706_S2006_PS5key - 7.06 Problem Set 5 - 2006 1. In the...

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1 7.06 Problem Set 5 -- 2006 1. In the first half of the course, we encountered many examples of proteins that entered the nucleus in response to the activation of a cell-signaling pathway. One example of such a protein was beta-catenin. Over spring break in the Bahamas, you identify a protein in an indigenous marine arthropod that is homologous to Drosophila beta-catenin. You find that this protein, which you name baha- catenin, is responsive to Wnt signaling and is phosphorylated by GSK3 in the absence of Wnt. However, phosporylated baha-catenin does not undergo proteasomal degradation (like beta- catenin does). You establish a line of cells that grow in culture from your indigenous marine arthropod. Using immunofluorescence on these cells, you find that baha-catenin localizes to the cytoplasm in the absence of Wnt and to the nucleus in the presence of Wnt. a) What additional information, if any, would you get by creating a fusion protein of baha- catenin to GFP and examining its localization in the presence and absence of Wnt? GFP fusion constructs would allow you to visualize protein movement in real time, thus providing information on the kinetics of baha-catenin localization in response to Wnt. For instance, you could determine how long after exposure to Wnt baha-catenin moves into the nucleus. b) Results from further experiments that you do confirms that phosphorylation and dephosphorylation of baha-catenin by GSK3 does indeed regulate the subcellular localization of baha-catenin. Your experiments are consistent with the model that phosphorylation of cytoplasmic baha-catenin in the absence of Wnt inhibits its import into the nucleus. Next, you create the following deletion constructs (each of which are mutant forms of baha-catenin fused to GFP at the C-terminus), express each one in your cell line, and examine baha-catenin localization in the absence and presence of Wnt. In the chart below, “ Δ ” means “deletion of.” Assume that all of the fusion constructs encode properly folded proteins. Construct –Wnt +Wnt Wash away Wnt Wild-type Cytoplasm Nucleus Cytoplasm Δ 1 Cytoplasm Nucleus Cytoplasm Δ 2 Cytoplasm Cytoplasm Cytoplasm Δ 3 Cytoplasm Nucleus Nucleus Δ 4 Cytoplasm Nucleus Cytoplasm Δ 5 Cytoplasm Nucleus Cytoplasm What conclusion(s) can you draw, looking only at the “–Wnt” and “+Wnt” results above? N 1 2 3 5 4 GFP C
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2 Region 2 of the protein encodes a peptide sequence that is necessary for baha-catenin localization to the nucleus in response to Wnt signaling. Region #2 therefore contains an NLS. c) How would you show that the peptide sequence you identified in part b) is sufficient to act as a nuclear localization sequence? You would generate a recombinant DNA construct that encoded some protein that was
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This note was uploaded on 01/23/2012 for the course BIOLOGY lsm1301 taught by Professor Seow during the Spring '11 term at National University of Singapore.

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706_S2006_PS5key - 7.06 Problem Set 5 - 2006 1. In the...

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