Margolis_guide

Margolis_guide - I.e., briefly outline the experiment in...

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Guide for Margolis et al. (1989) EGF induces tyrosine phosphorylation of phospholipase C-II: A potential mechanism for EGF receptor signaling. Cell 57:1101-1107. These questions are intended to help you read the paper in an inquisitive fashion and to help you focus on some of the issues you should understand. Please cover each of these questions during your reading and studying. Please submit the answers to 5 of these questions for your homework assignment. 1) Cell lysates were prepared to examine how EGF affects PLC-II. Orthovanadate, Na Fluoride, pyrophosphate and p-nitrophenyl phosphate were present in the lysis buffer. These inhibit phosphatase activities that are present in the lysate. Why was it important to inhibit dephosphorylation of proteins in the lysate? 2) How do the authors detect whether a protein is phosphorylated on tyrosine? How was it determined that PLC-II became phosphorylated on tyrosine after EGF treatment?
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Unformatted text preview: I.e., briefly outline the experiment in Figure 1. 3) What is the significance of the fact that PLCII becomes phophorylated within 30 seconds of EGF treatment? 4) Why does PLCII migrate more slowly in a SDS gel after treatment with EGF (Fig 2)? 5) What evidence indicates that EGF receptor and PLC-II are associated with one another in vivo (Fig 3 and Fig 5)? Do these experiments prove that they interact directly? 6) Does the EGF receptor interact with PLC-II in the absence of EGF (see Fig. 3)? 7) How does the use of tyrphostin allow the authors to argue that phosphorylation of PLC-II upon stimulation of EGF receptor is relevant to cell growth and proliferation (Figs 6, 7, 8, and 9)? 8) Inhibition of the EGF kinase activity with tyrphostin blocked the EGF-induced Ca++ increase in the cytoplasm. What pathway that leads to increase in Ca++ might be failing? 9) What is a mitogen?...
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