Development and application of specific inhibitors for cyclin-dependent protein kinases on the basis of their substrate specificities.

基于其底物特异性的细胞周期蛋白依赖性蛋白激酶特异性抑制剂的开发和应用。

• 批准号: 08680634
• 负责人: ANDO Shoji
• 金额: $ 1.41万
• 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: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08680634/
• 关键词: cyclin-dependent protein kinase; protein phosphorylation; substrate specificity; enzyme inhibitor

Cdc2 kinase or cdk5, a member of cyclin-dependent protein kinases, phosphorylates a Ser/Thr site immediately followed by a proline which acts as the substrate specificity determinant. We have examined structural features of proline which are essential for the substrate recognition by the kinase and found that the pyrrolidine ring of proline is primarily important. In addition, we observed that cdk5 has a stronger preference for a proline and basic residues on the carboxyl-terminal side of the phosphorylation site when compared to the preference exhibited by cdc2 kinase. These results gave useful information for developing spesific substrates and competitive inhibitors for each kinase. To obtain substrates/inhibitors which are resistant to enzymatic proteolysis, we undertook a synthesis of cyclic or retro-inverso peptides. Cyclic peptides which represent the site of histone or vimentin were effectively phosphorylated by cdc2 kinase. Thus, cyclization might be suitable for the purpose. We faced with some problems in the synthesis of retro-inverso peptides and are now testing conditions for obtaining objectives quantitatively.

Cdc 2激酶或cdk 5是细胞周期蛋白依赖性蛋白激酶的成员，其磷酸化丝氨酸/苏氨酸位点，紧接着是作为底物特异性决定簇的脯氨酸。我们已经研究了脯氨酸的结构特征，这是必不可少的底物识别的激酶，并发现脯氨酸的吡咯烷环是主要重要的。此外，我们观察到，cdk 5有一个更强的偏好脯氨酸和碱性残基的羧基端侧的磷酸化位点相比，由cdc 2激酶表现出的偏好。这些结果为开发特异性底物和竞争性抑制剂提供了有用的信息。为了获得对酶促蛋白水解具有抗性的底物/抑制剂，我们进行了环状或逆-倒位肽的合成。代表组蛋白或波形蛋白位点的环肽被cdc 2激酶有效磷酸化。因此，环化可能适合于该目的。我们面临着一些问题，在合成的逆向-反转肽，现在正在测试条件，以获得目标定量。

项目成果

Ando, S.: "Role of the pyrrolidine ring of proline in determining the substrate specificity of cdc2 kinase or cdk5" J.Biochem.122. 409-414 (1997)

Ando, S.：“脯氨酸吡咯烷环在确定 cdc2 激酶或 cdk5 底物特异性中的作用”J.Biochem.122。

Tokui,T.: "Enhancement of smooth muscle contraction with protein phosphatase inhibitor 1 : activation of inhibitor1 by cGMP-dependent protein kinase." Biochem.Biophys.Res.Commun.220・3. 777-783 (1996)

Tokui, T.：“蛋白磷酸酶抑制剂 1 增强平滑肌收缩：cGMP 依赖性蛋白激酶激活抑制剂 1”。Biochem.Biophys.Res.Commun.220·3 (1996)。

安藤 祥司: "タンパク質化学第6巻 細胞骨格と筋肉のタンパク質:中間径フィラメント結合タンパク質" 広川書店, 6 (1997)

安藤正司：《蛋白质化学第 6 卷细胞骨架和肌肉蛋白质：中间丝结合蛋白》广川书店，6 (1997)

Ando,S.: "Alterations in tyrosine phosphatase activity duringproliferation of Lewis lung carcinoma-derived cells,studied with syntehtic phosphopeptides as substrates." Peptide Chemistry 1995. 365-368 (1996)

Ando,S.：“以合成磷酸肽为底物研究路易斯肺癌衍生细胞增殖过程中酪氨酸磷酸酶活性的变化。”

Ando, S.: "Keratin 8 phosphorylation in vitro by cAMP-dependent protein kinase occurs within the amino-and carboxyl-terminal end domains." Biochem.Biophys.Res.Commun.221. 67-71 (1996)

Ando, S.：“cAMP 依赖性蛋白激酶在体外对角蛋白 8 进行磷酸化，发生在氨基和羧基末端结构域内。”