Tob belongs to the anti-proliferative Tob/BIG protein family. The expression level of Tob family proteins is strictly regulated both transcriptionally and through post-translational modification. Ubiquitin (Ub)/proteosome-dependent degradation of lob family proteins is critical in controlling cell cycle progression and DNA damage responses. Various Ub ligases (E3s) are responsible for degradation of lob protein. Here, we show that lob family proteins undergo monoubiquitination even in the absence of E3s in vitro. Determination of the ubiquitination site(s) in lob by mass spectrometric analysis revealed that two lysine residues (Lys48 and Lys63) located in lob/BTG homology domain are ubiquitinated. A mutant lob, in which both Lys48 and Lys63 are substituted with alanine, is more strongly polyubiquitinated than wild-type lob in vivo. These data suggest that monoubiquitination of lob family proteins confers resistance against polyubiquitination, which targets proteins for degradation. The strategy for regulating the stability of lob family proteins suggests a novel role for monoubiquitination. (C) 2011 Elsevier Inc. All rights reserved.