ACS DIVISION OF BIOLOGICAL CHEMISTRY

February 4th, 2015 by Corradini, E., Burgers, P. P., Plank, M., Heck, A. J. R., Scholten, A.

Protein-protein interactions are important in providing compartmentalization and specificity in cellular signal transduction. Many studies have hallmarked the well-designed compartmentalization of the cAMP-dependent protein kinase (PKA) through its anchoring proteins (AKAPs). Much less data is available on the compartmentalization of its closest homolog, cGMP-dependent protein kinase (PKG), via its own anchoring proteins (GKAPs). For the enrichment, screening and discovery of (novel) AKAPs a plethora of methodologies is available, including our previously described chemical proteomics approach based on immobilized cAMP or cGMP. Although this method was demonstrated to be effective, each immobilized cyclic nucleotide did not discriminate in the enrichment for either PKA or PKG, and their secondary interactors. Hence, with PKG signaling components being less abundant in most tissues, it turned out to be challenging to enrich and identify GKAPs. Here, we extend on this cAMP-based chemical proteomics approach, employing competitive concentrations of free cyclic nucleotides to isolate each kinase and its secondary interactors. Using this approach we identified Huntingtin associated protein 1 (HAP1) as a putative novel GKAP. Through sequence alignment with known GKAPs and secondary structure prediction analysis we defined a small sequence domain mediating the interaction with PKG Iβ, but not PKG Iα. In vitro binding studies and site directed mutagenesis further confirmed the specificity and affinity of HAP1 binding to the PKG Iβ N-terminus. These data fully support that HAP1 is a GKAP, anchoring specifically to the cGMP-dependent protein kinase isoform Iβ, and provide further evidence that also PKG spatio-temporal signaling is largely controlled by anchoring proteins.