Identification of an N-terminal glycogen synthase kinase 3 phosphorylation site which regulates the functional localization of polycystin-2 in vivo and in vitro

Abstract

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.

Cell biology of polycystin-2

“…Injection of wild type PKD2 cRNA had no significant effect on channel activity, whereas injection of the S812A mutant resulted in large Na + currents mediated by PKD2. In support of the idea that protein phosphorylation may regulate the amount of PKD2 in the plasma membrane, Streets et al [39] showed that constitutive phosphorylation of S76 of human PKD2 by GSK3 was necessary for its targeting to the plasma membrane (Fig. 2).…”

“…It is now accepted that PKD2 can function at the plasma membrane, but its activity there is under complex regulation involving shuttling between ER and plasma membrane, protein-protein interactions, and modes of activation. Specifically, it has been shown that the amount of PKD2 in the plasma membrane is dynamically regulated by interacting proteins (PKD1 [29,35], Polycystin-2 interactor, Golgi-and ER-associated protein-14 (PIGEA-14) [37]), posttranslational modifications (serine phosphorylation by casein kinase 2 (CK2) [38] and glycogen synthase kinase 3 (GSK3) [39]), interaction with other channel subunits in the plasma membrane (PKD1 [29,35,[40][41][42], TRPC1 [11], TRPV4 [43]) (Fig. 2), and finally, activation secondary to cell surface receptor activation (epidermal growth factor receptor (EGFR) [36]).…”

“…2). Mutation of S76 (or S80 in zebrafish) to A resulted in PKD2 accumulation in intracellular compartments and failed to rescue the cystic phenotype in zebrafish injected with a pkd2-specific antisense morpholino oligonucleotide [39]. Interestingly, the S76/S80 mutation did not affect the ciliary expression of PKD2, questioning the functional role of PKD2 in the primary cilium in zebrafish.…”

The cAMP Signaling Pathway and Direct Protein Kinase A Phosphorylation Regulate Polycystin-2 (TRPP2) Channel Function

“…Kinase phosphorylation of PC2 has been implicated in the selectivity of the channel to Ca 2ϩ signals (14), and regulates trafficking events that target the channel protein to various cellular domains (15). A stretch of acidic amino acids in the carboxyl terminus of PC2 mediates the interaction with PACS-1 and PACS-2 (16).…”

“…At least two evolutionarily conserved phosphorylation sites in PC2 were suggested to control its subcellular localization: serine residue 76 (Ser-76)/Ser-80 and Ser-812, which are phosphorylated by glycogen synthase kinase 3 (GSK3) (15) and casein kinase 2 (CK-2) (14), respectively. It has also been shown that PKC-dependent phosphorylation at Ser-801 is essential for the normal function of PC2 as an ER Ca 2ϩ release channel (22).…”