The properties of phosphodiesterase 11A4 GAF domains are regulated by modifications in its N‐terminal domain

Abstract

The tandem GAF domain of human phosphodiesterase 11A4 (hPDE11A4) requires 72 μm cGMP for half‐maximal effective concentration (EC50) of a cyanobacterial adenylyl cyclase used as a reporter enzyme. Here we examine whether modifications in the N‐terminus of PDE11A4 affect cGMP signalling. The N‐terminus has two phosphorylation sites for cyclic nucleotide monophosphate‐dependent protein kinases (Ser117, Ser168). Phosphorylation of both by cAMP‐dependent protein kinase decreased the EC50 value for cGMP from 72 to 23 μm. Phosphomimetic point mutations (S117D/S167D), which project complete phosphorylation, lowered the EC50 value to 16 μm. Structural and sequence data indicate that 196 amino acids precede the start of the GAF domain in hPDE11A4. Removal of 197 amino acids yielded unregulated cyclase activity, whereas truncation by 196 amino acids resulted in a cGMP‐regulated protein with a cGMP EC50 value of 7.6 μm. Truncation by 176 amino acids was required for cGMP EC50 values to decrease to below 10 μm; a construct truncated by 168 amino acids had an EC50 value of 224 μm. The decrease in EC50 values was accompanied by a sixfold increase in basal activity; the extent of cGMP stimulation remained unaffected, however. We conclude that N‐terminal modifications strongly affect cGMP regulation of hPDE11A4.

Functional Chimeras of the Phosphodiesterase 5 and 10 Tandem GAF Domains

“…As reported previously (7,16,30,31), we note that the basal activities of different constructs vary considerably. Thus, one may argue that the expression of the chimeras may result in polypeptides that are different, both structurally and functionally, from the wild-type constructs due to, for example, aberrant folding or domain assembly.…”

Activation of PDE10 and PDE11 Phosphodiesterases

“…In chimeric proteins composed of the N-terminal part of PDE11A4 including the GAF domains and the catalytic domain of Anabaena adenylyl cyclases, the N-terminal part preceding the GAF domains had a tremendous effect on the ability of cGMP to stimulate the chimera. Truncation of 196 N-terminal amino acids increased the cGMP affinity 20-fold (39). However, deletion of these 196 amino acids in the PDE11A4 holoenzyme abolished stimulation by the cGMP analogue but did not enable cGMP stimulation (data not shown).…”

Conformation Changes, N-terminal Involvement, and cGMP Signal Relay in the Phosphodiesterase-5 GAF Domain

“…small kinases will have better efficacy than large kinases. It is also interesting to note that the N terminus of PDE11A4 and the phosphorylation of its 2 serine residues impact the activities of PDE11-cyclase hybrids (36).…”