Carr D W, Stofko-Hahn R E, Fraser I D, Bishop S M, Acott T S, Brennan R G, Scott J D
Vollum Institute for Advanced Biomedical Research, Oregon Health Sciences University, Portland 97201-3098.
J Biol Chem. 1991 Aug 5;266(22):14188-92.
The type II cAMP-dependent protein kinase is localized to specific subcellular environments through the binding of the regulatory subunit (RII) dimer to RII-anchoring proteins. Computer-aided analysis of secondary structure, performed on four RII-anchoring protein sequences (the microtubule-associated protein 2, P150, and two thyroid proteins Ht 21 and Ht 31), has identified common regions of approximately 14 residues which display high probabilities of forming amphipathic helices. The potential amphipathic helix region of Ht 31 (Leu-Ile-Glu-Glu-Ala-Ala-Ser-Arg-Ile-Val-Asp-Ala-Val-Ile) lies between residues 494 and 507. A bacterially expressed 318-amino acid fragment, Ht 31 (418-736), containing the amphipathic helix region, was able to bind RII alpha. Site-directed mutagenesis designed to disrupt the secondary structure in the putative binding helix reduced binding dramatically. Specifically, substitution of proline for Ala-498 significantly diminished RII alpha binding, and similar mutation of Ile-502 or Ile-507 abolished interaction. Mutation of Ala-522 to proline, which is located outside the predicted amphipathic helix region, had no effect on RII alpha binding. These data suggest that anchoring proteins interact with RII alpha via an amphipathic helix binding motif.
II型环磷酸腺苷依赖性蛋白激酶通过调节亚基（RII）二聚体与RII锚定蛋白的结合而定位于特定的亚细胞环境。对四个RII锚定蛋白序列（微管相关蛋白2、P150以及两种甲状腺蛋白Ht 21和Ht 31）进行的二级结构计算机辅助分析，确定了大约14个残基的共同区域，这些区域显示出形成两亲性螺旋的高概率。Ht 31的潜在两亲性螺旋区域（亮氨酸-异亮氨酸-谷氨酸-谷氨酸-丙氨酸-丙氨酸-丝氨酸-精氨酸-异亮氨酸-缬氨酸-天冬氨酸-丙氨酸-缬氨酸-异亮氨酸）位于494至507位残基之间。一个细菌表达的包含两亲性螺旋区域的318个氨基酸的片段Ht 31（418-736）能够结合RIIα。旨在破坏假定结合螺旋中二级结构的定点诱变显著降低了结合。具体而言，用脯氨酸取代丙氨酸-498显著减少了RIIα的结合，而异亮氨酸-502或异亮氨酸-507的类似突变消除了相互作用。位于预测的两亲性螺旋区域之外的丙氨酸-522突变为脯氨酸对RIIα的结合没有影响。这些数据表明，锚定蛋白通过两亲性螺旋结合基序与RIIα相互作用。
