The amino-terminal cyclic nucleotide binding site of the type II cGMP-dependent protein kinase is essential for full cyclic nucleotide-dependent activation.

For the type I cGMP-dependent protein kinases (cGKIalpha and cGKIbeta), a high affinity interaction exists between the C2 amino group of cGMP and the hydroxyl side chain of a threonine conserved in most cGMP binding sites. To examine the effect of this interaction on ligand binding and kinase activation in the type II isozyme of cGMP-dependent protein kinase (cGKII), alanine was substituted for the conserved threonine or serine. cGKII was found to require the C2 amino group of cGMP and its cognate serine or threonine hydroxyl for efficient cGMP activation. Of the two binding sites, disruption of cGMP-specific binding in the NH(2)-terminal binding site had the greatest effect on cGMP-dependent kinase activation, like cGKI. However, ligand dissociation studies showed that the location of the rapid and slow dissociation sites of cGKII was reversed relative to cGKI. Another set of mutations that prevented cyclic nucleotide binding demonstrated the necessity of the NH(2)-terminal, rapid dissociation binding site for cyclic nucleotide-dependent activation of cGKII. These findings suggest distinct mechanisms of activation for cGKII and cGKI isoforms. Because cGKII mediates the effects of heat-stable enterotoxins via the cystic fibrosis transmembrane regulator Cl(-) channel, these findings define a structural target for drug design.