Hausken Z E, Coghlan V M, Hastings C A, Reimann E M, Scott J D
Vollum Institute, Portland, Oregon 97201-3098.
J Biol Chem. 1994 Sep 30;269(39):24245-51.
Compartmentalization of the type II cAMP-dependent protein kinase is maintained by association of the regulatory subunit (RII) with A-Kinase Anchor Proteins (AKAPs). In previous studies (Scott, J. D., Stofko, R. E., McDonald, J. R., Comer, J. D., Vitalis, E. A., and Mangili J. (1990) J. Biol. Chem. 265, 21561-21566) we have shown that dimerization of RII alpha was required for interaction with the cytoskeletal component microtubule-associated protein 2. In this report we show that the localization and dimerization domains of RII alpha are contained within the first thirty residues of each RII protomer. RII des-5 (an amino-terminal deletion mutant lacking residues 1-5) was unable to bind AKAPs but retained the ability to dimerize. RII alpha I3A,I5A (a mutant where isoleucines 3 and 5 were replaced with alanine) was unable to bind a variety of AKAPs. Mutation of both isoleucines decreased AKAP binding without affecting dimerization, cAMP binding, or the overall secondary structure of the protein. Measurement of RII alpha I3A,I5A interaction with the human thyroid AKAP, Ht 31, by two independent methods suggests that mutation of isoleucines 3 and 5 decreases affinity by at least 6-fold. Therefore, we propose that two isoleucine side chains on each RII protomer are principle sites of contact with the conserved amphipathic helix binding domain on AKAPs.
II型cAMP依赖性蛋白激酶的区室化是通过调节亚基（RII）与A激酶锚定蛋白（AKAPs）的结合来维持的。在先前的研究中（斯科特，J.D.，斯托夫科，R.E.，麦克唐纳，J.R.，科默，J.D.，维塔利斯，E.A.，和曼吉利，J.（1990）《生物化学杂志》265，21561 - 21566），我们已经表明RIIα的二聚化是与细胞骨架成分微管相关蛋白2相互作用所必需的。在本报告中，我们表明RIIα的定位和二聚化结构域包含在每个RII原体的前30个残基内。RII des - 5（一个缺少1 - 5位残基的氨基末端缺失突变体）无法结合AKAPs，但保留了二聚化的能力。RIIα I3A，I5A（异亮氨酸3和5被丙氨酸取代的突变体）无法结合多种AKAPs。两个异亮氨酸的突变降低了AKAP的结合，而不影响二聚化、cAMP结合或蛋白质的整体二级结构。通过两种独立方法测量RIIα I3A，I5A与人甲状腺AKAP，Ht 31的相互作用表明，异亮氨酸3和5的突变使亲和力降低了至少6倍。因此，我们提出每个RII原体上的两个异亮氨酸侧链是与AKAPs上保守的两亲性螺旋结合结构域接触的主要位点。
