A model for the inhibition of N-WASP by 187-1. Native N-WASP exists predominantly in a “closed” inactive conformation. Whereas binding to cdc42 stabilizes the “open” active conformation, 187-1 stabilizes the autoinhibited state. Competitive binding of 187-1 and cdc42 to N-WASP, therefore, drives the equilibrium between autoinhibited and activated states, respectively.

187-1, a 14-amino acid cyclic peptide, inhibits PIP₂-induced but not VCA-induced actin assembly in Xenopus cytoplasmic extract. (a) Polymerization kinetics of pyrene-actin after the addition of PIP₂-containing liposomes into extract containing the indicated concentrations of 187-1. (b) Immunofluorescence images of rhodamine-actin in extract ± 46 μM 187-1 induced to polymerize by PIP₂-containing liposomes (Upper) or 0.5 μM VCA polypeptide (Lower). (Scale bars, 100 μm.)

187-1 inhibits N-WASP activation of the Arp2/3 complex. (a) Schematic of the known components involved in signaling from PIP₂ to actin in Xenopus extract and their sequence of action. Note that PIP₂ has a primary role upstream of cdc42 but can also bind directly to and promote activation of N-WASP. (b–d) Pyrene-actin polymerization kinetics in the presence of the indicated purified components. (b) 187-1 does not affect actin polymerization downstream of VCA. (c) N-WASP-stimulated actin polymerization is inhibited by 187-1. (d) N-WASP activated by both cdc42 and PIP₂-containing liposomes is resistant to inhibition by 187-1.

187-1 binds to N-WASP and stabilizes the autoinhibited conformation. (a) 187-1 photo-crosslinks predominantly to N-WASP. A biotinylated photo-crosslinkable derivative of 187-1, 187-1(Bpa)* (Fig 1a) was incubated with an equimolar mixture of actin, the Arp2/3 complex, and N-WASP and was crosslinked by UV illumination. In some reactions (lanes 2, 3,5, 6, 8, 9, 11, and 12), we included a nonbiotinylated competitor, 187-1(Bpa) (Fig 1a). The concentrations of 187-1(Bpa)* and 187-1(Bpa) used are indicated. Reactions were analyzed by SDS/PAGE/Western blotting, and biotin was detected by using horseradish peroxidase-streptavidin and chemiluminescence. (b) 187-1 stabilizes the autoinhibited conformation of N-WASP. GST alone or GST-VCA fusion proteins were immobilized on glutathione agarose and incubated with the His₆-tagged WGP fragment of N-WASP (HT-WGP, amino acids 1–396) in the presence of GTPγS-bound cdc42 and 187-1 as indicated. After washing, the bound material was eluted in SDS/PAGE sample buffer, and HT-WGP was detected by SDS/PAGE/Western blotting by using anti-His antibodies and chemiluminescent detection. (c) 187-1 but not a diastereomer, F12, stabilizes N-WASP. GST-VCA-coated beads were incubated with HT-WGP as shown in b in the presence or absence of the indicated components, and bound HT-WGP was detected as described above.