Rapid in vitro conformational changes of the catalytic site of PKC alpha assessed by FIM-1 fluorescence

To study the activation process of protein kinase C (PKCalpha), we used a fluorescent probe, FIM-1, a bis-indolylmaleimide derivative, which binds to the ATP-binding site on the catalytic domain [Chen, C. S., and Poenie, M. (1993) J. Biol. Chem. 268, 15812]. This enabled us to directly observe the microenvironment of the ATP-binding site in vitro during the activation process. The FIM-1 binding affinity for PKCalpha (EC(50) between 6 and 10 nM) was affected neither by PKCalpha activating conditions nor by enzyme proteolysis. The fluorescence yield of the PKCalpha-FIM-1 complex depended on the PKCalpha activation state. This fluorescence yield was decreased upon proteolysis, which allowed us to study the rate of PKC proteolysis by mu-calpain and its modification by cofactors. Two binding sites were also observed for Ca2+ on the partially activated PKCalpha. After phorbol ester (TPA) application, PKC activation was characterized by biexponential kinetics, including a rapid phase completed within 5 min and a slow phase lasting at least 30 min, which reflected several activation steps. Two different binding sites for TPA were revealed on membrane-associated PKCalpha (EC(50) = 31 +/- 12 and 580 +/- 170 nM), and their modulation by phosphatidylserine and Ca2+ was characterized. The high-affinity TPA binding site was highly conserved, even on the soluble enzyme. Our study shows that binding of low concentrations of TPA triggers conformational changes in the soluble PKCalpha, which affect the microenvironment of its catalytic domain.