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Biochemical suppression of small-molecule inhibitors: a strategy to identify inhibitor targets and signaling pathway components.
Peterson JR
,
Lebensohn AM
,
Pelish HE
,
Kirschner MW
.
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Identification of small-molecule targets remains an important challenge for chemical genetics. We report an approach for target identification and protein discovery based on functional suppression of chemical inhibition in vitro. We discovered pirl1, an inhibitor of actin assembly, in a screen conducted with cytoplasmic extracts. Pirl1 was used to partially inhibit actin assembly in the same assay, and concentrated biochemical fractions of cytoplasmic extracts were added to find activities that suppressed pirl1 inhibition. Two activities were detected, separately purified, and identified as Arp2/3 complex and Cdc42/RhoGDI complex, both known regulators of actin assembly. We show that pirl1 directly inhibits activation of Cdc42/RhoGDI, but that Arp2/3 complex represents a downstream suppressor. This work introduces a general method for using low-micromolar chemical inhibitors to identify both inhibitor targets and other components of a signaling pathway.
Ballestrem,
Actin-dependent lamellipodia formation and microtubule-dependent tail retraction control-directed cell migration.
2000,
Pubmed
Benard,
Characterization of rac and cdc42 activation in chemoattractant-stimulated human neutrophils using a novel assay for active GTPases.
1999,
Pubmed
Block,
Purification of an N-ethylmaleimide-sensitive protein catalyzing vesicular transport.
1988,
Pubmed
Burdine,
Target identification in chemical genetics: the (often) missing link.
2004,
Pubmed
Cox,
Requirements for both Rac1 and Cdc42 in membrane ruffling and phagocytosis in leukocytes.
1997,
Pubmed
Desai,
The use of Xenopus egg extracts to study mitotic spindle assembly and function in vitro.
1999,
Pubmed
,
Xenbase
Ho,
Toca-1 mediates Cdc42-dependent actin nucleation by activating the N-WASP-WIP complex.
2004,
Pubmed
,
Xenbase
Hoffman,
Structure of the Rho family GTP-binding protein Cdc42 in complex with the multifunctional regulator RhoGDI.
2000,
Pubmed
Li,
Multicopy suppressors for novel antibacterial compounds reveal targets and drug efflux susceptibility.
2004,
Pubmed
Luesch,
A genome-wide overexpression screen in yeast for small-molecule target identification.
2005,
Pubmed
Ma,
Corequirement of specific phosphoinositides and small GTP-binding protein Cdc42 in inducing actin assembly in Xenopus egg extracts.
1998,
Pubmed
,
Xenbase
Millard,
Signalling to actin assembly via the WASP (Wiskott-Aldrich syndrome protein)-family proteins and the Arp2/3 complex.
2004,
Pubmed
Pelish,
Secramine inhibits Cdc42-dependent functions in cells and Cdc42 activation in vitro.
2006,
Pubmed
Peterson,
A chemical inhibitor of N-WASP reveals a new mechanism for targeting protein interactions.
2001,
Pubmed
,
Xenbase
Peterson,
Small molecules, big impact: a history of chemical inhibitors and the cytoskeleton.
2002,
Pubmed
Peterson,
Chemical inhibition of N-WASP by stabilization of a native autoinhibited conformation.
2004,
Pubmed
,
Xenbase
Ridley,
The small GTP-binding protein rac regulates growth factor-induced membrane ruffling.
1992,
Pubmed
Rohatgi,
The interaction between N-WASP and the Arp2/3 complex links Cdc42-dependent signals to actin assembly.
1999,
Pubmed
,
Xenbase
Rossman,
A crystallographic view of interactions between Dbs and Cdc42: PH domain-assisted guanine nucleotide exchange.
2002,
Pubmed
Rossman,
GEF means go: turning on RHO GTPases with guanine nucleotide-exchange factors.
2005,
Pubmed
Stockwell,
Chemical genetics: ligand-based discovery of gene function.
2000,
Pubmed
Tochtrop,
Target identification strategies in chemical genetics.
2004,
Pubmed
Verma,
Ubistatins inhibit proteasome-dependent degradation by binding the ubiquitin chain.
2004,
Pubmed
,
Xenbase
Wignall,
Identification of a novel protein regulating microtubule stability through a chemical approach.
2004,
Pubmed
,
Xenbase
Zhang,
The role of Mg2+ cofactor in the guanine nucleotide exchange and GTP hydrolysis reactions of Rho family GTP-binding proteins.
2000,
Pubmed
