Figure 2. Effect of (3-chloroacetyl)indol (3CAI) on AKT activity.

(A) 3CAI suppresses AKT1 kinase activity in vitro. The effect of 3CAI on AKT1, MEK1, JNK1, ERK1 or TOPK activity was assessed by an in vitro kinase assay using AKT1 (active, 100 ng), histone H2B (AKT substrate, 500 ng), MEK1 (active, 400 ng), inactive ERK2 (MEK1 substrate, 500 ng), JNK1 (active, 50 ng), c-Jun (JNK1 substrate, 500 ng), ERK1 (active, 400 ng), inactive RSK2 (ERK1 substrate, 500 ng), TOPK (active, 500 ng) or histone H2AX (TOPK substrate, 500 ng) with [γ-³²P]ATP (B) 3CAI inhibits PI3K kinase activity at the highest concentration in vitro. The inhibitory effect of 3CAI or LY294002 as a PI3K inhibitor on PI3K activity was assessed by an in vitro kinase assay. The conversion of PIP4 to PIP3 was determined by autoradiography. (C) 3CAI substantially inhibits AKT1 or (D) AKT2 kinase activity in a dose-dependent manner. The inhibitory effect of 3CAI, I3C or an AKT inhibitor VIII on AKT1 or AKT2 activity was assessed by an in vitro kinase assay. The ³²P-labeled substrate was visualized by autoradiography. Band density was measured using the image J program. All data are represented as means ± S.D. of values from three independent experiments. The asterisk (*) indicates a significant (p < 0.05) decrease caused by 3CAI, LY294002 or AKT inhibitor VIII compared to untreated control.