title: UCR1 and UCR2 domains unique to the cAMP-specific phosphodiesterase family form a discrete module via electrostatic interactions.
abstract: The cAMP-specific phosphodiesterases (PDE4) enzymes contain unique "signature" regions of amino acid sequence, called upstream conserved regions 1 and 2 (UCR1 and UCR2). UCR1 and UCR2 are located between the extreme amino-terminal region and the catalytic region of the PDE4 enzymes. The UCR1 of the PDE4D3 isoform was used as a "bait" in a two-hybrid screen, which identified a PDE4D cDNA clone containing UCR2 and the catalytic region but not UCR1. Two-hybrid and "pull down" analysis of constructs incorporating various regions of the PDE4D3 cDNA demonstrated that the carboxyl-terminal region of UCR1 interacted specifically with the amino-terminal region of UCR2. The interaction was blocked by mutations of two positively charged amino acids (Arg-98 and Arg-101 to alanine) located within an otherwise largely hydrophobic region of UCR1. Mutation of three negatively charged amino acids in UCR2 (Glu-146, Glu-147, and Asp-149, all to alanine) also blocked the interaction. The phosphorylation of UCR1 by cAMP-dependent protein kinase (PKA) in vitro attenuated the ability of UCR1 to interact with UCR2. Mutation of the PKA substrate site in UCR1 (Ser-54) to aspartic acid, which mimics the activation of PDE4D3 by PKA, profoundly reduced the interaction between UCR1 and UCR2. Our data are consistent with a model in which UCR1 and UCR2 act as independent domains whose interaction is determined by electrostatic interactions and which may be disrupted by PKA phosphorylation. We suggest that the UCR1 and UCR2 domains may form a module that interacts with and regulates the PDE4 catalytic region.
authors.affiliation: Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biology and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom.
authors.forename: M B
authors.initials: MB
authors.lastname: Beard
authors.forename: A E
authors.initials: AE
authors.lastname: Olsen
authors.forename: R E
authors.initials: RE
authors.lastname: Jones
authors.forename: S
authors.initials: S
authors.lastname: Erdogan
authors.forename: M D
authors.initials: MD
authors.lastname: Houslay
authors.forename: G B
authors.initials: GB
authors.lastname: Bolger
mesh_terms: D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D020134:Catalytic Domain; D003001:Cloning, Molecular; D017124:Conserved Sequence; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D018076:DNA, Complementary; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D055672:Static Electricity
chemical_list: D018076:DNA, Complementary; D011994:Recombinant Proteins; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; C517376:PDE4D protein, human