[Frontiers in Bioscience E3, 834-842, June 1, 2011]

AIG1 is a novel Pirh2-interacting protein that activates the NFAT signaling pathway

Gang Wu1, 2, Meiqian Sun3, Wei Zhang1, Keke Huo1

1 State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, 220 Handan Rd., Shanghai 200433, PR China, 2 Chinese Academy of Sciences and Max Planck Society (CAS-MPG) Partner Institute for Computational Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue Yang Rd., Shanghai 200031, PR China, 3 United Gene Bio-Pharma Inc. , 100 Handan Rd., Shanghai 200437, PR China

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Materials and methods
3.1. Yeast two-hybrid screening
3.2. GST pull-down assay
3.3. Cell culture and co-immunoprecipitation assay
3.4. In vivo ubiquitination assay
3.5. Subcellular localization analysis
3.6. Quantitative real-time reverse transcription-PCR
3.7. Pathway profiling assay
3.8. Statistical analysis
4. Results
4.1. Pirh2 interacts with AIG1 in vitro and in vivo
4.2. AIG1 is a conserved protein and can co-localize with Pirh2
4.3. Expression of AIG1 in HCCs compared to non-cancerous tissues
4.4. AIG1 activates the NFAT signaling pathway in a dose-dependent manner
5. Discussion
6. Acknowledgment
7. References

1. ABSTRACT

Pirh2 is an E3 ligase that negatively regulates p53 through both direct physical interaction and ubiquitin-mediated proteolysis. Here, we identified a novel Pirh2-interacting protein, AIG1, by yeast two-hybrid screening and confirmed its interaction with p53 both in vitro and in vivo. Quantitative real-time reverse transcription-PCR analysis showed that AIG1 expression levels were reduced in 50 out of 79 (63%) human hepatocellular carcinomas (HCCs) when compared to matched, non-cancerous liver tissue; levels were significantly different between HCCs with or without lymph node metastasis. Kaplan-Meier analysis indicated that the survival time of HCC patients down-regulated for AIG1 is much shorter than it is for patients up-regulated for AIG1 expression (p = 0.0313 as determined by the Log-rank test). Finally, AIG1 activated the nuclear factor of activated T cells (NFAT) signaling pathway in a dose-dependent manner when over-expressed in HEK293T cells. Our results suggest AIG1 could serve as a new biomarker for the diagnosis and prognostic evaluation of HCCs.