Analysis of the substrate specificities of cyclin-dependent protein kinases and application to development of specific inhibitors

Cdc2激酶或cdk5是周期蛋白依赖性蛋白激酶的一个成员，磷酸化丝氨酸/苏氨酸位点，紧接着磷酸化脯氨酸，脯氨酸作为激酶的底物特异性决定因素。我们发现cdc2激酶肽底物中的脯氨酸可以部分被肌氨酸取代，这表明脯氨酸的n -取代结构是该激酶识别底物的重要因素。为了更好地理解脯氨酸定向磷酸化，代表cdc2激酶vimentin位点的肽中的脯氨酸残基被各种n -甲基氨基酸或脯氨酸同源物取代。结果表明脯氨酸的环结构，特别是吡咯烷环结构对cdc2激酶对底物的识别和磷酸化起重要作用。此外，分子模型和生化结果表明，磷酸化的程度与每个肽在该位点周围的旋转结构的程度有关。脯氨酸可能需要形成和稳定一个回合结构，这可能更容易被cdc2激酶识别。与cdc2激酶相比，Cdk5也表现出类似但相对严格的脯氨酸特异性。这些结果将为开发周期蛋白依赖性蛋白激酶的特异性抑制剂提供有用的信息。

Cdc2 kinase or cdk5, a member ofcyclin-dependent protein kinases, phosphorylates a Ser/Thr site immediately followed by a proline which acts as the substrate specificity determinant for the kinases. We found that a proline in a peptide substrate for cdc2 kinase can be replaced partly by sarcosine, suggesting that the N-substituted structure of proline is an important factor for the substrate recognition by the kinase. To gain a better understanding the proline-directed phosphorylation, the proline residue in the peptide representing the site in vimentin for cdc2 kinase was replaced by various N-methylamino acids or proline homologs. The results obtained here suggested that the ring structure, especially the pyrrolidine ring structure of proline is important for the substrate recognition and phosphorylation by cdc2 kinase. Moreover, molecular modeling, together with the biochemical results, suggested that the extent of phosphorylation is related to how well each peptide can assume a turn structure around the site. Proline might be required to form and stabilize a turn structure, which might be preferable to be recognized by cdc2 kinase. Cdk5 also showed a similar but relatively strict proline-specificity compared to that of cdc2 kinase. These results would afford useful information for developing specific inhibitors for cyclin-dependent protein kinases.