03320 Exam 3 - Cell Cycle

sessions 21-

What are the main steps of the eukaryotic cell cycle (in order)

G1, S, G2, M

what are the steps involved in interphase

G1, S, G2

what is the START/restriction point

the point in G1 when the cell is committed to DNA synthesis after this point (S phase)

what are CDKs

Cyclin-dependent kinases. They drive progression through the cell cycle

CDKs are highly regulated. How? (3 major ways)

1. phosphorylation + dephos

2. molecular recognition/binding (like cyclins and CKIs - cyclin kinase inhibitors)

3. ubiquination mediated destruction

are Cyclins necessary or sufficient

necessary but not sufficient

what are cyclins, cdk and ckis

cyclins - bind to cdks to activate cdk

cdk.- cyclin-dependent kinases, kinases that require the binding of cyclin to be activated

cki - inhibits CDKs

what do cyclin-CDK complexes do

initiates different steps of the cell cycle

Describe the two G1 Cyclin-CDK complexes

1. CyclinD-CDK4/6 - G1 cyclin that helps initiate cell cycle

2. CyclinE-CDK2 - G1/S cyclin that prepares cell for S phase (moves them past restriction point

Describe the S phase Cyclin-CDK complex

Cyclin A-CDK2 - S phasecomplex that activates DNA rep.

Describe the G2 phase Cyclin-CDK complex

CyclinB-CDK1 - Mitotic CDKs that induce mitosis

How are Cyclin-CDK complex activity controlled?

Cyclin levels are manipulated to change the acivity of the complex

what are the two regulatory mechanisms of the cyclin-CDK complexes + what are they controlled by

1. cyclin accumulation - controlled by transcription of cyclin genes

2. cyclin disappearance - controlled by Ub-mediated proteosomal degredation

Explain the steps of the EGF signaling pathway (Stop at Ras/Rac, 5)

1. EGF binds to EGFR (type of RTK)

2. EGFR is phosphorylated

3. GRB2 binds to EGFR

4. Sos binds to GRB2 and acts as GEF

5. Ras/Rac is activated by Sos

Continue EGF pathway (from Ras activated stop before TF get involved, 3)

1. Ras activates RAF

2. RAF activates MEK

3. MEK activates ERK (MAPK)

Continue EGF pathway (from ERK activation,4)

1. ERK activates TCF which is a TF for the c-fos gene

2. c-fos is made and phosphorylated by ERK

3. c-fos works as a TF with c-Jun to transcribe c-myc

4. c-myc is phosphorylated by ERK to be a TF for c-myc gene like Cyclin D

Continue EGF pathway (from Rac activated,5)

1. Rac activates JIP1 including JNK

2. phosphorylated JNK activates TF for the c-jun gene

3. c-jun protein is made

4. c-jun is phosphorylated by JNK

5. c-jun with c-fos work as TF for c-myc

To review what does Rac pathway lead to and what does Ras pathway lead to (before proteins go in nucleus)

Rac - activation of JNK

Ras - activation of ERK

what does JNK do (2)

1. phosphorylate TF for c-jun gene

2. phosphorylate c-Jun

What does ERK do (3)

1. phosphorylate TCF (TF) for c-fos ene

2. Phosphorylate c-fos for c-myc gene

3. phosphorylate c-myc for c-myc regulated genes

wat does c-jun and c-fos do

act as TF (when phosphorylaed) to activate transcription of c-myc genes

what are anti-mitogens and what do they promote: (2)

mitogens that signal blocking mechanisms of the cell cycle

1. transcription of CKIp15INK4b

2. G1 arrest/entry into G0

What does CKIp15INK4b do

Acts as a CKI for CyclinD-CDK4/6 complex

what blocks the CKIp15INK4b

mitogens like growth factors

What is the target of Cyclin D-CDK4/6 kinase activity that we discussed

Rb

What does Rb do normally (2)

1. binds and blocks E2F activity

2. recruits chromatin modifying enzymes that promotes chromatin condensation that represses txn

what is E2F

family of transcription factors

what is the relationship between Cyclin D-CDK4/6 and Rb (2 things occur)

Rb is phosphorylated by the Cyclin D-CDK4/6 (kinase activity) which causes Rb to be exported to nucleus. This allows for release of Rb on E2F allowing for the txn of E2F target geneswh

what are E2F target genes? (3)

1. makes more E2F - positive feedback loop

2. genes encoding proteins needed for DNA synthesis

3. makes other cyclins

What cyclin is made specifically from E2F

cyclin E or A

What does Cyclin E-CDK2 do (generally)

promotes entry into S phase past restriction point.

how does Cyclin E-CDK2 promote entry into S phase (2, general)

1. promotes accumulation of S phase cyclins

2. inducing degradation of a CKI that inhibits S-phase CDKs

how specifically does Cyclin E-CDK2 promotes accumulation of S phase cyclins (2)

1. promotes their transcritipn

2. inhibiting Cdh1

what is Cdh1

substrate targeting co-activator —> specicity determing module

how does Cdh1 act as a substrate targeting co-activator?

binds to APC/C scaffold and works together with that and the catalytic module (holding ubiquitin)

What does Cdh1 usually do when not inhibited + when in cell cycle

• Acts during the mitosis stage as cell goes into G1

• targets S and M phase cyclins and ubiquitinates them —> degrade

How specifically does Cyclin E-CDK2 inactivate Cdh1

phosphorylates Cdh1

how does Cdh1 become reactivated

Cdc14 phosphatase is used to dephosphorylate

What stages in cell cycle is Cdh1 phos and which stages is it dephos

phos —> S phase, G2, mitosis

dephos —> exit from mitosis, G1

What is the second way that Cyclin E-CDK2 is able to induce S phase

inducing degradation of a CKI that inhibits S-phase CDKs

What is the s phase cyclin

cyclin A

What is the name of the CKI that regulates the S phase cyclin in yeast/p27 in metazoans

Sic1

What specifically does Cyclin E-CDK2 do to the Sic1(CKI)

phosphorylates at all 6 “sub-optimal” sites

what does phos of Sic1 do

allows for SCF ubiquitin ligase to bind and add Ub

what is SCF

E3 ubiquitin ligase

what happens to ubiquitinated Sic1

proteasomal degradation

what happens once the Sic1 is degraded

Cyclin A-CDK2 can induce S phase DNA replication

what happens if only one optimal site of Sic1 is phosphorylated vs 6 sub-optimal and when can each one occur

one - causes full deg. of Sic1 even at low G1/S CDK activity so there is a longer gradual increase of S phase CDK activity and entry into s phase

six - only partially phos at low G1/S CDK activity and fully phos when max G1/S CDk. So its only degraded at max G1/S CDK activity so there is a rapid flip entry into S-phase (rapid increase of S phase CDK activity)

What is the s phase cyclinCDK

Cyclin A-CDK2

How does Cyclin A-CDK2 promote a single round of DNA rep (2)

1. controlling initiation of replication specifically at the G1-S transition

2. preventing re-initiation at ORIs that have already been used in that S phase

What is ORI licensing (2) and when does it occur

during G1

1. ORC recognizes the ORIs and binds

2. Assembling replication initiation factors and the replicative helicase

What is the prerelication complex (pre-RC)

replication initiation factors + replicative helicase

What is ORI firing (3) and when does it occur

During S

1. recruiting helicase cofactors

2. activating helicase to unwind dna strands

   1. recruiting dna pol

what is the level of the Cyclin A-CDK2 during ORI licensing

low CDK state because we are still in G1 phase

what has to be true for the helicase to be loaded onto the DNA and what has to be true abt the level of Cyclin A-CDK2

helicase —> needs to be non phosphorylated and inactive

cyclin A-CDK2 —> low levels so helicase is not phos

how is helicase activated specifically

phosphorylated by Cyclin A-CDK2 and DKK (2nd kinase)

what does ORI licensing and firing ensure

that each ORI is only used once per cycle and therfore DNA is only replicated once per cycle

what is true abt Cyclin A-CDK and DDK levels during ORI firing

high

name all missing parts

What is order of the cyclins from G1—>S—>G2—>Mitosis (and nemonic)

DEAB

cyclin D —> E —> A —> B

what holds the sister chromatids together

cohesins

what is cohesin

a 4 subunit ring-shaped protein complex that acts like a physical embrace holding DNA strands together.

What are the 4 subunit of cohesin

Smc1, Smc3, Scc1, and Scc3.

What is Soroin

A regulatory protein that binds to bottom of cohesin and enables continual loaing and unloading of cohesins during G1W

Where are the cohesins located before dna rep (G1)

cohesins around individual homologous chromosome (each stick of double stranded DNA)

what happens to cohesins in S phase

they form cohesive complexes and contain the replicating sister chromatids

converting G1 cohesins into cohesive complexes (S) requires what (2)

1. acetylation of Smc3 by acetyl transferase (CoATs)

   1. Soronin stabilitization

what do G1 non-cohesive cohesins do (2)

1. partioning genomic dna ino discrete compartments (TAD)

2. facilitate DNA looping to regulate transcription

How is cyclin B-CDK1 activated

Active Cdc25B dephosphorylates Cyclin B-CDK1 to activate CDK1 kinase

where in the cell is cyclin B-CDK1 activated

on centrosome

How is Cdc25C activated

Active Cyclin B-CDK1 phosphorylates Cdc25C (blue knobs)

What is Cdc25C bound to

114-3-3 on multiple Ser/Thr knobs

what happens after Cdc25 is activated by Cyclin B-CDK1?

the phosphorlated sites on Cdc25C are recognition sites of Pin1 and Pik1