Abstract
AbstractARHI (DIRAS3) is an imprinted tumor suppressor gene whose expression is lost in the majority of breast and ovarian cancers. Unlike its homologs Ras and Rap, ARHI functions as a tumor suppressor. Our previous study showed that ARHI can interact with transcription activator Stat3 and inhibit its nuclear translocation in human breast and ovarian cancer cells. To identify proteins that interact with ARHI in nuclear translocation, we have performed proteomic analysis and identified several importins that can associate with ARHI. To further explore this novel finding, we have purified 10 GST-importin fusion proteins (importin 7, 8, 13, b1, a1, a3, a5, a6, a7 as well as mutant a1). Using a GST-pull down assay, we found that ARHI can bind strongly to most importins; however, its binding is significantly reduced with an importin a1 mutant which contains an altered nuclear localization signal (NLS) domain. In addition, an ARHI N-terminal deletion mutant (NTD) exhibits much less binding to all importins than does wild type ARHI ARHI and NTD proteins were purified and tested for their ability to inhibit nuclear importation of proteins in HeLa cells. ARHI protein inhibits interaction of Ran-importin complexes with GFP fusion proteins that contain an NLS domain and a beta-like import receptor binding domain, blocking their nuclear localization. Addition of ARHI also blocked nuclear localization of phosphorylated Stat3&x03B2;. By GST-pull down assays, we found that ARHI could compete for Ran-importins binding. Thus, ARHI-induced disruption of importin binding to cargo proteins including Stat3 could serve as an important regulatory mechanism that contributes to the tumor suppressor function of ARHI.
Highlights
Transport of macromolecules between the nucleus and cytoplasm is critical for the normal function of eukaryotic cells
Whereas importin α interacts with a nuclear localization signal (NLS) in the cargo, importin β binds to the autoinhibitory domain on importin α and mediates the transport of the trimeric complex from the cytoplasm to the nucleus through the nuclear pores
Our previous studies have shown that, when ARHI and Stat3 were both expressed in SKOv3 cells, ARHI formed a complex with Stat3 in the cytoplasm and prevented interleukin-6–induced Stat3 accumulation in the nucleus [17]
Summary
Transport of macromolecules between the nucleus and cytoplasm is critical for the normal function of eukaryotic cells. Aberrant nucleocytoplasmic transport of transcription factors (such as Stat and E2F1) [2, 3] and their regulatory kinases (such as Sgk and Erk/MAPK) [4] occurs through impaired nuclear import, enhanced export, suppression of degradation, and sequestration in protein aggregates. Secreted factors such as CCN proteins, EGF, FGFs and their receptors are often detected in the nucleus of cancer cells. Importin 13, a recently identified importin β family member, regulates nuclear import of the glucocorticoid receptor in airway epithelial cells [12]
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