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. 2021 May 13;296:100739. doi: 10.1016/j.jbc.2021.100739

Figure 1.

Figure 1

TRAF4 promotes IRS-1 ubiquitination through K29 linkage.A, Myc-TRAF4 interacted with FLAG-IRS1 in transiently transfected 293T cells. A coimmunoprecipitation (co-IP) experiment was performed using an anti-Myc antibody for immunoprecipitation, followed by Western blot. B, endogenous IRS1 interacted with endogenous TRAF4 in MCF-7 cells. Shown is a co-IP experiment using a TRAF4-specific antibody or IgG control for immunoprecipitation. C, IGF-1 treatment increases the interaction between endogenous IRS-1 and TRAF4. MCF-7 cells were serum starved for 24 h followed by treatment with or without IGF-1 (100 ng/ml) for 10 min. Shown is a co-IP experiment using an IRS-1-specific antibody or IgG control for immunoprecipitation. D, recombinant human IRS1 protein was incubated with purified UBE1, UbcH5a, HA-tagged ubiquitin in the absence or presence of purified TRAF4 protein. An immunoprecipitation was then carried out using an IRS1-specific antibody followed by Western blot analysis using an HA-specific antibody to detect IRS-1 ubiquitination. E, TRAF4 but not its RING domain deletion mutant promotes IRS-1 ubiquitination. 293T cells were cotransfected with IRS-1, HA-Ub, and TRAF4 constructs as indicated. FLAG-IRS-1 was immunoprecipitated using an anti-FLAG antibody, and the ubiquitinated IRS-1 was visualized by Western blot using an anti-HA antibody. F, IGF-1 induced endogenous IRS-1 ubiquitination in cells. MCF-7 cells were serum starved for 24 h followed by treatment with or without IGF-1 (100 ng/ml) for 15 min. Immunoprecipitation was then performed using an anti-IRS1 antibody followed by Western blot for detection of IRS1 ubiquitination using an anti-ubiquitin antibody. G, TRAF4 knockdown reduced IGF-1-induced IRS-1 ubiquitination. TRAF4 was knocked down in MCF-7 cells using a mixture of control siRNAs or TRAF4 siRNAs (top). After 48 h, cells were serum starved for 24 h followed by treatment with or without IGF-1 (100 ng/ml) for 15 min. Immunoprecipitation was performed using an anti-IRS1 antibody followed by Western blot for detection of IRS-1 ubiquitination using an anti-ubiquitin antibody (bottom). H, TRAF4 mediates IRS-1 ubiquitination through the K29 linkage. Ubiquitination assay was performed in 293T cells after transfection with FLAG-IRS-1, TRAF4, and WT or ubiquitin mutants. K6–K63 represent the ubiquitin mutants with all lysines mutated except the indicated lysine, whereas WT indicates wildtype ubiquitin.