Signalling Mechanisms Flashcards
Most adult cells are NOT constantly dividing - explain this, using reference to a molecule
In the ABSENCE of growth signals, they go into
• G0
OR
• Quiescent phase (e.g. liver hepatocytes)
c-Myc = ONCOGENE
• TF that stimulates expression of cell cycle genes
• [c-Myc] is LOW in quiescent phase
• [c-Myc] is HIGH when cell division is triggered
• OVERexpressed in many tumours
Key components of signalling pathways?
- Regulation of enzyme activity by protein phosphorylation (kinases)
- Adapter proteins
- Regulation by GTP-biding proteins
Explain how Signalling Pathways are stimulated by GFs
- Mitogenic GF (e.g. hepatocyte GF) binds to receptor
• receptor protein is tyrosine kinase - GF acts via. a small GTP-biding protein (Ras)
• triggers the kinase cascade - This triggers the activation of immediate, early genes required for progression of cells through the cell cycle
• c-Jun, c-Fos, c-Myc are the TFs
• this process is SLOWER as it requires transcription/translation
Explain how the Signalling Pathways are stimulated by Peptide GFs
- The phosphorylated RPTK recruits adaptor & signalling proteins (e.g. Grb2)
• RPTK = Receptor Protein Tyrosine Kinase - Dimeric GF binds to & draws together the RPTK
• cross-phosphorylation occurs using ATP - The phosphorylated domains acts as docking sites for adaptor proteins
• these proteins contribute to signalling downstream
What is an important adaptor protein used in signalling pathways?
Grb2
Explain what Herceptin is?
Anti-Her2 antibody
Blocks early growth
What are adaptor proteins?
APs are often modular
• different domains are mixed & matched to give the protein different properties
This property is important in molecular recognition
AND
Adaptor molecules have NO enzymatic function
• simply bring other proteins together
Explain the interactions that Grb2 has
Has ONLY 2 types of protein-protein interaction
- SH2 - binds to phosphorylated tyrosine of receptor (inducible)
- SH3 - bind to proline rich regions of other proteins (constitutive)
Explain how GTP-binding proteins are seen as molecular switches
Ras!
• Grb2 brings Ras onto the RTK
• It is a GTP-binding protein (powerful molecular switch)
• It is either ON (GTP-bound) OR OFF
Exchange factors = ON
GTPase activating proteins = OFF
Most Ras in a cell is OFF
• in cancer, the Ras protein may be mutated and constantly in the GTP-bound state –> signals
What type of proteins are GTP-binding proteins not?
They are NOT kinases!
Explain another way Ras can be activated using Adaptor Proteins
RPTKs signal to Ras
Grb2 is bound to RTPK via. SH2 domain
• Ras is ALWAYS bound to Grb2
AND
SH3 region is bound to Sos
• Sos allows exchange of GDP –> GTP (turns Ras ON)
• This changes conformation of Ras & activations of signals from Ras
Ras sits in the membrane of the cell - must bind to the plasma membrane to become activated
Ras can suffer from mutations - explain this, giving the 2 ways it can occur
Ras can be oncogenically activated by mutations that INCREASE the amount of active GTP-loaded Ras
2 ways this can happen
• V12Ras - glycine in position 12 –> valine
- PREVENTS GAP binding so prevents inactivation
• L61Ras - glutamine is position 61 –> leucine
- PREVENTS GTP hydrolysis
Once Ras has been switched on via. either of the 2 mechanisms, what then occurs?
Ras ACTIVATES ERK cascade (a form of a MAPK cascade)
• ERK = EC Signal-Regulated Kinase
• MAPK = Mitogen-Activated Protein Kinase
The Ras then DIRECTLY initiates the kinase cascade where EACH kinase activated ANOTHER kinase
What is specifically involved in the ERK cascade?
3 kinases are specific to the ERK cascade
• Raf (MAPKKK)
• MEK (MAPKKK)
• ERK (MAPK)
In general, what do PKs do?
STIMULATE
• changes in cell proteins
&
• gene expression
TO PROMOTE DIVISION
When the last kinase is phosphorylated, what occurs?
Several proteins change their activity
• e.g. TFs
which then regulate gene expression
E.g. of a gene that is stimulated is the c-Myc gene
Myc & Ras are KEY ONCOGENES that may be mutated in human tumours
What is Cell Cycle control based on?
Cdks
cyclin-dependent kinase
Describe Cdks
Cyclin-dependent kinase
• Present in proliferating cells throughout the cell cycle
Activity is regulated by i
• interaction with cyclins
&
• phosphorylation
What are Cdks not?
These are NOT TKs (these are serine-theronine kinase)
Describe Cyclins
Transiently expressed
• only expressed at specific points in the cell cycke
Regulated at level of expression
Once they are synthesised (i.e. activate a Cdks), they degrade
Explain the relationship between Cdks & cycline
Cyclin(s) bind to & activare Cdk(s), triggering different events in the cell cycle
• e.g. M-phase-promoting factor initiates mitosis
- Mitosis Promoting Factor = Cdk1 + Mitotic cyclin B
What do activated Cdks go on to do then?
They phosphorylate proteins (on Serine OR Theronine) to drive cell cycle progression
e. g. Nuclear lamins - causes NE breakdown
- Cdk1 + Cyclin B (mitotic cyclin)
e. g. Retinoblastoma protein - tumour supressor (inactivated in many cancers)
- Cdk2 + Cyclin E (G1 cyclin)
What regulates Cdks?
Although cyclin show a cyclical nature of expression, so can track synthesis & degradation in a predictable way:
PHOSPHORYLATION regulates Cdks
How are Cdks regulated?
PHOSPHORYLATION regulates Cdks
e.g. Cdk1 binds to Cyclin B which is INACTIVE until phosphorylated by 2 reactions:
• Cdk Activating Kinase (CAK)
= activating
&
• Wee1 = a Balancing Inhibitor (add phosphates)
= inhibitor
= Cdc25 then REMOVES the Wee1 inhibitory phosphate (to allow activation)
How is Wee1 then handled to allow for activation of Cdks?
Dephosphorylation of the inhibitor Wee1 phosphate
• activates Cdk1 at the end of interphase
Active MPF then phosphorylates Cdc25 to increase its activity
• drives a +VE feedback
• this helps drive mitosis as dephosphorylates Wee1
MPF = Mitosis Promoting Factor
General overview of Mitosis in terms of the signalling pathways?
MPF at the END of metaphase
• phosphorylates key substrates in the mitotic process
• puts mitosis on HOLD whilst substrates are phosphorylated
One the kinetochores are attached, a signal is released
• causes cyclin B to be degraded
• this causes Cdk1 to become inactive
• means the substrates are dephosphorylated
• mitosis can now PROCEED
Cyclins and Cdks at different stages of the cell cycle?
DIFFERENT cyclins & Cdks required at different stages of the cell cycle
• e.g. G1/S = Cdk2, Cyclin E
• e.g. S = Cdk2, Cyclin A
Note how the same Cdk is bound BUT with a diff. cyclin
• cyclin therefore changes the substrate specificity (to phosphorylate different substrates)
Using Cdks, how is the cell cycle triggered
Growth factor stimulation of signalling pathways promotes G0 to G1 transition:
• GF binds to the RPTK –> cascades via Ras
• transcription factor phosphorylation –> expression of c-Myc
• stimulate transcription of cyclin D
Cyclin D activates Cdk4 and Cdk6
• goes on to synthesis of Cyclin E
• this triggers the cell cycle
How is there regulated expression of cyclins/Cdks?
Each Cdk produced is involved in stimulating synthesis of the next Cdk by stimulating synthesis of genes required
• e.g. Cyclin D/Cdk4/6 –> Cyclin E
• gives direction and timing to cycle
Cyclins are susceptible to degradation, hence cyclical activation
Gene expression can also be regulated by Rb, explain how
pRb - protein retinoblastoma
pRb acts as a “brake” on the cell cycle.
• Cdks phosphorylate & progressively inactivate pRb
• Rb is a “tumour suppressor”.
MoA:
• In G0, Rb protein is resting with E2F transcription factors in the cell cytoplasm
• Cdk4/6-cyclin D kinase phosphorylates Rb and E2F is released and binds to gene promoters to drive transcription
Explain the relationship between Rb, E2F and cyclin E
One target of E2F is the cyclin E, the cyclin required to continue cell cycle progression
• Rb acts as a brake in the cell cycle by holding onto the E2F – this makes it a TUMOUR SUPRESSOR GENE.
Many tumours have reduced levels of Rb protein so can’t regulate E2F
Describe and explain the E2F TFs
E2F regulate proto-oncogenes
• includes Myc proteins
Cyclin E is one of the FIRST targets of E2F –> pushes the cell cycle forward
• Cyclin E –> binds to Cdk2
• “CyclinE-Cdk2 complex” phosphorylates Rb protein so another +ve feedback is initiated so more E2F is released by Rb
Explain the order that the cyclins and Cdks are switched on
Myc turns on cyclin D which complexes with Cdk4/6…
• Cyclins – D –> E –> A –> B.
• Cdks – 4/6 –> 2 –> 2 –> 1.
Ultimately leads to mitosis.
• more and more pRb is phosphorylated to release more E2F (which is what initially creates cyclin E).
• Cyclin A gene promoter is not activated until there is a HIGH concentration of E2F
Cdks are also regulated by something else - explain what it is and the two families
Cdk Inhibitors
Two families of Cdk-Is exist:
• INK4 family - e.g. p15(INK4b)
- G1-phase Cdk-I
- Inhibits Cdk4/6 by displacing Cyclin D
• CIP/KIP family - e.g. p21(C1P1)
- S-phase Cdk-I
- Inhibits all Cdks by binding to the Cdk/Cyc complex
For the cell cycle to progress, these inhibitors need to be DEGRADED
What type of genes are commonly LOST in cancers?
TUMOUR SUPRESSOR GENES
- Rb – inactivated in many cancers.
- P27KIP1 – under expression correlates with poor prognosis
What types of genes are commonly OVER-EXPRESSED in cancers?
ONCOGENES
- EGFR/HER2 – mutation activated in breast cancer – treated with Herceptin antibody (in HER2+ cancer)
- Ras – mutation activated – treated with membrane attachment inhibitors
- Cyclin D1 – overexpressed in 50% of breast cancer
- B-Raf – mutation activated in melanomas – treat with kinase inhibitors
- cMyc – overexpressed in many tumours
