Protein gel results of a receptor tyrosine kinase?

[Violagirl]

Homework Statement

You are studying a receptor tyrosine kinase and want to investigate the nature of the
autophosphorylation of this protein. To do this, you construct three forms of the receptor in an
expression vector: 1) a normal form with an active kinase domain and one tyrosine that is
phosphorylated upon activation, 2) a “kinase-dead” mutant that is larger than wildtype and carries an inactivating mutation in the kinase domain, and 3) a truncated form that has an active kinase domain but is missing the tyrosine residue (see Figure below). You express these forms in a cell line that lacks expression of this receptor (that is, there is no endogenous receptor at the plasma membrane in these cells). To test whether cis or trans phosphorylation occurs, you express the forms singly and in pairs (see lanes on gel below). You then treat these cells with ligand and radioactive ATP (32P on the γ-P) and then immunoprecipitate the receptor proteins from a cell extract. You analyze expression of the receptors by immunoblotting with an antibody that recognizes all three forms of the receptor and you detect phosphorylation by autoradiography. Your expression results are shown in the first blot
on the attached document.

Draw what the autoradiography results would look like if the receptor autophosphorylates in trans and in cis in two separate diagrams.

 

[Ygggdrasil]

Do you understand what the question is asking when it talks about autophosphorylation occurring "in trans" vs "in cis"?

 

[Violagirl]

I do. I know that autophosphorylation refers to how the enzyme can phosphorylate itself to isomerize to both cis and trans structures. In the cis state, its kinase activity is off and can only be turned on upon binding to a ligand, which would allow it to isomerize into a trans state and allow for it to act as a catalyst for the phosphorylation of other proteins. I didn't understand how you would go about showing the trans and cis structures would look like on an audioradiogram.

 

[Ygggdrasil]

Actually, that's not what cis and trans are referring to here. Receptor tyrosine kinases act as dimers, so two receptor molecules will come together to bind the ligand. These receptors can either be identical and form a homodimer or be two different variants of the receptor and form a heterodimer. When the dimer binds ligand, the kinase domains phosphorylate a tyrosine in the intracellular portion of the receptor, activating the receptor.

Let's call the two receptors in the dimer receptor A and receptor B. The question here is whether the kinase domain in receptor A phosphorylates the tyrosine in receptor A (the "in cis" model since the autophosphorylation occurs in an intramolecular reaction) or whether the kinase domain in receptor A can phosphorylate the tyrosine in receptor B (the "in trans" model since the autophosphorylation occurs in an intermolecular reaction).

Does this help?

 

[Violagirl]

That clarifies a lot for me, thank you! So in terms of thinking of how it would look on an gel assay, you would consider whether is it a homodimer or a heterodimer, right? So in terms of drawing expected autoradiography results for autophosphorylation of a receptor in a trans structure and if they only appear upon phosphorylation, would that mean you could only see the blots that show the expression of the "kinase dead mutant" that lacks the ligand binding site and truncated form containing the active kinase region but lacking the tyrosine residue and an absence of any homodimer structures forming? And vice versa for showing just the cis structures? I apologize for all the questions, thanks so much for going through this with me!

 

[Ygggdrasil]

Cis and trans do not refer to homodimer vs heterodimer in this case. Autophosphorylation in cis means that the autophosphorylation reaction occurs in an intramolecular reaction (the receptor phosphorylates its own tyrosine). Autophosphorylation in trans means that the autophosphorylation occurs in an intermolecular reaction (the receptor phosphorylates its neighbors tyrosine).

Perhaps consider the following analogy. Imagine a world where a law exists saying that everyone must tie their own shoes (all shoe tying must happen in cis). In this case, someone who lacks hands will never have their shoes tied. However, if you can ask your neighbor to tie your shoe (i.e. shoe tying can occur in trans), then the person lacking hands can have his shoes tied provided that his neighbor has hands as well.

The western blot will tell you which proteins get phosphorylated. Whether the proteins form homodimers or heterodimers is unimportant in the case of the gel since the SDS in the gel breaks apart the dimers.