The phorbol ester TPA markedly enhances the binding of calcium to the regulatory domain of protein kinase C beta 1 in the presence of phosphatidylserine.
Activation of protein kinase enzyme activity by Ca2+ and diacylglycerol or phorbol esters is a feature of certain isoforms of protein kinase C (PKC). Although the binding sites of phorbol ester on the regulatory domain of PKC have been extensively studied, little is known about the actual mechanisms of Ca2+ binding and how this leads to enzyme activation. We previously reported that high affinity binding of 45Ca2+ to the regulatory domain of PKC beta 1, expressed as a GST fusion protein in Escherichia coli, is dependent on the presence of phosphatidylserine (PS) or 12-O-tetradecanoylphorbol-13-acetate (TPA). In the present study we have used this system to further analyze Ca2+ binding. Using various deletions, we found that different domains in the regulatory domain of PKC beta 1 are involved in TPA-induced Ca2+ binding, depending on whether or not PS was also present in the binding assay. In addition, Ca2+ binding in the presence of TPA alone displayed very different kinetics than Ca2+ binding in the presence of TPA and PS. Scatchard analysis indicated that in the presence of TPA, the Kd value for Ca2+ binding was 51.9 microM. However, in the presence of both TPA and PS, the Kd value dropped to 0.23 microM. These results provide direct evidence that TPA activates certain isoforms of PKC by enhancing PS-dependent Ca2+ binding, thus decreasing the Kd value for Ca2+ binding to a physiological level.
Luo
J H
JH
Columbia-Presbyterian Cancer Center, Columbia University, New York, NY 10032, USA.
Xing
W Q
WQ
Weinstein
I B
IB
D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D007700:Kinetics; D010718:Phosphatidylserines / Q000494:pharmacology*; D011493:Protein Kinase C / Q000378:metabolism*; D064546:Protein Kinase C beta; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D010718:Phosphatidylserines; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium