Raptor binds the SAIN (Shc and IRS-1 NPXY binding) domain of insulin receptor substrate-1 (IRS-1) and regulates the phosphorylation of IRS-1 at Ser-636/639 by mTOR

Abstract

In normal physiological states mTOR phosphorylates and activates Akt. However, under diabetic-mimicking conditions mTOR inhibits phosphatidylinositol (PI) 3-kinase/Akt signaling by phosphorylating insulin receptor substrate-1 (IRS-1) at Ser-636/639. The molecular basis for the differential effect of mTOR signaling on Akt is poorly understood. Here, it has been shown that knockdown of mTOR, Raptor, and mLST8, but not Rictor and mSin1, suppresses insulin-stimulated phosphorylation of IRS-1 at Ser-636/639 and stabilizes IRS-1 after long term insulin stimulation. This phosphorylation depends on the PI 3-kinase/PDK1 axis but is Akt-independent. At the molecular level, Raptor binds the SAIN (Shc and IRS-1 NPXY binding) domain of IRS-1 and regulates the phosphorylation of IRS-1 at Ser-636/639 by mTOR. IRS-1 lacking the SAIN domain does not interact with Raptor, is not phosphorylated at Ser-636/639, and favorably interacts with PI 3-kinase. Overall, these data provide new insights in the molecular mechanisms by which mTORC1 inhibits PI 3-kinase/Akt signaling at the level of IRS-1 and suggest that mTOR signaling toward Akt is scaffold-dependent.

FIGURE 1.

mTORC1, but not mTORC2, regulates the phosphorylation of IRS-1 at Ser-636/639. A, HepG2 cells grown in the presence of serum and nutrients were lysed in 0.3% CHAPS and fractionated to equilibrium in 10–40% linear iodixanol gradient. Fractions were collected from the top and analyzed by Western blotting. B, HepG2 cells infected with the indicated lentiviruses were serum-starved overnight and stimulated for 30 min with serum (S) and insulin (I). Whole cell lysates were analyzed by Western blotting. C, MCF-7 cells infected with the indicated lentiviruses were analyzed by Western blotting. D, HEK293 cells stably expressing IRS-1 infected with the indicated lentiviruses were serum-starved overnight and stimulated for 30 min with serum and insulin. Whole cell lysates were analyzed by Western blotting. E, HEK293 cells stably expressing IRS-1 infected with the indicated lentiviruses were serum-starved overnight and stimulated for the indicated periods of time with insulin. Whole cell lysates were analyzed by Western blotting.

FIGURE 2.

Raptor knock down stabilizes IRS-1 after long term insulin stimulation. A, HepG2 cells and C2C12 myoblasts infected with the indicated lentiviruses were stimulated for 16 h with insulin. Whole cell lysates were analyzed by Western blotting. The graph shows the IRS-1 protein levels (as percentage of control) from three independent experiments (mean values ± S.E.). B, HepG2 cells infected with the indicated lentiviruses were treated with cycloheximide (40 μg/ml) for the indicated periods of time. The Western blots show the IRS-1 protein levels from two representative experiments. The graph shows the IRS-1 protein levels (as a percentage of control) normalized to tubulin from three independent experiments (mean values ± S.E.). *, p < 0.05.

FIGURE 3.

mLST8, but not S6K1 and mSin1, regulates serum- and insulin-stimulated phosphorylation of IRS-1 at Ser-636/639. A, HEK293 cells stably expressing IRS-1 transfected with the indicated siRNA were serum-deprived for 16 h and were stimulated with serum and insulin. Whole cell lysates were analyzed by Western blotting. B, HEK293 cells transfected with the indicated siRNA were serum-starved and then stimulated with serum (S) and insulin (I). Whole cells lysates were analyzed by Western blotting. C, C2C12 myoblasts were serum-starved overnight and pretreated with the indicated inhibitors for 45 min after by insulin stimulation. Whole cell lysates were analyzed by Western blotting. D, HEK293 cells stably expressing IRS-1 infected with the indicated lentiviruses were serum-starved and stimulated with serum and insulin. Whole cell lysates were analyzed by Western blotting. E, C2C12 myoblasts were serum-starved overnight and pretreated with the indicated inhibitors for 45 min followed by insulin stimulation. Whole cell lysates were analyzed by Western blotting. F, HEK293 cells stably expressing IRS-1 infected with the indicated lentiviruses were serum-starved and stimulated with serum and insulin. Whole cell lysates were analyzed by Western blotting.

FIGURE 4.

Raptor interacts with IRS-1 in a serum- and glucose-dependent manner. HEK293 cells stably expressing IRS-1 were transfected with equal amounts (A) or equal molarities (B) of the indicated Myc-tagged cDNA constructs. Whole cells lysates were immunoprecipitated (IP) with the Myc antibody, and the immunoprecipitated materials were analyzed by Western blotting. C, HEK293 cells stably expressing IRS-1 were transfected with Myc-tagged constructs of Raptor and PRAS40 followed by immunoprecipitation with the Myc antibody and analysis by Western blotting. D, HEK293 cells stably expressing IRS-1 transfected with Myc-tagged Raptor were either serum- or glucose-starved overnight. Whole cell lysates were immunoprecipitated with the Myc antibody and analyzed by Western blotting. E, HepG2 cells were serum-starved overnight followed by stimulation with insulin for 30 min. Cells were lysed in 0.3% CHAPS and fractionated to equilibrium in a 15–35% linear iodixanol gradient. Fourteen fractions were collected from the top of the gradient and analyzed by Western blotting.

FIGURE 5.

The SAIN domain of IRS-1 allosterically regulates the phosphorylation of IRS-1 at Ser-636/639. A, schematic diagram of the IRS-1 mutants. WT, wild type. B, HEK293 cells were transiently transfected with the indicated constructs of IRS-1. Whole cell lysates were analyzed by Western blotting. C, HEK293 cells were transiently transfected with the indicated constructs of Myc-tagged IRS-1. Whole cell lysates were immunoprecipitated (IP) with the Myc-antibody and probed for the presence of the p85a subunit of PI 3-kinase. D, schematic diagram of the GST-fused IRS-1 constructs used in pulldown experiments. E, the indicated GST-fused IRS-1 fragments were incubated with whole cell lysates derived from HEK293 cells stably expressing Raptor. The pulldown materials were analyzed for the presence of Raptor by Western blotting. PTB, phosphotyrosine binding.

FIGURE 6.

The SAIN domain of IRS-1 and IRS-2 interacts with Raptor. A, schematic diagram of the IRS-1 deletion mutants. WT, wild type. B, HEK293 cells transiently transfected with the indicated constructs of IRS-1 were analyzed by Western blotting. C, HEK293 cells were transiently transfected with the indicated Myc-tagged constructs of IRS-1. Whole cell lysates were immunoprecipitated (IP) with the Myc antibody and probed for the presence of Raptor. D, HEK293 cell stably expressing Myc-tagged IRS-2 were serum-starved overnight, pretreated with rapamycin as indicated, and stimulated with insulin. Myc-tagged IRS-2 was immunoprecipitated and probed with the 4E2 monoclonal antibody (Cell Signaling #9606, the antibody recognizes phosphoserine surrounded by Pro at the +2 position and Arg/Lys at the −3 position). E, the SAIN domains of IRS-1 and IRS-2 fused to GST protein were incubated with whole cell lysates derived from HEK293 cells. The pulldown materials were analyzed for the presence of Raptor by Western blotting. F, model of IRS-1 phosphorylation by mTORC1. Raptor binds the SAIN domain of IRS-1 and regulates the phosphorylation of IRS-1 at Ser-636/639 by mTOR. PTB, phosphotyrosine binding (domain).