Yen A, Varvayanis S
Department of Pathology, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853.
Exp Cell Res. 1994 Sep;214(1):250-7. doi: 10.1006/excr.1994.1255.
The cell cycle phase-specific phosphorylation status of the RB protein (retinoblastoma tumor suppressor gene product) during an elicited cellular program of G0 arrest and cell differentiation was characterized. The RB protein phosphorylation state is presumed to be an important determinant of cell proliferation or arrest. The cell cycle phase specificity of RB protein phosphorylation and dephosphorylation during HL-60 human leukemia cell proliferation and differentiation was determined using DNA-based fluorescence-activated cell sorting and Western analysis. The RB protein in proliferating G1 cells was phosphorylated, but at a relatively low level. The extent of phosphorylation increased in S phase cells and was maximum in G2 + M cells. After the cells were treated with retinoic acid or 1,25-dihydroxy vitamin D3, they began to accumulate in G1/0 and phenotypically convert. Significant unphosphorylated RB protein did not appear until after the first cells had arrested and differentiated. Dephosphorylation of the RB protein was first apparent at the beginning of G2 in the remaining cycling cells after onset of arrest and differentiation had already occurred. By the time the remaining cycling cells had divided and arrested, resulting in G0 cells, a majority of RB protein was dephosphorylated, but some remained phosphorylated. The data indicate that dephosphorylation of RB does not determine residence in G1/0. Rather dephosphorylation appears as one relatively late-occurring component of the metabolic cascade culminating in G0-arrested, phenotypically differentiated cells. Dephosphorylated RB appears as a feature of differentiated cells. The data are consistent with a role for hypophosphorylated RB not so much in deriving, but in possibly sustaining the differentiated state.
对视网膜母细胞瘤肿瘤抑制基因产物（RB蛋白）在诱导的G0期停滞和细胞分化细胞程序中的细胞周期阶段特异性磷酸化状态进行了表征。RB蛋白的磷酸化状态被认为是细胞增殖或停滞的重要决定因素。使用基于DNA的荧光激活细胞分选和蛋白质免疫印迹分析，确定了HL-60人白血病细胞增殖和分化过程中RB蛋白磷酸化和去磷酸化的细胞周期阶段特异性。增殖的G1期细胞中的RB蛋白发生了磷酸化，但水平相对较低。S期细胞中的磷酸化程度增加，在G2 + M期细胞中达到最大值。在用视黄酸或1,25-二羟基维生素D3处理细胞后，它们开始在G1/0期积累并发生表型转化。直到第一批细胞停滞并分化后，才出现明显的未磷酸化RB蛋白。在停滞和分化开始后，剩余循环细胞中RB蛋白的去磷酸化首先在G2期开始时明显。当剩余的循环细胞分裂并停滞，产生G0期细胞时，大多数RB蛋白去磷酸化，但仍有一些保持磷酸化状态。数据表明，RB蛋白的去磷酸化并不决定细胞在G1/0期的停留。相反，去磷酸化似乎是代谢级联反应中一个相对较晚出现的组成部分，最终导致细胞停滞在G0期并发生表型分化。去磷酸化的RB蛋白是分化细胞的一个特征。这些数据与低磷酸化RB蛋白的作用一致，即它不是在诱导分化状态，而是可能在维持分化状态方面发挥作用。
