FLUORESCENCE RESONANCE ENERGY-TRANSFER WITHIN A HETEROCHROMATIC CAMP-DEPENDENT PROTEIN-KINASE HOLOENZYME UNDER EQUILIBRIUM CONDITIONS - NEW INSIGHTS INTO THE CONFORMATIONAL-CHANGES THAT RESULT IN CAMP-DEPENDENT ACTIVATION

Previous studies of the ligand regulation of the cAMP-dependent protein kinase have demonstrated the cAMP-mediated dissociation of the holoenzyme by using nonequilibrium techniques; i.e., gel filtration, ion-exchange chromatography, and differential centrifugation. While physically mild, these could have caused weakly associated species to dissociate, thereby providing a potentially flawed interpretation of the mechanism of activation of the protein kinase. To assess this, the activation of the cAMP-dependent protein kinase has been monitored under equilibrium conditions using dipolar fluorescence energy transfer to measure changes in the proximity relations between the catalytic (C) and regulatory (R) subunits that compose the holoenzyme. Specifically, we prepared a heterochromatically labeled protein kinase type II holoenzyme, with the regulatory and catalytic subunits labeled with sulforhodamine and carboxyfluorescein, respectively, and monitored the exchange of electronic excitation energy between the C and R subunits by both donor lifetime and steady-state fluorescence. Biochemically, the heterochromatic holoenzyme was closely identical to the native protein with regard to cAMP-induced increase in catalytic activity, reassociation of C and R subunits, inhibition of catalytic activity by the specific protein kinase inhibitor (PKI), and observed dissociation examined by gel filtration upon cAMP addition. However, under equilibrium conditions, the energy-transfer measurements revealed that the addition of cAMP to this heterochromatic reporter complex promoted an estimated lo-A increase in the distance between the derivatization sites on C and R but not a dissociation of these subunits. Addition of PKI plus cAMP promoted full dissociation of the two subunits. Addition of a high-affinity substrate ((Ser 21)PKI(14-22)-amide) had no significant effect on energy transfer and therefore the distance between derivatization sites on C and R. These results demonstrate (1) that, in vitro, cAMP does not decrease the binding affinity between the subunits of the holoenzyme as much as is generally assumed and (2) that PKI, but not a high-affinity substrate, can affect holoenzyme dissociation. To what extent these regulatory events occur in viable cells is currently being examined.