Figure 3.

Suppression of PAK1 activity by p21-binding domain (PBD) binders in vitro. (a) Inhibition of Cdc42-dependent PAK1 activation. Cdc42•GTP, Wt-PAK1, myelin basic protein (MBP) and [γ-³²P]-ATP were incubated with various concentrations of the indicated compounds for 0.5 h. Immunoblotting of total MBP was performed as a loading control. (b) Direct inhibition of PAK1 activity by compounds in vitro. Active PAK1^T423E, MBP and [γ-³²P]-ATP were incubated with various concentrations of the indicated compounds for 0.5 h. Immunoblotting of total MBP was performed as a loading control. (c) Full-length PAK1^T423E/LL or PAK1^T423E was incubated with MBP and [γ-³²P]-ATP in the presence of dimethylsulphoxide (control) or the indicated compounds (40 μM) for 0.5 h. LL, H83L/H86L. (d) Activity of full-length PAK1^T423E or a single-substitution mutant was measured in the absence or presence of compounds as described in c. In all experiments, MBP phosphorylation was analyzed by autoradiography. All data are representative of at least three independent experiments.