Corradini Eleonora, Burgers Pepijn P, Plank Michael, Heck Albert J R, Scholten Arjen
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.
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
J Biol Chem. 2015 Mar 20;290(12):7887-96. doi: 10.1074/jbc.M114.622613. Epub 2015 Feb 4.
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.
蛋白质-蛋白质相互作用在细胞信号转导中提供区室化和特异性方面起着重要作用。许多研究都强调了环磷酸腺苷（cAMP）依赖性蛋白激酶（PKA）通过其锚定蛋白实现的精心设计的区室化。关于其最接近的同源物环磷酸鸟苷（cGMP）依赖性蛋白激酶（PKG）通过其自身的PKG锚定蛋白（GKAPs）进行区室化的数据则少得多。为了富集、筛选和发现（新型）PKA锚定蛋白，有大量方法可用，包括我们之前描述的基于固定化cAMP或cGMP的化学蛋白质组学方法。尽管该方法已被证明是有效的，但每种固定化环核苷酸在富集PKA或PKG及其二级相互作用蛋白时都没有区分能力。因此，由于PKG信号成分在大多数组织中含量较少，富集和鉴定GKAPs变得具有挑战性。在这里，我们使用游离环核苷酸的竞争浓度扩展了这种基于cAMP的化学蛋白质组学方法，以分离每种激酶及其二级相互作用蛋白。使用这种方法，我们鉴定出亨廷顿蛋白相关蛋白1（HAP1）作为一种推定的新型GKAP。通过与已知GKAPs的序列比对和二级结构预测分析，我们定义了一个小的序列结构域，该结构域介导与PKG Iβ而非PKG Iα的相互作用。体外结合研究和定点诱变进一步证实了HAP1与PKG Iβ N末端结合的特异性和亲和力。这些数据充分支持HAP1是一种GKAP，特异性锚定到cGMP依赖性蛋白激酶同工型Iβ，并提供了进一步的证据表明PKG的时空信号传导也在很大程度上受锚定蛋白控制。
