| Summary: | 博士 === 國防醫學院 === 生命科學研究所 === 94 === Many reports showed that proteins located at centrosome are usually involved in centrosome duplication, division and maturation. These proteins also affect cell division and may serve as microtubule spindle regulators. Centrosomal p4.1-associated protein (CPAP) was identified as a centrosomal protein that carries a novel microtubule-destabilizing domain. It is speculated that CPAP may participate a role in cell cycle progression through unclear mechanisms. In this thesis, I focus on characterization the protein property of CPAP and search for its regulatory proteins. To better investigation of CPAP, I firstly generated two specific anti-CPAP monoclonal antibodies (mAbs). These two CPAP antibodies against different region of CPAP and one of them may recognize CPAP of different species. Taking advantage of these specific mAbs, the effects of CPAP were analyzed by the RNA interference (RNAi). Several small conserved motifs were found in CPAP and various experiments were performed to confirm the predicted results. Firstly, I established the protein expression and modification profile of CPAP and found that the expression and phosphorylation level of CPAP are fluctuated during cell cycle progression. CPAP is phosphorylated at both interphase and mitosis. Like many mitotic factors, CPAP has a short half-life and appears to be degradated by proteasome when cells exit from mitosis. These results suggest that the endogenous CPAP level within the cells is regulated through a change of its expression and phosphorylation during cell cycle progression.
Since CPAP contains three PP1 binding motifs, inhibition of PP1 and PP2A activity by okadaic acid induces hyperphosphorylation of CPAP, suggesting that the dephosphorylation of CPAP is possibly mediated by PP1. The interaction between CPAP and PP1 was confirmed by GST pull-down and coimmunoprecipitation assays. PP1 constantly binds to CPAP through the three PP1-binding motifs. PP1 also dephosphorylate CPAP in vitro. The OA-induced phosphorylation sites of CPAP are located within residue 445-900 and these phosphorylations may modulate the protein stability of CPAP. In addition, I showed that CPAP is a novel binding partner of 14-3-3. Mutation of serine 1109 to alanine (S1109A) in the 14-3-3 binding motif and alkaline phosphatase treatment both abolished the association of CPAP with 14-3-3. Interestingly, the association between CPAP and 14-3-3 occurred in interphase and was significantly reduced during mitosis. These results show a direct interaction between CPAP and 14-3-3 and this interaction appears to be phosphorylation- and cell cycle-dependent.
Taking together, the above results implicated that PP1 and 14-3-3, two CPAP-interacting proteins, may participate the roles involved in the regulation of the protein stability and phosphorylation of CPAP during cell cycle progression.
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