PMID- 12176907
OWN - NLM
STAT- MEDLINE
DCOM- 20020913
LR  - 20210206
IS  - 0006-4971 (Print)
IS  - 0006-4971 (Linking)
VI  - 100
IP  - 5
DP  - 2002 Sep 1
TI  - 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.
PG  - 1835-44
AB  - 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.
FAU - Rabiet, Marie-Josephe
AU  - Rabiet MJ
AD  - Departement Reponse et Dynamique Cellulaires/Biochimie et Biophysique des 
      Systemes Integres, Grenoble, France. mjrabiet@cea.fr
FAU - Tardif, Marianne
AU  - Tardif M
FAU - Braun, Laurence
AU  - Braun L
FAU - Boulay, Francois
AU  - Boulay F
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - Blood
JT  - Blood
JID - 7603509
RN  - EC 1.6.3.- (NADPH Oxidases)
RN  - EC 3.6.5.2 (cdc42 GTP-Binding Protein)
RN  - F8VB5M810T (Tetracycline)
SB  - IM
MH  - Enzyme Activation
MH  - HL-60 Cells
MH  - Humans
MH  - NADPH Oxidases/*metabolism
MH  - Promoter Regions, Genetic
MH  - *Signal Transduction
MH  - Tetracycline/metabolism
MH  - Transcriptional Activation
MH  - cdc42 GTP-Binding Protein/genetics/*metabolism
EDAT- 2002/08/15 10:00
MHDA- 2002/09/14 10:01
CRDT- 2002/08/15 10:00
PHST- 2002/08/15 10:00 [pubmed]
PHST- 2002/09/14 10:01 [medline]
PHST- 2002/08/15 10:00 [entrez]
AID - S0006-4971(20)59271-9 [pii]
AID - 10.1182/blood-2001-12-0193 [doi]
PST - ppublish
SO  - Blood. 2002 Sep 1;100(5):1835-44. doi: 10.1182/blood-2001-12-0193.