JM Lecture 51: Introduction to Cell Cycle

Question 1

What are the techniques used to determine the number of cells undergoing various stages in the cell cycle?

Answer 1

mitotic index (mitotic cells/ cells undergoing division- uses non-specific staining)
Ki-67 (proliferating cells- uses immunohistochemistry)
flow cytometry (measuring DNA content- uses fluorescence)

Question 2

How do you calculate the mitotic index?

Answer 2

mitotic index = (number of mitotic cells) / (total number of cells)

Question 3

Where will the peaks for each phase in the cell cycle be located on a flow cytometer graph?

Answer 3

G1: about 100 DNA content (x1)
S: long peak inbetween 100 and 200
G2/M: small peak at about 200 DNA content (2x)

Question 4

What is the function of a cyclin-dependent kinase?

Answer 4

phosphorylate substrate on a serine or threonine that immediately precedes a proline residue

Question 5

What cyclin/cyclin-dependent kinase complex(es) are required for the G1 phase?

Answer 5

Cyclin D/ Cdk4 (early)
Cyclin D/ Cdk6 (early)
Cyclin E/ Cdk2 (late)

Question 6

What cyclin/cyclin-dependent kinase complex(es) are required for the S phase?

Answer 6

Cyclin A/ Cdk2

Question 7

What cyclin/cyclin-dependent kinase complex(es) are required for the G2/M phase?

Answer 7

Cyclin A/ Cdk1 (early G2)

Cyclin B/ Cdk1 (late G2/early M)

Question 8

What regulates cyclins?

Answer 8

upregulated transcriptionally

downregulated by protein degradation (via ubiquitin-targeting mechanism)

Question 9

Why does Cdk need cyclin binding?

Answer 9

1. provides part of the substrate binding site (allows for substrate specificity)
2. induces conformational change that allows the substrate to access the catalytic site
   * note that cyclin binding is necessary but NOT sufficient for Cdk activation

Question 10

How is Cdk activated?

Answer 10

1. cyclin binding (necessary but not sufficient for activation)
2. Cdk Activating Kinase (CAK) phosphorylates catalytic subunit of Cdk (on conserved threonine residue 160 of the t-loop- moves it out of the active site)

Question 11

How is Cdk inhibition activated/inactivated?

Answer 11

Wee1 (kinase) phosphorylates Cdk at threonine 14 and threonine 15 (adjacent to ATP binding pocket) inhibits it
removal of these phosphates by Cdc25 phosphatases allows it to be active again

Question 12

How are Cdc25 and Wee1 regulated by Cdks?

Answer 12

Cdks phosphorylate Cdc25 phosphatases (making them more active in removing inhibitory phosphates)
Cdks also phosphorylate Wee 1 (prevents it from adding inhibitory phosphates)
as Cdks become more active, there is a positive feedback to further enhance their activity

Question 13

What are CIP family inhibitors

Answer 13

CIP family (p21, p27, and p57)
inhibit all Cdks
bind the Cdk-cyclin complex and induce conformational change that prevents substrate binding

Question 14

What are INK4 family inhibitors?

Answer 14

INK4 (p15, p18, and p19)
specifically inhibit Cdk4 and Cdk6
binds Cdk subunit preventing it from interacting with cyclin

Question 15

What is ubiquitin?

Answer 15

a 76 amino acid peptide that serves as a signal for protein degradation
attached to lysine residues on proteins

Question 16

What is polyubiquitination?

Answer 16

signal that targets proteins for degradation by proteasome

mediated by ubiqutin ligases (E1, E2, and E3)

Question 17

What is the SCF (SKP-Cullin-F box) protein complex?

Answer 17

ubiquitin ligase for cyclins that are important to the G1 to S transition (Cyclin D and E)
activated by phosphorylation of cyclin by SKP ligase (always active)

Question 18

What is Anaphase Promoting Complex (APC) and what is its function?

Answer 18

ubiquitin ligase for cyclins that are important for mitotic progression (mainly Cyclin B)
activated by binding of co-factor (Cdh1 or Cdc20) to ACP