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Alternative titles; symbolsp31(COMET)CAUGHT BY MAD2; CMT2HGNC Approved Gene Symbol: MAD2L1BPCytogenetic location: 6p21.1 Genomic coordinates (GRCh38): 6:43,6...

Alternative titles; symbols

  • p31(COMET)

HGNC Approved Gene Symbol: MAD2L1BP

Cytogenetic location: 6p21.1 Genomic coordinates (GRCh38): 6:43,629,539-43,640,940 (from NCBI)

▼ Description
MAD2L1BP turns off the mitotic checkpoint and allows cells to exit mitosis by blocking recruitment of cytosolic MAD2 (601467) and promoting ATP-dependent disassembly of the mitotic checkpoint complex (MCC) (Teichner et al., 2011, Eytan et al., 2014).

▼ Cloning and Expression
By yeast 2-hybrid screening of a HeLa cell cDNA library using human MAD2 as bait, Habu et al. (2002) cloned MAD2L2BP, which they called CMT2. The deduced 274-amino acid CMT2 protein has a calculated molecular mass of 31 kD. Western blot analysis of transfected HeLa cells detected a 34-kD CMT2 protein. Immunofluorescence analysis showed that CMT2 was unevenly distributed throughout the nucleoplasm in HeLa cells. A fraction of CMT2 concentrated as speckles, and CMT2 speckles increased as mitosis progressed. From metaphase to anaphase, CMT2 concentrated on the spindle. After completion of chromosome segregation, the majority of CMT2 remained in the midzone.

Using live-cell imaging in transfected HeLa and PtK2 cells, Hagan et al. (2011) found that MAD2L2BP, which they called p31(COMET), was expressed at high levels on unattached kinetochores in prometaphase, at significantly lower levels as kinetochores became microtubule bound during metaphase, and in the cytosol at all cell-cycle stages.

▼ Mapping
Gross (2018) mapped the MAD2L1BP gene to chromosome 6p21.1 based on an alignment of the MAD2L1BP sequence (GenBank BC002904) with the genomic sequence (GRCh38).

▼ Gene Function
Using transfected HeLa cells, Habu et al. (2002) found that, at early mitosis, the majority of MAD2 formed a complex with p55CDC (CDC20; 603618). However, as the cell cycle progressed to mid-mitosis, the MAD2-p55CDC complex disassembled and the majority of MAD2 bound CMT2 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.

By analyzing extracts from nocodazole-arrested cells, Teichner et al. (2011) found that p31(COMET) bound to MAD2 in the MCC and triggered an alteration in MCC structure that caused dissociation of p55CDC from BUBR1 (BUB1B; 602860), accelerated release of the anaphase-promoting complex/cyclosome (APC/C) from checkpoint inhibition, and stimulated MCC disassembly. Further analysis showed that p31(COMET)-directed MCC disassembly required hydrolysis of ATP. Size-exclusion chromatography and immunoprecipitation analysis demonstrated that the combined action of p31(COMET) and ATP released p55CDC and MAD2 from the MCC as a subcomplex.

Using transfected HeLa cells, Hagan et al. (2011) showed that localization of p31(COMET) 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.

By database analysis, Date et al. (2013) found that p31(COMET) expression was upregulated in tumors originating from multiple tissues, including breast and lung, where MAD2 upregulation promotes tumorigenesis. Using an in vitro model of human mammary epithelial cell immortalization and transformation, the authors showed that the increased p31(COMET) expression was not due to increased proliferation of tumor cells, but that p31(COMET) and MAD2 were coordinately upregulated. Database analysis and exogenic expression of E2F transcription factors (see 189971) in HCT116 cells showed that E2F transcription factors bound to the p31(COMET) promoter and increased p31(COMET) expression. Consistent with this finding, depletion of the RB tumor suppressor (RB1; 614041) in 3T3 cells resulted in higher levels of p31(COMET) expression, confirming that p31(COMET) expression is regulated by the RB-E2F pathway. Targeting of the p31(COMET) gene was cell cycle regulated, and balanced expression of p31(COMET) and MAD2 was required for cellular proliferation.

Eytan et al. (2014) identified the AAA-ATPase TRIP13 (604507) as a factor that promoted ATP- and p31(COMET)-dependent disassembly of the p55CDC-MAD2 subcomplex in HeLa cell extracts. Using recombinant proteins, they demonstrated that p31(COMET) and TRIP13 acted together to dissociate the p55CDC-MAD2 subcomplex, disassemble the MCC, release the APC/C from checkpoint inhibition, and inactivate the mitotic checkpoint.

▼ Biochemical Features
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.

In combination with p31(comet), a spindle assembly checkpoint (SAC) antagonist, TRIP13 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.

Tags: 6p21.1