Harootunian A T, Adams S R, Wen W, Meinkoth J L, Taylor S S, Tsien R Y
Department of Pharmacology, University of California, San Diego, La Jolla 92093-0647.
Mol Biol Cell. 1993 Oct;4(10):993-1002. doi: 10.1091/mbc.4.10.993.
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.
环磷酸腺苷（cAMP）依赖性蛋白激酶（PKA）的催化（C）亚基先前已被证明，当细胞内cAMP升高和降低时，分别进入和离开细胞核。为了确定核转位机制，将荧光标记的C亚基以游离C亚基的形式或全酶（PKA）的形式注入活的REF52成纤维细胞中，其中催化亚基和调节亚基分别用荧光素和罗丹明标记。对细胞核和细胞质荧光强度的定量分析表明，游离C亚基的核积累与扩散进入细胞核的大分子最为相似。细胞质显微注射后，谷胱甘肽S-转移酶-C亚基融合蛋白未进入细胞核。刺穿核膜并未降低C亚基的核浓度，且C亚基进入细胞核似乎不饱和。冷却或耗尽细胞能量未能阻止C亚基进入细胞核。光漂白实验表明，即使在高[cAMP]下达到平衡后，C亚基的单个分子仍以与最初进入时大致相同的速率离开细胞核。这些结果表明，扩散足以解释C亚基亚细胞定位的大多数方面。
