706_S2006_PS5 - 7.06 Problem Set 5 - 2006 1. In the first...

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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? 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
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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_PS5 - 7.06 Problem Set 5 - 2006 1. In the first...

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