Table 1. The WAVE2 complex binds Rac-GTP or acidic phospholipids independently, but binding to both is required for activation; phosphorylation modulates lipid binding.
Liposome co-sedimentation assays containing 30 nM Arp2/3 complex, 5 nM WAVE2 complex from serum starved A-431 cells, and where indicated 20 μM (total lipid) liposomes composed of PC:PE (50:49 molar percentage) or PIP3 liposomes composed of PC:PI:PIP3 (44:45:10 molar percentage) (all liposomes contained 1% molar fraction Rhodamine PE for visualization purposes), and 50 nM prenylated Rac-GDP or GTP. Percentages of total WAVE2 bound (see experimental procedures for details) are from experiments with Rac2; comparable results were obtained with Rac1. The results of actin polymerization assays of equivalent composition are reported under “Activation”.
| WAVE2 complex | WAVE2 complex, PP2A treated | |||
|---|---|---|---|---|
| % bound | Activation | % bound | Activation | |
| PC:PE liposomes + Rac-GDP | 1 | No | Not tested | |
| PC:PE liposomes + Rac-GTP | 56 | No | 55 | No |
| PIP3 liposomes (no Rac) | 69 | No | 35 | No |
| PIP3 liposomes + Rac-GTP | 91 | Yes | 79 | No |