A Proteomics Investigation of Anchored PKA-RI Signaling

Abstract

Compartmentalization of kinases and phosphatases plays an important role in the specificity of second messenger mediated signaling events. Localization of the cAMP-dependent protein kinase is mediated by interaction of its regulatory subunit (PKA-R) with the versatile family of A-kinase anchoring proteins (AKAPs). Most AKAPs bind avidly to PKA-RII, while some have dual specificity for both PKA-RI and PKA-RII, however, no mammalian PKA-RI specific AKAPs have thus far been assigned. This was mainly attributed to the observation that PKA-RI is more cytosolic, as compared to the more heavily compartmentalized PKA-RII. Chemical proteomics screens of the cAMP interactome in mammalian heart tissue recently identified sphingosine kinase type 1-interacting protein (SKIP, SPHKAP) as a putative novel AKAP. Biochemical characterization now shows that SPHKAP can be considered as the first mammalian AKAP that preferentially binds to PKA-RIα. Recombinant human SPHKAP functions as an RI-specific AKAP that utilizes the for AKAPs characteristic amphipathic helix for interaction. Further chemical proteomic screening utilizing differential binding characteristics of specific cAMP-resins confirms SPHKAPs endogenous specificity for PKA-RI directly in mammalian heart and spleen tissue. Alignment of SPHKAPs amphipathic helix with peptide models of PKA-RI or PKA-RII specific anchoring domains shows that it has largely only PKA-RIα characteristics. To further investigate the role of the protein, we combined subcellular fractionation, confocal microscopy and LC-MS/MS analysis to study its subcellular localization and exact molecular environment. We found that SPHKAP localizes to the cytoplasm as well as to the mitochondrial inner membrane space and matrix. Here, it colocalizes with PKA-RIα and associates with a mitochondrial inner membrane organizing system (MINOS) complex that is responsible for maintaining crista structure. Additionally, SPHKAP associates with apoptosis-inducing factor (AIF) and the phosphatase PGAM5. We found that the protein N-terminus is required for its mitochondrial localization and overexpression of SPHKAP results in abnormal mitochondria. We show that SPHKAP is a substrate of PKA and that phosphorylation regulates the interaction between SPHKAP and AIF upon cAMP and apoptosis stimulation. Sequence analysis revealed that SPHKAP contains a 13-amino acid motif similar to the BH3 domain present in BCL-2 family proteins. As with other BH3-only proteins, co-immunoprecipitation and deletion of BH3 core amino acids demonstrated that SPHKAP interacts with the anti-apoptotic BCL-2 and BCL-xL through its BH3 domain. Taken together, The data suggest a putative role for the mitochondrial SPHKAP-PKA type I complex in regulating mitochondrial crista structure and mitochondrial-mediated apoptosis