III. Signal transduction and cell cycle | 42. Phosphatidylinositol signaling pathways

Question 1

I. Basics
1. What are the features of phospholipids?

Answer 1

• The plasma membrane contains phospholipids that are more than just structural.
• A phospholipid is amphiphilic, which means it has both hydrophobic and hydrophilic region

Question 2

I. Basics
2. Describe the structure of phospholipids

Answer 2

A phospholipid is amphiphilic, which means it has both hydrophobic and hydrophilic region:
- Hydrophilic head: glycerol bound to a negatively charged phosphate group by a phosphate ester bound. The glycerol has 3 carbons, each having one hydroxyl group (-OH) which binds the phosphate and the two fatty acid chains in the tail
- Hydrophobic tails: two fatty acid chains attached to the glycerol by ester bonds

Question 3

II. Phospholipase
1. What are Phospholipases?

Answer 3

Phospholipases are enzymes that hydrolyze phospholipids into fatty acids and phospholipids.

Question 4

II. Phospholipase
2. What is the target of Phospholipases?

Answer 4

The ester bonds between the glycerol and the R groups (phosphate and FAs) are the target of cleavage by different phospholipases.

Question 5

II. Phospholipase
3. What are 4 examples of phospholipase?

Answer 5

• Phospholipase A1
• Phospholipase A2
• Phospholipase C
• Phospholipase D

Question 6

II. Phospholipase
4. What is the role of Phospholipase A1?

Answer 6

Phospholipase A1 cleaves the 1st ester bond (FA)

Question 7

II. Phospholipase
5. What is the role of Phospholipase A2?

Answer 7

Phospholipase A2 cleaves the 2nd ester bond (FA), side chain R2 usually arachidonic acid

Question 8

II. Phospholipase
6. What is the role of Phospholipase C?

Answer 8

Phospholipase C cleaves the 3rd ester bond to the phosphate group

Question 9

II. Phospholipase
7. What is the role of Phospholipase D?

Answer 9

Phospholipase D cleaves the ester bond between the phosphate and the side chain R3

Question 10

III. Phospholipase C
1. What are the features of Phospholipase C?

Answer 10

Phospholipase C (PLC) in humans (animals) is specific for phosphatidylinositol 4,5-bisphosphate (PIP2), which is a phospholipid with an inositol bound to the phosphate group.

Question 11

III. Phospholipase C
2. What is the mechanism of Phospholipase C?

Answer 11

Activated phospholipase C will cleave PIP2, to generate two 2nd messengers: diacylglycerol (DAG) and inositol-triphosphate (IP3).

Question 12

III. Phospholipase C
3. Is phospholipase C a family of enzymes?
=> T/F?

Answer 12

TRUE!!!

Question 13

III. Phospholipase C
4. Phospholipase C is a family of enzymes
=> List 3 enzymes with their role

Answer 13

• PLCβ contains a G-protein interaction domain, which will be activated by Gq
• PLCδ functions in amplifying the signal
• PLCγ contains SH2 and SH3 domains, meaning it can be activated by receptor tyrosine kinases (e.g. insulin receptor)

Question 14

IV. IP3 signaling way
1. What is PLC activated by?

Answer 14

LC will be activated by a Gq G-protein, which will generate IP3 and DAG from PIP2.

Question 15

IV. IP3 signaling way
2. What is IP3?

Answer 15

IP3 is a water soluble molecule that leaves the plasma membrane and diffuses rapidly through the cytosol.

Question 16

IV. IP3 signaling way
3. What is the mechanism of IP3?

Answer 16

• IP3 is a water soluble molecule that leaves the plasma membrane and diffuses rapidly through the cytosol.
• When it reaches the ER, it binds and opens an IP3-gated Ca2+- channel (IP3 receptors) in the ER membrane. Ca2+ stored in the ER is released through the open channels, quickly raising the concentration of Ca2+ in the cytosol.

Question 17

IV. IP3 signaling way
4. What are the location and role of DAG?

Answer 17

DAG (=amphiphile – both hydrophilic & hydrophobic properties), which remains embedded in the plasma membrane, will in the presence of Ca2+ activate PKC.

Question 18

IV. IP3 signaling way
5. Describe the structure of PKC?

Answer 18

PKC consists of two domains: one regulatory domain (for DAG and Ca2+) and a catalytic domain (for ATP and substrate).

Question 19

IV. IP3 signaling way
6. What is the mechanism of PKC?

Answer 19

• PKC consists of two domains: one regulatory domain (for DAG and Ca2+) and a catalytic domain (for ATP and substrate).
• When PKC is activated by Ca2+, it is translocated to the plasma membrane where it binds phosphatidyl-serine residues and DAG.
• This activation causes the PKC to a conformation change and release of an inhibitory molecule.
• PKC will ‘’open up’’, so that its catalytic site is available to phosphorylate target proteins.
• PKC will be active for as along as the Ca2+-signal lasts, and will dissociate from the membrane after the activation.

Question 20

IV. IP3 signaling way
7. How is PLCγ activated?

Answer 20

PLCγ activated by receptor tyrosine kinases: leading the enzyme to the substrate

Question 21

IV. IP3 signaling way
8. What are the steps in IP3 signaling way?

Answer 21

• IP3 is a water soluble molecule that leaves the plasma membrane and diffuses rapidly through the cytosol.
• When it reaches the ER, it binds and opens an IP3-gated Ca2+- channel (IP3 receptors) in the ER membrane.
• Ca2+ stored in the ER is released through the open channels, quickly raising the concentration of Ca2+ in the cytosol.
• DAG (=amphiphile – both hydrophilic & hydrophobic properties), which remains embedded in the plasma membrane, will in the presence of Ca2+ activate PKC.
• PKC consists of two domains: one regulatory domain (for DAG and Ca2+) and a catalytic domain (for ATP and substrate).
• When PKC is activated by Ca2+, it is translocated to the plasma membrane where it binds phosphatidyl-serine residues and DAG.
• This activation causes the PKC to a conformation change and release of an inhibitory molecule.
• PKC will ‘’open up’’, so that its catalytic site is available to phosphorylate target proteins.
• PKC will be active for as along as the Ca2+-signal lasts, and will dissociate form the membrane after the activation.

Question 22

V. Signaling with PI 3-kinase
1. How does it signal with PI 3-kinase?

Answer 22

• Phosphoinositide 3-kinase (PI 3-kinase) is activated in different pathways, such as growth factor, insulin and integrin signaling, and is a part of the survival pathway
• Mainly phosphatidylinositol 3,4-bisphosphate (PI [3,4]-P2) and phosphatidylinositol 3,4,5-trisphosphate (PI [3,4,5]-P3)
• The lipid products generated by PI 3-kinase recruit PH domain proteins to the plasma membrane, for example PKB (Akt) and 3-phosphoinositide-dependent protein kinase-1 (PDK1)

Question 23

V. Signaling with PI 3-kinase
2. What are the 3 classes of PI 3-kinase? Describe

Answer 23

There are 3 classes of PI 3-kinases, where only class I is involved in signaling:
- Class I.A: contains a 85kDa regulatory subunit which has the SH2 domain that is phosphorylated by receptor tyrosine kinases and a 110kDa catalytic subunit which contains the catalytic domain
- Class I.B: consists of the βγ subunit of heterotrimeric G-proteins and a 110kDa catalytic subunit

Question 24

V. Signaling with PI 3-kinase
3. Describe Class I.A

Answer 24

Class I.A: contains a 85kDa regulatory subunit which has the SH2 domain that is
phosphorylated by receptor tyrosine kinases and a 110kDa catalytic subunit which
contains the catalytic domain

Question 25

V. Signaling with PI 3-kinase 4. Describe Class I.B

Answer 25

Class I.B: consists of the βγ subunit of heterotrimeric G-proteins and a 110kDa catalytic subunit

Question 26

V. Signaling with PI 3-kinase 5. How does activation of class I.A work?

Answer 26

- Class I.A PI 3-kinase can bind to an auto-phosphorylated receptor (tyrosine kinase) or GTP-bound Ras - Concurrent binding results in the strongest activation

Question 27

V. Signaling with PI 3-kinase 6. How does activation of class I.B work?

Answer 27

Can be activated by a receptor tyrosine kinase, for example insulin receptor

Question 28

V. Signaling with PI 3-kinase 7. What is the mechanism for the Activation of the effector enzymes by PI 3-kinases?

Answer 28

Inactive PDK1 is recruited to the plasma membrane and is phosphorylated by PI 3-kinases -> active PDK1 phosphorylates PKB (Akt) -> active PKB is released and will phosphorylate its substrates: - GSK3 (glycogen synthase kinase-3): which will inactive glycogen synthase enzyme (response to insulin when glucose is high) - BAD protein (survival pathway): will keep the BAD protein in the phosphorylated state, thereby releasing a protein called ‘’active apoptosis-inhibitory protein’’ which will prevent apoptosis from occurring

Question 29

V. Signaling with PI 3-kinase 8. How is PI 3-kinase encoded?

Answer 29

- PI 3-kinase is encoded by proto-oncogene, a gene that could become an oncogene due to mutation or increased expression. - Oncogene = a gene that has potential to cause cancer.

Question 30

V. Signaling with PI 3-kinase 8. How is PI 3-kinase encoded?

Answer 30

- PI 3-kinase is encoded by proto-oncogene, a gene that could become an oncogene due to mutation or increased expression. - Oncogene = a gene that has potential to cause cancer.

Question 31

V. Signaling with PI 3-kinase 9. What are the features of PTEN?

Answer 31

- PTEN acts as a phosphatase and is encoded by tumor suppressor gene (PTEN gene). - Mutations of this gene are a step in the development of many cancers. - PTEN will dephosphorylate PI (3,4,5)-P3 to PI (3,4)-P2, reversing the action of PI 3-kinases.