Screening for target Rabs of TBC (Tre-2/Bub2/Cdc16) domain-containing proteins based on their Rab-binding activity

Genes Cells. 2006 Sep;11(9):1023-37. doi: 10.1111/j.1365-2443.2006.00997.x.

Abstract

It has recently been proposed that the TBC (Tre2/Bub2/Cdc16) domain functions as a GAP (GTPase-activating protein) domain for small GTPase Rab. Because of the large number of Rab proteins in mammals, however, most TBC domains have never been investigated for Rab-GAP activity. In this study we established panels of the GTP-fixed form of 60 different Rabs constructed in pGAD-C1, a yeast two-hybrid bait vector. We also constructed a yeast two-hybrid prey vector (pGBDU-C1) that harbors the cDNA of 40 distinct TBC proteins. Systematic investigation of 2400 combinations of 60 GTP-fixed Rabs and 40 TBC proteins by yeast two-hybrid screening revealed that seven TBC proteins specifically and differentially interact with specific Rabs (e.g. OATL1 interacts with Rab2A; FLJ12085 with Rab5A/B/C; and Evi5-like with Rab10). Measurement of in vitro Rab-GAP activity revealed that OATL1 and Evi5-like actually possess significant Rab2A- and Rab10-GAP activity, respectively, but that FLJ12085 do not display Rab5A-GAP activity at all. These results indicate that specific interaction between TBC protein and Rab would be a useful indicator for screening for the target Rabs of some TBC/Rab-GAP domains, but that there is little correlation between the Rab-binding activity and Rab-GAP activity of other TBC proteins.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • COS Cells
  • Cell Cycle Proteins / chemistry*
  • Cell Cycle Proteins / metabolism*
  • Chlorocebus aethiops
  • GTPase-Activating Proteins / metabolism
  • Humans
  • Mice
  • Molecular Sequence Data
  • NIH 3T3 Cells
  • Nuclear Proteins / metabolism
  • Phylogeny
  • Protein Binding
  • Protein Structure, Tertiary
  • Transcription Factors / metabolism
  • Two-Hybrid System Techniques
  • rab GTP-Binding Proteins / metabolism*

Substances

  • Cell Cycle Proteins
  • EVI5 protein, human
  • Evi5 protein, mouse
  • GTPase-Activating Proteins
  • Nuclear Proteins
  • Transcription Factors
  • rab GTP-Binding Proteins

Associated data

  • GENBANK/AB232583
  • GENBANK/AB232584
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