The TOR signalling pathway

TOR substrates

S6K1 + 4EBP1. A TOR substrate dock in S6K1+4EBP1. A Tor substrate dock in S6k1 + 4EBP1 - TOR signalling or TOS motif

Larval fat body

some of the flies are expressing TOR transgene RNAi ( knock down of TOR in cell) - the TOR hypomorphs: have a small larval body + size;

TOR2

also function in actin polymerization

FRB

gate keeper

Genetic evidence

genetic evidence in flies and biochemical evidence in mammalian ells indicate that TSC1 and TSC2 protein complex inhibit TOR. TSC1 and TSC2 are products of tumor suppror genes TSC1 and TSC2 - mutations in which cause tuberous sclerosis. The TSC1/2 complex is inactivated in response to insulin via PKB mediated phosphorylation of 3 sites of TSC-2.

Tor1 cells

have only a mild phenotype.

Q. What are the mechanisms that mediate and integrate many parameters of cell growth?

ie. what controls when a cell grows at the right time and place ( The target of Rapamaycin)

Cyclosporin + cyclophillin

individually do not target PP2B ( they need to complex together)

TOR

is a central controller of growth.

Ragulator

is a trimeric protein complex, it interacts with RAG GTPase and it is essential for localizing them and mTORC1 to lysosomal surface, and is necessary for activation of mTORC1 pathway by aa. The Ragulator complex is the GEF for RAGs. GTP loading of RagB recruit mTORC1 to lysosome where Rheb resides.

TOR( target of rapamycin)

is conserved PI kinase related protein kinase.

The target of rapamycin TOR protein

is involved in cell growth regulating.

early 2000s

it was found that TOR1 and TOR2 ( separate signalling pathway) play a central role in controlling cell growth as part of two separate signalling branches.

TORC ( TORC is conserved)

like TOR, KOG and LST8 have obvious mammalian sequence homologs, raptor and mLST8 respectively. mTOR, raptor and mLST8 associate in complex with mTORC1.

Rapamycin + FK506

look alike

LST8

made of WD40 repeats.

mTOR architecture:

- HEAT repeats ( at the A subunit of PP2A) - FAT ( conserved among PIKK) - FRB - rapamycin binding domain - kinase domain - FATC - another conserved domain in PIKK ( Unknon function)

Reconstruction of TORC1

- LST8- made of WD40 repeats - mTOR( HEAT- FAT- FRB- kinase- FATC) - raptor - RAS40 -

RagGTPases copurify with Raptor/

...

GAP for RAGs

An 8 protein complex called GATOR was found associated with the RAG GTPase during co-immumnoprecipitation. One protein ( of 8) is GAP for RAGs and if GATOR function is lost it is insensitive to aa.

Q. What do the seemingly very different conditions of transplant rejection and cancer have in common in their underlying biology such that they can be treated with the same drug.

Both conditions are due to undesirable cell growth; suggesting thtat the target of rapamycin is the central controller of cell growth, but what does it target and how does it controls cell growth.

Multicellular organisms

In multicellular organisms, growth of individual cell is controlled relative to the overall body growth such that organs and tissue constituting the organism are properly proportioned.

Note

It was subsequently demonstrated that the FKBP rapamycin complex bind directly to TOR1 and TOR2 and is widely conserved both structurally and as the target of FKBP rapamycin.

TORC1 contain

KOG1, LST8, and either TOR1 +2. FKBP12-rapamycin binds to TORC1 and TORC1 disruption mimics rapamycin treatment suggesting that TORC1 mediate rapamycin sensitive temporal control control of cell growth.

Comparison

Like TOR in yeast cells, mTOR control cell growth in response to nutrients ( aa) . Thus, the TOR signalling pathway appear to be primordial pathway that has been conserved throughout eukaryotic evolution to control the fundamental processes of cell growth. In mammalian cells, growth is stimulated by a combination of nutrients and growth factors. The mTOR pathway mediate phosphorylation of inositol 3 kinase ( PI3K)- dependent growth factor signalling.

mammals express 4 proteins

RagA, RagB, RagC, RagD that form heterodimers consisting of RagA or RagB, with RagC or RagD. RagA and RagB like RagC and RagD are highly similar to each other and are functionally redundant. Rag heterodimers containing GTP bound RagB interact with mTORC! and aminoacids induce the mTOR Rag interaction by promoting loading of RagB with GTP, which enable it to directly interact with raptor component of mTORC1.

Rapamyscin

Rapamycin is produced by Streptomyces hygroscopicus, a bacterium isolated from soil. It has the following properies: - anti- fungal - immunosuppressant - used in organ transplant. - anti cancer.

Primary docking site for substrate recruitment

Raptor

RAG GTPases and amino acids

Recently, the RAG GTPases which are also members of the Ras family of GTP binding proteins were shown to be amino acid specific regulators of mTORC pathway.

RAG GTPases and amino acids

Recently, the Rag GTPases, which are also member of the Ras family of GTP binding proteins, where shown to be amino acid specific regulators of mTOR

FKBP12

Receptor for rapamycin - FKBP equivalent to yeast is FPR1.

TORC1

TORC1 is sensitive to rapamycin and regulates the temporal aspects of cell growth.

TSC1- TSC2

TSC-1 and TSC-2 is dispensable for the regulation of mTORC1 by aa ( and in cells lacking TSC2, the mTORC1 pathway is sensitive to aa starvation , but resistant to growth factor withdrawal.

TSC1/2

TSC1/2 is dispensable for regulation of mTORC1 by aa ( and in cells lacking TSC2, the mTOR is sensitive to aa starvation, but respondant to growth factor withdrawal.

FRB

The FRB act as a gate keeper to the active site. The active site is highly recessed owing to the FKBP12. rapamycin binding domain ( FRB domain). FRB domain act as a gate keeper, with its rapamycin binding site interacting with substrates to give them access to the restricted active site. Rapamycin- FKBP12 inhibit the kinase by directing blocking the rectricting the active site access. It is persumed that although TOS motif is primary means of substrate recruitment, the secondary FRB site may facillitate substrate entry ino the other wise restricted active site and may also provide an additional level of specificity ( at least for a subset of substrates)

Rheb

The TSC1/ TSC2 proteins form a tumor suppressor complex that transmits growth factors and energy signal to mTORC1 by regulating GTP loading state of Rheb, a Ras related GTP binding protein. Rheb is localized to lysosome wand when bound to GTP. Rheb interacts with and activate mTORC1 and appears to be necessary for teh activation of mTOR by all signals, including aa availability. Thus, mTOR come to lysosome to become activated.

mutants:

The WT+ rapamycin has the same phenotype as the tor1/tor2 mutant cells ( ie. large swollen cells = autophagy - they are smaller based on dry mass). Tor 2 mutant- sever phenotype ( their is distrubtion of actin when the yeast cells are budding)

The Tor pathway

The tor pathway control cell growth by activating an array of anabolic process including protein synthesis + transcriptional+ ribosomal biogensis and inhibition of catabolic process such as bulk protein turnover ( autophagy) and mRNA degradation, all in response to nutrients. Dysfunction of signalling pathways controlling cell growth results in cells of altered size + developmental errors+ wide variety of pathological conditions. Thus, an understanding of TOR signalling pathway may lead to nove drugs for treatment of cacner, diabetes, inflammation, muscle atropy, learning disability, depression, obesity and aging.

Nb.

These drugs form toxic compounds with immunophillins which have distinct molecular targets.

Ragulator

a GEF for the Rag GTPase

Experiment:

a) no aa starvation- the mTOR is fairly diffuse throughout the cell but not in the nucleus. b) aa starved for 50 mins + placed in aa for 10 mins. The Tor is localized in the same location as Rabs7 ( lysosome marker); in response to aa, TOR move to lysosome ( where RHEB is waiting). 3) RAG-GTP expression cells ( RAG- GTP is always bound , can not hydrolyze it when it bind to it. When there is no aa- Tor is sitting on lysosome already. ; when aa is starved and then added after 10 mins- everybody is sitting on lysosome if RAG-GTP is present because Rheb is always sitting on sufrace of lysosome waining for TOR to come to it+ activate teh signal downstream.

FKBP12/ Rapamycin

abolish TOR1 branch pathway.

TOR1 and TOR2

although structurally similar, TOR 1 and TOR2 are not functionally identical. These and other findings led to the model that TOR1 has 2 essential function: one function is redundant with TOR1 ( shared and other functions is unique to TOR2 ( TOR2- unique); these two TOR2 functions are two separate signalling branches that control cell growth in different ways. The above findings also led to the discovery that TOR control cell growth in response to nutrients. Subsequent studies demonstrated that TOR in higher eukarya also controlled cell growth in response to nutrients ( ie. TOR is conserved in structure and function).

Lysosomes

are a major site of protein degradation and aa recycling- vacuoles store aa@ high concentrations. Thus, mTORC! and its regulators may reside on lysosomal surface as to sense accuratrely unknown aspect of lysosomal function that refelect the interecellular pools of aa.

TOR1 and TOR2

are protein kinases. TOR1 and TOR2 are the founding members of the PI kinase related protein family (PIKK) - family of ser/threonine specific kinases ( including ATM, DNA-PK, TRRAP and ATR)

The location of RagGTPases, Ragulator, and mTORC1 on lysosomal surface implicates this organelle

as the site to be discovered sensing system that signal amino acid availability to the Ragulator-Rag complex- the lysosomal location of aa sensing branch of the mTORC1 pathway is consistent with the increasing evidence that lysosomes and yeast counterpart vacuoles are @ nexus of aa metabolism within the cell.

TORC1+2

biochemical purification of TOR1 and TOR2 from yeast led to the identification of two distinct TOR proteins complexes ( TORC1+TORC2)- which account for teh different sensitivity of TOR signalling to rapamycin

Ex.

cell growth is coordinated with division ( ie. before cell division, it duplicate its genome, its mass ( organelles) - these are thus checkpoints before they divide. growth is also controlled relative to cell division. In proliferating cells, growth is linked to the cell cycle such that most cells precisely double mass before division.

Cell growth

cell growth is highly regulated- cell respond to nutrients or other appropriate growth stimuli by upregulating macromolecule synthesis ( protein, RNA, glycogen), thereby increasing size+ duplication of DNA+ cell cycle. Coversly, cell respond to nutrient limitation or other types of stress by downregulating macromolecules synthesis and enhancing turnover of excess mass ( autophagy). Thus, the control of cell growth involves balancing positive regulation of anabolic processes with negative regulation of catabolic process.

RagB mutant

cells exppressing RagB mutant that is constitutively bound to GTP( RagB- GTP), the mTORC1 pathway is insensitive to aa starvation and mTORC1 reside in the Rab7 positive compartment even in the abscence o amino acids; it is the lysosomal suface that is the compartment where Rag protein reside in which mTORC1 moves in response to aa

Human or mammalian ( mTOR architecture)

conserved across eukarya ( mammals + yeast)

TSC-1 and TSC-2 proteins

form a tumor suppressor complex that transmit growth factor and energy signal to mTORC1 by regulating GTP binding state of Rheb - a Ras related GTP binding protein. Rheb is localized to lysosomes and activate mTORC1 and appear to be necessary for teh activation of mTOR by all singals, including aa availability, thus mTOR comes to lysosome to become activated.

The immunosuppressant rapamycin, FK-506, and cyclosporin

form toxin complexes with immunophillins which have distinct targets

Three classes of rapamycin yeast were uncovered:

fpr-1 - docks rapamycin ~ 96% tor-1 (~2%) tor-2( ~2%)

FRB

function as a gate keeper for active site.

TOR-1 or TOR-2

function in increased mass and block stress response.

RAPTOR

play a role in substrate recognition.

The TSC1/ TSC2

proteins form a tumor supprsesor complex that transmits growth factor and enrgy signal to mTORC! by regulating GTP loading site of Rheb, a Ras related GTP binding protein. Rheb is localized to lysosome and when bound to GTP, Rheb interacts with and activate mTORC1 and appear to be necessary for the activation of mTOR by all singals, including aa availability. Thus mTOR come to lysosome to become activated.

Rapamycin

rapamycin is currently used in prevention of organ rejection in kidney transplant patients. Furthermore, preclinical studies show that rapamycin and its derivative strongly inhibit the proliferation of tumor cells in culture and the two derivatives are now in clinical trials as anti-cancer agents. the candidate anti-cancer drug are performing particularly well against tumor deficient in tumor suppressor (TSC), PTEN, p53 ( TF deficient 50% of all cancer. Rapamycin is effective against tumor because it block growth of tumor cells directly and because of indirect effect of preventing the growth of new blood vessels ( angiogenesis) that supply oxygen and nutrietns to the tumor cells.

TOR and the control of growth in yeast ( fungus)

rapamycin is involved w/ FKBP12. It was found that rapamycin similarly inhibited cell cycle progression in yeast - the molecular target is the same in yeast + vertebral cells)

Rapamycin sensitive

rapamycin sensitive TOR signalling regulates when a cell grows

TOS motif

recognition of this apparent motif in S6K1 and 4EBP-1 result in biochemical experiments that revealed that this motif target protein to mTOR by raptor allow phosphorylation by mTOR.

RAPTOR

recognition of this apparent motif in S6k1 and 4EBP1 resulted in biochemical experiments that revealed that this motif target protein to mTOR by RAPTOR - allowing phosphorylation by mTOR.

How is TOR recruited on the lysosome?

recruitment of TOR on suface of lysosome to be activated by Rheb.

FRB

the FRB act as a gate keeper to the active site; the active site is highly recessed owing to the FKBP12.rapamycin binding domain ( FRB domain) - which act as a gate keeped with its rapamycin binding site interacting with subustrates to give them access to the restricteed active site.

The TOR-1 and TOR-2

the TOR-1 and TOR-2 genes were cloned, based on a dominant rapamycin resistant phenotype of the mutant alleles and sequenced. The TOR1/TOR2 proteins are both 28kDA in size and ~67% identical.

late endosome and lysosome

the activation of mTORC1 pathway by aa coorelate with movement of mTORC! from an undefined location to a compartment containing Rab7a comparement of both late endosome and lysosomes.

Solve structure of mTOR +LST8 as a complex.

the structure reveal an intrinsically active kinase conformation, with catalytic residues and catalytic mechanism remarkebly similar to canonical protein kinases. The interactions that FATC makes with the activation loop suggest that it has a role in stabilizing the activation loop; the activation loop is not phosphorylated like in other protein kinase; the kinase domain is squeezed into an active conformation.

WT+ rapamycin and tor1/2 mutant

they are large swollen cells= autophagy but based on dry mass they are smaller. They are large + swollen because they undergo autophag( self-digest + swell) but when weight down they are smaller.

yeast genetics

to identify the target of FKBP- rapamycin complex, rapamycin resistant yeast mutants were selected. As expected, fpr1 mutant defective in FKBP were recovered, but also mutants altered in two novel genes ( TOR1 and TOR2- target of rapamycin were involved) Yeast was grown in presence of 200 mM rapamycin + resistant colonies grown up and the mutated gene was identified.

Cyclosporin/ Cyclophillin

when they complex in the cell - they target PP2B ( know out its effect)

Rapamcin insensive TOR

where cell grows (TOR C2)

Yeast FRP1

yeast FPR1 ( FKBP12) was purified to homogenity and partially sequenced . The protein sequence information was used to isolate the FKBP gene. The predicted aminoacid sequence of yeast FKBP was 54% identical to that of the clone human FKBP12 ( mode of action of rapamycin is conserved from yeast to humans). Curiously, disruption of FKBP gene ( in yeast FPR1) revealed that FKBP is not essential for growth. The findings that FKBP disruptions are not lethal was puzzling because FKBP was believed to be invivo binding protein / target for teh toxic effect of rapamycin. However, the subsequent finding that an FPR1 disruption confer rapamycin resistance, combinding with the observation of some other drug analogs are not immunosuppressive despite still being able to bind and inhibit FKBP12 lead to the currently accepted model of immunosuppresive drug action : an immunophilin drug complex ( ex. FKBP- rapamycin ) gain a new toxic activity that act on another target molecule. This mode of drug also applies to the well know immunosuppressant cyclosporin A +FK506 ( from cyclophillin- cyclosporin A and FKBP/FK 506 is conserved from yeast to human)

Yeast have _________ TOR genes

yeast have 2 TOR genes ( TOR 1+2) while mammals have one TOR gene