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

Previous studies of the ligand regulation of the cAMP-dependent protei n kinase have demonstrated the cAMP-mediated dissociation of the holoe nzyme by using nonequilibrium techniques; i.e., gel filtration, ion-ex change chromatography, and differential centrifugation. While physical ly mild, these could have caused weakly associated species to dissocia te, thereby providing a potentially flawed interpretation of the mecha nism of activation of the protein kinase. To assess this, the activati on of the cAMP-dependent protein kinase has been monitored under equil ibrium conditions using dipolar fluorescence energy transfer to measur e changes in the proximity relations between the catalytic (C) and reg ulatory (R) subunits that compose the holoenzyme. Specifically, we pre pared a heterochromatically labeled protein kinase type II holoenzyme, with the regulatory and catalytic subunits labeled with sulforhodamin e and carboxyfluorescein, respectively, and monitored the exchange of electronic excitation energy between the C and R subunits by both dono r lifetime and steady-state fluorescence. Biochemically, the heterochr omatic holoenzyme was closely identical to the native protein with reg ard to cAMP-induced increase in catalytic activity, reassociation of C and R subunits, inhibition of catalytic activity by the specific prot ein 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 incr ease in the distance between the derivatization sites on C and R but n ot a dissociation of these subunits. Addition of PKI plus cAMP promote d full dissociation of the two subunits. Addition of a high-affinity s ubstrate ((Ser 21)PKI(14-22)-amide) had no significant effect on energ y transfer and therefore the distance between derivatization sites on C and R. These results demonstrate (1) that, in vitro, cAMP does not d ecrease the binding affinity between the subunits of the holoenzyme as much as is generally assumed and (2) that PKI, but not a high-affinit y substrate, can affect holoenzyme dissociation. To what extent these regulatory events occur in viable cells is currently being examined.