A variety of transcription factors are known to be modified by sumoylation (Elk-1, androgen receptor, c-Myb, AP-2, c-Jun and p53). In this study (Degerny C. et al. 2005 J. Biol. Chem. 280: 24330), we described the SUMO modification of ERM, a transcription factor of the Ets family, which is deregulated in metastatic human breast cancer cells and Neu-induced mouse mammary tumors. Four lysine residues at positions 89, 263, 293, and 350 are modified by SUMO. Sumoylation at these sites causes inhibition of ERM-dependent transcription without altering the subcellular localization, stability, and DNA-binding activity of ERM. ERM transcriptional activity has been previously described to be regulated by phosphorylation. This study shows that sumoylation also inhibits ERM transcription-enhancing activity.

Abgent provided our lab with antibodies against SUMO-1 (product Cat.# AP1222a) and SUMO-2/3 (product Cat.# AP1224a). We induced sumoylation of ERM by oxidative stress in presence of hydrogen peroxide in COS-7 cells expressing recombinant Flag-tagged ERM or Flag-tagged KR¹²³⁴⁵, an ERM protein mutated at all five sumoylation consensus sites. Upon immunoprecipitation of proteins with an anti-Flag antibody, the ERM, SUMO-1-modified ERM, and SUMO-2/3-modified ERM proteins were detected by western blotting with the respective antibody. The proteins migrating slower than ERM were identified as SUMO-2/3-modified ERM proteins. This experiment demonstrated that ERM can be sumoylated in vivo under stress by SUMO-2 or SUMO-3 proteins.

Figure legend: COS-7 cells were transfected for 24 hrs with a plasmid expressing FLAG-ERM (left panels) or FLAG-ERM KR¹²³⁴⁵ (right panels). Untreated (-) and H₂O₂-treated (+) cells were collected for immunoblot analysis. Top panels: cell lysates probed by western blot (WB) with an anti-ERM antibody. Center panels: cell lysates immunoprecipitated (IP) with an anti-FLAG antibody followed by WB with AP1222a SUMO-1 antibody. Bottom panels: cell lysates immunoprecipitated with an anti-FLAG antibody followed by WB with AP1224a SUMO-2/3 antibody. (*) represents immunoprecipitated ERM-like forms recognized by anti-SUMO antibodies.