• Duangnapa Kovanich, Marcel A G van der Heyden, Thin Thin Aye, Toon A B van Veen, Albert J R Heck, and Arjen Scholten.
• Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center forBiomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands.
• Chembiochem. 2010 May 3; 11 (7): 963-71.

AbstractThe 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.

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