Inhibition of tyrosine protein kinases by halomethyl ketones

Abstract

2-O-(Isobutoxycarbonyl)lactate chloromethyl ketone was previously shown to inhibit protein phosphorylation in plasma membranes of [mouse] Ehrlich ascites tumor cells. The present study shows that this inhibitor, as well as 3 halomethyl ketone dervatives of amino acids and peptides, specifically inhibits tyrosine protein kinase activity in intact plasma membranes and Triton extracts of plasma membrane of [human epidermoid carcinoma] A-431 tumor cells. The most effective inhibitor is a bromomethyl ketone derivative of leucine that inhibits the phosphorylation of a protein that migrates to the same position as the EGF [epidermal growth factor] receptor in sodium dodecyl sulfate-polyacrylamide gel elctrophoresis. Inhibition of phosphorylation took place in the presence or absence of added EGF, and the inhibitor did not interfere with the binding of EGF to the receptor nor with the dephosphorylation of the EGF-stimulated phosphoprotein. EGF-dependent phosphorylation in a Triton extact of plasma membranes from normal placenta was considerably less sensitive to the bromomethyl ketone derivative of leucine. The tyrosine protein kinase activity of the transformation gene product of Fujinami virus was particularly sensitive to the bromomethyl ketone derivative of leucine, while the src gene proudct of Rous sarcoma virus was comparatively less sensitive. The bromomethyl ketone inhibitor interfered with the phophorylation of the EGF receptor by [.gamma.-32P]-8-azido-ATP but much less with the light-sensitive binding. This observation and the lack of interference with EGF binding suggest that the inhibitor interacts with the protein kinase portion of the receptor complex.