Biochemical characterization of galloyl pedunculagin (ellagitannin) as a selective inhibitor of the beta-regulatory subunit of A-kinase in vitro.

The inhibitory effects of galloyl pedunculagin (GP) and eugeniin on the phosphorylation of histone H2B by cAMP-dependent protein kinase (A-kinase) and autophosphorylation of its beta-regulatory subunit (A-kinase beta) were examined in vitro. It was found that (i) GP (ID(50) = approx. 50 nM) effectively inhibits the activity of A-kinase (heterodimer), but high doses are required to inhibit the activities of the alpha-catalytic subunit (ID(50) = approx. 0.25 microM) and casein kinase II (CK-II, ID(50) = approx. 0.6 microM); (ii) GP inhibits the autophosphorylation of A-kinase beta in a dose-dependent manner with an ID(50) of approx. 6.6 nM, which is about 30-fold lower than that observed with CK-II beta; and (iii) GP reduces the suppressive effect of the beta-subunit on the activity of the alpha-subunit. In addition, purified bovine heart A-kinase precipitates when incubated with excess GP at pH 5.0. A similar precipitation of A-kinase was observed with eugeniin. These results show that the direct binding of GP to the beta-subunit prevents the physiological interaction between the beta- and alpha-subunits of A-kinase in vitro. This conclusion is presumably consistent with the binding affinity of proline-rich proteins with tannins, since A-kinase beta contains a proline-rich domain that interacts with GP or eugeniin. Therefore, GP will serve as a powerful inhibitor for in vitro and in vivo cellular studies of A-kinase beta-mediated signal transduction.