Signalling Mechanisms in growth and divisions: part 2 Flashcards
1
Q
Schematic of the Cell Cycle
A
Complex and strict regulation and check points
2
Q
How are Cdk’s controlled ?
What type of kinase are they?
A
- Cell cycle control is based on the Cdks
- They are actively regulated through their interaction with cyclins and phosphorylation
- This is a family of protein kinases called Cyclin-dependent kinases (Cdks)
- They are seen in different points of the cell cycle
- Synthesized and degraded really easy- therefore activation and deactivation is really important
- These are serine-threonine kinases (NOT tyrosine kinases)
- These Cdks are in the cell throughout the cell cycle but they are not activated until they bind to an activating protein called cyclin
- The Cdks are ALSO controlled by phosphorylation - this is an extra level of control
3
Q
Describe the relationship between Cyclins and Cdks
A
- Cdks are activated by binding to cyclins
- They are only expressed at certain points of the cell cycle
- Cyclins are transiently expressed during the cell cycle
- Once they have activated the Cdks, the cyclins are degraded
- The cyclins are regulated at the level of expression
4
Q
Describe how Different cyclin-Cdk complexes trigger different events in the cell cycle, and state an example.
A
- There are different cyclin-Cdk complexes that trigger different events in the cell cycle (at the beginning)
- The M-phase promoting factor controls the progression through mitosis - the Cdk is being activated by a mitotic cyclin
- Cdk1 + Mitotic Cyclin B: Mitosis Promoting Factor
- Once the Cdk has fulfilled their role, the cyclin is degraded and the Cdk is turned off
- At the start of DNA replication (bottom of the diagram) there is a Cdk that is turned on by binding to an S phase cyclin
- Then this cyclin is degraded once the Cdk has carried out its function
- There are different types and they are activated in different times
5
Q
Regulated Expression of Cyclins
A
- This shows the cyclical nature of the induction of the cyclins that are controlling mitosis
- It shows that the cyclins are found to become expressed during mitosis and then they go away (are degraded), then they come up again in the next mitosis
6
Q
Describe the nature of expression of cyclins and explain their promotion and inhibition.
A
- Cdk1 binds to cyclin B
- This Cdk-cyclin complex is usually inactive on its own
- It has another level of regulation, which is PHOSPHORYLATION
- There are TWO 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
- CAK puts an activating phosphorylation onto Cdk1
- Wee1 puts an inhibitory phosphorylation onto Cdk1
- This happens to ensure that everything is ready
- 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 (dephophorelates the phos[horylated CDK)
- Then you get an active MPF
- Overview of steps in activating MPF:
- Cyclin binding to Cdk
- Activating phosphorylation by CAK
- Removal of the inhibitory phosphate (that was put on by Wee1) by Cdc25
7
Q
Describe what happens with cyclins during Mitosis
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
- This is a form of positive feedback that drives mitosis
- As soon as you get some active Cdk1 then you activate more Cdc25, which, in turn, leads to more dephoshorylation of the inhibitory site and this positive feedback pushes the cell through mitosis
8
Q
Give an overview of Mitosis, and describe how it is controlled
A
- MPF, when active at the end of metaphase, phosphorylates a number of key substrates that are involved in the mitotic process
- This puts mitosis on hold when the substrates are phosphorylated
- They can’t progress to the next stage until a signal is sent saying that metaphase has been correctly achieved
- Once the kinetochores are correctly attached to the microtubule spindles, a signal is released that causes cyclin B to be degraded
- When cyclin B is degraded, Cdk1 becomes inactive and this means that the substrates (which were keeping mitosis on hold) are dephosphorylated so then mitosis can progress
9
Q
Describe the specificity of Cdks with cyclins
A
- Different cyclins and Cdks control different stages of mitosis
-
G1/S
- Cdk2
- Cyclin E
-
S
- Cdk2
- Cyclin A
- IMPORTANT NOTE: the same Cdk is being used in G1/S phase and S phase but they are doing different jobs
- Different ones are needed depending on what is needed
- This is because when cyclin binds to Cdk it actually changes its substrate specificity so that it can phosphorylate different substrates depending on which cyclins are bound to it
- It also changes substrate accessibility - the substrates available in G1/S will be different to those available in S phase
- This feature allows you to use the same Cdk in different stages of the cell cycle
10
Q
Describe transition between G0 to G1 transition
A
- Growth factors come and bind to the receptor protein tyrosine kinase
- Through Ras, this triggers a kinase cascade , and promote G0 to G1 trnasition
- This leads to the phosphorylation of transcription factors that turns on the expression of c-Myc
- Myc is a transcription factor and one its jobs is to stimulate the transcription of cyclin D
11
Q
Cyclin D activates Cdk4 and Cdk6 to stimulate synthesis of cyclin E
A
- This triggers the cell cycle
- RAS-Rf à myc à cyclin Dà CDK4/6à Rbà release E3fà is active and causes phosphorylation of the S cycle
12
Q
Regulated Expression of Cyclins/Cdks
A
- RECAP: growth factor leads to the production of Myc (a transcription factor), which, in turn, stimulates the synthesis of cyclin D. Cyclin D leads to the production of an active Cdk4/6-cyclin D complex, which will lead to the release of E3f from Rb which will release transcription factors and lead to changes in the DNA.
- This Cdk-cyclin complex then stimulates the synthesis of the next cyclin and they then become sequentially active - each cyclin is involved in stimulating the synthesis of the next cyclin (this is what gives direction to the circle)
- Degradation of one brings the activation of the other one
- It also gives timing because it takes time for the concentration of the cyclin to build up so the appropriate Cdk is activated
13
Q
What do Cdks do?
A
- MPF phosphorylates proteins involved in mitosis e.g. nuclear lamins (cause breakdown of the nuclear envelope, they are there, they need to be disambled )
- Breakdown of the nuclear envelope is caused by phosphorylation of the nuclear lamins
- Start kinase (from G1 to S) is a complex of Cdk2 and G1 cyclin (e.g. cyclin E) - this phosphorylates substrates needed for that phase
- The most important protein that is phosphorylated by start kinase is RETINOBLASTOMA
14
Q
Describe the regulation of Gene Expression by Retinoblastoma, how does it result in cancer?
A
- Retinoblastoma is a key protein in regulating the cell cycle - it is present throughout the cell cycle
- The active RB is gounf in the G0 and binds to the transcription of E2F and hold the transcription factor inactivated
- In the resting G0 state, retinoblastoma is unphosphorylated
- In this state, it binds to and sequesters a family of transcription factors called E2F
- When the E2F transcription factors are held in the cytoplasm by unphosphorylated retinoblastoma, everything is turned OFF
- Retinoblastoma is a target for Cdk4/6-cyclin D kinase (which becomes active following Myc induction)
- It starts to phosphorylate the retinoblastoma protein and as the protein becomes phosphorylated, it 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
- One of the targets of the E2F transcription factors is the gene for cyclin E - the next cyclin that is required for cell cycle progression
- Retinoblastoma acts as a brake in the cell cycle - when it is in unphosphorylated it sequesters the E2F transcription factors and prevents the gene expression needed for cell cycle progression
- When it is multiply phosphorylated, it becomes inactivated, releases the E2F transcription factors and allows cell cycle progression
- As it acts as a brake in the cell cycle, it is a TUMUOR SUPPRESSOR GENE
- Many tumours have reduced levels of retinoblastoma so it can’t regulate the E2F activity so it progresses through the cell cycle in an uncontrolled manner
15
Q
Which are the ways of production for Cyclin E?
A
- E2F transcription factors regulate proto-oncogenes including Myc proteins
- The cell need to maintain the Rb to hold the transcription factor in place
- They also control genes that are involved in the S phase e.g. thymidine kinase
- Cyclin E is one of the first targets of E2F transcription factors
- It will begin the transcription of cyclin E to push forwards the next phase of the cell cycle
- 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 in the cytoplasm increases
16
Q
Give an Overview of Cdk Activity (in which order do the Cyclins and Cdks work), when is the cyclin A gene promoter activated?
A
- Myc turns on cyclin D, which complexes with Cdk4/6
- This Cdk4/6-cyclin D complex phosphorylates retinoblastoma and allows E2F to start being released in the cytoplasm and then this stimulates the production of cyclin E
- Cyclin E forms a complex with Cdk2 and this complex then further phosphorylates retinoblastoma leading to a further increase in E2F
- The increase in E2F concentration means that it can now bind to targets with lower affinity
- There are multiple steps that a cell can regulate it, in tumours there are more than one step dysregulated
- The cyclin A gene promoter is not activated until the E2F concentration is high enough
- This cycle continues throughout the cell cycle
- Cyclins – D à E à A à B.
- Cdks – 4/6 à 2 à 2 à 1.
17
Q
How are Cyclin-dependent Kinases controlled. What are the families that the “controllers” can be divided into?
A
- Cdks are also regulated by inhibitors.
- YOU DO NOT NEED TO REMEMBER THE INDIVIDUAL NAMES BUT REMEMBER THE NAMES OF THE FAMILIES
- INK4 family is active in G1 - they inhibit Cdk4/6 by displacing cyclin D
- CIP/KIP family is active in S phase and inhibits the Cdk/cyclin complexes by binding to it (a bit like the diagram above)
- For the cell cycle to progress these inhibitors need to be degraded
- The degradation of the Cdk inhibitors have been superimposed
- They are regulated wih the kinases
18
Q
Which inhibitors are needed to be degraded to carry on which part of the cycle?
A
- Don’t need to remember the individual names but do remember the family names.
- For the cell cycle to progress, these inhibitors need to be degraded.
- Look to the left to see:
- INK4 family = G1 Cdk-I.
- CIP/KIP family = S Cdk-I.
20
Q
State the difference between oncogenes and prot-oncogenes, which is a common oncogene in breast cancer, what are the different Anti-cancer Drug targets?
A
- Many cell surface tyrosine kinase receptors are oncogenes
- Protooncogenic they are normal cell that in caner they become changes, they are mutationally activated
- A good example of a tyrosine kinase receptor that is an oncogene is Her2 which is mutationally activated and over-expressed in many breast cancers
- The Ras gene is mutationally activated in many cancers and it can be targeted by inhibitors of its membrane attachment
- The higher up the problem the more difficult it is to be controlled
21
Q
Anti-Cancer Drug Targets
A
You can target these proliferation pathways with various inhibitors
