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*

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.

The IRS-1 and IRS-2 proteins are closely related and represent substrates of the insulin and insulin-like growth factor receptors (9,10). They consist of several structurally and functionally distinctive domains. At their amino terminus lies a pleckstrin homology domain that is involved in coupling IRS proteins to the insulin receptor. Next to the pleckstrin homology domain are the phosphotyrosine binding domain and SAIN (Shc and IRS-1 NPXY binding) domain (9,11). The phosphotyrosine binding domain directly interacts with the phosphorylated NPXY motif of the ␤-subunit of the insulin and insulin-like growth factor receptors (12). The role of the SAIN domain in the physiology of IRS proteins is currently unknown. The carboxyl-terminal tail of IRS-1 contains a number of phosphotyrosine motifs that serve as docking sites for SH2-containing proteins, including enzymes, such as the PI 3-kinase and adapter molecules, such as Grb-2. mTOR bound to Rictor phosphorylates and activates Akt (4). However, diabetic-mimicking conditions (hyperglycemia and hyperinsulinemia) inhibit PI 3-kinase/Akt signaling in a Raptordependent manner via phosphorylation of IRS-1 at Ser-636/639 by mTOR (13). These serine residues lie next to the 632 YMPM motif that binds the PI 3-kinase upon insulin stimulation, and their phosphorylation inhibits the PI 3-kinase activity associated with IRS-1 (13). mTORC1 regulates additional inhibitory phosphorylations on IRS-1, such as Ser-307 and Ser-312, which interfere with the ability of IRS-1 to interact with the insulin receptor (13)(14)(15). Therefore, under diabetic-related conditions mTORC1 may inhibit in vivo glucose disposal by suppressing the PI 3-kinase activity associated with IRS-1. The physiological relevance of these findings is supported by recent studies that showed (a) the phosphorylation of IRS-1 at Ser-636/639 is increased in noninsulin-dependent diabetes mellitus subjects with a concomitant reduction in Akt activity (16), (b) rapamycin-mediated mTORC1 inhibition reduces in vivo the phosphorylation of IRS-1 at Ser-636/639 and stimulates insulin-mediated glucose uptake in skeletal muscle of human subjects (16,17), and (c) adipose-specific knock-out of raptor results in lean mice that are resistant to diet-induced obesity and exhibit insulin hypersensitivity with improved glucose tolerance (18).
The finding that Akt is regulated by mTOR through both positive (directly via mTORC2 (4)) and negative (indirectly via mTORC1 at the level of IRS-1 (13)) signals raises the question of signaling specificity. Therefore, it is important to determine (a) the role of individual mTORC1 and mTORC2 components in the phosphorylation of IRS-1 at Ser-636/39, (b) the role of PI 3-kinase/PDK1/Akt axis in the feedback inhibition of IRS-1 by mTORC1, and (c) the molecular mechanisms by which mTORC1 phosphorylates IRS-1. In this report I systematically knocked down all the known components of mTORC1 and mTORC2, and I demonstrate that the selective ability of mTORC1, but not mTORC2, to suppress the IRS-1-associated PI 3-kinase/Akt signaling depends on Raptor. Knockdown of mTOR, Raptor, and mLST8 abolishes serum-and insulin-induced phosphorylation of IRS-1 at Ser-636/639 and stabilizes IRS-1 after exposure to long term insulin stimulation. Knock down of Rictor and mSin1 did not affect the aforementioned phosphorylations. Knockdown of PI 3-kinase and PDK1, but not Akt1 or Akt2, suppresses the phosphorylation of IRS-1 at Ser-636/639 triggered by insulin. Mechanistically, Raptor, but not any other component of mTORC1 or mTORC2, interacts with the SAIN domain of IRS-1 and presents IRS-1 to mTOR. IRS-1 lacking the SAIN domain is not phosphorylated at Ser-636/639, and it is resistant to mTORC1-mediated inhibition of PI 3-kinase/Akt signaling. Further studies described herein also showed that the SAIN domain of IRS-2 interacts with Raptor, suggesting a common molecular mechanism by which mTORC1 regulates the phosphorylation of IRS proteins.
Cell Culture-MCF-7, HEK293, HepG2, and C2C12 myoblasts were from the American Tissue Culture Collection. The HEK293 cell line engineered to stably express wild type IRS-1 has been previously described (13,19). HEK293 cells stably expressing Raptor were generated by infecting HEK293 cells with a pMSCV-TAP-Raptor retrovirus (6) and selected for 1 week with 2 g/ml puromycin. All cell lines were grown in high glucose (25 mM) Dulbecco's modified Eagle's medium (#11995-065; Invitrogen) supplemented with 10% fetal bovine serum and antibiotics.
Cell Lysis, Immunoprecipitation, and Western Blotting-Cells were washed in ice-cold phosphate-buffered saline and solubilized in lysis buffer (50 mM Tris, pH 7.5, 200 mM NaCl, 1% Triton X-100, 10 mM Na 3 VO 4 , 50 mM NaF, 1 mM ␤-glycerophosphate, 1 mM sodium pyrophosphate, 1 mM EDTA, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 50 nM okadaic acid, supplemented with a mixture of protease inhibitors). The lysates were cleared by centrifugation for 10 min at 14,000 ϫ g at 4°C. For immunoprecipitation, 0.5-1 mg of a given lysate was mixed overnight via gentle agitation with 1-2 g of specific or nonspecific antibodies coupled to protein A-or G-Sepharose beads. After extensive washing with lysis buffer, beads were resuspended in SDS sample buffer, supplemented with 5% ␤-mercaptoethanol, boiled for 5 min, and subjected to Western blot analysis using standard Western blotting protocols.
Subcellular Fractionation-Cells were washed twice in icecold phosphate-buffered saline and homogenized in buffer A (0.3% CHAPS, 20 mM HEPES, pH 7.4, 1 mM EDTA, 2 mM MgCl 2 , 1 mM phenylmethylsulfonyl fluoride, 1 mM Na 3 VO 4 , supplemented with a mixture of protease and phosphatase inhibitors, Roche Applied Science). The homogenate was centrifuged at 4000 ϫ g for 10 min at 4°C, and the supernatant was layered on the top of a linear 10 -40% or 15-35% iodixanol gradient (Optiprep D1556) in 0.25 M sucrose, 1 mM EDTA, 20 mM HEPES and centrifuged to equilibrium at 30.000 rpm for more than 20 h at 4°C in a Beckman type SWTi41 rotor. Fractions were collected from the top and analyzed by Western blotting.
GST Pulldown Experiments-BL21 cells transformed with different GST-fused IRS-1/2 protein fragments were induced with 100 M isopropyl 1-thio-␤-D-galactopyranoside overnight at room temperature. BL21 cells were lysed in 50 mM Tris (pH 8.0, 200 mM NaCl, 1% Triton X-100, 2 mM dithiothreitol, 1 g/ml lysozyme, 0.1 mM phenylmethylsulfonyl fluoride, and 1ϫ Complete protease inhibitor mix (Roche Applied Science). The lysates were cleared by centrifugation at 10,000 ϫ g for 20 min at 4°C, and the proteins of interest were pulled down by incubating with 50 l of equilibrated glutathione beads for 2 h at 4°C. After extensive washing with lysis buffer, bound proteins were eluted in Laemmli buffer and analyzed by SDS-PAGE electrophoresis.
Lentivirus-mediated Short Hairpin RNA Silencing and Short Interference RNA-The oligonucleotides encoding the short hairpin RNA expression cassettes targeting human mTOR (pLKO1-shmTOR), human Raptor (pLKO1-shRaptor), and human Rictor (pLKO1-shRictor) transcripts have been previously described (4). Additional oligonucleotides encoding short hairpin RNA expression cassettes targeting different components of the mTORC1 and mTORC2 were as follows:

mTORC1 and Insulin Resistance
The oligonucleotides were annealed and subcloned into the pLKO.1 vector (#8453, Addgene) following the manufacturer's instructions. The pLKO.1-shGFP construct was used as the control (#12273, Addgene). For lentivirus production, HEK293T cells were co-transfected with the pLKO.1-based plasmid expressing the appropriate short hairpin RNA cassette and the packaging plasmids pCMV-dR8.2dvpr and pCMV-VSV-G with the calcium phosphate method. Virus-containing supernatants were collected at 48 h after transfection and used to infect different cell lines in the presence of 8 g/ml Polybrene (Sigma). Infected cells were selected for 2 days with 1-2 g/ml puromycin (Sigma) and analyzed 4 -6 days after infection. siRNA against the human mLST8 (#S100425460 and #S100425467) and mSin1 (#S100287861) were from Qiagen, and they were transfected using Lipofectamine 2000 (Invitrogen). Transfections were carried out using the final siRNA concentration of 80 nM. Transfection efficiency, measured with the use of a fluorescein-conjugated control nonspecific siRNA (Cell Signaling #6201), was higher than 80%. Cells were grown for 48 -60 h before each experiment.
Plasmid Construction-The pCMV-Myc-IRS-1 construct has been previously described (13). The amino acid numbering is based on the human IRS-1. To generate the IRS-1 constructs described in this study, the following primers were used. (i) For the pCMV-Myc-IRS-1 ⌬920 -1236 construct we used 5Ј-TTG GGG GAT CCC AAG GCA AGC-3Ј (forward) and 5Ј-AAG CTT CTA AAC TGA AGG GGA GCT ACG GGA AGT-3Ј (reverse). The PCR product was subcloned into the BamHI and HindIII restriction enzyme sites in pCMV-Myc-IRS-1. (ii) For the pCMV-Myc-IRS-1 ⌬TOS ( 942 MKMDLG 947 mutated to 942 ANAAAG 947 ) construct we used 5Ј-GAA GAG TAC GCG AAC GCG GCC GCG GGG CCA-3Ј (forward) and 5Ј-TGG CCC CGC GGC CGC GTT CGC GTA CTC TTC-3Ј (reverse). (iii) Statistical Analysis-Results are expressed as the means Ϯ S.E. Differences between two groups were assessed using the two-tailed Student's t test. Western blot band densitometry was done with the Igor Pro Software (Wavemetrics, Inc).

RESULTS AND DISCUSSION
Components of mTORC1 and mTORC2 Co-fractionate with IRS-1 in Equilibrium Density Gradients-Raptor interacts with IRS-1 and regulates the phosphorylation of IRS-1 at Ser-636/ 639 by mTOR (13). Since our previous report, studies by other groups showed that mTOR partitions between two distinct complexes (5,6,8,20), mTORC1 (mTOR, Raptor, mLST8, and PRAS40) and mTORC2 (mTOR, Rictor, mLST8, and mSin1). These findings raised the following questions of 1) whether additional components of mTORC1 are involved in the phosphorylation of IRS-1 and 2) whether mTORC2 participates in the regulation of IRS-1 phosphorylation either in a direct or indirect mechanism, e.g. by regulating the activity of Akt. To address this question the sedimentation profile of mTORC1 and mTORC2 components has been analyzed in equilibrium density gradients derived from HepG2 cells solubilized in the presence of 0.3% CHAPS to preserve the integrity of mTORassociated complexes. In HepG2 cells growing in nutrient-rich conditions (e.g. in the presence of serum, glucose, and amino acids) the sedimentation profile of IRS-1 peaks in fractions 4 -5 and 8, suggesting that it exists in two distinct subcellular compartments (Fig. 1A). The majority of Raptor and mTOR cofractionate with IRS-1 in fraction 8, consistent with the fact the IRS-1 interacts with Raptor (13). Although a significant amount

mTORC1 and Insulin Resistance
of IRS-1 is present in fraction 8, the majority of the protein is found in fractions 4 and 5. Fraction 8 also contains less amounts of p85␣ than fractions 4 and 5. This is consistent with the inhibitory role of mTORC1 in the assembly of IRS-1 with PI 3-kinase (p85␣/p110␣). In these experiments it has also been observed that additional components of mTORC1 (mLST8 and PRAS40) and mTORC2 (Rictor, mSin1, and mLST8) co-fractionate with IRS-1, suggesting that these proteins may be involved in the inhibition of IRS-1 by mTORC1 (Fig. 1A). Interestingly, under the same conditions only a tiny fraction of PRAS40 cofractionates with IRS-1/mTORC1 at fraction 8, presumably because of Akt-mediated phosphorylation of PRAS40, which leads to its dissociation from mTORC1. Last, only a tiny fraction of S6K1 co-fractionates with IRS-1/mTORC1 at fraction 8, whereas the vast majority of S6K1 co-fractionates with IRS-1 at fractions 4 and 5, which lack mTOR. Because both mTORC1 and S6K1 directly phosphorylate IRS-1, this finding may suggest that they target different pools of IRS-1.
mTORC1, but Not mTORC2, Regulates the Phosphorylation and Stability of IRS-1-To study the relative role of mTOR in the context of mTORC1 and/or mTORC2 in the regulation of IRS-1-associated PI 3-kinase/Akt signaling the endogenous mTOR, Raptor and Rictor were knocked down in different cell lines. Fig. 1B shows that in HepG2 cells Raptor knockdown attenuates the serum-and insulin-induced phosphorylation of IRS-1 at Ser-636/369 and stabilizes IRS-1. Decreased phosphorylation of IRS-1 at Ser-636/639 inversely correlates with the phosphorylation status of Akt. On the contrary, Rictor knockdown did not affect the phosphorylation of IRS-1 at Ser-636/639, although it decreased the phosphorylation of Akt at Ser-473 (4 -6). Similarly, in MCF-7 cells knockdown of Raptor reduced the phosphorylation of IRS-1 at Ser-636/ 639 and induced a marked upregulation in the phosphorylation of Akt (Fig. 1C). Knockdown of Rictor did not affect the phosphorylation of IRS-1 at Ser-636/639 and caused a marked reduction in the phosphorylation of Akt. Knockdown of mTOR affected the viability of MCF-7 cells and caused a dramatic decrease in the levels of a number of proteins, including IRS-1. This resulted in a reduction in the phosphorylation of Akt, mainly at Ser-473 (4 -6), and a marked up-regulation in the total levels of Akt. Because knockdown of mTORC1 and 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. AUGUST 21, 2009 • VOLUME 284 • NUMBER 34 JOURNAL OF BIOLOGICAL CHEMISTRY 22529 mTORC2 affected the levels of endogenous IRS-1 in different cell lines (Fig. 1, B and C, and data not shown), HEK293 cells were engineered to stably express IRS-1 to measure the stoichiometry of IRS-1 phosphorylated at Ser-636/639 upon mTOR, Raptor, and Rictor knockdown. Fig. 1D shows that knockdown of mTOR and Raptor attenuated the insulin-induced phosphorylation of IRS-1 at Ser-636/639, whereas knockdown of Rictor was without an effect. Last, Fig. 1E shows that Raptor knockdown did not alter the total levels of tyrosine phosphorylation of IRS-1. This finding suggests that mTORC1 inhibits the PI 3-kinase/ Akt signaling associated with IRS-1 by phosphorylating Ser/Thr residues, such as Ser-636/639, rather by inhibiting the ability of the insulin receptor to tyrosine phosphorylate IRS-1. Along the same lines, Raptor knockdown specifically enhanced the phosphorylation of Akt after insulin stimulation without affecting the phosphorylation of Erk (extracellular signal-regulated kinase) (Fig. 1E).

mTORC1 and Insulin Resistance
Long term insulin stimulation destabilizes IRS-1 and induces insulin resistance. This effect can be reversed by rapamycin, suggesting that inhibition of mTORC1 may stabilize IRS-1 (10,(21)(22)(23). However, it has been shown that long term rapamycin treatment also inhibits mTORC2 (24). Therefore, to address whether mTORC1 and/or mTORC2 regulate the stability of IRS-1 upon long term insulin stimulation, Raptor and Rictor were knocked down in HepG2 cells and C2C12 myoblasts. After stimulation with insulin for 16 h, it has been found that Raptor, but not Rictor, knock down stabilized IRS-1 in both cell lines ( Fig. 2A). Further experiments in HepG2 cells confirmed that Raptor knockdown increased IRS-1 half-life from 4 to Ͼ10 h compared with control cells (Fig. 2B). Taken together, the data presented in Figs. 1 and 2 suggest that mTORC1, but  mLST8, but Not mSin1 and S6K1, Regulates Serum-and Insulin-stimulated Phosphorylation of IRS-1 at Ser-636/639-mLST8 binds the kinase domain of mTOR and participates in the formation of both mTORC1 and mTORC2 complexes (5,20). mLST8 is required for mTORC1 and mTORC2 activity toward S6K1 (20) and Akt (5), respectively. mLST8 co-fractionates with mTORC1, mTORC2, and IRS-1 (Fig. 1A). This raises the question of whether mLST8 participates in the mTORC1-dependent phosphorylation of IRS-1. To address this question the endogenous mLST8 has been knocked down in HEK293 cells stably expressing IRS-1, and it has been found that mLST8 is required for both insulin-and serum-induced phosphorylation of IRS-1 at Ser-636/639 (Fig. 3A). Knockdown of mLST8 in HEK293 cells also suppressed serum-and insulin-dependent phosphorylations of both S6K1 and Akt. Therefore, mLST8 positively regulates mTOR kinase activity toward its substrates (IRS-1, S6K1, and Akt). Interestingly, mLST8 mRNA and protein levels are up-regulated in 3T3-L1 adipocytes after long term insulin stimulation in a concentration-dependent manner (25), suggesting that mLST8 may participate in the inhibitory effect of mTORC1 on IRS-1 signaling. mSin1 interacts with Rictor and positively regulates mTORC2-mediated Akt phosphorylation at Ser-473 (5). mSin1 co-fractionates with IRS-1, suggesting that it may be involved in the phosphorylation of IRS-1 (Fig. 1A). To address this question, mSin has been knocked down, and it has been found that although mSin1 is required for the phosphorylation of Akt at Ser-473, it did not affect the serum-and insulin-stimulated phosphorylation of IRS-1 at Ser-636/639 (Fig. 3A). S6K1, a downstream target of mTORC1, phosphorylates IRS-1 at multiple inhibitory serine residues (15,26). S6K1 directly phosphorylates IRS-1 at Ser-307 and inhibits its interaction with the insulin receptor (14). S6K1 knockout mice are deficient in phosphorylating IRS-1 at Ser-636/639 (26). However, by using a rapamycinresistant mutant of S6K1, we have shown that rapamycin is still capable of suppressing phosphorylation of IRS-1 at Ser-636/639, suggesting that mTOR/Raptor per se, rather than S6K1, are important for the aforementioned phosphorylation. Consistently, S6K1 does not phosphorylate IRS-1 at Ser-636/639 in vitro (15), whereas mTORC1 does (13). Moreover, Fig. 3B shows that knockdown of S6K1 in HEK293 cells stably expressing IRS-1 did not affect the phosphorylation of IRS-1 at Ser-636/639 upon serum or insulin stimulation. Therefore, S6K1 is not directly involved in the phosphorylation of IRS-1 at Ser-636/639. However, S6K1  AUGUST 21, 2009 • VOLUME 284 • NUMBER 34 participates in the negative feedback regulation of PI 3-kinase/Akt signaling by phosphorylating other rapamycinsensitive serine residues, such as Ser-307 (13)(14)(15).

mTORC1 and Insulin Resistance
mTORC1 Phosphorylation of IRS-1 at Ser-636/639 Is Akt-independent-Next, it has been addressed as to whether the PI 3-kinase/PDK1/Akt axis is involved in the regulation of IRS-1 phosphorylation at Ser-636/ 639. Two commonly used PI 3-kinase inhibitors, LY294002 and wortmannin, inhibited, in a dosedependent manner, the insulinstimulated phosphorylation of IRS-1 at Ser-636/639 in C2C12 myoblasts in a manner that parallels mTORC1 activity (Fig. 3C). However, LY294002 and wortmannin inhibit both mTOR and PI 3-kinase with similar IC 50 values (27). Therefore, the observed effect in Fig. 3C may reflect the inhibition of mTORC1 per se rather the inhibition of PI 3-kinase. To address this issue, the PI 3-kinase and PDK1 were knocked down in HEK293, and it has been found that both of them are required for the activation of the mTORC1 pathway as well as for the phosphorylation of IRS-1 at Ser-636/639 upon serum and insulin stimulation (Fig. 3D).
The PI 3-kinase/PDK1 axis and mTORC2 are involved in the activation of Akt. Moreover, Akt phosphorylates TSC2 (tuberous sclerosis complex) (28) and PRAS40 and activates mTORC1 (7,8). Therefore, it is likely, that Akt may regulate its attenuation after insulin stimulation by inducing the activation of mTORC1. To address this question, cells were treated with Akt inhibitors IV and VIII after insulin stimulation. Fig. 3E shows that both inhibitors failed to inhibit the insulin-stimulated phosphorylation of IRS-1 at Ser-636/639. Similarly, knockdown of either Akt1 or Akt2 in HEK293 cells did not interfere with the ability of mTORC1 to phosphorylate IRS-1 at Ser-636/ 639 (Fig. 3F). These findings suggest that (a) Akt1 and Akt2 have similar signaling abilities regarding the phosphorylation of IRS-1 by mTORC1 and (b) Akt1 and Akt2 have a minor role in the mTORC1-mediated feedback inhibition of the PI 3-kinase signaling associated with IRS-1. Raptor Interacts with IRS-1 in a Growth Factor-and Glucosedependent Manner-The finding that mLST8 (current study) and PRAS40 (7) regulate the phosphorylation of IRS-1 at Ser-636/639 and the observation that IRS-1 co-fractionates with components of both mTORC1 and mTORC2 in equilibrium density gradients raised the question as to whether those pro-

mTORC1 and Insulin Resistance
teins interact with IRS-1. To address this question we transfected HEK293 cells engineered to stably express IRS-1 with either equal amounts (Fig. 4A) or equal molarities (Fig. 4B) of Myc-tagged components of mTORC1 and mTORC2. Fig. 4, A, B, and C, show that Raptor is the only protein that interacts with IRS-1, suggesting that mLST8 and PRAS40 affect IRS-1 phosphorylation by regulating the kinase activity of mTOR. Interestingly, we found that the interaction between Raptor and IRS-1 is dynamic and is regulated both by growth factors and glucose (Fig. 4D) as is the case with the phosphorylation of IRS-1 at Ser-636/639 (13). Consistently, in serum-free media Raptor did not co-fractionate with IRS-1 (Fig. 4E, upper panel), whereas insulin stimulation induced the phosphorylation of IRS-1 at Ser-636/639 and its translocation to denser compartments along with mTOR and Raptor (fractions 5-8 in Fig. 4E,  lower panel).
The SAIN Domain of IRS-1 Interacts with Raptor and Regulates the Phosphorylation of IRS-1 at Ser-636/639-The domain of IRS-1 that interacts with Raptor is currently unknown. Raptor binds both S6K1 and 4E-BP1 proteins via their TOR-signaling motifs (TOS) (29 -34). 4E-BP1 has a TOS motif (FEMDI) in its carboxyl terminus, whereas S6K1 has a TOS motif (FDIDL) in its amino terminus (29 -34). A point mutation of the conserved Phe residue to Ala abolishes 4E-BP1 and S6K1 binding to Raptor and impairs their phosphorylation by mTOR. Sequence analysis revealed that IRS-1 has a putative TOS motif ( 937 GTEEYMKMDL 946 ) that may interact with Raptor (30,32). However, it lacks the critical Phe residue (instead it has a methionine). Fig. 5, A and B, show that deletion of the carboxyl terminus of IRS-1 (IRS-1 ⌬920 -1236) as well as mutation of the putative TOS motif did not affect the phosphorylation of IRS-1 at Ser-636/639. Surprisingly, in the same experiment it has been found that deletion of the SAIN domain (amino acids 250 -584) of IRS-1 attenuated the phosphorylation of IRS-1 at Ser-636/ 639 and increased IRS-1 binding to PI 3-kinase (Fig. 5, B and C). This is consistent with the inhibitory role of those phosphorylations in the interaction between IRS-1 and PI 3-kinase upon insulin stimulation (13). Last, the IRS-1 ⌬495-1236 mutant, which lacks the two SH2 domains, did not interact with PI 3-kinase and served as control.
To further characterize the importance of the SAIN domain, a series of IRS-1 constructs fused to GST protein were generated (Fig. 5D). These constructs were used in pulldown experiments using cell extracts obtained from HEK293 cells stably expressing Raptor. Fig. 5, D and E, show that the GST-IRS-1 260 -380 fragment efficiently pulled down endogenous as well exogenous Raptor, suggesting that this domain of IRS-1 directly interacts with Raptor. On the contrary, the GST-IRS-1 380 -500 fragment failed to pull down Raptor. Interestingly, we found that the GST-IRS-1 260 -500 and GST-IRS-1 260 -700 fragments that harbor the whole SAIN domain of IRS-1 exhibited higher ability to interact with Raptor in vitro, suggesting that additional elements of the SAIN domain are crucial for this interaction. Importantly, none of the GST-fused IRS-1 fragments pulled down mTOR or mLST8.
To assess in vivo the role of SAIN domain in the mTORC1mediated phosphorylation of IRS-1 at Ser-636/639, a series of IRS-1 mutants was generated that lacked different parts of the SAIN domain (Fig. 6A). Fig. 6B shows that deletion of the SAIN domain completely abolished the phosphorylation of IRS-1 at Ser-636/639. Interestingly, although in vitro a GST-IRS-1 260 -380 fragment is sufficient for the interaction with Raptor, deletion of the SAIN domain either in its amino or carboxyl terminus decreased phosphorylation of IRS-1 at Ser-636/639, suggesting that additional elements of the SAIN domain are required in vivo for the efficient phosphorylation of IRS-1 by mTORC1. This is consistent with the in vitro finding that the full length of SAIN domain significantly enhances the interaction with Raptor (Fig. 5E). Indeed, IRS-1 lacking the SAIN domain (IRS-1 ⌬250 -500) failed to co-immunoprecipitate with Raptor (Fig. 6C). Last, point mutations of Ser-307/312 to alanine, two well established negative phosphorylations mediated by S6K1 and JNK (c-Jun NH 2 -terminal kinase) (14,15,(35)(36)(37)(38)(39), respectively, did not affect the phosphorylation status of IRS-1 at Ser-636/639, suggesting that those phosphorylation events occur independently (Fig. 6B). Overall, data presented in Figs. 5 and 6 suggest that the domain of IRS-1 from amino acids 260 to 380 is required for the interaction between IRS-1 and Raptor, whereas the full-length SAIN domain (amino acids 250 -584) dramatically enhances in vitro and in vivo the interaction between IRS-1 and Raptor and the phosphorylation of IRS-1 at Ser-636/639 by mTORC1.
IRS-1 and IRS-2 proteins exhibit significant homology in their SAIN domains (9,11). Moreover, insulin triggers the phosphorylation of IRS-2 on serine residues (recognized by the phosphoserine 14-3-3 binding motif (4E2) monoclonal antibody, Cell Signaling #9606) and promotes the binding of p85␣/PI 3-kinase to IRS-2 in a rapamycin-dependent manner (Fig. 6D). This suggests that IRS-2 may also interact with Raptor via its SAIN domain. To address this hypothesis a GST-IRS-2 SAIN (amino acids 249 -534) fusion protein has been generated, and its ability to interact in vitro with Raptor has been examined. Fig. 6E indeed shows that the SAIN domain of IRS-2 interacts with Raptor, suggesting a common molecular mechanism by which mTORC1 regulates the phosphorylation of IRS proteins.
Overall, this report shows that 1) Raptor defines the selective ability of mTORC1 to interact with and to regulate the phosphorylation of IRS-1 at Ser-636/639, 2) mLST8 in the context of mTORC1 is required for the phosphorylation of IRS-1 at Ser-636/639, 3) Raptor knockdown stabilizes IRS-1 under diabeticmimicking conditions, and 4) the SAIN domains of IRS-1 binds to Raptor and allosterically regulates the phosphorylation of IRS-1 at Ser-636/639 by mTOR (Fig. 6F).