Phosphoinositides and PI3K

Question 1

How do phosphoinositides bind and what is its feature?

How can binding be described?

What two phosphoinositides are at the plasma membrane & can be included in coated pits?

What happens to the phosphoinositides further through the vesicular trafficking?

What phosphoinositide is present in vesicles & early endosomes? (separate)

What 2 phosphoinositides are in multi vesicular bodies?

Which phosphoinositide goes to the lysosome from here?

PI4P matures from the MVB - what is its fate?

Answer 1

PH domain - highly specifically

Lock and key

PIP3 & PI(4,5)P2 (PIP2)

Mature into different phosphoinositides

Vesicles = PI(3,4)P2
EE = PI3P

PI3P PI(3,5)P2

PI(3,5)P2

Enters golgi & then returns to plasma membrane

Question 2

How does PIP2 interact with the PLC delta PH domain?

What phosphoinositide does P40 phox PH domain bind?

What are 2 points about PH domains’ structure?

What about its homology?

What about its binding specificity? 2 points

Answer 2

it’s 4,5 phosphates makes contacts with the loop in PH domain

PIP3

1. Mixed alpha beta with loops for binding
2. 100-120 amino acids long

No sequence homology but structural 3D similarity - so must be individually characterised

1. Protein-lipid & protein-protein interactions
2. Differ in ligand affinity/specificity

Question 3

What 4 things make up the Grp1 PH domain for binding PIP3?

What other proteins have PH domains that allow them to bind to PIP3 and what are they involved in? 3

How could you produce an experiment to identify the PH domain proteins that bind to PIP3 in humans? 2 things

What does Akt require for its activation?

What are the following molecules in the Akt pathway and what is their function?

1. Foxo
2. GSK3
3. MDM2
4. TSC2

What are the 2 residues of Akt’s kinase domain is required for phosphorylation for its activation?

Answer 3

1. 7 beta strand sandwich forming core
2. c-terminal alpha helix 1
3. beta 6’ and 6’’ strands contribute to head group for phospholipid head group recognition
4. inositol(1,3,4,5)P4 loop binds to PIP3

o

1. Akt - cell proliferation/survival
2. PDK1 - translation & cell growth
3. Guanine nucleotide exchange factors - activate Rac/Rho for actin cytoskeleton & migration regulation

o

1. Link proteins with PH domains with fluorescent YFP
2. Place construct into cells & stimulate them to make PIP3 by PDGF

PIP3

1. Transcription factor - regulator
2. Protein kinase - activator
3. Ubiquitin ligase - triggers degradation of p53
4. GAP for Rheb - activates mTORC1

Threonine and serine

Question 4

What is the structure of Akt in its autoinhibited form?

In 3 steps how is Akt activated?

What does rapamycin do to mTORC?

What are the 3 proteins that bind to mTOR in mTORC2 and what are their functions?

Therefore, how is mTORC2 activated?

What are the 2 differences of proteins bound in mTORC1 and what do they do?

Therefore, where does mTORC1 activation occur?

What are the 4 steps for mTORC1 activation?

What kind of molecule is mTORC1 and what does it have roles in?

How else can mTORC1 be activated?

Answer 4

PH domain on top of the kinase domain

1. PIP3 at membrane recruits PDK1 and Akt
2. PDK1 phosphorylates threonine of Akt in kinase domain
3. mTORC2 phosphorylates the serine on tail region

PIP3

Inhibits them

1. Rictor - scaffold protein
2. Sin1 - PH domain for PIP3 binding & recruitment to membrane
3. Deptor - inhibits mTOR activity

o

1. Deptor AND PRAS40 inhibit mTOR activity
2. Raptor instead of Rictor & binds Rag G-proteins found on the lysosome

Lysosome

1. AKT is phosphorylated by PDK1 & mTORC2
2. AKT phosphorylates TSC complex for its inhibition - tuberous sclerosis complex 1 & 2 (Gap protein for Rheb)
3. GAP is inactive & Rheb-GTP builds up (due to no exchange factors for Rheb)
4. Rheb-GTP activates mTORC1 (kinase)

Kinase - anabolic metabolism, growth, proliferation, survival

Rag GTPases from amino acids & glucose

Question 5

What enzyme phosphorylates PI(4,5)P2 into PI(3,4,5)P3?

When PTEN dephosphorylates PIP3, what does it produce?

When SHIP dephosphorylates PIP3, what does it produce?

What can the product of SHIP dephosphorylation do?

What domain does PI3K have?

How are Class 1A and 1B of PI3K activated?

What kind of structure is PI3K and what are the subunits?

Answer 5

PI3K

PTEN = 3’phosphatase, so PI(4,5)P2

5’ phosphatase so PI(3,4)P2

Activate Akt

Ras binding domains

Class 1A by receptor tyrosine kinase activity
Class 1B by Ras-GTP activating G-proteins (GPCRs) and beta-gamma subunits activating

Heterodimer - p110 catalytic & p85 regulatory

Question 6

What 4 domains are present on the p85 subunit?

What are the 5 domains present on the p110 subunit of class 1A?

How many different subunits can be brought together for the Class 1A PI3K?

What does the regulatory subunits say about class 1A pI3K?

How is the catalytic subunit of class 1B different?

How many different subunits can be brought together for class 1B pI3K?

What are they regulated by?

What is the 2 step pathway for Class 1A PI3K activation?

Class 1B PI3K activation?

In which cells do you see either class of PI3K? 2 each

Answer 6

2 x SH2, SH3, p110 binding

Ras, C2 (to bind to phospholipids at membranes), helical, kinase and p85 binding

3 p110 and 5 p85

Regulated by receptor tyrosine kinases

Has no SH2 domain

2 regulatory & the 1 catalytic

Beta-gamma subunits from G proteins

1. Growth factor receptors are tyrosine phosphorylated & activated
2. Recruits the regulatory subunit & then catalytic subunit comes with it to convert PIP2 -> PIP3

o

1. Chemokine C5a activates neutrophils such that their GPCRs are activated & release beta-gamma subunits from Gi protein
2. B-g recruits both subunits of class 1B PI3K converts PIP2 -> PIP3

1A = T & B cells
1B = Neutrophils & platelets

Question 7

How is Class 1A PI3K activated by removing its autoinhibited state? 3 steps

What is its autoinhibited state?

What are the 2 helices involved in the autoinhibition?

What are the 2 activating mutations of class 1A PI3K?

What does this say about mutations effecting Class 1A?

What happens when PTEN/SHIP is mutated?

What can be said about PTEN/SHIP mutation in cancer?

How can Akt mutation contribute towards cancer?

Why is an Akt mutation less severe than PI3K?

Answer 7

1. Phosphotyrosine residue from receptor tyrosine kinase removes c-SH2 domain
2. Allows for flipping of ‘elbow region’ such that the phospholipid can bind to the p110
3. Removal of the n-SH2 by phosphotyrosine for full activation of PI3K

Regulatory arch of the elbow region on the p110 catalytic subunit is where the helices form an elbow at the lobe of p110 and c-SH2 of p85

ka11 & ka12

1. His-1047 of regulatory arch/elbow at C-lobe
2. Glu-545K of the helical domain

Mutations of the catalytic subunit & its autoinhibition mechanism results in constitutive activation

PIP3 isn’t degraded

Mutation such as they are lost

PH domain mutated - constitutive activation due to association with membrane

Further downstream