Question

“Protein A” is phosphorylated at one amino acid by a kinase, “protein B”, and dephosphorylated by a phosphatase, “protein C” You want to determine the mechanisms of protein B and C. What experimental information would you need in order to propose a reasonable mechanism? Explain.

Answer 1

Protein A phosphorylated .by protein B (kinase)

Protein A dephosphorylated by protein C (phosphatase)

Experiment

Suppose on phosphorylation protein A become active and lead to expression of gene and ATP is required for kinase activity of Protein B

Radioactive gamma phosphate ATP will be utilize and phosphatase inhibitor. Thus kinase protein B will phosphorylate gamma phosphate to protein A which is radioactive

Isolation of Protein A and detection of radioactivity will be indication of kinase activity of protein B

Note: normal phosphorylation can be detected of protein A by phosphorylation specific antibodies.

2 ) mutation in protein B or inhibitor of Protein B will result in absence of phosphorylation in protein A, thus no radioactivity will confirm that protein B is a kinase.

3 ) protein C is phosphatase which can be confirm by using phosphorylated protein A . Removal of radioactive phosphate will lead non radioactive protein A and also make it inactive thus no gene expression.

4 ) mutation in protein C or introduction in inhibitor for protein C will lead to radioactive protein A , confirming phosphatase activity of Protein C