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QuESTionS Grup
If you were to design a new post-translational modification system for signal transmission, what key features would you include? 1
Treatment of a cell with growth factor X leads to tyrosine phosphorylation of a number of target proteins, consistent with a model in which a tyrosine kinase is activated by
growth factor stimulation. Interestingly, treatment of the cell with vanadate (an inhibitor of phosphotyrosine phosphatases) also leads to the accumulation of the same set of
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tyrosine-phosphorylated species. Can you explain why this increase in target protein phosphorylation is observed with vanadate? What are the implications of your model for
how kinases and phosphatases operate as a regulatory system, and the speed at which signals will be turned on and off by this system?
You are given a purified preparation of protein kinase and test its ability to phosphorylate a peptide substrate at various concentrations of enzyme. You notice that at low
concentrations of enzyme, the rate of substrate phosphorylation is initially low but increases substantially over time. However, if you use a higher concentration of kinase, this
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apparent lag is not seen. How can you explain these result and how would you test your hypothesis?
In mammalian cells, the hydroxyl amino acids (serine, threonine, and tyrosine) are the major sites of regulatory phosphorylation. In bacteria, histidine phosphorylation
predominates in signaling. The free energy of hydrolysis (ΔG) of phosphohistidine is larger than for the phosphorylated hydroxyl amino acids, and the rate of spontaneous
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hydrolysis is much faster. What differences between eukaryotic and bacterial cells could account for the selective pressure for eukaryotic cells to use phosphorylation of the
hydroxyl amino acids instead of histidine for their signaling pathways?
Summarize how the gain of a single phosphate group in the GTP-bound state of a G protein (versus the GDP-bound state) can lead to changes in the interactions of the G
protein.
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G proteins are almost always active (and transmit a downstream signal) in their GTP-bound forms and inactive when bound to GDP. In principle, however, it would be possible
for the GDP-bound form to be active and the GTP-bound form to be inactive. How might you design a signaling system where the GDP-bound form is active in transmitting a
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signal? Explain how receptor binding would lead to activation of the G protein, and conversely how the G protein would eventually be down-regulated. In this system, what
kind of G protein mutants would be constitutively active (that is, active in the absence of receptor stimulation)?
Some of the most common mutations associated with cancer are found in the small G protein Ras. Oncogenic mutants of Ras lead to constitutive signaling. Suggest several
possible mechanisms for why these forms of Ras show constitutive signaling. Conversely, mutations of Ras have been isolated that have dominant negative activity (that is, they
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block signaling by the normal, endogenous G protein). Suggest possible mechanisms for how these mutants block signaling.