L9: PI3K

Question 1

PI3K mTOR

Answer 1

Phosphorylation of the inositol ring at the third position (PIP3) recruits proteins such as PKD1 or Akt that contain PH domains. These proteins are brought to the membrane because the inositol ring is now fully phosphorylated as PIP3.

Once recruited, PDK1 (a kinase) phosphorylates Akt, activating it. Akt is a serine/threonine kinase, meaning it adds phosphate groups to serine or threonine residues in target proteins, creating a kinase cascade.

In the cytoplasm, Akt is part of a larger complex that remains inactive until a critical GTPase, Rheb, is activated. Initially, Rheb is in its GDP-bound (inactive) state, and TSC1 and TSC2 proteins bind to Rheb, preventing it from being activated.

Akt then phosphorylates TSC2 within the complex. This phosphorylation disrupts the interaction between TSC1 and TSC2, causing the complex to fall apart.

Once the complex is disrupted, GEFs (guanine nucleotide exchange factors) can activate Rheb by promoting its exchange of GDP for GTP, turning it into its active GTP-bound form.

When Rheb is in the GTP-bound (active) state, it can bind to mTOR, triggering downstream signaling and promoting processes like protein synthesis and cell growth.

Question 2

generation of PIP3

Answer 2

Pagthway to mTOR: Generation of PIP3 by PI3K

PIP3 recruits PDK1 and Akt to the membrane via PH domains

Akt is activated by PDK1 (phosphorylation of Akt)

Active Akt phosphorylates TSC2 in the TSC2/TSC1/Rheb complex

Rheb is released from the complex

Rheb can become GTP loaded

GTP-Rheb can bind to and activate mTOR

Question 3

what is mTOR

Answer 3

Mammalian target of rapomyocin.
Mammalian Target Of Rapamycin

Serine / threonine kinase discovered via mechanism of Rapamycin action

3. Biological sensor : nutrients, stress etc.
   Two diff mtor complexes:
   mTORC1
   : raptor

mTORC2: rictor

Targets of mtorc1:
Activation f rheb and binding of rheb to this complex activates the kinase activity within this complex. Kinase activity held by mtor protein itself. That kinase activity feeds into these diff pathways. In terms of growth and proliferation- protein synthesis is really important.

Question 4

PIP

Answer 4

Lipids aren’t just structural (in membranes) — some act as second messengers.

Phosphoinositides (PIs) are membrane-tethered lipids built on phosphatidylinositol (PI).

The inositol ring of PI has free OH groups at positions 3, 4, and 5, which can be phosphorylated.

🧱 Basic Building Block: PI
Looks like this structurally:

A diacylglycerol (DAG) tail in the membrane

An inositol head group in the cytoplasm

🔁 Key Enzymes Modify PI
Enzyme Action Produces
PI3K Adds phosphate at position 3 PIP3
PI4K Adds at position 4 PI(4)P
PI5K Adds at position 5 PI(5)P

But most important in signalling is:

PI(4,5)P₂ → cleaved by PLC → gives:

DAG (remains in membrane) → activates PKC

IP₃ (goes to cytoplasm) → opens calcium channels in ER

Another important pathway:

PI(4,5)P₂ → phosphorylated by PI3K → PI(3,4,5)P₃ (PIP₃)

This is key in AKT signalling (important in cancer, growth factor response)

🧬 Protein Domains Recognise Specific PIPs
PH domains (Pleckstrin Homology): bind PIP3 or PIP2 at membranes

FYVE domains: bind PI(3)P

These help proteins localise to membranes and activate downstream signalling.

look at slide 13

Question 5

PI3Ks

Answer 5

PI3Kinases (PI3K)s

1988: PI3K activity discovered in oncogene complexes (Cantley + Downes et al.)

1991: cloning of the first PI3K gene (Parker, Waterfield et al.)

by 1997: 8 PI3K genes in mammals - fall in 3 classes - conserved in evolution

Class 1A PI3Ks: 3 isoforms — alpha, beta, delta. Involved in many cellular roles.

Class 2 and Class 3: More involved in intracellular processes like vesicle trafficking. Might be constitutively activated. Not much evidence of external activation (e.g., not sitting near growth factor receptors?).

Different classes of PI3K have preferred substrates.

⚙️ Substrate Preferences
PI3Ks deliver phosphorylation to the 3rd position on the inositol ring.

Class 1 prefers phosphorylating a ring that is already phosphorylated at positions 4 and 5 → that’s PIP2?

This generates PIP3, which is not the same as IP3 (you might’ve meant PIP3 here).

Class 2 and 3 seem to prefer a less modified inositol ring — not one that already has phosphate groups on it.

PIP2 is the preferred substrate for Class 1.

🧬 Class 1A PI3K Structure
PI3K is a complex of 2 subunits, both made from separate genes that are transcribed and translated in the cell, then come together.

The catalytic subunit is where the enzyme function sits — it adds phosphate to the 3-position on the inositol ring.

The regulatory subunit controls this activity.

The regulatory subunit is bound to p110 to form a fully functional kinase complex.

The regulatory subunit (e.g., p85) needs to be in a particular conformation to allow activation of the kinase.

🔗 Activation Mechanism
The SH2 domain in the p85 regulatory subunit recognises phosphorylated tyrosines on the cytoplasmic tail of tyrosine kinase receptors.

This receptor phosphorylation recruits the PI3K complex, which activates PI3K activity.

Recruitment brings PI3K to the membrane, where the inositol lipids are located.

The catalytic subunit also has a RAS-binding domain (RBD).

RAS is lipid-modified, which anchors it to the plasma membrane (PM).

All of this happens on the inner leaflet of the PM.

🎯 Relevance to Cancer
Either through growth factor receptors or RAS, both of these activation routes are relevant to tumorigenesis.

PH domain containing proteins can bind to PIP3
Some specificity to that

PI3K lipids bind to PH domains found
in a diverse set of molecules

Diff kinases e.g: PDK1, adaptor proteins like gab1, small dtpase modulators like GEFs abe ph domains.
Tyr kinases
adaptors
small GTPase modulators
Ser/Thr kinases
PDK1
DAPP1
GEFs
GAPs
Btk
Gab1
Itk
Rac
Arfs
Ras
GEFs:
PRex-1,2
p90 RSK
GEFs:
ARNO, Grp1,
cytohesin-1
GAPs:
centaurins, ARAPs
p70S6K
Akt/PKB
GAP1m
GAPIP4BP
AGC kinases

Question 6

The dephosphorylation event

Answer 6

Phosphorylation & Dephosphorylation: Lipids
For every phosphorylation event, there must be a dephosphorylation event to allow signal reset or regulation.

This applies to lipids too — not just proteins.

🧪 Lipid Kinases & Phosphatases
Lipid kinases like PI3K add phosphate groups (e.g., creating PIP3 from PIP2).

Lipid phosphatases remove those phosphates to turn off the signal or recycle the lipid.

🧼 PTEN
PTEN is a phosphatase that removes the 3-position phosphate from PIP3, converting it back to PIP2.

This resets the lipid for reuse if the cell receives a signal again.

🧽 SHIP2
SHIP2 removes a different phosphate (not the 3-position) — it acts on PIP3 to produce PI(3,4)P2.

This is another form of regulation — different phosphatases target different sites.

Question 7

mTOR and protein synthesis

Answer 7

🧬 mTOR & Protein Synthesis
mTORC1 phosphorylates 4E-BP1.

When 4E-BP1 is phosphorylated, it cannot bind to the translation complex (eIF4E complex).

This frees up the translation complex (e.g., eIF4E) to promote mRNA translation.

mTORC1 also activates S6 kinase, which phosphorylates components of the ribosome → enhances translation of specific mRNAs.

Overall: mTORC1 increases protein synthesis → supports cell growth and proliferation.

Question 8

pi3k in cancer

Answer 8

Deregulation of PI3K Signalling in Cancer
PI3K signalling often deregulated in cancer.

Mutations can occur in:

Catalytic domain

Regulatory domain

🔹 Types of Mutations:
Example: E545 or E542 mutations block regulation → kinase is active when it shouldn’t be (still at normal activity level, just unregulated).

Mutations in kinase domain → constitutively active, phosphorylating constantly.

Overall effect = increased PI3K activity when there should be none.

📈 Functional Consequences:
Drives cell growth, proliferation, and motility (important for invasion and metastasis).

Tumours with PI3Kα mutations are often addicted to this signalling → sensitive to PI3K inhibitors (oncogene addiction).

❌ Loss of Negative Regulation:
PTEN is the main phosphatase that dephosphorylates PIP3 to PIP2.

Loss of PTEN → PIP3 accumulates at the membrane.

Constant recruitment of PH domain proteins (like AKT).

Continuous activation of signalling pathways.

🧬 PTEN in Cancer:
PTEN = Phosphatase and tensin homologue deleted on chromosome 10

PTEN is lost in many tumour types.

PTEN +/- mice:

Develop autoimmunity and cancer with 100% penetrance.

🚨 Consequences of PTEN Loss:
↑ Proliferation

↑ Migration

↑ Cell growth

↓ Differentiation

↓ Cell death
→ Mainly because AKT pathway promotes survival and growth.

Question 9

targets

Answer 9

Target mtor rapamycin/rapalogs

(Specifically targeting mTor)
First generation of mTor inhibitors have entered the oncology clinic e.g.. Everolimus
🡪 solid tumours (kidney, breast, glioblastomas, prostate, head and neck) and haematopoietic malignancies (lymphoma)

Targeting the pi3k signalling network in cancer:
pharmacological PI3K inhibitors that inhibit all isoforms of PI3K –many are toxic in vivo

The class 1 isoform is mainly in haematpoetic cells and there are some inhibitors? In the clinic
STX-478, a Mutant-Selective, Allosteric PI3Ka Inhibitor Spares Metabolic Dysfunction and Improves Therapeutic Response in PI3Ka-Mutant Xenografts

therapeutic benefit of isoform-selective PI3Kα inhibition was established with alpelisib, which has equipotent activity against the wild-type and mutant enzyme.

However, alpelisib is associated with severe hyperglycemia and rash

STX-478, an allosteric PI3Kα inhibitor selectively targets mutant PI3Kα
No metabolic dysfunction observed with STX-478
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