The Target of Wiskostatin is NWASP

A central challenge of forward chemical genetic screens is the identification of protein targets of small-molecule inhibitors (Stockwell 2000; Burdine and Kodadek 2004; Tochtrop and King 2004). Although a number of conceptually distinct approaches have been used successfully for inhibitor target identification (Burdine and Kodadek 2004; Tochtrop and King 2004), no single method has proven universally applicable. To identify the target of wiskostatin, we therefore began with a candidate-testing approach. As described above, several proteins that mediate actin assembly downstream of Cdc42 have been discovered, and in vitro assays using purified preparations of these proteins have been developed that successfully measure their biochemical activities (Ma et al. 1998b; Rohatgi et al. 1999; Ho et al. 2004, 2006). We therefore used these assays to determine if wiskostatin inhibited any of the known proteins that mediate actin assembly downstream of Cdc42.

Initial experiments tested whether wiskostatin might inhibit PIP2-stimulated actin assembly by binding actin monomers directly and preventing their incorporation into growing filaments. The polymerization kinetics of purified native rabbit muscle actin was monitored by the fluorescence increase of added pyrene-labeled actin monomers. Whereas wiskostatin inhibited actin assembly in PIP2-stimulated extract with an EC50 of 4 | M, up to 20 | M wiskostatin did not affect the polymerization of purified actin. Higher wiskostatin doses, however, modestly inhibited the polymerization of purified actin directly. The apparent inability of wiskostatin to inhibit actin polymerization directly suggests that wiskostatin must target a signaling component upstream of actin itself.

We next tested if wiskostatin inhibited the actin filament nucleating activity of the Arp2/3 complex, an essential mediator of Cdc42-dependent actin assembly in HSS (Ma et al. 1998b). Purified bovine Arp2/3 complex is inactive, but can be activated experimentally by the addition of a recombinant polypeptide derived from the C-terminus of N-WASP termed the VCA segment (named for verprolin-homologous, cofilin-homologous, and acidic amino acid sequences) (Rohatgi et al. 1999). In the presence of VCA, the actin-nucleating activity of Arp2/3 complex is potently activated and can be monitored using the pyrene-actin fluorescence assay.

VCA-activated Arp2/3 complex was not inhibited by 10 ||M wiskostatin, indicating that the inhibitor must target some other component besides the Arp2/3 complex or the VCA segment of N-WASP.

Arp2/3 complex is activated in PIP2-stimulated HSS by its binding to the VCA region of endogenous N-WASP. Whereas the VCA segment is a potent activator of Arp2/3 complex, the activity of full-length N-WASP is sharply reduced due to autoinhibitory interactions between the VCA segment and an N-terminal domain called the GTPase-binding domain (GBD) (Kim et al. 2000; Prehoda et al. 2000; Rohatgi et al. 2000). Autoinhibition is relieved by the binding of active Cdc42 to the GBD, which releases the VCA segment, allowing VCA to activate Arp2/3 complex (Fig. 1a) (Prehoda et al. 2000; Rohatgi et al. 2000). We therefore tested whether wiskostatin inhibited N-WASP activation using purified pyrene-actin assays in the presence of purified Arp2/3 complex and recombinant N-WASP. Both the modest basal activity of autoinhibited N-WASP and the robust activity of N-WASP in the presence of PIP2 and Cdc42 were inhibited by wiskostatin. Thus, although wiskostatin did not inhibit Arp2/3 complex activation by the isolated VCA segment of N-WASP, it did inhibit Arp2/3 complex activation by full-length N-WASP. Importantly, wiskostatin inhibited N-WASP activity at doses consistent with its potency in HSS. Furthermore, several analogues of wiskostatin that were inactive at inhibiting PIP2-induced actin assembly in HSS also did not inhibit N-WASP activity in vitro, indicating that inhibition of N-WASP can explain wiskostatin inhibition of actin assembly in PIP2-stimulated HSS. The name "wiskostatin" was chosen for this compound based on its ability to inhibit a member of the Wiskott-Aldrich syndrome protein family.

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