β-catenin, was proposed to participate in such LRP6 phosphorylation. Here, we report a new class of membrane-associated kinases for LRP6 phosphorylation. We found that G protein-coupled receptor kinases 5 and 6 (GRK5/6), traditionally known to phosphorylate and desensitize 7TM G protein-coupled receptors, directly phosphorylate the PPPSP motifs on single transmembrane LRP6 and regulate Wnt/LRP6 signaling. GRK5/6-induced LRP6 activation is inhibited by the LRP6 antagonist Dickkopf. Depletion of GRK5 markedly reduces Wnt3A-stimulated LRP6 phosphorylation in cells. In zebrafish, functional knock-down of GRK5 results in reduced Wnt signaling, analogous to LRP6 knock-down, as assessed by decreased abundance of β-catenin and lowered expression of the Wnt target genes cdx4, vent, and axin2. Expression of GRK5 rescues the diminished β-catenin and axin2 response caused by GRK5 depletion. Thus, our findings identify GRK5/6 as novel kinases for the single transmembrane receptor LRP6 during Wnt signaling.

Highlights

Ceptors: the single transmembrane receptors low density lipoprotein receptor-related proteins 5 and 6 (LRP5 and LRP6)5 and seven transmembrane (7TM) receptors of the Frizzled family [1,2,3,4]
DKK1 inhibited the TOPflash luciferase activity stimulated by co-expression of GRK5 or GRK6 with LRP6, as well as the activity of LRP6 alone (Fig. 1B). These data suggest that GRK5 and GRK6 regulate canonical Wnt signaling in a kinase activity-dependent manner that requires LRP6
We have provided biochemical and physiological evidence to demonstrate that widely expressed GRK5 and GRK6 have an unexpected and important function during Wnt signaling as membrane-bound kinases phosphorylating LRP6

Summary

Introduction

Ceptors: the single transmembrane receptors low density lipoprotein receptor-related proteins 5 and 6 (LRP5 and LRP6)5 and seven transmembrane (7TM) receptors of the Frizzled family [1,2,3,4]. We report that GRK5 and GRK6 function as novel kinases responsible for LRP6 phosphorylation in Wnt signaling. Overexpression of either Wnt1 or LRP6 stimulated TOPflash reporter activity in HEK293 cells (Fig. 1A).

Results

Conclusion