Identification of an N-terminal glycogen synthase kinase 3 phosphorylation site which regulates the functional localization of polycystin-2 in vivo and in vitro.
PKD2 is mutated in 15% of patients with autosomal dominant polycystic kidney disease. Polycystin-2 (PC2), the PKD2 protein, is a non-selective Ca(2+)-permeable cation channel which may function at the cell surface and ER. Nevertheless, the factors that regulate the dynamic translocation of PC2 between the ER and other compartments are not well understood. Constitutive phosphorylation of PC2 at a single C-terminal site (Ser(812)) has been previously reported. As we were unable to abolish phospholabelling of PC2 in HEK293 cells by site-directed mutagenesis of Ser(812) or all five predicted phosphorylation sites in the C-terminus, we hypothesized that PC2 could also be phosphorylated at the N-terminus. In this paper, we report the identification of a new phosphorylation site for PC2 within its N-terminal domain (Ser(76)) and demonstrate that this residue is phosphorylated by glycogen synthase kinase 3 (GSK3). The consensus recognition sequence for GSK3 (Ser(76)/Ser(80)) is evolutionarily conserved down to lower vertebrates. In the presence of specific GSK3 inhibitors, the lateral plasma membrane pool of endogenous PC2 redistributes into an intracellular compartment in MDCK cells without any change in primary cilia localization. Finally, co-injection of wild-type but not a S76A/S80A mutant PKD2 capped mRNA could rescue the cystic phenotype induced by an antisense morpholino oligonucleotide to pkd2 in zebrafish pronephric kidney. We conclude that surface localization of PC2 is regulated by phosphorylation at a unique GSK3 site in its N-terminal domain in vivo and in vitro. This site is functionally significant for the maintenance of normal glomerular and tubular morphology.
Academic Nephrology Unit, Sheffield Kidney Institute, Division of Clinical Sciences, North, University of Sheffield, Sheffield, UK.
Andrew J
AJ
Streets
David J
DJ
Moon
Michelle E
ME
Kane
Tomoko
T
Obara
Albert C M
AC
Ong
D000818:Animals; D002460:Cell Line; D004285:Dogs; D038362:Glycogen Synthase Kinase 3 / Q000502:physiology*; D006801:Humans; D010446:Peptide Fragments / Q000378:metabolism*; D010766:Phosphorylation; D016891:Polycystic Kidney, Autosomal Dominant / Q000201:enzymology / Q000235:genetics*; D017434:Protein Structure, Tertiary; D050396:TRPP Cation Channels / Q000737:chemistry* / Q000378:metabolism*; D015027:Zebrafish
D038362:Glycogen Synthase Kinase 3; D010446:Peptide Fragments; D050396:TRPP Cation Channels