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.
Kosuge
S
S
Laboratory of Genetical Biochemistry, Graduate School of Medical Sciences, Kitasato University, Kitasato, Sagamihara 228-8555, Japan.
Maekawa
T
T
Saito
C
C
Tanaka
T
T
Kouno
I
I
Ohtsuki
K
K
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D001665:Binding Sites; D047390:Casein Kinase II; D020134:Catalytic Domain; D002417:Cattle; D011232:Chemical Precipitation; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology*; D005419:Flavonoids; D005707:Gallic Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D005960:Glucosides / Q000737:chemistry / Q000494:pharmacology; D006657:Histones / Q000378:metabolism; D047348:Hydrolyzable Tannins; D007700:Kinetics; D015394:Molecular Structure; D010636:Phenols / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011108:Polymers / Q000494:pharmacology; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D011794:Quercetin / Q000494:pharmacology; D013379:Substrate Specificity; D013552:Swine; D013634:Tannins / Q000737:chemistry* / Q000494:pharmacology*
D000975:Antioxidants; D004791:Enzyme Inhibitors; D005419:Flavonoids; D005960:Glucosides; D006657:Histones; D047348:Hydrolyzable Tannins; D010636:Phenols; D011108:Polymers; D021122:Protein Subunits; D013634:Tannins; C013515:ellagitannin; C110634:eugeniin; D005707:Gallic Acid; D011794:Quercetin; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases