A receptor tyrosine kinase (RTK) dimerizes and autophosphorylates in the presence of a ligand. A researcher prepares three constructs that express either the (A) full-length protein having a kinase domain as well as 3 tyrosine residues, (B) the RTK with a non-functional kinase domain but with the 3 tyrosine residues, and (C) the RTK lacking the 3 tyrosine residues but having a functional kinase domain. She expressed these constructs in cell lines lacking the RTK, either singly or in combinations as shown in the figure, breaks open the cell and added the ligand of the RTK in presence of radio-labelled ATP. She immunoprecipitated the RTK and analyzed the immunoprecipitated proteins by Coomassie staining as shown in the figure, followed by autoradiography.

Which one of the following autoradiograms would the researcher expect?

Correct option is A

Explanation:

Construct A:

• Full-length RTK with a functional kinase domain and 3 tyrosine residues.
• This construct can dimerize and autophosphorylate upon binding.
• When treated with ATP in the presence of a ligand, this construct will show strong bands in the autoradiogram due to autophosphorylation, indicating active signaling.

Construct B:

• RTK with a non-functional kinase domain but retains 3 tyrosine residues.
• While it can form dimers, it cannot autophosphorylate because the kinase domain is inactive.
• This construct will not show any autophosphorylation bands in the autoradiogram despite being present in the Coomassie stain because it cannot carry out phosphorylation.

Construct C:

• RTK that lacks the 3 tyrosine residues but has a functional kinase domain.
• Although it has an active kinase domain, it cannot perform autophosphorylation due to the absence of the necessary tyrosine residues.
• This construct may appear in the Coomassie-stained gel but will not show bands corresponding to autophosphorylation in the autoradiogram.

Analysis of the Expected Results:

Band Presence in Combinations:

• A + B:

  • A has a functional kinase domain and will autophosphorylate, generating a band in the autoradiogram.
  • B cannot phosphorylate itself, but it can be phosphorylated. Since B has tyrosine residues, it can receive phosphate groups from A when they are co-expressed.
  • Result: The band seen in A + B comes solely from A and B.

• A + C:

  • A will again autophosphorylate, generating a band.
  • C, although having a functional kinase domain, lacks tyrosine residues necessary for autophosphorylation, meaning it will not contribute any bands.
  • Result: The band seen in A + C also comes solely from A.

• B + C:

  • B has tyrosine residues but cannot autophosphorylate itself. However, it can potentially be phosphorylated by C, which has a functional kinase domain. C can phosphorylate B, leading to bands showing that C has acted on B.

Summary of Bands:

• A + B: Band due to A autophosphorylating and phosphorylating B.
• C: Band due to A autophosphorylating; C does not contribute to the band.
• B + C: Band due to C phosphorylating B.