Contribution of the Carboxyl-Terminal Region of the cAMP-Dependent Protein Kinase Type Iα Regulatory Subunit to Cyclic Nucleotide Interactions

The carboxyl-terminal 19 amino acids of the type Iα regulatory subunit (RIα) of cAMP-dependent protein kinase (PKA) were investigated to determine their contributions to cAMP selectivity. The parent RIα subunit contained an Ala to Thr mutation at position 334 so that it would bind both cAMP and cGMP with high affinity. Stop codons were introduced into the parent cDNA construct at positions corresponding to Val-375, Asn-372, Gln-370, and Cys-360. The purified, bacterially expressed proteins were characterized for their cAMP and cGMP dissociation properties. Site-selective cAMP analogs were used to compete against [3H]cAMP binding to the mutant RIα subunits to correctly assign fast and slow dissociationt1/2values to the A and B domains. A greater than 60-fold drop in B domaint1/2in the Asn-372-stop to Gln-370-stop transition implicated Tyr-371 as an important cAMP-binding determinant. A similar drop in [3H]cGMPt1/2for the same transition suggested that the cGMP/cAMP selectivity was not altered. To test this further, Tyr-371 was mutated to Ala, Phe, and Arg in the parent construct. The cAMP and cGMPt1/2values were determined, as were protein kinase activation constants (Ka) for holoenzymes formed from mutant RIα subunits and purified catalytic subunit. TheKadata suggested that mutation of Tyr-371 enhanced B domain cAMP selectivity. Isolated B domains containing Tyr-371-Arg or Tyr-371-Phe mutations were constructed, expressed, and purified to determine their relative inhibition constants (K′I) for cGMP vs cAMP. These data showed that B domain cAMP selectivity was minimally affected by alteration of Tyr-371. Based on these results, it is concluded that aromatic stacking is not important for determining B-domain cyclic nucleotide selectivity. It is proposed that the main function of Tyr-371 is stabilization of the B-domain cAMP-binding pocket through hydrogen bonding with Glu-324.