Designing isoform-specific peptide disruptors of protein kinase A localization
Designing isoform-specific peptide disruptors of protein kinase A localization
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DOI：
10.1073/pnas.2628038100
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发表时间：
2003-04-01
影响因子：
11.1
通讯作者：
Taylor, SS
中科院分区：
文献类型：
--
作者：
Burns-Hamuro, LL;Ma, YL;Taylor, SS
激酶锚定蛋白 (AKAP) 使用两亲性螺旋对接基序，通过结合和定位 cAMP 依赖性蛋白激酶 (PKA) 来协调 cAMP 介导的信号传导。模拟该螺旋的 PKA 定位肽干扰物已成功用于评估 PKA 在特定信号传导通路中的参与情况。然而，这些肽被开发为 II 型调节亚基 (RII) 的干扰物，尽管 RI 和 RII 亚型都可以与 AKAP 结合并具有离散的功能。为了评估每种局部异构体的作用，我们设计了特异性结合 RI 或 RII 的肽。使用肽阵列，我们定义了双特异性 AKAP 2 (D-AKAP2) 的最小结合序列，它与 RI 和 RII 紧密结合。通过使用肽取代阵列评估亲和力和同工型特异性的侧链要求，其中 D-AKAP2 的 A 激酶结合结构域的每个位置都被其他 19 个 L-氨基酸取代。该阵列包含 513 个 D-AKAP2 序列的单位点取代类似物。在定量荧光结合测定和基于细胞的共定位测定中评估含有单个和多个突变的肽。这一策略使我们能够设计出对 RIalpha 与 RIIalpha 具有高亲和力 (K-D = 1-2 nM) 和高特异性的肽。这些异构体特异性肽将成为评估局部 RI 和 RII PKA 之间功能差异的宝贵工具，并且是 RIalpha 特异性干扰剂。这种基于阵列的分析还为该对接基序的生物物理分析提供了基础。
A kinase-anchoring proteins (AKAPs) coordinate cAMP-mediated signaling by binding and localizing cAMP-dependent protein kinase (PKA), using an amphipathic helical docking motif. Peptide disruptors of PKA localization that mimic this helix have been used successfully to assess the involvement of PKA in specific signaling pathways. However, these peptides were developed as disruptors for the type II regulatory subunit (RII) even though both RI and RII isoforms can bind to AKAPs and have discrete functions. To evaluate the effects of each localized isoform, we designed peptides that specifically bind to either RI or RII. Using a peptide array, we have defined the minimal binding sequence of dual specific-AKAP 2 (D-AKAP2), which binds tightly to both RI and RII. Side-chain requirements for affinity and isoform specificity were evaluated by using a peptide substitution array where each position along the A kinase binding domain of D-AKAP2 was substituted by the other 19 L-amino acids. This array comprises 513 single-site substitution analogs of the D-AKAP2 sequence. Peptides containing single and multiple mutations were evaluated in a quantitative fluorescence binding assay and a cell-based colocalization assay. This strategy has allowed us to design peptides with high affinity (K-D = 1-2 nM) and high specificity for RIalpha versus RIIalpha. These isoform-specific peptides will be invaluable tools to evaluate functional differences between localized RI and RII PKA and are RIalpha-specific disruptors. This array-based analysis also provides a foundation for biophysical analysis of this docking motif.