7th Feb - Mitotic Spindle Assembly Checkpoint Flashcards
What are the cyclin/cdk complexes required for progression from metaphase to anaphase?
Cyclin A- CDK1
Cyclin B- CDK1
What are the kinases that regulate mitotic entry?
Plk1 Mps1 BubR1 Cdk1 Haspin NIMA-related kinases Auora B Aurora A
What is the function of Plk1?
Required for centrosome function, activation of Cdc25, cohesion phosphorylation, APC/C activation and cytokinesis
What is the function of Aurora A kinase?
Required for mitotic entry and spindle assembly
What is the function of Aurora B kinase?
Required for chromatin condensation and correction of improper MT-kinetochore attachments and mitotic exit
What is the function of NIMA-related Kinases?
Required for structural aspects of mitotic entry, centrosome function and spindle assembly
What is the function of Haspin?
Required for recruitment of Aurora B to chromatin
Outline the mechanism of Cdk1 activation
Aurora A –> Plk-1 –> Activates Cdc25 –> Cdk1 activation
POSITIVE FEEDBACK - Cdk1 phosphorylates Cdc25, activating it
Where does Cdk1 activation first occur?
At the centrosome which acts as a solid-phase signaling platform, thus increases speed and efficiency of signalling
Outline how mitotic entry is biochemically controlled
- Synthesis of cyclins A and B generates inactive Cdk1-cyclin complexes
- Cdk1 phosphorylated on T161 by CAK
- AuroraA activates Plk1 which phosphorylates Cdc25
- Cdc25 dephosphorylates T14/Y15 and partially activates Cdk1
- Partially activated Cdk1 further phosphorylates Cdc25, which further activates Cdk1
How is Cdk1 inhibited?
By phosphorylation on T14 and Y15 by Wee1
What are the two main criteria that must be filled before anaphase?
Full attachment of all chromosomes
Cdk1 must be switched off
Which two proteins must be destroyed before metaphase to anaphase transition?
Cyclin B
Securin
What is the evidence that cyclin B is destroyed before anaphase?
Mutant non-degradable cyclin B blocks mitotic exit
When Cdk1 is switched off cells revert to interphase state as competing phosphatases remove all the phosphates added by Cdk1
What is the evidence that securin is destroyed before anaphase?
Non-degradable securin prevents sister chromatid seperation
How is securin degraded?
By the cysteine protease seperase
How are both securin and cyclin B destroyed?
Targeted for proteasome destruction through the anaphase promoting complex (APC)/C Ub ligase
What is the APC/C?
Anaphase promoting complex which is a mitotic E3 Ub ligase, catalysing transfer of Ub from E2 to substrate.
It contains 15 core subunits and 2 adaptors: Cdc20 or Cdh1
What is the main function of the spindle asembly checkpoint?
It keeps APC/C off until all chromosomes are properly attached thus preventing anaphase entry
What are the key proteins involved in the spindle assembly checkpoint?
Mad1 Mad2 Bub1 BubR1/Mad3 Bub3 Mps1 Aurora B
Outline the spindle assembly checkpoint pathway
- MAD1 directly recognises the unrecruited kinetochore
- MAD1 recruits MAD2 and activates it
- MAD2 forms a complex with Cdc20 preventing Cdc20 from activating APC/C
OR
- BubR1 acts as a substrate binding to Cdc20 and blocking substrate recruitment through competitive inhibition
What is the function of Aurora B in the spindle assembly checkpoint?
Phosphorylates syntelic atttachments, causing the kinetochore to be released
What type of incorrect chromosome attachment is poorly recognised by the spindle assembly checkpoint?
Merotelic attachment
How was spindle assembly checkpoint signalling discovered?
Through laser ablation and micromanipulation
What is the KMN network?
Complex of KNL1, Mis12 and Ndc 80 which forms around kinetochores as the cells enter mitosis
What is the RZZ complex?
Zw10, Rod and Zuilch
What causes mosaic variegated aneuploidy?
A germline mutation in BubR1
How is Mad2 silenced in breast and other cancers?
Hypermethylated promoter
Other epigenetic mechanisms
What does Mad2 haploinsufficiency cause?
CIN
Give an example of a drug that interferes with microtubule dynamics
Taxanes e.g. Taxol
Vinca Alkaloids e.g. Vincristine, Vinblastine
How do Taxanes work?
They stabilize microtubules and thus prevent normal mitotic spindle dynamics and activate the spindle assembly checkpoint
How do vinca alkaloids work?
They depolymerise microtubules preventing spindle assembly
What types of cancer are anti-microtubule therapeutics commonly used to target?
Breast, ovarian, lung and haemotological cancers
Give an example of a side effect caused by an anti-microtubule therapeutic
Neurotoxicity (loss of sensation in the peripheral tissues) due to neuronal trafficking interference
Give an example of a TF that activates the cyclin B1 promoter
NF-Y
Fox 1
B-Myb
What is hysteresis?
the phenomenon in which the value of a physical property lags behind changes in the effect causing it, as for instance when magnetic induction lags behind the magnetizing force.
What is hysteresis in the SAC?
The threshold for activation is much higher than that for inactivation of anaphase
What possible targets have recently emerged for anti-mitotic therapy?
Kinesin spindle protein
Centromeric protein E
What is KSP?
Kinesin spindle protein which is required to establish mitotic-spindle bipolarity
Give an example of a KSP inhibitor?
Monatol
Isperinib
What is CENPE?
Centromeric protein E which is required for accurate chromosome congression at metaphase
