Question

You are a studying the progression of cells through the cell cycle. You are particularly interested in the role of the APC, which in a ubiquitin E3 ligase, in the control of the metaphase cyclin (cyclin B) and the timing of events. To look at this you start by synchronizing your cells in G1 and examining the levels of Cyclin B over time. The APC usually works on cyclin B during anaphase. RBX57 is a substrate for Cylcin B/CDK2. Given what you know about cyclin/CDK's draw what a. Time in hours you would expect for the CDK2 western blot. Describe why you have drawn this below 0 2 2.5 3 3.5 p-cyclin B Cyclin B b. Based on the data presented here, how does CDK2 phosphorylation affect cyclin B, is it activated or inactivated? Justify your answer using the data provided. p-RBX57 RBX57 B-actin At T-0 the majority of cells are in G1, by 2hrs they are exiting S phase. Given what you know of the function of Cyclin c. B, the mitotic cyclin and given the data presented here, what stages are the cells likely to be in at 2.5, 3 and 3.5 hours? Justify your theory using the data above. d. What is happening to cyclin B between 3 and 3.5 hours? Be sure to describe both what the Data shows and what you think that means is happening on a molecular scale. Given that you think that the APC is the E3 ubiquitin ligase What would you expect to see if you did the same experiment above in a cell that was treated with si RNA for the APC complex. Next to your figure describe why you e. drew what you did. Time in hours in cells treated with APC-siRNA 0 2 2.5 3.5 Cyclin B CDK2 B-actin

Answer 1

1) You are a studying the progression of cells through the cell cycle. You are particularly interested in the role of the APC, which in a ubiquitin E3 ligase, in the control of the metaphase cyclin (cyclin B) and the timing of events. To look at this you start by synchronizing your cells in G1 and examining the levels of Cyclin B over time. The APC usually works on cyclin B during anaphase. RBX57 is a substrate for Cylcia B/CDK2 (2 pts) Given what you know about cyclin/CDK's а. Time in hours draw what you would expect for the CDK2 western blot. 2.5 3 3.5 2 Describe why you have drawn this below p-cyclin B CDK 2, like every other cyclin dependent kinases are constitutively expressed, hence will be of same level in all the cell cycle phases Cyclin B CDK2 (4pts) Based on the data presented here, how does b. phosphorylation affect cyclin B, is it activated or inactivated? p-RBX57 Justify your answer using the data provided. RBX57 Cyclin B is inactivated as a result of phosphorylation, thats is why when the concentration of p-cyclin B (phospho cyclin B) is reduced, the concentration cyclin B (non phosphorylated) increases (2.5 and 3 hours). B-actin Also the concentration of phosphorylated RBX57 ( phosphorylated by cyclin B/CDK2) is increasing with increase in the level of cyclin B (and with decrease in level of phospho- cyclin B) at 2.5 hours) which means cyclin B is active when not phosphorylated. (4pts) At T-0 the majority of cells are in G1, by 2hrs they are exiting S phase. Given what you know of the function of C. Cyclin B, the mitotic cyclin and given the data presented here, what stages are the cells likely to be in at 2.5, 3 and 3.5 hours? Justify your theory using the data above. 0= G1 phase, 2 S phase, 2.5 G2 phase, 3 = M phase, 3.5= G1 phase Cyclin B is activated in S- G2 phase and its concentration slowly increases across S and G2 till it reaches its maximum level at M. Then it goes back to zero when the daughter cells are in G1 phase. d. (4pts) What is happening to cyclin B between 3 and 3.5 hours? Be sure to describe both what the Data shows and what you think that means is happening on a molecular scale. Cyclin B is decreasing in between these two time periods. It is because at the end of mitosis Cyclin B should decrease so that the cell can get out of mitosis. For this cyclin B will be ubiquinylated by APC and will undergo ubiquitin proteasomal degradation (3pts) Given that you think that the APC is the E3 ubiquitin ligase What would you expect to see if you did the same e experiment above in a cell that was treated with siRNA for the APC complex. Next to your figure describe why you drew what you did. Time in hours in cells treated with APC-siRNA In the absence of APC activity the cyclin B will not be degraded at the end of mitosis and the cells will still be in mitosis at 3.5 hours and the level of cyclinB will remain the same. CDK being constitutively expressed wil maintian the same level through out the different stages of cell cycle 0 2 2.5 3 3.5 Cyclin B CDK2 B-actin