PtdIns(3,4,5)P₃-dependent Activation of the mTORC2 Kinase Complex

Abstract

mTOR serves as a central regulator of cell growth and metabolism by forming two distinct complexes, mTORC1 and mTORC2. Although mechanisms of mTORC1 activation by growth factors and amino acids have been extensively studied, the upstream regulatory mechanisms leading to mTORC2 activation remain largely elusive. Here, we report that the PH domain of Sin1, an essential and unique component of mTORC2, interacts with the mTOR kinase domain to suppress mTOR activity. More importantly, PtdIns(3,4,5)P₃, but not other PtdInsP_n species, interacts with Sin1-PH to release its inhibition on the mTOR kinase domain, thereby triggering mTORC2 activation. Mutating critical Sin1 residues that mediate PtdIns(3,4,5)P₃ interaction inactivates mTORC2, whereas mTORC2 activity is pathologically increased by patient-derived mutations in the Sin1-PH domain, promoting cell growth and tumor formation. Together, our study unravels a PI3K-dependent mechanism for mTORC2 activation, allowing mTORC2 to activate Akt in a manner that is regulated temporally and spatially by PtdIns(3,4,5)P₃.

INTRODUCTION

The mechanistic target of rapamycin (mTOR) is an evolutionarily conserved Ser/Thr kinase essential for pivotal cellular and physiological functions including cell growth, proliferation and metabolism (1). Hyper-activation of the mTOR signaling is observed in virtually all human solid tumors and hematological malignancies, as well as diabetes and neurodegeneration (1). Structurally, mTOR serves as an indispensible catalytic subunit for two functionally distinct sub-complexes termed mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Both mTORC1 and mTORC2 share two common subunits mTOR and GβL, whereas Raptor is a unique component of mTORC1 (2) and both Rictor (3) and Sin1 (4–6) are restricted to mTORC2.

Importantly, both mTORC1 and mTORC2 sense cellular physiological cues, but with different specificities to ensure that cells proliferate only under favorable conditions. Specifically, activation of mTORC1 requires at least two types of stimulation. Triggered by amino acids or nutrients, mTORC1 is recruited to the surface of lysosomes in an amino-acid transporter SLC38A9 (7, 8), Rag-GTPase (9), Ragulator (10) and vacuolar adenosine triphosphatase (11)-dependent manner through an “inside-out” mechanism (12). However, full activation of mTORC1 also requires a lysosomal, GTP-loaded active form of Rheb, resulting from the release of TSC2 inhibitory effects on Rheb through phosphorylation of TSC2 by various growth signaling pathways, including PI3K/Akt signaling (13), RAS/ERK signaling (14) or Wnt signaling (15). Interestingly, amino-acid specific activation mechanisms were also identified, where leucine relies on the Rag-GTPase, while glutamine, depends on the adenosine-diphosphate-ribosylation-factor-1 GTPase instead of Rag for mTORC1 lysosome translocation and activation (16).

In contrast, mTORC2 is primarily activated by extracellular stimuli such as growth factors and insulin (17, 18). However, the detailed molecular mechanisms leading to mTORC2 activation have just begun to be explored. To this end, mTORC2 activation has been reported to require ribosome association (19) and to facilitate co-translational phosphorylation of Akt on Thr450 for stabilization of newly synthesized Akt protein (20). Furthermore, mTORC2 has been reported to locate proximal to ER, mitochondria, MAM (mitochondria-associated ER-membrane) or nucleus in different mammalian cells and near the plasma membrane in yeast (17). However, how growth factor signaling, which initiates from extracellular signals and occurs primarily at the plasma membrane, governs mTORC2 activation to subsequently activate Akt, is poorly understood.

Here, we report that mTORC2 is activated through a “release-of-inhibition” mechanism by growth signaling molecules in a PtdIns(3,4,5)P₃-dependent manner occurring at the plasma membrane proximity, which reveals a critical molecular mechanism underlying how aberrant alternation of the PtdIns(3,4,5)P₃/Sin1-PH/mTOR signaling pathway may contribute to pathological disorders.

RESULTS

The PH Domain of Sin1 Interacts with the mTOR Kinase Domain and Inhibits mTOR Catalytic Activity towards Phosphorylating Akt

Notably, the recently reported partial mTOR/GβL crystal structure (21) suggests an “active-site restriction” model for mTOR activity control (22), where the mTOR active site is relatively accessible, but the ability of mTOR substrates to enter the mTOR kinase active site is in part governed by mTOR-associated proteins, such as Raptor (22). Inspired by this hypothesis, we evaluated whether the two mTORC2 specific subunits, Rictor and Sin1 can function as possible mTOR kinase activity regulators. Interestingly, aside from the reported GβL interaction with the LBE motif (21) of the mTOR kinase domain (Fig. S1A–B), we found that only Sin1, but not Rictor, interacted with the catalytic C-loop of the mTOR kinase domain (amino acids 2298–2518) that in large exerts mTOR kinase activity (23) in cells (Fig. S1C). These results indicate Sin1 as a possible modulator of mTORC2 kinase activity. Consistently, although Sin1 is essential for mTORC2 complex integrity and kinase activity (4–6), it was previously identified as an endogenous inhibitor for various stress-induced signaling components by binding directly to their catalytic domains, including MEKK2 (24), JNK (25) and Ras (26) (Fig. S1D). Notably, Sin1 binds the mTOR kinase domain (mTOR-KD, aa 2186–2431) primarily through its carboxyl-terminal PH domain and to a much less extent, its N-terminus (Fig. 1A–B). Importantly, Sin1-PH, but not Sin1-N, inhibited mTOR’s ability to phosphorylate Akt-S473 in vitro (Fig. 1C and Fig. S1E) and in cells (Fig. 1D and Fig. S1F–J), demonstrating the importance of the Sin1-PH domain in suppressing mTOR kinase activity. Surprisingly, expression of full-length or N-deleted Sin1, but not PH-deleted Sin1, led to reduced Akt-S473 phosphorylation in a Sin1 dose-dependent manner (Fig. 1E and Fig. S1K–O), further supporting a negative role of the Sin1-PH domain in regulating mTORC2 activity. Therefore, in the remainder of the study we focused on examining mechanistically how Sin1-PH may function to suppress mTORC2 activation, although we cannot exclude the possibility that the Sin1-N may also play a role in mTORC2 regulation.

Fig. 1.

The PH domain of Sin1 binds the mTOR kinase domain and inhibits mTORC2 catalytic activity.

A, A schematic illustration of the Sin1 domain structures.

B, The Sin1-PH domain binds the mTOR kinase domain (KD, aa 2180–2431). Flag-mTOR-KD was transfected into HEK293 cells and affinity purified by Flag-M2 beads 48 hr post-transfection. Then Flag-mTOR-KD immunoprecipitates (IP) were used to pull down indicated Sin1 truncations expressed in HEK293 cells after serum starvation for 24 hr and subjected to immunoblot (IB) analyses.

C, Sin1-PH suppresses mTOR to phosphorylate Akt in vitro. In vitro kinase assays of recombinant active mTOR kinases with GST-Akt1-tail (aa 409–480) as a substrate, in the presence of increasing doses of bacterially purified GST or GST-Sin1-PH proteins.

D, Sin1-PH suppresses mTORC2 activity in cells. IB of whole cell lysates (WCL) derived from MDA-MB-231 cells transfected with increasing amounts of HA-Sin1-PH.

E, Expression of full-length, but not PH domain-deleted Sin1, leads to reduced Akt-pS473 in cells. IB analysis of WCLs derived from HEK293T cells transfected with increasing doses of HA-Sin1.

F, Binding of Sin1-PH to mTOR-KD does not disrupt mTORC2 complex integrity. IB of Flag-IP and WCLs derived from HEK293T cells transfected with HA-Sin1, Flag-mTOR and increasing doses of HA-Sin1-PH.

G, Sin1-PH competes with full-length Sin1 for binding mTOR-KD. IB analysis of WCLs and Flag-IPs derived from HEK293T cells transfected with indicated constructs.

To gain further insights into how Sin1-PH mediates suppression of the mTOR activity, we next examined the specific region(s) of mTOR that interact(s) with Sin1-PH. Consistent with previous reports that Sin1 prefers binding enzymatic domains (24), we observed that Sin1-PH mainly bound the kinase domain of mTOR (Fig. S1P). Moreover, Sin1-PH interacted with the large portion of the mTOR kinase domain (N-FATC, aa 2115–2549), but not the FRB region located in the N-terminus of mTOR kinase domain (aa 2001–2114) (Fig. S1A and S1Q). Furthermore, the mTOR catalytic C-loop, but not the GβL-interacting LBE domain (22) (Fig. S1A), was necessary for mediating the Sin1-PH interaction with mTOR-KD (Fig. S1R–S) and this interaction was recently confirmed by cross-linking experiments (27). Notably, both PFAM and Phosphosite Plus algorithms, as well as structural homology-based modeling of mTOR to the PIKK superfamily members (23) indicate a conserved kinase domain present in mTOR (termed as KD, aa 2186–2431), which displays similar affinity as KD-L (aa 2115–2518) in associating with the Sin1-PH domain in cells (Fig. S1T). Therefore, to possibly bypass the conformational constraints caused by utilizing only the C-loop of the mTOR kinase domain, we mainly used the mTOR-KD (Fig. S1A) in subsequent experiments. Consistent with a critical role of Sin1-PH in suppressing mTOR-KD, deletion of the PH domain compromised Sin1 interaction with mTOR-KD (Fig. S1U), but not full-length mTOR (Fig. S1V), possibly through an mTOR-independent manner, as Sin1 could bind multiple mTORC2 components including Rictor and GβL other than mTOR itself (28, 29). Notably, expression of Sin1-PH did not interfere with the mTORC2 complex integrity (Fig. 1F and Fig. S1W–X), but competed with full-length Sin1 for mTOR-KD interaction (Fig. 1G). Together, these data demonstrate that Sin1-PH interacts with mTOR-KD to suppress mTOR kinase activity (Fig. S1Y).

The Sin1-PH Motif is a Physiological PH Domain that can Functionally Replace the Akt1-PH Domain in Cells

The PH (pleckstrin homology) domain is characterized by its affinity and specificity for binding PtdInsP_ns (phosphatidylinositol phosphates) with at least one pair of adjacent phosphates within the inositol headgroups (30). However, only ~10% of all PH domains display PtdInsP_n-binding specificity and affinity (30). In support of a unique feature for Sin1-PH in suppressing mTOR kinase activity, expression of either the Akt1-PH domain (Fig. S2A) or the PDK1-PH domain (Fig. S2A–B) did not significantly affect Akt-S473 phosphorylation in cells. To gain further insights into the physiological role of Sin1-PH in cells, we found that deletion of the Akt1-PH domain significantly abrogated Akt phosphorylation (Fig. 2A–B), whereas substitution of Akt1-PH with Sin1-PH (Fig. 2A) could in large functionally reconstitute phosphorylation of Akt-S473 but not Akt-T308 in cells (Fig. 2B) upon stimulation (Fig. 2C–D and Fig. S2C), suggesting a potential physiological PtdInsP_n-binding function for the Sin1-PH domain.

Fig. 2.

The PH domain of Sin1 largely replaces the PH domain of Akt1 for function in cells.

A, A schematic illustration of the Akt1-WT (left) and the Sin1-PH-Akt1 chimera domain structures (right).

B, Deleting the PH domain of Akt1 leads to attenuated Akt phosphorylation. Immunoblot (IB) analysis of whole cell lysates (WCLs) derived from DLD1-Akt1/2^−/− cells transfected with indicated constructs.

C–D, Sin1-PH in large functionally replaces Akt1-PH in cells. IB of WCLs derived from Akt1/2^−/− MEFs (C) or HeLa cells (D) transfected with indicated constructs. Where indicated, cells were serum starved for 36 hr before adding insulin (100 nM) for 30 min or EGF (100 ng/ml) for 10 min (C) or IGF-1 (100 ng/ml) for the indicated time periods (D).

PtdIns(3,4,5)P₃ Specifically Interacts with the Sin1-PH Domain to Promote Akt Activation at the Plasma Membrane

To examine the phosphoinositide-binding specificity of the Sin1-PH domain, we first used PtdInsP_n overlay assays to map that Sin1-PH bound preferentially in vitro to PtdIns(3,4,5)P₃ and PtdIns(3,5)P₂, and to a lesser extend, PtdIns5P and PA (Fig. 3A). However, as all PtdInsP_ns are immobilized in these overlay assays, these experiments may not faithfully mimic physiological conditions. Therefore, we further examined the specificity of agarose-beads coupled “PIPsomes” that better mimic physiological PtdInsP_n species in interacting with Sin1. Importantly, under this relatively more physiological condition, only PIPsomes containing PtdIns(3,4,5)P₃, but not those with PtdIns(3,5)P₂, were able to pull down Sin1 (Fig. 3B). Notably, other mTORC2 components were not pulled down in the triton buffer used, which disrupts the integrity of the mTORC2 complex (31). Furthermore, Sin1 mainly interacted with PtdIns(3,4,5)P₃, but not other PtdInsP_n species examined (Fig. 3C), highlighting that like Akt-PH (32), Sin1-PH is largely a PtdIns(3,4,5)P₃-binding motif. In keeping with PtdIns(3,4,5)P₃ as a critical upstream mediator for mTORC2 function, inhibition of pan-PI3K (using wortmannin, LY2940002 and Plk90), or p110α and p110β (using BKM120), both of which produce PtdIns(3,4,5)P₃, but not PIKFYVE (by YM201636) that produces PtdIns(3,5)P₂ (33), led to reduced Akt phosphorylation in cells (Fig. 3D and Fig. S3A–B). Consistently, depletion of p110α (PIK3CA) (Fig. 3E–F and Fig. S3C), but not PIKFYVE (Fig. 3G–H and Fig. S3D), resulted in reduced Akt-S473 phosphorylation. Taken together, these data demonstrate that PtdIns(3,4,5)P₃, but not PtdIns(3,5)P₂, is the major physiological PtdInsP_n species that governs mTORC2 activation in cells.

Fig. 3.

PI(3,4,5)P₃ directly interacts with the Sin1-PH domain and recruits mTORC2 to plasma membrane proximity.

A, PIP_n overlay assays indicate that GST-Sin1-PH mainly interacts with PI(3,5)P₂ and PI(3,4,5)P₃ under 2-dimentional conditions.

B, PI(3,4,5)P₃, but not PI(3,5)P₂-coupled beads pull down ectopically expressed Sin1, but not other mTORC2 components in cells. Immunoblot (IB) of indicated PIP beads pulldowns and whole cell lysates (WCLs) derived from HEK293T cells transfected with indicated constructs.

C, PI(3,4,5)P₃, but not other PIPs examined, pulled down ectopically expressed Sin1. IB of WCLs and PIP beads pulldowns derived from HEK293T cells transfected with indicated constructs. Please note that various Sin1 mutants were included here to screen for critical residues mediating the PI(3,4,5)P₃ interaction with Sin1-PH. The detailed description of these mutants can be found in Fig. 4E and Fig. 5 or associated text.

D, Inhibition of PI(3,4,5)P₃, but not PI(3,5)P₂ generation, leads to reduced Akt-S473 phosphorylation in cells. IB of WCLs derived from HeLa cells treated with indicated inhibitors for 2 hr. Drug doses used: wortmannin (100 nM), LY2940002 (1 µM), Plk90 (20 nM), BKM120 (100 nM) and YM201636 (500 nM).

E–H, Depletion of endogenous PIK3CA, but not PIKFYVE, leads to attenuated mTORC2 activity towards phosphorylating Akt-S473 in cells. IB of WCLs derived from primary foreskin fibroblasts (E, G) or PC3 (F, H) cells infected with shPIK3CA (E, F) or shPIKFYVE (G, H) lenti-viruses. 72 hrs post-puromycin selection (1 µg/ml), cells were harvested for IB analysis.

I, PI(3,4,5)P₃ mainly binds the PH domain of Sin1. IB of PIP₃ beads pulldowns and WCLs derived from HEK293T cells transfected with indicated constructs.

J, PI(3,4,5)P₃ pulls down intact mTORC2 complexes. IB of PIP₃ pulldowns (in CHAPS buffers) and WCLs derived from HEK293T cells transfected with indicated constructs.

K, Representative confocal images to illustrate that GFP-Sin1-PH enriches in plasma membrane proximity upon insulin (100 nM) stimulation for 15 min, which was abolished by inhibiting PIP₃ generation via 20 nM Plk90 or 100 nM BKM120, but not by inhibiting PI(3,5)P₂ generation with 500 nM YM201636.

In agreement with the notion that the Sin1-PH domain interacts with PtdIns(3,4,5)P₃, we observed that PtdIns(3,4,5)P₃ primarily interacted with the PH motif, but not other domains of Sin1 in cells (Fig. 3I). Moreover, the Sin1-PH domain binds Ins(1,3,4,5)P₄ (the soluble headgroup of PtdIns(3,4,5)P₃) (32) with a Kd comparable to that of Akt1-PH in vitro (Fig. S3E–F). Consistently, in CHAPS buffers that retain mTORC2 integrity, PtdIns(3,4,5)P₃ beads were able to pull down intact mTORC2 complexes (Fig. 3J), in a manner that is in large mediated by Sin1, as genetic ablation of Sin1 led to compromised Rictor interaction with PtdIns(3,4,5)P₃ beads (Fig. S3G). More importantly, the mTORC2 complexes isolated by PtdIns(3,4,5)P₃ pulldowns were able to phosphorylate Akt-S473 in vitro (Fig. S3H), and this phosphorylation could be antagonized by pharmacological inhibition of mTOR kinase (Fig. S3I), suggesting that PtdIns(3,4,5)P₃-associated mTORC2 complexes are catalytically active.

Given that the mTORC2 substrate Akt is activated primarily on the plasma membrane (PM), and mTORC2 has also been observed localized on plasma membrane in both mammalian cells (34) and yeast (35, 36), we next examined whether mTORC2 is activated on PM, such that its activation may subsequently trigger phosphorylation of PM-associated Akt on Ser473. To this end, we observed that although dispersed under serum-deprived conditions, the Sin1-PH domain accumulated proximal to the plasma membrane upon insulin stimulation (Fig. 3K). Furthermore, this localization could be blocked by pre-treatment with the PI3K inhibitor, Plk90 or the PIK3CA inhibitor, BKM120, but not the PIKFYVE inhibitor, YM201636 (Fig. 3K). These results suggest that the Sin1-PH domain may be recruited to PM at regions with PtdIns(3,4,5)P₃, but not PtdIns(3,5)P₂ synthesis. Consistently, depletion of endogenous p110α, but not PIKFYVE, resulted in impaired PM localization of Sin1-PH induced by insulin (Fig. S3J). Moreover, endogenous Rictor was slightly enriched on PM upon insulin stimulation in Sin1^+/+ but not Sin1^−/− MEFs (Fig. S3K–L), highlighting a critical role for Sin1 in the PM localization of the mTORC2 complex for its kinase activation.

PtdIns(3,4,5)P₃ Interacts with the PH Domain of Sin1 via Three Critical Residues Including R393, K428 and K464

To gain further mechanistic insights into PtdIns(3,4,5)P₃-mediated activation of mTORC2, we performed in vitro kinase assays in a cell free system. Under these conditions, consistent with a previous report (37), PtdIns(3,4,5)P₃, but not other PtdInsP_n-containing poly-PIPsomes, was able to directly trigger activation of inactive mTORC2 complexes immunoprecipitated from serum-starved cells, as measured by Akt-S473 phosphorylation in vitro (Fig. 4A–B). Previous studies on PtdInsP_n-dependent activation of Akt (32) inspired us to postulate that similar to PtdIns(3,4,5)P₃-mediated Akt activation, PtdIns(3,4,5)P₃ may also activate mTORC2 by releasing Sin1-PH inhibition on mTOR-KD. Consistently, the Sin1-PH domain interaction and suppression of the mTOR catalytic domain could be released in response to physiological stimuli that trigger mTORC2 activity to phosphorylate Akt at S473, such as insulin (Fig. 4C) or EGF (Fig. S4A), or by accumulated PIP₃ species through PTEN loss (Fig. S4B)

Fig. 4.

PI(3,4,5)P₃ promotes mTORC2 activation to phosphorylate Akt-S473.

A, In vitro kinase assays indicating that mTORC2 complexes immunoprecipitated (IP) by HA-Rictor are active upon insulin stimulation in phosphorylating Akt-S473.

B, In vitro kinase assays showing that PI(3,4,5)P₃-polysomes activate the purified inactive mTORC2 complexes in vitro. HA-Rictor containing mTORC2 complexes were immunoprecipitated from HEK293 cells and serum starved for 36 hr before harvest in CHAPS buffers. 25 µL of 1 mM polyPIPsomes containing 5% indicated PIP species were incubated with 10 µL of HA-Rictor precipitates in kinase assays.

C, Insulin treatment attenuated Sin1-PH interaction with mTOR-KD-L. Immunoblot (IB) analysis of whole cell lysates (WCLs) and GST-pulldowns derived from 293 cells transfected with indicated constructs. Where indicated, cells were serum-starved for 24 hr and stimulated by 100 nM insulin for 30 min before harvesting.

D, An illustration of solved Akt1-PH/IP₄ co-crystal structure (PDB: IUNQ) by PyMOL.

E, An illustration of possible Sin1-PH/IP₄ complex structure by super-imposing IP₄ into solved Sin1-PH structure (PDB: 3VOQ) by PyMOL.

We therefore examined the critical residues in the Sin1-PH domain that mediate its interaction with PtdIns(3,4,5)P₃. By comparing the Akt1-PH/Ins(1,3,4,5)P₄ crystal structure (PDB: 1H10) with a computer modeled Sin1-PH/Ins(1,3,4,5)P₄ structure through superimposing Ins(1,3,4,5)P₄ into a characterized Sin1-PH domain structure (38) (Fig. 4D–E), we identified several potential critical residues including R393, K428 and K464 within the Sin1-PH domain that might mediate Ins(1,3,4,5)P₄ binding. The triple R393C/K428A/K464A mutant (termed CAA), but not any of the single mutants alone, was impaired in promoting Akt-S473 phosphorylation in cells in response to IGF-1 (Fig. 5A) or insulin (Fig. S5A) in a time (Fig. 5B) and a dose (Fig. S5B) dependent manner. Notably, this deficiency was not due to disrupted mTORC2 integrity by various Sin1 mutations (Fig. S5C), suggesting that loss of PtdIns(3,4,5)P₃ binding (Fig.3C and 5C) is the major reason for the failure of Sin1-CAA containing mTORC2 complexes to phosphorylate Akt in cells (Fig. 5A–B and Fig. S5A–B) and in vitro (Fig. 5D and Fig. S5D). Notably, upon insulin stimulation, Sin1-CAA expressing HAP1-Sin1^−/− cells displayed a reduced, but not completely loss of Akt-pS473 (Fig. 5B and S5B), suggesting that in addition to PIP₃, other mechanisms may also account for mTORC2-mediated activation of Akt triggered by insulin, such as ribosome association with Sin1 (19) (Fig. S5E), or association with Akt that may bring mTORC2 to PM (27).

Fig. 5.

PI(3,4,5)P₃ mainly interacts with the Sin1-PH domain via R393, K428 and K464 residues to govern mTORC2 activation.

A, The Sin1-R393C/K428A/K464A mutant is deficient in phosphorylating Akt-S473. Immunoblot (IB) analysis of whole cell lysates (WCLs) derived from Sin1^−/− MEFs transfected with indicated constructs. Cells were serum-starved for 24 hr before 100 ng/ml IGF-1 was added for 30 min.

B, Sin1-CAA is deficient in activating mTORC2 upon insulin stimulation. HAP1-Sin1^−/− cells were infected with MSCV-Sin1-HA-WT or CAA retro-viruses, selected with 1 µg/ml puromycin for 3 days to eliminate non-infected cells and serum-starved for 24 hr and stimulated with 100 nM insulin for indicated periods before harvesting for IB analyses.

C, PI(3,4,5)P₃ loses its interaction with Sin1-CAA. IB of PIP₃ beads pulldowns derived from HEK293T cells transfected with indicated constructs.

D, In vitro kinase assays demonstrating that PI(3,4,5)P₃ pulls down active mTORC2 complexes. IB of in vitro kinase assays derived from incubating PI(3,4,5)P₃ beads pulldowns from HEK293T cells transfected with indicated constructs, using GST-Akt1-tail (aa 409–480) as a substrate.

E, Sin1-PH-CAA is deficient in functionally replacing Akt1-PH. IB analyses of WCLs derived from Akt1/2^−/− MEFs transfected with indicated constructs. Where indicated, cells were serum-starved for 36 hr before stimulated by insulin (100 nM) for 30 min or EGF (100 ng/ml) for 10 min.

F, Sin1-CAA binds the mTOR kinase domain. IB analysis of HA-IPs and WCLs derived from HEK293T cells transfected with indicated constructs.

G, Sin1-PH-CAA retains its ability to suppress mTOR kinase in vitro. In vitro kinase assays of recombinant active mTOR kinases with GST-Akt1-tail (aa 409–480) as a substrate, in the presence of increasing doses of bacterially purified GST-Sin1-PH-CAA recombinant proteins.

H, Representative confocal images to illustrate that GFP-Sin1-PH-WT, but not GFP-Sin1-PH-CAA, enriches and co-localizes with Akt1-PH at plasma membrane proximity upon insulin (100 nM) stimulation.

I, Sin1-CAA is deficient in activating Akt in cells. IB analysis of WCLs derived from Sin1-depleted OVCAR5 cells stably expressing MSCV-Sin1-WT-HA or MSCV-Sin1-CAA-HA. Where indicated, cells were serum-starved for 36 hr before stimulation by EGF (100 ng/ml) for 10 min.

J–K, Soft agar assays using OVCAR5 cell lines generated in (I).

Moreover, compared with Sin1-WT (Fig. S3F), the Sin1-CAA mutant displayed a significantly lower affinity with Ins(1,3,4,5)P₄ in vitro (Fig. S5F), further supporting that R395, K428 and K464 may be the major residues that mediate Sin1-PH interaction with Ins(1,3,4,5)P₄. In agreement with this model, unlike Sin1-PH-WT (Fig. 2B–D), the PtdIns(3,4,5)P₃ binding-deficient Sin1-PH-CAA mutant was largely incapable of functionally substituting Akt1-PH to restore Akt phosphorylation in cells (Fig. 5E and Fig. S5G–H). Furthermore, similar to Sin1-PH-WT, Sin1-PH-CAA largely retained its ability to bind mTOR-KD (Fig. 5F), thereby inhibiting mTOR-mediated phosphorylation of Akt-S473 in vitro (Fig. 5G). Mechanistically, this may be in part due to compromised Sin1-PH-CAA co-localization with Akt1-PH on PM, due to its deficiency to properly interact with PtdIns(3,4,5)P₃ present on the plasma membrane (Fig. 5H and Fig. S5I–J).

To further examine how loss of PtdIns(3,4,5)P₃ binding to Sin1 may influence mTORC2-mediated cellular responses, we stably expressed Sin1-WT or the Sin1-CAA mutant in OVCAR5 cells depleted of endogenous Sin1. Under these conditions, we found that mTORC2 activity was attenuated in Sin1-CAA expressing cells, as indicated by reduced Akt-S473 phosphorylation upon EGF (Fig. 5I) or insulin (Fig. S5K) stimulation in Sin1-CAA expressing cells. More importantly, Sin1-CAA expressing cells also formed fewer colonies on soft agar (Fig. 5J–K), indicating that loss of PtdIns(3,4,5)P₃ binding leads to impaired mTORC2 kinase activity, which subsequently suppresses cell transformation phenotypes. Notably, expression of Sin1-CAA did not significantly affect the cellular responses to various DNA damaging agents inducing apoptotic drugs such as doxorubicin and cisplatin (Fig. S5L–M), suggesting that although Akt can be activated upon DNA damage (Fig. S5N), possibly mediated through DNA-PK (39), this mechanism is likely to be independent of PI3K/PtdIns(3,4,5)P₃-mediated mTORC2 activation.

PtdIns(3,4,5)P₃ Binds the PH Domain of Sin1 and Releases Sin1-PH-mediated Inhibition on the mTOR Kinase Domain

Notably, mTOR-KD (Fig. 6A and Fig. S1A) or mTOR-KDL (Fig. S6A and Fig. S1A), but not the full-length mTOR (Fig. 6B), competed with PtdIns(3,4,5)P₃ for binding Sin1, suggesting that PtdIns(3,4,5)P₃ may directly bind Sin1-PH to release Sin1-PH inhibition on the mTOR catalytic domain, therefore exposing the mTOR active site to mTOR substrates. On the other hand, in addition to the mTOR-KD/Sin1-PH interaction, mTOR might have additional interactions with Sin1 either directly or indirectly through GβL or Rictor, since Sin1-PH expression (Fig. 1F and Fig. S1W) or PtdIns(3,4,5)P₃ interaction with Sin1-PH (Fig. 6B) does not disrupt the mTORC2 holo-enzyme complex. This model is further reinforced by the observation that PtdIns(3,4,5)P₃-poly-PIPsomes gradually disturbed the mTOR-KD interaction with Sin1-PH-WT, but not Sin1-PH-CAA, in a dose-dependent manner (Fig. 6C). Notably, although Sin1-CAA is deficient in binding PtdIns(3,4,5)P₃, it still binds mTOR-KD with a similar affinity to Sin1-WT (Fig. 5F). Together, these data indicate that both PtdIns(3,4,5)P₃ and mTOR-KD bind the Sin1-PH domain in a possibly mutually exclusive manner, such that PtdIns(3,4,5)P₃ binding releases Sin1-PH from binding the mTOR catalytic domain and activates mTORC2.

Fig. 6.

PI(3,4,5)P₃ releases Sin1-PH-mediated inhibition on mTOR-KD, leading to mTORC2 activation.

A–B, PI(3,4,5)P₃ competes with mTOR-KD (A), but not full-length mTOR (B) in binding Sin1. Immunoblot (IB) analysis of PIP₃ pulldowns, Flag-immunoprecipitates (IPs) and whole cell lysates (WCLs) derived from HEK293T cells transfected with indicated constructs.

C, PI(3,4,5)P₃-ploysomes attenuate mTOR-KD interaction with Sin1-PH-WT, but not Sin1-PH-CAA. IB analysis of GST pulldowns in the presence of increasing amounts of PIP-polysomes (0,10 or 20 µl) in CHAPS buffers.

D, Compared to Sin1-WT, ectopic expression of Sin1-D412G leads to elevated Akt-pS473 in cells. IB analysis of WCLs derived from Sin1^−/− MEFs transfected with indicated Sin1 constructs.

E, Compared to Sin1-WT, ectopic expression of Sin1-D412G leads to elevated Akt-pS473 under indicated experimental conditions. IB analysis of WCLs derived from Sin1^−/− MEFs transfected with indicated Sin1 constructs. Cells were serum-starved for 24 hr before stimulation by insulin (100 nM) for 30 min.

F–G, Sin1-D412G loses interaction with mTOR-KD in cells (F) and in vitro (G). IB of HA-IPs and WLCs derived from HEK293T cells transfected with indicated constructs (F), or GST pulldowns in the presence of indicated PIP-polysomes (G).

H, Compared to WT-Sin1, Sin1-D412G leads to an elevated Akt activation upon EGF stimulation. IB of WCLs derived from endogenous-Sin1-depleted OVCAR5 cells stably expressing indicated Sin1 constructs. Where indicated, cells were serum-starved for 36 hr before stimulation by EGF (100 ng/ml) for 10 min.

I, Compared to WT-Sin1, Sin1-D412G results in reduced apoptosis. IB of WCLs derived from endogenous-Sin1-depleted OVCAR5 cells stably expressing indicated Sin1 constructs. Where marked, cells were treated with etoposide for 24 hr before collection.

J–K, Compared to WT-Sin1, Sin1-D412G expressing cells display an elevated resistance to etoposide (J) or cisplatin (K) challenges. Various cell lines generated in (H) were cultured in 10% FBS-containing medium with the indicated concentrations of etoposide (J) or cisplatin (K) for 24 hr before performing cell viability assays. Data was shown as mean ± SD for three independent experiments. * indicates p<0.05 (student’s t-test).

L–M, Compared to WT-Sin1, Sin1-D412G expressing cells display enhanced colony formation (L) and soft-agar growth abilities (M). Data was shown as mean ± SD for three independent experiments. * indicates p<0.05 (student’s t-test).

N–P, Compared to WT-Sin1, Sin1-D412G expressing cells display enhanced tumor formation in a xenograft mouse model. 3×10⁶ of the generated cells in (H) were injected into nude mice (n=10 for each group) and monitored for tumor formation (O). Formed tumors were dissected (N) and weighed (P). As indicated, * represents p<0.05 calculated by student’s t-test.

Q, Compared to WT-Sin1, elevated Akt-pS473 levels were observed in Sin1-D412G expressing xenograft tumors. IB analysis of WCLs derived from xenografted tumors obtained in (N).

Cancer Patient-derived Mutations in the PH Domain of Sin1 Display Elevated Oncogenic Ability to Activate Akt through Losing the Sin1-PH Interaction with the mTOR Kinase Domain

In order to pinpoint the Sin1-PH/mTOR-KD interaction patch, we examined cancer patient-derived Sin1-PH mutations, assuming that loss of the Sin1-PH/mTOR-KD interaction might lead to elevated Akt activation, thus facilitating tumorigenesis. Analysis of TCGA data sets (cBio and Cosmic) reveals 6 somatic mutations in the Sin1-PH domain (Fig. S6B). Aside from two of these, most of the mutations in Sin1-PH compromised their interactions with mTOR-KD (Fig. S6C), leading to elevated mTORC2 activity towards phosphorylating Akt-Ser473 in cells (Fig. S6D–E). Importantly, as the D412G mutant robustly enhanced mTORC2 activity to elevate Akt-S473 phosphorylation in cells (Fig. 6D–E and Fig. S6D–E), we chose this mutant for further mechanistic and functional studies.

Notably, compared to WT-Sin1, D412G-Sin1-containing mTORC2 complexes also exhibited elevated activity towards phosphorylating Akt in vitro under starved or normal conditions (Fig. S6F), and this is in part due to its compromised binding to mTOR-KD (Fig. 6F–G). In addition, Sin1-D412G displayed enhanced mTORC2 kinase activity even under non-stimulated conditions (Fig. 6E and Fig. S6F), suggesting that Sin1-D412G containing mTORC2 may be more active through exposing the mTOR catalytic site. Collectively, these results reveal the D412 residue as part of a critical binding patch for mediating Sin1-PH interactions with the mTOR catalytic domain, in a manner that is distinct from the Sin1-PH/PtdIns(3,4,5)P₃-binding patch that includes R393, K428 and K464 (Fig. 4E).

To gain further insights into whether by losing inhibition of mTOR-KD, Sin1-D412G exerts oncogenic roles in cells via elevating mTOR kinase activity, we reintroduced Sin1-D412G or Sin1-WT in endogenous Sin1-depleted OVCAR5 ovarian cancer cells (Fig. S6G), since this cancerous mutation was identified in an ovarian cancer patient (sample ID: TCGA-13-1509). Compared to Sin1-WT, Sin1-D412G expressing cells displayed increased Akt phosphorylation (Fig. 6H) and correspondingly reduced cellular apoptosis triggered by etoposide (Fig. 6I), as well as enhanced resistance to chemotherapeutic drugs such as etoposide (Fig. 6J and Fig. S6H), cisplatin (Fig. 6K), doxorubicin (Fig. S6I) or Taxol (Fig. S6J). In addition to its anti-apoptotic function, elevated Akt activity in Sin1-D412G expressing cells also resulted in enhanced colony formation ability (Fig. 6L), soft agar growth (Fig. 6M) and increased tumor growth in a xenograft assay (Fig. 6N–Q and Fig. S6K). Furthermore, other Sin1-PH cancerous mutations including S449I, A451E and T456M that displayed significantly reduced interactions with mTOR-KD (Fig. S6C) also exhibited elevated and sustained Akt activation (Fig. S6L–M), as well as enhanced colony formation and soft agar growth ability (Fig. S6 N–Q). These results together support a model in which these Sin1 PH mutants may form a critical Sin1-PH/mTOR-KD interaction patch, and they are more oncogenic than Sin1-WT, in part due to elevated mTORC2/Akt oncogenic signaling resulted from their loss of inhibitory effects on mTOR-KD.

This is corroborated by depletion of endogenous Akt1 (Fig. S6R) or Akt2 (Fig. S6S) in Sin1-D412G expressing OVCAR5 cells, which sensitized cells to treatments with chemotherapeutic drugs including cisplatin (Fig. S6T) and doxorubicin (Fig. S6U), as well as reduced colony formation (Fig. S6V) and soft agar growth (Fig. S6W). Notably, compared with Sin1-WT, expression of Sin1-D412G did not lead to significant changes in cell cycle profiles (Fig. S6X–Z), highlighting that the oncogenic activity of Sin1-D412G is largely due to direct activation of mTORC2, rather than a secondary cell cycle effect (40) to augment the Akt oncogenic signaling.

Addition of the KRas-CAAX Sequence to the Carboxyl-terminus of Sin1 Generates a Relatively Constitutively Active mTORC2

Intrigued by the fact that membrane-targeted Akt mutants are constitutively active (41), analogously we generated two putative constitutively-active Sin1 mutants, one with a Src myristoylation tag at the N-terminus and the other one fused in frame with a KRas-CAAX motif at the carboxyl-terminus immediately after its PH motif (Fig. 7A), since the CAAX motif at the C-terminus of Ras creates a farnesyl moiety necessary for RAS membrane association and activation (42).

Fig. 7.

C-terminal tagging of the KRas-CAAX sequence to Sin1 partially rescues the deficiency of Sin1-CAA towards activating Akt.

A, A schematic illustration of the Myr-Sin1 or Sin1-CAAX chimera structure.

B, C-terminal addition of the KRas-CAAX sequence in part rescues Sin1-CAA in phosphorylating Akt-S473 in cells. Immunoblot (IB) analysis of whole cell lysates (WCLs) derived from Sin1^−/− MEFs transfected with indicated constructs. Where indicated, cells were serum starved for 24 hr and stimulated by 100 nM insulin for indicated time periods.

C–D, Addition of KRas-CAAX sequence does not affect mTORC2 complex integrity (C) or Sin1 interaction with Akt1 (D). IB of Flag-IPs and WCLs derived from 293T cells transfected with indicated constructs.

E–F, The CAAX tag alleviates Sin1-PH interaction with mTOR-KD. IB of GST pulldowns and WCLs derived from 293T cells transfected with indicated constructs.

G, Addition of a N-terminal-Myr tag, but not a C-terminal-CAAX tag largely rescues Akt phosphorylation in Akt1-R25C mutant. IB analysis of WCLs derived from DLD1-Akt1/2^−/− cells transfected with indicated constructs.

H, A proposed model for the PI(3,4,5)P₃-mediated mTORC2 activation mechanism.

Notably, the Sin1-CAAX mutant, but not the Myr-Sin1 mutant, led to elevated mTORC2 activity in transfected cells (Fig. S7A), and in part rescued Sin1-CAA towards phosphorylating Akt in cells (Fig. 7B) and in vitro (Fig. S7B), suggesting that spatially, C-terminal rather than N-terminal attachment of Sin1 to PM is critical for activating mTORC2. Moreover, the CAAX moiety did not influence mTORC2 complex integrity (Fig. 7C) or interaction with its substrate Akt (Fig. 7D), but in part attenuated the inhibitory binding of Sin1-PH to mTOR-KD (Fig. 7E–F), supporting the notion that the C-terminal CAAX tag could in part compensate for the loss of PtdIns(3,4,5)P₃ binding in the Sin1-CAA mutant, mainly through both releasing Sin1-PH-mediated inhibition on mTOR-KD and directly recruiting the Sin1 to PM (Fig. S5J and Fig. S7C–D). Similarly, the addition of an N-terminal Myr-tag to Akt1 in large rescued phosphorylation of the PtdIns(3,4,5)P₃-binding deficient Akt1-R25C mutant (Fig. 7G), while the C-terminal tagging of a CAAX sequence completely abrogated Akt activity, likely interfering with phosphorylation of the C-terminus of Akt (40) (Fig. 7G and Fig. S7E). Thus, similar to Myr-Akt, Sin1-CAAX could in part functionally phosphorylate Akt-S473 independent of PI(3,4,5)P₃ (Fig. S7F–G).

Collectively, our results support a model in which the Sin1-PH domain interacts with and inhibits the mTOR kinase domain, and the direct binding of PtdIns(3,4,5)P₃ with the Sin1-PH motif releases the inhibitory effect of Sin1, leading to mTORC2 activation and Akt phosphorylation (Fig. 7H).

DICUSSION

Although hyper-activation of the mTORC2/Akt pathway has been commonly observed in various human cancers (43, 44), where and how the mTORC2 kinase complex is activated upon growth stimulation has been remained elusive for decades. Distinct from mTORC1 that depends on proteins including Rag GTPase and Rheb GTPase for lysosomal recruitment and activation (12), in this study, we identified a non-protein molecule, PtdIns(3,4,5)P₃ as a direct upstream activator for mTORC2. Mechanistically, consistent with the crystal insights from the partial active mTOR complex (21), the active site of mTOR in the mTORC2 complex is largely governed by the PH domain of Sin1, and the binding of Sin1-PH to mTOR-KD under non-stimulated conditions blocks the access of the mTORC2 substrate, Akt to the mTOR active site for phosphorylation and activation. Upon insulin or growth stimulation, activated PI3K generates PtdIns(3,4,5)P₃ that directly binds Sin1-PH in part via R393, K428 and K464 residues, leading to attenuated Sin1-PH interaction with the mTOR kinase domain and subsequent exposure of the mTOR active site to Akt for its activation. Given that Sin1 is also the mTORC2 component responsible for recruiting certain mTORC2 substrates including SGK and Akt (45), it seems that Sin1 plays dual roles in mediating Akt activation by mTORC2: Sin1 controls the timing of the exposure of mTOR catalytic core and spatially recruits Akt to mTORC2 complex proximity for Akt-S473 phosphorylation and activation. However, the detailed molecular mechanisms underlying how these two events are coordinated warrants further investigation.

Interestingly, cancer patient-derived Sin1 oncogenic mutations (D412G characterized in this study and R81T characterized in (28)), appear to be mutually exclusive with either Akt1 gene amplification (Fig. S7H) or PIK3CA oncogenic mutations (E545K and H1047R) (46) (Fig. S7I). These observations support that PI3K/Sin1/Akt may be in the same genetic pathway, highlighting the critical role of PI3K in governing mTORC2 activation. As previously ribosome association has been shown to be critical for mTORC2 activation (19), and mTORC2 was observed at various cellular components including nucleus, mitochondria, ER and plasma membrane (17), whether there are populations of mTORC2 sub-complexes at various cellular locations, as well as how and whether mTORC2 shuttles between ribosomes and plasma membrane or other cellular components for activation warrant further exploration. Interestingly, two recent studies suggest that both mTORC2 and mTORC1 form dimers for function (27, 47), echoing previous discoveries in yeast that yeast TOR complexes are dimmers (48). Consistently, dimerization of other members of the PIKK super-families including DNA-PK (49) and ATM (50) have been observed and shown to be critical for their activation. This dimerization or even oligomerization feature for the mTOR complex potentially adds additional layers of regulations for the mechanistic studies, which may require a patch of phospholipids, or lipid rafts enriched with PtdIns(3,4,5)P₃ to hook mTORC2 for activation.

METHODS

Cell Lines

All cells were maintained in DMEM or RPMI supplemented with 10% FBS and 100 U/mL penicillin and streptomycin. HAP1-Sin1^−/− and matching parental cells were purchased from Horizon in June 2015. All other cell lines were routinely maintained and examined in the lab but have not been further authenticated.

Cell Culture

Cell transfection was performed using lipofectamine and plus reagents as described previously (40). Packaging of lentiviral shRNA viruses and retroviral MSCV-Sin1 expressing viruses, as well as subsequent infection of various cell lines were performed according to the protocol described previously (51). Kinase inhibitors wortmannin (Selleck S2758), LY2940002 (Cell Signaling Technology 9901), Plk90 (a kind gift from the Shepherd Lab), BKM120 (Selleck S2247) and YM201636 (Santa Cruz, SC 204193) were used at the dose as indicated. PIP strips (P6001), PIP polysomes (Y0000, Y-P0000, Y-P0003, Y-P0004, Y-P0005, Y-P034, Y-P0035, Y-P0039 and Y-P045) and PIP beads (P-B00Ss) were purchased from Echelon Biosciences. EGF (Sigma E9644), insulin (Invitrogen 41400-045), IGF-1 (Sigma I3769), etoposide (Sigma E1383), cisplatin (Selleck S1166), doxorubicin (Sigma D1515) and taxol (Sigma T7191) were used at the indicated doses.

Antibodies

All antibodies were used at a 1:1000 dilution in 5% non-fat milk for western blotting. Anti-phospho-Ser473-Akt antibody (mouse mAb 4051 and rabbit mAb 4060), anti-phospho-Thr308-Akt antibody (2965), anti-phospho-Thr450-Akt antibody (12178), anti-Akt1 antibody (2938), anti-Akt total antibody (4691), anti-phospho-Ser9-GSK3β antibody (5558), anti-GSK3β antibody (12456), anti-Rictor antibody (9476), anti-mTOR antibody (2983), anti-GβL antibody (3274), anti-Sin1 antibody (12860 or K87 generated in the Su lab), anti-phospho-NDRG1 (3217), anti-phospho-FOXO1(Thr24) /FOXO3a(Thr32) antibody (9464), anti-FOXO1 antibody (9454), anti-phospho-T246-PRAS40 antibody (13175), anti-PRAS40 antibody (2691), anti-PIK3CA (p110α) antibody (4249), anti-GST antibody (2625), polyclonal anti-Myc-Tag antibody (2278), monoclonal anti-Myc-Tag (2276), anti-cleaved PARP antibody (5625), and anti-cleaved caspase-3 antibody (9661) were purchased from Cell Signaling Technology. Polyclonal anti-HA antibody (sc-805) was purchased from Santa Cruz. Anti-Tubulin antibody (T-5168), anti-Vinculin antibody (V-4505), polyclonal anti-Flag antibody (F-2425), monoclonal anti-Flag antibody (F-3165, clone M2), anti-Flag agarose beads (A-2220), anti-HA agarose beads (A-2095), peroxidase-conjugated anti-mouse secondary antibody (A-4416) and peroxidase-conjugated anti-rabbit secondary antibody (A-4914) were purchased from Sigma. Monoclonal anti-HA antibody (901502) was purchased from BioLegend. The anti-phospho-Ser657-PKC antibody (sc-12356) was purchased from Santa Cruz Biotechnology. The anti-PIKFYVE antibody was generated by Cell Signaling Technology.

PIP Strip Overlay Assays

The overlay assays were performed according to manufacture’s instructions with minor modifications. Briefly, PIP membranes were blocked with 3% BSA in 1xTBST (0.1% Tween-20) at room temperature for 1 hr. 2 µg/ml purified indicated GST or GST-Sin1 proteins were incubated with PIP membranes at room temperature for another 2 hr, followed by gentle washing in 1xTPST for 3×5 min. Anti-GST antibody (1:1000 from CST 2625) was prepared in 3% BSA in 1xTBST and incubated with the membranes for 1 hr at room temperature. After gentle washing in 1xTPST for 3×5 min, membranes were subjected to western blotting.

Immunoblots and Immunoprecipitation

Cells were lysed as indicated in EBC buffer (50 mM Tris pH 7.5, 120 mM NaCl, 0.5% NP-40) or CHAPS (50 mM Tris pH 7.5, 120 mM NaCl, 0.3% CHAPS) supplemented with protease inhibitors (Complete Mini, Roche) and phosphatase inhibitors (phosphatase inhibitor cocktail set I and II, Calbiochem). The protein concentrations of lysates were measured by the Beckman Coulter DU-800 spectrophotometer using the Bio-Rad protein assay reagent. Same amounts of whole cell lysates were resolved by SDS-PAGE and immunoblotted with indicated antibodies. For immunoprecipitation, 1000 µg lysates were incubated with the indicated antibody (1–2 µg) for 3–4 hr at 4 °C followed by 1 hr incubation with Protein A/G sepharose beads (GE Healthcare). Immunoprecipitants were washed five times with NETN buffers (20 mM Tris, pH 8.0, 100 mM NaCl, 1 mM EDTA and 0.5% NP-40) or CHAPS buffers before being resolved by SDS-PAGE and immunoblotted with indicated antibodies.

In vitro Kinase Assays

mTOR in vitro kinase assays were adapted from described previously (40). Briefly, the reaction contains 100 ng mTOR (EMD/Calbiochem 475987, contains truncated mTOR kinase-domain with amino acids 1360–2549), 2 µg of GST or GST-Akt1-tail (aa 409–480) and 100 µM cold ATP (with 1 µCi/mL γ-³²P-ATP for hot assays) in the kinase assay buffers (50 µM HEPES pH 7.5, 10 µM MnCl₂, 10 µM MgCl₂, 2µM DTT and 0.5 µM EGTA). As illustrated in the figures, indicated amounts of GST or GST-Sin1-PH recombinant proteins were added to the reactions for competition assays. The reactions were incubated at 30°C for 30 min. The reaction was stopped by the addition of SDS containing lysis buffer and resolved by SDS-PAGE. Phosphorylation of GST-Akt1-tail was detected by autoradiography.

mTORC2 in vitro kinase assays were performed as described below. Briefly, HA immunno-precipitation in CHAPS buffer was performed in HEK293 cells transfected with HA-Rictor under serum starvation (for 48 hr) or insulin stimulation (100 nM insulin for 30 min) conditions. HA-Rictor IPs were washed extensively in CHAPS buffer and supplied as the kinase sources for in vitro kinase assays. 10 µL of HA-Rictor IPs were incubated with 2 µg of GST-Akt1-tail (aa 409–480) and 200 µM cold ATP in the kinase assay buffer (50 µM HEPES pH 7.5, 10 µM MnCl₂, 10 µM MgCl₂, 2µM DTT, 0.5 µM EGTA), in the presence of indicated amount of PIP-polysomes at 30°C for 1 hr. The reactions were gently tapped every 10 min to mix the reactions well and the reactions were stopped by addition of SDS containing lysis buffer and resolved by SDS-PAGE. Phosphorylation of GST-Akt1-tail was detected by western blotting using the Akt-pS473 antibody.

Immunofluorescence and Confocal Analysis

Cells were grown on glass coverslips for 24 hr and fixed with 3.7% formaldehyde in 1xPBS for 15 min at room temperature and permeabilized with 0.1% Triton X-100 in 1xPBS for 5 min. Samples were rinse three times in 1xPBS with 5 min each wash. Coverslips were then blocked for 30 min with 5% BSA and incubated with primary antibodies for 60 min. After 3×5 min 1xPBST (0.1% Tween-20) washes, the coverslips were incubated with Alexa-488 conjugated goat anti Rabbit secondary antibody or Alexa-594 conjugated goat anti mouse secondary antibody (Invitrogen) for 60 min and washed three times with 1xPBST. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI) for 10 min. Coverslips were rinsed 2×3 min with 1xPBS and mounted onto slides using prolong gold anti-fade reagent (Invitrogen).

Mouse Xenograft Assays

All animal experiments were approved by the BIDMC IACUC review board. Briefly, 2.5×10⁶ endogenous Sin1-depleted OVCAR5 cells stably expressing MSCV-Sin1-WT-HA or MSCV-Sin1-D412G-HA were mixed with matri-gel (1:1) and injected into the flank of 10 female nude mice (5 weeks old). Tumor size was measured every three days with a caliper, and the tumor volume was determined with the formula: L*W²*0.52, where L is the longest diameter and W is the shortest diameter. After 18 days, mice were sacrificed and xenografted solid tumors were dissected, then tumor weights were measured and recorded.

SIGNIFICANCE.

The Sin1-PH domain interacts with the mTOR kinase-domain to suppress mTOR activity, and PtdIns(3,4,5)P₃ binds the Sin1-PH domain to release its inhibition on the mTOR kinase-domain, leading to mTORC2 activation. Cancer patient-derived Sin1-PH domain mutations gain oncogenicity by loss of suppressing mTOR activity as a means to facilitate tumorigenesis.

Abbreviations

KD

kinase domain

PH

pleckstrin domain

PIP₃

PtdIns(3,4,5)P₃