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Pharmacology > PI3K signalling > Flashcards

PI3K signalling Flashcards

1
Q

What does PI3K catalyse?

A

Production of PIP3 (phosphatidylinositol-3,4,5-triphosphate) from PIP2 (phosphatidylinositol-4,5-diphosphate) in response to growth factors and hormones

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2
Q

What does PIP3 act as?

A

A membrane anchor for various signalling proteins, including Rac and Rho GTPases, TEC tyrosine kinases, PLC-gamma and PKB/Akt

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3
Q

PIP3 acts as a memrane anchor for various signalling proteins. Name 4/5.

A
  1. Rac and Rho GTPases
  2. TEC tyrosine kinases
  3. PLC-gamma
  4. PKB/Akt
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4
Q

How are the signalling proteins Rac and Rho GTPases, TEC tyrosine kinases, PLC-gamma and PKB/Akt recruited to the membrane? Where are they usually located in unstimulated cells?

A

Via PIP3. They are usually in the cytosol of unstimulated cells

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5
Q

Multiple isoforms of PI3K exist. How many classes?

A

3: Class I, Class II, Class III

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6
Q

Which class of PI3Ks are the best understood?

A

Class Ia (alpha, beta and delta)

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7
Q

Which PI3K isoforms are in Class Ia?

A

alpha, beta and delta

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8
Q

Which class of PI3Ks are primarily responsible for the formation of PIP3 in response to growth factors?

A

Class Ia (alpha, beta, delta)

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9
Q

What is the downstream effector enzyme of Class Ia PI3Ks?

A

PKB (also known as Akt)

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10
Q

What is PKB also known as?

A

Akt

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11
Q

Class I PI3K is a heterodimeric enzyme. What are its two subunits?

A

Catalytic

Regulatory

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12
Q

Class I PI3K, in mammals, contains how many different types of each of its two subunits?

A
  1. 4 different catalytic subunits (p110-alpha, p110-beta, p110-delta and p110-gamma)
  2. 3 different regulatory subunits (p85-alpha, p85-beta, p55-gamma)
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13
Q

Which receptors are PI3Ks typically activated by?

A

Tyrosine kinase receptors:

1) EGFR,
2) HER-2,
3) PDGFR-alpha,
4) IGF-1R,
5) VEGFR2

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14
Q

How is the catalytic subunit of PI3K maintained in a low-activity state in the cytoplasm in unstimulated cells?

A

By the NEGATIVE INFLUENCE of the regulatory subunit

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15
Q

How is the negative influence of the regulatory subunit on the catalytic subunit of PI3K removed?

A

Upon cell stimulation, the regulatory subunit interacts directly with phosphotyrosine residues on activated tyrosine kinase receptors r on phosphorylated adaptor proteins linked to receptors.
This alleviates the negative influence on the catalytic subunit

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16
Q

How does PI3K become activated?

A

The regulatory subunit binds to phosphotyrosines on activated TRKs/phosphorylated adaptor proteins linked to the receptors

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17
Q

What 2 functions does the binding of the PI3K regulatory subunit to phosphotyrosines have?

A
  1. alleviates negative influence on the catalytic subunit

2. brings catalytic domain to its substrate (PIP2), allowing PIP3 to be produced

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18
Q

What is the substrate for the PI3K catalytic domain?

A

PIP2

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19
Q

PIP3 acts as an anchor for a range of signalling proteins. Which is one of its major effectors?

A

Akt.

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20
Q

What happens when Akt binds to PIP3?

A
  1. Localises Akt to the plasma membrane, allowing it to be phosphorylated (and hence activated) by the kinase PDK1 (which also binds PIP3).
  2. Phosphorylated Akt moves into the cytoplasm and subsequently phosphorylated many proteins critical to cell growth and survival.
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21
Q

Which enzyme activates Akt (PKB)?

A

PDK1

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22
Q

How does PDK1 activate Akt(PKB)?

A

They are both bound to PIP3 at the plasma membrane, and PDK1 phosphorylates PKB

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23
Q

Once phosphorylated by PDK1 at the plasma membrane, what happens to PI3K?

A

It moves to the cytoplasm and phosphorylates many proteins critical to cell growth and survival

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24
Q

How is PI3K recruited to the plasma membrane?

A

Its regulatory subunit binds to phosphotyrosines

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25
Q

Activated Akt moves to the cytoplasm and phosphorylates many proteins critical to cell growth and survival. Give three examples of such proteins, and how phosphorylation affects them.

A
  1. Pro-apoptotic proteins such as BAD and caspase 9 are inhibited
  2. Transcription of the forkhead-related (FKHR) family of transcription factors is blocked
  3. GSK3 is inhibited, resulting in activation of pathways usually inhibited by GSK3
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26
Q

How is PI3K signalling terminated?

A

Degraded by 2 different types of phosphatases:

  1. SHIP
  2. PTEN
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27
Q

Which 2 types of phosphatases are responsible for the degradation of PI3K (and thus termination of its signalling)?

A
  1. SHIP (Src-homology 2 (SH2)-containing phosphatases)
    - SHIP1 (restricted to haematopoietic cells)
    - SHIP2 (ubiquitous expression)
  2. PTEN (phosphatase and tensin homolog deleted on Chr 10)
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28
Q

What is SHIP, and what is the difference between its two types?

A

Src-homology 2 (SH2)-containing phosphatases
SHIP1 is restricted to hematopoietic cells
SHIP2 is ubiquitous

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29
Q

Several PI3K cancer activating mutations have been identified. Particularly in which subunit?

A

The p110 catalytic subunit

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30
Q

Give two examples of mutations in PI3K that have been linked to cancer.

A

H1047R: increases plasma membrane binding
E545K: decreases inhibition from p85a regulatory subunit

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31
Q

PTEN has tumour-suppressor properties. Which of its abilities is this attributed to?

A

Its ability to dephosphorylate PIP3

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32
Q

What is PTEN’s role in PI3K signalling?

A

It dephosphorylates PIP3 thus terminating PI3K signalling

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33
Q

What diseases are somatic mutations in PTEN frequently associated with?

A

Cancer

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34
Q

Mutations in PTEN are associated with 2 inherited tumour syndromes. What are these?

A

1) Cowden disease

2) Bannayan-Zonana syndrome

35
Q

How can studies be carried out to study the activity of PKB/Akt in cancers?

A

Using phospho-specific antibodies for activated PKB/Akt

36
Q

PKB/Akt activity has been shown to be elevated in a range of cancers. List 3.

A
  1. breast
  2. colon
  3. ovarian
  4. pancreatic
  5. prostate
37
Q

What type of cancer is mutations in SHIP1 associated with?

A

Leukaemia

38
Q

Give two examples of p110-alpha-specific inhibitors that have been developed and are in clinical trials.

A
  1. BYL-719

2. A66

39
Q

How is the PI3K pathway being targeted to treat cancer?

A

Selective inhibitors are currently in trials

40
Q

Phosphatidylinositol constitutes what percentage of membrane lipids?

A

5-10%

41
Q

Where on PIP2 does PI3K add the phosphate group?

A

The 3’ -OH position of the inositol ring

42
Q

How are membrane phospholipids classified?

A

According to their polar head group (e.g. choline or inositol)

43
Q

What plays a critical role in determining the downstream signalling effect of a phosphatidyinositol?

A

The location of phosphates on the inositol ring

44
Q

What are the two different forms of PIP2 from which PI(3,4,5)P3 can be generated?

A

PtdIns(3,4)P2 and PtdIns(4,5,)2

45
Q

Which kinases make PIP3 from:

1) PI(3,4)P2
2) PI(4,5)P2

A

1) PI5K

2) PI3K (I)

46
Q

Which enzyme catalyses the degradation of PIP3 into:

1) PI(4,5)P2
2) PI(3,4)P2

A

1) PTEN

2) SHIP1 + 2

47
Q

Which domains are found in the regulatory (p85) subunit of PI3K?

A

2 x SH2

SH3

48
Q

Which domains are found in the catalytic subunit of PI3K?

A 
p85 binding domain
Ras binding domain
HR3 (C1-like, possible plays a role in membrane binding)
HR2 (acts as scaffold)
HR1 (kinase core)
49
Q

Where in the catalytic subunit of PI3K is the catalytic activity?

A

In the amino terminal (in HR1 domain)

50
Q

How does the catalytic subunit of PI3K bind to the regulatory subunit?

A

Via the catalytic subunit’s p85 binding domain

51
Q

How is PI3K activated via growth factor receptors?

A

1) activation of growth factor receptor causes AUTOPHOSPHORYLATION producing docking sites for adaptor proteins and PI3K
2) Interactions between p85 with phosphorylated receptor and between activated RAS and the p110 subunit recruits PI3K to the membrane, bringing into contact with its substrate

52
Q

In leukocytes, PI3Ks can be activated by non-receptor kinases. Give an example.

A

Janus kinases (JAKs), Syk or ZAP70

53
Q

Class Ib PI3Ks consist of a p110-gamma catalytic subunit and do not interact with p85 adaptor molecules. What do they interact with instead?

A

The Beta-Gamma subunits of G-proteins

54
Q

p85, in addition to interaction with the phosphorylated growth factor receptor, may also interact with non receptor proteins at the membrane in order to bring p110 into contact with PIP2. Give an example of this.

A

A complicated signalling scaffold formed by the adaptor proteins Shc, Grb2 and Gab2 can recruit PI3K to the plasma membrane in response to the cytokines IL-2 and IL-3

55
Q

Proteins with what types of domains bind to PIP3?

A

Pleckstrin homology (PH) domains

56
Q

How do signallinng proteins accumulate at the sites of PI3K activation?

A

By binding to PIP3 via their pleckstrin homology (PH) domains

57
Q

How does PI3K affect cell growth, survival and movement?

A

Through the signalling proteins that bind to PIP3 through their PH domains

58
Q

Give 6 examples of proteins that mediate PI3K signalling through being recruited to PIP3 via their PH domains. Briefly explain what they do.

A
  1. Rac/Rho GTPases (involved in cytoskeletal changes and regulation of MAPKs)
  2. TEC (tyrosine kinases involved in regulating gene expression)
  3. ARF6 (ADP-ribosylating factor 6)
  4. PLC-gamma
  5. PKB
  6. PDK1
59
Q

What sort of enzyme is PKB?

A

serine/threonine kinase

60
Q

PKB is a serine/threonine kinase belonging to which superfamily?

A

The AGC superfamily

61
Q

How many isoforms of PKB exist in mammals?

A

3

62
Q

What are the three functionally distinct regions of PKB?

A
  1. N-term PH domain
  2. C-term catalytic domain
  3. C-term hydrophobic motif
63
Q

What happens to most targets of PKB?

A

Inhibited

64
Q

Whilst PKB normally inhibits the targets it phosphorylates, what is usually the effect on the signalling pathway?

A

Often the protein being inhibited is an inhibitor itself, so PKB causes stimulatory effect in signalling pathway

65
Q

What does activation of PKB require?

A

Phosphorylation by PDK

66
Q

In the case of PKB, PDK acts as a simple ‘on/off’ switch. How does it act on PKC?

A

Phosphorylation by PDK acts as a ‘priming step’, promoting autophosphorylation that generates a catalytically competent PKC form that requires only DAG/Ca2+ binding for full activation

67
Q

How does PKB promote cell survival?

A

Through inhibiting pro-apoptotic proteins (BAD and Caspase 9)

68
Q

How does SHIP terminate PI3K signalling?

A
  1. The PH domain of PKB is able to bind both PI(3,4)P2 and PI(3,4,5)P3 with equal affinity
  2. thus, upregulation of SHIP activity would remove PIP3 binding sites for PH domain containing proteins with PIP3 selectivity leaving PI(3,4)P2 sites that PKB could bind to.
69
Q

How does PTEN terminate PI3K signalling?

A

Removes both PIP3 and PI(3,4)P2 sites, having a more negative impact on PKB

70
Q

What is the effect of the E545K mutation in the Type Ia PI3K p110-alpha subunit?

A

Decreases inhibition from the p85-alpha regulatory subunit

71
Q

What is the advantage of ISOTYPE-SELECTIVE PI3K inhibitors?

A

They are specific to LIMIT POTENTIAL SIDE-EFFECTS

72
Q

What was a main problem with PI3-K inhibitors?

A

Not very specific - side effects

73
Q

Give an example of a PI3K inhibitor that is currently in clinical trials

A

XL147

74
Q

What is PI3K?

A

Phosphoinositide 3-kinase. An enzyme that catalyses the production of phosphatidylinositol-3,4,5-triphoshate (PIP3) from phosphatidyl-4,5-diphosphate (PIP2)

75
Q

Which PI3K catalytic subunit isotypes are involved in:

1) PI3K signalling through the insulin receptor
2) lymphocyte signalling

A

1) p110-alpha

2) p110-gamma and p-110-delta

76
Q

p110-alpha plays an important role in PI3K signalling from the insulin receptor. As such, why might you want to target the p110-beta isotype in targeting the PI3K signalling pathway to treat cancer?

A

To limit HYPERGLYCAEMIA as a side effect

77
Q

Why might targeting p110-gamma and p110-delta to treat cancer have negative side effects?

A

They both play important roles in lymphocyte signalling. An inhibitor could cause immunological side effects

78
Q

What part of the PI3K signalling pathway does the drug CAL-101 target?

A

p110-delta

79
Q

How does the drug CAL-101 work?

A

It is a selective inhibitor of p110-delta. As this isotype is important in antiapoptotic signalling pathways in some forms of leukaemia, inhibiting p110-delta could induce apoptosis to treat leukemia

80
Q

Which cancer does CAL-101 target?

A

Leukemia

81
Q

Which PI3K catalytic subunit isotype does CAL-101 target?

A

p110-delta

82
Q

Name two inhibitors of PKB that are currently under development

A
  1. Perifosine - inhibits ability of Akt to localise to plasma membrane
  2. MK-2206 - allosteric inhibitor of Akt
83
Q

How does Perifosine work?

A

Inhibits ability of Akt to localise to plasma membrane

Pharmacology (13 decks)

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