Photoaffinity labelling of the ATP-binding site of the epidermal growth factor-dependent protein kinase

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Abstract
Epidermal growth factor (EGF), after binding to its receptor, activates a tyrosine-specific protein kinase which phosphorylates several substrates, including the EGF receptor itself. The effects of a photoaffinity analog of ATP, arylazido-.beta.-alanyl-ATP, on the EGF-dependent protein kinase in A431 human [epidermoid] tumor cell plasma membrane vesicles was investigated. This analog was capable of inactivating the EGF-receptor kinase in a photodependent manner. Partial inactivation occurred at an analog concentration of 1 .mu.M and complete inactivation occurred at 10 .mu.M when a 2-min light exposure was used. Arylazido-.beta.-alanine at 100 .mu.M and ATP at 100 .mu.M were incapable of inactivating the enzyme with 2 min of light exposure. The photodependent inactivation of the enzyme by the analog could be partially blocked by 20 mM-ATP and more effectively blocked by either 20 mM-adenosine 5''-[.beta..gamma.-imido]triphosphate or 20 mM-guanosine 5''-[.beta..gamma.-imido]triphosphate, indicating nucleotide-binding site specificity. Arylazido-.beta.-alanyl-[.alpha.-32P]ATP was capable of labeling membrane proteins in a photodependent manner. Numerous proteins were labeled, the most prominent of which ran with an apparent MW of 53,000 on polyacrylamide-gel electrophoresis. A band of minor intensity was seen of MW corresponding to the EGF receptor (170,000). Immunoprecipitation of affinity-labeled and solubilized membranes with an anti-(EGF receptor) monoclonal antibody demonstrated that the MW 170,000 receptor protein was photoaffinity labeled by the analog. The MW 53,000 peptide was not specifically bound by the anti-receptor antibody. The affinity labeling of the receptor was not enhanced by EGF, suggesting that EGF stimulation of the kinase activity does not result from changes in the affinity of the kinase for ATP. Arylazido-.beta.-alanyl-ATP evidently interacts with the ATP-binding site of the EGF-receptor kinase with apparent high affinity. This analog is an effective photoaffinity label for the kinase. The EGF receptor, identified by using monoclonal antibodies evidently contains an ATP-binding site, providing further confirmation that the EGF receptor and EGF-dependent protein kinase are domains of the MW 170,000 protein.