4: Signalling Mechanisms in Growth and Division Flashcards
(47 cards)
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)
2
Q
What are the key components of this signalling pathway?
A
- Regulation of enzyme activity by protein phosphorylation (kinases)
- Adapter proteins
- Regulation by GTP-binding proteins
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
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
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)
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
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
8
Q
Summarise the RPTK signal to Ras
A
- ligand binds to TK-receptor (GF to GFR)
- this causes ic changes in the TK receptors (cross phosphorylation)
- Adaptor proteins bind (e.g. Grb2) to c-terminus phosphorylated tyrosine
- 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.
- Ras activation causes further transmission of the signal
- Ras is inactivated by GTPases and GAPs (GTPase activating proteins)
9
Q
Sos
A
- mutated in many different cancers
- exchange factor that is a part in activating Ras
- bound to Grb2 via SH3 domains
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.
11
Q
What is the ERK cascade?
A
= extracellular-signal regulated cascade
- activated by Ras
- this is a specific one, MAPK is generic
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
13
Q
Summarise the steps of the Erk cascade
A
- Activation via Ras
- phosphorylation od Raf (MAPKKK) to….
- …Mek; phosphorylation of Mek (MAPKK) to…..
- ….. Erk (MAPK)
- 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)
14
Q
Raf
A
- b-Raf is am oncogene (upregulated in melanoma)
- =MAPKKK
- first kinase in the Erk cascade
15
Q
What are Rys and Myc?
A
Oncogenes
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
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
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
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
Growth factor stimulation of signaling pathways promotes G0 to G1 transition
- 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
Cyclin D1
- oncogene
- over expressed in 50% of breast cancers
- c-myc acts on CycD
27
How does the ERK cascade regulate gene expression and leads to the progression through G1?
- 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
Regulation of gene expression by Rb
- 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
Regulated expression of Cyclins and Cdks - how does it work?
- 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
Which genes are regulated by ET2?
```
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
E2F and Cdk activity throughout the cell cycle
- 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
CKI
- 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
What are the two families of CKIs? What separates them?
- 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
Ras mutations
- 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
What is the activity of Cdks regulated by?
- interaction with cyclins
- phosphorylation
They are present in the cell throughout the cell cycle.
36
When are cyclins present?
- 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
How are Cdks activated?
- 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
What is the function of Cdk1 and Cyclin B?
- Cdk1/cycB active: Mitosis “on hold” – key substrates phosphorylated
- act as mitosis promoting factor
39
What leads to the degradation of cdk1/cycB?
- 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
What is the function of APCC?
= 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
Different cyclins and Cdks at different parts of the cell cycle
- Cyclins activate Cdks but also alter substrate specificity
| - substrate accessibility changes through cell cycle
42
What promotes G0 to G1 transition?
- 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
What does Cmyc stimulate?
- e.g. transcription of Cyclin D1 (which is needed for the transition from G0 to G1
44
Which cyclins and Cdks do what in which part of the cell cycle?
- 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
Nuclear lamins and MPF
- The nuclear lamins are phosphorylated by MPF (Cdk1 and CycB)
- this leads to breakdown of the nuclear envelope
46
Give an action of Cdk2/CycE
phosphorylation of Retinoblastoma protein (pRb)
| Tumour suppressor - inactivated in many cancers
47
CKI
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.
