Signalling mechanisms of growth and division Flashcards
In what phase are most adult cells?
G0 (quiescent phase)
What is the role of c-Myc?
Transcription factor that stimulates expression of cell cycle genes
What are Myc conc levels like during the quiescent G0 phase and when cell division is triggered?
Very low in G0 phase but rapid increase in Myc when cell division is triggered which then plateaus at an intermediate level- this correlates with cells moving out of G0 and into G1
What stimulates the cell cycle by signalling pathways?
Growth factor
What sort of receptor does growth factor usually bind to?
Tyrosine kinase type receptors
What does growth factor act via after binding to the receptor?
Small GTP-binding protein - Ras
What does Ras trigger?
Kinase cascade
How quick is the early stage of the cell cycle triggering?
Very fast
What does the kinase cascade trigger?
Activation of genes that are required for the progression of cells through the cell cycle
How does the speed of the gene activation compare to the earlier stage of the cell cycle triggering?
It is slower because it requires transcription and translation to take place
What is an example of a mitogenic growth factor?
Hepatocyte growth factor
What does the phosphorylated receptor protein tyrosine kinase recruit?
Adapter and signalling proteins (Grb2)
What sort of receptors normally sit on the plasma membrane in terms of oligomerisation?
Monomers
What are most growth factors in terms of oligomerisation?
Dimers
What happens when the dimeric growth factor binds to the two receptor tyrosine kinase molecules?
It brings them closer together
What is able to happen when the two receptors are close together?
The tyrosine kinase domain is able to cross-phosphorylate the partner receptor
What do the tyrosine kinases use to phosphorylate the tyrosine residues in proteins?
Gamma phosphate of ATP
What do the phosphorylated domains on the tyrosine kinase receptors act as?
Docking sites for adaptor proteins which contribute to downstream signalling
What does the antibody herceptin do?
Inhibits the her2 receptor tyrosine kinase
Why is herceptin’s function important?
It can be used to treat a number of tumours- it blocks the early stage of growth stimulation
What is one of the important adaptor molecules that is recruited?
Grb2
What does the fact that adaptor proteins are often modular mean?
There are different domains that are mixed and matched to give the protein different properties which are important in molecular recognition
What other roles do adaptor proteins have apart from binging proteins together?
None! No enzymatic activity
What are the only two types of Grb protein-protein interactions?
SH2- binds to phosphorylated tyrosine of the receptor
SH3 (two copies)- binds to the proline rich regions of other proteins
What else does Grb bind to as well as RPTK via its SH2 domain?
A protein called Sos through its SH3 domains (Grb is always bound to Sos)
What is Sos?
An exchange factor for Ras (a signalling molecule that sits in the membrane of the cell)
When RPTK becomes activated, what happens?
Phosphorylation of the receptor
What happens after phosphorylation of the receptor?
Grb2 (with Sos attached) binds to the phosphorylated tyrosine domains and Sos is then close enough to the membrane to activate Ras
How does Sos activate Ras?
It 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 into an active state that can signal downstream and can allow the propagation of the signal
For Ras to work, what has to happen and how is this information used clinically?
It has to bind with the plasma membrane
Interfering with the membrane binding of the Ras, you can make good anti-cancer therapy
What sort of protein is Ras?
GTP binding proteins
What are GTP binding proteins very effective at?
Being molecular switches:
On- GTP bound
Off- GDP bound
Why is GTP replacing GDP to make Ras active not phosphorylation?
It is merely an exchange of GDP for GTP (catalysed by Sos)
What is intrinsic GTP hydrolysis capability?
The GTP binding protein is able to hydrolyse GTP to GDP to turn itself off
What else can stimulate the hydrolysis of the GTP?
GTPase activating proteins (GAPs)
What is the cycle of GTP binding proteins almost always controlled by?
Turn it on: Exchange factors (e.g. Sos)
Turn it off: GTPase activating proteins (GAPs)
What is the main G-protein in case of growth factor stimulatory pathways?
Ras
Why is it very important that you are able to turn off the signalling activity of Ras protein?
Prevent uncontrolled division- Ras is mutated in ways that cause the Ras protein to constantly be in the GTP bound form
What is V12Ras?
Glycine residue in position 12 of the Ras protein has changed to valine due to a Ras gene mutation- side chain has gone from being a simple hydrogen (in glycine) to a hydrophobic side chain (valine)
What effect does the change in structure between Ras and V12Ras have?
It prevents GAPs from binding to Ras thus meaning that Ras can’t turn off very easily
What is L61Ras?
Glutamine in position 61 is converted to leucine- single base change in the genome- side chain goes from being an amide to a hydrophobic side chain
What effect does the change in structure between Ras and L61Ras have?
It inhibits the intrinsic GTPase activity of the Ras protein so it ends up being constantly in the GTP bound state and giving growth stimulatory signals
What does Ras activate?
Protein kinase cascade
How does the protein kinase cascade work?
GTP bound Ras binds to a kinase and then that kinase activates several other kinases etc
What is the kinase cascade that is involved in growth stimulatory signalling known as?
ERK cascade (extracellular signal-regulated kinase cascade)
What is the name of this family of kinase cascades?
MAPK cascades (mitogen-activated protein kinase cascade)
What are the three kinases specific to ERK in order?
Raf
MEK
EPK
What do protein kinases do?
Stimulate changes in cell proteins and gene expression to promote division
What does the last kinase (EPK) of the cascade do?
Phosphorylates a number of proteins and changes their activity including gene regulatory proteins (transcription factors) which then go on to regulate gene expression
What effect does activating the growth factor pathway have on c-Myc production?
The kinase cascade leads to activation of a gene regulatory protein which stimulates c-Myc production as it is a key molecule in stimulating growth
What sort of genes are Myc and Ras?
Oncogenes
What is cell cycle control based on?
Cyclically activated protein kinases
What does Cdk stand for?
Cyclin-dependent kinase
What sort of kinase are Cdks?
Serine-threonine kinases
Where are Cdks found?
Present in proliferating cells throughout cell cycle
What is Cdk activity regulated by?
Interaction with cyclins to become activated and phosphorylation
Where are cyclins found?
They are transiently expressed at specific points in the cell cycle
What happens to cyclins after they’ve activated Cdks?
Cyclins are degraded
What are cyclins regulated by?
Level of expression
Why are there different cyclin-cdk complexes?
They trigger different effects in the cell cycle
What controls the progression through mitosis?
M-phase promoting factor
What turns on the Cdk at the start of DNA replication?
An S phase cyclin binding
What does the mitosis promoting factor consist of?
Cdk1 and mitotic cyclin (usually cyclin B)
What does Cdk1 bind to?
Cyclin B
In what state is the Cdk1-cyclin B complex normally?
Inactive on its own
What is the other level of regulation of Cdk-cyclin complexes?
Phosphorylation- there are 2 phosphorylation reactions
Cdk has to be activated at specific sites
What performs the activation of Cdk?
Cdk activating kinase (CAK)
What puts an inhibitory phosphorylation on Cdk1 and opposes the action of CAK?
Wee1- an inhibitory kinase
What removes the inhibitory phosphate that Wee1 puts on hence allowing Cdk1 to function?
Cdc25
Give an overview of the steps involved in activating MPF
Cyclin binding to Cdk
Activating phosphorylation by CAK
Removal of inhibitory phosphate (put on by Wee1) by Cdc25
When does the dephosphorylation that activates Cdk1 occur?
At the end of interphase
What effect does active MPF have on Cdc25?
It can phosphorylate it to upregulate its activity- positive feedback
What does MPF do when active at the end of metaphase?
Phosphorylates a number of key substrates that are involved in the mitotic process
What effect does MPF phosphorylating the key substrates involved in mitosis have?
It puts mitosis on hold- they can’t progress to the next stage until a signal is sent saying that metaphase has been achieved
What happens once metaphase has been achieved and the kinetochores are correctly attached to the microtubule spindles?
A signal is released that causes cyclin B to be degraded which means Cdk1 becomes inactive so the substrates keeping mitosis on hold are dephosphorylated so mitosis can then progress
What Cdks and cyclins are involved in G1/S phase?
Cdk2
Cyclin E
What Cdks and cyclins are involved in just S phase?
Cdk2
Cyclin A
Even though Cdk2 is involved in G1 and S phase, why does it carry out different jobs in both?
When cyclin binds to cdk it actually changes its substrate specificity so it can phosphorylate different substrates depending on which cyclins are bound to it. It also changes substrate accessibility
What does cyclin D activate and what does that stimulate?
Cdk4 and Cdk6 which stimulates synthesis of cyclin E
What effect does each cyclin being involved with stimulating the synthesis of the next cyclin have?
It gives direction to the cell cycle and gives timing because it takes time for the concentration of cyclin to build up so appropriate Cdk is activated
What do Cdks do?
Phosphorylate proteins to drive cell cycle progression
What causes breakdown of the nuclear envelope?
Phosphorylation of nuclear laming by MPF
What is the start kinase?
(From G1 to S)- complex of Cdk2 and G1 cyclin (Cyclin E)- phosphorylates substrates needed for that phase
What is the most important protein that is phosphorylated by start kinase?
Retinoblastoma- key protein in regulating cell cycle (present throughout)
In the G0 resting state, what is retinoblastoma like and what does it do?
Unphosphorylated
It binds to and sequesters a family of transcription factors called E2F
What happens when E2F transcription factors are held in the cytoplasm by unphosphorylated retinoblastoma?
Everything is turned off
What is retinoblastoma protein a target for?
Cdk4/6-cyclin D kinase (which becomes active following Myc induction)
What effect does Cdk4/6-cyclin D kinase have on retinoblastoma?
It phosphorylates retinoblastoma and as it becomes phosphorylated it loses affinity for E2F so releases E2F
What does E2F transcription factors do after being released?
Bind to promoters in the nucleus of genes that are involved in cell cycle progression
What is one of the main targets for E2F transcription factors?
Gene for cyclin E- next cyclin required for cell cycle progression
What do retinoblastoma act as in the cell cycle?
A brake- when it’s unphosphorylated, it sequesters the E2F transcription factors and prevents gene expression needed for progression
What sort of gene is retinoblastoma?
Tumour supressor gene
What is retinoblastoma like in tumour cells?
Many tumour cells have reduced levels of retinoblastoma so it can’t regulate E2F activity so it progresses through cell cycle in an uncontrolled manner
What genes are regulated by E2F transcription factors?
Protooncogenes (including Myc)
Involved in the S phase e.g. thymidine kinase
Involved in the cell cycle e.g. cyclin A and E (first target)
What does Myc do?
Turns on cyclin D which complexes with Cdk4/6
What does the Cdk4/6 complex do?
Phosphorylates retinoblastoma and allows E2F to start being released in the cytoplasm which stimulates the production of cyclin E
What does cyclin E form and what does that do?
Forms a complex with Cdk2 and this complex then further phosphorylates retinoblastoma leading to a further increase in E2F
What does the increase in E2F concentration caused by cyclin E mean?
It can now bind to targets with lower affinity
Until what is the cyclin A gene promoter not activated?
Until the E2F concentration is high enough
What regulates Cdks?
Cdk inhibitors
What are the two Cdk inhibitor families and when are they found?
INK4 family- active in G1
CIP/KIP family- active in S phase
How do the INK4 and CIP families work?
INK4- inhibits Cdk4/6 by displacing cyclin D
CIP/KIP- inhibits the Cdk/cyclin complexes by binding to it
Which genes are commonly over expressed/produced and lost
Lost- Tumour supressor
More- Oncogenes
What cell cycle oncogenes are there?
Cell surface tyrosine kinase receptors- EGFR/HER2 Ras Cyclin D1 B-Raf c-Myc
What cell cycle tumour suppressors are there?
Rb
p27KIP1
