The F-box protein Fbxo7 interacts with human inhibitor of apoptosis protein cIAP1 and promotes cIAP1 ubiquitination

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Abstract

Human cIAP1 protein is a member of the inhibitor of apoptosis proteins (IAPs) that are involved in apoptosis regulation and an increasing number of other functions, including cell cycle and intracellular signal transduction. In order to identify novel proteins involved in cIAP1 regulation, we performed a yeast two-hybrid screen and identified an F-box protein Fbxo7 as a cIAP1 interacting protein. Co-immunoprecipitation assay showed that cIAP1 can interact with Fbxo7 in human cells. When co-expressed in cells, cIAP1 and Fbxo7 co-localized remarkably both in the cytoplasm and nucleus, and considerable amounts of these often co-localized at one or few distinct Golgi-like structures close to the nucleus. Furthermore, we showed that overexpression of Fbxo7 promotes the ubiquitination of cIAP1. Since F-box proteins are specificity determining subunits of SCF ubiquitin protein ligases, our results suggest that Fbxo7 can mediate the ubiquitination of cIAP1 by SCF ubiquitin protein ligase and thus have important implication in the regulation of cIAP1 function.

Section snippets

Materials and methods

Plasmid constructs. Human cIAP1 cDNA fragment from the second BIR domain to the C-terminus (aa 183–618) was obtained by PCR amplification from human brain cDNA (BD Biosciences). The sequences of the PCR primers used are 5′-GGAATTCACTGAAGAAGCCAGATT-3′ and 5′-GGAATTCTTAAGAGAGAAATGTACG-3′. The PCR product was cut with EcoRI and inserted into the EcoRI site of the Gal4 DNA-binding domain vector pAS2-1 to generate pAS2-1-cIAP1-B2T as the bait for yeast two-hybrid screen. Human full-length cIAP1 cDNA

Identification of Fbxo7 as a cIAP1 interacting protein by yeast two-hybrid screen

In order to identify novel proteins involved in cIAP1 regulation, a yeast two-hybrid screen was performed. To avoid a transcriptional activating sequence present in cIAP1 protein (our unpublished data), the bait construct consists of the cIAP1 region from the second BIR domain to the C-terminus (aa 183–618) fused to the Gal4 DNA binding domain. A human lymphocyte Matchmaker cDNA library fused to the Gal4 activation domain was screened. Yeast colonies, which grew on media lacking Trp, Leu, and

Discussion

In this paper, we demonstrate that the inhibitor of apoptosis protein cIAP1 interacts with an F-box protein, Fbxo7. Furthermore, we show that overexpression of Fbxo7 promotes the ubiquitination of cIAP1. cIAP1 has been reported to interact with several proteins, including caspases-3 and -7, TRAF2, Smac, RIP, and IKKγ [6], [15], [16], [17], [18]. All these proteins have known functions in apoptosis signaling or execution pathways, and most of them are downstream targets subjected to cIAP1

Acknowledgments

This work was supported in part by the grants from the National Science Council, Taiwan, ROC, to C.-Y. Yuo and from the Kaohsiung Medical University to Y.-F. Chang. We are grateful to Yu-Tzu Chan and Jian-Liang Lin for experimental assistance.

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