Figure 8.
ACK1 is an ubiquitin-binding protein. (A) Alignment of the putative ACK1-Uba domain with the Uba domains of Cbl-b, c-Cbl, and Rad23. The conserved residues are boxed. (B) The Uba domain of ACK1 interacts with proteasomes and ubiquitinated proteins. The GST-ACK1-Uba, GST-Cbl-b-Uba, GST-c-Cbl-Uba, or GST-Hrs-UIM domain was expressed in E. coli and purified by affinity pulldown with glutathione-agarose beads. The bead-bound GST-fusion proteins (∼20 μg) were incubated with COS7 cell lysates (∼0.5 mg). The coprecipitated proteins were subjected to SDS-PAGE electrophoresis and immunoblotting with anti-proteasome S1 (top panel), anti-proteasome S10B (second panel), or anti-ubiquitin antibody (third panel). The loaded GST-ACK1-Uba, GST-Cbl-b-Uba, GST-c-Cbl-Uba, or GST-Hrs-UIM proteins were shown in the bottom panel by Coomassie blue staining. (C) Both mono-ubiquitin and poly-ubiquitin bind to ACK1. The bead-bound GST mono-ubiquitin or poly-ubiquitin (Ubi)4 was incubated with Myc-tagged ACK1-overexpressed COS7 cell lysates. The coprecipitated Myc-ACK1 was detected by immunoblotting with an anti-Myc antibody (top panel). (D) ACK1 has a high binding affinity to poly-ubiquitin. Myc-tagged ACK1, HA-tagged c-Cbl, or Hrs was transfected into COS7 cells for expression. The cell lysates were incubated with bead-bound GST-(Ubi)4. The GST-(Ubi)4 beads were precipitated by centrifugation. The coprecipitated ACK1, c-Cbl, or Hrs was detected by immunoblotting with anti-Myc or anti-HA antibodies (top panel). The input amount of ACK1, c-Cbl, or Hrs in lysates was shown in the bottom panel.