L16: UPS in G1/S and G2/M transition

Question 1

Ubiquitin proteosome system (name the 3 components, describe process of Ub loading)

Answer 1

• E1 (Ub activating enzyme)
• E2 (conjugating enzyme)
• E3 (Ub ligase)

Ub conjugated by Eq in an ATP-dependent reaction. Ub transferred to E2, becomes covalently attached - binds to E3 along w/ target protein, Ub transferred to substrate by E2/E3

Question 2

Polyubiquitination basic role

Answer 2

Usually leads to destruction of proteins
-> aids directionality (permanently switch off target protein activity)

Question 3

Ubiquitin ligases as different cell cycle stages (G1 and S-Phase Ub ligase, G2-Anapase) w/ key function

Answer 3

• G1 Ub ligase = APC-Cdh1 (maintains a low cdk activity level)
• S-phase Ub ligase = SCF (Degrades replication complex assembly proteins to prevent re-replication)
• G2-Anaphase = APC-Cdc20 (Coordinates mitotic timing and degradation of Cyclin B (MPF))

Question 4

Switching from APC-Cdh1 to SCF (how is it done; 2 key ways. How is irreversibility ensured; 2 ways)

Answer 4

APC-Cdh1 maintains low cdk activity by destroying S-phase cyclin; must increase cdk activity (i.e. increase cyclin E expression) to activate SCF…
1. p27: p27 is a CDKI, which keeps cdk activity low and prevents G2 exit to S-phase; it is phosphorylated by SCF and degraded
(aka Sic1 in yeast)
2. APC-Cdh1: APC-Cdh1 is phosphorylated by G1/S-Cdk. pCdh1 is polyubiquitylated by SCF, targeting it for degradation by proteasome. Leaves APC inactive.

Question 5

SCF

Answer 5

• Made up of Skp, Cullin, F-box
• Acts to degrade PreRC component (pCdt1), preventing re-replication (L15)
• Cdt1 phosph. by cdks prior to this

Question 6

MPF

Answer 6

• aka Cyclin B-CDK1
• Triggers mitosis
• Activates APC-Cdh1 (cyclin-degrading enzyme) -> inactivated
• Also regulated by Wee1 vs CAK

Question 7

Wee1 and CAK at G2/M (and their +ve feedback loops)

Answer 7

• Opposing action (Wee1 inhibits, AK activates)
• Ensure that mitosis only occurs at correct stage of cell cycle (M-cyclin-cdk1 is inactive until phosph.)
• For M-cdk to be activated by MPF, MPF must be phosph. by Cdc25 (+ve feedback loop 1)
• Phosph. M-cdk inhibits Wee1 (+ve feedback loop 2)
• Wee1 phosph. Tyr15, CAK phosph. Thr160 or 161 (depending on organism)

Question 8

MPF and mitosis

Answer 8

• Targets cohesin complexes which hold sister chromatids together
• Degradation of cohesin allows resolution of centromeres and segregation of sister chromatids

Question 9

The moderators of cohesin, process of cohesin degradation and implications for mitosis

Answer 9

• Separase and securin
  1. Separase inhibited by binding of securin
  2. During late metaphase, APC activated by mitotic Cdk/Cdc20 (APC promotes separating of sister chromatids during this process)
  3. Securin poly-Ub, subject to proteosomal degradation
  4. Activated separase cleaves Scc1 (cohesin complex component)
  5. Sister chromatids pulled apart by mitotic spindle

Question 10

MPF and Cdc20 inactivation

Answer 10

• MPF able to promote the transition back to G1; promotes formation of APC-Cdh1 which degrades cyclin B
  -> inactivation of cdk activity in G1 phase
  -> maintains low level through continual degradation of cyclin B, cyclin A