_____________________ Name Bio336/391M Exam 2 Mar. 7, 2008
1. (15 points) State whether the following types of mutations would be potentially oncogenic or not. Briefly explain your reasoning. a. A mutation that results in overexpression of a ligand of a receptor tyrosine kinase (RTK). Oncogenic. Autocrine expression of an RTK ligand could lead to constitutively on signaling. b. A mutation in Ras that results in a loss of its catalytic activity. Oncogenic. Loss of GTPase activity (Ras's only catalytic activity) will lead to always-on signaling through generation of RasGTP c. A mutation in an RTK that results in a loss of its catalytic activity. .Loss of tyrosine kinase activity will result in no signaling, which should NOT be oncogenic.
d. Deletion of both alleles of a gene encoding a phosphatase that dephosphorylates an activated RTK. Oncogenic; this will result in an inability to down-regulate an activated RTK. e. Gene amplification of a chromosomal region encoding a mitogen activated protein kinase (MAP kinase).
Oncogenic. Overexpression of a MAP kinase can lead to up-regulated signaling downstream of Ras.
2. (12 points) Design an experiment that would determine whether the transforming potential of an oncogenic Ras protein (i.e., a codon 12 mutant) is dependent on farnesylation. (Include how you might determine that the protein(s) of interest are actually farnesylated). Take an immortalized cell line such as NIH3T3 cells, which are sensitive to transformation by codon 12-mutated Ras ((we know this from Weinberg "great experiment"). We create a mutated oncogenic Ras gene that has the cysteine codon four codons from the end of the protein altered to encode an alanine. This version of the protein cannot by farnesylated (or geranylated). We assay this version of the gene, as well as the parental codon-12 (oncogenic) Ras for transformation of NIH3T3 cells. If they both transform, then farnesylation is not required for transformation. If the cysteine mutant does not transform, then farnesylation may be required for transformation. We can also make GFP (green fluorescent protein) fusions to both of these genes and determine their localization in the cell ( we can also assay the GFP versions for transformation); the cysteine mutant should is not expected to be localized to the plasma membrane, while the normal oncogenic Ras protein should be. I has many answers that proposed using previously characterized farnesyl transferase inhibitors to address the question. A major problem with this is that even if farnesylation is required for transformation it might not be revealed in this experiment, since geranylation is often observed when farnesylation is blocked (particularly for K-Ras). I also had many answers for the localization that relied on immunoprecipitation and SDS-PAGE gels to determine if the protein was farnesylated. This is tricky since proteins with a large lipid molecule covalently attached will not behave well by SDS-PAGE. Also, I had a lot of people say they would use an antibody against the farnesyl group to immunoprecipitate the modified protein; this is also unlikely to work since it is unlikely that you could isolate an antibody against a large lipid molecule. But I graded this pretty easily.
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Name _____________________
3. (18 points) Post-translational protein modifications play a major role in controlling the activities of many oncoproteins. a. Names three types of protein modifications that we've talked about so far in class b. For each of these, give a specific example of how this type of modification is important for the function or regulation of a specific cancer-related protein c. Draw a chemical structure (with as much detail as possible) representative of each of these protein modifications.
a. Ubiquitination, phosophorylation (ser/thr and/or tyrosine), various lipid modifications (farnesylation or geranylation, myristilation, parlmitoylation), proteolytic cleavage. b. I won't go through all the possibilities here. c. I wanted to see correct chemical bonds at the right places and correct amino acid side chains.
4. (12 points) a. What chemical reaction is catalyzed by Ras (indicate the substrate and the products)? GTP to GDP + Pi
b. The human neurofibromatosis type 1 (NF1) gene encodes a GTPase Activating Protein for Ras. Is NF1 is putative dominant oncoprotein or a recessive oncogene/tumor suppressor? Explain.
Tumor suppressor. Loss of both alleles would result in an inability of Ras to be activated to catalyze the reaction in (a) above; therefore Ras would always be in its GTP-bound form and always signaling.
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Name _____________________
c. The human Vav gene encodes a guanine nucleotide exchange factor for Rho GTPases, and Vav mutations are associated with hematopoietic malignancies. Is Vav a putative dominant oncoprotein or a recessive tumor suppressor? Explain.
Dominant oncoprotein. Overexpression of Vav would result in pushing Ras into its GTP-bound form in the absence of appropriate Pharmaceutical signals.
d. companies have invested millions of dollars towards the development of farnesyltransferase inhibitors to block Ras modification. Why haven't they invested similar amounts of money to develop inhibitors of Src myristylation? Because Src is a very minor player in human cancer (even though the lessons it taught us, from chickens, were very important)
5. (12 points) As you know, the v-Src protein from Rous sarcoma virus (RSV) lacks tyrosine 527 normally found in c-Src. Let's say you are a researcher and you have isolated two new RSV-like viruses (call them RSV-2 and RSV-3), which, like the original, have picked up the Src gene. You clone and sequence the v-Src genes from RSV-2 and RSV-3. You discover that both viruses do indeed encode mutated versions of Src, and you confirm that these mutations correlate with transforming activity. It is interesting, however, that the region surrounding tyrosine 527 is completely normal in both proteins. Predict two different regions of the proteins where you might expect to find the v-Src-2 and v-Src-3 transforming mutations. (Explain). The SH2 domain could be altered so that it could no longer bind phosphotyrosine 527; this would push Src toward its "open" active conformation. The SH3 domain and/or the PPLP sequence in the linker region might be altered; this would also push Src toward its "open" active conformation. The SH1 (catalytic) domain might be altered such that phosphorylation of Tyr 416 is no longer necessary for activation; i.e., a mutation at the active site could result in always-on kinase activity.
. 6. (9 points). The human p73 tumor suppressor protein received its name based on the fact that it migrated on an SDS-PAGE gel as a 73 kilodalton protein. Based on this information: a. Approximately how many amino acids is the protein composed of? b. Approximately how many base pairs would the open reading frame be that encodes this protein? c. How large would the gene be? I told you this was coming.... a. You needed to know that the average MW of the 20 amino acids is about 115. 73 kD = 73,000 daltons; 73,000 divided by 115 is about 634 amino acids.
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Name _____________________ b. 634 amino would be encoded by exactly 634 x 3 nucleotides = 1,904 bp. c. Impossible to know, but given that nearly all human genes have introns, it is safe to guess that the gene is much larger than 1,904 bp.
7. (8) a. (6) Retroviruses have been associated with tumorigenesis in both animals and humans. State three general mechanisms by which retroviruses have been shown to promote tumorigenesis. they might have picked up a viral version of a cellular proto-oncogene (as in RSV) they might insert themselves near a proto-oncogene in such as way that they result in up-regulated of this gene (enhancer or promotoer insertions) they might encode a viral protein (which is not a counterpart to a cellular gene) that influences cell proliferation/control decisions (e.g., as with HTLV-1 Tax)
b.
(2) Which mechanism is most relevant to human cancer? (Explain) The last one, above.
8. The ubiquitin system (9 points). a. (6) There are two ATP-dependent steps in ubiquitin-mediated protein degradation. What is the energy being used for in each of these steps? Be specific (i.e., don't tell me that ATP is being to drive specificity of protein degradation).
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The E1-catalyzed reaction, which chemically activiates ubiquitin, is ATP-dependent (formation of Ubadenylate intermediate) The proteasome contains ATPases which are used to unfold and translocate target proteins into the proteasome.
b. (3) Small molecule inhibitors of the proteasome (i.e., drugs) are currently on the market for treatment of multiple myeloma. Do you think these drugs might also be useful for treating HTLV-1-associated leukemia? Explain. Seems like a reasonable idea. This would stabilize IkappaB, which is normally being targeted for ubiquitin-mediated degradation as a result of phosphorylation, which is in turn a result of activation of IKK (IkappaB kinase) by Tax.
9. (5 points) Otto Warburg (Nobel Prize, 1933) was wrong when he claimed that cancer could be cured by exposure to high oxygen concentrations, but he was correct that most cancer cells behave unusually with respect to energy production and oxygen utilization. What is the "Warburg Effect", and how is this taken advantage of in cancer detection/imaging?
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Name _____________________ The Warburg effect is the observation that many cancer cells continue to undergo glycolysis even in the presence of high oxygen concentation. Therefore, to create the same amount of ATP, a cancer cell would need to utilize much more glucose than a normal cell (which would utilize oxidative phosphorylation under these conditions). This results in a high "glucose flux" in the cancer cells compared to surrounding normal cells. This can be picked up in positron emission tomography when patients are injected with radioactively labeled glucose.
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