Actin Polymerization Induced by PIP2 and Cdc42

PIP2 promotes the nucleation of actin filaments in Xenopus egg extract via a Cdc42-dependent pathway (Ma et al. 1998a; Pelish et al. 2006; Peterson et al. 2006). PIP2 has also been linked to activation of Cdc42 in mammalian cells (Martin-Belmonte et al. 2007). PIP2 activation of Cdc42 is accomplished in part by promoting the membrane recruitment of Cdc42 through a direct interaction with a poly-basic stretch of amino acids in the C-terminal region of Cdc42 (Heo et al. 2006) or, alternatively, through indirect recruitment by the PIP2-binding protein annexin 2 (Martin-Belmonte et al. 2007). In addition, PIP2 may promote guanine nucleotide exchange on Cdc42 either directly (Zheng et al. 1996) or indirectly through the regulation of guanine nucleotide exchange factors (Baumeister et al. 2006). PIP2 can also contribute to actin assembly by binding and promoting the activation of a ubiquitously expressed Cdc42 effector called N-WASP (neural Wiskott-Aldrich syndrome protein) (Miki et al. 1996; Prehoda et al. 2000; Rohatgi et al. 2000, 2001; Papayannopoulos et al. 2005) or by preventing capping protein from capping actin filament barbed ends (Schafer et al. 1996). It should be noted, however, that these latter two roles for PIP2 appear to be minor in Xenopus HSS compared to its role in activating Cdc42 (Rohatgi et al. 2000; Ho et al. 2004).

Cdc42 has been shown in many systems to direct the recruitment and activation of proteins that mediate the nucleation and polymerization of actin filaments (Rohatgi et al. 1999; Taunton et al. 2000; Lechler et al. 2001; Sokac et al. 2003). Studies concurrent with our work used an activity-based biochemical purification to identify proteins in Xenopus egg extract required for mediating actin polymerization downstream of Cdc42 (Ma et al. 1998b; Rohatgi et al. 1999; Ho et al. 2004). These studies identified three protein factors required for actin nucleation by Cdc42: the Cdc42-binding scaffolding protein N-WASP, a second Cdc42-binding protein known as Toca-1 (transducer of Cdc42-mediated actin assembly), and the ubiquitous actin filament nucleating protein complex, the Arp2/3 complex. Cdc42, together with Toca-1, binds N-WASP and promotes N-WASP activation (Rohatgi et al. 2000; Ho et al. 2006). Active N-WASP, in turn, binds and stimulates the actin filament nucleation activity of the Arp2/3 complex (Rohatgi et al. 1999). Both N-WASP and Arp2/3 complex are now appreciated as important for actin filament nucleation in many contexts (Millard et al. 2004; Vartiainen and Machesky 2004; Stradal and Scita 2006; Takenawa and Suetsugu 2007).

To identify proteins linking PIP2 signaling to actin filament assembly as well as to develop reagents for studying this signaling pathway in other systems, we screened unbiased libraries of chemical compounds for their ability to inhibit actin assembly in PIP2-stimulated HSS. These compounds included both structurally diverse, drug-like small molecules and a library of cyclic peptides. Over 26,000 compounds were tested for their ability to inhibit actin assembly induced by 10 |M liposomes containing PIP2 (PIP2: phosphatidylcholine:phosphatidylinositol, 4:48:48 molar ratio) in HSS. Actin assembly was measured in HSS by supplementing the HSS with trace amounts of rabbit muscle actin monomers covalently labeled with the flourophore pyrene. Incorporation of pyrene-actin into actin filaments results in a dramatic increase in pyrene fluorescence that can be measured in the intact HSS (Kouyama and Mihashi 1981; Ma et al. 1998a; Peterson et al. 2001; Lebensohn et al. 2006). Compounds were tested in a high-throughput manner utilizing 384-well plates. Active compounds identified by the screen were ranked based on their potency determined in dose-response experiments using the primary screening assay. Among the most potent compounds identified were a 14-amino-acid cyclic peptide called 187-1 (Peterson et al. 2001), a tetracyclic indole called pirl1 (Peterson et al. 2006), and an N-alkylated carbazole derivative that we subsequently termed wiskostatin (Peterson et al. 2004; and see below).

Wiskostatin is a 3,6-dibromocarbazole derivative, N-alkylated with a dimethyl-aminopropan-2-ol group. In addition to wiskostatin, an otherwise similar 3,6-dichlorocarbazole variant of wiskostatin was identified by the primary screen, although it was slightly less potent. Wiskostatin inhibited actin polymerization in PIP2-stimulated Xenopus egg extract with an EC50 of ~4 ||M. Thus, the maximal rate of actin polymerization was slowed by 50% relative to solvent-alone controls in the presence of this dose of wiskostatin. Wiskostatin concentrations >10 | M completely suppressed actin assembly stimulated by PIP2-containing liposomes.

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