Abstract
In eukaryotes, the mitotic spindle assembly checkpoint provides a monitor for the fidelity of chromosomal segregation. In this context, the mitotic arrest deficiency protein 2 (MAD2) censors chromosomal mis-segregation by monitoring microtubule attachment/tension, a role that requires its attachment to kinetochores. Studies in yeast have shown that binding of MAD1 to MAD2 is important for the checkpoint function of the latter. The interactions between human MAD1 (hsMAD1) and human MAD2 (hsMAD2) have, however, remained poorly characterized. Here we report that two leucine zipper domains (amino acids 501-522 and 557-571) in hsMAD1 are required for its contact with hsMAD2. Interestingly, in several cancer cell lines, we noted the frequent presence of a coding single nucleotide Arg to His polymorphism at codon 558 located within the second leucine zipper of hsMAD1. We found that hsMAD1H558 is less proficient than hsMAD1R558 in binding hsMAD2 and in enforcing mitotic arrest. We also document a first example of loss-of-heterozygosity for a spindle checkpoint gene (at the hsMAD1 558 locus) in a human breast cancer. Based on our findings, it is possible that hsMAD1H558 could be an at-risk polymorphism that contributes to attenuated spindle checkpoint function in human cells.
Highlights
From the Molecular Virology Section, Laboratory of Molecular Microbiology, NIAID, National Institutes of Health, Bethesda, Maryland 20892-0460
We report that two leucine zipper domains in hsMAD1 are required for its contact with hsMAD2
It is possible that hsMAD1H558 could be an at-risk polymorphism that contributes to attenuated spindle checkpoint function in human cells
Summary
Vol 277, No 34, Issue of August 23, pp. 31005–31013, 2002 Printed in U.S.A. A POLYMORPHIC CHANGE IN AN hsMAD1 LEUCINE ZIPPER AFFECTS MAD1-MAD2 INTERACTION AND SPINDLE CHECKPOINT FUNCTION*. The high frequency of aneuploidy in cancers has prompted the thought that loss-of-function mutations in human BUB/ MAD proteins might occur rather commonly. Binding of the spindle effector protein, hsMAD2, to kinetochores is a critical step in mitotic checkpoint function. Earlier it was thought that oligomerized hsMAD2 was wholly sufficient to produce cell cycle arrest; recent data [17] suggest that binding of hsMAD2 to hsMAD1 rather than oligomerization is the critical step required for spindle checkpoint function. We show that binding of full-length MAD2 by full MAD1 requires two leucine zipper domains (amino acids 501–522 and 557–571) in hsMAD1 One of these two leucine zipper domains is disrupted by an Arg-558 to His-coding single nucleotide polymorphism in mad allele frequently found in many human cancer cell lines
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