The Regulation Of HDAC2 Autophosphorylation On Its SUMO E3 Ligase Activity Function And Mechanism

Research background and purpose:Histone deacetylases (HDACs) are a family of zinc ion dependent metalloprotease, with their active domain located at the N-terminal. HDACs induce deacetylation of histone or other proteins, especially the deacetylation of nucleosome histones, which leads to chromosome condensation. Reformation of nucleosome structure leads to inhibition of transcription activation of target genes including anti-oncogene, apoptosis gene, and differentiation gene, hence induces tumorgenesis. Therefore HDACs are acknowledged widely as effective anti-tumor targets.18 HDACs have been discovered until now. They can be divided into 4 major types according to structure and function. Type I HDACs include HDAC1,2,3 and 8, are widely expressed in all kinds of eukaryotic cells, playing a vital role for cell proliferation, growth, apoptosis and differentiation, therefore closely related to tumorgenesis.HDAC2 is highly expressed in colorectal cancer, lung cancer and other tumors, the expression level is significantly correlated with tumor prognosis. Inhibition of HDAC2 activity or expression can significantly inhibit the proliferation and growth of colorectal tumor cells, and promote apoptosis of cancer cells. However, studies found continuously applying deacetylases inhibitors can induce acquired drug resistance to skin T cell lymphoma cells, colorectal cancer cells and lung cancer cells. The drug resistance mechanism is still unclear. But if in combined use of HDAC inhibitors and mTOR pathway inhibitor sirolimus, the tumor cytotxicity-effect of HDAC inhibitors (HDACi) to colorectal cancer can be enhanced. It suggests that HDAC2 plays an important role in colorectal cancer and lung cancer genesis, development and drug resistance, although its mechanism is still unclear.In addition, our preliminary study also found that, HDAC2 has SUMO E3 ligase activity apart from other functions such as deacetylation, down regulation of nucleosome histone acetylation, silencing expression of anti-proliferation/pro-apoptotic genes like p53, p21, Bax and NOXA, or directly deacetylation of these proteins hence playing a critical role in colorectal tumorgenesis and development. Through its SUMO E3 ligase activity, HDAC2 can up regulate sumoylation of translation initiation factor eIF4E, activating cap-dependent mRNA translation, up regulate expression of pro-proliferation/anti-apoptotic genes like ODC, C-myc, Survivin or Bcl-2, hence performing vitally in regulation of proliferation and apoptosis of colorectal cancerHowever, although we found and confirmed HDAC2 functions as SUMO E3 ligase, and proved this novel function can activate cap-dependent mRNA translation through regulating sumoylation of eukaryotic translation initiation factor eIF4E, the biological meaning is still unclear, especially about how this novel function selectively regulates expression of genes which are beneficial for tumorgenesis, proliferation, growth and apoptosis-related by associating deacetylation activity. And it’s implied, simply blocking HDAC2 deacetylation activity has deficiency on tumor therapy, and this could be the main reason of tumor resistance. Therefore we think the strategy of HDAC2 dual enzyme co-suppression may have better efficiency in the treatment of cancer. However, at present all HDAC2 targeting anti-tumor drugs are specifically designed for acetylases activity. Inhibitors targeting to SUMO E3 ligase activity remain to be developed. We must firstly understand the activation regulatory mechanism of this enzyme in order to design HDAC2 SUMO E3 ligase targeting inhibitor.The activity of HDACs are regulated by multiple mechanisms, including post-translation modifications (PTMs), subcellular localization and corepressor protein complex interactions. Among them, PTMs do not only directly influence HDACs activity, but also impact HDACs subcellular localization and protein complex interactions, thus indirectly regulate its transcriptional inhibition activity. PTMs mainly include phosphrylation, acetylation, ubiquitination and sumoylation, etc. Phosphorylation is the main PTMs of HDAC2. Five Serine sites:S394, S407, S411, S422 and S424 located at C-terminal can be phosphorylated. In addition, analysis show phosphorylation region of HDAC2 is just located within its SUMO E3 ligase structural domain, and there is a close link between its phosphorlation and sumoylation. Therefore, we speculate that HDAC2 autophosphorylation plays an important regulatory role in SUMO E3 ligase function, yet to be confirmed.In vivo, HDAC2 phosphorylation kinases are mainly CKII. CKII can also induce in vitro phosphorylation of HDAC2 serine S394, S422, and S424. Consistent with this, CKII can also induce HDAC1 phosphorylation. Mutation of HDAC1 phosphoryaltion sites S421A and S423A can eliminate its phosphorylation modification, thus promoting sumoylation of HDAC1. It indicates that CKII induced HDAC2 autophosphorylation may also have neccessary links with its own sumoylation, which is key to its SUMO E3 ligase activity. However, there has been no report yet about regulatory function of CKII induced HDAC2 phosphorylation on its own SUMO E3 ligase activity.Therefore, this study will mainly apply gene point mutation technology, primarily search the influence of HDAC2 autophosphorylation on its SUMO E3 ligase function, and clarify regulatory mechanism of autophorylation on HDAC2 SUMO E3 ligase, establishing the foundation of designing HDAC2 SUMO E3 ligase targeting inhibitors.MethodsThe study will firstly construct HDAC2 S394A, S407A, S411A, S422A and S424A phosphorylation mutants through gene point mutation technology, then apply reporter gene to test the HADC2 SUMO E3 ligase activity; secondly applying CK2 kinase inhibitor tetrabromobenzotriazole (TBB) to block CK2 activity, confirm indirectly weakening HDAC2 autophosphorylation has impacts on HDAC2 SUMO E3 ligase activity. Thirdly, in the presence of HDAC2, new phosphorylation sites are analyzed by bioinformatics analysis, further new mutants were then constructed, afterwards reporter gene experiments, Western-blot experiment and cell proliferation experiment were applied to explore HDAC2 specific sites phosphorylation’s impact on its SUMO E3 ligase activity, the mechanism as well as the biological meaning.Results1. Successfully constructed eukaryotic expression vector of known phosphorylation region truncation as well as five phosphorylation sites negative mutants of HDAC2.2. Reporter gene experiment confirmed, HDAC2’s known phosphorylation region truncation mutants do not impact its SUMO E3 ligase activity, HDAC2’s five known serine sites phosphorylation has no regulation on its SUMO E3 ligase activity.3. Tetrabromobenzotriazole (TBB) can inhibit CKII induced HDAC2 autophosphorylation, and, this inhibitor has negative regulation to HDAC2 induced cap-dependent translation of reporter gene LUC.4. Bioinformatics prospect found that T477 and T480 sites of HDAC2 are two potential autophosphoryltion site, and in comparison with wild type, T477A phosphrylation negative mutants of this site significantly the expression of HDAC2 SUMO E3 ligase reporter gene (4.24 times of wild type), hence indicating phosphorylation of this site negatively regulates HDAC2 SUMO E3 ligase activity.5. IP, western blot and cell proliferation experiments confirm, T477A negative mutants of HDAC2 significantly increase HDAC2’s own sumoylation and sumoylation of eIF4E, increase translation level of target proteins (such as Bcl-2, cyclin D1, C-myc, Survivin and ODC) which regulate eIF4E, and promote colorectal tumor cell proliferation.ConclusionThe autophosphorylation of HDAC2 participates the adjustment of its SUMO E3 ligase activity, the regulatory mechanism is related to the phosphorylation of T477 site of HDAC2, and irrelevant with phosphorylation of S394, S407, S411, S422 and S424. Meanwhile, the phosphorylation of HDAC2 T477 site has negative regulation of its SUMO E3 ligase activity, which means, HDAC2 T477A mutants can significantly increase HDAC2’s own sumoylation, sumoylation of eIF4E, and increase the expression level of Bcl-2, cyclin D1, C-myc, Survivin and ODC at translation level, and promote colorectal cancer cell proliferation.