The ASB2ß Ubiquitin-interacting motif is involved in its monoubiquitination.

ASB2 proteins are E3 ubiquitin (Ub) ligases that ubiquitinate filamins. There are two ASB2 splice variants, ASB2α and ASB2ß. ASB2ß has a ubiquitin-binding motif (UIM) at the N-terminal region but ASB2α does not. Here, we provide the first evidence that ASB2ß but not ASB2α is monoubiquitinated and that this monoubiquitination involves the UIM. Myc-tagged ASB2ß 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 ASB2ß protein band that had both a Myc and a HA tag. The molecular weight of this protein was larger than that of ASB2ß, and the difference in molecular weight between these two proteins corresponded to the molecular weight of monoubiquitin, strongly implying that monoubiquitinated ASB2ß is produced in cells. ASB2ß with mutations in the UIM motif; either Glu·Asp·Glu27-29Ala·Ala·Ala mutations (ASB2ß M1) or a Ser38Ala mutation, (ASB2ß M2) were not monoubiquitinated, suggesting the importance of the UIM for ASB2ß monoubiquitination. Furthermore, an ASB2ß mutant that lacked a SOCS box (ASB2ß ΔC) and did not show E3 Ub ligase activity was monoubiquitinated to the same extent as the wild-type ASB2ß. In contrast, an ASB2ß mutant that lacked the UIM-containing domain (ASB2ß ΔN) was not monoubiquitinated. These results suggest that ASB2ß but not ASB2α might be monoubiquitinated and that the ASB2ß UIM motif, but not its E3 Ub ligase activity, plays a pivotal role in this monoubiquitination.

Comprehensive trapping of polyubiquitinated proteins using the UIM peptide of ASB2a.

An alternative splicing variant of E3 ubiquitin ligase ASB2, termed ASB2a, has a distinct N-terminal sequence containing a ubiquitin-interacting motif (UIM) consensus sequence. Examination of the minimal essential region for binding to polyubiquitinated proteins indicated that the UIM consensus sequence (residues 26-41) alone is not enough, and that amino acids 12-41 from the N-terminus of ASB2a is essential for binding. ASB2a(12-41) peptide was chemically synthesized and coupled to Sepharose 4B via disulfide bonds. This ASB2a(12-41) peptide-coupled affinity resin bound both K48- and K63-linked polyubiquitinated proteins in cell lysates and comprehensively captured polyubiquitinated proteins, including polyubiquitinated ß-catenin, I-κB, and EGF receptor, which were eluted with 2-mercaptoethanol under non-denaturing conditions. These results indicate that this UIM affinity purification (designated as ubiquitin-trapping) is a useful method to discover polyubiquitinated proteins and their associated proteins.

ASB proteins interact with Cullin5 and Rbx2 to form E3 ubiquitin ligase complexes.

The ankyrin repeat and SOCS box (ASB) family is composed of 18 proteins from ASB1 to ASB18 and belongs to the suppressor of cytokine signaling (SOCS) box protein superfamily. ASB2 was recently shown to interact with a certain Cul-Rbx module to form an E3 ubiquitin (Ub) ligase complex, but the functional composition of the ASB-containing E3 Ub ligase complexes remains to be characterized. Here, we show that ASB proteins interact with Cul5-Rbx2 but neither Cul2 nor Rbx1 in cells. Mutational analysis revealed that the highly conserved amino acid sequences of the BC box and Cul5 box in the SOCS box of ASB proteins were essential for the interaction with Cul5-Rbx2. Although ASB proteins show slight divergences from the consensus sequences of the BC box and Cul5 box, all five tested ASB proteins bound to Cul5-Rbx2. Furthermore, all three tested ASB complexes containing Cul5-Rbx2 were found to have E3 Ub ligase activity. These findings suggest that the ASB family proteins interact with Cul5-Rbx2 to form E3 Ub ligases and play significant roles via a ubiquitination-mediated pathway.