Cancer 5: Signaling + mechanisms in growth and division Flashcards
a) What is the significance of c-Myc?
b) How is it upregulated?
c) What happens when c-myc = mutated?
a) - c-Myc is an oncogene
- acts as transcription factor - stimulates expression of cell cycle genes
- -> growth factor increases [Myc]
b) How it is upregulated:
cell receives signal –> stimulates an intracellular sginalling cascade –> causes upregulation of transcription factors e.g c-Myc
• c-Myc stimulates the expression of cell cycle genes, and has an important role in G1
c) Mutated Ras and c-Myc act as oncogenes, leading to uncontrolled cell division and tumour proliferation
What are 3 key components of signaling pathways?
- Regulation of enzyme activity by protein phosphorylation (kinases)
- Adapter proteins
- Regulation by GTP-binding proteins
Describe the process of growth factor stimulation of signaling pathways?
This can be divided into two phases:
• growth factor binding to the cell surface
receptor protein tyrosine kinase (RPTK)
o RPTK = phosphorylated –> activates kinase cascade
- which provides additional docking sites + activates Ras (small G protein).
o Ras binding stimulates –> early-response gene expression from MAPK/ERK cascade, eg c-Myc expression
–> activation of the cell-cycle control system
E.g. anti-HER2 Ab Herceptin used in HER2-positive metastatic breast cancer
EGFR/HER2, mutationally activated or overexpression can cause: ____ ____
breast cancer
What is RAS?
and what does it do to kinase cascades?
RAS = oncogene
activates kinase cascades
How does peptide growth factors take part in signaling?
- Phosphorylated receptor tyrosine kinase recruits adaptor + signalling proteins
- Intracellular signalling proteins bind to phosphorylated tyrosines –> allows adaptor proteins to bind to them
- E.g. anti-HER2 Ab Herceptin used in HER2-positive metastatic breast cancer
- -> blocks binding of ligand
Following RPTK phosphorylation, describe the process of Ras complex activation
Following RPTK phosphorylation, additional docking sites are formed. adapter proteins such as Grb2 (growth-factor receptor binding protein 2) binds (not Ras)
• Binding of Grb2 recruits inactive Ras protein to the plasma membrane.
–> The inactive Ras protein = associated with a GDP molecule
- Ras-activating proteins –> then activates Ras. (using exchange factor Sos - which exchanges a phosphate from a GTP (from ras-activating protein) –> for GDP (from Ras)
- The Ras complex with GTP is now active
How does receptor protein TKs signal to RAS?
- Sos binds Grb2 (adapter) via SH3 domains –> Sos exchanges GDP for GTP –> activates Ras
- Ras must bind plasma membrane to become activated
How is RAS activated oncogenically?
what re the 2 mutations involved in this process?
Activated by mutations increasing amount of active GTP-loaded Ras
2 mutations involved:
o V12Ras = glycine –> valine at position 12 –> prevents GAP binding
o L61Ras = glutamine –> leucine at position 61 –> prevents GTP hydrolysis
How does active ras activate protein kinase cascade?
- Active Ras binds + activates kinase –> causes kinase cascade
2 key ones:
o Specifically - Extracellular signal-regulated kinase (ERK) cascade
o Generically - Mitogen-activated protein kinase (MAPK) cascade
What is a ERK Cascade?
what effect does it have on the cell cycle ?
When active: Ras activates protein kinase cascade
• Generically, this is referred to as a
mitogen-activated protein cascade
(MAPK).
• More specifically, Ras activates an
Extracellular signal-regulated kinase cascade (ERK)
ERK –> stimulate changes in cell proteins + gene expression (eg c-Myc) –> to promote cell division
How do kinases stimulate changes in cell proteins/ gene expression to promote division?
Myc + Ras = can be oncogenes if mutation/ expressed in high amount
o Phosphorylate proteins (non-kinases) –> promote division
o Phosphorylate gene regulatory proteins –> change gene expression e.g. c-Myc that stimulates cell proliferation
What are cyclin dependent kinases (Cdks) ?
what are they regulated by?
Cdks = controls cell cycle
activity of Cdks = regulated by:
o interaction with cyclins
o Phosphorylation
What is the significance of Cyclins?
- proteins which activate Cdks.
- transiently expressed at specific points in the cell cycle
- form cyclin-Cdk complexes –> different complexes triggers different events in the cell cycle
What do activated Cdks do:?
- They phosphorylate proteins (on Serine or Threonine) –> to drive cell cycle progression
e. g CDK1 (w/ mitotic-cyclin) –> phosphorylates substrates at mitosis e.g. nuclear lamins (form nuclear envelope); when phosphorylated –> envelope broken down
e. g CDK2 (w/ cyclin E) –> start kinase (tumour suppressor)
e. g Cdk1 + Mitotic Cyclin B–> forms MPF: –> stimulates mitosis
Note: Cdk1 + Mitotic Cyclin B–> forms MPF: –> stimulates mitosis
- Mitotic Cyclin (B) forms a complex with Cdk1, acting as the mitosis promoting factor (MPF)
How is Cdks regulated via phosphorylation?
inactive MPF = formed by CDK1 + cyclin B –> activated by phosphorylation by:
o CDK-activating kinase (CAK)
o Inhibitory kinase (Wee1)
a) These 2 kinases phosphorylate separate residues = one activated + one inhibited
b) Then phosphatase removes inhibited phosphate –> to form a fully active MPF
requires BOTH:
- activating phosphorylation + removal of inactivating phosphorylation
Eg 1. MPF targets nuclear lamins; the phosphorylation causes breakdown of nuclear envelope (key step in prophase)
• Eg 2. Cdk4/6-Cyclin D targets retinoblastoma protein (pRB); phosphorylation inactivates the protein.
–> which releases E2F transcription factor, driving gene transcription of Cyclin E and allowing progression of the cell cycle from G1 > S phase
Different cyclins and different Cdks are required at different stages of the cell cycle
• G1 involves Cyclin ___ and Cdk___
• S phase involves cyclin ____ and Cdk____
Different cyclins and different Cdks are required at different stages of the cell cycle
• G1 involves Cyclin E and Cdk2
• S phase involves cyclin A and Cdk2
What is the function of Anaphase Promoting Complex C
what is it activated by?
Anaphase Promoting Complex C:
Ub ligase that catalyses Ubn + degradation of cycB.
o Activated by Cdc20 (binds phosphorylated APC/C, phosn. done by MPF)
o So, Cdk1/cycB promotes own degradation.
o Active APC/C-Cdc20 released from attached kinetochores
Cyclin D binds/activates CDK4/6 to stimulate synthesis of cyclin E:
C-myc –> activates cyclin D
LOOK at diagram
Regulation of gene expression by Rb:
- important tumour suppressor
- at G0, RB = activated
holds onto transcription factor E2F (inactive) - in proliferating cell
- cdk4/6 - cyclin D –> phosphorylate Rb protein
- inactivates rb protein
- release e2f
- bind to dna
- start transcribing next cyclin requires for the cycle
Cyclin E
in a similar method,
then Cyclin E acts on cdk2 –> release E2F –> forms cyclin A –> release TF –> forms cyclin B which acts on cdk1–> cause mitosis etc.
describe the regulation of Rb in cell cycle
- C-Myc stimulates cyclin D production
- CDK4/6- D phosphorylates pRb –> release some E2F–>cyclin E production etc.
- When pRb phosphorylated further –> further inhibition –> larger E2F release
Cdks are also regulated by CDK inhibitors (CKI)
How do CKI work?
CKIs
- -> inhibits Cdks
- -> prevent progression through the cell cycle (which is important in maintaining integrity of the cell cycle)
What are the 2 CKI FAMILIES?
- G1 phase CKIs (INK4) = inhibit CDK4/6 by displacing cyclin D, prevent entry into G1
- S phase CKIs (CIP/KIP) = inhibit all CDKs by binding to CDK/cyclin complex, prevent entry into S-phase (hold in G1)
–> regulated by degradation
Grb 2 = contains SH3 + SH2 + SH3
SH = src homology regions
SH2 = recognizes phosphorylated tyrosine
- SH3 = proline rich regions –> docking site for other proteins
recruits ras
ras binds to memb –> becomes activated –> then transmits signals downstream
What are characteristics of Cyclin?
- transiently expressed at spcf point in the cell cycle
- regulated at level of expression
- synthesized –> then degraded
- susceptible to degradation –> which causes cyclical activation
NOTE: Signal from fully attached kinetochores
causes cyclin B to be degraded:
- Cdk1 inactivated
- key substrates dephosphorylated
- mitosis progresses
-
P27KIP1 = tumour suppressor –> reduced expression = poorer prognosis in malignancy
-
How is Ras deactivated?
GTP-binding (G) proteins
- G- proteins act as a molecular switch
- turns Ras off
- by GTP hydrolysis (of the GTP molecule associated with the active Ras) using GTPase activating proteins (GAP proteins)
NOTE: ONCOGENES
- EGFR/HER2, mutationally activated or overexpressed in many breast cancers (Herceptin antibody for the treatment of HER2-positive metastatic breast cancer)
- Ras, mutationally activated in many cancers (inhibitors of membrane attachment)
- Cyclin D1, overexpressed in 50% of breast cancers
- B-Raf, mutationally activated in melanomas (kinase inhibitors in trials)
- c-Myc, overexpressed in many tumours
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