title: Huntingtin-associated protein 1 (HAP1) is a cGMP-dependent kinase anchoring protein (GKAP) specific for the cGMP-dependent protein kinase Iβ isoform.
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.
authors: From the Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Science Faculty, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands and Netherlands Proteomics Centre, Padualaan 8, 3584 CH Utrecht, The Netherlands.
authors: Eleonora
authors: E
authors: Corradini
authors: From the Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Science Faculty, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands and Netherlands Proteomics Centre, Padualaan 8, 3584 CH Utrecht, The Netherlands.
authors: Pepijn P
authors: PP
authors: Burgers
authors: From the Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Science Faculty, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands and Netherlands Proteomics Centre, Padualaan 8, 3584 CH Utrecht, The Netherlands.
authors: Michael
authors: M
authors: Plank
authors: From the Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Science Faculty, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands and Netherlands Proteomics Centre, Padualaan 8, 3584 CH Utrecht, The Netherlands a.j.r.heck@uu.nl.
authors: Albert J R
authors: AJ
authors: Heck
authors: From the Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Science Faculty, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands and Netherlands Proteomics Centre, Padualaan 8, 3584 CH Utrecht, The Netherlands.
authors: Arjen
authors: A
authors: Scholten
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D040901:Proteomics; D017386:Sequence Homology, Amino Acid
chemical_list: C096896:HAP1 protein, human; D007527:Isoenzymes; D009419:Nerve Tissue Proteins; D017869:Cyclic GMP-Dependent Protein Kinases
keywords: Chemical Proteomics; Cyclic GMP (cGMP); HAP1; Mass Spectrometry (MS); Protein Kinase G (PKG); Proteomics; Signal Transduction
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