706_F06pset2_ans - 7.06 FALL 2006 Problem Set#2 1 De s i gn...

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7.06 Fall 2006 PSet 2 answers 1 of 4 7.06 FALL 2006 Problem Set #2 1. Design an experiment that would show that Raf lies downstream of Ras. What kind of results would you expect? Culture cells containing a constitutively active Ras D protein and a mutant nonfunctional Raf. Although these cells will be stimulated uncontrollably by the constitutively active Ras D , there will be no downstream signal (because Raf is not functional). This is consistent with Raf being downstream of Ras. 2. You wonder whether Ras and Raf interact directly (i.e. Do they bind to each other?). Design an experiment that could answer this question and explain what kind of results you would expect. One possible experiment: set up an in vitro binding assay using recombinant proteins. Incubate Ras with varying concentrations of Raf and run on a non-denaturing gel. If Ras and Raf are able to bind, you will see a new band corresponsing to the complex on your gel. Alternatively, you could Co-IP them. 3. What would be the effect of the following mutations on the RAS signaling pathway activated by Epidermal Growth Factor (EGF)? a.) RAS protein can bind but not hydrolyze GTP. (Dominant) activation of the pathway - will always be in GTP bound (active) state. b.) SOS (GEF) protein with mutated proline rich region. (Recessive) inactivation of the pathway - will not bind to SH3 domain of GRB2. c.) EGF Receptor (EGFR) with all Tyrosines mutated to Phenylaline. (Recessive) inactivation - no phosphorylation of phenylalines (in place of tyrosine) possible. 4. You are working on a new growth hormone. You have cloned a receptor and found a novel kinase, FAB2, which seems to be activated by addition of the growth hormone. You think FAB2 may be involved in the MAP kinase signaling pathway. a.) How could you determine if the MAPK is the direct target protein for FAB2? You could perform an in vitro assay using your kinase and MAPK and look for incorporation of radiactive phosphate. You will need recombinant proteins for this. b.) How could you identify the residues on MAPK phosphorylated by FAB2 activity? Individually mutate each putative phosphorylation site in MAPK and repeat the above experiment. When you no longer have incorporation of the label, you have likely mutated the target residue.
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7.06 Fall 2006 PSet 2 answers 2 of 4 5. As you know, yeast comes in two different mating types: a and alpha. Each secretes a different mating factor recognized by receptors on the cell surface which in turn results in
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