Epidermal growth factor is a mitogen, because it stimulates cells to divide

Question

(1) Explain the role of Ras in epidermal growth factor signaling that results in cell division. (2) Design two experiments to prove that Ras is a key player in the EGF-activated pathway. (3) What assay other than cell division could you use to assess whether the EGF-Ras-MAPK pathway has been activated?
What will be an ideal response?

Answer 1

Answer:
(1) Ras plays a key role in regulating the EGF signaling pathway. Ras is a G protein that is activated to the GTP-bound "on" form by proteins recruited to the membrane by EGF binding to its receptor. When on, Ras activates a kinase cascade ending with MAPK activation, which stimulates expression of early genes necessary for the cell to enter S phase and eventually divide. Ras turns off the signaling pathway by hydrolyzing GTP to GDP.
(2) Experiments
a. One experiment could be to use any of a number of systems to knock down or knock out Ras expression in a cell and confirm that EGF no longer stimulates cell division.
b. A reciprocal experiment could be to produce a cell that contains a mutant form of Ras that is unable to hydrolyze its GTP to GDP and therefore is locked in the "on" state. This could be done by microinjecting the permanently activated Ras protein, or by transforming the cell with a plasmid encoding the mutant Ras so the cell expresses that protein. In either case, the cell should be stimulated to divide even when not signaled by EGF.
(3) The EGF-Ras-MAPK pathway activates expression of "early" genes, so an assay for Jun/Fos activation would indicate whether the pathway is activated or not.