You are interested in studying the cell cycle and have joined a lab to do so. A

Question

You are interested in studying the cell cycle and have joined a lab to do so. A student in the lab has taken the genes encoding for Cdk and M-cyclin, expressed them separately in bacteria, purified large quantities of both proteins, and then added them together to form a pure M cyclin-Cdk complex. All these purification steps have occured smoothly, as assayed by protein gels he has run, and he is able to see a nice cyclin-Cdk complex forming. He decides to inject the purified M cyclin-Cdk complex into oocytes that are not undergoing mitosis (like the experiment described in class). He sees no effect of injecting purified M cyclin-Cdk into oocytes. As a control, he also repeats the experiment described in class and injects mitotic extract into oocytes and finds that the oocytes assemble a mitotic spindle (as expected). Given what you know about M cyclin-Cdk regulation, propose a reason for why his experiment may not have worked? Be sure to explain your answer.

Explanation / Answer

Points to remember:

Cdk is active if all the below mentioned conditions are met.

1) Cdk is dephosphorylated at threonine 14 and tyrosine 15 by phosphatase. In a cell, Cdk is exists in an inactive form with phosphorylation at threonine 14 and tyrosine 15.

2) Cdk is in active form if it is bound to cyclin i.e. it exists as Cdk- cyclin complex.

3) Cdk is phosphorylated by cyclin activated kinase (CAK) at threonine 161 which lies in the active site of Cdk.

Considering experiment 1 where purified Cdk and cyclin were used to prepare cyclin-Cdk complex. In this experiment, the purified Cdk exists in an inactive state i.e. phosphorylated at threonine 14 and tyrosine 15. The experiment was not substituted with phosphatase. Secondly, the experiment doesn’t use CAK that phosphorylates Cdk at threonine 161. Hence, absence of phosphatase and CAK results in presence of phosphate at threonine 14 and tyrosine 15 of Cdk and absence of phosphate at threonine 161 which maintains Cdk in an inactive conformation despite the existence of Cdk-cyclin complex. In the absence of active cyclin- Cdk complex, the oocytes fail to assemble mitotic spindle.

Considering experiment 2 in which cell lysate is injected into the oocytes results in the formation of mitotic spindle. In this case, the cell lysate will contain phosphatases and CAK.   Phosphatases will facilitate dephosphorylation of Cdk at threonine 14 and tyrosine 15 while CAK will phosphorylate threonine 161 resulting in the active cyclin-Cdk complex which is functional and hence is able to support the formation of mitotic spindle.

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