Rac1 regulates heat shock responses by reorganization of vimentin filaments: identification using MALDI-TOF MS.
Rac1 has been implicated in a wide variety of biological processes, including actin remodeling and various signaling cascades. Here we have examined whether Rac1 might be involved in heat shock-induced cell signaling. We found that Rat2 stable cells expressing a dominant negative Rac1 mutant, RacN17 (Rat2-RacN17), were significantly more tolerant to heat shock than control Rat2 cells, and simultaneously inhibited the activation of SAPK/JNK by heat shock compared to control Rat2 cells. However, no discernible effect was observed in typical heat shock responses including total protein synthesis and heat shock protein synthesis. To identify the proteins involved in this difference, we separated the proteins of both Rat2 and Rat2-RacN17 cell lines after heat shock using two-dimensional gel electrophoresis and identified the differentially expressed proteins by matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) after in-gel trypsin digestion. Differentially expressed proteins between two cell lines were identified as vimentin. Rat2-RacN17 cells showed significant changes in vimentin as well as marked changes in vimentin reorganization by heat shock. The vimentin changes were identified as N-terminal head domain cleavage. These results suggest that Rac1 plays a pivotal role in the heat shock-induced signaling cascade by modifying intermediate vimentin filaments.
Lee
S Y
SY
The Center for Cell Signaling Research, Division of Molecular Life Sciences and College of Pharmacy, Ewha Womans University, Seoul, 120-750, Korea.
Song
E J
EJ
Kim
H J
HJ
Kang
H J
HJ
Kim
J H
JH
Lee
K J
KJ
D000818:Animals; D002460:Cell Line / Q000166:cytology / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D018869:Heat-Shock Response / Q000502:physiology*; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009154:Mutation / Q000502:physiology; D051381:Rats; D015398:Signal Transduction / Q000502:physiology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D014746:Vimentin / Q000302:isolation & purification / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*
D014746:Vimentin; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D020830:rac1 GTP-Binding Protein