The small GTPase Cdc42 is necessary for primary ciliogenesis in renal tubular epithelial cells

J Biol Chem. 2011 Jun 24;286(25):22469-77. doi: 10.1074/jbc.M111.238469. Epub 2011 May 4.

Abstract

Primary cilia are found on many epithelial cell types, including renal tubular epithelial cells, where they participate in flow sensing. Disruption of cilia function has been linked to the pathogenesis of polycystic kidney disease. We demonstrated previously that the exocyst, a highly conserved eight-protein membrane trafficking complex, localizes to primary cilia of renal tubular epithelial cells, is required for ciliogenesis, biochemically and genetically interacts with polycystin-2 (the protein product of the polycystic kidney disease 2 gene), and, when disrupted, results in MAPK pathway activation both in vitro and in vivo. The small GTPase Cdc42 is a candidate for regulation of the exocyst at the primary cilium. Here, we demonstrate that Cdc42 biochemically interacts with Sec10, a crucial component of the exocyst complex, and that Cdc42 colocalizes with Sec10 at the primary cilium. Expression of dominant negative Cdc42 and shRNA-mediated knockdown of both Cdc42 and Tuba, a Cdc42 guanine nucleotide exchange factor, inhibit ciliogenesis in Madin-Darby canine kidney cells. Furthermore, exocyst Sec8 and polycystin-2 no longer localize to primary cilia or the ciliary region following Cdc42 and Tuba knockdown. We also show that Sec10 directly interacts with Par6, a member of the Par complex that itself directly interacts with Cdc42. Finally, we show that Cdc42 knockdown results in activation of the MAPK pathway, something observed in cells with dysfunctional primary cilia. These data support a model in which Cdc42 localizes the exocyst to the primary cilium, whereupon the exocyst then targets and docks vesicles carrying proteins necessary for ciliogenesis.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Adaptor Proteins, Signal Transducing / deficiency
  • Adaptor Proteins, Signal Transducing / genetics
  • Adaptor Proteins, Signal Transducing / metabolism
  • Animals
  • Cilia / metabolism*
  • Cytoskeletal Proteins / metabolism
  • Dogs
  • Enzyme Activation
  • Epithelial Cells / cytology*
  • Epithelial Cells / enzymology*
  • Epithelial Cells / metabolism
  • Gene Knockdown Techniques
  • Humans
  • Kidney Tubules / cytology*
  • MAP Kinase Signaling System
  • Mitogen-Activated Protein Kinases / metabolism
  • Protein Transport
  • RNA, Small Interfering / genetics
  • Vesicular Transport Proteins / deficiency
  • Vesicular Transport Proteins / genetics
  • Vesicular Transport Proteins / metabolism
  • cdc42 GTP-Binding Protein / metabolism*

Substances

  • Adaptor Proteins, Signal Transducing
  • Cytoskeletal Proteins
  • EXOC5 protein, human
  • RNA, Small Interfering
  • Vesicular Transport Proteins
  • Mitogen-Activated Protein Kinases
  • cdc42 GTP-Binding Protein