Despite their potential importance, the functions of phosphatases in cell cycle regulation are still mysterious. The lack of specific inhibitors is one of the biggest obstacles leading to the slow progress in phosphatase studies. To tackle this problem, our laboratory has recently constructed a short hairpin RNA library to specifically knockdown individual phosphatases by RNA interference. This loss of function approach was used to study the functions of the phosphatases in intracellular pathways. Several lines of evidence are accumulating to suggest that phosphatases play important roles in the regulation of anaphase-promoting complex/cyclosome (APC/C) and p53, vital cell cycle regulators for triggering anaphase onset and maintaining genomic integrity, respectively. In this study, I co...[ Read more ]

Despite their potential importance, the functions of phosphatases in cell cycle regulation are still mysterious. The lack of specific inhibitors is one of the biggest obstacles leading to the slow progress in phosphatase studies. To tackle this problem, our laboratory has recently constructed a short hairpin RNA library to specifically knockdown individual phosphatases by RNA interference. This loss of function approach was used to study the functions of the phosphatases in intracellular pathways. Several lines of evidence are accumulating to suggest that phosphatases play important roles in the regulation of anaphase-promoting complex/cyclosome (APC/C) and p53, vital cell cycle regulators for triggering anaphase onset and maintaining genomic integrity, respectively. In this study, I constructed several in vivo reporter systems for monitoring the changes in APC/C and p53 activities. These systems were extensively characterized and validated to serve as cell models for screening APC/C- and p53-regulatory phosphatases.

Primary screening of APC/C-regulatory phosphatases identified twenty potential phosphatases that could be involved in APC/C regulation. The knockdown of these phosphatases mainly led to enhanced APC/C activity in G₁ phase, implying that phosphatases mainly act as APC/C inhibitors and dephosphorylation might be one of the key steps to inactivate APC/C during G₁-S transition. Intriguingly, one pair of shRNAs caused a robust upregulation of APC/C activity in G₂/M boundary. In normal conditions, the APC/C activity is suppressed by several mechanisms in G₂/M phase which include the direct bindings of inhibitors to APC/C. My finding shows that phosphatase is capable to interfere these APC/C inhibitory signals, implicating the presence of a new APC/C regulatory mechanism in G₂/M phase. In the final stage, eight pairs of shRNAs were randomly selected for validation tests. The results showed that five pairs of these shRNAs led to a reduction in endogenous APC/C substrate levels, which showed that the APC/C was really affected by these shRNAs. These results underscore the importance of APC/C regulation by phosphatases.