Document Detail


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.
MedLine Citation:
PMID:  12176907     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Marie-Josèphe Rabiet; Marianne Tardif; Laurence Braun; François Boulay
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Blood     Volume:  100     ISSN:  0006-4971     ISO Abbreviation:  Blood     Publication Date:  2002 Sep 
Date Detail:
Created Date:  2002-08-14     Completed Date:  2002-09-13     Revised Date:  2008-11-21    
Medline Journal Info:
Nlm Unique ID:  7603509     Medline TA:  Blood     Country:  United States    
Other Details:
Languages:  eng     Pagination:  1835-44     Citation Subset:  AIM; IM    
Affiliation:
Département Réponse et Dynamique Cellulaires/Biochimie et Biophysique des Systèmes Intégrés, Grenoble, France. mjrabiet@cea.fr
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MeSH Terms
Descriptor/Qualifier:
Enzyme Activation
HL-60 Cells
Humans
NADPH Oxidase / metabolism*
Promoter Regions, Genetic
Signal Transduction*
Tetracycline / metabolism
Transcriptional Activation
cdc42 GTP-Binding Protein / genetics,  metabolism*
Chemical
Reg. No./Substance:
60-54-8/Tetracycline; EC 1.6.3.1/NADPH Oxidase; EC 3.6.5.2/cdc42 GTP-Binding Protein

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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