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.
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-1alpha 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 PLCbeta2 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.
Rabiet
Marie-Josèphe
MJ
Département Réponse et Dynamique Cellulaires/Biochimie et Biophysique des Systèmes Intégrés, Grenoble, France. mjrabiet@cea.fr
Tardif
Marianne
M
Braun
Laurence
L
Boulay
François
F
D004789:Enzyme Activation; D018922:HL-60 Cells; D006801:Humans; D019255:NADPH Oxidases / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D015398:Signal Transduction; D013752:Tetracycline / Q000378:metabolism; D015533:Transcriptional Activation; D020764:cdc42 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
D019255:NADPH Oxidases; D020764:cdc42 GTP-Binding Protein; D013752:Tetracycline