Function Of HPLIC-2 In Ubiquitin-Proteasome Pathway And The Interaction Between HPLIC-2 And C-Abl

Human PLIC-2(hPLIC-2), the homologues of yeast Dsk2 and mouse PLICs (proteins link IAP and cytoskeleton, PLICs), contains an amino-terminal ubiquitin-like (ubl) domain and a carboxyl-terminal ubiquitin-associated (uba) domain. The ubiquitin-like hPLIC-2 protein can associate with proteasomes, and hPLIC-2 overexpression can specifically interfere with ubiquitin-mediated proteolysis. Because hPLIC-2 can also interact with certain E3 ubiquitin protein ligases, they may provide a link between the ubiquitination and proteasomal degradation machineries. However, the concrete function of hPLIC-2 in ubiquitin- proteasome pathway is unclear. It was shown by the yeast two-hybrid experiment using full length c-Abl (cellular-Abelson gene)as bait protein that hPLIC-2 may be one of the potential c-Abl. c-Abl, the ubiquitously expressed non-receptor tyrosine kinase, can inhibit the activity of proteasome. In this experiment, the function of hPLIC-2 in the ubiquitin- proteasome pathway and the interaction between hPLIC-2 and c-Abl were studied.Molecular biologic technology, such as RNA interference, cell transfection,protein purification, immunoblot, immunoprecipitation, were applied to this study. Firstly, a cell line in which endogenous hPLIC-2 was stably inhibited have been established. Anti-hPLIC-2 polyclonal antibody was prepared after expressing GST-hPLIC-2 fusion protein. Further study by Immunoblot and Immunoprecipitation authenticated that hPLIC-2 overexpression can advance total protein ubiquitination in vivo. While, when hPLIC-2 was inhibited, the degree of ubiquitination in vivo decreased clearly. The amount of hPLIC-2 binding ubiquitinated proteins was increased in the presence of c-Abl. The result suggested that hPLIC-2 and c-Abl act in concert to advance total protein ubiquitination in vivo. Isotopic...