(a) Examine the Figure below relating to the S.pombe cell cycle:
1) Describe the function of Cdc25 and Wee1 and why deficit of Cdc25 or excess of Wee1 can cause G2 arrest
2) b) Describe how deficit of Wee1 or excess of Cdc25 may result in abnormally small cells
(b) The activities of Wee1 tyrosine kinase and Cdc25 tyrosine phospatase determine the state of phosphorylation of tyrosine 15 in the Cdk1 component of mitotic cyclin-Cdk complexes (MPF). When tyrosine 15 is phosphorylated, M-Cdk is inactive; when tyrosine 15 is not phosphorylated, M-Cdk is active. Just as the activity of M-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 tyrosine kinase is active and Cdc25 tyrosine phosphatase is inactive. As a result M-Cdk is inactive because its Cdk1 component is phosphorylated on tyrosine 15. M-Cdk in these extracts can be rapidly activated by addition of okadaic acid, which is a potent inhibitor of serine/threonine phosphatases. Using antibodies specific for each component it is possible to examine their phosphorylation states by changes in mobility upon gel electrophoresis.
(Phosphorylated proteins generally run slower than their non phosphorylated counterparts).
1) Based on the results with okadaic acid, decide whether the active forms of Wee1 and Cdc25 are phosphorylated or unphosphorylated. In the figure indicate the phosphorylated forms of Wee 1 and Cdc25 and label the arrows connecting their active and inactive forms to show which transitions are controlled by protein kinases and which by protein phosphatases.
2) Are the protein kinases and phosphatases that control Wee1 and Cdc25 specific for serine/threonine side chains or for tyrosine side chains? How do you know?
3) How does addition of okadaic acid cause an increase in phosphorylation of Wee1 and Cdc25, but a decrease in phosphorylation of Cdk1?
4) If you assume that Cdc25 and Wee1 are targets for phosphorylation by active M-Cdk, can you explain how the appearance of a small amount of active M-Cdk would lead to its rapid and complete activation?