Biology Forums - Study Force

Can someone please explain to me in GREAT specific detail how signal transduction pathways are discovered? I want to specifically know how the Ras-raf-mek-erk pathway was figured out. Was immunoprecipiation used? Was radiolabeled Phospates added onto atp and then someone used to figure out how the phospate was transfered by the kinases? I need to know step by step. No copy and paste jobs please.

Also, if you were trying to figure out binding affinity to mutant ras by different compounds.  Where would you go about finding these compounds to try out? I'm not just talking binding to the effector loop, but potential covalent binding sites that could have an allosteric effect.

Start with this link:

https://www.ncbi.nlm.nih.gov/pubmed/7935352

This link explains some some common pathways:

https://www.ncbi.nlm.nih.gov/books/NBK10043/

I know this doesn't answer your question, but I'd assume *a lot* of work goes in it. :sweat:

Thank you for the links. :) That first link doesn't show the study, but just the abstract. Am I missing something there. I don't have any money to pay for the study. The second link is really nice at showing pathways. I have weinberg's cancer biology book which shows those pathways as well.


Something really confusing me. I read in cell biology by alberts that to figure out a cell transduction pathway,you use radiolabeled phosphates attached to atp, put them in the cell, and then add in a mitogenic growth factor and see what gets a phosphate attached to it by running a gel elecrophoresis and separating out the proteins and seeing which one has a radiolabeled phosphate. I'm guessing you'd have to run a 2D gel?


Questions:
1) How do you get the radiolabeled ATP's into the Cell? Would the negatively charged phosphates repel against the negatively charged phospholipid bi-layer? You transfect DNA by using a liposome or by using a calcium phospahte buffer, which works for unknown reasons. You can also use electroporation, but all of this works for dna. Atp is different.


2)You're dealing with THOUSANDS of proteins in a living cell where kinases are constantly phosphorylating different proteins. How can you be sure that any protein that has the radiolabeled phospahate was actually caused by the binding of the growth factor and part of the pathway and not part of some other normally occuring metabolic pathway?

3)When you co-immunoprecipitate a protein and it's partner out of solution how can you be sure that it is part of the pathway? Again thousands of proteins and the interactions are transient. So how do you get the timing down right as well?

4)How do you figure out the ORDER of the pathway? Doesn't all of this happen very rapidly?

This just seems like a total mess(bordering on impossible in my mind!) to figure out. Does anyone else feel perplexed HOW these pathways were figured out?