The checkpoint protein MAD2 and the mitotic regulator CDC20 form a ternary complex with the anaphase-promoting complex to control anaphase initiation.
The spindle assembly checkpoint mechanism delays anaphase initiation until all chromosomes are aligned at the metaphase plate. Activation of the anaphase-promoting complex (APC) by binding of CDC20 and CDH1 is required for exit from mitosis, and APC has been implicated as a target for the checkpoint intervention. We show that the human checkpoint protein hMAD2 prevents activation of APC by forming a hMAD2-CDC20-APC complex. When injected into Xenopus embryos, hMAD2 arrests cells at mitosis with an inactive APC. The recombinant hMAD2 protein exists in two-folded states: a tetramer and a monomer. Both the tetramer and the monomer bind to CDC20, but only the tetramer inhibits activation of APC and blocks cell cycle progression. Thus, hMAD2 binding is not sufficient for inhibition, and a change in hMAD2 structure may play a role in transducing the checkpoint signal. There are at least three different forms of mitotic APC that can be detected in vivo: an inactive hMAD2-CDC20-APC ternary complex present at metaphase, a CDC20-APC binary complex active in degrading specific substrates at anaphase, and a CDH1-APC complex active later in mitosis and in G1. We conclude that the checkpoint-mediated cell cycle arrest involves hMAD2 receiving an upstream signal to inhibit activation of APC.
Fang
G
G
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Yu
H
H
Kirschner
M W
MW
D000705:Anaphase / Q000187:drug effects / Q000502:physiology*; D064173:Anaphase-Promoting Complex-Cyclosome; D000818:Animals; D001704:Biopolymers / Q000494:pharmacology; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism*; D002474:Cell-Free System / Q000187:drug effects / Q000502:physiology; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D004625:Embryo, Nonmammalian / Q000187:drug effects / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D008025:Ligases / Q000737:chemistry / Q000378:metabolism*; D008938:Mitosis / Q000502:physiology; D015394:Molecular Structure; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins; D051899:Smad2 Protein; D008941:Spindle Apparatus / Q000502:physiology; D013329:Structure-Activity Relationship; D015534:Trans-Activators; D043743:Ubiquitin-Protein Ligase Complexes; D044767:Ubiquitin-Protein Ligases; D014981:Xenopus; D029867:Xenopus Proteins
D001704:Biopolymers; C577495:CDC20 protein, S cerevisiae; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; C497782:SMAD2 protein, human; D029701:Saccharomyces cerevisiae Proteins; D051899:Smad2 Protein; C497785:Smad2 protein, Xenopus; D015534:Trans-Activators; D029867:Xenopus Proteins; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D044767:Ubiquitin-Protein Ligases; D008025:Ligases