Morrione A, Plant P, Valentinis B, Staub O, Kumar S, Rotin D, Baserga R
Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
J Biol Chem. 1999 Aug 20;274(34):24094-9. doi: 10.1074/jbc.274.34.24094.
We have utilized the yeast two-hybrid system to identify proteins interacting with mouse Grb10, an adapter protein known to interact with both the insulin and the insulin-like growth factor-I receptors. We have isolated a mouse cDNA clone containing the C2 domain of mouse Nedd4, a ubiquitin protein ligase (E3) that also contains a hect (homologous to the E6-AP carboxyl-terminus) domain and three WW domains. The interaction with Grb10 in the two-hybrid system was confirmed using the full-length Nedd4, and it was abolished by deleting the last 148 amino acids of Grb10, a region that includes the SH2 domain and the newly identified BPS domain. The interaction between Grb10 and Nedd4 was also reproduced in vivo in mouse embryo fibroblasts, where endogenous Nedd4 co-immunoprecipitated constitutively with both the endogenous and an overexpressed Grb10. This interaction was Ca(2+)-independent. Grb10 interacting with Nedd4 was not ubiquitinated in vivo, raising the possibility that this interaction may be used to target other proteins, like tyrosine kinase receptors, for ubiquitination.
我们利用酵母双杂交系统来鉴定与小鼠Grb10相互作用的蛋白质，Grb10是一种已知能与胰岛素及胰岛素样生长因子-I受体相互作用的衔接蛋白。我们分离出了一个小鼠cDNA克隆，其包含小鼠Nedd4的C2结构域，Nedd4是一种泛素蛋白连接酶（E3），还包含一个hect（与E6-AP羧基末端同源）结构域和三个WW结构域。使用全长Nedd4在双杂交系统中证实了其与Grb10的相互作用，并且通过缺失Grb10的最后148个氨基酸（该区域包括SH2结构域和新鉴定的BPS结构域），这种相互作用被消除。Grb10与Nedd4之间的相互作用在小鼠胚胎成纤维细胞中也在体内重现，内源性Nedd4与内源性和过表达的Grb10均组成性地共免疫沉淀。这种相互作用不依赖于Ca(2+)。与Nedd4相互作用的Grb10在体内未被泛素化，这增加了这种相互作用可能用于将其他蛋白质（如酪氨酸激酶受体）靶向泛素化的可能性。
