Your findings indicate that substance X abolishes the activity or activation of CDK2, but does not alter CDK2 levels.

12. Which of the following mechanisms may explain these results ? (Choose wrong or possible) [Go to CDK Regulation.]

A. Substrate X inhibits phosphorylation of Y15 on CDK2 by Wee1, and thus inhibits the activation of CDK2.
B. Substrate X inhibits cyclin E binding to CDK2, and thus inhibits the activation of CDK2.
C. Substrate X inhibits p27 binding to CDK2-cyclin E complex, and thus inhibits the activation of CDK2.
D. Substrate X inhibits the phosphorylation of T160 on CDK2 by cdc25A, and thus inhibits the activation of CDK2.
E. Substrate X blocks CDK2 substrate binding site and serves as an inhibitor of the activity of the kinase itself.

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You now perform the following experiment:
Cells are synchronized to G0 by starvation. CS is than added for 12 hrs. Cells are
exposed to PBS or substance X at t=8-12, and are harvested at t=12. (Why are cells exposed to substance X during t=8-12 and not t=8-16 ?) CDK2 antibodies are used to immunoprecipitate CDK2 containing immunocomplexes. You run the samples on SDS-PAGE gels and use anti cyclin E, anti CDK2, anti p27 and anti p21 antibodies in western blots to determine the levels of these proteins in CDK2 containing immunocomplexes.

Here are your results:

13. You may draw the following conclusions (true or false):

A. Following substance X treatment cyclin E levels are reduced. Since CDK2 levels are also reduced the stichiometry of the complex is not altered.
B. Following substance X treatment p27 levels in CDK2 immunocomplexes are slightly reduced.
C. Following substance X treatment p21 levels are not changed whereas cyclin E levels are significantly reduced. That means that there is more p21 in each complex, and this causes the observed reduction in CDK2 kinase activity.

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You now perform the following experiment:
Cells are synchronized to G0 by starvation. CS is than added for 12 hrs. Cells are
exposed to PBS or substance X at t=8-12, and are harvested at t=12. Samples of cell lysates (crude extracts) are run on SDS-PAGE gels and subjected to western blot analysis using anti cyclin E, anti CDK2, anti p27 and anti p21 antibodies.

14. What is the purpose of this experiment?

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Here are your results:

15. What conclusions may you draw from this experiment?

16. Can you suggest additional experiments that are essential to elucidate the mechanism of substance X effect on cell cycle progression?

Please submit your answers to Dr. M. Brandeis by next week.