Facilitator Questions

1. Discuss the basic features of a mass spectrometer, with an emphasis on the methods used to identify Cdk1 substrates in the experiments described in this lecture.


2. Most phosphorylation events in the cell are not permanent: protein kinases are generally opposed by protein phosphatases that remove phosphates. Why are phosphatases critical for the success of the mass spectrometry approach to finding Cdk targets?


3. Imagine that you have discovered a protein kinase that you suspect helps regulate an essential cellular process such as mitotic spindle assembly. (a) Discuss the methods you might use to assess the importance of your protein kinase in mitotic spindle assembly. (b) Discuss some of the approaches you might use to identify the relevant targets of this kinase.


4. Imagine that you have identified a candidate target for your favorite protein kinase. How do you prove that this candidate is a physiologically relevant substrate of your protein kinase in the cell?


5. Imagine that your studies reveal that your favorite protein kinase has hundreds of potential substrates, as in the case for the Cdks described in this lecture. How do you assess the importance of phosphorylation of so many candidates?


6. The experiments described in this lecture led to the identification of several Cdk substrates that are phosphorylated more rapidly by one cyclin-Cdk complex (Clb5-Cdk1) than another (Clb2-Cdk1). Describe some experiments that would allow you to test if Clb5 specificity for these targets is important in the cell.


7. Fission yeast cells are able to survive reasonably well when they contain just a single cyclin that drives both S phase and M phase. It has been proposed that early eukaryotes also controlled their cell cycle with a single cyclin-Cdk complex. What are the potential problems that might arise when the cell cycle is controlled by a single cyclin-Cdk complex, and how did the evolution of multiple cyclins help solve these problems?


8. Cyclins are destroyed in mitosis (as discussed in Lecture 1), leading to Cdk inactivation; this allows phosphatases to dephosphorylate Cdk substrates. Dephosphorylation of Cdk substrates is essential for the completion of many late mitotic events. Interestingly, all cyclins are not destroyed at the same time in mitosis: Clb5 is destroyed earlier (in metaphase) than most Clb2 (in late anaphase). How might the timing of destruction of different cyclins influence the time at which different Cdk substrates are dephosphorylated?


9. Several Cdk substrates contain clusters of phosphorylation sites that seem randomly scattered in poorly conserved regions. What experiment might you do to test whether the positioning of phosphates in these regions is important for their regulatory function?