Question

A signaling pathway exists so that an extracellular signaling molecule X binds to a receptor tyrosine kinase (RTK) in the cell’s plasma membrane. The RTK activates Ras, which phosphorylates and activates protein kinase 1, which in turn phosphorylates and activates protein kinase 2. Protein kinase 2 phosphorylates a transcription factor, activating it. This transcription factor is necessary and sufficient for the expression of a gene that promotes exocytosis.

a) State whether exocytosis would be increased or decreased in the following scenarios.

i. One of the tyrosine amino acids meant to be phosphorylated in the RTK is replaced with an isoleucine.     

ii. Ras loses its ability to hydrolyze GTP.  

iii. The extracellular binding site of the RTK is mutated so it can no longer bind to signaling molecule X.       

iv. The transcription factor lacks the amino acid that is phosphorylated.        

b) You know that protein kinases 1 and 2 can be two possible proteins, Protein A and Protein B, but you don’t know which letter corresponds to which protein kinase. You do some experiments to find out and come to the following conclusions:

• When Protein A is mutated so it cannot be phosphorylated but Protein B is normal, there is no exocytosis.
• When Protein B is mutated so it cannot be phosphorylated but Protein A is normal, there is no exocytosis.
• When Protein A is mutated so it cannot be phosphorylated but Protein B is mutated so it is constitutively active, exocytosis occurs.

Which letter corresponds to which protein? How do you know?

Answer 1

I. The replacement of tyrosine amino acid with isoleucine would reduce the auto phosphorylation event and thus reduced activation of receptor and exocytosis.

Ii. Ras looses ability to hydrolyse GTP, in this case the signal always remain active, thus increasing the exocytosis.

III. If extracellular molecule cannot bind to RTk, then exocytosis decreases.

IV. If transcription factor is not phosphorylation then the genes are not activated. So the exocytosis decreases.

The protein A and B question.

The condition 3 says that protein A is mutated so it won’t work. But protein B is constitutively expressed, so exocytosis occurs. In order to cause effect the protein B has to be acting below protein A. If not, it cannot phosphorylation protein A and no exocytosis would occur.

So protein A is protein kinase 1, and protein B is protein kinase 2.