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 Ca²⁺-sensitive adenylyl cyclase 8 (AC8) contains a Ca²⁺/calmodulin binding site on the N terminus that is essential for stimulation of activity by the capacitative entry of Ca²⁺ 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 Ca²⁺/calmodulin. However, PP2A_C and Ca²⁺/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.