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Cell cycle-linked MeCP2 phosphorylation modulates adult neurogenesis involving the Notch signalling pathway.

Li H, Zhong X, Chau KF, Santistevan NJ, Guo W, Kong G, Li X, Kadakia M, Masliah J, Chi J, Jin P, Zhang J, Zhao X, Chang Q - Nat Commun (2014)

Bottom Line: Neuronal activity regulates the phosphorylation states at multiple sites on MeCP2 in postmitotic neurons.However, it is unknown whether phosphorylation at any of the previously identified sites on MeCP2 can be induced by signals other than neuronal activity in other cell types, and what functions MeCP2 phosphorylation may have in those contexts.Our findings suggest MeCP2 S421 phosphorylation may function as a general epigenetic switch accessible by different extracellular stimuli through different signalling pathways for regulating diverse biological functions in different cell types.

View Article: PubMed Central - PubMed

Affiliation: 1] Waisman Center, University of Wisconsin-Madison, Madison, Wisconsin 53705, USA [2] Genetics Training Program, University of Wisconsin-Madison, Madison, Wisconsin 53705, USA.

ABSTRACT
Neuronal activity regulates the phosphorylation states at multiple sites on MeCP2 in postmitotic neurons. The precise control of the phosphorylation status of MeCP2 in neurons is critical for the normal development and function of the mammalian brain. However, it is unknown whether phosphorylation at any of the previously identified sites on MeCP2 can be induced by signals other than neuronal activity in other cell types, and what functions MeCP2 phosphorylation may have in those contexts. Here we show that in neural progenitor cells isolated from the adult mouse hippocampus, cell cycle-linked phosphorylation at serine 421 on MeCP2 is directly regulated by aurora kinase B and modulates the balance between proliferation and neural differentiation through the Notch signalling pathway. Our findings suggest MeCP2 S421 phosphorylation may function as a general epigenetic switch accessible by different extracellular stimuli through different signalling pathways for regulating diverse biological functions in different cell types.

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Aurora kinase B is required for MeCP2 S421 phosphorylation in the aNPCs(a,b) Western blot analysis and quantification of the relative MeCP2 S421 phosphorylation level in aNPCs: 1) DMSO, 2) 36 hours of nocodazole (150ng/ml) treatment, 3) 24 hours of hesperadin (2 μM) treatment after pre-synchronization of the cells by nocodazole for 12 hours. (n=3 in each group) (c,d) Western blot analysis and quantification of the relative MeCP2 S421 phosphorylation level in EGFP-shRNA or AURKB-shRNA lentivirus infected aNPCs, which are treated with DMSO or nocodazole for 24 hours. (e,f) Western blot analysis reveals that MeCP2 and AURKB are co-immunoprecipitated reciprocally. (g) Western blot analysis reveals endogenous interaction between MeCP2 and AURKB in aNPCs. (h) In vitro kinase assay followed by Western blot demonstrates that AURKB can phosphorylate S421 on MeCP2. Numbers next to Western blots are molecular weight markers.
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Figure 2: Aurora kinase B is required for MeCP2 S421 phosphorylation in the aNPCs(a,b) Western blot analysis and quantification of the relative MeCP2 S421 phosphorylation level in aNPCs: 1) DMSO, 2) 36 hours of nocodazole (150ng/ml) treatment, 3) 24 hours of hesperadin (2 μM) treatment after pre-synchronization of the cells by nocodazole for 12 hours. (n=3 in each group) (c,d) Western blot analysis and quantification of the relative MeCP2 S421 phosphorylation level in EGFP-shRNA or AURKB-shRNA lentivirus infected aNPCs, which are treated with DMSO or nocodazole for 24 hours. (e,f) Western blot analysis reveals that MeCP2 and AURKB are co-immunoprecipitated reciprocally. (g) Western blot analysis reveals endogenous interaction between MeCP2 and AURKB in aNPCs. (h) In vitro kinase assay followed by Western blot demonstrates that AURKB can phosphorylate S421 on MeCP2. Numbers next to Western blots are molecular weight markers.

Mentions: Since the consensus sequence for the phosphorylation site of a CDK substrate, [serine/threonine]-proline-any amino acid-[lysine/arginine], is not found around S421, CDKs are unlikely to be the direct kinase for S421 in aNPCs. To search for the kinase that is directly responsible for phosphorylating S421 in aNPCs, we performed an in silico screen of consensus substrate sequences to identify a list of potential kinases, which was followed by a pharmacological screen in nocodazole-treated N2A cells. Aurora kinase B was the only candidate coming out of this two-step screen (Supplementary Fig. 2i). Consistent with previous reports that aurora kinase B is upregulated at the G2/M phase and downstream of CDKs17,18, we found that its level increased significantly in nocodazole-treated aNPCs, which was blocked by roscovitine (Fig. 1e). The phosphorylation level of serine 10 on histone 3, a known substrate of aurora kinase B19, closely followed the changes in the level of aurora kinase B in aNPCs (Fig. 1e). Hesperadin, a highly specific inhibitor of aurora kinase B, efficiently blocked cell cycle-linked S421 phosphorylation in aNPCs (Fig. 2a–b and Supplementary Fig. 2j–k), suggesting the aurora kinase B is required for S421 phosphorylation in these cells. To confirm this finding with an independent method, we generated lentivirus encoding shRNA specific for aurora kinase B (Supplementary Fig. 2l), and found it also significantly blocked cell cycle-linked S421 phosphorylation in aNPCs (Fig. 2c–d). As a negative control, a lentivirus encoding shRNA specific for EGFP had no effect on aurora kinase B level and S421 phosphorylation in aNPCs (Fig. 2c–d). To test whether MeCP2 and aurora kinase B physically interact with each other, we overexpressed epitope-tagged MeCP2 and aurora kinase B in Neuro2A cells and performed reciprocal co-immunoprecipitation followed by Western blot experiments. We found that flag-tagged aurora kinase B, but not flag-tagged EGFP, could pull down MeCP2 (Fig. 2e). Reversely, flag-tagged MeCP2, but not flag-tagged EGFP, could pull down aurora kinase B (Fig. 2f). Furthermore, the physical interaction between MeCP2 and aurora kinase B, as well as that between aurora kinase B and its known substrate-histone H3, can be detected in aNPCs arrested at the G2/M phase (Fig. 2g). Finally, when purified MeCP2 protein was incubated with aurora kinase B (but not an inactive form of the kinase-AURKB-DN) in the presence of ATP in an in vitro kinase assay, S421 was readily phosphorylated (Fig. 2h). Taken together, these results identify aurora kinase B as the direct kinase of MeCP2 S421 phosphorylation in aNPCs.


Cell cycle-linked MeCP2 phosphorylation modulates adult neurogenesis involving the Notch signalling pathway.

Li H, Zhong X, Chau KF, Santistevan NJ, Guo W, Kong G, Li X, Kadakia M, Masliah J, Chi J, Jin P, Zhang J, Zhao X, Chang Q - Nat Commun (2014)

Aurora kinase B is required for MeCP2 S421 phosphorylation in the aNPCs(a,b) Western blot analysis and quantification of the relative MeCP2 S421 phosphorylation level in aNPCs: 1) DMSO, 2) 36 hours of nocodazole (150ng/ml) treatment, 3) 24 hours of hesperadin (2 μM) treatment after pre-synchronization of the cells by nocodazole for 12 hours. (n=3 in each group) (c,d) Western blot analysis and quantification of the relative MeCP2 S421 phosphorylation level in EGFP-shRNA or AURKB-shRNA lentivirus infected aNPCs, which are treated with DMSO or nocodazole for 24 hours. (e,f) Western blot analysis reveals that MeCP2 and AURKB are co-immunoprecipitated reciprocally. (g) Western blot analysis reveals endogenous interaction between MeCP2 and AURKB in aNPCs. (h) In vitro kinase assay followed by Western blot demonstrates that AURKB can phosphorylate S421 on MeCP2. Numbers next to Western blots are molecular weight markers.
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Related In: Results  -  Collection

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Figure 2: Aurora kinase B is required for MeCP2 S421 phosphorylation in the aNPCs(a,b) Western blot analysis and quantification of the relative MeCP2 S421 phosphorylation level in aNPCs: 1) DMSO, 2) 36 hours of nocodazole (150ng/ml) treatment, 3) 24 hours of hesperadin (2 μM) treatment after pre-synchronization of the cells by nocodazole for 12 hours. (n=3 in each group) (c,d) Western blot analysis and quantification of the relative MeCP2 S421 phosphorylation level in EGFP-shRNA or AURKB-shRNA lentivirus infected aNPCs, which are treated with DMSO or nocodazole for 24 hours. (e,f) Western blot analysis reveals that MeCP2 and AURKB are co-immunoprecipitated reciprocally. (g) Western blot analysis reveals endogenous interaction between MeCP2 and AURKB in aNPCs. (h) In vitro kinase assay followed by Western blot demonstrates that AURKB can phosphorylate S421 on MeCP2. Numbers next to Western blots are molecular weight markers.
Mentions: Since the consensus sequence for the phosphorylation site of a CDK substrate, [serine/threonine]-proline-any amino acid-[lysine/arginine], is not found around S421, CDKs are unlikely to be the direct kinase for S421 in aNPCs. To search for the kinase that is directly responsible for phosphorylating S421 in aNPCs, we performed an in silico screen of consensus substrate sequences to identify a list of potential kinases, which was followed by a pharmacological screen in nocodazole-treated N2A cells. Aurora kinase B was the only candidate coming out of this two-step screen (Supplementary Fig. 2i). Consistent with previous reports that aurora kinase B is upregulated at the G2/M phase and downstream of CDKs17,18, we found that its level increased significantly in nocodazole-treated aNPCs, which was blocked by roscovitine (Fig. 1e). The phosphorylation level of serine 10 on histone 3, a known substrate of aurora kinase B19, closely followed the changes in the level of aurora kinase B in aNPCs (Fig. 1e). Hesperadin, a highly specific inhibitor of aurora kinase B, efficiently blocked cell cycle-linked S421 phosphorylation in aNPCs (Fig. 2a–b and Supplementary Fig. 2j–k), suggesting the aurora kinase B is required for S421 phosphorylation in these cells. To confirm this finding with an independent method, we generated lentivirus encoding shRNA specific for aurora kinase B (Supplementary Fig. 2l), and found it also significantly blocked cell cycle-linked S421 phosphorylation in aNPCs (Fig. 2c–d). As a negative control, a lentivirus encoding shRNA specific for EGFP had no effect on aurora kinase B level and S421 phosphorylation in aNPCs (Fig. 2c–d). To test whether MeCP2 and aurora kinase B physically interact with each other, we overexpressed epitope-tagged MeCP2 and aurora kinase B in Neuro2A cells and performed reciprocal co-immunoprecipitation followed by Western blot experiments. We found that flag-tagged aurora kinase B, but not flag-tagged EGFP, could pull down MeCP2 (Fig. 2e). Reversely, flag-tagged MeCP2, but not flag-tagged EGFP, could pull down aurora kinase B (Fig. 2f). Furthermore, the physical interaction between MeCP2 and aurora kinase B, as well as that between aurora kinase B and its known substrate-histone H3, can be detected in aNPCs arrested at the G2/M phase (Fig. 2g). Finally, when purified MeCP2 protein was incubated with aurora kinase B (but not an inactive form of the kinase-AURKB-DN) in the presence of ATP in an in vitro kinase assay, S421 was readily phosphorylated (Fig. 2h). Taken together, these results identify aurora kinase B as the direct kinase of MeCP2 S421 phosphorylation in aNPCs.

Bottom Line: Neuronal activity regulates the phosphorylation states at multiple sites on MeCP2 in postmitotic neurons.However, it is unknown whether phosphorylation at any of the previously identified sites on MeCP2 can be induced by signals other than neuronal activity in other cell types, and what functions MeCP2 phosphorylation may have in those contexts.Our findings suggest MeCP2 S421 phosphorylation may function as a general epigenetic switch accessible by different extracellular stimuli through different signalling pathways for regulating diverse biological functions in different cell types.

View Article: PubMed Central - PubMed

Affiliation: 1] Waisman Center, University of Wisconsin-Madison, Madison, Wisconsin 53705, USA [2] Genetics Training Program, University of Wisconsin-Madison, Madison, Wisconsin 53705, USA.

ABSTRACT
Neuronal activity regulates the phosphorylation states at multiple sites on MeCP2 in postmitotic neurons. The precise control of the phosphorylation status of MeCP2 in neurons is critical for the normal development and function of the mammalian brain. However, it is unknown whether phosphorylation at any of the previously identified sites on MeCP2 can be induced by signals other than neuronal activity in other cell types, and what functions MeCP2 phosphorylation may have in those contexts. Here we show that in neural progenitor cells isolated from the adult mouse hippocampus, cell cycle-linked phosphorylation at serine 421 on MeCP2 is directly regulated by aurora kinase B and modulates the balance between proliferation and neural differentiation through the Notch signalling pathway. Our findings suggest MeCP2 S421 phosphorylation may function as a general epigenetic switch accessible by different extracellular stimuli through different signalling pathways for regulating diverse biological functions in different cell types.

Show MeSH