5 - Membrane Phospholipids and Phosphoinositide Signals - Gray

Question 1

What is an additional source of second messengers (ie not ones that are synthesised by enzymes eg cAMP) ?

Answer 1

phosphorylated membrane lipids - which are cleaved to release their 2nd messengers

Question 2

what is the predominant function of phosphatidylinositol? what else can it be used for?

Answer 2

PI used for membrane structure, approx 1% used for signalling

Question 3

What is PLC? What are its iso forms and how are these activated

Answer 3

• phospholipase C
• effector enzyme
  PLCy activated through RTKs (Ras Independent RTK linked signalling)
  PLCB activated through Gqa proteins downstream of GPCRs

Question 4

What does PLC act upon and what are the products?

Answer 4

phosphatidyl inositol 4,5-bisphosphate (PIP2)

cleavage releases 2nd messengers - diacylglycerol (DAG - membrane bound) and Inositol 1,4,5-triphosphate (IP3)

Question 5

What are the results of IP3 and DAG 2nd messengers on the cell as a whole?

Answer 5

IP3;

• causes rise intracellular Ca2+ conc through release from intracellular stores
• induces many effects eg on smooth muscle cells causing constriction (binding to a1 adrenergic R, M3 acetylcholine R)

DAG;
- remains in membrane and together with Ca2+ activates PKC which regulates metabolic enzymes and TFs through P

Question 6

Draw a diagram and explain how GPCRs cause downstream activation of enzymes.

Answer 6

• GPCR activation -> Gqa activation -> activation of PLC
• cleavage of PIP2 -> IP3 and DAG (remains in membrane)
• IP3 binds to Ca2+ ion channels eg the endoplasmic reticulum IP3 gated channel causing release of Ca2+
• causes more Ca channels to open, releasing > Ca
• rise in intracellular Ca levels
• Ca interacts with soluble PKC which interacts with membrane via binding to DAG (sticky patch). now active
• leading to the P of metabolic enzymes and TFs etc

Question 7

describe how the signal activating PKC is turned off

Answer 7

• Ca2+ activated DAG kinase ends signal by phosphorylating DAG to phosphatidic acid

Question 8

how is PIP2 phosphorylated to PIP3?

Answer 8

activated RTK or cytokine R recruits PI-3 kinase (phosphatidylinositol) to membrane via SH2 domains. PI3 kinase P PIP2 -> PIP3

Question 9

what is the function of PIP3?

Answer 9

recruitment of other protein kinases to act for further signalling via PH domains

Question 10

name ALL of the 2nd messengers that originate from PIP2

Answer 10

• DAG
• IP3
• PIP3

Question 11

how is PI3 kinase recruited to the membrane and what does its activation lead to?

Answer 11

recruited to the membrane through activated RTK/cytokine receptors
- promotes cell survival by inhibiting apoptosis

Question 12

give the mechanism of how PI3K promotes cell survival

Answer 12

• PI3K at membrane converts PIP2 -> PIP3
• PIP3 interacts with Protein kinase B via PH domain and brings it to the membrane
• PKB Ps its substrates eg BAD protein. its substrates are invovled in the caspase cascade leading to apoptosis so phosphorylation of substrates leads to the sequestering so no longer involved in apoptosis

Question 13

what is the name of the protein invovled in allowing the caspase cascade to occur? (hint; this protein is found mutated in a number of cancers)

Answer 13

• PTEN
• phosphatase which converts PIP3 back to PIP2
• PIP3 can no longer recruit PKB to membrane so loses its ability to P and sequester components of the caspase cascade
• leading to apoptosis

Question 14

under what conditions would cause the Dictyostelim amoebae to come together? what is the signal for this?

Answer 14

• conditions of starvation they would aggregate -> fruiting body
• cAMP = chemotactic signal for this

Question 15

draw a diagram of PIP3 localisation of when we have increasing gradients of cAMP

Answer 15

343 - 5 word

Question 16

what determines the presence of PTEN @ the membrane ?

Answer 16

PIP2 presence = recruits PTEN

PIP3 = destabilises PTEN from the membrane

Question 17

describe the experiment that allowed dictyostelium polarisation in response to a cAMP gradient to be studied and describe their observations
draw diagrams

Answer 17

• GFP used to locate several different proteins
• cAMP binds to receptor, activates the G protein all over the cell surface
• cAMP presence leads to higher levels of PI3K activity @ one side of the cell relative to the other
• PI3K = PIP2 -> PIP3. more PIP3 @ one side leading to more destabilisation of PTEN at one side. relatively > PTEN at the back of the cell where lower cAMP
• PTEN converts PIP2 > PIP3 so we get even less PIP3 at the back than the front
• actin cytoskeleton forms at the leading edge and myosin contraction results at the rear

Question 18

what happens when the cAMP gradient is removed when studying dictyostelium aggregation?

Answer 18

cell polarisation is not stable