The Development of the Strategy for the Presumption of the Tertiary Structure of Protein Kinases by Specific Inhibitors

蛋白激酶是将π转化为某些蛋白质的酶。这些反应已被揭示参与许多细胞功能的调节。目前蛋白激酶的数量已超过100种，从结构和功能的角度对其进行研究，阐明每种蛋白激酶的生理功能是十分必要的。我们的研究目的是：(1)推测蛋白激酶功能域的三级结构，(2)开发新的特异性蛋白激酶抑制剂，(3)最终目的是阐明蛋白激酶的生理功能。利用Ca2+/calmodulin dependent protein kinase II（CaM kinase II）的特异性抑制剂KN-62，我们成功地揭示了CaM kinase II参与平滑肌收缩、全身压力的中枢调节、胰岛素或内皮素的分泌等。H-89是cAMP依赖性蛋白激酶（a -激酶）的特异性抑制剂，发现a -激酶参与c-fos基因的转录调控，并参与立即早期基因的诱导调控。我们成功合成了CaM激酶II或MAP激酶激活的新型蛋白激酶特异性抑制剂，该蛋白激酶是调节各种细胞外刺激诱导的信号的关键分子。研究发现，KN-62对钙调蛋白依赖性蛋白激酶CaM kinase V具有抑制作用。在此基础上，我们认为CaM kinase II的钙调素结合域与CaM kinase V的钙调素结合域非常相似。综上所述，我们成功地阐明了蛋白激酶的功能，开发了新的特异性抑制剂，并明确了CaM kinase II的钙调素结合域与CaM kinase V的三级结构的相似性。

Protein kinases are enzymes which tansfer Pi into some proteins. These reactions have been revealed to be involved in the regulation of many cellular function. now that the number of protein kinases has reached to over 100, it is essential to study them with structural and functional approach to clarify the physiological function of each protein kinase. The aim of our study is, (1) to presume the tertiary structure of the functional domain of protein kinase, (2) to develop the new specific protein kinase inhibitors, and (3), it is final aim, to clarify the physiological function of protein kinases. Using specific inhibitor for Ca2+/calmodulin dependent protein kinase II(CaM kinase II), KN-62, we succeeded in revealing the involvement of CaM kinase II in smooth muscle contraction, the central regulation of systemic pressure, secretion of insulin or endotherin and so on. H-89, specific inhibitor for cAMP dependent protein kinase(A-kinase), revealed the involvement of the A-kinase in the regulation of transcription of c-fos gene, and in the regulation of induction of immediately early genes. We succeeded in synthesizing the novel specific inhibitor for CaM kinase II or for the novel protein kinase activated by MAP kinase, which is a key molecule for regulating the signals induced by various extracellular stimuli. KN-62 was revealed to inhibit CaM kinase V which was a novel Ca2+/calmodulin dependent protein kinase with respect with calmodulin. On the basis of this result, it will be suggested that the calmodulin binding domain of CaM kinase II is quite similar to that of CaM kinase V.Taken together, we succeeded in clarifying the function of protein kinases, in developing the new specific inhibitors, and in clearing the similarity between the tertiary structure of calmodulin binding domain of CaM kinase II and CaM kinase V.