GSK3β-Dzip1-Rab8 cascade regulates ciliogenesis after mitosis

PLoS Biol. 2015 Apr 10;13(4):e1002129. doi: 10.1371/journal.pbio.1002129. eCollection 2015 Apr.

Abstract

The primary cilium, which disassembles before mitotic entry and reassembles after mitosis, organizes many signal transduction pathways that are crucial for cell life and individual development. However, how ciliogenesis is regulated during the cell cycle remains largely unknown. Here we show that GSK3β, Dzip1, and Rab8 co-regulate ciliogenesis by promoting the assembly of the ciliary membrane after mitosis. Immunofluorescence and super-resolution microscopy showed that Dzip1 was localized to the periciliary diffusion barrier and enriched at the mother centriole. Knockdown of Dzip1 by short hairpin RNAs led to failed ciliary localization of Rab8, and Rab8 accumulation at the basal body. Dzip1 preferentially bound to Rab8GDP and promoted its dissociation from its inhibitor GDI2 at the pericentriolar region, as demonstrated by sucrose gradient centrifugation of purified basal bodies, immunoprecipitation, and acceptor-bleaching fluorescence resonance energy transfer assays. By means of in vitro phosphorylation, in vivo gel shift, phospho-peptide identification by mass spectrometry, and GST pulldown assays, we demonstrated that Dzip1 was phosphorylated by GSK3β at S520 in G0 phase, which increased its binding to GDI2 to promote the release of Rab8GDP at the cilium base. Moreover, ciliogenesis was inhibited by overexpression of the GSK3β-nonphosphorylatable Dzip1 mutant or by disabling of GSK3β by specific inhibitors or knockout of GSK3β in cells. Collectively, our data reveal a unique cascade consisting of GSK3β, Dzip1, and Rab8 that regulates ciliogenesis after mitosis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing
  • Animals
  • Cilia*
  • DNA-Binding Proteins / physiology*
  • Glycogen Synthase Kinase 3 / physiology*
  • Glycogen Synthase Kinase 3 beta
  • Mice
  • Mitosis*
  • NIH 3T3 Cells
  • Phosphorylation
  • rab GTP-Binding Proteins / physiology*

Substances

  • Adaptor Proteins, Signal Transducing
  • DNA-Binding Proteins
  • Dzip1 protein, mouse
  • Rab8 protein, mouse
  • Glycogen Synthase Kinase 3 beta
  • Gsk3b protein, mouse
  • Glycogen Synthase Kinase 3
  • rab GTP-Binding Proteins

Grant support

This work was supported by funds to CMZ from the State Key Basic Research and Development Plan (2014CB138402 and 2010CB833705) and the National Natural Science Foundation of China (NSFC, www.nsfc.gov.cn) (31430051, 31371365, 91313302 and 31030044). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.