Characterization of the neuronal targeting protein spinophilin and its interactions with protein phosphatase-1
Characterization of the neuronal targeting protein spinophilin and its interactions with protein phosphatase-1
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DOI：
10.1021/bi982900m
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发表时间：
1999-04-06
期刊：
影响因子：
2.9
通讯作者：
Greengard, P
中科院分区：
文献类型：
--
作者：
Hsieh-Wilson, LC;Allen, PB;Greengard, P
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、细胞周期进展和神经传递。PP1的定位和底物特异性是由一类被称为靶向亚基的蛋白质决定的。在目前的研究中，PP1和嗜脊髓蛋白（一种将PP1靶向树突棘的神经元蛋白）之间的相互作用已经被表征。缺失分析显示，一个高亲和力的结合域位于嗜spinophilin残基417-494。该结构域在氨基酸447和451 （R- k - i - h - f）之间包含一个五肽基序（R/K-R/K-V/I-X-F），在其他PPI调控亚基中保守。通过共沉淀、覆盖和竞争结合实验观察到，苯丙氨酸-451 （F451A）突变或保守基序的缺失会破坏嗜脊髓蛋白结合PP1的能力。此外，417-442或474-494区域的缺失，无论是单独的还是联合的，都削弱了嗜脊髓蛋白共沉淀PP1的能力。一项对嗜脊髓蛋白肽结合和抑制特性的比较表明，不同的嗜脊髓蛋白亚结构域负责结合和调节PPI活性。调控亚域的突变分析显示，嗜spinophin与PP1相互作用的机制不同于细胞质抑制剂DARPP-32（多巴胺和camp调节的磷酸化蛋白，m - r32 000）和抑制剂-1。最后，对嗜脊髓蛋白和PP1之间相互作用的表征有助于设计能够破坏嗜脊髓蛋白-PP1相互作用的肽拮抗剂。这些研究支持了嗜脊髓蛋白在体内作为神经元PP1靶向亚基的概念，通过将酶定向到突触后密度并调节其对生理底物的活性。
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 PPI 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 PPI 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, M-r 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.