BS3054 L7 - Phosphoinositides

Phospholipid structure

2 fatty acids, 1 glycerol, 1 phosphate group

- hydrophilic head and hydrophobic tail = amphipathic

X group on phosphate of phospholipids

- can add groups

e.g. add choline to make phosphatidylcholine

e.g. add inositol to make phosphatidylinositol

phospholipids classified according to their polar head group and their abundance

- phosphatidylcholine = 50% of membrane lipids

- phosphatidylserine = 2-10%

- phosphatidylethanolamine = 15-35%

- phosphatidylinositol = 5-10%

Other denotations of phosphatidylinositol

PI or PtdIns

How is PI modified

- phosphorylation of any of the 6 carbons on the inositol group make other signalling molecules e.g. PIP2

Variations of PI

- phosphatidylinositol = PI

- phosphatidylinositol 4-phosphate = PIP

- phosphatidylinositol (4,5) - bisphosphate = PIP2

- phosphatidylinositol (3,4,5) - triphosphate = PIP3

why is PIP2 called phosphatidylinositol 4,5-bisphosphate not called phosphatidylinositol 4,5-diphosphate

- because the phosphates are on different carbons they are not organised in a chain where it would be referred to as diphosphate

Which carbons is PIP2 phosphorylated on

carbons 4 and 5

phosphatidylinositol structure

what enzymes turn phospholipids into signalling second messenger molecules

phospholipases

variations of phospholipases

- PLA1

- PLA2

- PLD

- PLC

= same substrate different outcomes

Role of phospholipase C

- cleaves PIP2 between the oxygen on the glycerol backbone and the phosphate

- forms DAG and IP3 = both second messengers

what is IP3

inositol 1,4,5-triphosphate

Receptor mediated signalling through PLC

- agonist binds receptor = conformational change

- alpha subinit dissociation and GDP/GTP exchange

- recruitment of PLC = cleaves PIP2 = DAG + IP3

- IP3 stimulates release of calcium from IC stores (ER) by binding to IP3 receptor

- DAG and calcium activates PKC

IP3 receptor

- acts as coincidence detector

- requires both IP3 to bind and calcium to be present for ion channel to open and cause calcium release into cytoplasm = calcium induced calcium release

families and isoforms of PLC

6 families, 13 isoforms (30 splice variants):

- PLC-beta (4 isoforms)

- PLC - gamma (2 isoforms)

- PLC - delta (3 isoforms)

- PLC - epsilon (1 isoform)

- PLC - zeta (1 isoform)

- pLC - eta (2 isoforms)

why is it important for there to be different isoforms of PLC

- to allow precise control so signalling can be regualted between cell types/locations

PLC-beta

- has 4 isoforms PLC-b1-4

- X and Y catalytic domains

- PH domain = allows localisation = bind PIs

- 4 tandem EF-hand domains = calcium function but unclear

- C2 domain = Ca2+ binding

- CC domain and PDZ domain = protein-protein interactions with PLC

homology between isoforms of PLC-beta

- 60% homology of the catalytic domain between isoforms

what is PLC-b most commonly associated with regulating

GPCRs

PLC-beta isoforms tissue distribution

B1 and B3 = fairly widespread

B2 = immune/haematopoietic

B4 = retina and certain neurons

What can activate PLC-beta

- Gaq subunit

- G beta-gamma subunit

- Ca2+

what domains does G-beta-gamma subunit associate with on PLC-beta

- PH domain (localisation of PIs)

- catalytic region

- almost always beta-gamma subunit from Gai/10 proteins as they are the most abundant

PLC-beta role other than phospholipase activity

- GTPase activating proteins

- can bind to alpha subunit and speed up intrinsic GTPase activity to cause reassociation between alpha and beta-gamma subunit

- same role as RGS proteins

coincidence detection with G-proteins and PLC-B3

- Gaq and Gbeta-gamma both activate PLC-B3 = synergistic activation

how is inositol recycled

- dephosphorylation of IP3 creates inositol

- inositiol fed back into membrane where it is phosphorylated into PIP2 again by kinases

- PIP2 can be turned into IP3 and DAG again or phosphorylated by phosphoinositide 3-kinase into PIP3 = signalling

phosphoinositide kianses

- phosphorylate PIs at 3,4,5 positions on inositol

- phosphatases dephosphorylate these

e.g. phosphoinositide 3-kinase phosphorylates in the 3 carbon position

PI3Ks

= phosphatidylinositol 3-kinases

= phosphorylate in the 3-OH position of the inositol ring in PIs

- 3 main classes = I, II, III

- activated by diverse cell surface receptors mainly RTKs and some GPCRs through Src transactivation of RTKs

- its preferred substrate in vivo is PIP2 = converts it to PIP3

GPCR singalling via Src transactivation of RTKs

- agonist binding to GPCRs can cause Src activation

- Src can then phosphorylate and activate RTKs

- transactivation amplifies the signal by integrating different pathways onto the RTK cascade

Structure and function of PI3-kinase

Regulatory subunit:

- p85 with SH2 and SH3 domains associated = allows recruitment to RTKs or adaptor proteins

Catalytic subunit

- p85 binding domain

- Ras binding domain - Ras binding can activate catalytic subunit

- HR3 = membrane binding

- HR2 = scaffold for other proteins to bind to it

- HR1 = kinase core

Why is PI3k associated with cancer

- Ras binding to PI3k causes cell proliferation

activation of PI3 kinase via RTKs

- growth factor binds to RTK causinf autophosphorylation and dimerisation

- allows docking of Grb2, SOS, RAS and GTP

- PI13k docks via Ras domain

= production of PIP3 on membrane

PIP3 as an anchor for signalling proteins

- signalling proteins with PH domains accumulate at sites of PI3K activation by binding to PIP3

- these proteins regulate cell growth, survival and movement

- examples of proteins containing PH domains are PKB (Akt) and PDK1

PKB (Akt)

- serine/threonine kinase

- growth factor pathway

- activated by PI3K and PDK1/2

PDK1

phosphoinositide dependent kinase 1 (involved in activation of PKB)

PKB activation via PDK1

- PIP3 in the membrane recruits PKB (Akt) and PDK1

- PDK1 phosphorylates PKB to partially activate it

- mTORC2 complex further phosphorylates AKT to fully activate it

- activated AKT then inhibits the TSC complex leading to activation of mTORC1 = controls protein synthesis and growth

What is PKB/Akt generally associated with

anti-apoptosis, growth, proliferation and migration

Termination of PI3-kinase signalling

- SHIP proteins remove binding sites for proteins with PIP selective PH domain

- SHIP proteins generate PIP2 from PIP3 that PKB can bind to

- PH domain of PKB binds PIP2 and PIP3 with equal affinity

- PTEN turns PIP3 into PIP2 and PIP2 into PI

PI3-kinase signalling and cancer

- numerous oncogenes activate type IA PI3-kinase

- activating mutation of PI3-kinase described in cancer

- PTEN has tumour supressor properties - mutations in PTEN associated with cancer

- mutations in SHIP1 recently associated with some leukaemias

therapeutic attempts to inhibit PI3-kinase in cancers

- small non-specific molecules

- wortmannin

- LY294002

- copanlisib and apelisib - only active on one kinase (class I PI1K inhibitors)

- most approved drugs have since been withdrawn due to side effects

Theoretical ideal therapeutic targets for PI3K in cancer

Isotype selective PI3K inhibitors:

- inhibitors that target specific p110 catalytic subunits - many minimise side effects

Inhibitors of Akt (not yet apporoved)

- inhibition of downstream signalling from PI3K activation many be beneficial

- two examples of Akt inhibitors:

1. ipatasertib = binds ATP binding site of Akt (breast cancer)

2. afuresertib = competitive inhibitor