Ludlow J W, Shon J, Pipas J M, Livingston D M, DeCaprio J A
Dana-Farber Cancer Institute, Boston, Massachusetts 02115.
Cell. 1990 Feb 9;60(3):387-96. doi: 10.1016/0092-8674(90)90590-b.
Synchronized monkey cells pulse-labeled with [35S]-methionine and chased for various lengths of time were extracted, and immunoprecipitations were performed using monoclonal antibodies directed against the retinoblastoma protein (RB) and SV40 T antigen (T). By following a discrete population of these two proteins through the cell cycle, the following information was obtained. RB, which is wholly unphosphorylated in G1, became phosphorylated at the beginning of S and remained phosphorylated through S and G2. RB was, then, completely dephosphorylated between the end of G2 and the beginning of G1. Second, while all of the detectable unphosphorylated RB can be found complexed with T, these complexes present during G1 dissociated in S and reformed again in M or early G1. Finally, T molecules appeared to oligomerize prior to binding RB. Thus, complex formation between T and RB may be regulated in part by the cell cycle-dependent phosphorylation and dephosphorylation of RB and by the quaternary structure of T.
用[35S]-甲硫氨酸进行脉冲标记并追踪不同时长的同步化猴细胞被提取出来，然后使用针对视网膜母细胞瘤蛋白（RB）和SV40 T抗原（T）的单克隆抗体进行免疫沉淀。通过在细胞周期中追踪这两种蛋白的离散群体，获得了以下信息。在G1期完全未磷酸化的RB，在S期开始时被磷酸化，并在S期和G2期一直保持磷酸化状态。然后，RB在G2期末尾和G1期开始之间完全去磷酸化。其次，虽然所有可检测到的未磷酸化RB都能与T形成复合物，但这些在G1期存在的复合物在S期解离，并在M期或早期G1期再次形成。最后，T分子在结合RB之前似乎会发生寡聚化。因此，T和RB之间的复合物形成可能部分受RB的细胞周期依赖性磷酸化和去磷酸化以及T的四级结构调控。
