Please answer with a good explanation Page 5 initials Short Answer. You can (and

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Please answer with a good explanation Page 5 initials Short Answer. You can (and must) answer in the space provided. #19(5) Suppose that investigators isolated src and mixed it with a cytoplasmic fraction of rat liver that contained a milieu of proteins and small molecules including a known protein substrate of investigators were also able to isolate a known src phosphatase from another source. When they incubated src the src phosphatase, and the cytosolic fraction with labeled ATP, they found that both src and its substrate were labeled with phosphorous. When they omitted the src phosphatase, they found that neither sre nor the sre src. The substrate were labeled. In the next experiment, investigators replaced the above src phosphatase with a newly discoved phosphatase (phosphotase II) to see if it could also activate src. When they incubated src, the phosphatase II, and the cytosolic fraction with labeled ATP, they found that neither src nor its substrate were labeled with phosphorous. One of their control experiments included both the both the original src phosphotase and phosphatase II along with the cytosolic fraction and labeled ATP. In this case they unexpectedly found that that neither src nor its substrate were labeled with phosphorous. How could you explain these results? Be very specific. 2

Explanation / Answer

The activity of src tyrosine kinase requires dephosphorylation of the carboxyl-terminal inhibitory tyrosine 527 phosphorylation site. The src phosphatase dephosphorylates the src kinase at the tyrosine residue. Hence, the inhibition is removed and the src kinase is activated. There is another phosphotyrosine 416 residue that is autophosphorylated to activate the enzyme and occurs only when the src phosphatase acts on src kinase.

1. In cells that have src, src phosphates and the cystolic fraction with ATP, the src phosphatase dephosphorylates the tyrosine residue of src. This results in activation of src kinase. Kinases are enzymes that phosphorylate or transfers phosphates from ATP to the substrate. Hence, the substrate is dephosphorylated at phosphotyrosine 527. Activation of src leads to autophosphorylation at tyr 416. The kinase will now phosphorylate the substrate. Hence, both src and the substrate are labeled with phosphorus. Omission of src phosphatase will not result in autophosphorylation. Hence, enzyme is not phosphorylated and neither is the substrate.

2. If src phosphatase is eliminated, the inhibitory phosphotyrosine 527 is not dephoshorylated. Hence, there is no autophoshorylation to activate the kinase. As a result, the substrate is not phosphorylated.

3. The phosphatase II (a ubiquitous serine/threonine phosphatase) will dephosphorylate all the tyrosine residue on the kinase. Hence, the kinase is not activated, and no phosphorous labeling will be seen in substrate and kinase.

4. In the cocktail that has both phosphatase II and src phosphatase, although src phosphatase activates the enzyme by dephosphorylating 527, there will also be no autophosphorylation of tyr 416 due to activity of phosphatase II. Hence, src kinase and substrate are not labeled with phosphorus.

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