Identification of phosphorylation sites of TOPORS and a role for serine 98 in the regulation of ubiquitin but not SUMO E3 ligase activity.
TOPORS is the first example of a protein that possesses both ubiquitin and SUMO E3 ligase activity. The ubiquitination activity maps to a conserved RING domain in the N-terminal region of the protein, which is not required for sumoylation activity. Similar to other E3 ligases, it is likely that the ubiquitin and sumoylation activities of TOPORS are regulated by post-translational modifications. Therefore, we employed mass spectrometry to identify post-translational modifications of TOPORS. Several putative phosphorylated regions were identified in conserved regions of the protein. We investigated the role of phosphorylation of serine 98, which is adjacent to the RING domain, in both cells and in vitro. Mutation of serine 98 to aspartic acid resulted in an increase in the ubiquitin ligase activity of TOPORS both in cells and in vitro. In addition, this mutation increased the binding of TOPORS to the E2 enzyme UbcH5a both in vitro and in cells. Conversely, a phospho-deficient mutant (S98A) exhibited little change in ubiquitin ligase activity compared to wild-type TOPORS, both in cells and in vitro. Neither of the mutants affected the localization of TOPORS to punctate nuclear regions. In addition, neither mutant affected the SUMO ligase activity of TOPORS in cells or in vitro. Molecular modeling studies support a role for serine 98 in regulating TOPORS-E2 interactions. Our findings indicate that phosphorylation of serine 98 regulates the ubiquitin but not the SUMO ligase activity of TOPORS, consistent with a potential binary switch function for TOPORS in protein ubiquitination versus sumoylation.
Park
Hye-Jin
HJ
Departments of Pharmacology and Medicine, The Cancer Institute of New Jersey, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, 195 Little Albany Street, New Brunswick, New Jersey 08901, USA.
Zheng
Haiyan
H
Kulkarni
Diptee
D
Kerrigan
John
J
Pungaliya
Pooja
P
Saleem
Ahamed
A
Rubin
Eric H
EH
D001665:Binding Sites; D045744:Cell Line, Tumor; D006801:Humans; D013058:Mass Spectrometry; D020125:Mutation, Missense; D009363:Neoplasm Proteins / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding / Q000235:genetics; D011499:Protein Processing, Post-Translational; D012694:Serine; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*
D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; D025801:Ubiquitin; D012694:Serine; C120157:TOPORS protein, human; D044767:Ubiquitin-Protein Ligases