The properties of phosphodiesterase 11A4 GAF domains are regulated by modifications in its N-terminal domain.
The tandem GAF domain of human phosphodiesterase 11A4 (hPDE11A4) requires 72 microm cGMP for half-maximal effective concentration (EC(50)) 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 EC(50) value for cGMP from 72 to 23 microm. Phosphomimetic point mutations (S117D/S167D), which project complete phosphorylation, lowered the EC(50) value to 16 microm. 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 EC(50) value of 7.6 microm. Truncation by 176 amino acids was required for cGMP EC(50) values to decrease to below 10 microm; a construct truncated by 168 amino acids had an EC(50) value of 224 microm. The decrease in EC(50) 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.
Gross-Langenhoff
Marco
M
Pharmazeutisches Institut, Universität Tübingen, Morgenstelle 8, Tübingen, Germany.
Stenzl
Arnulf
A
Altenberend
Florian
F
Schultz
Anita
A
Schultz
Joachim E
JE
D015106:3',5'-Cyclic-GMP Phosphodiesterases; D020816:Amino Acid Motifs; D006152:Cyclic GMP / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010768:Phosphoserine / Q000378:metabolism; D017434:Protein Structure, Tertiary
D010768:Phosphoserine; D010727:Phosphoric Diester Hydrolases; D015106:3',5'-Cyclic-GMP Phosphodiesterases; C406737:PDE11A protein, human; D006152:Cyclic GMP