mTOR Signaling: PI3K/AKT Upstream Activation, mTORC1/mTORC2 Architecture, and Nutrient Sensing in Research

# mTOR Signaling: PI3K/AKT Upstream Activation, mTORC1/mTORC2 Architecture, and Nutrient Sensing in Research

For Research Use Only (RUO). Not for use in humans or animals.

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The mechanistic target of rapamycin (mTOR) kinase operates as the central integrator of nutrient availability, growth factor signals, energy status, and stress inputs, translating these diverse cues into coordinated anabolic and catabolic outputs. mTOR functions within two structurally and functionally distinct multiprotein complexes — mTORC1 and mTORC2 — that differ in subunit composition, upstream regulation, substrate specificity, and rapamycin sensitivity. The PI3K/AKT pathway provides the primary growth factor input to mTORC1, while the Ragulator/RAG GTPase system relays amino acid sufficiency. Understanding the mechanistic architecture of this network — from receptor tyrosine kinase activation through TSC1/TSC2 and RHEB to S6K1, 4EBP1, and ULK1 substrates — is essential for interpreting experiments with rapamycin, Torin1, and PI3K/AKT inhibitors in research contexts.

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mTOR Complex Architecture

mTORC1: The Anabolic Effector Complex

mTORC1 comprises six components:

• •mTOR: 289 kDa Ser/Thr kinase; PIKK family; N-terminal HEAT repeats (protein-protein interactions) -> FAT domain -> FRB domain (FKBP12-rapamycin binding site) -> kinase domain -> FATC domain
• •Raptor (Regulatory Associated Protein of mTOR): scaffold subunit; contains TOS motif-binding domain that docks TOS (TOR signaling) motif-containing substrates (S6K1 FDIDL; 4EBP1 FEMDI); required for substrate recruitment and mTORC1 activity at lysosomes
• •mLST8 (mammalian Lethal with Sec Thirteen 8; also GbetaL): WD40 repeat protein; stabilizes the kinase domain activation loop; present in both mTORC1 and mTORC2
• •PRAS40 (Proline-Rich AKT Substrate of 40 kDa): endogenous inhibitor; competes with substrates for Raptor TOS-binding; phosphorylated by AKT (T246) to relieve inhibition
• •DEPTOR (DEP Domain-Containing mTOR Interacting Protein): endogenous mTOR inhibitor; present in both complexes; its phosphorylation by mTORC1 creates a degradation signal
• •Tti1/Tel2: chaperone complex that stabilizes PIKK family members including mTOR

mTORC1 resides predominantly on lysosomal membranes when active — this compartmentalization is essential for RHEB-mediated activation (RHEB is also lysosomal) and amino acid sensing via the Ragulator-RAG system.

mTORC2: The AKT Activator

mTORC2 shares mTOR, mLST8, DEPTOR, and Tti1/Tel2 with mTORC1, but contains unique subunits:

• •Rictor (Rapamycin-Insensitive Companion of mTOR): defining mTORC2 subunit; no TOS-binding activity; interacts with mSin1 and scaffolds mTORC2 substrates including AKT and SGK1
• •mSin1 (Mammalian Stress-Activated Map Kinase-Interacting Protein 1): contains PH domain that binds PIP3; recruited to membranes by PI3K activation; PH domain gates mTORC2 activity
• •Protor1/2 (Protein Observed With Rictor 1/2): Rictor-interacting proteins; required for mTORC2-mediated SGK1 (Ser422) phosphorylation; dispensable for AKT-HM phosphorylation

mTORC2 is insensitive to acute rapamycin treatment (FKBP12-rapamycin binds FRB on mTOR but cannot access mTORC2 due to Rictor steric exclusion). Prolonged rapamycin treatment can indirectly suppress mTORC2 by sequestering newly synthesized mTOR before it assembles into mTORC2.

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PI3K/AKT Upstream Activation

Class I PI3K: Catalytic and Regulatory Subunits

Class IA PI3Ks are obligate heterodimers of a p110 catalytic subunit (p110alpha/PIK3CA, p110beta/PIK3CB, p110delta/PIK3CD) and a p85 regulatory subunit (p85alpha/p85beta/p55alpha/p55beta/p50alpha). Receptor tyrosine kinases (RTKs) activate PI3K through two mechanisms: (1) direct phosphotyrosine recruitment -- p85 SH2 domains bind pYXXM motifs on activated RTKs (e.g., PDGFR, c-Kit) or IRS1/IRS2 adaptors (downstream of InsR/IGF1R); (2) RAS-GTP binding -- p110alpha contains a RAS-binding domain (RBD) that directly recruits activated RAS, allosterically stimulating p110alpha kinase activity.

Upon recruitment to the membrane, p110 catalyzes phosphorylation of PtdIns(4,5)P2 (PIP2) to PtdIns(3,4,5)P3 (PIP3). PIP3 is the key second messenger that recruits PH domain-containing proteins including AKT and PDK1 to the inner plasma membrane leaflet.

PTEN (Phosphatase and Tensin Homolog) is the primary negative regulator: its lipid phosphatase domain dephosphorylates PIP3 back to PIP2, directly opposing PI3K. PTEN is among the most commonly lost tumor suppressors (glioblastoma ~40%, prostate ~40%, endometrial ~50%), and its loss drives constitutive PI3K/AKT/mTOR activation.

PIK3CA hotspot mutations (H1047R, E545K, E542K) occur in ~30% of breast cancers, ~20% of endometrial cancers, and ~10% of colorectal cancers. H1047R lies in the kinase domain activation loop and increases catalytic activity; E545K/E542K are in the helical domain and disrupt inhibitory p85 contacts.

AKT: Three-Step Activation

AKT (also PKB) family has three isoforms (AKT1/2/3) with overlapping but distinct substrate specificities. Full AKT activation requires three events:

1. PIP3 recruitment: AKT PH domain binds PIP3 (Kd ~50 nM); conformational change exposes T308 (activation loop) and S473 (hydrophobic motif, HM)

2. PDK1 phosphorylation of T308: PDK1 is also recruited to PIP3 via its PH domain; phosphorylates AKT T308 in the activation loop, activating kinase ~100-fold; PDK1 alone is insufficient for full AKT activity

3. mTORC2 phosphorylation of S473: mTORC2 phosphorylates the HM of AKT at S473; this phosphorylation stabilizes the active conformation and enables phosphorylation of a subset of substrates (e.g., FOXO3a, TSC2 T1462) that require dual phosphorylation

AKT phosphorylates a broad substrate consensus (RxRxxS/T): TSC2 (T1462, S939, S981), PRAS40 (T246), GSK3alpha/beta (S21/S9), FOXO1/3a/4 (multiple sites causing nuclear export), BAD (S136), MDM2 (S166/S186, nuclear translocation promotes p53 degradation), and many others.

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TSC1/TSC2 Complex and RHEB GTPase

TSC Complex as Signal Integrator

The TSC1 (hamartin)-TSC2 (tuberin)-TBC1D7 trimeric complex is the central negative regulator of mTORC1. TSC2 contains a GAP (GTPase Activating Protein) domain that stimulates intrinsic GTPase activity of RHEB (Ras Homolog Enriched in Brain), converting active RHEB-GTP to inactive RHEB-GDP.

Multiple upstream kinases converge on the TSC complex:

mTORC1-activating signals (phosphorylate/inhibit TSC2):

• •AKT: phosphorylates TSC2 at T1462, S939, S981 -> 14-3-3 sequestration -> TSC2 dissociation from lysosomes -> RHEB-GTP accumulates -> mTORC1 active
• •ERK1/2: phosphorylates TSC2 at S664/S540 -> TSC complex destabilization
• •RSK1 (downstream of ERK): phosphorylates TSC2 at S1798
• •IKKbeta: phosphorylates TSC1 at S487/S511 -> TSC complex disruption (inflammatory mTOR activation)

mTORC1-inhibiting signals (activate TSC complex):

• •AMPK: phosphorylates TSC2 at S1387 (activating GAP activity) AND Raptor at S792 (directly inhibiting mTORC1)
• •REDD1 (regulated by hypoxia/HIF-1alpha and glucocorticoids): promotes TSC1-TSC2 interaction, activating the GAP complex
• •DYRK1A: phosphorylates TSC2 at S1005, activating the complex
• •GSK3beta: phosphorylates TSC2 at S1337/T1341 (priming by AMPK at S1345 required)

RHEB GTPase

RHEB is a small GTPase (Ras superfamily) constitutively farnesylated and associated with lysosomal membranes. RHEB-GTP directly activates mTOR kinase by binding the mTOR N-lobe and allosterically stimulating its catalytic activity -- the precise molecular mechanism involves RHEB-GTP destabilizing the FKBP38 inhibitory interaction with the mTOR FRB domain.

RHEB has no known GEF (Guanine nucleotide Exchange Factor); its nucleotide exchange rate is intrinsically fast, so it cycles rapidly to GTP unless TSC2-GAP accelerates GTP hydrolysis. Tuberous sclerosis complex (TSC1/TSC2 germline mutations) causes constitutive RHEB-GTP and mTORC1 hyperactivation, leading to benign tumor formation (hamartomas) in brain, kidney, lung, skin.

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mTORC1 Substrates: S6K1, 4EBP1, and ULK1

S6K1 (RPS6KB1): Translational Regulation

S6K1 is phosphorylated by mTORC1 at T389 (HM) -- this is the primary readout of mTORC1 activity (detected by Western blot with anti-pS6K1-T389). T389 phosphorylation enables PDK1 to phosphorylate S6K1 T229 (activation loop) for full kinase activation. Active S6K1 phosphorylates:

• •RPS6 (ribosomal protein S6) at S235/S236/S240/S244/S247 -- facilitates ribosome biogenesis
• •eIF4B (S422) -- promotes 5-prime-cap mRNA unwinding helicase activity of eIF4A
• •PDCD4 (S67/S71/S76) -- targets PDCD4 for SCF-betaTrCP-mediated ubiquitination and degradation
• •eEF2K (S366) -- inhibits eEF2K, relieving eEF2 phosphorylation and promoting elongation
• •IRS1 (S307, S636/S639, S1101) -- negative feedback on insulin/IGF1 signaling

The S6K1->IRS1 negative feedback is critical: mTORC1 activation paradoxically reduces PI3K/AKT/mTORC1 signaling by degrading IRS1. This feedback is disrupted by rapamycin -- a key reason why rapamycin can paradoxically increase AKT activity in some cancer cells.

4EBP1 (EIF4EBP1): Cap-Dependent Translation Gate

4EBP1 is the primary translational repressor controlled by mTORC1. Hypophosphorylated 4EBP1 binds eIF4E (the cap-binding protein) with high affinity, preventing eIF4E association with eIF4G and assembly of the eIF4F complex (eIF4E-eIF4G-eIF4A). mTORC1 phosphorylates 4EBP1 sequentially:

1. T37/T46 (priming phosphorylation, first)

2. S65, T70 (subsequent)

3. S83 (final)

Hyperphosphorylated 4EBP1 dissociates from eIF4E, liberating eIF4E to form eIF4F and drive cap-dependent translation of 5-prime-TOP mRNAs encoding ribosomal proteins and translation factors, and structured 5-prime-UTR mRNAs encoding oncoproteins (cyclin D1, MYC, MCL-1, survivin).

Key distinction: Rapamycin partially inhibits 4EBP1 phosphorylation (incomplete and variable) but fully suppresses S6K1-T389. Catalytic mTOR inhibitors (Torin1, PP242/torkinib, AZD2014/vistusertib) fully suppress both -- 4EBP1 dephosphorylation (especially T37/46) correlates better with translational and anti-proliferative effects than S6K1 alone.

ULK1: Autophagy Initiation

mTORC1 directly phosphorylates ULK1 (Unc-51-Like Autophagy Activating Kinase 1) at S757 (in humans), which disrupts ULK1 interaction with AMPK and inhibits ULK1 kinase activity. When mTORC1 is inhibited (nutrient deprivation, Torin1, rapamycin), ULK1 is dephosphorylated and activated, initiating autophagy:

• •Active ULK1 phosphorylates BECLIN-1 (S14), ATG13, FIP200, and ATG101 within the ULK1 complex
• •ULK1 also phosphorylates PI3KC3/VPS34 complex components (BECLIN-1, ATG14L) to generate PI3P at the phagophore assembly site
• •This coordinates with the ATG12-ATG5-ATG16L1 complex and LC3 lipidation (LC3-I -> LC3-II) for autophagosome membrane elongation

AMPK and mTORC1 act antagonistically on ULK1: AMPK phosphorylates S317 and S777 to activate ULK1, while mTORC1 at S757 prevents this. Direct AMPK-mTOR crosstalk occurs at the mTORC1 level as well (Raptor S792 phosphorylation by AMPK).

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Amino Acid Sensing: Ragulator-RAG GTPase Axis

The most mechanistically intricate upstream input to mTORC1 is amino acid sensing, which acts through a lysosome-based signaling platform independent of TSC2/RHEB.

RAG GTPases

Four RAG GTPases exist in two heterodimeric pairs: RAGA/RAGB (functionally redundant) dimerize with RAGC/RAGD (functionally redundant). The RAG heterodimer is constitutively associated with the lysosomal surface via the Ragulator complex (LAMTOR1-5). When amino acids are sufficient:

• •RAGA/B is loaded with GTP (active state) -- facilitated by the FLCN-FNIP2 complex acting as a GAP for RAGC/D (making RAGC/D-GDP) and by SLC38A9 acting as a GEF for RAGA/B
• •RAGC/D is loaded with GDP (permissive state)
• •The RAGA/B-GTP--RAGC/D-GDP heterodimer recruits mTORC1 (via Raptor interaction with RAGA/B-GTP) to the lysosomal surface where RHEB resides

When amino acids are depleted:

• •GATOR1 complex (DEPDC5-NPRL2-NPRL3) acts as a GAP for RAGA/B, converting RAGA/B to GDP-bound inactive state
• •mTORC1 dissociates from lysosomes and is inactivated
• •GATOR2 complex (WDR24-WDR59-MIOS-SEH1L-SEC13) inhibits GATOR1; GATOR2 is itself inhibited by SESN1/2 (sensing leucine) and CASTOR1/2 (sensing arginine)

Specific Amino Acid Sensors

Leucine sensing: SESN2 (Sestrin 2) directly binds leucine (Kd ~20 uM); leucine binding disrupts SESN2 interaction with GATOR2, releasing GATOR2 to inhibit GATOR1, allowing RAGA/B-GTP accumulation and mTORC1 activation.

Arginine sensing: CASTOR1 (Cellular Arginine Sensor for mTORC1) directly binds arginine; arginine disrupts CASTOR1 homodimer interaction with GATOR2, similarly releasing GATOR2 brake. SLC38A9, a lysosomal arginine transporter, also senses intralysosomal arginine.

Lysosomal v-ATPase: The lysosomal vacuolar H+-ATPase (v-ATPase) is required for mTORC1 activation by amino acids -- it interacts with Ragulator and senses intralysosomal amino acid levels. v-ATPase inhibition (Bafilomycin A1, ConA) blocks amino acid-dependent mTORC1 activation.

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AMPK-mTOR Crosstalk

AMPK (AMP-activated protein kinase) is the cellular energy sensor: it is activated when AMP:ATP ratio rises (energy stress), by LKB1 (STK11) and CaMKKbeta kinases. AMPK inhibits mTORC1 through two parallel mechanisms:

1. TSC2 phosphorylation (S1387): activates TSC GAP, converting RHEB to GDP-bound inactive state

2. Raptor phosphorylation (S792): creates a 14-3-3 binding site on Raptor; 14-3-3 binding inhibits mTORC1 catalytic activity directly

Additionally, AMPK activates ULK1 (S317/S777) to initiate autophagy under energy stress -- opposed by mTORC1 (ULK1-S757 phosphorylation).

Metformin activates AMPK through Complex I inhibition (raising AMP/ATP). AICAR (aminoimidazole-4-carboxamide ribonucleotide) is an AMPK activator widely used in research (converted intracellularly to ZMP, an AMP analog).

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Research Tools for mTOR/PI3K/AKT Investigation

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Experimental Protocols

Protocol 1: mTORC1 Activity Western Blot Panel (Rapamycin vs Torin1 Comparison)

This protocol establishes the differential sensitivity of mTORC1 substrates to allosteric (rapamycin) vs catalytic (Torin1) mTOR inhibition -- critical for distinguishing rapamycin-sensitive from rapamycin-resistant mTORC1 outputs.

Reagents: Rapamycin (100 nM), Torin1 (250 nM), RIPA-based lysis buffer with PhosSTOP + protease inhibitors, antibodies: pS6K1-T389, total S6K1, p4EBP1-T37/46, total 4EBP1, pAKT-S473, pAKT-T308, total AKT, beta-actin

Protocol:

1. Seed cells in 6-well plates; grow to 70-80% confluence; serum-starve 4 h to reduce basal signaling

2. Treat: DMSO vehicle, rapamycin 100 nM, Torin1 250 nM; 1 h at 37 degrees C; then restimulate with serum (10% FBS) or insulin (100 nM) for 30 min

3. Aspirate media; add 150 ul ice-cold lysis buffer per well; scrape; collect; rotate 15 min 4 degrees C; centrifuge 13,000xg 15 min; collect supernatant

4. Quantify protein (BCA); normalize 30-50 ug per lane; denature (95 degrees C, 5 min, reducing); resolve on 10-12% SDS-PAGE; transfer to PVDF

5. Block 5% BSA in TBST 1 h; primary antibodies overnight 4 degrees C

6. Wash; HRP secondary; ECL detection

7. Expected pattern: rapamycin abolishes pS6K1-T389 but partially preserves p4EBP1-T37/46 and elevates pAKT-S473 (IRS1 feedback relief); Torin1 abolishes both pS6K1-T389 and p4EBP1-T37/46, reduces pAKT-S473

8. Quantify band intensities (ImageJ); normalize phospho/total; calculate fold-change vs vehicle

Protocol 2: Amino Acid Withdrawal and Re-Addition mTORC1 Activation Assay

Tests the RAG/Ragulator amino acid sensing pathway distinct from RHEB/TSC-mediated growth factor regulation.

Reagents: Amino acid-free DMEM or EBSS, MEM amino acid solution 50x (Sigma M5550), Bafilomycin A1 (100 nM), lysis buffer, pS6K1-T389/p4EBP1 antibodies

Protocol:

1. Incubate cells in amino acid-free medium (EBSS) 1 h to deplete mTORC1

2. Re-add amino acids (MEM AA solution, 1x final) for 15, 30, 60 min time course (without serum) to assess amino acid-specific mTORC1 re-activation via RAG GTPases

3. Parallel experiment: add Bafilomycin A1 (100 nM) 30 min before amino acid re-addition -- should block mTORC1 re-activation by disrupting v-ATPase/Ragulator signaling

4. Leucine dose-response: add individual amino acids (leucine 0.1-10 mM, arginine 0.1-5 mM, glutamine 0.5-10 mM) to AA-depleted cells; determine EC50 for mTORC1 activation per amino acid

5. Western blot for pS6K1-T389 and p4EBP1-T37/46 at each time point and dose

6. Control: Raptor siRNA knockdown confirms mTORC1 specificity of re-activation signal

Protocol 3: AKT Phosphorylation by Flow Cytometry (Single-Cell Resolution)

Quantifies pAKT-S473 and pAKT-T308 at single-cell resolution for heterogeneous populations.

Reagents: 16% formaldehyde, methanol (-20 degrees C), anti-pAKT-S473-Alexa488 (CST #4058), anti-pAKT-T308-PE (CST), Fc block, FACS buffer (PBS + 0.5% BSA + 0.02% sodium azide)

Protocol:

1. Treat cells with PI3K/AKT inhibitors (MK-2206 1-10 uM, BYL719 1 uM, BEZ235 100 nM) or stimulate with EGF (100 ng/ml), insulin (100 nM) for desired time

2. Fix: add formaldehyde to 1.6% final in cell culture medium; 10 min RT; pellet 400xg 5 min

3. Permeabilize: resuspend in ice-cold methanol; 30 min -20 degrees C (phosphoepitope preservation); wash 2x with FACS buffer

4. Block: Fc block 10 min; then primary antibodies (anti-pAKT-S473-Alexa488 + anti-pAKT-T308-PE, 1:50 each) 1 h RT

5. Wash 2x; resuspend in FACS buffer; acquire on flow cytometer; record 10,000-50,000 events

6. Analysis: gate singlets; plot pAKT-S473 vs pAKT-T308; calculate MFI; inhibitor IC50 by MFI vs dose

7. MK-2206 (AKT allosteric) suppresses both S473 and T308; BYL719 suppresses both in PIK3CA-mutant but not PTEN-null cells

Protocol 4: mTOR Lysosomal Localization by Immunofluorescence

Visualizes mTOR recruitment to LAMP1-positive lysosomes -- direct readout of RAG/Ragulator activation.

Reagents: Anti-mTOR antibody (CST #2983, rabbit), anti-LAMP1 (mouse, DSHB H4A3), secondary antibodies (anti-rabbit Alexa488, anti-mouse Alexa555), 4% paraformaldehyde, 0.1% saponin, DAPI

Protocol:

1. Seed cells on glass coverslips in 24-well plates; grow overnight

2. Treat: amino acid withdrawal 1 h (mTOR diffuse, cytoplasmic) vs re-addition 30 min (mTOR lysosomal); or Torin1 (mTOR remains cytoplasmic, not lysosomal)

3. Fix: 4% PFA in PBS 15 min RT; wash 3x PBS

4. Permeabilize: 0.1% saponin in PBS 10 min; block 5% BSA + 0.1% saponin 30 min

5. Primary antibodies: anti-mTOR (1:200) + anti-LAMP1 (1:100); 2 h RT or overnight 4 degrees C

6. Secondary antibodies: anti-rabbit Alexa488 + anti-mouse Alexa555; 1 h RT; DAPI; mount with ProLong Gold

7. Confocal imaging: z-stack acquisition; 0.3 um step size; 63x oil objective

8. Measure Pearson correlation coefficient of mTOR/LAMP1 co-localization (ImageJ JACoP); amino acid replete: r >0.6; withdrawn: r <0.2; Torin1 does not alter lysosomal localization (mTOR localizes but is kinase-inactive)

Protocol 5: 4EBP1 Phosphorylation State Shift Assay (Phos-tag Gel)

Resolves all 4EBP1 phosphorylation states by mobility shift -- superior to standard Western blot for complete 4EBP1 phosphorylation status.

Reagents: Phos-tag Acrylamide (Wako AAL-107, 50 uM final), MnCl2 (100 uM), 10% acrylamide gel mix, EDTA wash buffer (transfer step), anti-4EBP1 antibody (CST #9644)

Protocol:

1. Prepare 10% Phos-tag SDS-PAGE: add Phos-tag acrylamide (50 uM) and MnCl2 (100 uM) to standard acrylamide mix; cast gel; allow 1 h polymerization

2. Load 20-30 ug protein per lane; run at 20 mA constant current (~3-4 h for complete resolution)

3. CRITICAL: Before transfer, incubate gel 2x 15 min in transfer buffer + 1 mM EDTA (chelates Mn2+); then standard wet transfer 2 h at 100V

4. Western blot with anti-4EBP1 total antibody; ECL detection

5. Band pattern: active mTORC1 conditions show alpha, beta, gamma, delta phosphoforms (slowest migration = hyperphosphorylated delta/gamma); rapamycin shifts partially toward faster forms; Torin1 shifts all 4EBP1 to fastest (hypophosphorylated alpha/beta forms)

6. Quantify each form as % total 4EBP1; hyperphosphorylated fraction inversely correlates with translational repression

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Feedback Regulation and Resistance Mechanisms

mTORC1->IRS1 feedback (S6K1 arm): Chronic mTORC1 activation leads to S6K1-mediated IRS1 phosphorylation and degradation, attenuating PI3K/AKT/mTORC1 signaling. Rapamycin relieves this brake, paradoxically increasing pAKT-S473 (via mTORC2) and pAKT-T308 (via released IRS1/PI3K) -- a pro-survival rebound seen in multiple cancer cell line settings.

mTORC2->AKT->mTORC1 input: mTORC2 phosphorylates AKT-S473, which in turn phosphorylates TSC2-T1462, activating mTORC1 -- a feedforward loop. Dual mTOR inhibitors (Torin1, AZD2014) break this loop by inhibiting both mTORC1 and mTORC2.

4EBP1 hyperphosphorylation as resistance mechanism: In some cancer cells, mTORC1 hyperactivity or alternative kinases (CDK1, ERK1/2) contribute to 4EBP1 T37/46 phosphorylation, maintaining 4EBP1 phosphorylation even under rapamycin treatment -- explaining why translational outputs (MYC, cyclin D1, survivin protein levels) are often rapamycin-resistant while S6K1-T389 is fully suppressed.

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Disease Contexts

TSC1/TSC2 Mutations (Tuberous Sclerosis Complex): Germline loss-of-function mutations in TSC1 or TSC2 cause tuberous sclerosis complex -- hamartomas in brain (tubers, subependymal giant cell astrocytomas/SEGA), kidney (angiomyolipomata), lung (lymphangioleiomyomatosis/LAM), and skin. mTORC1 is constitutively hyperactive (RHEB-GTP accumulates). Rapalogs (everolimus) reduce SEGA and AML volumes in research models.

PIK3CA/PTEN-Driven Cancers: Breast cancer PIK3CA H1047R mutations are studied with BYL719 (alpelisib) -- selectively blocks PI3Kalpha, suppressing pAKT-T308/S473 in PIK3CA-mutant but not KRAS-mutant cells. PTEN-null prostate and endometrial cancer cell lines serve as models of maximal PI3K/AKT/mTOR pathway activation.

IDH-mutant Glioma: 2-HG from IDH1/2 mutations activates mTORC1 in part by generating NADPH imbalance and promoting PI3K activity -- linking oncometabolite biology to mTOR. Additionally, mTORC1-dependent cap-dependent translation of HIF-1alpha contributes to cell survival under hypoxia.

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