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

CDC2K或CDK5是细胞周期蛋白依赖性蛋白激酶的一员，它立即磷酸化Ser/Thr位点，然后是作为底物特异性决定因素的Pro。我们研究了对底物识别至关重要的Pro的结构特征，发现Pro的吡咯烷环是最重要的。此外，我们观察到，与cdc2激酶相比，cdk5对磷酸化位点的羧基末端的脯氨酸和碱性残基有更强的偏好。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。为了获得抗酶蛋白降解的底物/抑制剂，我们进行了环肽或逆转录多肽的合成。代表组蛋白或波形蛋白位置的环肽可被cdc2激酶有效地磷酸化。因此，环化反应可能适合于此目的。我们在逆转录多肽的合成中遇到了一些问题，目前正在测试定量获得目标的条件。

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.