Large-scale analysis of lysine SUMOylation by SUMO remnant immunoaffinity profiling.
Small ubiquitin-related modifiers (SUMO) are evolutionarily conserved ubiquitin-like proteins that regulate several cellular processes including cell cycle progression, intracellular trafficking, protein degradation and apoptosis. Despite the importance of protein SUMOylation in different biological pathways, the global identification of acceptor sites in complex cell extracts remains a challenge. Here we generate a monoclonal antibody that enriches for peptides containing SUMO remnant chains following tryptic digestion. We identify 954 SUMO3-modified lysine residues on 538 proteins and profile by quantitative proteomics the dynamic changes of protein SUMOylation following proteasome inhibition. More than 86% of these SUMOylation sites have not been reported previously, including 5 sites on the tumour suppressor parafibromin (CDC73). The modification of CDC73 at K136 affects its nuclear retention within PML nuclear bodies on proteasome inhibition. In contrast, a CDC73 K136R mutant translocates to the cytoplasm under the same conditions, further demonstrating the effectiveness of our method to characterize the dynamics of lysine SUMOylation.
1] Institute for Research in Immunology and Cancer, Université de Montréal, PO Box 6128, Station Centre-ville, Montréal, Québec, Canada H3C 3J7 [2] Department of Chemistry, Université de Montréal, PO Box 6128, Station Centre-ville, Montréal, Québec, Canada H3C 3J7.
Frédéric
F
Lamoliatte
Institute for Research in Immunology and Cancer, Université de Montréal, PO Box 6128, Station Centre-ville, Montréal, Québec, Canada H3C 3J7.
Danielle
D
Caron
Institute for Research in Immunology and Cancer, Université de Montréal, PO Box 6128, Station Centre-ville, Montréal, Québec, Canada H3C 3J7.
Chantal
C
Durette
1] Institute for Research in Immunology and Cancer, Université de Montréal, PO Box 6128, Station Centre-ville, Montréal, Québec, Canada H3C 3J7 [2] Department of Biochemistry, Université de Montréal, PO Box 6128, Station Centre-ville, Montréal, Québec, Canada H3C 3J7.
Louiza
L
Mahrouche
Université Paris Descartes, INSERM UMR-S1124, Paris 75006, France.
Mohamed Ali
MA
Maroui
Institute for Research in Immunology and Cancer, Université de Montréal, PO Box 6128, Station Centre-ville, Montréal, Québec, Canada H3C 3J7.
Olivier
O
Caron-Lizotte
Institute for Research in Immunology and Cancer, Université de Montréal, PO Box 6128, Station Centre-ville, Montréal, Québec, Canada H3C 3J7.
Eric
E
Bonneil
Université Paris Descartes, INSERM UMR-S1124, Paris 75006, France.
Mounira K
MK
Chelbi-Alix
1] Institute for Research in Immunology and Cancer, Université de Montréal, PO Box 6128, Station Centre-ville, Montréal, Québec, Canada H3C 3J7 [2] Department of Chemistry, Université de Montréal, PO Box 6128, Station Centre-ville, Montréal, Québec, Canada H3C 3J7 [3] Department of Biochemistry, Université de Montréal, PO Box 6128, Station Centre-ville, Montréal, Québec, Canada H3C 3J7.
Pierre
P
Thibault
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002846:Chromatography, Affinity; D008401:Gas Chromatography-Mass Spectrometry; D057809:HEK293 Cells; D006801:Humans; D006825:Hybridomas / Q000378:metabolism; D008239:Lysine / Q000378:metabolism*; D010455:Peptides / Q000276:immunology / Q000378:metabolism; D020543:Proteome / Q000378:metabolism; D011817:Rabbits; D025841:Small Ubiquitin-Related Modifier Proteins / Q000276:immunology / Q000378:metabolism*; D058207:Sumoylation; D025521:Tumor Suppressor Proteins / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; C454900:CDC73 protein, human; D010455:Peptides; D020543:Proteome; D025841:Small Ubiquitin-Related Modifier Proteins; D025521:Tumor Suppressor Proteins; D008239:Lysine