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. 2008 May 9;436(2):239-44.
doi: 10.1016/j.neulet.2008.03.029. Epub 2008 Mar 15.

Analysis of SUMO-1 modification of neuronal proteins containing consensus SUMOylation motifs

Kevin A Wilkinson  1 , Atsushi Nishimune, Jeremy M Henley
Affiliations

Analysis of SUMO-1 modification of neuronal proteins containing consensus SUMOylation motifs

Kevin A Wilkinson et al. Neurosci Lett. .

Abstract

SUMOylation is emerging as an important mechanism for modulating protein function in many cell types. A large variety of proteins have been proposed as SUMO targets based on the presence of a consensus SUMOylation core motif (Psi-K-x-D/E). In neurons these include multiple synaptic proteins but it has not been established whether proteins carrying this motif are SUMOylated either in vitro or in vivo. Here we use a bacterial SUMOylation assay to systematically test for SUMO-1 modification of a selection of neuronal proteins containing one or more amino acid sequences predicted as high-probability SUMOylation sites in computer-based searches. Of the 39 proteins analysed only 14 sites were posttranslationally modified by SUMO-1, including the group III metabotropic glutamate receptors and the kainate receptor subunit GluR7. These results identify new candidate proteins that may be involved in the SUMO regulation of synaptic activity and also demonstrate that the presence of the Psi-K-x-D/E motif is not sufficient to indicate that a protein can be SUMOylated in this bacterial system.

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Figures

Fig. 1
Fig. 1
Efficient SUMOylation of known substrate proteins. Vectors encoding GST, or known SUMOylation substrates were transformed into bacteria with or without the SUMOylation plasmid (indicated by ‘+’ or ‘−’). Crude bacterial lysates were then subjected to Western blotting for GST or 6×His, as appropriate. SUMOylated species are indicated by an arrow. (A) GST alone, (B) GST-tagged PML (residues 485–495), (C) 6×His-tagged RanGAP (entire C2 domain), (D) GST-tagged GluR6a (intracellular C-terminus), (E) GST-p53 (full length), (F) GST-p53 was purified on glutathione beads and probed for GST (upper panel) or SUMO-1 (lower panel), confirming the band shift seen in (E) is due to SUMOylation of p53.
Fig. 2
Fig. 2
Proteins identified as SUMOylation substrates. (A) The intracellular C-termini of the kainate receptor subunits GluR5–7 were expressed as GST-fusion proteins in bacteria with (+) or without (−) the SUMOylation plasmid. Crude bacterial lysates were Western blotting for GST. (B) Proteins showing a band shift when co-expressed with the SUMOylation plasmid in (A) were purified on glutathione beads and subjected to western blotting for GST and SUMO-1, to confirm the band shift seen corresponded to SUMO-1 modification. (C) GST-fusions of mGluR intracellular C-termini. (D) GST-mGluR C-termini showing a band shift in (C) were purified and Western blotting for GST and SUMO-1.
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