DIRECT CYTOCHEMICAL-LOCALIZATION OF CATALYTIC SUBUNITS DISSOCIATED FROM CAMP-DEPENDENT PROTEIN-KINASE IN REUBER H-35 HEPATOMA-CELLS .2. TEMPORAL AND SPATIAL KINETICS
DIRECT CYTOCHEMICAL-LOCALIZATION OF CATALYTIC SUBUNITS DISSOCIATED FROM CAMP-DEPENDENT PROTEIN-KINASE IN REUBER H-35 HEPATOMA-CELLS .2. TEMPORAL AND SPATIAL KINETICS
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DOI：
10.1083/jcb.93.3.727
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发表时间：
1982-01-01
影响因子：
7.8
通讯作者：
FLETCHER, WH
中科院分区：
文献类型：
--
作者：
BYUS, CV;FLETCHER, WH
cAMP依赖性蛋白激酶的激活是cAMP导致多种细胞功能改变的主要模式。激酶活化导致催化亚基的释放，该催化亚基作为游离酶对多种特异性蛋白质底物具有磷酸转移酶活性。用一种灵敏和特异的细胞荧光技术，监测了培养的H35肝癌细胞中游离催化亚基的出现，这些细胞在与N6- 1“-O-二丁酰-cAMP（DBcAMP）、8-溴腺苷-3”：5“-环磷酸（8-BrcAMP）和胰高血糖素孵育后。细胞化学方法采用游离催化亚基的热稳定抑制剂，该抑制剂已与异硫氰酸荧光素（F：PKI）结合，并如所述进行了验证。通过增加DBcAMP、8-BrcAMP和胰高血糖素的浓度，研究了cAMP依赖性蛋白激酶激活后游离催化亚基的时间和空间动力学。在类似的条件下，通过测定蛋白激酶活性比，在H35细胞上清液中生化地评估蛋白激酶活化。用DBcAMP（0.1 mM）孵育肝癌细胞导致活性比从对照培养物中的0.2增加到1至2小时内接近1.0的值。在同一时期内，使用F：PKI探针，细胞质和核仁荧光的显着增加，指示释放的游离催化亚基被巧合地观察到。与加入DBcAMP后5-15 min内细胞质和核仁中催化亚基的快速出现相反，直到1 h后才出现可辨别的核质荧光。与用DBcAMP处理的细胞相比，用8-BrcAMP（0.01-1.0 mM）孵育的H35细胞培养物表现出细胞化学测量的蛋白激酶的更快活化。与加入类似物1分钟内未刺激的细胞相比，与8-BrcAMP孵育的培养物具有显著增加的细胞质和核仁荧光，并在15分钟内达到最大水平。在刺激后1、5、15和60 min，随着8-BrcAMP浓度从0.01-1.0 mM增加，观察到游离催化亚基）。向培养物中加入胰高血糖素（10-6 M）导致cAMP依赖性蛋白激酶的活化，这通过活性比的增加来确定。在整个孵育期间，细胞质和核仁荧光的显著升高抑制了这种增加。显然，环核苷酸类似物和多肽激素导致cAMP依赖性蛋白激酶在这些细胞中类似的细胞内隔室中的激活。
The activation of cAMP-dependent protein kinase was the predominant mode by which cAMP leads to alterations of a large variety of cellular functions. Kinase activation results in the release of the catalytic subunit which as the free enzyme possesses phosphotransferase activity for a variety of specific protein substrates. Using a sensitive and specific cytofluorometric technique the appearance of free catalytic subunit was monitored in Reuber H35 [rat] hepatoma cells in culture after incubation with N6-1''-O-dibutyryl-cAMP (DBcAMP), 8-bromoadenosine-3'':5''-cyclic monophosphate (8-BrcAMP), and glucagon. The cytochemical method employs the heat-stable inhibitor of the free catalytic subunit which has been conjugated to fluorescein isothiocyanate (F:PKI) and was validated as described. The temporal and spatial kinetics of the free catalytic subunit was studied following activation of cAMP-dependent protein kinase by increasing concentrations of DBcAMP, 8-BrcAMP and glucagon. Under similar conditions protein kinase activation was assessed biochemically in H35 cell supernatants by assaying the protein kinase activity ratio. Incubation of the hepatoma cells with DBcAMP (0.1 mM) led to an increase in the activity ratio from 0.2 in control cultures to a value of nearly 1.0 within a 1- to 2-h period. During this same period using the F:PKI probe, a significant increase in cytoplasmic and nucleolar fluorescence indicative of the release of the free catalytic subunit was coincidentally observed. In contrast to the rapid appearance of catalytic subunit in the cytoplasm and nucleolus of the cell within 5-15 min of the addition of DBcAMP, discernible nucleoplasmic fluorescence did not occur until after 1 h. H35 cell cultures incubated with 8-BrcAMP (0.01-1.0 mM) exhibited a more rapid activation of the protein kinase measured cytochemically compared to the cells treated with DBcAMP. Cultures incubated with 8-BrcAMP had significantly increased cytoplasmic and nucleolar fluorescence compared to unstimulated cells within 1 min of the addition of the analogue and reached a maximal level within 15 min. By employing microspectrophotometry a distinct dose-dependent increase in cellular flourescence (i.e., free catalytic subunit) was oberved as the concentration of 8-BrcAMP was increased from 0.01-1.0 mM at 1, 5, 15 and 60 min following stimulation. The addition of glucagon (10-6 M) to the culture led to the activation of cAMP-dependent protein kinase as determined by an increase in the activity ratio. This increase was paralleled throughout the incubation period by a marked elevation in cytoplasmic and nucleolar fluorescence. Evidently, both cyclic nucleotide analogues and a polypeptide hormone lead to the activation of cAMP-dependent protein kinase in similar intracellular compartments in these cells.