Nakayama T, Nishizawa K, Sato C
Laboratory of Experimental Radiology, Aichi Cancer Center Research Institute, Nagoya, Japan.
Exp Cell Res. 1988 Aug;177(2):360-71. doi: 10.1016/0014-4827(88)90469-7.
We previously reported that treatment of SV-3Y1 cells in an exponential growth state with 1 mM db-cAMP plus 1 mM theophylline induced reversible disappearance of nuclear dots stained by monoclonal anti-microtubule-associated protein (MAP)-1 antibody [T. Nakayama, K. Nishizawa, G. Kimura, and C. Sato (1986) Exp. Cell Res. 163, 246]. In the present study, we examined the relation between the intracellular localization and phosphorylation of 300- to 350-kDa proteins that are intracellular antigens for our anti-MAP-1 and -2 antibodies. Treatment with 1 mM db-cAMP plus 1 mM theophylline was found to result in a reversible decrease in immunofluorescent staining of the nucleus with polyclonal MAP-1 or -2 antibody, and a reversible increase in that of the cytoplasm. Simultaneous treatment with 2.5 microM colchicine, 2.5 microM colcemid, 20 microM putrescine, or 3 mM alpha-naphthyl phosphate in the presence of db-cAMP plus theophylline almost prevented this effect of db-cAMP plus theophylline. We examined the cytoplasmic and nuclear fractions by immunoperoxidase staining, immunoprecipitation, and 125I-protein A with anti-MAP-1 and -2 antibodies. Treatment with db-cAMP plus theophylline resulted in the increase of 300- to 350-kDa proteins in the cytoplasm and a decrease in the nucleus. This treatment also caused the dephosphorylation of 300- to 350-kDa proteins. The present research indicated that treatment with db-cAMP plus theophylline resulted in the reversible translocation of 300- to 350-kDa proteins from the nucleus to the cytoplasm accompanied by the dephosphorylation of these proteins.
我们之前报道过，用1 mM二丁酰环磷腺苷（db-cAMP）加1 mM氨茶碱处理处于指数生长期的SV-3Y1细胞，会导致用单克隆抗微管相关蛋白（MAP）-1抗体染色的核点可逆性消失[T. 中山、K. 西泽、G. 木村和C. 佐藤（1986年）《实验细胞研究》163, 246]。在本研究中，我们检测了300至350 kDa蛋白的细胞内定位与磷酸化之间的关系，这些蛋白是我们抗MAP-1和-2抗体的细胞内抗原。发现用1 mM db-cAMP加1 mM氨茶碱处理会导致用多克隆MAP-1或-2抗体对细胞核进行免疫荧光染色可逆性降低，而对细胞质的免疫荧光染色可逆性增加。在db-cAMP加氨茶碱存在的情况下，同时用2.5 microM秋水仙碱、2.5 microM秋水仙酰胺、20 microM腐胺或3 mM α-萘基磷酸处理几乎可防止db-cAMP加氨茶碱的这种作用。我们用抗MAP-1和-2抗体通过免疫过氧化物酶染色、免疫沉淀和125I-蛋白A检测了细胞质和细胞核部分。用db-cAMP加氨茶碱处理导致细胞质中300至350 kDa蛋白增加，细胞核中减少。这种处理还导致300至350 kDa蛋白去磷酸化。本研究表明，用db-cAMP加氨茶碱处理导致300至350 kDa蛋白从细胞核可逆性转运至细胞质，并伴随这些蛋白的去磷酸化。
