Progression through the cell cycle depends on the timely degradation of regulatory proteins through the ubiquitin pathway. The anaphase-promoting complex (APC), in association with its activators Cdc2O or Cdh1, acts as the ubiquitin ligase (E3) in the mitotic degradation system. APCCdC2O ubiquitinates anaphase inhibitors to allow the separation of sister-chromatids, while APCcdh1 mediates the proteolysis of mitotic cyclins, permitting the exit from mitosis. Research in this proposal focuses on the cell cycle-regulation of the APCcdh1 ubiquitin ligase activity in vertebrates.
Aim 1 studies the roles of the mammalian spindle checkpoint proteins Mad2 and its newly identified homolog, Mad2b, in the regulation of APCcdh1. At pro-metaphase, the activity of APCCdC2O is blocked by Mad2. However, it is unclear whether APCCdh1 is also inhibited by the same pathway. Our preliminary data indicate that both Mad2 and Mad2b inhibit APCcdh1 in vitro. Overexpression of Mad2b causes a G2/M delay in mammalian cells. The specificity of Mad2 and Mad2b toward Cdc2O or Cdh1 will be further analyzed in vitro and in vivo. The upstream signals that control Mad2b will also be studied. The focus of Aim 2 is the regulation of APCcdh1 in late anaphase by the hCdcl4 phosphatase. Phosphorylation of Cdh1 by cdc2 inhibits APCCdh1 while hCdcl4 dephosphorylates Cdh1 and activates APCcdh1. The hCdcl4 protein localizes to the centrosomes and exists as a 500 kD complex in mammalian cells. The mechanism by which phosphorylation inhibits APCcdh1, the substrate specificity of hCdcl4, and the cell cycle-regulation of hCdcl4 will be elucidated.
Aim 3 is to characterize the biochemical function of a novel hCdcl4-binding protein (14BP1). Sequence analysis reveals that I4BP1 might localize to the nucleolus and be functionally linked to Bub2-like proteins. The effects of 14BP1 on the phosphatase activity and the cellular localization of hCdcl4 will be tested. Additional proteins that interact with hCdcl4 and 14BP1 will be identified. Mutations of spindle assembly checkpoint genes have recently been found in human cancers, and malfunction of this checkpoint may contribute to genetic instability of tumor cells. Research on the regulation of APCcdh1 will provide insights into how the mitotic checkpoint halts the cell cycle and present novel molecular targets for the design of agents that interfere with this pathway.
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