All members of the EPI64 subfamily of TBC/RabGAPs also have GAP activities towards Ras

J Biochem. 2013 Mar;153(3):283-8. doi: 10.1093/jb/mvs147. Epub 2012 Dec 17.

Abstract

The importance of interconnective signalling networks between distinct GTPases and their regulators is being recognized. EPI64C/TBC1D10C/carabin, a haematopoietically enriched GTPase-activating protein (GAP) for Rab35, has been shown to exhibit RasGAP activity. Owing to the diverged Rab specificities among the EPI64 members (EPI64A-C) and the relatively weak sequence conservation between EPI64A/B and EPI64C in their catalytic TBC domains, it is difficult to predict whether EPI64A and B will also have RasGAP activities. Therefore, in this study, we examined the RasGAP activities of all three EPI64 subfamily members. We found that EPI64A-C exhibited in vivo GAP activities towards Ras using three independent methods, spectrofluorometry with Förster resonance energy transfer (FRET) sensors, the Bos' pull-down assay and time-lapse FRET imaging. EPI64A and B were predominantly localized at the periphery of COS-7 cells. In COS-7 cells, confocal FRET imaging showed that H-Ras activity was higher at the Golgi than at the plasma membrane. Thus, we propose that EPI64A and B, which are ubiquitously expressed members of the EPI64 subfamily, inactivate Ras and certain Rabs at the periphery of cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing / genetics
  • Adaptor Proteins, Signal Transducing / metabolism
  • Animals
  • COS Cells
  • Cell Membrane / metabolism
  • Chlorocebus aethiops
  • Epidermal Growth Factor / pharmacology
  • Fluorescence Resonance Energy Transfer
  • GTPase-Activating Proteins / genetics
  • GTPase-Activating Proteins / metabolism*
  • Golgi Apparatus / metabolism
  • HEK293 Cells
  • Humans
  • Immunoblotting
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Microscopy, Confocal
  • Microtubule-Associated Proteins / genetics
  • Microtubule-Associated Proteins / metabolism*
  • Mitogen-Activated Protein Kinase 1 / metabolism
  • Phosphorylation / drug effects
  • Proto-Oncogene Proteins p21(ras) / genetics
  • Proto-Oncogene Proteins p21(ras) / metabolism*
  • Time-Lapse Imaging / methods
  • rab GTP-Binding Proteins / genetics
  • rab GTP-Binding Proteins / metabolism

Substances

  • Adaptor Proteins, Signal Transducing
  • GTPase-Activating Proteins
  • Luminescent Proteins
  • Microtubule-Associated Proteins
  • RABGAP1 protein, human
  • TBC1D10A protein, human
  • TBC1D10B protein, human
  • TBC1D10C protein, human
  • Epidermal Growth Factor
  • MAPK1 protein, human
  • Mitogen-Activated Protein Kinase 1
  • RAB35 protein, human
  • HRAS protein, human
  • Proto-Oncogene Proteins p21(ras)
  • rab GTP-Binding Proteins