Alternative titles; symbols
HGNC Approved Gene Symbol: MAD2L1
Cytogenetic location: 4q27 Genomic coordinates (GRCh38) : 4:120,055,623-120,066,848 (from NCBI)
Li and Benezra (1996) reviewed mitotic checkpoint control mechanisms and noted that these mechanisms check the cells preparedness to undergo division. Through these mechanisms cell cycle progression is blocked before the irreversible events associated with anaphase if either the mitotic spindle apparatus is not properly assembled or the kinetochore is not properly attached to the spindle. Mitotic arrest-deficient-2 (MAD2) is one of 6 yeast genes that are required for execution of the mitotic checkpoint. Dysfunction of MAD2 may lead to malignancy or degeneration of cells (Li and Nicklas, 1995; Li and Benezra, 1996).
Li and Benezra (1996) isolated a human homolog of MAD2 (MAD2L1) in a screen for high copy-number suppressors of thiabendazole sensitivity in yeast lacking CBF1, a component of the kinetochore. (Thiabendazole is a mitotic spindle assembly inhibitor.) The gene encodes a 205-amino acid polypeptide. DNA sequence determination revealed that the open reading frame of the human clone is 60% identical to the yeast MAD2 gene. They used antibody electroporation experiments to demonstrate that the human MAD2 gene was a necessary component of the mitotic checkpoint in HeLa cells. Through immunofluorescence studies they demonstrated that the human MAD2 protein is localized at the kinetochore after chromosome condensation but that it is no longer observed at the kinetochore in metaphase. Based on this observation they proposed that MAD2 monitors the completeness of the spindle kinetochore attachment. Li and Benezra (1996) demonstrated that a human breast tumor cell line T47D has reduced MAD2 expression and that it failed to arrest in mitosis after nocodazole treatment. They proposed that loss of MAD2 function might also lead to aberrant chromosome segregation in mammalian cells.
Chen et al. (1996) isolated a Xenopus homolog of yeast MAD2. They reported that the product of this gene plays an essential role in spindle checkpoint assembly. The protein associated with unattached kinetochores in prometaphase and nocodazole treated cells and disappeared from kinetochores at metaphase.
Using a yeast 2-hybrid analysis with the cytoplasmic tails of several a disintegrin and metalloproteinase domain (ADAM) proteins as bait, Nelson et al. (1999) found that MAD2L1 interacts strongly with TACE (ADAM17; 603639) but not with ADAM9 (602713), which interacts with MAD2L2, or with other ADAMs tested. Further binding analyses defined a 35-amino acid stretch of TACE containing a proline-rich SH3-ligand domain (PXPXXP) as the interaction site for MAD2L1.
Luo et al. (2002) showed that RNA interference-mediated suppression of MAD1 (602686) function in mammalian cells caused loss of MAD2 kinetochore localization and impairment of the spindle checkpoint. MAD1 and CDC20 (603618) contain MAD2-binding motifs that share a common consensus, and the authors identified a class of MAD2-binding peptides (MBPs) with a similar consensus. Binding of one of these ligands, MBP1, triggered an extensive rearrangement of the tertiary structure of MAD2. MAD2 also underwent a similar striking structural change upon binding to a MAD1 or CDC20 binding motif peptide. These data suggested that, upon checkpoint activation, MAD1 recruits MAD2 to unattached kinetochores and may promote binding of MAD2 to CDC20.
Using transfected HeLa cells, Habu et al. (2002) found that, at early mitosis, the majority of MAD2 formed a complex with p55CDC (CDC20). However, as the cell cycle progressed to mid-mitosis, the MAD2-p55CDC complex disassembled and the majority of MAD2 bound CMT2 (MAD2L1BP; 618136) upon completion of spindle formation. Overexpression of CMT2 in HeLa cells arrested by nocodazole revealed that formation of the MAD2-CMT2 complex resulted in abrogation of the arrest maintained by the spindle checkpoint. Inactivation of CMT2 in Hell cells caused a delay in transition from metaphase to anaphase, followed by cell death.
Hernando et al. (2004) showed that MAD2 is a direct E2F (see 189971) target and, as a consequence, is aberrantly expressed in cells with Rb (614041) pathway defects. Concordantly, Mad2 is overexpressed in several tumor types, where it correlates with high E2F activity and poor patient prognosis. Generation of Rb pathway lesions in normal and transformed cells produced aberrant Mad2 expression and mitotic defects leading to aneuploidy, such that elevated Mad2 contributed directly to these defects. Hernando et al. (2004) concluded that their results demonstrated how chromosome instability can arise as a byproduct of defects in cell cycle control that compromise the accuracy of mitosis, and suggested a new model to explain the frequent appearance of aneuploidy in human cancer.
Using an unbiased screen, Guardavaccaro et al. (2008) demonstrated that REST (600571) is an interactor with the F-box protein beta-TRCP (603482). REST is degraded by means of the ubiquitin ligase beta-TRCP during the G2 phase of the cell cycle to allow transcriptional derepression of Mad2, an essential component of the spindle assembly checkpoint. The expression in cultured cells of a stable REST mutant, which is unable to bind beta-TRCP, inhibited Mad2 expression and resulted in a phenotype analogous to that observed in Mad2 heterozygous cells. In particular, Guardavaccaro et al. (2008) observed defects that were consistent with faulty activation of the spindle checkpoint, such as shortened mitosis, premature sister-chromatid separation, chromosome bridges and missegregation in anaphase, tetraploidy, and a faster mitotic slippage in the presence of a spindle inhibitor. An indistinguishable phenotype was observed by expressing the oncogenic REST-FS mutant, which does not bind beta-TRCP. Thus, beta-TRCP-dependent degradation of REST during G2 permits the optimal activation of the spindle checkpoint, and consequently it is required for the fidelity of mitosis.
Sotillo et al. (2010) showed that induction of chromosome instability by overexpression of the mitotic checkpoint gene Mad2 in mice does not affect the regression of Kras (190070)-driven lung tumors when Kras is inhibited. However, tumors that experience transient Mad2 overexpression and consequent chromosome instability recur at markedly elevated rates. The recurrent tumors are highly aneuploid and have varied activation of proproliferative pathways. Thus, Sotillo et al. (2010) concluded that early chromosomal instability may be responsible for tumor relapse after seemingly effective anticancer treatments.
PCID2 (613713) is expressed in immature and early-stage B lymphocytes. Nakaya et al. (2010) found that knockdown of PCID2 in HeLa cells by small interfering RNA (siRNA) induced abnormalities in cell cycling, with an increase of apoptotic and hyperploid cells. Knockdown of PCID2 also suppressed MAD2 expression, but not expression of other cell cycle checkpoint proteins, such as MAD1 (MAD1L1; 602686) and BUBR1 (BUB1B; 602860), or cell cycle-associated proteins, such as cyclin A (CCNA2; 123835), cyclin B1 (CCNB1; 123836), and CDK1 (116940). In situ hybridization showed that MAD2 expression was markedly decreased in the cytoplasm compared with the nucleus after PCID2 siRNA treatment, suggesting that PCID2 regulates MAD2 mRNA metabolism. Nakaya et al. (2010) concluded that regulation of MAD2 by PCID2 may be a key event at the mitotic checkpoint in B cells.
Using transfected HeLa cells, Hagan et al. (2011) showed that localization of p31(COMET) (MAD2L1BP) to unattached kinetochores was dependent on MAD2. Overexpression of p31(COMET) or depletion of MAD2 caused premature anaphase, whereas p31(COMET) depletion or MAD2 overexpression arrested cells in mitosis. Depleting p31(COMET) did not interfere with chromosome-microtubule attachment, nor did it prevent normal dissociation of checkpoint proteins from kinetochores as mitosis proceeded. Further analysis showed that cell arrest following depletion of p31(COMET) required MAD2. Moreover, p31(COMET) acted downstream of the timer function of MAD2 but upstream of kinetochore function, and it trafficked on and off kinetochores by rapidly associating and dissociating from a MAD1-MAD2 scaffold of kinetochores.
Crystal Structure
Yang et al. (2007) determined the crystal structure of the human MAD2-p31(COMET) complex at 2.3-angstrom resolution. The structure of p31(COMET) contained 3 central alpha helices sandwiched by a 7-stranded beta sheet on one side and a short helix on the other. The overall folding topology of p31(COMET) was similar to that of ligand-bound MAD2, and p31(COMET) bound at the dimerization interface of MAD2. The C-terminal segment of MAD2 underwent rearrangement and provided the structural basis for binding specificity of p31(COMET) to different MAD2 conformers. Mutagenesis studies validated the findings from the crystal structure and showed that mutations of p31(COMET) that disrupted MAD2 binding also disrupted the ability of p31(COMET) to overcome spindle checkpoint-dependent mitotic arrest. The authors noted that, in mitosis, a MAD1 (MAD1L1; 602686)-MAD2 core complex recruits cytosolic MAD2 to kinetochores through MAD2 dimerization and converts MAD2 to a conformer amenable to p55CDC binding. By overlaying MAD2 molecules in the MAD1-MAD2 and MAD2-p31(COMET) structures, Yang et al. (2007) constructed a structural model of the MAD1-MAD2-p31(COMET) ternary complex. They found that the MAD2-p31(COMET) binding mode in the MAD2-p31(COMET) complex was compatible with formation of the MAD1-MAD2-p31(COMET) ternary complex, and as a result, p31(COMET) blocked MAD1-assisted recruitment of cytosolic MAD2 by acting as a structural mimic of MAD2.
Cryoelectron Microscopy
In combination with p31(comet) (MAD2L1BP), a spindle assembly checkpoint (SAC) antagonist, TRIP13 (604507) remodels active closed MAD2 (C-MAD2) into inactive open MAD2 (O-MAD2). Alfieri et al. (2018) determined cryoelectron microscopy structures of the TRIP13-p31(comet)-C-MAD2-CDC20 complex, which revealed that p31(comet) recruits C-MAD2 to a defined site on the TRIP13 hexameric ring, positioning the N terminus of C-MAD2 to insert into the axial pore of TRIP13 and distorting the TRIP13 ring to initiate remodeling. Molecular modeling suggested that by gripping C-MAD2 within its axial pore, TRIP13 couples sequential ATP-driven translocation of its hexameric ring along MAD2 to push upwards on, and simultaneously rotate, the globular domains of the p31(comet)-C-MAD2 complex. This unwinds a region of the alpha-A helix of C-MAD2 that is required to stabilize the C-MAD2 beta-sheet, thus destabilizing C-MAD2 in favor of O-MAD2 and dissociating MAD2 from p31(comet). Alfieri et al. (2018) concluded that their study provided insights into how specific substrates are recruited to AAA+ ATPases through adaptor proteins and suggested a model of how translocation through the axial pore of AAA+ ATPases is coupled to protein remodeling.
Xu et al. (1997) mapped the MAD2L1 gene to 5q23-q31 by fluorescence in situ hybridization (FISH). However, Krishnan et al. (1998) mapped the gene to 4q27, using a combination of somatic cell hybrid analysis, radiation hybrid mapping, and FISH. By analysis of a radiation hybrid panel, Cahill et al. (1999) confirmed that the MAD2L1 gene maps to 4q27. They mapped a related gene, MAD2L2 (604094), to 1p36, and a MAD2 pseudogene to 14q21-q23.
The initiation of chromosome segregation at anaphase is linked by the spindle assembly checkpoint to the completion of chromosome-microtubule attachment during metaphase. To determine the function of the Mad2 protein during normal cell division, Dobles et al. (2000) knocked out the Mad2 gene in mice. They found that embryonic cells lacking Mad2 at embryonic day 5.5, like mad2 yeast, grew normally but were unable to arrest in response to spindle disruption. At embryonic day 6.5, the cells of the epiblast began rapid cell division, and the absence of a checkpoint resulted in widespread chromosome missegregation and apoptosis. In contrast, the postmitotic trophoblast giant cells survived without Mad2. Thus, the spindle assembly checkpoint is required for accurate chromosome segregation in mitotic mouse cells and for embryonic viability, even in the absence of spindle damage.
Shonn et al. (2000) characterized the spindle checkpoint in meiosis of S. cerevisiae by comparing wildtype and mad2-deficient yeast. In the absence of the checkpoint, the frequency of meiosis I missegregation increased with increasing chromosome length, reaching 19% for the longest chromosome. Meiosis I nondisjunction in spindle checkpoint mutants could be prevented by delaying the onset of anaphase. In a recombinant-defective mutant, the checkpoint delayed the biochemical events of anaphase I, suggesting that chromosomes that are attached to microtubules but are not under tension can activate the spindle checkpoint. Spindle checkpoint mutants reduced the accuracy of chromosome segregation in meiosis I much more than that in meiosis II, suggesting that checkpoint defects may contribute to Down syndrome (190685). Shonn et al. (2000) showed that the budding yeast spindle checkpoint, which is largely dispensable in wildtype mitosis, plays a critical role in meiotic chromosome segregation. They suggested that the difference may reflect the different chromosome linkages in mitosis and meiosis I. In mitosis, sister chromatid cohesion forces sister kinetochores to face opposite spindle poles. In meiosis I, homologs are linked at sites of recombination that can be far from the kinetochores, creating a floppy linkage. If the nearest recombination event is further from the centromere on long chromosomes, this idea may explain why long chromosomes preferentially nondisjoin in checkpoint-defective cells.
Michel et al. (2001) reported that deletion of one MAD2 allele results in a defective mitotic checkpoint in both human cancer cells and murine primary embryonic fibroblasts. Checkpoint-defective cells show premature sister chromatid separation in the presence of spindle inhibitors and an elevated rate of chromosome missegregation events in the absence of these agents. Furthermore, Mad2 +/- mice develop lung tumors at high rates after long latencies, implicating defects in the mitotic checkpoint in tumorigenesis.
By propagating mouse embryonic fibroblasts deficient in both Mad2 and p53 (TP53; 191170), Burds et al. (2005) showed that lack of spindle checkpoint is nonlethal when combined with p53 inactivation. Mouse blastocysts deficient in both Mad2 and p53 were also viable in culture. However, the simultaneous loss of spindle checkpoint and p53 function created chromosome instability.
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