Identification of phosphorylation sites of TOPORS and a role for serine 98 in the regulation of ubiquitin but not SUMO E3 ligase activity.
Identification of phosphorylation sites of TOPORS and a role for serine 98 in the regulation of ubiquitin but not SUMO E3 ligase activity.
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鉴定 TOPORS 磷酸化位点以及丝氨酸 98 在调节泛素而非 SUMO E3 连接酶活性中的作用。
DOI：
10.1021/bi801904q
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发表时间：
2008
期刊：
影响因子：
2.9
通讯作者：
Rubin,EricH
中科院分区：
文献类型：
--
作者：
Park,Hye-Jin;Zheng,Haiyan;Kulkarni,Diptee;Kerrigan,John;Pungaliya,Pooja;Saleem,Ahamed;Rubin,EricH
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.
