The ubiquitin-associated domain of hPLIC-2 interacts with the proteasome

Abstract

The ubiquitin-like hPLIC proteins can associate with proteasomes, and hPLIC overexpression can specifically interfere with ubiquitin-mediated proteolysis (Kleijnen et al., 2000). Because the hPLIC proteins can also interact with certain E3 ubiquitin protein ligases, they may provide a link between the ubiquitination and proteasomal degradation machineries. The amino-terminal ubiquitin-like (ubl) domain is a proteasome-binding domain. Herein, we report that there is a second proteasome-binding domain in hPLIC-2: the carboxyl-terminal ubiquitin-associated (uba) domain. Coimmunoprecipitation experiments of wild-type and mutant hPLIC proteins revealed that the ubl and uba domains each contribute independently to hPLIC-2-proteasome binding. There is specificity for the interaction of the hPLIC-2 uba domain with proteasomes, because uba domains from several other proteins failed to bind proteasomes. Furthermore, the binding of uba domains to polyubiquitinated proteins does not seem to be sufficient for the proteasome binding. Finally, the uba domain is necessary for the ability of full-length hPLIC-2 to interfere with the ubiquitin-mediated proteolysis of p53. The PLIC uba domain has been reported to bind and affect the functions of proteins such as GABAA receptor and presenilins. It is possible that the function of these proteins may be regulated or mediated through proteasomal degradation pathways.

Figure 2.

The hPLIC-2 uba domain can bind proteasomes in a GST pull-down experiment. (A) List of GST-hPLIC-2 fusion constructs. (B) GST alone (lane 1) and the indicated GST-hPLIC-2 fusion proteins (lanes 2–8) were bacterially expressed, purified, bound to glutathione-Sepharose beads, and analyzed by Coomassie staining (M, marker lane). (C) The beads were incubated with HeLa cell lysate, pulled down, and assayed for proteasome binding by blotting for the α2 proteasomal core subunit.

Figure 3.

Specificity of the hPLIC-2 uba domain–proteasome interaction. (A) The hPLIC-2 uba domain but not the c-cbl uba domain can interact with proteasomes. GST fusions of the uba domains of hPLIC-2 (lane 2) and c-cbl (lane 3) were compared for their ability to pull down proteasomes. GST fusions were mixed with HeLa cell lysate, and proteasome proteins were analyzed by Western blotting for the α2 proteasomal core subunit. (B) Uba domain binding to polyubiquitin chains does not correlate with proteasome pull down. The indicated GST fusion proteins were tested for their ability to pull down polyubiquitinated proteins from HeLa cell lysate, in the same experimental setup as described above. The ability of these GST constructs to pull down proteasomes was tested in a separate experiment (bottom). (C) The ubl and uba domains of hPLIC-2 fused to GST can bind purified yeast proteasomes, whereas the uba domain of c-cbl cannot. Proteasome components precipitating with the GST-fusion proteins were detected by Western blotting for the Rpt2 proteasomal cap subunit. (D) Both the ubl and uba domains of hPLIC-2 can bind to in vitro synthesized proteasomal cap subunit S5a. In vitro translated ³⁵S-labeled S5a was mixed with the indicated GST fusion proteins, pulled down, analyzed by SDS-PAGE, and exposed to film.

Figure 5.

The uba domain is required for interfering with the degradation of p53 in HeLa cells upon hPLIC-2 overexpression. (A) List of expression vectors used. (B) FLAG-tagged hPLIC-2, full-length or lacking the uba domain, either wild type or with the I75A/A77S/H99A triple mutation in the ubl domain, was transfected into HeLa cells. Total cell lysates were analyzed by Western blotting for p53 levels. hPLIC expression levels were checked with the FLAG antibody.

Figure 1.

The hPLIC-2 I75A/A77S/H99A triple mutation in the ubl domain retains binding to the proteasome. (A) Schematic representation of hPLIC-2 depicting the amino-terminal ubl domain (shaded), the collagen-like domain (CL) (hatched), and the carboxyl-terminal uba domain (dotted). (B) FLAG-tagged, full-length, wild-type hPLIC-2 (lane 3), full-length hPLIC-2 containing a I75A/A77S/H99A triple mutation in the ubl domain (lane 2), or vector alone (lane 1) was transfected into HeLa cells and treated with proteasome inhibitor before cell lysis. hPLIC-2 was immunoprecipitated and assayed by Western blotting with both a FLAG tag antibody and the α2 proteasomal core subunit antibody. LC and HC depict the IgG light chain and heavy chain, respectively.

Figure 4.

Both the ubl domain and the uba domain contribute to the interaction of hPLIC-2 with the proteasome. (A) List of expression vectors used. (B) The indicated constructs were transfected into HeLa cells and treated with proteasome inhibitor before cell lysis. Coimmunoprecipitation of proteasomes with hPLIC-2 was assayed by blotting for the α2 proteasomal core subunit and the FLAG tag. LC and HC depict the IgG light chain and heavy chain, respectively.