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. 2004 Jul 19;166(2):213–223. doi: 10.1083/jcb.200403069

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Copyright © 2004, The Rockefeller University Press

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Figure 6. — S6K phosphorylation blocks IRS-1 function. (A) IRS-1 tyrosine phosphorylation and association with PI3K is suppressed in TSC2 ^−/− MEFs and rescued by inhibition of S6K. IRS-1 immunoprecipitates in unstimulated or insulin-stimulated TSC2 ^+/+ or TSC2 ^−/− MEFs after rapamycin treatment for the indicated times, immunoblotted for phosphotyrosine (panel 1), or the coimmunoprecipitation of the p85 subunit of PI3K (panel 2). The total levels of IRS-1 and p85 in cell lysates are indicated in panels 3 and 4, respectively. (B) Quantitation of insulin- stimulated IRS-1 tyrosine phosphorylation in untreated TSC2 ^−/− MEFs or the same cells after rapamycin treatment for the indicated times. An arbitrary value was obtained representing the difference between unstimulated and the corresponding insulin-stimulated level of phosphotyrosine in IRS-1 immunoprecipitations as detected on scanned autoradiographs. The values from three independent experiments were averaged and plotted on a bar chart, with the error bars showing the SEM. (C) Phosphorylation of immobilized GST-IRS-1^108–516 PTB domain by S6K2 inhibits insulin receptor (InR) interaction. The interaction with tyrosine-phosphorylated InR (top) was determined in vitro by the addition of glutathione beads containing the indicated amounts of S6K2-phosphorylated or unphosphorylated GST-IRS-1^108–516 or GST to lysates prepared from insulin-stimulated CHO-IR cells. An immunoblot of the level of GST-IRS-1 fusion protein or GST is indicated on the bottom. DP indicates degradation products of the GST-IRS-1 protein. (D) Quantitation of InR binding to GST-IRS-1^108–516 (WT) or a mutant GST-IRS-1^108–516 in which serine 302 is mutated to alanine (S302A) after in vitro phosphorylation by S6K2. Error bars show the SEM from three experiments. (E) InR binding of wild-type GST-IRS-1^108–516, or S302A and S302E mutants in a pull-down assay. Top, InR; bottom, anti-GST. (F) Quantitation of InR binding to GST-IRS-1^108–516 (WT) or S302A and S302E mutants. An arbitrary value of 100 was assigned for the binding of wild-type GST-IRS-1^108–516, and the binding of the mutant IRS-1 proteins was expressed relative to this value. Error bars show the SEM from three experiments.

Figure 6. — S6K phosphorylation blocks IRS-1 function. (A) IRS-1 tyrosine phosphorylation and association with PI3K is suppressed in TSC2 ^−/− MEFs and rescued by inhibition of S6K. IRS-1 immunoprecipitates in unstimulated or insulin-stimulated TSC2 ^+/+ or TSC2 ^−/− MEFs after rapamycin treatment for the indicated times, immunoblotted for phosphotyrosine (panel 1), or the coimmunoprecipitation of the p85 subunit of PI3K (panel 2). The total levels of IRS-1 and p85 in cell lysates are indicated in panels 3 and 4, respectively. (B) Quantitation of insulin- stimulated IRS-1 tyrosine phosphorylation in untreated TSC2 ^−/− MEFs or the same cells after rapamycin treatment for the indicated times. An arbitrary value was obtained representing the difference between unstimulated and the corresponding insulin-stimulated level of phosphotyrosine in IRS-1 immunoprecipitations as detected on scanned autoradiographs. The values from three independent experiments were averaged and plotted on a bar chart, with the error bars showing the SEM. (C) Phosphorylation of immobilized GST-IRS-1^108–516 PTB domain by S6K2 inhibits insulin receptor (InR) interaction. The interaction with tyrosine-phosphorylated InR (top) was determined in vitro by the addition of glutathione beads containing the indicated amounts of S6K2-phosphorylated or unphosphorylated GST-IRS-1^108–516 or GST to lysates prepared from insulin-stimulated CHO-IR cells. An immunoblot of the level of GST-IRS-1 fusion protein or GST is indicated on the bottom. DP indicates degradation products of the GST-IRS-1 protein. (D) Quantitation of InR binding to GST-IRS-1^108–516 (WT) or a mutant GST-IRS-1^108–516 in which serine 302 is mutated to alanine (S302A) after in vitro phosphorylation by S6K2. Error bars show the SEM from three experiments. (E) InR binding of wild-type GST-IRS-1^108–516, or S302A and S302E mutants in a pull-down assay. Top, InR; bottom, anti-GST. (F) Quantitation of InR binding to GST-IRS-1^108–516 (WT) or S302A and S302E mutants. An arbitrary value of 100 was assigned for the binding of wild-type GST-IRS-1^108–516, and the binding of the mutant IRS-1 proteins was expressed relative to this value. Error bars show the SEM from three experiments.