Short answer questions Flashcards
A single amino acid change in RAS eliminates its ability to hydrolyze GTP, even in the presence of
a GTPase-activating protein (GAP). Roughly 30% of human cancers have this change in RAS. You
have just identified a small molecule that prevents the dimerization of a receptor tyrosine kinase
that signals via RAS. Would you expect this molecule to be effective in the treatment of cancers
that express this common, mutant form of RAS? Why or why not? Only one of these two
outcomes is possible. (4 pts)
RAS is active in its GTP-bound state. Since this amino acid change eliminates The ability of RAS to hydrolyze GTP, it is stuck in its active state.
RAS is found downstream in the pathway, blocking the RTK signalling uptream cannot be effective in stopping the mutant RAS since is it locked in an active state.
Stopping RTK activation and signalling will not be effective in this treatment of cancer since RAS is in its active state downstream, leading to RAS signallng to the nucleus.
- A ligand binds to the extracellular domain of its target receptor tyrosine kinase (RTK) that
promotes proliferation.
A) What are the two processes that must occur for the signal to be transmitted to the other
side of the plasma membrane? (ie. What leads to the formation of the phosphor-tyrosine
residues) (1 pts)
B) High-affinity docking sites (phosphor-tyrosine residues) for intracellular signaling proteins
are created on the intracellular domain of the RTK. What type of domain must these
intracellular proteins contain to dock at an activated RTK? (1 pt)
C) Not all intracellular proteins that bind to the high-affinity docking sites of RTKs act to relay
the signal onward. The c-Cbl protein causes receptor recycling by targeting the RTK to
lysosomes. What post-translational modification does c-Cbl perform on the RTK to signal its
targeting to lysosomes? (1 pt)
D) What would you expect to happen to a person who acquired an inactivating mutation in c-
Cbl? (1 pts)
A). Ligand receptor binding causes receptor dimerization and cross-phosphorylation called trans-autophosphorylation
*IGF-Dimerization brings kinase domains closer together –> brings kinase domains closer together –> kinase domains can phosphorylate eachother –> leads to increased kinase activity –> phosphorylation of other tyrosines –> creates phospho-tyrosine residues
*EGF-Binding triggers conformational change –> activates kinase domain of receptor = phosphorylation of other tyrosines generating phospho-tyrosine residues
B). SH2 domain/PTB domain - binds to phospho-tyrosine residues
C). c-Cbl protein covalently adds a single ubiquitin to one or more sites n the RTK
Monoubiquitylation to one or more sites promotes endocytosis and degredation of RTKs by targeting to clathrin-coated vesicles and to lysosome
D). Mutations that inactivate c-Cbl dependant RTK down-regulation causes prolonged RTK signaling, leads to cancer
(c-Cbl is a growth inhibitor)
3) Lateral inhibition is critical for the maintenance of proliferative progenitor populations in many
tissues. This involves cross talk between the Notch and WNT signaling pathways. Describe how
activation of the Notch-delta pathway leads to lateral inhibition in the development of neural
cells in drosophila (5 marks)
- Notch is processed/cleaved at the golgi (but both pieces stay associated with eachother)
- this cleavage event causes notch to be inserted into the plasma membrane
- it binds delta from a neighboring cell which causes the release of the extracellular domain and it gets
taken up into the delta expressing cell (which will differentiate into a neuron) - On the cell originally expressing notch there are 2 more cleavage events that happen which releases
the cytoplasmic tail, it translocated to the nucleus where acts as a transcription factor - It will activate transcription of genes that will ensure the cells remains undifferentiated
- important so that when one cell differentiates into a neuron the surrounding cells remain
undifferentiated to be able to replenish the tissue when new cells are needed
4) Describe how a mutation in the APC subunit of the GSK3Beta complex can lead to colon cancer.
Begin with describing the normal signaling pathway and then how APC mutation impacts on its
activity. (hint: this is the WNT pathway, 5 marks)
Without Wnt:
*CK1 and GSK3B bound by APC and axin (Dishevelled is inactie) –> 2 kinases (CK1 and GSK3B phosphorylate B-catenin and target for degredation
*Groucho inhibits Wnt gene transcription
With Wnt:
*Binds to Frizzled rec –> recruits LRP –> activates Dishevelled –> Dishevelled dissociates APC complex (pulls away axin)
*B-catenin moves to nucleus and displaces Groucho
*This is latent proteolysis/activation since B-catenin is always there are ready to go UNLESS degraded
Mutations in APC complex would cause APC complex to get split up in other ways, which will prevent the degredation of B-catenin, leading to constant WNT gene transcription which can cause cancerl
5) Insulin binds to its RTK activating the PI3-K/AKT pathway to promote growth. Explain how the
activation of the RTK leads to activation of AKT and how AKT promotes cell growth. (5 marks)
*Insulin binds to RTK, causes dimerization leads to formation of phospho-tyrosine residues
*Recruits adaptor protein with SH2 domain–> has kinase domain –> phosphorylates Pi –> PIP3
*PIP3 is membrane bound, acts as a docking site –> recruits PDK1 and AKT
*PDK partially phosphorylates to partially activate AKT –> AKT further phosphorylates and fully activated by mTOR
*AKT dissociates from membrane and phosphorylates BAD –> BAD is bound to apoptosis inhibitory protein
*BAD releases inhibitory protein when activated –> apoptosis is inhibited (stops cell death, promotes growth)
6) What are the 4 mechanisms through which i–Smads negatively regulate the Smad pathway? (4
marks)
*Compete with Smads for binding sites on receptor
*Recruit ubiquitin ligases called Smurfsm ubiquitylate the receptor
*Recruit protein phosphates - remove phosphate groups
*Bind to co-Smad (Smad4) preventing Smad 2/3 from binding or promoting its ubiquitylation
- The critical concentration of actin in a test tube is less than 1 μM. However, the concentration
of actin in cells is 50-100 times greater than the critical concentration observed for pure actin in a
test tube.
(A) How is this possible? Why isn’t actin always polymerizing in cells? (2 pts)
(B) Why is this advantageous? (1 pts)
A).Thymosin limits amout of actin filaments available to be added to polymer
B).
- Dynamic instability causes microtubules either to grow or to shrink rapidly. Consider an
individual microtubule that is in its shrinking phase.
A) What is this shrinking phase called? (1pt)
B) What must happen at the end of a microtubule in order for it to stop shrinking and start
growing? (1pt)
C) How would an increase in the tubulin concentration affect this switch from shrinking to
growing? (1pt)
D) What would happen if GDP, but no GTP, were present in the solution? (1pt)
A). MT catastrophe
*Happens when you run out of soluble units or they’re low enough that the hydrolysis rate is faster than addition of units –> loss of GTP cap, rapid shrinkage, release of D forms
B). D form dimers (that were released during catastrophe) could have their GDP exchanged for GTP –> GTP concentration increased beyond critical concentration –> rapid growth (MT rescue)
C). When the concentration of
D). GDP bound tubulin causes curvature in filament, less stable than GTP.
- In the polymerization of in vitro of actin filaments from their subunits,
A) What does the “lag phase” correspond to? (1 pt)
B) How do you eliminate the lag phase? (1 pt)
C) Your actin filaments have an elongation rate shown as a dashed line. Which one of the
other five lines (A to E) would you think better shows what happens when a plus-end
capping protein such as CapZ is present? Explain. (2 pts)
D) Name a protein that you could add to your actin filaments to aid in their disassembly.
How does it function? (1 pt)
A). Nucleation
B). Actin subunits have to come into contact randomly on their own to merge and form small oligomers. By adding oligomers in the beginning, you get same rate of growth but critical concentration reached in less time, therefore bypassing the lag step.
C).
D). Cofilin - destabalizes actin filaments by binding and forcing filament to twist more tightly. Preferentially binds to old ATP-containing filaments. Puts more stress on bonds and increases chance of breaking
4) A muscle contraction relies on calcium released from 2 sources, explain how a muscle
contraction occurs, start with an action potential that triggers the small influx of calcium and
explain how this results in sarcomere shortening. (be sure to explain how Calcium facilitates
myosin binding to the actin filament and the resulting contraction, do not go into details about
the structure and phosphorylation of myosin). (4 marks)
- Action potential triggers the opening of voltage gated ion channels on the plasma membrane
allowing a small amount of calcium into the cell - This small amount of calcium binds to calcium release channels on the sarcoplasmic reticulum
causing a massive release of calcium - this calcium binds to troponin c which allows for the dissociation of tropomyosin from the actin
filament reveling the myosin binding sites - the myosin head hydrolyzes ATP, but the ADP and Pi remain bound to the myosin which forms a weak
bond with the actin filament which causes the release of the Pi that leads to a large conformational
change and a tight binding with the actin filament which leads to the dissociation of the ADP and the
myosin is tightly bound to the actin in rigor
5) The picture below shows a kinesin molecule binding to a MT
a) Label the leading and lagging heads, indicate with an arrow which way the kinesin is
travelling. (1.5 marks)
b) Explain how the kinesin protein walks along the microtubule through ATP hydrolysis. (3
marks)
c) If the cargo needed to be moved towards the minus end of the microtubule which
motor protein would be used? (0.5 marks)
A). on page
B).
*At the start of each step: rear head is tightly bound to MT & ATP (lagging head) front head loosely bound to MT & ADP (leading head)
*ATP in the lagging head is hydrolyzed to ADP + Pi, drives head to displace
*ATP replaces ADP on front head
*Pi released, causes change in orientation of rear head (rear head now pointing forward) by shifting neck
-Rear head is pulled forward, binds to MT and completes step.
C). Dyenin
