GTPgammaS-induced actin polymerisation in vitro: ATP- and phosphoinositide-independent signalling via Rho-family proteins and a plasma membrane-associated guanine nucleotide exchange factor.

Details

Serval ID
serval:BIB_D936C376BC6E
Type
Article: article from journal or magazin.
Collection
Publications
Title
GTPgammaS-induced actin polymerisation in vitro: ATP- and phosphoinositide-independent signalling via Rho-family proteins and a plasma membrane-associated guanine nucleotide exchange factor.
Journal
Journal of Cell Science
Author(s)
Katanaev V.L., Wymann M.P.
ISSN
0021-9533 (Print)
ISSN-L
0021-9533
Publication state
Published
Issued date
1998
Volume
111 ( Pt 11)
Pages
1583-1594
Language
english
Abstract
In a cell-free system from neutrophil cytosol GTP(&ggr ;)S can induce an increase in the number of free filament barbed ends and massive actin polymerisation and cross-linking. GTP(&ggr ;)S stimulation was susceptible to an excess of GDP, but not Bordetella pertussis toxin and could not be mimicked by aluminium fluoride, myristoylated GTPgammaS.Gialpha2 or Gbeta1gamma2 subunits of trimeric G proteins. In contrast, RhoGDI and Clostridium difficile toxin B (inactivating Rho family proteins) completely abrogated the effect of GTPgammaS. When recombinant, constitutively activated and GTPgammaS-loaded Rac1, RhoA, or Cdc42 proteins alone or in combination were probed at concentrations >100 times the endogenous, however, they were ineffective. Purified Cdc42/Rac-interactive binding (CRIB) domain of WASP or C3 transferase did not prevent actin polymerisation by GTPgammaS. The action of GTPgammaS was blocked by mM [Mg2+], unless a heat- and trypsin-sensitive component present in neutrophil plasma membrane was added. Liberation of barbed ends seems therefore to be mediated by a toxin B-sensitive cytosolic Rho-family protein, requiring a membrane-associated guanine nucleotide exchange factor (GEF) for its activation by GTPgammaS under physiologic conditions. The inefficiency of various protein kinase and phosphatase inhibitors (staurosporine, genistein, wortmannin, okadaic acid and vanadate) and removal of ATP by apyrase, suggests that phosphate transfer reactions are not required for the downstream propagation of the GTPgammaS signal. Moreover, exogenously added phosphoinositides failed to induce actin polymerisation and a PtdIns(4,5)P2-binding peptide did not interfere with the response to GTPgammaS. The speed and simplicity of the presented assay applicable to protein purification techniques will facilitate the further elucidation of the molecular partners involved in actin polymerisation.
Keywords
Actins/chemistry, Adenosine Triphosphate/physiology, Dimerization, GTP-Binding Proteins/physiology, Guanine Nucleotide Exchange Factors, Guanosine 5'-O-(3-Thiotriphosphate)/pharmacology, Humans, Membrane Proteins/physiology, Neutrophils/chemistry, Neutrophils/physiology, Phosphatidylinositols/physiology, Proteins/physiology, Signal Transduction/physiology, rhoB GTP-Binding Protein
Pubmed
Web of science
Create date
09/07/2012 9:13
Last modification date
20/08/2019 15:58
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