title: Phosphodiesterase-8A binds to and regulates Raf-1 kinase.
abstract: 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.
authors.affiliation: Institute of Cardiovascular and Medical Science, Cellular and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom.
authors.forename: Kim M
authors.initials: KM
authors.lastname: Brown
authors.forename: Jon P
authors.initials: JP
authors.lastname: Day
authors.forename: Elaine
authors.initials: E
authors.lastname: Huston
authors.forename: Bastian
authors.initials: B
authors.lastname: Zimmermann
authors.forename: Kornelia
authors.initials: K
authors.lastname: Hampel
authors.forename: Frank
authors.initials: F
authors.lastname: Christian
authors.forename: David
authors.initials: D
authors.lastname: Romano
authors.forename: Selim
authors.initials: S
authors.lastname: Terhzaz
authors.forename: Louisa C Y
authors.initials: LC
authors.lastname: Lee
authors.forename: Miranda J
authors.initials: MJ
authors.lastname: Willis
authors.forename: David B
authors.initials: DB
authors.lastname: Morton
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Beavo
authors.forename: Masami
authors.initials: M
authors.lastname: Shimizu-Albergine
authors.forename: Shireen A
authors.initials: SA
authors.lastname: Davies
authors.forename: Walter
authors.initials: W
authors.lastname: Kolch
authors.forename: Miles D
authors.initials: MD
authors.lastname: Houslay
authors.forename: George S
authors.initials: GS
authors.lastname: Baillie
mesh_terms: D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D017931:DNA Primers / Q000235:genetics; D004331:Drosophila melanogaster; D017353:Gene Deletion; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D020935:MAP Kinase Signaling System / Q000235:genetics / Q000502:physiology*; D013058:Mass Spectrometry; D051379:Mice; D018345:Mice, Knockout; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D020349:Surface Plasmon Resonance
chemical_list: D017931:DNA Primers; D019908:Proto-Oncogene Proteins c-raf; D015105:3',5'-Cyclic-AMP Phosphodiesterases; C520790:Pde8a protein, mouse