Koh W S, Jeon Y J, Herring A C, Kaminski N E
Department of Pharmacology and Toxicology, Michigan State University, East Lansing 48824, USA.
Life Sci. 1997;60(6):425-32. doi: 10.1016/s0024-3205(96)00667-4.
Cyclic AMP regulates a variety of cellular responses through activation of cAMP-dependent protein kinase (PKA). The catalytic subunit of PKA, in turn, activate cAMP responsive element (CRE) and nuclear factor-kappa B (NF-kappa B) binding proteins. In this study, we demonstrated that binding activity to both CRE and kappa B sites in nuclear extracts from spleen cells is modulated by PKA in a time-dependent manner. Electrophoretic mobility shift assays showed that binding by transcription factors to either the CRE or kappa B motif was rapidly up-regulated by cAMP, with maximum binding detected at 30 min in response to forskolin stimulation of splenocytes. This was followed by a steady decline in CRE and kappa B thereafter reaching basal levels by 2 hr. This up-regulation in CRE and kappa B binding was closely associated with an enhancement of PKA activity which was maximum at 30 min following forskolin stimulation. However, unlike the binding of regulatory factors to CRE and kappa B motifs which was very transient, peak PKA activity was sustained for 2 hr. Interestingly, okadaic acid, a protein phosphatase inhibitor, prevented the decline in protein binding to CRE and kappa B motifs 2 hr following forskolin stimulation and actually produced a slight increase at 30 min. These data suggest that binding by transcription factors to CRE and kappa B sites are up-regulated concomitantly with PKA activation but subsequently down-regulated by a protein phosphatase.
环磷酸腺苷（cAMP）通过激活依赖cAMP的蛋白激酶（PKA）来调节多种细胞反应。反过来，PKA的催化亚基会激活cAMP反应元件（CRE）和核因子-κB（NF-κB）结合蛋白。在本研究中，我们证明了脾细胞核提取物中与CRE和κB位点的结合活性受PKA以时间依赖性方式调节。电泳迁移率变动分析表明，转录因子与CRE或κB基序的结合被cAMP迅速上调，在用福斯可林刺激脾细胞后30分钟检测到最大结合。随后，CRE和κB的结合稳步下降，到2小时后达到基础水平。CRE和κB结合的这种上调与PKA活性的增强密切相关，PKA活性在福斯可林刺激后30分钟达到最大值。然而，与调节因子与CRE和κB基序的结合非常短暂不同，PKA的峰值活性持续了2小时。有趣的是，蛋白磷酸酶抑制剂冈田酸可防止福斯可林刺激2小时后与CRE和κB基序的蛋白结合下降，并且在30分钟时实际上略有增加。这些数据表明，转录因子与CRE和κB位点的结合与PKA激活同时上调，但随后被一种蛋白磷酸酶下调。
