PROLIFERATING CELL NUCLEAR ANTIGEN BOUND TO DNA-SYNTHESIS SITES - PHOSPHORYLATION AND ASSOCIATION WITH CYCLIN-D1 AND CYCLIN-A
PROLIFERATING CELL NUCLEAR ANTIGEN BOUND TO DNA-SYNTHESIS SITES - PHOSPHORYLATION AND ASSOCIATION WITH CYCLIN-D1 AND CYCLIN-A
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DOI：
10.1006/excr.1994.1341
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发表时间：
1994-12-01
影响因子：
3.7
通讯作者：
BIANCHI, L
中科院分区：
文献类型：
--
作者：
PROSPERI, E;SCOVASSI, AI;BIANCHI, L
有证据表明，人成纤维细胞中增殖细胞核抗原（PCNA）与核染色质的关联与该蛋白的磷酸化状态有关。使用低渗裂解程序提取可溶形式的 PCNA，结果表明，剩余的核结合形式（主要在 S 期细胞中）高度磷酸化。处于早期 G(1) 或 G(2) + M 期的细胞含有基础水平的仅弱磷酸化的结合形式的蛋白质。使用分级免疫沉淀技术，发现 PCNA 在可溶性和不溶性级分中与细胞周期蛋白 A 相关。相比之下，在可溶性级分中发现了 PCNA 与细胞周期蛋白 D1 的关联，而在不溶性级分中则没有可检测到的水平。细胞周期蛋白 D1 和细胞周期蛋白 A 的细胞周期分布的免疫荧光标记和流式细胞术分析表明，与 PCNA 一样，两种蛋白的最大水平在 G(1)/S 相边界处与核结构结合。这些结果表明 PCNA 与 DNA 合成位点的结合发生在磷酸化之后。与细胞周期蛋白 D1 和细胞周期蛋白 A 的关联可能发生在 G(1)/S 相边界处组装的大分子复合物中，以驱动 DNA 复制因子的激活。 (C) 1994 学术出版社
Evidence is presented that association of proliferating cell nuclear antigen (PCNA) with nuclear chromatin in human fibroblasts is related to the phosphorylation status of the protein. Using a hypotonic lysis procedure to extract the soluble form of PCNA, it has been shown that the remaining nuclear-bound form, predominantly in S-phase cells, is highly phosphorylated. Cells in early G(1), or in G(2) + M phases, contain basal levels of the bound form of the protein that is only weakly phosphorylated. Using fractionated immunoprecipitation techniques, PCNA was found to be associated with cyclin A in both soluble and insoluble fractions. In contrast, association of PCNA with cyclin D1 was found in the soluble fraction, while no detectable levels were present in the insoluble fraction. Immunofluorescence labeling and flow cytometric analysis of the cell cycle distribution of cyclin D1 and cyclin A showed that, like PCNA, maximal levels of both proteins were bound to nuclear structures at the G(1)/S phase boundary. These results suggest that binding of PCNA to DNA synthesis sites occurs after phosphorylation. Association with cyclin D1 and cyclin A might occur in a macromolecular complex assembled at the G(1)/S phase boundary to drive activation of DNA replication factors. (C) 1994 Academic Press,Inc.