Schillace R V, Scott J D
Howard Hughes Medical Institute, L-474 Vollum Institute, Oregon Health Sciences University, 3181 S.W. Sam Jackson Park Road, Portland, Oregon 97201-3098, USA.
Curr Biol. 1999 Mar 25;9(6):321-4. doi: 10.1016/s0960-9822(99)80141-9.
The cyclic AMP (cAMP)-dependent protein kinase (PKA) and the type 1 protein phosphatase (PP1) are broad-specificity signaling enzymes with opposing actions that catalyze changes in the phosphorylation state of cellular proteins. Subcellular targeting to the vicinity of preferred substrates is a means of restricting the specificity of each enzyme [1] [2]. Compartmentalization of the PKA holoenzyme is mediated through association of the regulatory subunits with A-kinase anchoring proteins (AKAPs), whereas a diverse family of phosphatase-targeting subunits directs the location of the PP1 catalytic subunit (PP1c) [3] [4]. Here, we demonstrate that the PKA-anchoring protein, AKAP220, binds PP1c with a dissociation constant (KD) of 12.1 +/- 4 nM in vitro. Immunoprecipitation of PP1 from cell extracts resulted in a 10.4 +/- 3.8-fold enrichment of PKA activity. AKAP220 co-purified with PP1c by affinity chromatography on microcystin sepharos Immunocytochemical analysis demonstrated that the kinase, the phosphatase and the anchoring protein had distinct but overlapping staining patterns in rat hippocampal neurons. Collectively, these results provide the first evidence that AKAP220 is a multivalent anchoring protein that maintains a signaling scaffold of PP1 and the PKA holoenzyme.
环磷酸腺苷（cAMP）依赖性蛋白激酶（PKA）和1型蛋白磷酸酶（PP1）是具有相反作用的广谱信号酶，它们催化细胞蛋白磷酸化状态的变化。亚细胞靶向至优选底物附近是限制每种酶特异性的一种方式[1][2]。PKA全酶的区室化是通过调节亚基与A激酶锚定蛋白（AKAPs）的结合来介导的，而多种磷酸酶靶向亚基家族则指导PP1催化亚基（PP1c）的定位[3][4]。在此，我们证明PKA锚定蛋白AKAP220在体外以12.1±4 nM的解离常数（KD）结合PP1c。从细胞提取物中免疫沉淀PP1导致PKA活性富集10.4±3.8倍。通过在微囊藻毒素琼脂糖上的亲和色谱法，AKAP220与PP1c共纯化。免疫细胞化学分析表明，该激酶、磷酸酶和锚定蛋白在大鼠海马神经元中具有不同但重叠的染色模式。总体而言，这些结果提供了首个证据表明AKAP220是一种多价锚定蛋白，它维持着PP1和PKA全酶的信号支架。
