Prosperi E, Scovassi A I, Stivala L A, Bianchi L
Centro di Studio per l'Istochimica del C.N.R., Dipartimento di Biologia Animale, Università di Pavia, Italy.
Exp Cell Res. 1994 Dec;215(2):257-62. doi: 10.1006/excr.1994.1341.
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 G1, or in G2 + 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 G1/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 G1/S phase boundary to drive activation of DNA replication factors.
有证据表明，人成纤维细胞中增殖细胞核抗原（PCNA）与核染色质的结合与该蛋白的磷酸化状态有关。使用低渗裂解程序提取PCNA的可溶性形式，结果表明，剩余的主要存在于S期细胞中的核结合形式高度磷酸化。处于早期G1期或G2 + M期的细胞中，该蛋白结合形式的基础水平仅被微弱磷酸化。使用分级免疫沉淀技术发现，PCNA在可溶性和不溶性组分中均与细胞周期蛋白A相关。相反，PCNA与细胞周期蛋白D1的结合仅在可溶性组分中被发现，而在不溶性组分中未检测到。对细胞周期蛋白D1和细胞周期蛋白A的细胞周期分布进行免疫荧光标记和流式细胞术分析表明，与PCNA一样，这两种蛋白的最大水平在G1/S期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的结合可能发生在G1/S期边界处组装的大分子复合物中，以驱动DNA复制因子的激活。
