Phosphodiesterase-8A binds to and regulates Raf-1 kinase.

V-raf-1 murine leukemia viral oncogene homolog 1 (Raf-1) is a key activator of the ERK pathway and is a target for cross-regulation of this pathway by the cAMP signaling system. The cAMP-activated protein kinase, PKA, inhibits Raf-1 by phosphorylation on S259. Here, we show that the cAMP-degrading phosphodiesterase-8A (PDE8A) associates with Raf-1 to protect it from inhibitory phosphorylation by PKA, thereby enhancing Raf-1's ability to stimulate ERK signaling. PDE8A binds to Raf-1 with high (picomolar) affinity. Mapping of the interaction domain on PDE8A using peptide array technology identified amino acids 454-465 as the main binding site, which could be disrupted by mutation. A cell-permeable peptide corresponding to this region disrupted the PDE8A/Raf-1 interaction in cells, thereby reducing ERK activation and the cellular response to EGF. Overexpression of a catalytically inactive PDE8A in cells displayed a dominant negative phenotype on ERK activation. These effects were recapitulated at the organism level in genetically modified (PDE8A(-/-)) mice. Similarly, PDE8 deletion in Drosophila melanogaster reduced basal ERK activation and sensitized flies to stress-induced death. We propose that PDE8A is a physiological regulator of Raf-1 signaling in some cells.

Arrestin-dependent localization of phosphodiesterases.

Many G-protein-coupled receptors trigger the synthesis of cAMP in order to transduce signals from the membrane into the cell cytoplasm. As stimulation of each receptor type results in a specific physiological outcome, compartmentalization of proteins that make, break, and are activated by cAMP underpin receptor-specific responses. Until 2002, it was thought that static compartmentalization of phosphodiesterase 4 (PDE4), conferred by N-terminal targeting sequences, was one way to shape intricate cAMP gradients that formed after receptor activation. Discovery of the PDE4-ß-arrestin complex represented a major breakthrough in cAMP signaling, as it spurred the initial realization that PDE4s could be transported to sites of high cAMP to orchestrate destruction of the second messenger at the same time as the receptor's signal to the G-protein is silenced. This chapter charts the scientific process that led to the discovery and characterization of the PDE4-ß-arrestin interaction and discusses the known functions of this signaling complex.