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Prepublished online as a Blood First Edition Paper on May 13, 2002; DOI 10.1182/blood-2001-12-0193.
Submitted December 6, 2001
Accepted April 11, 2002
INHIBITORY EFFECTS OF A DOMINANT-INTERFERING FORM OF THE RHO-GTPASE CDC42 IN THE CHEMOATTRACTANT-ELICITED SIGNALING PATHWAYS LEADING TO NADPH OXIDASE ACTIVATION IN DIFFERENTIATED HL-60 CELLS
Marie-Joseph Rabiet, Marianne Tardif, Laurence Braun, and Francois Boulay*
DRDC/BBSI, CEA-Grenoble, Grenoble, France
* Corresponding author; email: fboulay{at}cea.fr.
A tetracycline-controlled expression system was adapted to the human promyelocytic HL-60 cell line by placing the transactivator (tTA-off) sequence under the control of the human EF-1 promoter region. Constitutively active and dominant-inhibitory forms of Cdc42 (Cdc42V12 and Cdc42N17, respectively) were conditionally expressed in this system. The expression of Cdc42V12 had no marked effect on chemoattractant-mediated superoxide production, corroborating previous results indicating that the GTP-bound form of Cdc42 is ineffective in directly activating NADPH oxidase in a cell-free system. However, the N17 mutant potently inhibited chemoattractant-induced superoxide production. The expression of Cdc42N17 interfered with the GTP-loading of Rac and Ras, and with the activation of the MAP-kinase pathway. A drastic reduction of chemoattractant-induced inositol-1,4,5-trisphosphate formation and calcium mobilization was observed, corroborating previous in vitro studies identifying PLCß2 as a Rac/Cdc42 effector. Cdc42N17 was also found to inhibit the translocation of Ras-GRF2, a guanine nucleotide exchange factor for Ras and Rac but not for Cdc42. Thus, the dominant-inhibitory mutant Cdc42N17 was found to interfere at multiple levels in the signaling pathways. The pleiotropic inhibitory effects of Cdc42N17 illustrate the potential pitfalls of using dominant inhibitory proteins to study the function of Ras-family GTPases. In this regard, a number of conclusions drawn from the use of dominant inhibitory mutants in myeloid cells might have to be reconsidered.
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