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Vol. 18, Issue 8, 2949-2959, August 2007

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The M-Ras-RA-GEF-2-Rap1 Pathway Mediates Tumor Necrosis Factor-–dependent Regulation of Integrin Activation in Splenocytes

Yoko Yoshikawa^*, Takaya Satoh^*, Takashi Tamura^*, Ping Wei^*, Shymaa E. Bilasy^*, Hironori Edamatsu^*, Atsu Aiba, Koko Katagiri, Tatsuo Kinashi, Kazuki Nakao, and Tohru Kataoka^*

*Division of Molecular Biology, Department of Biochemistry and Molecular Biology, and Division of Molecular Genetics, Department of Physiology and Cell Biology, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan; Department of Molecular Genetics, Institute of Biomedical Science, Kansai Medical University, Osaka 570-8506, Japan; and Laboratory of Animal Resources and Genetic Engineering, Riken Center for Developmental Biology, Kobe 650-0047, Japan

Submitted March 16, 2007; Revised May 14, 2007; Accepted May 18, 2007
Monitoring Editor: Richard Assoian

The Rap1 small GTPase has been implicated in regulation of integrin-mediated leukocyte adhesion downstream of various chemokines and cytokines in many aspects of inflammatory and immune responses. However, the mechanism for Rap1 regulation in the adhesion signaling remains unclear. RA-GEF-2 is a member of the multiple-member family of guanine nucleotide exchange factors (GEFs) for Rap1 and characterized by the possession of a Ras/Rap1-associating domain, interacting with M-Ras-GTP as an effector, in addition to the GEF catalytic domain. Here, we show that RA-GEF-2 is specifically responsible for the activation of Rap1 that mediates tumor necrosis factor- (TNF-)-triggered integrin activation. In BAF3 hematopoietic cells, activated M-Ras potently induced lymphocyte function–associated antigen 1 (LFA-1)-mediated cell aggregation. This activation was totally abrogated by knockdown of RA-GEF-2 or Rap1. TNF- treatment activated LFA-1 in a manner dependent on M-Ras, RA-GEF-2, and Rap1 and induced activation of M-Ras and Rap1 in the plasma membrane, which was accompanied by recruitment of RA-GEF-2. Finally, we demonstrated that M-Ras and RA-GEF-2 were indeed involved in TNF-–stimulated and Rap1-mediated LFA-1 activation in splenocytes by using mice deficient in RA-GEF-2. These findings proved a crucial role of the cross-talk between two Ras-family GTPases M-Ras and Rap1, mediated by RA-GEF-2, in adhesion signaling.

Address correspondence to: Tohru Kataoka (kataoka{at}people.kobe-u.ac.jp).

Abbreviations used: ES, embryonic stem; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GEF, guanine nucleotide exchange factor; HA, hemagglutinin; HBSS, Hanks' balanced salt solution; ICAM-1, intercellular adhesion molecule 1; IL, interleukin; LFA-1, lymphocyte function–associated antigen 1; PBS, phosphate-buffered saline; PLC, phospholipase C; PMA, phorbol myristate acetate; RA, Ras/Rap1-associating; RID, RalGDS-Ras–interacting domain; RNAi, RNA interference; SDF-1, stromal cell–derived factor-1; siRNA, small interfering RNA; TNF-, tumor necrosis factor-; TRAF, TNF receptor–associated factors.