Insulin induced phosphorylation of prohibitin at tyrosine114 recruits Shp1
Insulin induced phosphorylation of prohibitin at tyrosine114 recruits Shp1
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DOI：
10.1016/j.bbamcr.2009.05.008
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发表时间：
2009-08-01
影响因子：
5.1
通讯作者：
Mishra, Suresh
中科院分区：
文献类型：
--
作者：
Ande, Sudharsana R.;Gu, Yuanyuan;Mishra, Suresh
抑制素（PHB 1）是一种进化上保守的、广泛表达的多功能蛋白质，存在于多种细胞区室中。磷酸化的PHB已被认为是在其各种功能的调节的潜在机制之一，但确切的磷酸化位点仍有待确定。为了更好地了解磷酸化的功能相关性，我们已经探索了潜在的磷酸化位点，结合使用的方法，包括磷酸化氨基特异性免疫印迹，蛋白水解，双向凝胶电泳，磷酸化氨基酸分析和定点突变技术和报告，酪氨酸114（Tyr（114））的磷酸化在响应于胰岛素刺激。此外，使用活性胰岛素受体（IR）和合成的生物素化的PHB肽（PHB 107 -121），我们已经表明，IR也磷酸化酪氨酸（114）在体外激酶测定。通过使用抗磷酸化Tyr（114）特异性抗体的免疫印迹来确认在Tyr（114）处的PHB磷酸化。此外，我们证明，SH 2结构域含有酪氨酸磷酸酶-1（Shp 1）与抗血清免疫沉淀后，胰岛素诱导的磷酸化的聚羟基丁酸酯。Tyr（114）磷酸化的生物素化-PHB 107 -121肽也能够在下拉测定中下拉Shp 1。非磷酸化的PHB 107 -121肽、相应的PHB 2（121-135）肽和Tyr 114 Phe突变体-PHB-Shp 1不能被拉低。总之，我们已经确定了酪氨酸（114）在聚羟基丁酸作为一个重要的磷酸化位点和磷酸化在这个残基创建一个结合位点的Shp 1在体内和体外。(C)2009 Elsevier B. V.保留所有权利。
Prohibitin (PHB or PHB1) is an evolutionarily conserved ubiquitously expressed multifunctional protein and is present in various cellular compartments. Phosphorylation of PHB has been suggested as one of the potential mechanisms in the regulation of its various functions however exact sites of phosphorylation remain to be determined. To better understand the functional relevance of phosphorylation of PHB, we have explored the potential sites of phosphorylation using combination of approaches including phosphoamino specific immunoblotting, proteolysis, two-dimensional gel electrophoresis, phosphoamino acid analysis and site-directed mutagenesis techniques and report that tyrosine 114 (Tyr(114)) in PHB is phosphorylated in response to insulin stimulation. In addition, using active insulin receptor (IR) and synthetic biotinylated PHB peptide (PHB107-121) we have shown that IR also phosphorylates Tyr(114) in an in vitro kinase assay. Phosphorylation of PHB at Tyr(114) was confirmed by immunoblotting using antiphosphoTyr(114) specific antibody. Furthermore, we demonstrate that SH2 domain containing tyrosine phosphatase-1 (Shp1) co-immunoprecipitate with PHB antiserum after insulin induced phosphorylation of PHB. Biotinylated-PHB107-121 peptide phosphorylated at Tyr(114) was also able to pull down Shp1 in pull down assays. Non-phosphorylated PHB107-121 peptide, corresponding PHB2(121-135) pepticle and Tyr114Phe mutant-PHB fail to pull down Shp1. In summary, we have identified Tyr(114) in PHB as an important site of phosphorylation and phosphorylation at this residue creates a binding site for Shp1 both in vivo and in vitro. (C) 2009 Elsevier B.V. All rights reserved.