[PubMed] [CrossRef] [Google Scholar] 4. study identifies a previously unfamiliar signaling pathway by which GP78 stimulates ERK activation via DUSP1 degradation to mediate EGFR-dependent malignancy cell proliferation and invasion. ubiquitination assay. Purified Flag-DUSP1-fused His tag and GST-GP78 proteins were mixed, followed by addition of E1, E2, ATP, and Ub, and then incubated at 30C for 30?min. Samples were resolved by SDS-PAGE and subjected to immunoblot analysis with anti-Flag and GP78 antibodies. (E) Recognition of DUSP1 ubiquitination site. HEK293T cells were transfected with HA-Ub and the crazy type (WT) or one of mutant constructs of DUSP1 for 24?h. The K230R, K280R, and K289R constructs have a single mutation, while the 3M create consists of all three mutations (K230R, K280R, and K289R). Monoubiquitinated DUSP1 levels were calculated based on the molecular people of DUSP1-v5 amino acids plus HA-Ub amino acids. (F) Effect of GP78 on monoubiquitination of the K280R DUSP1 mutant. HEK293T cells were transfected with K280R and GP78-myc for 48?h. The total cell lysates were immunoprecipitated with His antibody and analyzed by Western blotting with ubiquitin antibody. Figures to the left of the gels are kilodaltons. To confirm the part of GP78 in mediating DUSP1 ubiquitination inside a cell-free system, a glutathione DUSP1 ubiquitination assay was performed using both purified Rabbit Polyclonal to APLF GST-tagged GP78 C terminus and His-tagged Flag-DUSP1 in the presence of UbcH5B or Ubc7. Of notice, UbcH5B and Ubc7 were previously used for GP78 ubiquitination (2). As demonstrated in Fig. 2D, 2-HG (sodium salt) Flag-DUSP1 was ubiquitinated in the presence of GST-GP78, confirming that GP78 can act as an E3 ligase to result in DUSP1 ubiquitination. Next, we asked which specific lysine residue(s) on DUSP1 is the site for its ubiquitination. By analyzing its protein sequence with the algorithm UbPred (www.ubpred.org), we identified 10 lysine (K) residues on DUSP1: K57, K97, K122, K138, K192, K221, K230, 2-HG (sodium salt) K248, K280, and K289. In addition, a quantitative-proteomics approach showed that DUSP1 is frequently revised at K230, K280, and K289 (www.phosphosite.org). On the basis of these findings, we performed site-directed mutagenesis on DUSP1 to replace K230, K280, and K289 with arginine (R) and showed that a mutation in K230R did not impact DUSP1 poly- and monoubiquitination (Fig. 2E). In contrast, a mutation in K280R or K289R led to a significant decrease in DUSP1 polyubiquitination, and DUSP1 monoubiquitination was abolished in K280R but not in K289R. Consistently, DUSP1 mono- and polyubiquitination were completely abolished in the 3?M construct, which contains three mutated lysines (i.e., K230R, K280R, and K289R). Interestingly, GP78 cotransfection slightly induced K280R polyubiquitination without influencing its monoubiquitination (Fig. 2F). These data suggest that K280 and K289 are responsible for DUSP1 polyubiquitination and that K280 is definitely a priming site for DUSP1 ubiquitination, including its monoubiquitination. DUSP1 literally interacts with GP78. Promoting DUSP1 ubiquitination suggests that GP78 actually interacts with DUSP1. Consequently, we performed coimmunoprecipitation (co-IP) experiments with lysates of HEK293T cells transfected with pcDNA3-GP78-myc and pcDNA3-DUSP1-v5. Number 3B demonstrates GP78-myc coimmunoprecipitated with DUSP1-v5 when whole-cell lysates were incubated with V5 antibody. Reciprocally, co-IP with Myc antibody exposed that DUSP1-v5 coimmunoprecipitated with GP78-myc. In addition, we found that GP78 could interact with DUSP4 (data not demonstrated), 2-HG (sodium salt) another member of the DUSP family (18). We then queried the region that was responsible for this observed connection. GP78 offers four main practical domains, i.e., transmembrane, Ring, Cue, and G2BR domains (Fig. 3A). We generated two deletion constructs in pcDNA6-v5 that communicate either amino acids (aa) 1 to 239 or.
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