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A molecular basis for phosphorylation-dependent SUMO conjugation by the E2 UBC9

Nature Structural & Molecular Biology volume 16, pages 945–952 (2009)Cite this article

Abstract

Phosphorylation and small ubiquitin-like modifier (SUMO) conjugation contribute to the spatial and temporal regulation of substrates containing phosphorylation-dependent SUMO consensus motifs (PDSMs). Myocyte-enhancement factor 2 (MEF2) is a transcription factor and PDSM substrate whose modification by SUMO drives postsynaptic dendritic differentiation. NMR analysis revealed that the human SUMO E2 interacted with model substrates for phosphorylated and nonphosphorylated MEF2 in similar extended conformations. Mutational and biochemical analysis identified a basic E2 surface that enhanced SUMO conjugation to phosphorylated PDSM substrates MEF2 and heat-shock transcription factor 1 (HSF1), but not to nonphosphorylated MEF2 or HSF1, nor the non-PDSM substrate p53. Mutant ubiquitin-conjugating enzyme UBC9 isoforms defective in promoting SUMO conjugation to phosphorylated MEF2 in vitro and in vivo also impair postsynaptic differentiation in organotypic cerebellar slices. These data support an E2-dependent mechanism that underlies phosphorylation-dependent SUMO conjugation in pathways that range from the heat-shock response to nuclear hormone signaling to brain development.

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Figure 1: Phosphorylation-dependent SUMO conjugation is mediated by the SUMO E2.
Figure 2: Model for PDSM recognition by the E2 UBC9.
Figure 3: Kinetic and mutational analyses for UBC9 amino acid residues involved in PDSM discrimination.
Figure 4: UBC9 Lys65 is important for PDSM discrimination of HSF1 but not for the non-PDSM substrate p53.
Figure 5: Amino acid side chains that constitute the basic surface on UBC9 are important for PDSM discrimination of MEF2 in vivo.
Figure 6: UBC9 mutants deficient in PDSM discrimination are also deficient for dendritic claw differentiation in the cerebellar cortex.

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Acknowledgements

We thank the NMR staff, particularly K. Dutta, at the New York Structural Biology Center (NYSBC) for assistance in NMR data collection and processing. We also thank A. Shalizi and A. Yunus for helpful discussions, and N. Arango for help with 293T transfection assays. NMR resources at NYSBC are supported by the US National Institutes of Health (NIH) grant P41 GM66354. The work was supported in part by NIH grants GM075695 (A.D.C.), NS041021 (A.B.) and GM065872 (C.D.L.).

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Authors and Affiliations

  1. Program in Structural Biology, Sloan-Kettering Institute, New York, New York, USA

    Firaz Mohideen, Allan D Capili & Christopher D Lima

  2. Department of Pathology, Harvard Medical School, New Research Building, Boston, Massachusetts, USA

    Parizad M Bilimoria, Tomoko Yamada & Azad Bonni

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  1. Firaz Mohideen
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Contributions

F.M. designed, performed and interpreted the experiments in Figures 1,2,3,4,5 and in the Supplementary Information, except for NMR experiments; A.D.C. designed, performed and interpreted NMR experiments; P.M.B. and T.Y. designed, performed and interpreted the experiments in Figure 6; A.B. supervised the research by P.M.B. and T.Y. and made contributions intellectually and to manuscript editing; C.D.L. supervised F.M. and A.D.C., designed experiments and interpreted data; F.M. and C.D.L. wrote the manuscript.

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Correspondence to Christopher D Lima.

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Mohideen, F., Capili, A., Bilimoria, P. et al. A molecular basis for phosphorylation-dependent SUMO conjugation by the E2 UBC9. Nat Struct Mol Biol 16, 945–952 (2009). https://doi.org/10.1038/nsmb.1648

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