Sphingosine Kinase Interacting Protein is an A‐Kinase Anchoring Protein Specific for Type I cAMP‐Dependent Protein Kinase

Abstract

The 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 has mainly been attributed to the observation that PKA-RI is more cytosolic than 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-RIalpha. Recombinant human SPHKAP functions as an RI-specific AKAP that utilizes the characteristic AKAP 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. Immunolocalization studies revealed that recombinant SPHKAP is expressed in the cytoplasm, where PKA-RIalpha also mainly resides. Alignment of SPHKAPs' amphipathic helix with peptide models of PKA-RI- or PKA-RII-specific anchoring domains shows that it has largely only PKA-RIalpha characteristics. Being the first mammalian PKA-RI-specific AKAP with cytosolic localization, SPHKAP is a very promising model for studying the function of the less explored cytosolic PKA-RI signaling nodes.

Protein Kinase A Opposes the Phosphorylation-dependent Recruitment of Glycogen Synthase Kinase 3β to A-kinase Anchoring Protein 220

“…For example, the multivalent scaffolding protein yotiao/ AKAP450 contains two PKA binding sites and constrains a range of protein kinases and phosphatases at the Golgi and centrosomes (56,57). Likewise, the sphingosine kinase interacting protein (SKIP) is an RI-selective anchoring protein that tethers a pair of type I PKA holoenzymes with preferred substrates at the inner face of the mitochondrial matrix (43,44). Dimerization of anchoring proteins is another means to increase the local concentration of PKA as exemplified by membrane-associated AKAP79/150 (58).…”

“…Selectivity-Although the majority of anchoring proteins preferentially interact with type II PKA, a few type I PKA-selective AKAPs have also been reported (2,(43)(44)(45). Another subset, designated dual function AKAPs, can interact with either RI or RII (46,47).…”

An entirely specific type I A-kinase anchoring protein that can sequester two molecules of protein kinase A at mitochondria

“…2. Resolving the subtype selectivity of this AKAP is important in light of conflicting reports claiming that SKIP is an RII or RI anchoring protein (30,45,46).…”

Huntingtin-associated Protein 1 (HAP1) Is a cGMP-dependent Kinase Anchoring Protein (GKAP) Specific for the cGMP-dependent Protein Kinase Iβ Isoform

“…PKG localization via binding to protein kinase G anchoring proteins (GKAPs) is mediated by its N terminus, and the handful of GKAPs found thus far indeed show specificity for different PKG isoforms (12)(13)(14)(15)(16). This is again similar to how AKAPs interact with respect to different isoforms of PKA regulatory subunits (5,17,18).…”

“…Particularly, immobilized cAMP on agarose beads played a pivotal role in the identification and characterization of several new AKAPs. However, all these studies focused on PKA and AKAPs (18,28,29) and not on PKG and new GKAPs.…”