Institute of Plant Biology, Biological Research Center, Hungarian Academy of Sciences, Temesvári krt. 62, H-6726 Szeged, Hungary.
J Exp Bot. 2011 Mar;62(6):2155-68. doi: 10.1093/jxb/erq413. Epub 2010 Dec 31.
Plant retinoblastoma-related (RBR) proteins are primarily considered as key regulators of G(1)/S phase transition, with functional roles in a variety of cellular events during plant growth and organ development. Polyclonal antibody against the C-terminal region of the Arabidopsis RBR1 protein also specifically recognizes the alfalfa 115 kDa MsRBR protein, as shown by the antigen competition assay. The MsRBR protein was detected in all cell cycle phases, with a moderate increase in samples representing G(2)/M cells. Antibody against the human phospho-pRb peptide (Ser807/811) cross-reacted with the same 115 kDa MsRBR protein and with the in vitro phosphorylated MsRBR protein C-terminal fragment. Phospho-MsRBR protein was low in G(1) cells. Its amount increased upon entry into the S phase and remained high during the G(2)/M phases. Roscovitine treatment abolished the activity of alfalfa MsCDKA1;1 and MsCDKB2;1, and the phospho-MsRBR protein level was significantly decreased in the treated cells. Colchicine block increased the detected levels of both forms of MsRBR protein. Reduced levels of the MsRBR protein in cells at stationary phase or grown in hormone-free medium can be a sign of the division-dependent presence of plant RBR proteins. Immunolocalization of the phospho-MsRBR protein indicated spots of variable number and size in the labelled interphase nuclei and high signal intensity of nuclear granules in prophase. Structures similar to phospho-MsRBR proteins cannot be recognized in later mitotic phases. Based on the presented western blot and immunolocalization data, the possible involvement of RBR proteins in G(2)/M phase regulation in plant cells is discussed.
植物视网膜母细胞瘤相关 (RBR) 蛋白主要被认为是 G1/S 期转换的关键调节剂，在植物生长和器官发育过程中的各种细胞事件中具有功能作用。针对拟南芥 RBR1 蛋白 C 端区域的多克隆抗体也特异性识别苜蓿 115 kDa MsRBR 蛋白，如抗原竞争测定所示。在所有细胞周期相中都检测到 MsRBR 蛋白，在代表 G2/M 细胞的样品中中度增加。针对人磷酸化 pRb 肽 (Ser807/811) 的抗体与相同的 115 kDa MsRBR 蛋白和体外磷酸化 MsRBR 蛋白 C 端片段发生交叉反应。磷酸化 MsRBR 蛋白在 G1 细胞中含量较低。进入 S 期后其含量增加，并在 G2/M 期保持高水平。罗司渥丁处理消除了苜蓿 MsCDKA1;1 和 MsCDKB2;1 的活性，并且处理细胞中的磷酸化 MsRBR 蛋白水平显着降低。秋水仙碱阻断增加了两种形式的 MsRBR 蛋白的检测水平。在静止期或在无激素培养基中生长的细胞中 MsRBR 蛋白水平降低可能是植物 RBR 蛋白存在于细胞分裂中的标志。磷酸化 MsRBR 蛋白的免疫定位在标记的间期中核中显示出数量和大小可变的斑点，并且在前期中核颗粒的信号强度高。在后期有丝分裂阶段无法识别与磷酸化 MsRBR 蛋白相似的结构。基于呈现的 Western blot 和免疫定位数据，讨论了 RBR 蛋白在植物细胞 G2/M 期调节中的可能参与。
