706_S2006_PS3 - 7.06 Problem Set #3, 2006 1. You are...

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7.06 Problem Set #3, 2006 1. You are studying the EGF/Ras/MAPK pathway in cultured cells. When the pathway is activated, cells are signaled to proliferate. You generate various mutants described below. (Assume that none of the mutations affect the ability of the proteins to fold properly.) Assume that, when you add ligand (EGF), it is present in abundance. You perform each of your studies in two ways: (i) Overexpression of the mutant while the wild-type endogenous gene is present (ii) Replacement of the endogenous gene with the mutant gene In each case, predict the outcome and explain your reasoning. Your choices are: constitutive signaling (+/- ligand), no signaling (+/- ligand), or no effect (proliferation in the presence of ligand and no proliferation in the absence of ligand). a) Mutation of the EGF receptor so that dimerization is not possible. b) Mutation of the proline-rich domain of Sos to an alanine-rich domain. c) Mutation of the SH2 domain of Grb2 so that it is non-functional. d) Mutation of the SH3 domain of Grb2 such that it is non-functional. e) A serine-to-alanine mutation at each of the serines in the N-terminal regulatory domain of Raf to which 14-3-3 binds. f) A serine-to-aspartate mutation at each of the serines in the N-terminal regulatory domain of Raf to which 14-3-3 binds. g) Mutation of the two most C-terminal cysteine residues in Ras to alanine. (Hint: Think back to problem set one.)
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2. You are working on a hormone named Insulin-like growth factor 1 (IGF-1). Without IGF-1, the pancreatic cells you are working with undergo apoptosis (programmed cell death). This apoptotic response can be prevented by adding IGF-1 to the cell culture medium. Immediately, you realize that IGF-1 turns on an anti-apoptotic (i.e. cell survival) signaling pathway and you are very excited to understand more about this pathway. Using affinity labeling (remember back to Unit One), you identify the receptor for IGF-1 and name this receptor IGF-1R. You discover that, upon IGF-1 stimulation, IGF-1Rs are phosphorylated on several tyrosine residues. a) Describe how you would perform a genetic screen to identify other proteins involved in this anti-apoptotic ( or cell survival ) pathway. b) After performing the experiment described in part a) , you discover that you have isolated loss-of-function mutants in two genes involved in the cell survival pathway: IRS-1 and Pi3K.
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706_S2006_PS3 - 7.06 Problem Set #3, 2006 1. You are...

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