Phosphorylation of the spindle checkpoint protein Mad2 regulates its conformational transition.
Regulated conformational changes of proteins are critical for cellular signal transduction. The spindle checkpoint protein Mad2 is an unusual protein with two native folds: the latent open conformer (O-Mad2) and the activated closed conformer (C-Mad2). During mitosis, cytosolic O-Mad2 binds to the Mad1-Mad2 core complex at unattached kinetochores and undergoes conformational activation to become C-Mad2. C-Mad2 binds to and inhibits Cdc20, an activator of APC/C, to prevent precocious anaphase onset. Here, we show that the conformational transition of Mad2 is regulated by phosphorylation of S195 in its C-terminal region. The phospho-mimicking Mad2(S195D) mutant and the phospho-S195 Mad2 protein obtained using intein-mediated semisynthesis do not form C-Mad2 on their own. Mad2(S195D) fails to bind to Cdc20, a low-affinity ligand, but still binds to high-affinity ligands, such as Mad1 and MBP1, forming ligand-bound C-Mad2. Overexpression of Mad2(S195D) in human cells causes checkpoint defects. Our results indicate that Mad2 phosphorylation inhibits its function through differentially regulating its binding to Mad1 and Cdc20 and establish that the conformational change of Mad2 is regulated by posttranslational mechanisms.
Department of Pharmacology, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, 6001 Forest Park Road, Dallas, TX 75390-9041, USA.
Soonjoung
S
Kim
Hongbin
H
Sun
Haydn L
HL
Ball
Katja
K
Wassmann
Xuelian
X
Luo
Hongtao
H
Yu
D000595:Amino Acid Sequence; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000378:metabolism*; D005799:Genes, Dominant / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008024:Ligands; D064246:Mad2 Proteins; D008938:Mitosis; D008954:Models, Biological; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism*; D008941:Spindle Apparatus / Q000378:metabolism*; D013329:Structure-Activity Relationship
D002135:Calcium-Binding Proteins; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins; D008024:Ligands; C501384:MAD1L1 protein, human; C577697:MAD2L1 protein, human; D064246:Mad2 Proteins; D050505:Mutant Proteins; D009687:Nuclear Proteins; D012097:Repressor Proteins; C577488:CDC20 protein, human; D010768:Phosphoserine