15 - Lipid Phosphorylation

Question 1

What feature of a pyridine lipid (inositol lipid) helps to generate signalling molecules?

Answer 1

The six carbon ring has a number of hydroxyl groups on it which can be targeted by kinases which can add phosphates to generate signalling molecules

Question 2

Which carbons on the carbon ring are important in the pyridine lipid?

Answer 2

In this particular phospholipid it tends to be carbons 3,4 and 5 that are targeted with particular kinases
o 3: PI3 kinase
o 4: PI4 kinase
o 5: PI5 kinase
o PI3 kinase is the most important in regulating cell growth as it has a link to cancer

Question 3

What are the PI3 kinase substrates?

Answer 3

If we look at the action of PI3 kinase, its substrates include phosphatidyl inositol (PtdIns) which is the lipid with the ring with no phosphates on it, its other substrates are phosphatidyl inositol 4-phosphate which has a phosphate on the 4 position, and phosphatidyl inositol 4,5-bisphosphate

Question 4

What happens when PI3 kinase is activated?

Answer 4

If we activate PI3 kinase it can phosphorylate PtdIns on the 3 position making PtdIns3P, PtdIns4P can be converted into PtdIns(3, 4)P2, and PtdIns(4, 5)P2 can be coverted into PtdIns(3, 4,5)P3
o This is all brought about by the PI3 enzyme
o These all lead to biological responses

Question 5

What is PtdIns (4, 5)P2 a target of?

Answer 5

PtdIns (4, 5)P2 is also a target of hydrolysis by the enzyme phospholipase C, so this can be cleaved releasing InsP3 and DAG which both activate an enzyme leading to further biological responses

Question 6

PI vs PIP3

Answer 6

o PI does not have any phosphorylation on the sugar rings other than the one linking it to the backbone
o PIP3 is phosphorylated on the 3,4 and 5 positions
- Involved in growth factor signalling

Question 7

What are the different classes of PI3 kinases?

Answer 7

There are number of different classes of PI3 kinases: alpha, beta, delta and gamma

Question 8

What is the substrate for PI3 kinases?

Answer 8

The substrate is always PtdIns(4,5)P2 and they all phosphorylate on the 3 position to generate PIP3

Question 9

What receptors can activate PI3 kinases?

Answer 9

The receptors that can activate PI3 kinases are receptor tyrosine kinases which can activate through RAS or directly (alpha beta and delta)
- A separate class of PI3 kinases (gamma) regulated by GPCR

Question 10

What was used to study PI3 kinases?

Answer 10

In the 1990, one major tool used to study PI3 kinases was wortmannin which is an inhibitor of PI3 kinases
o It was used to test whether any biological actions in the cells were mediated by PI3 kinases

Question 11

What is the structure of PI3 kinases like?

Answer 11

o The structure of PI3 kinases are relatively similar, have an activation domain which requires an adaptor protein p85
o The principal structure between the alpha, beta, gamma are relatively similar as they have the Ras activation domain, the C2 domain and the lipid kinase domain

Question 12

Inputs to PI3K family

Answer 12

• cytokines
• thrombin
• growth factors
• t-cell antigen
• g-proteins
• PROTEIN TYROSINE KINASES

Question 13

PI3K outputs

Answer 13

• PtdIns(3)P
• PtdIns (3, 4)P(2)
• PtdIns(3, 4, 5)P(3)

Question 14

Outputs of PtdIns(3)P, PtdIns (3, 4)P(2) and PtdIns(3, 4, 5)P(3)

Answer 14

• DNA synthesis
• cell survival
• membrane ruffling
• chemotaxis
• oxidative burst
• cytoskeletal rearrangements
• oocyte maturation
• receptor internalisation
• GLUCOSE TRANSPORT
• VESICLE TRAFFICKING

Question 15

What feature do some cytosolic proteins have?

Answer 15

Some cytosolic proteins have DOMAINS that BIND to the polar head groups of lipids that transiently exist in the cell membrane
o E.g., PLECKSTRIN HOMOLOGY (PH) domains bind to phosphorylated derivatives of phosphatidylinositol (PI-3-P, PI-3,4_P2, PI-3,4,5-P3)
o These PH domains interact with the 3 positions

Question 16

What do enzymes like PI3 kinase provide a mechanism for?

Answer 16

Enzymes like PI3 kinase provide a mechanism for modulating affinity of certain cytosolic proteins for the membrane surface

Question 17

What happens when insulin expressing cells are stimulated?

Answer 17

Cells that express the insulin receptor when stimulated, glucose transporters move from intracellular stores of the cell membrane and these then uptake glucose from the bloodstream into the cells where they can be used or stored
o This translocation of glucose requires PI3 kinase

Question 18

What happens when the insulin receptor is activated

Answer 18

• once the insulin receptor is activated, it recruits a number of proteins, including proteins which trigger the activation of PI3 kinase with Ras
• this leads to the generation of PIP3 through phosphorylation of PtdIns(4,5)P2 leading to the recruitment of PDK1 to the membrane
• this leads to other action of PKB which results in the movement of GluT4 to the cell surface through unknown mechanisms

Question 19

How is the insulin mechanism switched off?

Answer 19

PTEN lipid phosphatase switches off this action by dephosphorylating PIP3