Question

You have isolated an epithelial cell line which is unresponsive to EGF (normal epithelial derived cell lines require EGF in the culture media for growth). You suspect that EGF receptor signaling in this cell line is impaired, and you designate this a mutant cell line. As an assay for EGFR signaling, you have stably transfected this mutant cell line and a normal cell line with a luciferase-based reporter construct that is responsive to the transcription factor Elk-1. You want to determine what component of the signaling pathway is impaired.

8. (5 pts) Expression of a constitutively active raf results in the following:

Luciferase Activity (A.U. + 10%)

-EGF +EGF

Mutant 192 190

Normal 186 189

Briefly state what the results tell you and indicate whether the impaired component in the mutant cell line lies upstream or downstream in the EGF signaling cascade, or if the component indicated is in fact the impaired component.

Answer 1

EGF activates Elk-1 TF by MAP kinase pathway. In the series of reaction raf is one of the product use for activation of Elk-1.

In this experiment we use constitutively active raf, it activates transcription factor Elk-1 always so we get Luciferase Activity always.

With out EGF both mutant and normal cell line express luciferase activity so raf express is occur. Mutant strain the activity is high.

When addition EGF factor the mutant strain luciferase activity is decreasing, So EGF directly inhibts the activity of Raf protein shows downstream mutation..Mean while normal cell line there is increasing in luciferase activity shows normal activity of cell.