Terbium as a luminescent probe of metal-binding sites in protein kinase C.
In the present report, we demonstrate that Tb3+ binds to protein kinase C and serves as a luminescent reporter of certain cationic metal-binding sites. Tb3+ titration of 50 nM protein kinase C results in a 20-fold enhancement of Tb3+ luminescence which is half-maximal at 12 microM Tb3+. A Kd of approximately 145 nM was determined for Tb3+ binding to the enzyme. The excitation spectrum of bound Tb3+ exhibits a peak at 280 nm characteristic of energy transfer from protein tryptophan or tyrosine residues. The luminescence of this complex can be markedly decreased by other metals, including Pb2+ (IC50 = 25 microM), La3+ (IC50 = 50 microM), Hg2+ (IC50 = 300 microM), Ca2+ (IC50 = 6 mM), and Zn2+ (IC50 greater than 10 mM), and chelation of Tb3+ by 2 mM EGTA. Tb3+ binding to protein kinase C is correlated with its inhibition of protein kinase activity (IC50 = 8 microM), r = 0.99) and phorbol ester binding (IC50 = 15 microM, r = 0.98). Tb3+ inhibition of protein kinase C activity cannot be overcome by excess Ca2+, but can be partially overcome with excess phosphatidylserine or by chelation of Tb3+ with EGTA. Tb3+ noncompetitively inhibits phorbol ester binding by decreasing the maximal extent of binding without significantly altering binding affinity. The results suggest that the Tb3(+)-binding site is at or allosterically related to the enzyme's phosphatidylserine-binding site, but is distinct from the phorbol ester-binding domain and the Ca2(+)-binding site that regulates enzyme activity.
Walters
J D
JD
College of Dentistry, Ohio State University Medical Center, Columbus 43210.
Johnson
J D
JD
D001665:Binding Sites; D001667:Binding, Competitive; D004533:Egtazic Acid / Q000494:pharmacology; D004735:Energy Transfer; D006801:Humans; D008163:Luminescent Measurements; D008670:Metals / Q000378:metabolism* / Q000494:pharmacology; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism; D010718:Phosphatidylserines / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013053:Spectrophotometry; D013725:Terbium / Q000378:metabolism* / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D008670:Metals; D010718:Phosphatidylserines; D013725:Terbium; D015240:Phorbol 12,13-Dibutyrate; D004533:Egtazic Acid; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate