In vitro effects of oxygen-derived free radicals on type I and type II cAMP-dependent protein kinases

Abstract

Oxygen free radicals may act as second messengers in signal transduction pathways and contribute to inflammatory diseases. We studied the action in vitro of radiolytically generated hydroxyl radicals (.OH) and superoxide radicals (O-2) on the cAMP-dependent protein kinases, I and II (PKAI and -II, respectively). The effects of the gasses O2 and N2O used to produce O-2 or .OH radicals by gamma-radiolysis of the water were also studied. PKAI is more sensitive than PKAII to oxygen gas (10 mM sodium formate) and to hydroxyl and superoxide radicals. Hydroxyl radicals decreased the kinase phosphotransferase activities stimulated either by cAMP or its site-specific analogs for both PKAI and PKAII; however, PKAI was more affected. The binding of [3H]cAMP and of 8-N3-[32P]cAMP to RI regulatory subunits was decreased. .OH caused a loss of tryptophan 260 fluorescence at site A of PKAI and of bityrosine production. Superoxide radicals affected only PKAI. O-2 modified both cAMP-binding sites A and B of the regulatory subunit but had a smaller effect on the catalytic subunit. The catalytic subunit was more sensitive to radicals when free than when part of the holoenzymes during exposure to the oxygen free radicals. These results suggest that oxygen free radicals alter the structure of PKA enzymes. Thus, oxidative modifications may alter key enzymes, including cAMP-dependent protein kinases, in certain pathological states.