Figure 6.

Lysine 446 of HSPA5 is a site of MUL1-mediated K48-linked ubiquitination. (A) HSPA5 interacted with MUL1. At 24 h after cotransfection with the indicated plasmids, FaDu cells were treated with MG132 (10 μM) for 8 h before cell harvest and then subjected to immunoprecipitation with an anti-GFP antibody, followed by western blot with the indicated antibodies. (B) MUL1 induced K48-linked ubiquitination of HSPA5. FaDu cells were cotransfected with plasmids as indicated. At 24 h after cotransfection, cells were treated with MG132 (10 μM) for 8 h and further subjected to ubiquitination assay. (C) K446 is a putative ubiquitination site for MUL1. Each indicated plasmid was transfected into FaDu cells together with or without EGFP-MUL1 and 24 h later, cells were treated with MG132 (10 μM) for 8 h and further subjected to Ni-NTA His affinity-isolation ubiquitination assay. (D) The effect of MUL1 on HSPA5 protein stability was inhibited in the HSPA5^K446R mutant, compared to wild-type HSPA5. K446R mutant was inhibited MUL1-dependent protein stability compared with wild type of HSPA5. WT HSPA5-MYC-His or HSPA5^K446R-MYC-His plasmids were transfected into FaDu cells together with or without EGFP-MUL1 dose dependently and 24 h later, the level of each indicated protein was determined by western blot. (E) The half-life of the K446R mutant protein was compared with the wild type of HSPA5. WT HSPA5-MYC-His or HSPA5^K446R-MYC-His plasmids were transfected into BEAS-2B cells and 24 h later, treatment with CHX (20 μg/ml) was included for each indicated time. Endogenous MUL1 or exogenous HSPA5 were detected using anti-MUL1 or anti-MYC antibodies. (F) K446R did not induce ubiquitination from the NTS. Cells transfected with each indicated plasmid, were treated with NTS for 24 h and the ubiquitinated form of HSPA5 was detected by Ni-NTA His affinity isolation assay.