Fig 1: TRAF4 regulates the interaction of IRS-1 with IGFR and IRS-1 tyrosine phosphorylation upon IGF-1 stimulation.A, TRAF4 overexpression increased IGF-1-induced IRS-1-IGFR interaction and IRS-1 tyrosine phosphorylation. MCF-7 cells were transfected with FLAG-IRS-1 with and without TRAF4, serum starved for 24 h, and then treated with or without IGF-1 (100 ng/ml) for 15 min. Immunoprecipitation was performed using an anti-FLAG antibody followed by Western blot (left). TRAF4 and FLAG-IRS-1 overexpression and endogenous expression of indicated proteins were determined by Western blot. GAPDH was used as internal control (right). B, TRAF4 does not alter IRS-1 membrane recruitment in the presence of IGF-1. MCF-7 cells were transfected with vector control or TRAF4 expression plasmid, serum starved, and then stimulated with IGF-1 for 10 min before harvesting. Cytosol and membrane fractions were prepared using a Mem-PER plus membrane protein extraction kit. Western blot was performed to determine the distribution of IRS-1 in cytosol and membrane fraction using anti-IRS1 antibody. GAPDH was used as loading control for cytosol and whole-cell lysate fraction, whereas E-cadherin was used as loading control for the membrane fraction. C, TRAF4 does not alter the protein level and the phosphorylation level of IGF-1Rβ. MCF-7 cells were transfected with either vector control or TRAF4 plasmids. These cells were serum starved for 24 h followed by treatment with or without IGF-1 (100 ng/ml) for 5 min. Cell lysate was prepared followed by Western blot to detect phosphorylation levels of IGF-1Rβ. D, deletion of TRAF4 abolished IGF-1-induced interaction between IRS-1 and IGFR. MCF-7 cells were transfected with a pool of two different siRNAs against TRAF4 or control siRNA. MCF-7 cells were then 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-IRS-1 antibody followed by Western blot (left). TRAF4 knockdown efficiency and endogenous expression of indicated proteins were determined by Western blot. GAPDH was used as internal control (right).
Fig 2: TRAF4 regulates IRS-1-mediated IGF-1 signaling.A, depletion of TRAF4 or IRS-1 inhibits IGF-1-induced downstream ERK and AKT phosphorylation. IRS-1 or TRAF4 was depleted in MCF-7 cells using a pool of two different siRNAs against IRS-1 or TRAF4, respectively. MCF-7 cells were then serum starved for 24 h and treated with or without IGF-1 (100 ng/ml) for 15 min. Gene knockdown efficiency and ERK and AKT phosphorylation were determined by Western blot. B, depletion of TRAF4 or IRS-1 inhibits MCF-7 cell proliferation. TRAF4 or IRS-1 was knocked down using two different TRAF4 or IRS-1 siRNAs, respectively, or transfected with control siRNA in MCF-7 cells. Cell proliferation was determined for indicated period of time using MTS assay. ∗p < 0.05 by two-way ANOVA with Tukey’s multiple comparisons test. Data are presented as mean ± SD. C, IRS-1 or TRAF4 depletion reduced IGF-1-induced cell proliferation. These MCF-7 cells transfected with control, IRS-1 or TRAF4 siRNA were serum starved and treated with or without IGF-1 followed by cell proliferation assay. Cell proliferation was determined for indicated periods using an MTS assay. ∗p < 0.05 by two-way ANOVA with Tukey’s multiple comparisons test. Data are presented as mean ± SD.
Fig 3: 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.
Fig 4: Mutation of TRAF4-targeted IRS-1 ubiquitination sites inhibits IGF-1 signaling and TRAF4-promoted IRS-1 tyrosine phosphorylation.A, IRS-1 ubiquitination mutant failed to induce downstream ERK and AKT phosphorylation upon IGF-1 stimulation. MCF-7 cells were transfected either with control vector, FLAG-IRS-1 WT or mutant IRS-1 (K1186_1189R). MCF-7 cells were then serum starved for 24 h and treated with or without IGF-1 (100 ng/ml) for 15 min. Western blot was performed to determine the level of ERK and AKT phosphorylation. B, mutation of K1186 and K1189 of IRS-1 inhibits TRAF4-promoted IRS-1 tyrosine phosphorylation and its interaction with IGFR. 293T cells were cotransfected with FLAG-tagged wildtype or mutant IRS-1 in the absence or presence of TRAF4. The cells were then serum starved for 24 h and treated with IGF-1 (100 ng/ml) for 15 min. The wildtype or mutant IRS-1 was then immunoprecipitated using an anti-FLAG antibody. Western blot was then performed to determine IRS-1 phosphorylation level and its interaction with IGFR (top). Overexpression of TRAF4, IRS-1 WT, and mutant was determined by Western blot. GAPDH was used as an internal control (bottom). C, IRS-1 wildtype but not the ubiquitination mutant rescued the effect of siIRS-1 on cell proliferation. Shown is a cell proliferation assay in MCF-7 cells after IRS-1 knockdown and transfection with either WT IRS-1 or IRS-1 mutant. ∗p < 0.05 by two-way ANOVA with Tukey’s multiple comparisons test. Data are presented as mean ± SD.
Fig 5: TRAF4 targets K1186 and K1189 of IRS-1 for ubiquitination.A, schematic representation of IRS-1 WT and the mutants. The C-terminal last 62 amino acid sequences are shown with K1186 and K1189 in red letter. B, deletion of the C-terminal (1181–1243) region of IRS-1 abolished TRAF4-mediated ubiquitination. FLAG-IRS-1 and deletion mutant was cotransfected with TRAF4 and HA-Ub into 293T cells. The IRS-1 was immunoprecipitated using a FLAG antibody, and the ubiquitinated IRS-1 was detected using an anti-HA antibody by Western blot. C, mutation of lysine 1186 and 1189 to arginine in IRS-1 abolished TRAF4-mediated ubiquitination. FLAG-IRS-1 WT or K1186_1189R mutant was cotransfected with TRAF4 and HA-Ub into 293T cells. The IRS-1 was immunoprecipitated using a FLAG antibody, and ubiquitinated IRS-1 was detected using an anti-HA antibody by Western blot. D, IRS-1 ubiquitination site mutation does not abolish the interaction between IRS-1 and TRAF4. Top, Myc-TRAF4 interacted with FLAG-IRS-1 WT or TRAF4-ubiquitin mutant IRS-1 (IRS-1 mut) in transiently transfected 293T cells. A coimmunoprecipitation experiment was performed using an anti-FLAG antibody for immunoprecipitation, followed by Western blot. Bottom, input control.
Supplier Page from Abcam for Recombinant Human IRS1 protein