Hsieh-Wilson L C, Allen P B, Watanabe T, Nairn A C, Greengard P
Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York 10021, USA.
Biochemistry. 1999 Apr 6;38(14):4365-73. doi: 10.1021/bi982900m.
Protein phosphatase-1 (PP1) plays an important role in a variety of cellular processes, including muscle contraction, cell-cycle progression, and neurotransmission. The localization and substrate specificity of PP1 are determined by a class of proteins known as targeting subunits. In the present study, the interaction between PP1 and spinophilin, a neuronal protein that targets PP1 to dendritic spines, has been characterized. Deletion analysis revealed that a high-affinity binding domain is located within residues 417-494 of spinophilin. This domain contains a pentapeptide motif (R/K-R/K-V/I-X-F) between amino acids 447 and 451 (R-K-I-H-F) that is conserved in other PP1 regulatory subunits. Mutation of phenylalanine-451 (F451A) or deletion of the conserved motif abolished the ability of spinophilin to bind PP1, as observed by coprecipitation, overlay, and competition binding assays. In addition, deletion of regions 417-442 or 474-494, either singly or in combination, impaired the ability of spinophilin to coprecipitate PP1. A comparison of the binding and inhibitory properties of spinophilin peptides suggested that distinct subdomains of spinophilin are responsible for binding and modulating PP1 activity. Mutational analysis of the modulatory subdomain revealed that spinophilin interacts with PP1 via a mechanism unlike those used by the cytosolic inhibitors DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, Mr 32 000) and inhibitor-1. Finally, characterization of the interactions between spinophilin and PP1 has facilitated the design of peptide antagonists capable of disrupting spinophilin-PP1 interactions. These studies support the notion that spinophilin functions in vivo as a neuronal PP1 targeting subunit by directing the enzyme to postsynaptic densities and regulating its activity toward physiological substrates.
蛋白磷酸酶-1（PP1）在多种细胞过程中发挥重要作用，包括肌肉收缩、细胞周期进程和神经传递。PP1的定位和底物特异性由一类称为靶向亚基的蛋白质决定。在本研究中，已对PP1与亲棘蛋白（一种将PP1靶向树突棘的神经元蛋白）之间的相互作用进行了表征。缺失分析表明，亲棘蛋白的高亲和力结合域位于其417 - 494位氨基酸残基内。该结构域在447至451位氨基酸之间包含一个五肽基序（R/K - R/K - V/I - X - F）（R - K - I - H - F），此基序在其他PP1调节亚基中保守。通过共沉淀、覆盖和竞争结合试验观察到，苯丙氨酸-451（F451A）突变或保守基序缺失消除了亲棘蛋白结合PP1的能力。此外，单独或联合缺失417 - 442或474 - 494区域会损害亲棘蛋白共沉淀PP1的能力。亲棘蛋白肽的结合和抑制特性比较表明，亲棘蛋白的不同亚结构域负责结合和调节PP1活性。调节亚结构域的突变分析表明，亲棘蛋白与PP1相互作用的机制不同于胞质抑制剂多巴胺和cAMP调节的磷蛋白（DARPP - 32，分子量32 000）和抑制剂-1所采用的机制。最后，亲棘蛋白与PP1相互作用的表征有助于设计能够破坏亲棘蛋白 - PP1相互作用的肽拮抗剂。这些研究支持这样一种观点，即亲棘蛋白在体内作为神经元PP1靶向亚基发挥作用——将该酶导向突触后致密区并调节其对生理底物的活性。
