de Bruijn Diederik R H, dos Santos Nuno R, Kater-Baats Ellen, Thijssen José, van den Berk Lieke, Stap Jiska, Balemans Matthé, Schepens Marga, Merkx Gerard, van Kessel Ad Geurts
Department of Human Genetics, University Medical Center Nijmegen, Nijmegen, The Netherlands.
Genes Chromosomes Cancer. 2002 Jul;34(3):285-98. doi: 10.1002/gcc.10073.
The SSX gene family is composed of at least five functional and highly homologous members, SSX1 to SSX5, that are normally expressed in only the testis and thyroid. SSX1, SSX2, or SSX4 may be fused to the SYT gene as a result of the t(X;18) translocation in synovial sarcoma. In addition, the SSX1, SSX2, SSX4, and SSX5 genes were found to be aberrantly expressed in several other malignancies, including melanoma. The SSX proteins are localized in the nucleus and are diffusely distributed. In addition, they may be included in polycomb-group nuclear bodies. Other studies have indicated that the SSX proteins may act as transcriptional repressors. As a first step toward the elucidation of the cellular signaling networks in which the SSX proteins may act, we used the yeast two-hybrid system to identify SSX2-interacting proteins. By doing so, two novel human proteins were detected: RAB3IP, the human homolog of an interactor of the Ras-like GTPase Rab3A; and a novel protein, SSX2IP. RAB3IP did not interact with either SSX1, SSX3, or SSX4 in the yeast two-hybrid system, whereas SSX2IP interacted with SSX3 but not with either SSX1 or SSX4. Further analysis of deletion mutants showed that both RAB3IP and SSX2IP interact with the N-terminal moiety of the SSX2 protein. Immunofluorescence analyses of transfected cells revealed that the RAB3IP protein is normally localized in the cytoplasm. However, coexpression of both RAB3IP and SSX2 led to colocalization of both proteins in the nucleus. Likewise, the SSX2IP protein was found to be colocalizing with SSX2 in the nucleus. By performing glutathione-S-transferase pull-down assays, we found that both RAB3IP and SSX2IP interact directly with SSX2 in vitro. These newly observed protein/protein interactions may have important implications for the mechanisms underlying normal and malignant cellular growth.
SSX基因家族由至少五个功能高度同源的成员组成，即SSX1至SSX5，它们通常仅在睾丸和甲状腺中表达。由于滑膜肉瘤中的t(X;18)易位，SSX1、SSX2或SSX4可能与SYT基因融合。此外，发现SSX1、SSX2、SSX4和SSX5基因在包括黑色素瘤在内的其他几种恶性肿瘤中异常表达。SSX蛋白定位于细胞核且呈弥漫性分布。此外，它们可能包含在多梳蛋白组核小体中。其他研究表明，SSX蛋白可能作为转录抑制因子发挥作用。作为阐明SSX蛋白可能参与的细胞信号网络的第一步，我们使用酵母双杂交系统来鉴定与SSX2相互作用的蛋白。通过这样做，检测到了两种新的人类蛋白：RAB3IP，即类Ras GTP酶Rab3A的一个相互作用分子的人类同源物；以及一种新蛋白SSX2IP。在酵母双杂交系统中，RAB3IP不与SSX1、SSX3或SSX4相互作用，而SSX2IP与SSX3相互作用，但不与SSX1或SSX4相互作用。对缺失突变体的进一步分析表明，RAB3IP和SSX2IP都与SSX2蛋白的N端部分相互作用。对转染细胞的免疫荧光分析显示，RAB3IP蛋白通常定位于细胞质中。然而，RAB3IP和SSX2的共表达导致两种蛋白在细胞核中共定位。同样，发现SSX2IP蛋白与SSX2在细胞核中共定位。通过进行谷胱甘肽-S-转移酶下拉试验，我们发现RAB3IP和SSX2IP在体外都直接与SSX2相互作用。这些新观察到的蛋白/蛋白相互作用可能对正常和恶性细胞生长的潜在机制具有重要意义。
