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 phos phorylation. In these proteins, a conserved region is located between the a mino-terminal domain, which is the target for phosphorylation, and the cata lytic domain. Previous studies have indicated that nested deletions encompa ssing 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 inc rease in hydrolytic activity was observed when PDE4D3 from FRTL-5 cells was incubated with the endoproteinase Lys-C, The activation was abolished by p rotease inhibitors and was absent when a phosphorylated enzyme was used, We stern blot analysis with PDE4D-specific antibodies indicated the Lys-C trea tment separates the catalytic domain of PDE4D3 from the inhibitory domain. Incubation with antibodies recognizing an epitope within this domain caused a 3- to 6-fold increase in activity of native or recombinant PDE4D3, Again , PDE activation by these antibodies had properties similar to, and not add itive with, the activation by protein kinase A phosphorylation. An interact ion between the inhibitory domain and both regulatory and catalytic domains of PDE4D3 was detected by the yeast two-hybrid system. Mutations of Ser(54 ) to Ala in the regulatory domain decreased or abolished this interaction, whereas mutations of Ser(54) to the negatively charged Asp strengthened it. These data strongly support the hypothesis that an inhibitory domain is pr esent in PDE4D and that phosphorylation of the regulatory domain causes act ivation of the enzyme by modulating the interaction between inhibitory and catalytic domains.