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Activation of the cAMP-specific Phosphodiesterase PDE4D3 by Phosphorylation
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 encompa...
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Published in: | The Journal of biological chemistry 1999-07, Vol.274 (28), p.19677-19685 |
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Main Authors: | , , |
Format: | Article |
Language: | English |
Citations: | Items that this one cites Items that cite this one |
Online Access: | Get full text |
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Summary: | 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 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 present
in PDE4D and that phosphorylation of the regulatory domain causes activation of the enzyme by modulating the interaction between
inhibitory and catalytic domains. |
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ISSN: | 0021-9258 1083-351X |
DOI: | 10.1074/jbc.274.28.19677 |