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Abstract: FR-PO568

Dephosphorylation Facilitates Trafficking of Mutant Polycystin-2 to Cilia

Session Information

Category: Genetic Diseases of the Kidneys

  • 1201 Genetic Diseases of the Kidneys: Cystic

Authors

  • Cai, Yiqiang, Yale University Department of Internal Medicine, New Haven, Connecticut, United States
  • Dong, Ke, Yale University Department of Internal Medicine, New Haven, Connecticut, United States
  • Spitzer, Max, Yale University Department of Internal Medicine, New Haven, Connecticut, United States
  • Geiges, Linda, Yale University Department of Internal Medicine, New Haven, Connecticut, United States
  • Tian, Xin, Yale University Department of Internal Medicine, New Haven, Connecticut, United States
  • Krappitz, Matteus, Yale University Department of Internal Medicine, New Haven, Connecticut, United States
  • Diggs, Lonnette, Yale University Department of Internal Medicine, New Haven, Connecticut, United States
  • Wei, Zemeng, Yale University Department of Internal Medicine, New Haven, Connecticut, United States
  • Cordido, Adrian, Yale University Department of Internal Medicine, New Haven, Connecticut, United States
  • Pei, Steven Lim Cho, Yale University Department of Internal Medicine, New Haven, Connecticut, United States
  • Fedeles, Sorin V., Yale University Department of Internal Medicine, New Haven, Connecticut, United States
  • Somlo, Stefan, Yale University Department of Internal Medicine, New Haven, Connecticut, United States
Background

ADPKD is characterized by mutations in polycystin-1 (PC1) and polycystin-2 (PC2) that result in progressive cyst formation in the kidneys and liver. The steady state expression and ciliary trafficking of PC1 requires the presence of PC2. Missense mutations resulting in alterations in trafficking properties of PC1 and PC2 underlie a subset of cases of ADPKD. Here, we investigate the role of phosphorylation of PC2 in its localization to cilia.

Methods

Unbiased phosphoproteomic analysis of mouse kidney lysates was performed to identify the phosphorylation sites of native PC2. mNeonGreen-tagged PC2 (PC2NG) constructs of wild type or dephosphorylated forms were generated and expressed in IMCD3 cells lacking endogenous Pkd1 or Pkd2 or both. Ciliary expression of PC2 was analyzed via live-cell imaging and by immunofluorescent cell staining. Knock-in mice carrying dephosphorylated Pkd2S829A and a novel anti-phospho-specific PC2 antibody were generated and utilized as part of this study.

Results

Two phosphorylation sites in native PC2 were identified from native protein in mouse kidneys: Ser812 and Ser829. We investigated the functional effects of Ser829 phosphorylation. We found that expression of PC2 in cilia was regulated by phosphorylation at Ser829. While dephosphorylation at the Ser829 was not a prerequisite for PC2 ciliary expression, the phosphorylation deficient Ser829Ala (S829A) form of PC2 showed quantitatively enhanced expression in cilia that did not require the presence of PC1. Mice carrying a homozygous knockin phosphorylation deficient Pkd2S829A allele have no phenotype indicating that the absence of phosphorylation at Ser829 does not result in reduced PC2 function. Introduction of S829A into a PC2 construct containing a human pathogenic mutant that otherwise does not appear cilia resulted in expression in cilia, indicated that dephosphorylation rescued the trafficking defect of the human mutation.

Conclusion

Dephosphorylation of PC2 at Ser829 retains normal function in vivo. Dephosphorylation at Ser829 enhances the steady state expression of PC2 in cilia and it overcomes cilia trafficking defect of a human pathogenic missense mutant of PC2. The phosphorylation state of PC2 has a functional role in expression of the polycystin complex in cilia.

Funding

  • NIDDK Support