A protein phosphatase-1γ1 isoform selectivity determinant in dendritic spine-associated neurabin
A protein phosphatase-1γ1 isoform selectivity determinant in dendritic spine-associated neurabin
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DOI：
10.1074/jbc.m402261200
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发表时间：
2004-05-21
影响因子：
4.8
通讯作者：
Colbran, RJ
中科院分区：
文献类型：
--
作者：
Carmody, LC;Bauman, PA;Colbran, RJ
蛋白磷酸酶-1（PP 1）催化亚基亚型与不同的蛋白质相互作用，通常含有典型的（R/ K）（V/ I）XF基序。尽管共享相似的90%的氨基酸序列同一性，PP 1 β和PP 1 γ 1具有不同的亚细胞定位，可以通过与PP 1结合蛋白的选择性相互作用来确定。脑和肌肉提取物的免疫沉淀研究表明，PP 1 γ 1选择性地与F-肌动蛋白靶向蛋白--亲棘素和神经肽相互作用，而PP 1 β选择性地与横纹肌糖原靶向亚基--G（M）/ R-GL相互作用。含有神经肽残基146 - 493（GST-Nb-（146 - 493））或G（M）/ R-GL残基1 - 240（GST-G（M）-（1 - 240））的谷胱甘肽S-转移酶（GST）融合蛋白在结合和磷酸酶活性抑制测定中重现了这些同种型选择性。定点诱变表明，这种同种型选择性不是由于典型的PP 1结合基序（神经肽，（KIKF 460）-K-457; G（M）/ R-GL，（RVSF 68）-R-65）之间的序列差异。一种嵌合GST融合蛋白，其含有融合至神经肽残基457 - 493的G（M）/ R-GL的残基1 - 64（GST-G（M）/ Nb）选择性结合并抑制PP 1 γ 1，而含有Nb-（146 - 460）与G（M）-（69 - 240）融合，选择性地与PP 1 β相互作用并弱抑制PP 1 β，暗示（R/ K）（V/ I）XF基序的C-末端结构域是PP 1同种型选择性的决定因素。删除neurabin（Delta PI）中的Pro（464）和Ile（465），使（R/ K）（V/ I）XF基序中的保守氨基酸簇与G（M）/ R-GL相同，严重损害了neurabin结合和抑制两种亚型的能力，但不影响PP 1 γ 1的选择性。对一系列C-末端截短的GST-Nb-（146 - 493）蛋白的进一步分析鉴定了neurabin的残基473 - 479含有关键的PP 1 γ 1-选择性决定簇。结合，这些数据确定了一种新的PP 1 γ 1选择性相互作用域的neurabin，可能允许选择性调节和/或亚细胞靶向PP 1亚型。
Protein phosphatase- 1 ( PP1) catalytic subunit isoforms interact with diverse proteins, typically containing a canonical ( R/ K)( V/ I) XF motif. Despite sharing similar to 90% amino acid sequence identity, PP1beta and PP1gamma1 have distinct subcellular localizations that may be determined by selective interactions with PP1- binding proteins. Immunoprecipitation studies from brain and muscle extracts demonstrated that PP1gamma1 selectively interacts with spinophilin and neurabin, F- actin- targeting proteins, whereas PP1beta selectively interacted with G(M)/ R-GL, the striated- muscle glycogen- targeting subunit. Glutathione S- transferase ( GST) fusion proteins containing residues 146 - 493 of neurabin ( GST- Nb-( 146 - 493)) or residues 1 - 240 of G(M)/ R-GL ( GST-G(M)-( 1 - 240)) recapitulated these isoform selectivities in binding and phosphatase activity inhibition assays. Site- directed mutagenesis indicated that this isoform selectivity was not due to sequence differences between the canonical PP1- binding motifs ( neurabin, (KIKF460)-K-457; G(M)/ R-GL, (RVSF68)-R-65). A chimeric GST fusion protein containing residues 1 - 64 of G(M)/ R-GL fused to residues 457 - 493 of neurabin ( GST- G(M)/ Nb) selectively bound to and inhibited PP1gamma1, whereas a GST- Nb/ G(M) chimera containing Nb-( 146 - 460) fused to G(M)-( 69 - 240) selectively interacted with and weakly inhibited PP1 beta, implicating domain( s) C- terminal to the ( R/ K)( V/ I) XF motif as determinants of PP1 isoform selectivity. Deletion of Pro(464) and Ile(465) in neurabin ( Delta PI) to equally space a conserved cluster of amino acids from the ( R/ K)( V/ I) XF motif as in G(M)/ R-GL severely compromised the ability of neurabin to bind and inhibit both isoforms but did not affect PP1gamma1 selectivity. Further analysis of a series of C- terminal truncated GST- Nb-( 146 - 493) proteins identified residues 473 - 479 of neurabin as containing a crucial PP1gamma1- selectivity determinant. In combination, these data identify a novel PP1gamma1- selective interaction domain in neurabin that may allow for selective regulation and/ or subcellular targeting of PP1 isoforms.