Contribution of the carboxyl-terminal regional of the cAMP-dependent protein kinase type I alpha regulatory subunit to cyclic nucleotide interactions.
The carboxyl-terminal 19 amino acids of the type I alpha regulatory subunit (RI alpha) of cAMP-dependent protein kinase (PKA) were investigated to determine their contributions to cAMP selectivity. The parent RI alpha 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 RI alpha subunits to correctly assign fast and slow dissociation t1/2 values to the A and B domains. A greater than 60-fold drop in B domain t1/2 in the Asn-372-stop to Gln-370-stop transition implicated Tyr-371 as an important cAMP-binding determinant. A similar drop in [3H]cGMP t1/2 for 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 cGMP t1/2 values were determined, as were protein kinase activation constants (Ka) for holoenzymes formed from mutant RI alpha subunits and purified catalytic subunit. The Ka data 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.
Kapphahn
M A
MA
Department of Biochemistry and Molecular Biology, University of North Dakota School of Medicine and Health Sciences, Grand Forks 58202-9037, USA.
Shabb
J B
JB
D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D002417:Cattle; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism*; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006860:Hydrogen Bonding; D007700:Kinetics; D008297:Male; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D013737:Testis / Q000201:enzymology
D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; D010446:Peptide Fragments; D011994:Recombinant Proteins; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D006152:Cyclic GMP