Lecture 16 - The role of the ubiquitin proteosome system in cell cyle regulation Flashcards
What is ubiquitination?
Ubiquitination/Ubiquitylation: Interchangeable terms referring to the joining of ubiquitin onto a target molecule. (Post-translational modification altering protein function.)
Enzymes Involved:
* Ubiquitin Activating Enzyme (E1): Initiates the ubiquitin transfer relay chain. Ub is conjugated to the E1 enzyme in an ATP dependent reaction.
* Ubiquitin Conjugating Enzyme (E2): E1 transfers the Ubiquitin to the E2, covalently attaching Ub to E2. E2 Transfers ubiquitin to the substrate.
* Ubiquitin Ligase (E3): E2 binds to the E3 as does the target protein. E3 works with E2 to add ubiquitin chains onto target proteins.
Successive rounds of ubiquitylation label substrates for proteasomal mediated degradation, additional ubiquitin chains are added to a lysine group on the ubiquitin molecule itself Once the substrate is polyubiquitylated it is released from the ternary complex and recognized by the proteasome where it is degraded. Also referred to the ubiquitin-proteosome system.
How does proteasomal mediated degradation of cyclins regulate cell cycle transitions?
Cell Cycle Regulation:
Ubiquitin Ligase Families:
* Anaphase Promoting Complex (APC): Works in G1 phase, degrades mitotic and S-phase cyclins.
* SCF (Skp-Cullin-F-box): S-phase-specific ubiquitin ligase.
G1/S Phase transition
* APC-Cdh1 complex - During the G1 phase it degrades both mitotic and S-phase cyclins which ensures that the cell remains in G1 by preventing cyclin accumulation
* SCF - During the G1/S transition it works as an S-phase ubiquitin ligase which allows the controlled degradation of specific proteins, facilitating the transition to S phase.
In G1 phase CDK activity is maintained at a low level by APC-Cdh1 which prevents entry into the S phase.
The switch to SCF (Skp-Cullin-F-box) is regulated by increasing CDK activity. This is achieved by:
* Phosphorylation and degradation of the CDK inhibitor by protein p27
a. S-phase CDK is inhibited by binding of p27
b. P27 is phosphorylated on multiple sides by G1/S CDK
c. This marks them for degradation by SCF
d. S phase CDK is now activated which promotes DNA replication initiation
* Phosphorylation and destruction of Cdh1 (Subunit of APC-CDH1 complex)
APC-Cdh1 is phosphorylated by G1-S CDK and poly-ubiquitylated by SCF leading to degradation of Cdh1.
The destruction of Cdh1 promotes S-Cyclin (cyclin A) accumulation
The degradation of the CDK inhibitor p27 enforces directionality
CDK activity is low in G1-phase due to inhibition of the S-phase CDK. The CDK inhibitor protein is present until the end of G1 phase The degradation of the CDK inhibitor protein is concomitant with G1 exit and is mediated by its phosphorylation and ubiquitylation at the end of G1 phase. Now released, SPF is able to promote S-phase entry
Preventing premature replication licensing at high CDK activity is mediated by SCF
* ORC, Cdc6 and Cdt1 are required to recruit MCM2-7 helicase
* CDK mediated phosphorylation of Cdt1 leads to its dissociation from chromatin
* SCF recognises phosphorylated proteins, polyubiquitylates for proteosome degradation
* pCdt1 (phosphorylated) is degraded by SCF in S-phase, preventing replication licensing until the next cell cycle
G1 Phase and CDK Activity Regulation:
During G1 phase, the cell prepares for DNA replication, but CDK (Cyclin-Dependent Kinase) activity is kept low to prevent premature entry into S phase.
APC-Cdh1 complex plays a crucial role in maintaining low CDK activity during G1 phase by degrading both mitotic and S-phase cyclins, preventing cyclin accumulation and inhibiting CDK activity.
Transition to S Phase:
As the cell prepares to transition from G1 to S phase, CDK activity needs to increase. This increase is regulated by the switch from APC-Cdh1 to SCF ubiquitin ligase.
SCF becomes active during the G1/S transition and targets specific proteins for degradation, facilitating the transition to S phase.
Regulation of CDK Inhibitor p27:
In G1 phase, CDK activity is inhibited by the CDK inhibitor protein p27. This inhibition prevents premature entry into S phase.
Phosphorylation of p27 by G1/S CDK marks it for degradation by SCF, thereby releasing the inhibition on CDK activity and allowing progression into S phase.
Regulation of APC-Cdh1 Activity:
APC-Cdh1 is inactivated during the G1/S transition by phosphorylation and subsequent degradation.
G1/S CDK phosphorylates APC-Cdh1, marking it for polyubiquitylation by SCF and subsequent degradation.
Degradation of APC-Cdh1 allows for the accumulation of S-Cyclin (cyclin A), promoting the initiation of DNA replication in S phase.
Preventing Premature Replication Licensing:
Before DNA replication can occur, a process called replication licensing must take place. This involves the recruitment of proteins such as ORC (Origin Recognition Complex), Cdc6, and Cdt1 to initiate the assembly of the pre-replication complex.
CDK activity needs to be regulated to prevent premature replication licensing. CDK-mediated phosphorylation of Cdt1 leads to its dissociation from chromatin, preventing the formation of the pre-replication complex.
SCF recognizes and targets phosphorylated Cdt1 for degradation by the proteasome, preventing replication licensing until the next cell cycle.
How does MPF regulate mitosis?
What is the role of APC?
The regulation of MPF, also known as cyclin B, involves several mechanisms:
1. Expression and Accumulation of Cyclin B: Cyclin B accumulates and binds to its binding partner CDK1, forming the maturation-promoting factor (MPF). MPF allows cells to bypass the G2 checkpoint and enter mitosis.
2. Activation of Cyclin-Degrading Enzyme: MPF activates the cyclin-degrading enzyme, a combination of APC (Anaphase Promoting Complex) with CDC20 and CDH1. These components play different roles but are coordinated.
3. Cyclin B Destruction: Once activated, APC-CDC20 targets cyclin B for destruction, leading to the inactivation of MPF. CDK1 is then recycled into the next cell cycle.
The activity of Cyclin B-CDK1 (MPF) is restricted to the G2/M transition. Several regulatory steps control its activation:
* Phosphorylation: Cyclin B-CDK1 complex requires phosphorylation on threonine 161 for activation and removal of an inhibitory phosphorylation on tyrosine 15. This phosphorylation balance is maintained by kinases (CAK) and the balance between inhibitory kinase Wee1 and protein phosphatase CDC25.
* Feedback Mechanisms: Positive feedback loops involve the phosphorylation of CDC25, enhancing its activity, and the inhibition of Wee1, promoting CDK1 activity.
What are the targets of MPF
Proteins involved in chromosome adhesion or cohesion
Once activated MPF targets factors that ensure chromosome adhesion.
* Cohesin complex loops around sister chromatid arms and aids the formation of the mitotic spindle
* Degradation of Cohesin enables the resolution of centromeres and segregation of sister chromosomes.
Cohesin is targeted by MPF for destruction through phosphorylation of another protein, separase.
The metaphase checkpoint ensures proper chromosome alignment. Activation of APC-CDC20 and separase leads to the degradation of securin, initiating Cohesin destruction and sister chromatid separation during anaphase.
1. Separase is a protease inhibited by direct binding of securin
2. At late metaphase APC is activated by the mitotic CDK
3. Securin is polyubiquitinated and subject to proteasomal degradation
4. Activated separase cleaves target protein Scc1, a component of the Cohesin complex
5. Sister chromatids are immediately pulled apart b mechanical forces generate by the mitotic spindle.
The degradation of Cyclin B imposes irreversibility, preventing cells from going back into mitosis until the next cell cycle.
APC-CDH1 takes over from APC-CDC20 and targets Cyclin B for degradation.
