Abstract: FR-PO959
Homeostasis of Phosphate Kinase PIPKIγ and Phosphatase INPP5E Modulates the Ciliary Accumulation of Polycystin2 in ADPKD
Session Information
- PKD Cellular Pathogenesis, ARPKD, ADTKD, Ciliopathies
October 26, 2018 | Location: Exhibit Hall, San Diego Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Genetic Diseases of the Kidney
- 1001 Genetic Diseases of the Kidney: Cystic
Author
- Chen, Chuan, Mayo Clinic, Rochester, Minnesota, United States
Background
Primary cilia defects lead to a panel of genetic human disorders, collectively termed as ciliopathies. Polycystin2 as a transmembrane protein functions to interact with PKD1 and maintains normal orientationof tubular architecture. Mutations in either PKD1 or PKD2 causes autosomal dominant polycystic kidney disease (ADPKD), which is also considered to be among the ciliopathy diseases.
The function of phosphoinositides in mediating acute response, and acting as constitutive signals to help define organelle identity, are also emphasized by various human diseases. Recently, several studies implicated the involvement of PI species in ciliopathies. Also, Homeostasis of Phosphoinositide PI(4)P and PI(4,5)P2 functions significantly in cilia, and mutations in the phosphatases INPP5E can significantly increase ciliary PI(4,5)P2. But the relationship between regulated homoeostasis of PIs and ciliopathy proteins still remain to explore in the future.
Methods
Cell culture and transfection of DNA constructs and siRNA
Immunofluorescence microscopy
Immunoprecipitation and pull-down assay
Results
Phosphatidylinositol phosphate kinase PIPKIγ and phosphatase INPP5E cordinate to regulate homeostasis of PI(4)P and PI(4,5)P2 in cilia. Recently, we discovered In INPP5E knock-out MEF cells, endogenous ciliary pool of polycystin2 is significantly increased, while PIPKIγ depletion can partially reduce the accumulation of polycystin2. Also, in GANAB cell which always exhibits the absent ciliary localization of polycystin2, after knocking down INPP5E, we also observed increased PC2 accumulation. To verify this observation, we treated GANAB cell with the INPP5E inhibitor and found similar ciliary accumulation of PC2 after 24h treatment. As mutations in INPP5E increased ciliary PI(4,5)P2, we hypothesized that the enriched PI(4,5)P2 promoted PC2 accumulation. By overexpressing PIPKIγ kinase domain or its activator protein HYLS1 in GANAB cells, the increased PC2 signal in cilia also convinced our hypothesis.
Conclusion
By activating PIPKIr or inhibiting INPP5E, we found enriched PI(4.5)P2 is associated with ciliary PC2 accumulation. As reduced level of PC2 can always lead to kidney disease ADPKD, increased dosage of ciliary PC2 by PI(4,5)P2 may provide some new targets to understand the relationship between PIs and kidney diseases and also new methods for studying ADPKD.