title: A direct interaction between the N terminus of adenylyl cyclase AC8 and the catalytic subunit of protein phosphatase 2A.
abstract: Although protein scaffolding complexes compartmentalize protein kinase A (PKA) and phosphodiesterases to optimize cAMP signaling, adenylyl cyclases, the sources of cAMP, have been implicated in very few direct protein interactions. The N termini of adenylyl cyclases are highly divergent, which hints at isoform-specific interactions. Indeed, the Ca(2+)-sensitive adenylyl cyclase 8 (AC8) contains a Ca(2+)/calmodulin binding site on the N terminus that is essential for stimulation of activity by the capacitative entry of Ca(2+) in the intact cell. Here, we have used the N terminus of AC8 as a bait in a yeast two-hybrid screen of a human embryonic kidney (HEK) 293 cell cDNA library and identified the catalytic subunit of the serine/threonine protein phosphatase 2A (PP2A(C)) as a binding partner. Confirming the highly specific nature of this novel interaction, glutathione-S-transferase fusion proteins containing the full-length N terminus of AC8 affinity precipitated catalytically active PP2A(C) from both HEK293 and mouse forebrain membranes-the latter a normal source of AC8. The scaffolding subunit of PP2A (PP2A(A); 65 kDa) was also precipitated by the N terminus of AC8, indicating that AC8 may occur in a complex with the PP2A core dimer. The interaction between the N terminus of AC8 and PP2A(C) was antagonized by Ca(2+)/calmodulin. However, PP2A(C) and Ca(2+)/calmodulin did not share identical binding specificities in the N terminus of AC8. PKA-mediated phosphorylation did not influence either calmodulin or PP2A(C) association with AC8. In addition, both PP2A(C) and AC8 occurred in lipid rafts. These findings are the first demonstration of an association between adenylyl cyclase and any downstream element of cAMP signaling.
authors.affiliation: Department of Pharmacology, University of Cambridge, UK.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Crossthwaite
authors.forename: Antonio
authors.initials: A
authors.lastname: Ciruela
authors.forename: Timothy F
authors.initials: TF
authors.lastname: Rayner
authors.forename: Dermot M F
authors.initials: DM
authors.lastname: Cooper
mesh_terms: D000262:Adenylyl Cyclases / Q000032:analysis / Q000378:metabolism*; D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D002147:Calmodulin / Q000378:metabolism; D020134:Catalytic Domain; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D006801:Humans; D021962:Membrane Microdomains / Q000201:enzymology; D051379:Mice; D010749:Phosphoprotein Phosphatases / Q000032:analysis / Q000378:metabolism*; D010766:Phosphorylation; D016548:Prosencephalon / Q000201:enzymology; D025941:Protein Interaction Mapping; D054648:Protein Phosphatase 2
chemical_list: D002147:Calmodulin; D017868:Cyclic AMP-Dependent Protein Kinases; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; D000262:Adenylyl Cyclases; C118292:adenylyl cyclase 8