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Isolation and characterization of a mammalian gene encoding a high-affinity cAMP phosphodiesterase.

ABSTRACT: A rat brain cDNA library has been constructed in a Saccharomyces cerevisiae expression vector and used to isolate genes that can function in yeast to suppress the phenotypic effects of RAS2val19, a mutant form of the RAS2 gene analogous to an oncogenic mutant of the human HRAS gene. One cDNA, DPD, was cloned and its genetic and biochemical properties were characterized. A DPD product would share 80% amino acid sequence identity with the Drosophila melanogaster dunce-encoded protein over an extended region. We have shown that the DPD protein is a high-affinity cAMP-specific phosphodiesterase.

SUBMITTER: Colicelli J

PROVIDER: S-EPMC287185 | biostudies-other | 1989 May

REPOSITORIES: biostudies-other

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Isolation and characterization of a mammalian gene encoding a high-affinity cAMP phosphodiesterase.

Colicelli J J Birchmeier C C Michaeli T T O'Neill K K Riggs M M Wigler M M

Proceedings of the National Academy of Sciences of the United States of America 19890501 10

A rat brain cDNA library has been constructed in a Saccharomyces cerevisiae expression vector and used to isolate genes that can function in yeast to suppress the phenotypic effects of RAS2val19, a mutant form of the RAS2 gene analogous to an oncogenic mutant of the human HRAS gene. One cDNA, DPD, was cloned and its genetic and biochemical properties were characterized. A DPD product would share 80% amino acid sequence identity with the Drosophila melanogaster dunce-encoded protein over an exten ...[more]

PMID: 2542941

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Project description:Cyclic AMP-dependent pathways mediate the communication between external stimuli and the intracellular signaling machinery, thereby influencing important aspects of cellular growth, morphogenesis and differentiation. Crucial to proper function and robustness of these signaling cascades is the strict regulation and maintenance of intracellular levels of cAMP through a fine balance between biosynthesis (by adenylate cyclases) and hydrolysis (by cAMP phosphodiesterases). We functionally characterized gene-deletion mutants of a high-affinity (PdeH) and a low-affinity (PdeL) cAMP phosphodiesterase in order to gain insights into the spatial and temporal regulation of cAMP signaling in the rice-blast fungus Magnaporthe oryzae. In contrast to the expendable PdeL function, the PdeH activity was found to be a key regulator of asexual and pathogenic development in M. oryzae. Loss of PdeH led to increased accumulation of intracellular cAMP during vegetative and infectious growth. Furthermore, the pdeHDelta showed enhanced conidiation (2-3 fold), precocious appressorial development, loss of surface dependency during pathogenesis, and highly reduced in planta growth and host colonization. A pdeHDelta pdeLDelta mutant showed reduced conidiation, exhibited dramatically increased (approximately 10 fold) cAMP levels relative to the wild type, and was completely defective in virulence. Exogenous addition of 8-Br-cAMP to the wild type simulated the pdeHDelta defects in conidiation as well as in planta growth and development. While a fully functional GFP-PdeH was cytosolic but associated dynamically with the plasma membrane and vesicular compartments, the GFP-PdeL localized predominantly to the nucleus. Based on data from cAMP measurements and Real-Time RTPCR, we uncover a PdeH-dependent biphasic regulation of cAMP levels during early and late stages of appressorial development in M. oryzae. We propose that PdeH-mediated sustenance and dynamic regulation of cAMP signaling during M. oryzae development is crucial for successful establishment and spread of the blast disease in rice.

Project description:Trypanosoma cruzi, the causative agent of Chagas disease, encodes a number of different cAMP-specific PDE (phosphodiesterase) families. Here we report the identification and characterization of TcrPDEB1 and its comparison with the previously identified TcrPDEB2 (formerly known as TcPDE1). These are two different PDE enzymes of the TcrPDEB family, named in accordance with the recent recommendations of the Nomenclature Committee for Kinetoplast PDEs [Kunz, Beavo, D'Angelo, Flawia, Francis, Johner, Laxman, Oberholzer, Rascon, Shakur et al. (2006) Mol. Biochem. Parasitol. 145, 133-135]. Both enzymes show resistance to inhibition by many mammalian PDE inhibitors, and those that do inhibit do so with appreciable differences in their inhibitor profiles for the two enzymes. Both enzymes contain two GAF (cGMP-specific and -stimulated phosphodiesterases, Anabaena adenylate cyclases and Escherichia coli FhlA) domains and a catalytic domain highly homologous with that of the T. brucei TbPDE2/TbrPDEB2 family. The N-terminus+GAF-A domains of both enzymes showed significant differences in their affinities for cyclic nucleotide binding. Using a calorimetric technique that allows accurate measurements of low-affinity binding sites, the TcrPDEB2 N-terminus+GAF-A domain was found to bind cAMP with an affinity of approximately 500 nM. The TcrPDEB1 N-terminus+GAF-A domain bound cAMP with a slightly lower affinity of approximately 1 muM. The N-terminus+GAF-A domain of TcrPDEB1 did not bind cGMP, whereas the N-terminus+GAF-A domain of TcrPDEB2 bound cGMP with a low affinity of approximately 3 muM. GAF domains homologous with those found in these proteins were also identified in related trypanosomatid parasites. Finally, a fluorescent cAMP analogue, MANT-cAMP [2'-O-(N-methylanthraniloyl)adenosine-3',5'-cyclic monophosphate], was found to be a substrate for the TcPDEB1 catalytic domain, opening the possibility of using this molecule as a substrate in non-radioactive, fluorescence-based PDE assays, including screening for trypanosome PDE inhibitors.

Dataset Information

Isolation and characterization of a mammalian gene encoding a high-affinity cAMP phosphodiesterase.

Publications

Isolation and characterization of a mammalian gene encoding a high-affinity cAMP phosphodiesterase.

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