Rampalli A M, Gao C Y, Chauthaiwale V M, Zelenka P S
Laboratory of Molecular and Developmental Biology, National Eye Institute, NIH, Bethesda, Maryland 20892, USA.
Oncogene. 1998 Jan 22;16(3):399-408. doi: 10.1038/sj.onc.1201546.
During growth arrest and differentiation, activity of the E2F family of transcription factors is inhibited by interactions with pRb and the related proteins, p107 and p130. To determine which members of the E2F and pRb families may contribute to growth arrest as lens epithelial cells differentiate into fiber cells, we examined the expression of individual E2F species and characterized the E2F protein complexes formed in rat lens epithelia and fibers. RT/PCR detected all five known members of the E2F family in lens epithelial cells, but only E2F-1, E2F-3, and E2F-5 in fiber cells. Proteins extracted from lens epithelia of newborn rats formed at least two specific complexes with an E2F consensus oligonucleotide. Proteins from lens fiber cells formed three specific complexes, one of which comigrated with an epithelial cell complex. Incubation of epithelial and fiber cell extracts with an antibody specific for p107 demonstrated that two fiber cell complexes and one epithelial cell complex contained p107. Although the remaining fiber cell complex did not react with antibodies to pRb or p130 in this assay, a strong reaction with pRb antibody was observed when the electromobility shifted complexes were subsequently immunoblotted (shift/Western assay). Immunocytochemistry confirmed that pRb protein is present in the nuclei of both epithelial cells and fiber cells. Immunoblotting of whole cell extracts with pRb antibody showed multiple, phosphorylated forms of pRb in the epithelial cells, but predominantly hypophosphorylated pRb in the fiber cells. None of the complexes formed with E2F were recognized exclusively by the p130 antibody, although the previously identified p107 complexes reacted weakly. The absence of p130/E2F complexes was correlated with the presence of multiple ubiquitinated forms of p130, especially in the fiber cells. Thus, although p130/E2F complexes are implicated in the terminal differentiation of many cell types, in differentiating lens fiber cells pRb and p107 seem to be the primary regulators of E2F activity.
在生长停滞和分化过程中，E2F转录因子家族的活性通过与pRb以及相关蛋白p107和p130的相互作用而受到抑制。为了确定E2F家族和pRb家族的哪些成员可能在晶状体上皮细胞分化为纤维细胞时对生长停滞起作用，我们检测了各个E2F种类的表达，并对大鼠晶状体上皮和纤维中形成的E2F蛋白复合物进行了表征。逆转录/聚合酶链反应（RT/PCR）在晶状体上皮细胞中检测到了E2F家族的所有五个已知成员，但在纤维细胞中仅检测到E2F-1、E2F-3和E2F-5。从新生大鼠晶状体上皮中提取的蛋白质与E2F共有寡核苷酸形成了至少两种特异性复合物。晶状体纤维细胞中的蛋白质形成了三种特异性复合物，其中一种与上皮细胞复合物迁移情况相同。用针对p107的特异性抗体孵育上皮细胞和纤维细胞提取物表明，两种纤维细胞复合物和一种上皮细胞复合物含有p107。尽管在该检测中剩余的纤维细胞复合物不与针对pRb或p130的抗体反应，但当对电泳迁移率变动的复合物进行随后的免疫印迹分析（迁移率变动/蛋白质免疫印迹法）时，观察到与pRb抗体有强烈反应。免疫细胞化学证实pRb蛋白存在于上皮细胞和纤维细胞的细胞核中。用pRb抗体对全细胞提取物进行免疫印迹显示，上皮细胞中有多种磷酸化形式的pRb，但纤维细胞中主要是低磷酸化的pRb。与E2F形成的复合物中没有一种仅被p130抗体识别，尽管先前鉴定的p107复合物反应较弱。p130/E2F复合物的缺失与多种泛素化形式的p130的存在相关，尤其是在纤维细胞中。因此，尽管p130/E2F复合物与许多细胞类型的终末分化有关，但在分化的晶状体纤维细胞中，pRb和p107似乎是E2F活性的主要调节因子。
