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Prepublished online as a Blood First Edition Paper on May 13, 2002; DOI 10.1182/blood-2001-12-0193.
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Blood, 1 September 2002, Vol. 100, No. 5, pp. 1835-1844
PHAGOCYTES
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-Josèphe Rabiet,
Marianne Tardif,
Laurence Braun, and
François Boulay
From the Département Réponse et Dynamique
Cellulaires/Biochimie et Biophysique des Systèmes
Intégrés (Unité Mixte de Recherche 5092 Commissariat
à l'Energie Atomique/Centre National de la Recherche
Scientifique/Université Joseph Fourier), Grenoble, France.
A tetracycline-controlled expression system was adapted to the
human promyelocytic HL-60 cell line by placement of 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 guanosine 5'-triphosphate (GTP)-bound form
of Cdc42 is ineffective in directly activating nicotinamide adenine
dinucleotide phosphate (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 study results identifying
PLC 2 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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