Structure-Function Relationship and Phosphorylation-Dependent Regulation of Intermediate Filament Proteins.

中间丝蛋白的结构-功能关系和磷酸化依赖性调节。

• 批准号: 10680674
• 负责人: ANDO Shoji
• 金额: $ 1.79万
• 依托单位: Saga Medical School
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10680674/
• 关键词: Intermediate Filaments; Cytoskeleton; Protein Phosphorylation; Protein Engineering; Protein-Protein Interaction

The amino-terminal head domain of vimentin is the target site for several protein kinases and phosphorylation induces disassembly of the vimentin intermediate filaments in vivo and in vitro. To better understand molecular mechanisms involved in phosphorylation-dependent disassembly, we examined domain interactions involving the head domain and the effect of phosphorylation on the interaction, using surface plasmon resonance. We observed that the head domain binds to the carboxyl-terminal helix 2B in the rod domain, under physiological ionic strength. This interaction was interfered with by A-kinase phosphorylation of the head domain. Deletion of the carboxyl-terminal twenty amino acids of helix 2B resulted in loss of the interaction. Furthermore, peptide representing the carboxyl-terminal twenty residues of helix 2B had a substantial affinity with the head domain but not with the phosphorylated one. These findings support the idea that the interaction between the head domain and the last twenty residues of helix 2B is essential for association of vimentin tetramers into the intermediate filaments and that the phosphorylation-dependent disassembly is the result of loss of the interaction.

波形蛋白的氨基末端头结构域是多种蛋白激酶的靶位点，磷酸化可诱导体内和体外波形蛋白中间丝的解体。为了更好地理解磷酸化依赖性分解所涉及的分子机制，我们使用表面等离子体共振检查了涉及头域的域相互作用以及磷酸化对相互作用的影响。我们观察到，在生理离子强度下，头结构域与杆结构域中的羧基末端螺旋 2B 结合。这种相互作用受到头域 A-激酶磷酸化的干扰。螺旋 2B 羧基末端 20 个氨基酸的缺失导致相互作用丧失。此外，代表螺旋2B羧基端20个残基的肽与头部结构域具有显着的亲和力，但与磷酸化结构域不具有显着的亲和力。这些发现支持这样的观点，即头部结构域和螺旋 2B 的最后 20 个残基之间的相互作用对于波形蛋白四聚体与中间丝的结合至关重要，并且磷酸化依赖性分解是相互作用丧失的结果。

项目成果

S.Ando et al.: "Phosphorylation of synthetic peptides containing proline homologs by cdc2 kinase or cdk5."Peptide Science-Present and Future. 364-366 (1999)

S.Ando 等人：“通过 cdc2 激酶或 cdk5 对含有脯氨酸同系物的合成肽进行磷酸化。”肽科学——现在和未来。

S.Ando et al.: "Measurment of protein tyrosine phosphatase activity using as substrates synthetic phosphotyrosine-containing peptides"Research Communications in Biochemistry, Cell and Molecular Biology. (in press).

S.Ando 等人：“使用合成的含磷酸酪氨酸肽作为底物测量蛋白质酪氨酸磷酸酶活性”生物化学、细胞和分子生物学研究通讯。

R.Gohara et al.: "Phosphorylation of vimentin head domain inhibits interaction with the carboxyl-terminal end of α-helical rod domain studied by surface plasmon resonance measurements"FEBS Letters. 489・2-3. 182-186 (2001)

R. Gohara 等人：“通过表面等离子共振测量研究波形蛋白头结构域的磷酸化抑制与 α-螺旋杆结构域的羧基末端的相互作用” FEBS Letters 489・2-186 (2001)。

S.Ando et al.: "Phosphorylation of Synthetic peptides containing proline homologs by cdc2 kinase or cdk5"Peptide Science-Present and Future. 364-366 (1999)

S.Ando 等人：“通过 cdc2 激酶或 cdk5 磷酸化含有脯氨酸同源物的合成肽”肽科学 - 现在和未来。

安藤祥司,稲垣昌樹: "プロテインキナーゼドメインとリン酸化特異性"実験医学. 18・1. 7-12 (2000)

安藤正司、稻垣正树：“蛋白质激酶结构域和磷酸化特异性”《实验医学》18・1（2000）。