Figure 5
Inhibition of kinase activity. (a) TKI incubation versus spiking. Prior to lysis, HCT116 cells were incubated in vitro ±2 μM sunitinib, and subsequent lysates were spiked ±2 μM sunitinib (drug incubation/drug spiking). Signal intensity of the top 15 substrates is represented from high to low. Although inhibition patterns were similar, inhibition potency increased for sunitinib-incubated versus spiked samples, while additional spiking of previously incubated samples did not increase the degree of inhibition. (b) Competition with ATP. The HCT116 lysate was spiked with 4 μM sunitinib, 25 μM sorafenib or 10 nM dasatinib in the presence of increasing ATP concentrations. Despite average phosphorylation inhibition of 65–85% at 100 μM ATP, increasing ATP concentrations induced signal intensity and attenuated the inhibitory effects of sunitinib and sorafenib. With dasatinib, signal intensity increased to a lesser extent, suggesting partial ATP-independent inhibition of kinase activity. (c and d) Substrate-specific inhibition. Microarrays were incubated with 125 ng ml of recombinant Src or 500 ng ml Axl kinase±85 nM and 2 μM of the Src-inhibitor dasatinib and Axl-inhibitor R428, respectively. Both drugs resulted in near-complete inhibition of phosphorylation. The results of the top 25 phosphorylated substrates are shown.
