Poyurovsky Masha V, Jacq Xavier, Ma Charles, Karni-Schmidt Orit, Parker Peter J, Chalfie Martin, Manley James L, Prives Carol
Department of Biological Sciences, Columbia University, New York, NY 10027, USA.
Mol Cell. 2003 Oct;12(4):875-87. doi: 10.1016/s1097-2765(03)00400-3.
The RING domain of Mdm2 contains a conserved Walker A or P loop motif that is a characteristic of nucleotide binding proteins. We found that Mdm2 binds adenine-containing nucleotides preferentially and that nucleotide binding leads to a conformational change in the Mdm2 C terminus. Although nucleotide binding is not required for Mdm2 E3 ubiquitin ligase activity, we show that nucleotide binding-defective P loop mutants are impaired in p14(ARF)-independent nucleolar localization both in vivo and in vitro. Consistent with this, ATP-bound Mdm2 is preferentially localized to the nucleolus. Indeed, we identify a unique amino acid substitution in the P loop motif (K454A) that uncouples nucleolar localization and E3 ubiquitin ligase activity of Mdm2 and leads to upregulation of the E3 activity both in human cells and in Caenorhabditis elegans. We propose that nucleotide binding-facilitated nucleolar localization of Mdm2 is an evolutionarily conserved regulator of Mdm2 activity.
Mdm2的环状结构域包含一个保守的沃克A或P环基序，这是核苷酸结合蛋白的一个特征。我们发现Mdm2优先结合含腺嘌呤的核苷酸，并且核苷酸结合会导致Mdm2 C末端的构象变化。虽然核苷酸结合对于Mdm2 E3泛素连接酶活性不是必需的，但我们表明，核苷酸结合缺陷的P环突变体在体内和体外的p14(ARF)非依赖性核仁定位中均受损。与此一致的是，结合ATP的Mdm2优先定位于核仁。事实上，我们在P环基序中鉴定出一个独特的氨基酸取代（K454A），它使Mdm2的核仁定位与E3泛素连接酶活性脱钩，并导致人细胞和秀丽隐杆线虫中E3活性上调。我们提出，核苷酸结合促进的Mdm2核仁定位是Mdm2活性的一种进化保守调节因子。
