Conformation of the C1 phorbol-ester-binding domain participates in the activating conformational change of protein kinase C

The fluorescent phorbol ester 12-N-methylanthraniloylphorbol 13-acetate [sa pintoxin D (SAPD)] was used as both the activator and the probe for the act ivating conformational change of the C I domain of recombinant protein kina se C (PKC)alpha. Fluorescence emission spectra and steady-state anisotropy measurements of SAPD in fully active membrane-associated PKC show that ther e is a relatively hydrophobic environment and restricted motional freedom c haracterizing the phorbol-ester-binding site. SAPD also interacts with the membrane lipids so that it was necessary to resort to time-resolved anisotr opy measurements to resolve the signals corresponding to PKC-bound SAPD fro m that associated with buffer and lipid. In the presence of membrane lipids (unilamellar vesicles of phosphatidylcholine and phosphatidylserine, 4:1 m olar ratio) and Ca2+, at a concentration sufficient to activate the enzyme fully, a long correlation time characteristic of highly restricted motion w as observed for PKC-associated SAPD. The fraction of SAPD molecules display ing this restricted motion, in comparison with the total SAPD including tha t in lipids and in buffer, increased with increasing concentrations of Ca2 and paralleled the appearance of enzyme activity, whereas the rotational c orrelation time remained constant. This could be rationalized as an increas e in the number of active PKC conformers in the total population of PKC mol ecules. It therefore seems that there is a distinct conformation of the C1 activator-binding domain associated with the active form of PKC. The additi on of SAPD and dioleoyl-sn-glycerol together produced an activity higher th an that achievable by either activator alone both at concentrations that al one induced maximal activity for the respective activator; this higher acti vity was associated with a further restriction in SAPD motion. Increasing t he cholesterol concentration, the phosphatidylethanolamine concentration, t he sn-2 unsaturation in phosphatidylcholine and the vesicle curvature each also elevated SAPD-induced PKC activity and again increased the PKC-associa ted SAPD rotational correlation time. In summary, the rotational correlatio n time of PKC-bound SAPD, extractable from a single time-resolved fluoresce nce anisotropy measurement, provides a novel probe for the involvement of i nteractions between the C1 domain and phorbol eater in the modulation of PK C activity.