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

The inhibitory effects of galloyl pedunculasn (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 (ID50 = 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 (ID50 = approx, 0.25 muM) and casein kinase II (CK-II, ID50 = approx. 0.6 muM); (ii) GP inhibits the auto-phosphorylation of A-kinase beta in a dose-dependent manner with an ID50 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.