Mobilization of the A-kinase N-myristate through an isoform-specific intermolecular switch

the catalytic (C) subunit of cAMP-dependent protein kinase is N-myristylate d, it is a soluble protein, and no physiological role has been identified f or its myristyl moiety. To determine whether the interaction of the two reg ulatory (R) subunit isoforms (R-I and R-II) with the N-myristylated C subun it affects its ability to target membranes, the effect of N-myristylation a nd the R-I and (II) subunit isoforms on C subunit binding to phosphatidylch oline/ phosphatidylserine liposomes was examined. Only the combination of N -myristylation and R-II subunit interaction produced a dramatic increase in the rate of liposomal binding. To assess whether the R-II subunit also inc reased the conformational flexibility of the C subunit N terminus, the effe ct of N-myristylation and the R-I and R-II subunits on the rotational freed om of the C subunit N terminus was measured. Specifically, fluorescein male imide was conjugated to Cys-16 in the N-terminal domain of a K16C mutant of the C subunit and the time-resolved emission anisotropy was determined. Th e interaction of the R-II subunit. but not the R-I subunit significantly in creased the backbone flexibility around the site of mutation and labeling, strongly suggesting that R-II subunit binding to the myristylated C subunit induced a unique conformation of the C subunit that is associated with an increase in both the N-terminal flexibility and the exposure of the N-myris tate, R-II subunit thus appears to serve as an intermolecular switch that d isrupts of the link between the N-terminal and core catalytic domains of th e C subunit to expose the N-myristate and poise the holoenzyme for interact ion with membranes.