Abstract
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. Much less data are available on the compartmentalization of its closest homolog, cGMP-dependent protein kinase (PKG), via its own PKG anchoring proteins (GKAPs). For the enrichment, screening, and discovery of (novel) PKA anchoring proteins, 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 this cAMP-based chemical proteomics approach using 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 spatiotemporal signaling is largely controlled by anchoring proteins.
Highlights
Protein kinase compartmentalization through anchoring proteins provides spatiotemporal specificity
Competitive Chemical Proteomics Enables the Selective Identification of GKAPs—Previous chemical proteomics experiments based on various immobilized cAMP or cGMP analogs revealed not much specificity for either protein kinase (PKA) or PKG [41,42,43]
The search for novel GKAPs using a chemical proteomics methodology is severely hampered by co-purification of the more abundant PKA pathway components
Summary
Protein kinase compartmentalization through anchoring proteins provides spatiotemporal specificity. At the N terminus of PKG, the monomers are held together by a heptad repeat leucine zipper, whereas PKA-R dimerizes with a very different fold called the X-type four-helix bundle This suggests that, PKA and PKG are close homologs, they differ entirely in the domains mediating their intracellular localization through binding to anchoring proteins. PKG localization via binding to protein kinase G anchoring proteins (GKAPs) is mediated by its N terminus, and the handful of GKAPs found far 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]. Follow-up experiments presented here establish HAP1 as a novel GKAP and highlight the potential of our novel chemical proteomics methodology for discovery of GKAPs in other cells and tissue
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