Activation of the cAMP-specific phosphodiesterase PDE4D3 by phosphorylation. Identification and function of an inhibitory domain.
Splicing variants of type 4 phosphodiesterases (PDE4) are regulated by phosphorylation. In these proteins, a conserved region is located between the amino-terminal domain, which is the target for phosphorylation, and the catalytic domain. Previous studies have indicated that nested deletions encompassing this region cause an increase in catalytic activity, suggesting this domain exerts an inhibitory constraint on catalysis. Here, we have further investigated the presence and function of this domain. A time-dependent increase in hydrolytic activity was observed when PDE4D3 from FRTL-5 cells was incubated with the endoproteinase Lys-C. The activation was abolished by protease inhibitors and was absent when a phosphorylated enzyme was used. Western blot analysis with PDE4D-specific antibodies indicated the Lys-C treatment separates the catalytic domain of PDE4D3 from the inhibitory domain. Incubation with antibodies recognizing an epitope within this domain caused a 3- to 4-fold increase in activity of native or recombinant PDE4D3. Again, PDE activation by these antibodies had properties similar to, and not additive with, the activation by protein kinase A phosphorylation. An interaction between the inhibitory domain and both regulatory and catalytic domains of PDE4D3 was detected by the yeast two-hybrid system. Mutations of Ser54 to Ala in the regulatory domain decreased or abolished this interaction, whereas mutations of Ser54 to the negatively charged Asp strengthened it. These data strongly support the hypothesis that an inhibitory domain is present in PDE4D and that phosphorylation of the regulatory domain causes activation of the enzyme by modulating the interaction between inhibitory and catalytic domains.
Lim
J
J
Division of Reproductive Biology, Department of Gynecology and Obstetrics, Stanford University School of Medicine, Stanford California 94305-5317, USA.
Pahlke
G
G
Conti
M
M
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000235:genetics* / Q000378:metabolism; D017398:Alternative Splicing / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D001665:Binding Sites; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism*; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D004789:Enzyme Activation; D008666:Metalloendopeptidases / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation; D011480:Protease Inhibitors; D051381:Rats; D011994:Recombinant Proteins; D012441:Saccharomyces cerevisiae; D013379:Substrate Specificity
D000906:Antibodies; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D000242:Cyclic AMP; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase