Enslen H, Tokumitsu H, Soderling T R
Vollum Institute, Oregon Health Sciences University, Portland 97201.
Biochem Biophys Res Commun. 1995 Feb 27;207(3):1038-43. doi: 10.1006/bbrc.1995.1289.
Previous reports have shown that CaM-kinase IV can phosphorylate the transcription factor CREB in vitro on Ser133. Furthermore, transfected CaM-kinase IV can activate CREB-dependent transcription, but at a lower efficiency than the cAMP-kinase. In this paper we examine the kinetics and site-specificity of CREB phosphorylation in vitro by CaM-kinase IV after its phosphorylation and activation by a newly discovered brain CaM-kinase IV kinase. Our results show that activated CaM-kinase IV has the same Km (1-5 microM) for CREB phosphorylation, but the Vmax is about 30-fold higher than with non-activated CaM-kinase IV. Activated CaM-kinase IV still shows specificity for phosphorylation of Ser133, the site necessary for transactivation by CREB. It is likely that the lower efficiency of transcriptional activation by transfected CaM-kinase IV in previous studies was due to the fact that the CaM-kinase IV was not activated by CaM-kinase IV kinase.
先前的报道表明，钙调蛋白依赖性蛋白激酶IV（CaM-kinase IV）在体外可使转录因子CREB的133位丝氨酸发生磷酸化。此外，转染的CaM-kinase IV可激活CREB依赖性转录，但效率低于环磷酸腺苷激酶（cAMP-kinase）。在本文中，我们研究了新发现的脑CaM-kinase IV激酶对CaM-kinase IV进行磷酸化和激活后，其在体外对CREB磷酸化的动力学和位点特异性。我们的结果表明，激活的CaM-kinase IV对CREB磷酸化的米氏常数（Km）相同（1-5微摩尔），但最大反应速度（Vmax）比未激活的CaM-kinase IV高约30倍。激活的CaM-kinase IV对133位丝氨酸的磷酸化仍具有特异性，该位点是CREB反式激活所必需的。在先前的研究中，转染的CaM-kinase IV转录激活效率较低，可能是因为CaM-kinase IV未被CaM-kinase IV激酶激活。
