title: RED, a spindle pole-associated protein, is required for kinetochore localization of MAD1, mitotic progression, and activation of the spindle assembly checkpoint.
abstract: The spindle assembly checkpoint (SAC) is essential for ensuring the proper attachment of kinetochores to the spindle and, thus, the precise separation of paired sister chromatids during mitosis. The SAC proteins are recruited to the unattached kinetochores for activation of the SAC in prometaphase. However, it has been less studied whether activation of the SAC also requires the proteins that do not localize to the kinetochores. Here, we show that the nuclear protein RED, also called IK, a down-regulator of human leukocyte antigen (HLA) II, interacts with the human SAC protein MAD1. Two RED-interacting regions identified in MAD1 are from amino acid residues 301-340 and 439-480, designated as MAD1(301-340) and MAD1(439-480), respectively. Our observations reveal that RED is a spindle pole-associated protein that colocalizes with MAD1 at the spindle poles in metaphase and anaphase. Depletion of RED can cause a shorter mitotic timing, a failure in the kinetochore localization of MAD1 in prometaphase, and a defect in the SAC. Furthermore, the RED-interacting peptides MAD1(301-340) and MAD1(439-480), fused to enhanced green fluorescence protein, can colocalize with RED at the spindle poles in prometaphase, and their expression can abrogate the SAC. Taken together, we conclude that RED is required for kinetochore localization of MAD1, mitotic progression, and activation of the SAC.
authors.affiliation: Institute of Medical Sciences, Tzu-Chi University, Hualien 97004, Taiwan.
authors.forename: Pei-Chi
authors.initials: PC
authors.lastname: Yeh
authors.forename: Chang-Ching
authors.initials: CC
authors.lastname: Yeh
authors.forename: Yi-Cheng
authors.initials: YC
authors.lastname: Chen
authors.forename: Yue-Li
authors.initials: YL
authors.lastname: Juang
mesh_terms: D059447:Cell Cycle Checkpoints; D018797:Cell Cycle Proteins / Q000378:metabolism*; D016207:Cytokines / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D055785:Gene Knockdown Techniques; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D018386:Kinetochores / Q000378:metabolism*; D059566:M Phase Cell Cycle Checkpoints; D008938:Mitosis; D009687:Nuclear Proteins / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D021381:Protein Transport; D034622:RNA Interference; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D059010:Single-Cell Analysis; D059008:Time-Lapse Imaging; D020798:Two-Hybrid System Techniques
chemical_list: D018797:Cell Cycle Proteins; D016207:Cytokines; C090444:IK protein, human; C501384:MAD1L1 protein, human; D009687:Nuclear Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins