Instructions: What is the difference between scaffolding and docking functions? Support your answer with by specifically citing experimental data.This is based upon Bardwell, et al, 2001 (posted as an attached file). Be specific as possible in your answers. When asked to cite experimental data you must report specifically on experimental data reported in the paper and indicate where I the paper you obtained the data (e.g. Figure 1). Part A: Short answer/definition questionsAnswer 10 of the following 14 questions.1. What are two different mechanisms of target activation by MAPKs?2. What are the four defined MAPK signaling pathways in yeast?3. What is an ortholog? Give a specific example of an ortholog of yeast Ste7.4. Explain why yeast may serve as a model organism for studies of mammalian MAPKs.5. What is a combinatorial library? 6. Explain the limitation of using the consensus sequence, -(Ser/Thr)-Pro-, as a determinant of kinase specificity.7. Why is the high affinity binding site (docking site) under investigation unlikely to represent a prototypical enzyme-substrate interaction site?8. Typically, non catalytic domains are not well conserved relative to catalytic domains across species. However, the docking site is unique in that it is a non catalytic domain and conserved across many MEK orthologs. Speculate on why this domain, in contrast to the majority of residues in the non catalytic domain, is conserved.9. Referring to figure 2A, why were Kss1 and Hog1 radiolabeled?10. Referring to figure 2A, why did the authors include the GST alone condition?11. The MEK2-ERK2 interaction had a Kd of ~ 9µM. Define Kd and describe what 9uM is specifically referring to.12. What is lacZ? How is it used as a reporter?13. What is a halo assay?14. What is an adaptor protein?Part B: Long answer questionsAnswer 10 of the following 16 questions.1. After figure 2 the authors conclude that the docking site is sufficient for binding. What do they mean by this? Is the docking site the only site on Ste7 or MEK1 that interacts with their targets? Cite specific experimental data to support your conclusions.2. The Ste7-ERK1 interaction had a Kd of ~1µM. Explain why this is surprising. Would you expect the Kd of the Ste7-ERK2 interaction to be higher or lower then Ste7-JNK1? Support your answer by citing experimental data.3. What is an IC50 value? How might the IC50 values derived from figure 5 be related to the Kd values determined earlier in the paper?4. Using figure 1 and figure 4, propose a MEK1 mutant with an alteration of TWO and only TWO specific amino acids that would result in the largest decrease in ERK2 binding relative to WT.5. Which protein would you predict to have greater homology with ERK2, Kss1 or Hog1? Support your answer by citing experimental data.6. Why did the authors test the ability of peptides to inhibit PKA?7. Why did the authors use a Ste7 deletion strain to examine the function of docking defective mutants?8. Ste7 docking mutants had substantial inhibition of binding Fus3 in vitro. Why then was there only a minor effect of Ste7 docking mutants in vivo? How did the authors address this curious result experimentally?9. What is the difference between scaffolding and docking functions? Support your answer with by specifically citing experimental data.10. Provide a biological explanation for the synergistic effect of combining Ste5D746D and Ste72-19 deletion mutants.11. Provide a biological explanation for the reduced β-gal activity in the absence of Ste5 reported in figure 6D.12. Referring to figure 7, construct a similar diagram indicating the interactions between Ste5, Ste11, Ste7, and Fus3 (this may be hand drawn).13. Evaluate the authors’ conclusion that the MAPK-docking site is both necessary and sufficient for the formation of stable MEK-MAPK complexes. Do the in vitro and in vivo data both support the authors’ conclusion?14. How might the existence of modular MAPK-docking sites on multiple proteins contribute to signal-response specificity?15. The authors report differences in the calculated Kd for the Ste7-Fus3 interaction by two difference assays, immunoprecipitation and cosedimentation. Provide an explanation for this discrepancy.16. This work was supported by a Special Fellow Award from the Leukemia and Lymphoma Society to the senior author Lee Bardwell. Why would a organization interested in developing cancer therapeutics fund research on yeast and this work specifically?Part C: The authors use 135 mM salt in the buffer used for the GST-binding assays (Cook, et al, 1996). Why did the authors use this concentration of salt?
