Cell Bio Review

Mitosis

Cdk1/cyclin A and B, consists of prophase, prometaphase, metaphase, anaphase, and telophase (basically chromosome condensation, alignment and separation)

G1

Cdk4-6/cyclin D and Cdk2/cyclin E, the gap/growth period after mitotis with high metabolic activity

S

Cdk2/cyclin A, synthesis of new DNA / the cell goes from 2n to 4n

G2

Cdk1/cyclin A, second gap/ growth period after S phase, checking to make DNA replication went correctly

In the experiments of Blow & Laskey studying DNA replication in a Xenopus oocyte extract, what protein had to be synthesized in order to create active MPF?

-B & L: cyclin B had to be synthesized
-Cyclin B promotes the disassembly of nuclear lamina which makes up the nuclear envelope
Cyclin B as part of MPF promotes nuclear lamina dissociation

What are two distinct means by which phosphorylation regulates the activity of the cyclin dependent kinase holoenzyme?

What are the four enzymes responsible for the balance of phosphorylation on the cyclin-dependent kinase? Note that only three were explicitly identified in the lecture slides.

Threoninie161 phosphorylation activates Cdk, tyrosine15 phosphorylation inhibits Cdk
-Wee1, Cdc25, CAK, any other serine/threonine phosphatase

How does phosphorylation of the cyclin-dependent kinase inhibitors (p21Cip1 and p27Kip1) affect those proteins.

-p21Cip1 and p27Kip are inhibited when they are phosphorylated
-since you an inhibiting an inhibitor of cell cycle progression, the phosphorylation event will permit cell-cycle progression
Phosphorylation leads to ubiquitinylation of CKIs and their degradation, which is more than just inhibition.

Cdk1/cyclin B can phosphorylate Cdc25 and Wee1. The result of these phosphorylation events is the explosive activation of Cdk1/cyclin B through positive feedback. Were both Cdc25 and Wee1 activated by this phosphorylation? If not, which one was activated?

( In G2, Cdk1/cyclin B is kept inactive because Wee1 activity > Cdc25 activity but towards the end of G2 (to push cell into M), Cdk1/cyclin A phosphorylates Cdc25 such that its activity > Wee1 and now Cdk1/cyclin B is active. ) Once Cdk1/cyclin B is active, it inhibits Wee1 and further activates Cdc25 and this is what caused the positive feedback and explosive activation of Cdk1/cyclin B.

The targets phosphorylated by MPF include cyclin A, cyclin B, Cdc25 and subunits of the APC/C complex. Does this contribute to the positive or the negative feedback pathways that impinge on the control of MPF activity? Why?

Negative feedback since the phosphorylation of cyclin A and cyclin B lead to proteolysis so the cell can exit mitosis and move on to cytokinesis

Why is p16Ink4a considered to be a tumor suppressor when p21Cip1 does not have that same label even though they both prevent cell cycle progression?

Since p16Ink4a inhibits entry into the cell cycle and the cell will continue to remain in G0. While p21Cip1 only prevents cell cycle progression, p21Cip1 can bind to Cdk4-6/cyclin D and not inhibit it so the cell cycle still proceeds. This "loophole" of Cdk4-6/cyclin D could potentially lead to unregulated progression of the cell cycle which could lead to cancer

All very rapid or "explosive" events tend to be the product of positive feedback. Describe how the inhibition that prevents anaphase is released and how positive feedback is achieved to accelerate the explosive separation of sister chromatids at anaphase.

Separase is inhibited by securin when there is no APC/C activity (securin is activated by CDk1/cyclin B). Then activation of APC/c ubiquitinates Cdk1/cyclin B and stops separase from inhibiting securin so now securin is free to disrupt sister chromatid cohesion and proceed to anaphase.
Separase activated Cdc14 which dephosphorylates securin and increase the explosive separation of sister chromatids at anaphase.

How do mechanisms that respond to damaged DNA contribute to the progression of DNA replication during S-phase?

DDK responds to DNA damage and will allow for more DNA synthesis by activating pre existing, dormant, origins of replication to begin replication again and fix the damage.

You are using C. elegans as a model system to study programmed cell death. You perform a mutational analysis on the BCL-2 homolog CED-9 that results in loss of function of this gene. What is the function of CED-9? What do you expect to observe in terms of the development of C. elegans after loss of function of CED-9?

CED-9 is an inhibitor of apoptosis that stops the activation of pro-apoptotic CED-4. CED-4 then activates CED-3 which is responsible for the execution of cell death. If CED-9 is lost, the C. elegans would die because there is no inhibition of cell death.

What would be the impact of a mutation on the P4-P2 positions on caspase recognition of a substrate target?

The caspase would lose its specificity since P4 through P1 is what the caspases recognize in their substrates

Identify one apoptosis-related event associated with caspase cleavage for each of the following sites.
1. Nucleus
2. Plasma membrane
3. Mitochondria

1. chromatin condensation
2. plasma membrane blebbing
3. ATP levels fall / NDUFS1