Question #1 The activities of the Weel tyrosine kinase and the Cde25 tyrosine ph
ID: 220204 • Letter: Q
Question
Question #1 The activities of the Weel tyrosine kinase and the Cde25 tyrosine phosphatase determine the state of phosphorylation of The regulation of these various regulatory activities can be studied in extracts of frog oocyte eytoplasm. In these extracts tyrosine 15 of Cdk1component of MPF and control its activity. Just as MPF activity is controlled by kinases and phosphatases, the activities of Weel and Cdc25 are controlled by phosphorylation. MPF can be activated by the addition of okadaic acid, which is a potent inhibitor of serine/threonine phosphatases. Using antibodies to each component, it is possible to examine their phosphorylation states by changes in mobility after electrophoresis and Western blotting (phosphorylated proteins run slower than unphosphorylated counterparts). Okadaic Acid: 100kDa 100kDa 80kDa- 60kDa 100kDa B0kDa 60kDa 80kDa |- 60kDa 40kDa 30kDa 20kDa 40kDa 30kDa 20kDa 10kDa 30kDa Probed for: Cdk 1 Wee1 Cdc25 a. Based on the okadaic acid data, what are the active forms of the Weel kinase and Cde phosphatase (circle on the gels)? b. How does okadaic acid cause phosphorylation of Weel and Cde25, yet dephosphorylation of Cdk1? c. Assume that Weel and Cdc25 are actual targets of active MPF. Might the appearance of a small initiating amount of active MPF lead to rapid and complete activation? How?Explanation / Answer
Ans 1.
a) The levels of cdk phosphatase is constant, whereas the level of wee-1 was significantly decreased. cdc formed new bands which are characterized by slower electrophoretic mobility that disappeared after treatment with phosphatase. When tyrosine 15 is phosphorylated MPF is inactive; when tyrosine 15 dephosphorylated, MPF is active. As, activity of MPF is controlled by phosphorylation, so too are the activities of Wee1 kinase and Cdc25 phosphatase controlled by phosphorylation. So, in this situation, Wee1 kinase is active and Cdc25 phosphatase is inactive. That is why, MPF is inactive because its Cdc2 component is phosphorylated on tyrosine 15.
b) In G2 - M phase, activation of the mitotic kinase Cdc2 and complex is done by Wee1 -mediated phosphorylation of Cdc. Mitotic phases are performed by the complex factors which can inactivate Wee1, activate Cdc25, and promotes Cdc2/Cyclin B1 activation. Cdc2/Cyclin B1 activation are influenced by positive and negative feedback loops which can activate Cdc25 and can suppress Wee1. So, it can be said that the transition in mitosis is triggered by involvement of the integration of Cdc2/Cyclin B1 inhibitory and activating factors.
Wee1 and Myt1 inhibition leads to increase in Cdk1 activation in the normal mitotic entry, even in the absence of G2. Low levels of Cdc25 phosphatases prevents Wee1 and Myt1 inhibition and activation of Cdk1/cyclin B kinase and substrate phosphorylation induces a mitotic collapse, which leads to dephosphorylation of mitotic substrates. And this is blocked by (PP1/PP2A phosphatase inhibitor), okadaic acid. By this finding it can be said that the positive feedback in Cdk activation overcome the activity of Cdk-opposing phosphatases and stops the forward progression in mitosis.
c) MPF is composed of a catalytic CDK1 and cyclin B subunit and cyclin B are responsible for the MPF activity. MPF in all cells are governed by the regulatory activity of Wee1 kinases, which cause an inhibitory phosphorylation of CDK1 and the CDC25 phosphatases, causes dephosphorylation activity of CDK1 at the same sites. High levels of CDC25 and low Wee-1 activity are needed for switching on the CDK1 component of MPF. In mitosis cycle MPF is restricted to the cytoplasm, by cytoplasmic retention sequence, which contains nuclear export signal. When cells completes the mitosis cycle, cyclin B1 is phosphorylated with its cytoplasmic retention sequence, and leads to rapid accumulation of cyclin B1 and MPF within the nucleus.
Ans 2. Cell cycle phases is mechanized by activations of cyclin-dependent kinases (CDKs), which triggers the DNA synthesis(S phase) and mitosis (M phase), and anaphase-promoting complex (APC), which promotes exit from mitosis and return to G1 phase.