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Year 2 - MCD - Cancer > 4: Signalling Mechanisms in Growth and Division > Flashcards

4: Signalling Mechanisms in Growth and Division Flashcards

1
Q

c-myc

A
  • oncogene
  • over expressed in many tumours
  • c-myc is a TF: stimulates the expression of cell cycle genes (e.g. cyclin D1)
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2
Q

What are the key components of this signalling pathway?

A
  1. Regulation of enzyme activity by protein phosphorylation (kinases)
  2. Adapter proteins
  3. Regulation by GTP-binding proteins
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3
Q

What are the effects of GF binding?

A
  • in the presence of a ligand the receptor dimerises and is activated via phosphorylation at tyrosine kinase domains
  • tyrosine phosphorylation provides docking sites for adapter proteins -> ic signalling proteins bound to phosphorylated tyrosines
  • signal relayed into the cells interior
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4
Q

Herceptin

A
  • anti-HER2 AB
  • used in treatment of HER2 positive metastatic breast cancer
  • binds to the ec domain of the receptor and prevents all the signalling by EGFR
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5
Q

Grb2

A
  • adaptor protein involved in signal transduction
  • has 2 Src homology regions (SH) (Src = oncogene)
    • SH3 (x2): binds to proline rich regions (constitutive)
    • SH2: binds to phosphorylated tyrosines (inducible, specific sequence context)
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6
Q

Adaptor proteins

A
  • Tyrosine phosphorylation provides docking sites for adapter proteins
  • Protein-protein interactions: protein binding – bringing proteins together
  • Proteins are modular and contain domains, i.e. functional and structural units that are copied in many proteins
  • Some domains are important in molecular recognition – have no enzymatic function of their own, simply bring other proteins together
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7
Q

Ras

A
  • GTPase
  • GTP binding protein
  • transmits signals
  • activated by GTP binding (after a signal comes in) via exchange factor (e.g. Sos)
  • deactivated by GTPase activating proteins that stimulate the hydrolysis of bound GTP to GDP
  • must bind to the plasma membrane to be activated
  • NOT kinases!!
  • major oncogene
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8
Q

Summarise the RPTK signal to Ras

A
  1. ligand binds to TK-receptor (GF to GFR)
  2. this causes ic changes in the TK receptors (cross phosphorylation)
  3. Adaptor proteins bind (e.g. Grb2) to c-terminus phosphorylated tyrosine
  4. An adaptor protein that is bound to Sos (= exchange factor) is responsible for the activation of Ras (via GTP binding) -> proximity is important, only the Ras that is close by will be activated.
  5. Ras activation causes further transmission of the signal
  6. Ras is inactivated by GTPases and GAPs (GTPase activating proteins)
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9
Q

Sos

A
  • mutated in many different cancers
  • exchange factor that is a part in activating Ras
  • bound to Grb2 via SH3 domains
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10
Q

Common Ras mutations in cancer

A

V12Ras

  • glycine12 -> valine
  • prevents GAP binding (=prevents inactivation)
  • constitutively active

L61Ras

  • glutamine61->Leucine
  • prevents GTP hydrolysis
  • constitutively active

Trasnient activation and inactivation of these pathways is important for homeostasis -> problem if mutated in cancer.

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11
Q

What is the ERK cascade?

A

= extracellular-signal regulated cascade

  • activated by Ras
  • this is a specific one, MAPK is generic
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12
Q

ERK vs MAPK

A

ERK = extracellular-signal regulated (ERK) cascade
-> specifically

MAPK = Mitogen-activated protein kinase (MAPK) cascades
-> generically

2 names for the same group of proteins that are activated downstream of Ras

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13
Q

Summarise the steps of the Erk cascade

A
  1. Activation via Ras
  2. phosphorylation od Raf (MAPKKK) to….
  3. …Mek; phosphorylation of Mek (MAPKK) to…..
  4. ….. Erk (MAPK)
  5. further phosphorylation causes: a) changes in gene expression and b) change in protein activity via phosphorylation of molecules e.g. proteins or gene regulatory proteins.
    - > this causes e.g. c-myc, cell proliferation

(ATP in, ADP out, Men has 1 P attached, Erk has 2 Ps attached)

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14
Q

Raf

A
  • b-Raf is am oncogene (upregulated in melanoma)
  • =MAPKKK
  • first kinase in the Erk cascade
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15
Q

What are Rys and Myc?

A

Oncogenes

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16
Q

What controls the cell cycle? Why does it have to be controlled?

A
  • Cyclin-dependent kinases (Cdks) -> cyclically active protein kinases

Important because cell division..

  • is complex
  • requires tract regulation
  • checkpoints: need correct timing and sequence
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17
Q

Cdks

A

= Cyclin dependant kinases

  • control the cell cycle
  • present in proliferating cells throughout cell cycle
  • activity is regulated by a) interaction with cyclins and b) phosphorylation
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18
Q

Cyclins

A
  • key regulators of cyclin dependant kinases
  • Transiently expressed at specific points in the cell cycle
  • regulated at level of expression -> regulated expression, constant synthesis and degradation (can be seen on gels)
  • Cyclin(s) bind to and activate Cdk(s) triggering different events in the cell cycle
  • they also alter target specificity of the Cdks
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19
Q

MPF

A

mitosis promoting factor

20
Q

What do activated Cdk’s do?

A
  • They phosphorylate proteins (on Serine or Threonine) to drive cell cycle progression
  • e.g. Cdk1/CycB: Nuclear lamins (causes breakdown of nuclear envelope -> chromosomes can migrate)
  • e.g. Retinoblastoma protein (pRb)
    Tumour suppressor - inactivated in many cancers
  • Different cyclins/cdks target different substrates at different cell locations during the cell cycle.
21
Q

How are Cdk’s activated?

A
  • Requires activating phosphorylation AND removal of inactivating phosphorylation
  • Cdk1 and CycB form a complex -> Cdk activating kinase phosphorylates it -> inactive complex with an activating and inhibitory site -> phosphatase removes the inactivating site -> active Cdk1 (at the end of interpahse)

-> goes through 3 controls, strictly regulated.

22
Q

How are Cdk activating kinases (CAKs) activated?

A
  • via phosphorylation

- there is a positive feedback loop by the active Cdk product

23
Q

What is the Cdk signalling in entry into mitosis?

A
  • before mitosis Cdk1/CycB is active
  • Mitosis is ‘on hold’
  • key substrates are phoshporylated
  • at the anaphase checkpoint there are signals from fully attached kinetochores
  • these signals cause CycB degradation
  • Cdk1 is inactivated
  • key substrates are dephosphorylated
  • Mitosis progresses.
24
Q

What are the different Cdk’s and Cyclins in the different parts of the cell cycle?

A
  • Cdk1/CycB = MCdk (MPF) > promotes entry into mitosis
  • Cdk2/CycE = G1/S Cdk
  • Cdk2/CycA = S-Cdk
  • Cdk4/6 and CycD = G1Cdk (progression from G0->G1)
  • > Cdk2 binds to different cyclins
  • Cyclins activate Cdks but also alter substrate specificity
  • substrate accessibility changes through cell cycle
25
Q

Growth factor stimulation of signaling pathways promotes G0 to G1 transition

A
  • GF binding
  • MAPK cascade
  • Immediate early gene transcription factors (e.g. c-jun, c-Fos, c-Myc)
  • Stimulate transcription of other genes (e.g. cyclin D)
26
Q

Cyclin D1

A
  • oncogene
  • over expressed in 50% of breast cancers
  • c-myc acts on CycD
27
Q

How does the ERK cascade regulate gene expression and leads to the progression through G1?

A
  • GF binds to GFR
  • dimerisation of R, tyrosine kinases, adaptor proteins bind
  • Ras activation
  • Ras activates the MAPK cascade
  • MAPK cascade up regulates protein activation and gene expression incl. c-myc
  • c-myc is a TF for cyclin D which is responsible for the progression from G0 -> G1
28
Q

Regulation of gene expression by Rb

A
  • Rb is a ts-gene
  • active Rb binds to E2F which is a TF and maintains it inactivated
  • therefore it inhibits transcripton of Cycle
  • pRb acts as a brake on the cell cycle
  • when CycD is activated it phosphorylates Rb releasing. E2F which binds to different genes
  • pRb is active when it is unphosphorylated ad it is activated (holds E2F) when it is not phosphorylated
29
Q

Regulated expression of Cyclins and Cdks - how does it work?

A
  • Cdks become sequentially active and stimulate synthesis of genes required for next phase, e.g. cyclin D/Cdk4/6 stimulates expression of cyclin E – gives direction and timing to cycle
  • cyclins susceptible to degradation, hence cyclical activation
  • Sequence of events, degradation of one activates the other one (see slide for specific Cdks and Cycs at different parts of the cycle)
30
Q

Which genes are regulated by ET2?

A 
Protooncogenes:
c-Myc
N-Myc
B-Myb
IGF-1
Cell Cycle:
E2F-1,2,3
pRb
p107
cyclin A
cyclin E
CDK4
CDK2
DNA Synthesis:
Thymidine kinase
Thymidine synthetase
Dihydrofolate reductase (DHFR)
DNA Polymerase

-> constitutive activation of Rb is disastrous for the cell because of how many things it activates downstream (Seen in some cancers)

31
Q

E2F and Cdk activity throughout the cell cycle

A
  • c-Myc stimulates CycD and Cdk4/6 binding, phosphorylation of pRb comes with it.
  • Phosphorylation pRb means that it is inactivated and releases E2F -> CycE+Cdk2 bind
  • this causes further phosphorylation of pRb (2p now) and - this releases a larger amount of E2F and CycA+Cdk2 are stimulated
  • This causes further phosphorylation of pRb (3p now) which increases TF and causes CycB and Cdk1 to stimulate mitosis

(see slide 34)

32
Q

CKI

A
  • Cdk inhibitors
  • Cdks are also regulated by those.
  • inactivate Cdks by e.g. binding to the cyc/cdk complex or by displacing cyclin
  • CKI must be degraded to allow cell cycle progression
33
Q

What are the two families of CKIs? What separates them?

A
  • sparateed by the timing of the cell cycle
  • CKI must be degraded to allow cell cycle progression
INK4 family: G1 phase CKIs (inhibit Cdk4/6 by displacing cyclin D) -> degraded dutring G1 phase
	p15INK4b	
	p16INK4a
	p18INK4c
	p19INK4d

CIP/KIP family: S phase CKIs (inhibit all Cdks by binding to the Cdk/cyclin complex) -> degraded during S-phase

p21CIP1/WAF1
p27KIP1	
p57KIP2

p27KIP1 tumour suppressor – reduced expression correlates with poor prognosis in many malignancies

34
Q

Ras mutations

A
  • Mutations exist where Ras is ontogenically activated.
  • V12 Ras: prevents GAP binding (inactivation) -> glycine to valine
  • L61 Ras: prevents GTP hydrolysis -> glutamine to leucine
35
Q

What is the activity of Cdks regulated by?

A
  • interaction with cyclins
  • phosphorylation

They are present in the cell throughout the cell cycle.

36
Q

When are cyclins present?

A
  • Transiently expressed at specific points in the cell cycle
  • regulated at the level of expression
  • Synthesised, then degraded (slowly goes up and then rapid decline/fall)
37
Q

How are Cdks activated?

A
  • Cdk and cyclin bind together (inactive)
  • an inhibitory phosphate ( e.g. wee1) and an activating phosphate (CAK - cyclin activating kinase) are added to the Cdk (inactive)
  • a phosphatase (e.g. cdc25) removes the inhibitory phosphate
  • the Cdk is then active
  • Dephosphorylation activates Cdk1 at the end of interphase
  • positive feedback reinforces activation of MPF and drives mitosis (inactive cdc25->active cdc25 (phosphorylated))
38
Q

What is the function of Cdk1 and Cyclin B?

A
  • Cdk1/cycB active: Mitosis “on hold” – key substrates phosphorylated
  • act as mitosis promoting factor
39
Q

What leads to the degradation of cdk1/cycB?

A
  • Signal from fully attached kinetochore causes cyclin B to be degraded:
  • Cdk1 inactivated
  • key substrates dephosphorylated
  • mitosis progresses
  • Anaphase Promoting Complex C (APC/C) is a Ub ligase that catalyses Ubn and degradation of cycB.
  • APC/C is activated by Cdc20 which binds to phosphorylated APC/C - this phosphorylation is carried out by Cdk1/cycB.
  • So, Cdk1/cycB promotes it’s own degradation by activating APC/C-Cdc20.
  • Active APC/C-Cdc20 (amongst other things) is released from attached kinetochores to signal cycB degradation.
40
Q

What is the function of APCC?

A

= anaphase promoting complex

-> transition from metaphase to anaphase

  • binds to cdc20
  • activated by cdk1/cycB -> promotes its own degradation
  • activated by correctly attached kinetochores.
41
Q

Different cyclins and Cdks at different parts of the cell cycle

A
  • Cyclins activate Cdks but also alter substrate specificity

- substrate accessibility changes through cell cycle

42
Q

What promotes G0 to G1 transition?

A
  • GF stimulation of signalling pathways
  • > Immediate early gene TFs (e.g. c-jun, c-Fos, c-Myc)
  • > stimulate the transcription of other genes (e.g. cyclin D
43
Q

What does Cmyc stimulate?

A
  • e.g. transcription of Cyclin D1 (which is needed for the transition from G0 to G1
44
Q

Which cyclins and Cdks do what in which part of the cell cycle?

A
  • Cdk4/6 + CycD: G0 -> G1 (CycD stimulated by c-myc)
  • Cdk2/CycE: G1 - > S
  • Cdk2/CycA: S -> G2
  • Cdk1-CycB: G2 -> M (MPF)
  • APC/C-Cdc20: drive metaphase -> anaphase transition
45
Q

Nuclear lamins and MPF

A
  • The nuclear lamins are phosphorylated by MPF (Cdk1 and CycB)
  • this leads to breakdown of the nuclear envelope
46
Q

Give an action of Cdk2/CycE

A

phosphorylation of Retinoblastoma protein (pRb)

Tumour suppressor - inactivated in many cancers

47
Q

CKI

A

cyclin kinase inhibitor

2 families:
- INK4: G1 phase CKIs
Inhibit Cdk4/6 by displacing cyclin D 
- CIP/KIP family: S phase CKIs
Inhibit all Cdks by binding to the Cdk/cyclin complex

CKI must be degraded to allow cell cycle progression

Families are separated by the timing of the cell cycle.

Year 2 - MCD - Cancer (15 decks)

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