Biology 2382B Lecture Notes - Hela, Nuclear Membrane, Cdk Inhibitor

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coralimpala296

3 Mar 2014

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Wee1 is a kinase that phosphorylates cdk leading to its inactivation, while cdc25 is a phosphatase that removes the inhibitory phosphate group (left by wee1) from cdk and promotes its activation. In the recessive elongated fission yeast cells, these mutants were long and unable to engage mitosis (increased g2), because their mutation either resulted in an excess of wee1 or a deficit of cdc25 (leading to inappropriately inactive mpf). In the dominant premature fission yeast cells, these mutants were short and engaged mitosis unprepared (decreased g2), because their mutation either resulted in an excess of cdc25 or a deficit of wee1 (leading to prematurely activated mpf). When mitotic cyclin binds to the cdk, it initially forms an inactive mitosis promoting factor. Wee1, an inhibitory kinase, introduces a phosphate group to the tyrosine at position 15 (y15) on bound cdk, so the mpf complex remains inactive (key to discouraging premature mitosis).

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QUESTION 1:

(a)Examine the Figure below relating to the S.pombe cellcycle:

1) Describe the function of Cdc25 and Wee1and why deficit of Cdc25 or excess of Wee1 can cause G2arrest

2) b) Describe how deficit of Wee1 or excess ofCdc25 may result in abnormally small cells

(b) Theactivities of Wee1 tyrosine kinase and Cdc25 tyrosine phospatasedetermine the state of phosphorylation of tyrosine 15 in the Cdk1component of mitotic cyclin-Cdk complexes (MPF). When tyrosine 15is phosphorylated, M-Cdk is inactive; when tyrosine 15 is notphosphorylated, M-Cdk is active. Just as the activity of M-Cdkitself is controlled by phosphorylation, so too are the activitiesof Wee1 kinase and Cdc25 phosphatase. The regulation of thesevarious activities can be studied in extracts of frog oocytes. Insuch extracts Wee1 tyrosine kinase is active and Cdc25 tyrosinephosphatase is inactive. As a result M-Cdk is inactive because itsCdk1 component is phosphorylated on tyrosine 15. M-Cdk in theseextracts can be rapidly activated by addition of okadaic acid,which is a potent inhibitor of serine/threonine phosphatases. Usingantibodies specific for each component it is possible to examinetheir phosphorylation states by changes in mobility upon gelelectrophoresis.

(Phosphorylated proteins generally run slower than theirnon phosphorylated counterparts).

1) Based on theresults with okadaic acid, decide whether the active forms of Wee1and Cdc25 are phosphorylated or unphosphorylated. In the figureindicate the phosphorylated forms of Wee 1 and Cdc25 and label thearrows connecting their active and inactive forms to show whichtransitions are controlled by protein kinases and which by proteinphosphatases.

2) Are the proteinkinases and phosphatases that control Wee1 and Cdc25 specific forserine/threonine side chains or for tyrosine side chains? How doyou know?

3) How doesaddition of okadaic acid cause an increase in phosphorylation ofWee1 and Cdc25, but a decrease in phosphorylation ofCdk1?

4) If you assumethat Cdc25 and Wee1 are targets for phosphorylation by activeM-Cdk, can you explain how the appearance of a small amount ofactive M-Cdk would lead to its rapid and completeactivation?

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Biology 2382B Lecture Notes - Hela, Nuclear Membrane, Cdk Inhibitor

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