Modulation of nuclear cyclic AMP-dependent protein kinase in dibutyryl cyclic AMP-treated rat H4IIE hepatoma cells.
Modulation of nuclear cyclic AMP-dependent protein kinase in dibutyryl cyclic AMP-treated rat H4IIE hepatoma cells.
复制标题
二丁酰环 AMP 处理的大鼠 H4IIE 肝癌细胞中核环 AMP 依赖性蛋白激酶的调节。
DOI：
10.1042/bj2600673
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发表时间：
1989
期刊：
影响因子：
--
通讯作者：
Jungmann,RA
中科院分区：
文献类型：
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作者：
Squinto,SP;Jungmann,RA
进行了生化和免疫化学研究，以量化环腺苷酸对环腺苷酸依赖性蛋白激酶亚基水平的影响，在H4IIE肝癌细胞核。二丁酰环AMP（10 μ M）引起了显着的双相（刺激后10和120分钟）增加总核蛋白激酶活性。在二丁酰环AMP刺激后10分钟观察到的增加主要是由于约100 μ mol/L的cAMP。3-催化（C）亚基活性增加了一倍，而刺激后120分钟观察到的变化包括C亚基和环AMP非依赖性蛋白激酶活性的增加。通过用8-叠氮环[32 P] AMP进行光亲和标记的核蛋白提取物的分析仅鉴定了II型调节亚基（RII），但未鉴定I型调节亚基（RI）。通过二维凝胶电泳对核RII变体的分析表明，二丁酰环AMP引起两种RII变体形式的出现，这两种形式不存在于未受刺激的细胞的核中。使用亲和纯化的多克隆抗体和免疫印迹程序，我们确定了一个约。2-在双丁酰环腺苷酸处理的肝癌细胞的核提取物中，RII和C亚基增加了一倍。最后，通过e.l.i.s.a.表明二丁酰环腺苷酸双相增加核RII和C亚基水平，在初始刺激后10和120 min达到峰值。核RI亚基水平不受影响。这些结果提供了定性和定量的证据，由环腺苷酸的调节大鼠H4IIE肝癌细胞的核RII和C亚基的水平，并表明一个相对快速，但暂时有限的二丁酰环腺苷酸诱导易位的RII和C亚基的核网站。
Biochemical and immunochemical studies were undertaken to quantify the effects of cyclic AMP on cyclic AMP-dependent protein kinase subunit levels in nuclei of H4IIE hepatoma cells. Dibutyryl cyclic AMP (10 microM) caused a significant biphasic (10 and 120 min after stimulation) increase in total nuclear protein kinase activity. The increase observed 10 min after dibutyryl cyclic AMP stimulation was primarily due to an approx. 3-fold increase of catalytic (C) subunit activity, whereas the change observed 120 min after stimulation consisted of an increase in both C subunit and cyclic AMP-independent protein kinase activities. Analysis of nuclear protein extracts by photoaffinity labelling with 8-azido cyclic [32P]AMP identified only the type II regulatory subunit (RII), but not the type I regulatory subunit (RI). Analysis of nuclear RII variants by two-dimensional gel electrophoresis demonstrated that dibutyryl cyclic AMP caused the appearance of two RII variant forms which were not present in the nuclei of unstimulated cells. Using affinity-purified polyclonal antibodies and immunoblotting procedures, we identified an approx. 2-fold increase in the RII and C subunits in nuclear extracts of dibutyryl cyclic AMP-treated hepatoma cells. Finally, the RI, RII and C subunits were quantified by an e.l.i.s.a. which indicated that dibutyryl cyclic AMP increased nuclear RII and C subunits levels biphasically, reaching peak values 10 and 120 min after the initial stimulation. Nuclear RI subunit levels were not affected. These results provide qualitative as well as quantitative evidence for a modulation by cyclic AMP of the nuclear RII and C subunit levels in rat H4IIE hepatoma cells, and indicate a relatively rapid but temporarily limited dibutyryl cyclic AMP-induced translocation of the RII and C subunits to nuclear sites.