MAD2
Encyclopedia
MAD2 is an essential spindle checkpoint
protein
. The spindle checkpoint system is a regulatory system that restrains progression through the metaphase
-to-anaphase
transition. The Mad2 gene was first identified in the yeast S. cerevisiae
in a screen for genes which when mutated would confer sensitivity to microtubule
poisons. The human orthologues of Mad2 (MAD2L1
and MAD2L2
) were first cloned in a search for human cDNAs that would rescue the microtubule poison-sensitivity of a yeast strain in which a kinetochore
binding protein was missing. The protein was shown to be present at unattached kinetochores and antibody inhibition studies proved it was essential to execute a block in the metaphase-to-anaphase transition in response to the microtubule poison nocodazole
. Subsequent cloning of the Xenopus laevis orthologue, facilitated by the sharing of the human sequence, allowed for the characterization of the mitotic checkpoint in egg extracts.
. The cell cycle
surveillance mechanism that prevents sister-chromatid separation and transition into anaphase is called the spindle checkpoint. As a safeguard against chromosome segregation errors, the spindle assembly checkpoint (SAC) delays anaphase until all sister chromatid pairs have become bipolarly attached.
Once microtubules attach to kinetochores, chromosomes are aligned on the metaphase plate, and proper bi-orientation has been achieved, the SAC stopping mechanisms are removed. Entrance into anaphase is mediated by APCCdc20 activation. APCCdc20 is a ubiquitin-protein ligase that tags the protein, securin, for destruction. Securin destruction liberates and activates its bound protease partner, separase. Separase bound to securin remains inhibited; however, when inhibition is relieved, activated separase cleaves the cohesin complex which links the sister chromatids together.
Without Cdc20, the anaphase-promoting complex (APC) cannot become activated and anaphase is not triggered. Mad2 was shown to inhibit the activity of the APC by direct physical interaction in a ternary complex with Cdc20. Kinetochores that remain unattached to microtubules catalyze the sequestration of Cdc20 by Mad2. In fact, when metaphase mammalian cells are treated with the spindle-depolymerizing agent nocodazole, Mad2 proteins become localized at the kinetochores of all sister-chromatid pairs.
or Cdc20
and, thus, can only bind one of the two proteins at a time.
A model, which accounts for Mad2 adopting a conformation capable of binding Cdc20, relies upon the formation of Mad1-Mad2 core complex first. In this model, external Open Mad2 is recruited to the Mad1:Mad2 template. This Mad2:Mad2 interaction is thought to enable a conformational change which allows the peripherally bound Open Mad2 to interact with Cdc20. Cdc20:Mad2 then dissociates and Mad1:Mad2 is enabled to bind a free cytosolic Mad2 again.
It is speculated that once formed, Cdc20:Mad2 complexes can amplify the anaphase wait signal by stimulating further conversion of cytosolic Open Mad2 and free Cdc20 into more Cdc20:Closed Mad2 complexes. This diffusible signal propagation away from the kinetochore complexes could account for how vacancy of just one tiny kinetochore site can completely shut down the metaphase-to-anaphase transition.
, BubR1
, and Bub3
. BubR1 and Bub3 can also form complexes with Cdc20, but it remains to be seen if these proteins facilitate Cdc20 binding to Open Mad2.
It is also quite unclear how p31comet antagonizes the checkpoint and promotes the dissociation of Mad2-Cdc20. De Antoni et al. in conjunction with the “Mad2 Template” suggest that p31comet competes with Open Mad2 for binding to Closed Mad2:Mad1. Testing is underway in order to illuminate how p31comet may silence the spindle checkpoint.
Spindle checkpoint
In order to preserve one cell's identity and its proper functioning, it is necessary to maintain constant the appropriate number of chromosomes after each cell division...
protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
. The spindle checkpoint system is a regulatory system that restrains progression through the metaphase
Metaphase
Metaphase, from the ancient Greek μετά and φάσις , is a stage of mitosis in the eukaryotic cell cycle in which condensed & highly coiled chromosomes, carrying genetic information, align in the middle of the cell before being separated into each of the two daughter cells...
-to-anaphase
Anaphase
Anaphase, from the ancient Greek ἀνά and φάσις , is the stage of mitosis or meiosis when chromosomes move to opposite poles of the cell....
transition. The Mad2 gene was first identified in the yeast S. cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae is a species of yeast. It is perhaps the most useful yeast, having been instrumental to baking and brewing since ancient times. It is believed that it was originally isolated from the skin of grapes...
in a screen for genes which when mutated would confer sensitivity to microtubule
Microtubule
Microtubules are a component of the cytoskeleton. These rope-like polymers of tubulin can grow as long as 25 micrometers and are highly dynamic. The outer diameter of microtubule is about 25 nm. Microtubules are important for maintaining cell structure, providing platforms for intracellular...
poisons. The human orthologues of Mad2 (MAD2L1
MAD2L1
Mitotic spindle assembly checkpoint protein MAD2A is a protein that in humans is encoded by the MAD2L1 gene.-Interactions:MAD2L1 has been shown to interact with MAD2L2, CDC20, BUB1B, Estrogen receptor beta, ADAM17, UBD, CDC27 and Mad1....
and MAD2L2
MAD2L2
Mitotic spindle assembly checkpoint protein MAD2B is a protein that in humans is encoded by the MAD2L2 gene.-Interactions:MAD2L2 has been shown to interact with ADAM9, REV1, MAD2L1 and REV3L.-Further reading:...
) were first cloned in a search for human cDNAs that would rescue the microtubule poison-sensitivity of a yeast strain in which a kinetochore
Kinetochore
The kinetochore is the protein structure on chromatids where the spindle fibers attach during cell division to pull sister chromatids apart....
binding protein was missing. The protein was shown to be present at unattached kinetochores and antibody inhibition studies proved it was essential to execute a block in the metaphase-to-anaphase transition in response to the microtubule poison nocodazole
Nocodazole
Nocodazole is an anti-neoplastic agent which exerts its effect in cells by interfering with the polymerization of microtubules. Microtubules are one type of fibre which constitutes the cytoskeleton, and the dynamic microtubule network has several important roles in the cell, including vesicular...
. Subsequent cloning of the Xenopus laevis orthologue, facilitated by the sharing of the human sequence, allowed for the characterization of the mitotic checkpoint in egg extracts.
Metaphase-to-anaphase transition
Progression from metaphase to anaphase is marked by sister chromatid separationSister chromatid exchange
Sister chromatid exchange is the exchange of genetic material between two identical sister chromatids.It was first discovered by using the Giemsa staining method on one chromatid belonging to the sister chromatid complex before anaphase in mitosis...
. The cell cycle
Cell cycle
The cell cycle, or cell-division cycle, is the series of events that takes place in a cell leading to its division and duplication . In cells without a nucleus , the cell cycle occurs via a process termed binary fission...
surveillance mechanism that prevents sister-chromatid separation and transition into anaphase is called the spindle checkpoint. As a safeguard against chromosome segregation errors, the spindle assembly checkpoint (SAC) delays anaphase until all sister chromatid pairs have become bipolarly attached.
Once microtubules attach to kinetochores, chromosomes are aligned on the metaphase plate, and proper bi-orientation has been achieved, the SAC stopping mechanisms are removed. Entrance into anaphase is mediated by APCCdc20 activation. APCCdc20 is a ubiquitin-protein ligase that tags the protein, securin, for destruction. Securin destruction liberates and activates its bound protease partner, separase. Separase bound to securin remains inhibited; however, when inhibition is relieved, activated separase cleaves the cohesin complex which links the sister chromatids together.
Without Cdc20, the anaphase-promoting complex (APC) cannot become activated and anaphase is not triggered. Mad2 was shown to inhibit the activity of the APC by direct physical interaction in a ternary complex with Cdc20. Kinetochores that remain unattached to microtubules catalyze the sequestration of Cdc20 by Mad2. In fact, when metaphase mammalian cells are treated with the spindle-depolymerizing agent nocodazole, Mad2 proteins become localized at the kinetochores of all sister-chromatid pairs.
Mad2 conformers
Mad2 is capable of forming multimers and adopts at least two structural conformations. Open Mad2 differs from closed Mad2 in the positioning of the 50 residue C-terminal segment. This “safety belt” is held tightly against the right side of the protein in the open conformation. Upon loosening, the safety belt can be re-positioned around a binding partner. In the closed conformation, the safety belt wraps around the bound ligand and interacts with a different region of Mad2. Binding partners of Mad2 include either Cdc20 or Mad1. Mad1 and Cdc20 bind Mad2 in an identical fashion. Mad2 uses the same site to bind either Mad1Mad1
Mitotic spindle assembly checkpoint protein MAD1 is a protein that in humans is encoded by the MAD1L1 gene.MAD1L1 is also known as Human Accelerated Region 3. It may therefore have played a key role in differentiating Humans from Apes.-Interactions:...
or Cdc20
CDC20
The cell-division cycle protein 20 is an essential regulator of cell division that is encoded by the CDC20 gene in humans. To the best of current knowledge its most important function is to activate the anaphase promoting complex , a large 11-13 subunit complex that initiates chromatid separation...
and, thus, can only bind one of the two proteins at a time.
Mad2 activation in the spindle assembly checkpoint
Since unattached kinetchores establish and maintain the SAC, Mad2 is recruited to prevent these misaligned sister chromatids from separating. When the checkpoint/braking process is activated, Mad2 binds Mad1 to form Closed-Mad2-Mad1 complexes. Given that Mad1:Mad2 is a stable complex and Cdc20 and Mad1 bind Mad 2 in the very same binding site, it is highly unlikely that Closed Mad2 releases Mad1 to bind Cdc20.A model, which accounts for Mad2 adopting a conformation capable of binding Cdc20, relies upon the formation of Mad1-Mad2 core complex first. In this model, external Open Mad2 is recruited to the Mad1:Mad2 template. This Mad2:Mad2 interaction is thought to enable a conformational change which allows the peripherally bound Open Mad2 to interact with Cdc20. Cdc20:Mad2 then dissociates and Mad1:Mad2 is enabled to bind a free cytosolic Mad2 again.
It is speculated that once formed, Cdc20:Mad2 complexes can amplify the anaphase wait signal by stimulating further conversion of cytosolic Open Mad2 and free Cdc20 into more Cdc20:Closed Mad2 complexes. This diffusible signal propagation away from the kinetochore complexes could account for how vacancy of just one tiny kinetochore site can completely shut down the metaphase-to-anaphase transition.
Future work
Much remains to be explained about spindle checkpoint signaling and the contribution of other spindle checkpoint assembly proteins such as Bub1BUB1
Mitotic checkpoint serine/threonine-protein kinase BUB1 also known as BUB1 is an enzyme that in humans is encoded by the BUB1 gene....
, BubR1
BUB1B
Mitotic checkpoint serine/threonine-protein kinase BUB1 beta is an enzyme that in humans is encoded by the BUB1B gene.-Interactions:BUB1B has been shown to interact with AP2B1, HDAC1, BUB3, MAD2L1, Gamma-synuclein, BRCA2 and CDC20.-Further reading:...
, and Bub3
BUB3
Mitotic checkpoint protein BUB3 is a protein that in humans is encoded by the BUB3 gene.Bub3 is a protein involved with the regulation of the Spindle Assembly Checkpoint ; though BUB3 is non-essential in yeast, it is essential in higher eukaryotes...
. BubR1 and Bub3 can also form complexes with Cdc20, but it remains to be seen if these proteins facilitate Cdc20 binding to Open Mad2.
It is also quite unclear how p31comet antagonizes the checkpoint and promotes the dissociation of Mad2-Cdc20. De Antoni et al. in conjunction with the “Mad2 Template” suggest that p31comet competes with Open Mad2 for binding to Closed Mad2:Mad1. Testing is underway in order to illuminate how p31comet may silence the spindle checkpoint.