Methyl vinyl ketone (MVK) is an α,β-unsaturated carbonyl compound contained in smoke and exhaust gas. This compound is known to covalently bind to proteins via Michael reaction. However, the target proteins are fully unsolved yet. We found that MVK suppresses phosphatidylinositol 3-kinase (PI3K) –Akt signaling. This pathway is essential for various biological regulation, including cell survival, glucose metabolism, and autophagy. In this study, we tried to clarify the mechanism by which MVK interferes with PI3K–Akt signaling.
Initially, we investigated the effects of MVK on the phosphorylation of epidermal growth factor receptor (EGFR), PI3K and Akt by treatment with EGF in A549 cells. MVK significantly attenuated the levels of pAkt and pPI3K, but not pEGFR formation. In addition, co-immunoprecipitation analysis revealed that MVK inhibits the interaction of PI3K with EGFR. Interestingly, exposure to MVK did not change the levels of phosphatidylinositol 3,4,5-trisphosphate indicating that the catalytic domain in PI3K is not a target of MVK. Next, we employed LC-MS/MS analysis to determine the modification sites in PI3K. We identified that both Cys146 and 656 residues in PI3K p85 subunit are modified with MVK. To confirm if these sites are essential for the EGF signaling, we substituted each Cys residue with Ser. We found that EGF treatment fails to activate the phosphorylation of PI3K p85 C656S mutant but not the C146S mutant. These results indicated that MVK may be a novel type of PI3K inhibitor via modification of p85 subunit.