Kinesin-2 mediates physical and functional interactions between polycystin-2 and fibrocystin

Abstract

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in PKD1, encoding polycystin-1 (PC1), or PKD2 (polycystin-2, PC2). Autosomal recessive PKD (ARPKD) is caused by mutations in PKHD1, encoding fibrocystin/polyductin (FPC). No molecular link between ADPKD and ARPKD has been determined. Here, we demonstrated, by yeast two-hybrid and biochemical assays, that KIF3B, a motor subunit of kinesin-2, associates with PC2 and FPC. Co-immunoprecipitation experiments using Madin-Darby canine kidney (MDCK) and inner medullary collecting duct (IMCD) cells and human kidney revealed that PC2 and KIF3B, FPC and KIF3B and, furthermore, PC2 and FPC are endogenously in the same complex(es), though no direct association between the PC2 and FPC intracellular termini was detected. In vitro binding and Far Western blot experiments demonstrated that PC2 and FPC are in the same complex only if KIF3B is present, presumably by forming a PC2-KIF3B-FPC complex. This was supported by our observation that altering KIF3B level in IMCD cells by over-expression or siRNA significantly affected complexing between PC2 and FPC. Immunofluorescence experiments showed that PC2, FPC and KIF3B partially co-localized in primary cilia of over-confluent and perinuclear regions of sub-confluent cells. Furthermore, KIF3B mediated functional modulation of purified PC2 channels by FPC in a planer lipid bilayer electrophysiology system. The FPC C-terminus substantially stimulated PC2 channel activity in the presence of KIF3B, whereas FPC or KIF3B alone had no effect. Taken together, we discovered that kinesin-2 is a linker between PC2 and FPC and mediates the regulation of PC2 channel function by FPC. Our study may be important for elucidating common molecular pathways for PKD of different genotypes.

Biliary and Pancreatic Dysgenesis in Mice Harboring a Mutation in Pkhd1

“…44 Fibrocystin is localized to the cilium and has been recently found in the same protein complex as PC2. 18,45 This suggests that fibrocystin and the autosomal dominant disease proteins may share a common pathway. We addressed the question whether lack of PC1 or PC2 might influence location or level of fibrocystin expression by examining fibrocystic expression in orthologous cystic models because of mutation in either Pkd1 or Pkd2.…”

Ciliary dysfunction in polycystic kidney disease: an emerging model with polarizing potential

“…Studies have shown that polycystin-1, polycystin-2, and fibrocystin localize to the primary cilium. These three proteins appear to be in the same complex (47,48). Furthermore, these proteins have been proposed to have a role in mediating flow dependent mechanosensation (47,(49)(50)(51)(52)(53)(54), suggesting a common pathway in cyst development.…”

Fibrocystin/Polyductin Modulates Renal Tubular Formation by Regulating Polycystin-2 Expression and Function

“…Several in vitro studies from our group and others have shown that FPC and PC2 co-localize to the basal bodies/primary cilia of renal epithelial cells and are able to form a molecular complex and function in the same signaling pathway. 31,36 For further validation of this finding in vivo, the phenotypic effects of transheterozygosity for Pkhd1 and Pkd2 were examined. We intercrossed Pkhd1 ϩ/Ϫ and Pkd2 ϩ/Ϫ mutant mice to produce cohorts of age-matched littermates.…”

“…31,36 In addition, other polyclonal antibodies and mAb were purchased: Anti-acetylated ␣-tubulin, anti-␥-tubulin, anti-␤-actin, anti-Flag, and anti-HA mAb (Sigma, St. Louis, …”

“…31,36,39 For performance of co-IP and Western analysis, the entire intracellular termini of FPC and PC2 were constructed into Flagtagged and HA-tagged pCMV expression vectors 53 and were named FPC-C-HA (amino acids 3872 to 4074), PC2-N-HA (amino acids 1 to 221), and PC2-C-Flag (amino acids 682 to 968). To index cystic disease severity, we calculated the total number of cysts using four histologic sections from each kidney.…”