BOG, a Novel pRb Binding Protein Affecting Cell-cycle Control and Transformation

BOG(RBBP-9)是一种新的细胞蛋白，通过LXCXE基序与pRb、p107和p130相互作用。用谷氨酰胺取代LXCXE序列(Bog D)中的亮氨酸足以破坏bog与pRb/p107/p130之间的相互作用。在这里，我们通过亚细胞分离、免疫组织化学和Bog-EGFP1融合构建表明，在各种细胞系中，bog蛋白主要位于细胞核中。为了验证bog蛋白的核定位是否依赖于其pRB结合基序，我们在几个小鼠、大鼠和人细胞系中表达了bog-EGFP和bog D-EGFP融合蛋白。在含有功能不活跃的pRB的Saos-2细胞和pRB-/-小鼠胚胎成纤维细胞中，融合蛋白bog-EGFP仍定位于细胞核。然而，与野生型不同的是，bog D-EGFP不能在细胞核内蓄积，并表现出更大的细胞质定位。这些结果表明，LXCXE基序对于BOG与RB家族成员的结合和蛋白质的核定位都是至关重要的，这表明在没有PRB的情况下，与PRB相关蛋白p107和/或p130的结合可能足以支持蛋白质的核积累。

BOG (RBBP-9) is a novel cellular protein which interacts with pRb, p107 and p130 through the LXCXE motif. Substitution of a leucine with glutamine in the LXCXE sequence (BOG D) is sufficient to disrupt the interaction between BOG and pRb/p107/p130. Here we show by employing subcellular fractionation, immunohistochemistry and BOG-EGFP1 fusion constructs, that the BOG protein was primarily located in the nucleus in a variety of cell lines. To examine whether the nuclear localization of the BOG protein is dependent on its pRb binding motif, we expressed the fusion proteins BOG-EGFP and BOG D-EGFP, in several mouse, rat and human cell lines. In Saos-2 cells, which contain a functionally inactive pRb, and in pRb -/- mouse embryo fibroblasts, the fusion protein BOG-EGFP was still localized to the nucleus. However, unlike the wild-type, BOG D-EGFP failed to accumulate in the nuclei, and demonstrated a greater cytoplasmic localization. These results indicate that LXCXE motif is critical for both binding of BOG to members of the Rb family and the nuclear localization of the protein, suggesting that the binding with the pRb related proteins p107 and/or p130 may be sufficient to support the nuclear accumulation of the protein in the absence of pRb.