UCR1 and UCR2 domains unique to the cAMP-specific phosphodiesterase familyform a discrete module via electrostatic interactions

The cAMP-specific phosphodiesterases (PDE4) enzymes contain unique "signatu re" regions of amino acid sequence, called upstream conserved regions 1 and 2 (UCR1 and UCR2), UCR1 and UCR2 are located between the extreme amino-ter minal 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 identifie d a PDE4D cDNA clone containing UCR2 and the catalytic region but not UCR1. Two-hybrid and "pull down" analysis of constructs incorporating various re gions of the PDE4D3 cDNA demonstrated that the carboxyl terminal region of UCR1 interacted specifically with the amino-terminal region of UCR2, The in teraction was blocked by mutations of two positively charged amino acids (A rg-98 and Arg-101 to alanine) located within an otherwise largely hydrophob ic 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 interacti on. The phosphorylation of UCR1 by cAMP-dependent protein kinase (PKA) in v itro 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 acti vation of PDE4D3 by PKA, profoundly reduced the interaction between UCR1 an d UCR2. Our data are consistent with a model in which UCR1 and UCR2 act as independent domains whose interaction is determined by electrostatic intera ctions and which may be disrupted by PKA phosphorylation, We suggest that t he UCR1 and UCR2 domains may form a module that interacts with and regulate s the PDE4 catalytic region.