Cancer 4 Flashcards
1
Q
what phase are cells in if they are not dividing?
A
- they will be in G0
- quiescent phase
2
Q
what is Mcy and what is its role?
A
- Mcy is a transcription factor (protein that controls the expression of other genes)
- the concentration of Myc is very low when the cell is in
the quiescent G0 phase
- when cell division is triggered by adding a growth factor there is a rapid and dramatic rise in Myc
- This correlates with cells moving out of G0 and into G1
- Myc controls many genes in the cell cycle
3
Q
What are the three key components of signaling pathways?
A
- regulation of enzyme activity by protein phosphorylation (kinases)
- the role of the adapter proteins
- regulation by GTP - binding proteins
4
Q
how are growth factors stimulated by signaling pathways?
A
- The growth factor arrives and it binds to a receptor (normally tyrosine kinase type receptors)
- it then acts via a small GTP binding protein (Ras)
- this then triggers a kinase cascade (very fast)
- this then triggers the activation of genes that are required for the progression of cells through the cell cycle (slower - 1 hour)
- one of the genes triggered early in the kinase cascade is c - Myc which goes on to regulate the expression of many other genes
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5
Q
explain signaling by peptide growth factors?
A
- the phosphorylated receptor protein tyrosine kinase recruits adapter and signaling proteins
- the receptors normally sit on the plasma membrane as monomers but most growth factors are dimetric
- the dimetric growth factor binds to x2 tyrosine kinase and brings them close
- when the receptors are close the tyrosine kinase cross - phosphorylates with the partner receptor
- Tyrosine kinases use the gamma phosphate of ATP to phosphorylate tyrosine residues in proteins
- The phosphorylated domains on the tyrosine kinase receptors act as docking sites for adaptor proteins
- The adaptor proteins contribute to downstream signalling
6
Q
give an example of interfering with growth factor signalling?
A
- there is an antibody called Herceptin that inhibits the her2 receptor tyrosine kinase
- this is important in blocking the early stages of growth stimulation
- that can be used in tumors eg. breast cancer
7
Q
give an example of an important adaptor molecule?
A
Grb2
8
Q
what do adaptor proteins bind to?
what is the structure of an adapter protein?
A
- the adapter proteins bind on to the phosphorylated tyrosines which act as docking sites
- adaptor proteins are often modular
- they are made up of domains that are mixed and matched
- These different domains are important in molecular recognition
9
Q
do adapter proteins have enzymic function?
A
- The adaptor molecules have no enzymatic function = they don’t do anything other than bringing other proteins together
10
Q
what are the 3 types of protein-protein interactions of Grb2?
A
- SH2 = binds to the phosphorylated tyrosines of the receptor
binds to RPTK
- SH3 (there are two copies) = bind to the proline-rich regions of other proteins
binds to Sos
11
Q
- Grb is bound to the RPTK via its SH2 domain and it binds to a protein called Sos through its SH3 domains
- Sos is an exchange factor for Ras
- Grb2 is always bound to Sos
- when RPTK is activated there is phosphorylation of the receptor
- Then Grb2 (with Sos attached) binds to these phosphorylated tyrosine domains
- Sos then is close enough to activate Ras
- Sos allows the exchange of `GDP for GTP in ras to form a GTP bound form of Ras
- this changes the conformation of Ras which puts it in an active state that is able to signal and allow the propagation of the signal
- it is important that the Ras protein binds with the plasma membrane
A
12
Q
why might interfering with the membrane binding of Ras be helpful?
A
- can make a good anti-cancer therapy
13
Q
what turns Ras off and on?
A
Ras is on when it is GTP bound
Ras is off when it is GDP bound
14
Q
what catalyses the replacement of GDP with GTP?
A
sos
15
Q
how does Ras turn itself on and off?
A
- This is a self-regulating system so Ras can turn itself off
- the GTP binding protein is able to hydrolyze GTP to GDP to turn itself off = ( intrinsic GTP hydrolysis capability )
- but it is very slow
16
Q
what external proteins turn ras on?
what external proteins turn ras off?
A
- Exchange factors (e.g. Sos) that turn it ON
- GTPase activating proteins (GAPs) that turn it OFF
17
Q
what is the main function of ras?
why is it important that it is carefully controlled?
A
- it is very important in growth factor stimulatory pathways
- it is important that ras is switched off in order to prevent uncontrolled division
18
Q
what might happen to Ras in cancer?
A
- in cancer the ras protein is mutated in ways that cause the Ras protein to constantly be in the GTP bound form
19
Q
show how RPTKs signal to Ras?
A
- there are dimeric activated receptors
- adaptor protein Grb2
- Grb2 binds to phosphorylated tyrosine
- this binds tightly to the exchange factor sos (activating protein)
- sos then mediates the action of Ras at the membrane
- allowing ras to transmit signal downstream
20
Q
give 2 example of point mutations in cancer affecting ras?
A
- V12Ras
- L61Ras
21
Q
explain the V12Ras mutation:
A
- the glycine in position 12 of Ras is changed to valine due to a point mutation
- the side chain changes from hydrogen to a hydrophobic side chain
- this prevents GAPs from binding to Ras
- this means Ras cannot be turned off very easily
22
Q
explain the L61Ras mutation:
A
- glutamine in position 61 is converted to leucine
- this is a single base change in the genome
- the side chain switches from being an amide to being a hydrophobic side chain
- This mutation inhibits the intrinsic GTPase activity of the Ras protein
- Ras is then constantly on and always giving growth stimulatory signals
23
Q
how does Ras activate the protein kinase cascade?
A
- GTP bound Ras binds to a kinase and then that kinase activates other kinases
- there is a chain of kinase phosphorylation
- The kinase cascade that is specifically involved in the growth stimulatory signaling is called the ERK cascade
- ERK cascades belong to the family of MAPK cascades
ERK cascade (extracellular signal regulated kinase cascade)
MAPK cascades (mitogen activated protein kinase cascade)
24
Q
what kinases make up the ERK kinase?
A
25
Q
what is the end result of the ERK cascade?
A
- at the end of the cascade, the last kinase phosphorylates a number of proteins and changes their activity
- among the proteins being phosphorylated there are gene regulatory proteins (transcription factors)
- once phosphorylated the transcription factors go on to regulate gene expression
- the most important gene to be turned on by the ERK pathway is c- Myc gene
- Myc and Ras are key molecules in stimulating growth
- Myc and Ras are oncogenes
26
Q
what are Cyclin-dependent kinases?
have are they controlled?
A
- These are serine-threonine kinases
- when they bind to an activating protein called cyclin they are activated
- The Cdks are ALSO controlled by phosphorylation
27
Q
what is the role of cyclins?
A
- cdks are activated by binding to cyclins
- Cyclins are transiently expressed during the cell cycle
- once they have activated the Cdks the cyclins are degraded
28
Q
when is cyclin necessary within the cell cycle?
A
At the start of mitosis
- the M phase promoting factor controls mitosis
- there is a release of mitotic cyclin which activates the Cdk
- once the Cdk has completed its role it is degraded and the Cdk is chopped up
Just before DNA replication
- At the start of DNA replication, there is a Cdk that is turned on by binding to an S phase cyclin
- again once the Cdk has carried out its function it is degraded
29
Q
what does the mitosis promoting factor consist of?
A
- cdk1
- mitotic cyclin (cyclin B)
30
Q
show on a graph what the pattern of cyclin release is?
A
- cyclical nature
- it shows that cyclins are expressed during mitosis and then they are degraded and this happens in a cyclical nature
31
Q
how are cdks regulated by phosphorylation?
A
- Cdk1 binds to cyclin B
- the Cdk- cyclin complex is usually inactive on its own
- the complex undergoes 2 phosphorylation reactions that regulate Cdk activity
- Cdk has to be activated at specific sites to become activated
- This activation is performed by Cdk activating kinase (CAK)
- Balancing this is an inhibitory kinase called Wee1
- Even though Cdk1 is bound to cyclin, it needs the inhibitory phosphate to be taken off before it can function
- Cdc25 takes off the inhibitory phosphate that was put on by Wee1
- only then is the MPF active
32
Q
how does dephosphorylation activate Cdk1?
A
- Dephosphorylation of the inhibitory site of Cdk1 by Cdc25 activates it towards the end of interphase
- Active MPF is able to phosphorylate Cdc25 to increase its activity
- Cdc25 takes off more inhibitory phosphate
- this is a form of positive feedback that drives mitosis
33
Q
how do cyclins and Cdk 1 help with mitosis checkpoints?
A
- whilst MPF is active at the end of metaphase it phosphorylates a number of key substrates that are involved in the mitotic process
- this puts mitosis on hold
- mitosis cannot progress to anaphase until the signal has been released
- Once the kinetochores are correctly attached to the microtubule spindles at the end of metaphase
- a signal is released that causes cyclin B to be degraded
- When cyclin B is degraded, Cdk1 becomes inactive
- this means that the substrates (which were keeping mitosis on hold) are dephosphorylated so then mitosis can progress
34
Q
what cyclins and cdks are needed at different stages of mitosis?
A
- different cyclins and cdks control different stages of mitosis
- the same cdk is being used but for different purposes
- this is because when cyclin binds to Cdk it actually changes its substrate specificity
- It also changes substrate accessibility
- this feature allows you to use the same Cdk in different stages of the cell cycle
35
Q
how does growth factor stimulation promote the transition of G1 to G2?
A
- growth factors come and bind to the receptor protein tyrosine kinase
- through Ras this triggers a kinase cascade
- this leads to phosphorylation of transcription factors that turns on the expression of c-Myc
- Myc is a transcription factor and one of its jobs is to stimulate the transcription of cyclin D
36
Q
how does Myc result in the synthesis of cyclin E?
A
- Myc is a transcription factor that stimulates the transcription of cyclin D
- Cyclin D activates Cdk4 and Cdk6 to stimulate the synthesis of cyclin E
- this triggers the cell cycle
37
Q
how do cyclins and cdks give timing and direction to the cell cycle?
A
- production of Myc leads to the synthesis of cyclin D
- cyclin D leads to production of active Cdk4/6 / cyclin D complex
- This Cdk- cyclin complex then stimulates the synthesis of the next cyclin
- each cyclin is involved in stimulating the synthesis of the next cyclin
- this gives the cell cycle both timing and direction
38
Q
what is the function of nuclear lamins?
A
- nuclear lamins cause the breakdown of the nuclear envelope
- Breakdown of the nuclear envelope is caused by phosphorylation of the nuclear lamins
39
Q
what does start kinase do?
A
- start kinase is a complex of cdk2 and cyclin E
- it phosphorylates substrates needed for G1
- one of the most important proteins that is phosphorylated by start kinase is retinoblastoma
40
Q
explain how retinoblastoma regulates the cell cycle?
A
- Retinoblastoma is a key protein in regulating the cell cycle
- In the resting G0 state, retinoblastoma is unphosphorylated and active
- at G0 it is bound to a family of transcription factors called E2F
- whilst the retinoblastoma is bound to E2F everything is turned off
- Retinoblastoma is a target for Cdk4/6 cyclin D kinase (which becomes active following Myc induction)
- as the protein becomes phosphorylated, it becomes inactive and loses its affinity for E2F so it releases E2F
- E2F transcription factors can then bind to promoters in the nucleus of genes that are involved in cell cycle progression eg. cyclin E
- Retinoblastoma acts as a brake in the cell cycle, when it is unphosphorylated it prevents the movement of the cell cycle
- it is a TUMUOR SUPPRESSOR GENE
- Many tumours have reduced levels of retinoblastoma
so it can’t regulate the E2F activity
41
Q
what other genes are regulated by E2F?
A
- E2F transcription factors regulate proto=oncogenes eg. Myc
- They also control genes that are involved in the S phase eg. thymidine kinase
- Cyclin E is one of the first targets of E2F transcription factors
- it begins the transcription of cyclin E to bring on the next phase of the cell cycle
42
Q
what happens after the inital release of E2F?
A
- The initial release of E2F after phosphorylation will allow the transcription of cyclin E
- Cyclin E will then form a complex with Cdk2
- Retinoblastoma is also a target for phosphorylation by the active Cdk2 - cyclin E complex
- This means that retinoblastoma becomes further phosphorylated and it releases more E2F
- so the concentration of E2F is increased
43
Q
overview of Cdk activity?
A
- Myc turns on cyclin D, which complexes with Cdk4/6
- This Cdk4/6=cyclin D complex phosphorylates retinoblastoma
- this allows E2F to start being released in the cytoplasm and stimulates cyclin E
- Cyclin E forms a complex with Cdk2 and this complex further phosphorylates retinoblastoma leading to increase in E2F
- The increase in E2F concentration means that it can now bind to targets with lower affinity
- The cyclin A gene promoter is not activated until the E2F concentration is high enough
- cycle continues
44
Q
how are CDKs inhibitors?
A
- there are 2 families of inhibitors called Cdk inhibitors that add another level of regulation
- INK4 family
- CIP/KIP family
45
Q
what does the INK4 family do?
A
- INK4 family is active in G1
- they inhibit Cdk4/6 by displacing cyclin D
46
Q
what does the CIP/KIP family do?
A
- active in S phase and inhibits the Cdk/cyclin complexes by binding to it
47
Q
what must happen to the inhibitors for the cell cycle to progress?
A
- For the cell cycle to progress these inhibitors need to be degraded
- The degradation of the Cdk inhibitors have been superimposed
48
Q
what is the importance of cell cycle regulators in cancer?
A
- Genes that are commonly lost in cancers are tumour suppressor genes
- Genes that are commonly over=expressed/over=produced in cancers are oncogenes
49
Q
what are oncogenes?
A
- derived from normal protooncogenes that become mutated
- Many cell surface tyrosine kinase receptors are oncogenes
- when they are overexpressed they cause excessive growth
50
Q
A
51
Q
what happens to tumour supressors in cancer?
A
- they become inactivated
