AKAP121 is a multifunctional scaffold that couples PKA /cAMP signaling to regulation of mitochondrial structure and physiological function

Abstract

The intracellular location of PKAII is a key determinant for the specificity of serial signaling reactions that are triggered by hormones that activate adenylate cyclase at the cell surface. Localization of PKA is mediated by AKAPs (A Kinase Anchor Proteins). AKAPs avidly bind the regulatory subunits (RII) of PKAII via a hydrophobic tethering domain. Distinct AKAPs contain similar RII binding sites, but have unique targeting domains that place the catalytic subunit of PKA in proximity with target substrates that are enriched in intracellular microdomains. In previous studies, the Rubin laboratory discovered a novel AKAP (designated AKAP121) that routes RII (PKAII) to mitochondria. An N terminal targeting domain (∼30 amino acids) is essential for association of an anchor protein-PKAII complex with a mitochondrion. In my investigations, I demonstrated that AKAP121 is targeted exclusively to the outer mitochondrial membrane by using immuno-electron microscopy. I discovered that RII and RI binding activities of AKAP121 are confined to a predicted amphipathic alpha-helix region generated by amino acid 306 to 327. Mutation of V317 to N disrupted both RI and RII binding; substitution of 1314 with A had no effect on sequestration of either isoform. However, substitution of L313 with A abrogated RI ligation by AKAP121, but had no effect on generation of AKAP121-RII complexes. Thus, despite evident overlap in function, the RI and RII ligation sites are not identical. The N terminal mitochondrial targeting region and R binding site of AKAP 121 are associated with a C terminal Tudor domain. Tudor domains, which were discovered in a Drosophila RNA binding protein, are conserved amino acid sequences and secondary structures of unknown function. I documented a novel function for Tudor domains: Ligation of NSF (N-Ethylmaleimide Sensitive Factor) by AKAP121 is mediated by the Tudor domain of the anchor protein; NSF or related AAA (ATPases Associated with various cellular Activities) family chaperones may be ligands for various Tudor domains. Finally, I discovered a novel and dramatic effect of cAMP/PKA/AKAP on mitochondria. Anchor protein-PKAII complexes control the balance between mitochondrial fusion and fission. This suggests that AKAP121 plays an important role in energy homeostasis and mitochondrial metabolism.