Guowei Fang - US grants

DESCRIPTION (provided by applicant): This proposal concerns the molecular mechanism of the spindle assembly checkpoint in the mammalian system. This mitotic checkpoint ensures the fidelity of chromosome segregation; inactivation of this checkpoint causes mis-segregation of chromosomes and aneuploidy, leading to tumorigenesis. The checkpoint mechanism prevents premature anaphase initiation by inhibiting the anaphase-promoting complex (APC)/cyclosome, a ubiquitin ligase that controls sister chromatid separation. The activity of APC at metaphase is controlled by its activator CDC20 and by its inhibitor MAD2, a checkpoint protein. We propose experiments here to address the function of two checkpoint proteins, MAD2 and BUBR1. Our long-term goal is to identify all the components in the mitotic checkpoint pathway, to understand the biochemistry of each signaling step and to reconstitute the signaling pathway in vitro using purified metaphase chromosomes. 1. To detect a checkpoint-dependent chance in MAD2 structure. We will develop a mammalian cell-free checkpoint system using purified metaphase chromosomes and determine the biochemical basis of the checkpoint signaling by the MAD2 protein. 2. To define the role of BUBR1 in control of CDC20 activity. CDC20 is a substrate of the checkpoint kinase BUBR1. We will analyze the biochemical effect and physiological function of phosphorylation of CDC20 by BUBR1, both in vitro and in vivo. 3. To investigate the regulation of BUBR1 by checkpoint pathway. BUBR 1 is activated by the checkpoint pathway. We will study the molecular mechanism that regulates BUBR1 kinase activity in response to checkpoint activation. Results from proposed studies will provide a molecular pathway for the mitotic checkpoint control in mammalian cells. Since the checkpoint pathway is inactivated in several types of cancer, a better understanding of the biochemical pathway for the checkpoint signaling is not only essential to our understanding of the basic workings of the cell cycle machinery and regulated proteolysis in all eukaryotic cells, but also central for the development of novel strategies for cancer diagnosis and treatment.

Anaphase; CRISP; Chromosome Segregation; Chromosomes; Class; Computer Retrieval of Information on Scientific Projects Database; Frequencies (time pattern); Frequency; Funding; Grant; Institution; Investigators; Kinetochores; M Phase; M phase (cell cycle); Metaphase; Micro-tubule; Microtubule Polymerization; Microtubules; Mitosis; Mitosis Stage; Mitotic; Mitotic Anaphase; Mitotic Metaphase; Mitotic spindle; NIH; National Institutes of Health; National Institutes of Health (U.S.); Phenocopy; Proteins; Racial Segregation; Rate; Recruitment Activity; Research; Research Personnel; Research Resources; Researchers; Resources; SIS; Sister; Source; United States National Institutes of Health; driving force; functional genomics; gene product; recruit; segregation

20S Catalytic Proteasome; 20S Core Proteasome; 20S Proteasome; 20S Proteosome; Anaphase; CRISP; Cell Cycle Progression; Cell Cycle Proteins; Cell Division Cycle Proteins; Cell-Cycle Regulatory Proteins; Computer Retrieval of Information on Scientific Projects Database; EC 2.7; Funding; Genetic Alteration; Genetic Change; Genetic defect; Grant; Institution; Investigators; Kinases; Kinetochores; M Phase; M phase (cell cycle); Macropain; Macroxyproteinase; Mediating; Metaphase; Microtubule Polymerization; Mitosis; Mitosis Stage; Mitotic; Mitotic Anaphase; Mitotic Metaphase; Multicatalytic Proteinase; Mutation; NIH; National Institutes of Health; National Institutes of Health (U.S.); Phosphotransferases; Physiologic; Physiological; Prosome; Proteasome; Proteasome Endopeptidase Complex; Protein Cleavage; Proteins; Proteolysis; Proteomics; Proteosome; Racial Segregation; Regulation; Research; Research Personnel; Research Resources; Researchers; Resources; SIS; Sister; Sister Chromatid; Source; Transphosphorylases; United States National Institutes of Health; Variant; Variation; beta-TrCP; beta-Transducin Repeat-Containing Proteins; betaTrCP; cdc Proteins; functional genomics; gene product; genome mutation; multicatalytic endopeptidase complex; segregation; ubiquitin ligase

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A central question in the study of cell proliferation is, what controls cell- cycle transitions. Although the accumulation of mitotic cyclins drives the transition from the G2 phase to the M phase in embryonic cells, the trigger for mitotic entry in somatic cells remains unknown. We report that the synergistic action of Bora and the kinase Aurora A (Aur-A) controls the G2-M transition. Bora accumulates in the G2 phase and promotes Aur-A-mediated activation of the Polo-like kinase 1 (Plk1), leading to the activation of the cyclin-dependent kinase 1 (Cdk1) and mitotic entry. Mechanistically, Bora interacts with Plk1 and controls the accessibility of its activation loop for phosphorylation and activation by Aur-A. Thus, Bora and Aur-A control mitotic entry, which provides a mechanism for one of the most important, and yet ill-defined events in the cell cycle.