ASB2 proteins are E3 ubiquitin (Ub) ligases that ubiquitinate filamins. There are two ASB2 splice variants, ASB2 alpha and ASB2 beta. ASB2 beta has a ubiquitin-binding motif (UIM) at the N-terminal region but ASB2 alpha does not. Here, we provide the first evidence that ASB2 beta but not ASB2 alpha is monoubiquitinated and that this monoubiquitination involves the UIM. Myc-tagged ASB2 beta and hemagglutinin (HA)-tagged Ub were co-expressed in HEK293 cells using the pCMV expression vector. Immunoprecipitation with an anti-Myc antibody followed by immunoblotting with anti-Myc and anti-HA antibodies showed an additional ASB2 beta protein band that had both a Myc and a HA tag. The molecular weight of this protein was larger than that of ASB2 beta, and the difference in molecular weight between these two proteins corresponded to the molecular weight of monoubiquitin, strongly implying that monoubiquitinated ASB2 beta is produced in cells. ASB2 beta with mutations in the UIM motif; either Glu.Asp.Glu27-29Ala.Ala.Ala mutations (ASB2 beta M1) or a Ser38Ala mutation, (ASB2 beta M2) were not monoubiquitinated, suggesting the importance of the UIM for ASB2 beta monoubiquitination. Furthermore, an ASB2 beta mutant that lacked a SOCS box (ASB2 beta Delta C) and did not show E3 Ub ligase activity was monoubiquitinated to the same extent as the wild-type ASB2 beta. In contrast, an ASB2 beta mutant that lacked the UIM-containing domain (ASB2 beta Delta N) was not monoubiquitinated. These results suggest that ASB2 beta but not ASB2 alpha might be monoubiquitinated and that the ASB2 beta UIM motif, but not its E3 Ub ligase activity, plays a pivotal role in this monoubiquitination. (C) 2012 Published by Elsevier Inc.