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 (cGKI alpha and cGKI beta), a high affinity interaction exists between the C2 amino group of cGMP and th e hydroxyl side chain of a threonine conserved in most cGMP binding sites. To examine the effect of this interaction on ligand binding and kinase acti vation in the type II isozyme of cGMP-dependent protein kinase (cGKII), ala nine 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, disruptio n of cGMP-specific binding in the NH2-terminal binding site had the greates t effect on cGMP-dependent kinase activation, like cGKI. However, ligand di ssociation studies showed that the location of the rapid and slow dissociat ion sites of cGKII was reversed relative to cGKI, Another set of mutations that prevented cyclic nucleotide binding demonstrated the necessity of the NH2-terminal, rapid dissociation binding site for cyclic nucleotide-depende nt activation of cGKII. These findings suggest distinct mechanisms of activ ation for cGKII and cGKI isoforms, Because cGKII mediates the effects of he at-stable enterotoxins via the cystic fibrosis transmembrane regulator Cl- channel, these findings define a structural target for drug design.