Degradation of the human mitotic checkpoint kinase Mps1 is cell cycle-regulated by APC-cCdc20 and APC-cCdh1 ubiquitin ligases.
Mps1 is a dual specificity protein kinase with key roles in regulating the spindle assembly checkpoint and chromosome-microtubule attachments. Consistent with these mitotic functions, Mps1 protein levels fluctuate during the cell cycle, peaking at early mitosis and abruptly declining during mitotic exit and progression into the G(1) phase. Although evidence in budding yeast indicates that Mps1 is targeted for degradation at anaphase by the anaphase-promoting complex (APC)-c(Cdc20) complex, little is known about the regulatory mechanisms that govern Mps1 protein levels in human cells. Here, we provide evidence for the ubiquitin ligase/proteosome pathway in regulating human Mps1 levels during late mitosis through G(1) phase. First, we showed that treatment of HEK 293T cells with the proteosome inhibitor MG132 resulted in an increase in both the polyubiquitination and the accumulation of Mps1 protein levels. Next, Mps1 was shown to co-precipitate with APC and its activators Cdc20 and Cdh1 in a cell cycle-dependent manner. Consistent with this, overexpression of Cdc20 or Cdh1 led to a marked reduction of endogenous Mps1 levels during anaphase or G(1) phase, respectively. In contrast, depletion of Cdc20 or Cdh1 by RNAi treatment both led to the stabilization of Mps1 protein during mitosis or G(1) phase, respectively. Finally, we identified a single D-box motif in human Mps1 that is required for its ubiquitination and degradation. Failure to appropriately degrade Mps1 is sufficient to trigger centrosome amplification and mitotic abnormalities in human cells. Thus, our results suggest that the sequential actions of the APC-c(Cdc20) and APC-c(Cdh1) ubiquitin ligases regulate the clearance of Mps1 levels and are critical for Mps1 functions during the cell cycle in human cells.
Cui
Yongping
Y
From the Key Laboratory of Cellular Physiology, Ministry of Education, Taiyuan, Shanxi 030001, China; Department of Cell Biology and Genetics, Taiyuan, Shanxi 030001, China. Electronic address: cuiy0922@yahoo.com.
Cheng
Xiaolong
X
Department of Anatomy, Shanxi Medical University, Taiyuan, Shanxi 030001, China.
Zhang
Ce
C
From the Key Laboratory of Cellular Physiology, Ministry of Education, Taiyuan, Shanxi 030001, China.
Zhang
Yanyan
Y
Department of Cell Biology and Genetics, Taiyuan, Shanxi 030001, China.
Li
Shujing
S
Department of Cell Biology and Genetics, Taiyuan, Shanxi 030001, China.
Wang
Chuangui
C
School of Life Science, East China Normal University, Shanghai 200062, China.
Guadagno
Thomas M
TM
Department of Molecular Oncology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida 33612. Electronic address: Thomas.Guadagno@yahoo.com.
D020816:Amino Acid Motifs; D015703:Antigens, CD; D015820:Cadherins / Q000235:genetics / Q000378:metabolism*; D064199:Cdc20 Proteins; D002453:Cell Cycle / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D018385:Centrosome / Q000201:enzymology; D004795:Enzyme Stability / Q000235:genetics; D006801:Humans; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*
D015703:Antigens, CD; C513384:CDH1 protein, human; D015820:Cadherins; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins; C577488:CDC20 protein, human; D044767:Ubiquitin-Protein Ligases; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; C075840:TTK protein, human