2. (1.5pts) One critically important aspect of cell cycle control is ensuring that transitions such as entry into mitosis occur rapidly and completely. The regulation of Cyclin-Cdk by Wee1 tyrosine kinase and Cdc25 (tyrosine phosphatase) gives a good appreciation of how this control can be achieved. The balance of activities of Wee1 and Cdc25 determines the state of phosphorylation of tyrosine 15 of cdk. When tyrosine 15 is phosphorylated, Cyclin-cdk is inactive; when tyrosine 15 is not phosphorylated, Cyclin-cdk is active. Just as the activity of Cyclin-cdk itself is controlled by phosphorylation, so too are the activities of Wee1 kinase and Cdc25 phosphatase!
The regulation of these various activities can be studied in extracts of frog oocytes. In such extracts Wee1 kinase is active and Cdc25 phosphatase is inactive. As a result Cyclin-cdk is inactive because tyrosine 15 of cdk is phosphorylated. Cyclin-cdk in these extracts can be rapidly activated by okadaic acid, which is a potent inhibitor of serine/threonine protein phosphatases. Using antibodies specific for each component (Cdk, Wee1, and Cdc25), it is possible to examine their phosphorylation state by changes in mobility during gel electrophoresis (phosphorylated proteins generally run slower than non phosphorylated proteins).
Ladder
107
70
34
– +
– +
– +
Okadaic acid:
Cdk Wee Cdc25
Ladder
107
70
34
– +
– +
– +
Okadaic acid:
Cdk Wee Cdc25
• In the figure above, circle the phosphorylated forms of each protein. (0.25, all or none)
• Based on the results with okadaic acid (+ lane)…..
• The active form of Wee1 is phosphorylated or not? (0.125, all or none)
• The active form of CDC25 is phosphorylated or not? (0.125, all or none)
• Explain how an increase in the phosphorylated forms of Wee-1 and CDC25 can cause a decrease in the phosphorylation of Cdk? Rubric (0.5): Apply what you know about activation of CDK by phosphorylation (0.25) to whether Wee1 and CDC25 have been activated or inhibited by phosphorylation (0.25).
• If you assume that Cdc25 and Wee1 are targets for phosphorylation by Cdk in active cdk-cyclin complex, can you explain how the appearance of a small amount of active cdk-cyclin would lead to its rapid and complete activation? Rubric (0.5): Remember, Cdk-cyclin is a kinase! Explain how MPK might regulate Wee1 and Cdc25 (0.25). Based on the first part of your answer, explain the reason for why a rapid and complete activation of CDK-Cyclin (MPF) is achieved (0.25).
3. (2pts) We will again check out the review article on CHK2 used in problem set 1. CHK2 is activated by DNA damage. Remember, CHK2 is a kinase. See any proteins you recognize below? Let’s think about the effects of CHK2 activation on p53 (TP53), CDC25, and BRCA1.
a. CHK2 phosphorylates p53 when DNA damage occurs. Why does p53 phosphorylation lead to increased levels of p53 protein in a cell? Rubric (0.5pts): Explanation of normal p53 production and regulation of protein levels and effect of phosphorylation on these processes.
b. Explain why activation of p53 will pause DNA replication. You will need to include affects on transcription, Kinase activity, and components of DNA replication to be complete. Rubric (0.5pts): Effect of p53 activation on downstream events ending in DNA replication inhibition.
c. “CHK2-mediated phosphorylation of CDC25A phosphatase at S123 inhibits dephosphorylation of the cyclin-dependent kinase 2 (CDK2)-cyclin E complex, halting the cell cycle before entry into the S phase.” Unpack this sentence.
i. Do you think CDK and CHK2 are phosphorylating the same location in CHK2, why or why not? Rubric (0.25pts): Yes or no with explanation. Just yes or no = zero pts
ii. Phosphorylation of CDC25A by CHK2 inhibits CDC25. Why would this action pause the cell cycle pausing DNA replication (S phase)? Rubric (0.25pts): Effect of CDC25 inhibition on downstream events ending in DNA replication inhibition.
d. BRCA1 is also phosphorylated by CHK2 at serine 988. Discuss the effects on cell cycle progression for the two discussed effects in the review article:
i. BRCA1 phosphorylation modulates activity from NHEJ to HR for DNA repair. Why would this be beneficial when DNA damage occurs? Rubric (0.25pts): Look up the difference between these two types of DNA repair. Which is more accurate?
ii. BRCA1 also accumulates at the centrosome and inhibits nucleation of microtubules. Why would inhibition of nucleation of microtubules pause the cell cycle? Rubric (0.25pts): Link failure to nucleate microtubules with specific stages of the cell cycle that have to happen. UNIT 2!
4. (3pts) In class we discussed the multihit hypothesis in detail.
a. Define the multihit hypothesis as it relates to cancer. This definition has expanded since the first week of class! We now know specific functions of genes that increase cancer risk. A 20 point question on your final will ask you to tell a story about how a cell in your body became cancer using mutations in genes that encode for proteins you learned in Unit 1, 2, 3, 4, and 5! Let’s practice here.
Illustrate your definition using the gene that is associated with your own cancer that I assigned to you in cancer journal #1. Do some quick research and see if the gene is a proto-oncogene or a tumor suppressor.
So the gene above will be 1-2 hits depending on what you find. To get to the number of hits associated with cancer look back at genes/proteins we have already learned about in this course.
Now, use the gene I gave you and some of the genes you have already learned about to illustrate the number of hits that would have made one cell in your body a cancer cell. First, define the multi-hit hypothesis adding examples of genes that are “hits”. Second, Tell a story about one cell in your body becoming cancer that illustrates your definition. Rubric (3): Complete definition-updated with what we know now! (1) with example (2).
Gene: MDM2
