MOVEMENT OF THE FREE CATALYTIC SUBUNIT OF CAMP-DEPENDENT PROTEIN-KINASE INTO AND OUT OF THE NUCLEUS CAN BE EXPLAINED BY DIFFUSION
MOVEMENT OF THE FREE CATALYTIC SUBUNIT OF CAMP-DEPENDENT PROTEIN-KINASE INTO AND OUT OF THE NUCLEUS CAN BE EXPLAINED BY DIFFUSION
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DOI：
10.1091/mbc.4.10.993
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发表时间：
1993-10-01
影响因子：
3.3
通讯作者：
TSIEN, RY
中科院分区：
文献类型：
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作者：
HAROOTUNIAN, AT;ADAMS, SR;TSIEN, RY
环磷酸腺苷(CAMP)依赖的蛋白激酶(PKA)的催化亚基(C)亚基在细胞内cAMP升高和cAMP降低时分别进入和离开细胞核。为了探讨核转位的机制，将荧光标记的C亚基以游离C亚基或全酶(PKA)的形式注入活的REF52成纤维细胞，其中催化和调节亚基分别用荧光素和罗丹明标记。细胞核和细胞质荧光强度的定量显示，游离C亚基的核积累与扩散到核内的大分子最相似。胞质显微注射后，谷胱甘肽-S-转移酶-C亚单位融合蛋白未进入细胞核。穿透核膜并没有降低C亚基的核浓度，C亚基进入细胞核似乎不是饱和的。冷却或耗尽能量的细胞不能阻止C亚单位进入细胞核。光漂白实验表明，即使在高[cAMP]达到平衡后，C亚基的单个分子继续以与它们最初进入的速度大致相同的速度离开细胞核。这些结果表明，扩散足以解释C亚单位亚细胞定位的大部分方面。
The catalytic (C) subunit of cyclic AMP (cAMP) dependent protein kinase (PKA) has previously been shown to enter and exit the nucleus of cells when intracellular cAMP is raised and lowered, respectively. To determine the mechanism of nuclear translocation, fluorescently labeled C subunit was injected into living REF52 fibroblasts either as free C subunit or in the form of holoenzyme (PKA) in which the catalytic and regulatory subunits were labeled with fluorescein and rhodamine, respectively. Quantification of nuclear and cytoplasmic fluorescence intensities revealed that free C subunit nuclear accumulation was most similar to that of macromolecules that diffuse into the nucleus. A glutathione S-transferase-C subunit fusion protein did not enter the nucleus following cytoplasmic microinjection. Puncturing the nuclear membrane did not decrease the nuclear concentration of C subunit, and C subunit entry into the nucleus did not appear to be saturable. Cooling or depleting cells of energy failed to block movement of C subunit into the nucleus. Photobleaching experiments showed that even after reaching equilibrium at high [cAMP], individual molecules of C subunit continued to leave the nucleus at approximately the same rate that they had originally entered. These results indicate that diffusion is sufficient to explain most aspects of C subunit subcellular localization.