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Background  Thymoquinone (TQ) was shown to reduce tumor growth in several cancer models both  in vitro  and  in vivo . So far only a few targets of TQ, including protein kinases have been identified. Considering that kinases are promising candidates for targeted anticancer therapy, we studied the complex kinase network regulated by TQ.    Methods  Novel kinase targets influenced by TQ were revealed by  in silico  analysis of peptide array data obtained from TQ-treated HCT116wt cells. Western blotting and kinase activity assays were used to determine changes in kinase expression patterns in colorectal cancer cells (HCT116wt, DLD-1, HT29). To study the viability/apoptotic effects of combining the PAK1 inhibitor IPA-3 and TQ, crystal violet assay and AnnexinV/PI staining were employed. Interactions between PAK1 and ERK1/2 were investigated by co-immunoprecipitation and modeled by docking studies. Transfection with different PAK1 mutants unraveled the role of TQ-induced changes in PAK1 phosphorylation and TQ´s effects on PAK1 scaffold function.    Results  Of the 104 proteins identified, 50 were upregulated ≥2 fold by TQ and included molecules in the AKT-MEK-ERK1/2 pathway. Oncogenic PAK1 emerged as an interesting TQ target. Time-dependent changes in two PAK1 phosphorylation sites generated a specific kinase profile with early increase in pPAK Thr212  followed by late increase in pPAK Thr423 . TQ induced an increase of pERK1/2 and triggered the early formation of an ERK1/2-PAK1 complex. Modeling confirmed that TQ binds in the vicinity of Thr212 accompanied by conformational changes in ERK2-PAK1 binding. Transfecting the cells with the non-phosphorylatable mutant T212A revealed an increase of pPAK Thr423  and enhanced apoptosis. Likewise, an increase in apoptosis was observed in cells transfected with both the kinase-dead K299R mutant and PAK1 siRNA. Using structural modeling we suggest that TQ interferes also with the kinase domain consequently disturbing its interaction with pPAK Thr423 , finally inhibiting MEK-ERK1/2 signaling and disrupting its prosurvival function. pERK1/2 loss was also validated  in vivo .    Conclusions  Our study shows for the first time that the small molecule TQ directly binds to PAK1 changing its conformation and scaffold function. Because TQ affects the central RAF/MEK/ERK1/2 pathway, the combination of TQ with targeted therapies is worth considering for future anticancer treatments.    Electronic supplementary material  The online version of this article (doi:10.1186/1476-4598-13-201) contains supplementary material, which is available to authorized users.

Thymoquinone-induced conformational changes of PAK1 interrupt prosurvival MEK-ERK signaling in colorectal cancer