Chapter Four Flashcards
What does the term EGF stand for?
Is it the only type?
Epidermal growth factor.
Absolutely not
What are the general events in cell division?
- The reproductive signal
(could be EGF) - Replication of DNA
- Segregation (anaphase/telophase)
- Cytokinesis: the actual division - separation into the two new cells
Why do eukaryotes not replicate in masse when there is enough nutrients like prokaryotes?
They replicate to suite the needs of the whole organism.
What must eukaryotes respond to facilitate division?
Some sort of intracellular signal.
What are the three types of amino acids that are typically phosphorylated?
Tyrosine
Serine
Threonine
What are some things that are involved in transducing a growth factor signal?
- The growth factor itself
- The growth factor receptor
- Signal transducers within the cell
- Nuclear transcription factors.
What is dimerization?
What is the significance of it?
Dimerization is when two separate polypeptides form a quaternary structure that facilitates a specific function.
GFRs need to dimerize before they do what they are supposed to.
Most GFRs are _______.
Explain how they typically transmit their signal intracellularly.
Kinases
The gamma phosphate is transferred to the hydroxyl groups of specific (formerly mentioned) amino acids.
True or false:
EGFs are overexpressed in cancer for some reason.
True
What type of kinases are EGFRs?
Tyrosine kinases
What are the four members of the EGFR fam?
- Her1 - normal
- Her2 - no ligand
- Her3 - no action
- Her4 - normal
Explain the first step in EGF signalling
EGF binding
The extracellular (hydrophilic amino acids) domains of two EGFR family members form a binding pocket
Explain the second step in EGF signalling.
Dimerization
When the EGF binds to the EGFR, it causes a conformational change that allows the dimers to bind.
A similar binding spot is seen when another receptor binds to EGF (homo or heterodimers)
What is the third step in EGF signalling called?
Without EGF what happens to the pathway?
With it?
Autophosphorylation
Normally pathway is blocked, but binding of the dimers allows this to be overcome.
What does the conformational change (dimerization) cause?
What happens next?
The conformational change allows ATP and a substrate to access the kinase domain of the EGFR dimer
Each kinase domain of the receptor will phosphorylate the other at multiple tyrosines.
What is responsible for recruiting substrate proteins?
The phosphorylation
Kinase activity at the dimers must be turned off at a certain time.
What are the four ways this can happen?
- Add another phosphate - conformational change
- Tyrosine phosphatases
- Negative regulators - something competes with phosphate
- Engulf whole receptor complex and degrade it
What is the fourth step in EGF signalling called?
Activation of Intracellular Transducers.
What do some tyrosine-phosphorylated complexes attract?
They create high affinity binding sites for Src domains?
What are the two Src proteins we talked about?
What do they bind to?
SH2 - their C terminal binds to phosphorylated tyrosine
SH3 - binds to residues on partner proteins (SOS)
What is the complex with one SH2 and two SH3s called?
Grb2
What is SOS?
What is RAS?
SOS is a nucleotide exchange factor that activates RAS, meaning it exchanges GDP with GTP to activate RAS.
RAS is a GTP binding protein that acts to activate serine and threonine kinases when it is bound to GTP - activates downstream kinases
Where do RAS proteins reside?
What directs them there?
What are the three types of RAS?
They reside within the cell membrane.
Farn proteins (farnesylation) attachment of hydrophobic groups.
The three types are N, H, K
What is the inactive state of RAS proteins?
What is the active state of RAS proteins?
When GDP is bound
When GTP is bound
What are GAPases and how are they important for regulating RAS?
They are enzymes that catalyze the hydrolysis of GTP to GDP to terminate the signal.
RAS proteins even have their own GTPase activity for self-regulation.
What is the fifth step of EGF signalling called?
Activation of the Ser/Thr Cascade.
What is Raf protein?
What does it do?
It is a serine/threonine kinase that is activated by a RAS when it has GTP bound to it. (Sometimes it is called MAPKKK
It acts to phosphorylate MEK protein.
What is MEK (sometimes referred to as MAPKK) protein?
What does it do?
It is another kinase that is activated by Raf.
It can add kinases to tyrosine, serine or threonine.
Once it gets phosphorylated by Raf, it acts to phosphorylate MAPK
What does MAPK stand for?
Mitogen-activated protein kinase.
From which family of kinases is MAPK from?
It is from the ser/thr kinase family.
What does MAPK do once it is activated by MEK?
It travels to the nucleus, and phosphorylates transcription factors to regulate their activity.
What is the sixth step of EGF signalling?
The regulation of transcription factors by MAPK
Explain what the Myc family of TFs are responsible for and how MAPK would influence their activity.
They are responsible for regulating the genes that are responsible for proliferation.
If MAPK phosphorylates any of the TFs in the Myc family, it will positively regulate genes (for proliferation) that are regulated by Myc.
(It will allow Myc TFs to bind to the DNA, promoting expression)
Explain briefly what AP1 TFs are important for and how MAPK influences their activity.
AP1 TFs are involved in growth, difference and death.
Remember that AP1 TFs involves Jun and Fos dimers - can be homo or hetero.
MAPK will phosphorylate Fos and alters the DNA binding activity - probably making it more likely to bind to the DNA.
Why would expression of genes important for proliferation, regulated by Myc likely target tumor suppressors like p-53?
It would cause rapid proliferation and stress on the cell to which the tumor suppressor would respond.
What is the relationship between Myc TFs and N-Ras?
Myc increases N-ras activity and we know that the Ras family transmits signals that eventually result in phosphorylation of Myc transcription factors.
Remember the Src protein that was involved early on in the EGF signalling pathway?
What is a Src protein?
It is a intracellular tyrosine kinase that has both SH2 and SH3 domains
What else did we learn that interacts with Src proteins?
Focal Adhesion Kinase.
FAK
What is FAK important for?
It is important for cell shape, adherence and motility.
How are FAKs regulated?
In their active state they may phosphorylate stuff to facilitate their action.
To inactivate them, the tyrosine kinase must be phosphorylated by something else, causing interaction of the phosphorylated tyrosine and the SH2 domain, keepin it in the inactive orientation.
What are six ways to stop the EGF pathway?
- endocytosis of receptor
- downregulation
- RAS itself (GAP
- Phosphatases
- Kinase activity (inhibit)
- More phosphates
How many oncogenes vs. tumor suppressors have been identified?
there has been over 100 oncogenes discovered and 15 tumor suppressors.
Do mutations seen in the regulatory regions of oncogenes cause higher levels of expression of the oncogene?
Yes.
What are the ways that proto-oncogenes become onco-genes?
- Point mutations and/or deletions in the coding sequences will cause the structure and function to change.
- Point mutations and/or deletions in the regulatory sequences will cause overexpression
- Chromosomal translocations will cause fusion proteins that have new and different functions
- Insertional mutagenesis by viral integration can cause expression that is not normally seen
- Gene amplification on both chromosomal DNA as well as extra chromosomal DNA can result in the increase of gene dose and protein production.
What makes ecDNA display non-Mendelian inheritance patterns?
What is significant about this?
They segregation results in uneven distribution of DNA meaning that some cells will have more onco-gene expression than others.
ecDNA is less compact and more accessible.
Where did our understanding of oncogenes come from?
They came from our studies of retroviruses
What do retroviruses do?
They inject RNA into the host, which then gets reverse transcribed into DNA
What is a provirus?
The DNA form of a retrovirus after it has been reverse transcribed.
What happens to the provirus?
It is randomly integrated into the host genome, and is replicated, transcribed, and translated by the host.
What is significant about viruses in how they relate to oncogenes?
They can acquire fragments of genes from host at sites of integration - “they are constantly changing and picking up new stuff”
When was the viral (v-src) found?
When was the cellular (c-src) found?
1911
1976
What does PDGF do?
Platelet derived growth factor stimulates the cells around the wound edge to proliferate and repair the damage.
PDGF was one of the first discovered proto-oncogenes.
What does the oncogene of this do?
It results in PDGF that isn’t secreted but instead kept in the cytoplasm.
This results in the inappropriate activation and unregulated growth.
Is EGFR a proto-oncogene?
What is different about the oncogene form?
Yes
It is shorter and doesn’t have an extracellular domain. This results in constitutive action.
If there is no binding spot for EGF, what keeps the tyrosine kinase on?
Mutations that:
- Simply keep the tyrosines on
- Allow for dimerization without EGF
- Bind to something like EGF
What is the most commonly mutated oncogene in human cancers?
What does the mutation cause?
Ras
It cause the Ras to lose its GTPase activity, so it can no longer be inactivated to RAS-GDP.
This results in the constitutive activation of RAS protein.
What type of cancer commonly has the B -Raf point mutation?
Melanoma
What does this result in?
It results in constitutive kinase activity and insensitivity to feedback mechanisms.
What is ABL?
It is the nuclear threonine kinase that is involved in DNA damage-induced apoptosis
What happens to ABL in the formation of the Philidelphia chromosome?
It places the ABL (from chromosome 9) via translocation to the 22nd chromosome next to BCR
What is the result of the BCR-ABL fusion protein?
tyrosine kinase activity is constitutive in the cytoplasm and can access many different substrates.
What is AP-1 and what its components?
It is a transcription factor.
The components are Jun and Fos
The components of AP-1 are proto-oncogenes.
What happens to make them become oncogenes?
What does this result in?
A mutation in Fos results in a truncation (shortening) that eliminates a motif that is involved in the stability of the mRNA
The mutation causes the mRNA to have a longer half life which results in more Jun and Fos and ultimately more AP-1 meaning there will be an increase in the transcription of AP-1 regulated genes.
Why are many specific kinase inhibitors made when the structure of the catalytic domains of the kinase are very similar when active?
You want to find the differences to treat specific ones for specific disorders.
How are EGFRs targeted?
Herceptin is used which is a humanized monoclonal antibody that is created to bind to the Her 2 or ErbB2 receptor which results in:
- Increased receptor degradation
- Inhibition of angiogenesis
- Recruitment of immune cells
What is Herceptin used for?
It is used for treatment of metastatic breast cancers that overexpress ErbB2
Another way to target EGFR is the drug Tarceva.
What does it do?
It competes for the binding of ATP
Shows promise, but eventually stops working because of new mutations.
What does Gleevec do?
It is used to treat CML that express the BCR-ABL fusion protein.
It binds to the ATP binding pocket of the catalytic domain of ABL.
Also works in the early stages, but eventually the mutations lead to relapse.
What is oncogene addiction?
When a cancer has multiple genetic alterations and oncogenes, but grows “addicted” to the signalling pathway activated by a specific oncogene.
What happens when researchers try to target Ras?
They have been unsuccessful and it has been deemed undrugable.
