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

Cdc 2激酶或cdk 5是细胞周期蛋白依赖性蛋白激酶的一个成员，它磷酸化一个丝氨酸/苏氨酸位点，紧接着是一个脯氨酸，脯氨酸作为激酶的底物特异性决定簇。我们发现，脯氨酸在cdc 2激酶的肽底物中可以部分地被肌氨酸取代，这表明脯氨酸的N-取代结构是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，将代表波形蛋白中cdc 2激酶位点的肽中的脯氨酸残基替换为各种N-甲基氨基酸或脯氨酸同系物。本文获得的结果表明，脯氨酸的环结构，尤其是吡咯烷环结构对于cdc 2激酶的底物识别和磷酸化非常重要。此外，分子模拟和生物化学结果表明，磷酸化的程度与每个肽在该位点周围呈现转角结构的程度有关。可能需要脯氨酸来形成和稳定转角结构，这可能更适合于被cdc 2激酶识别。与cdc 2激酶相比，cdk 5也表现出类似但相对严格的脯氨酸特异性。这些结果将为开发特异性细胞周期蛋白依赖性蛋白激酶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.