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The upstream conserved regions (UCRs) mediate homo- and hetero-oligomerization of type 4 cyclic nucleotide phosphodiesterases (PDE4s).

ABSTRACT: PDE4s (type 4 cyclic nucleotide phosphodiesterases) are divided into long and short forms by the presence or absence of conserved N-terminal domains termed UCRs (upstream conserved regions). We have shown previously that PDE4D2, a short variant, is a monomer, whereas PDE4D3, a long variant, is a dimer. In the present study, we have determined the apparent molecular masses of various long and short PDE4 variants by size-exclusion chromatography and sucrose density-gradient centrifugation. Our results indicate that dimerization is a conserved property of all long PDE4 forms, whereas short forms are monomers. Dimerization is mediated by the UCR domains. Given their high sequence conservation, the UCR domains mediate not only homo-oligomerization, but also hetero-oligomerization of distinct PDE4 long forms as detected by co-immunoprecipitation assays and FRET microscopy. Endogenous PDE4 hetero-oligomers are, however, low in abundance compared with homo-dimers, revealing the presence of mechanisms that predispose PDE4s towards homo-oligomerization. Oligomerization is a prerequisite for the regulatory properties of the PDE4 long forms, such as their PKA (protein kinase A)-dependent activation, but is not necessary for PDE4 protein-protein interactions. As a result, individual PDE4 protomers may independently mediate protein-protein interactions, providing a mechanism whereby PDE4s contribute to the assembly of macromolecular signalling complexes.

SUBMITTER: Xie M

PROVIDER: S-EPMC4315173 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

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The upstream conserved regions (UCRs) mediate homo- and hetero-oligomerization of type 4 cyclic nucleotide phosphodiesterases (PDE4s).

Xie Moses M Blackman Brigitte B Scheitrum Colleen C Mika Delphine D Blanchard Elise E Lei Tao T Conti Marco M Richter Wito W

The Biochemical journal 20140501 3

PDE4s (type 4 cyclic nucleotide phosphodiesterases) are divided into long and short forms by the presence or absence of conserved N-terminal domains termed UCRs (upstream conserved regions). We have shown previously that PDE4D2, a short variant, is a monomer, whereas PDE4D3, a long variant, is a dimer. In the present study, we have determined the apparent molecular masses of various long and short PDE4 variants by size-exclusion chromatography and sucrose density-gradient centrifugation. Our res ...[more]

PMID: 24555506

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Project description:BACKGROUND: Leishmania represent a complex of important human pathogens that belong to the systematic order of the kinetoplastida. They are transmitted between their human and mammalian hosts by different bloodsucking sandfly vectors. In their hosts, the Leishmania undergo several differentiation steps, and their coordination and optimization crucially depend on numerous interactions between the parasites and the physiological environment presented by the fly and human hosts. Little is still known about the signalling networks involved in these functions. In an attempt to better understand the role of cyclic nucleotide signalling in Leishmania differentiation and host-parasite interaction, we here present an initial study on the cyclic nucleotide-specific phosphodiesterases of Leishmania major. RESULTS: This paper presents the identification of three class I cyclic-nucleotide-specific phosphodiesterases (PDEs) from L. major, PDEs whose catalytic domains exhibit considerable sequence conservation with, among other, all eleven human PDE families. In contrast to other protozoa such as Dictyostelium, or fungi such as Saccharomyces cerevisiae, Candida ssp or Neurospora, no genes for class II PDEs were found in the Leishmania genomes. LmjPDEA contains a class I catalytic domain at the C-terminus of the polypeptide, with no other discernible functional domains elsewhere. LmjPDEB1 and LmjPDEB2 are coded for by closely related, tandemly linked genes on chromosome 15. Both PDEs contain two GAF domains in their N-terminal region, and their almost identical catalytic domains are located at the C-terminus of the polypeptide. LmjPDEA, LmjPDEB1 and LmjPDEB2 were further characterized by functional complementation in a PDE-deficient S. cerevisiae strain. All three enzymes conferred complementation, demonstrating that all three can hydrolyze cAMP. Recombinant LmjPDEB1 and LmjPDEB2 were shown to be cAMP-specific, with Km values in the low micromolar range. Several PDE inhibitors were found to be active against these PDEs in vitro, and to inhibit cell proliferation. CONCLUSION: The genome of L. major contains only PDE genes that are predicted to code for class I PDEs, and none for class II PDEs. This is more similar to what is found in higher eukaryotes than it is to the situation in Dictyostelium or the fungi that concomitantly express class I and class II PDEs. Functional complementation demonstrated that LmjPDEA, LmjPDEB1 and LmjPDEB2 are capable of hydrolyzing cAMP. In vitro studies with recombinant LmjPDEB1 and LmjPDEB2 confirmed this, and they demonstrated that both are completely cAMP-specific. Both enzymes are inhibited by several commercially available PDE inhibitors. The observation that these inhibitors also interfere with cell growth in culture indicates that inhibition of the PDEs is fatal for the cell, suggesting an important role of cAMP signalling for the maintenance of cellular integrity and proliferation.

Dataset Information

The upstream conserved regions (UCRs) mediate homo- and hetero-oligomerization of type 4 cyclic nucleotide phosphodiesterases (PDE4s).

Publications

The upstream conserved regions (UCRs) mediate homo- and hetero-oligomerization of type 4 cyclic nucleotide phosphodiesterases (PDE4s).

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