Nucleotide binding by the Mdm2 RING domain facilitates Arf-independent Mdm2 nucleolar localization.
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.
Department of Biological Sciences, Columbia University, New York, NY 10027, USA.
Masha V
MV
Poyurovsky
Xavier
X
Jacq
Charles
C
Ma
Orit
O
Karni-Schmidt
Peter J
PJ
Parker
Martin
M
Chalfie
James L
JL
Manley
Carol
C
Prives
D021581:Active Transport, Cell Nucleus / Q000502:physiology; D000227:Adenine Nucleotides / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence / Q000502:physiology; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D017173:Caenorhabditis elegans; D002451:Cell Compartmentation / Q000502:physiology; D045744:Cell Line, Tumor; D002466:Cell Nucleolus / Q000378:metabolism*; D019143:Evolution, Molecular; D006801:Humans; D008968:Molecular Conformation; D009154:Mutation / Q000235:genetics; D009687:Nuclear Proteins; D010766:Phosphorylation; D017434:Protein Structure, Tertiary / Q000502:physiology; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D051736:Proto-Oncogene Proteins c-mdm2; D025803:Tumor Suppressor Protein p14ARF / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D015854:Up-Regulation / Q000502:physiology
D000227:Adenine Nucleotides; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D025803:Tumor Suppressor Protein p14ARF; D000255:Adenosine Triphosphate; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; D044767:Ubiquitin-Protein Ligases