Nuclear import of proteins with nuclear localization signals (NLSs) is mediated by shuttling carriers, the importins. Some cargoes display more than a single NLS, and among these are homeodomain proteins such as Arx, which is critical for development of multiple tissues. Arx has two functional NLSs. The present studies show that several pathways can import Arx via its NLS2, which is within its DNA binding homeodomain. Using an in vitro nuclear import assay, we show that import of Arx via NLS2 can be mediated by importin ␤1, importin 9, or importin 13, with binding being strongest to importin ␤1. All binding is sensitive to RanGTP. Experiments based on precise domain deletions indicate that NLS2 binds imp␤1, imp9, and imp13 and includes both an importin binding subdomain and a regulatory subdomain with arginine residues being important for function. Moreover, Arx can be co-precipitated with these importins when NLS2 is present. Although nuclear import of Arx can be mediated by these three importin ␤s, importin ␤1 seems to play the major role judging from in vivo small interfering RNA ablations and the in vitro import assay. This is the first evidence to show the role of importin ␤1 in nuclear import of paired-type homeodomain proteins. We propose a novel and possibly quite general mechanism for nuclear import of paired-type homeodomain proteins which is critical for development.Precise nucleocytoplasmic distribution of the homeodomain superfamily of transcription factors is critical for development. Several studies have demonstrated that NLS activity resides within the 60-amino acid DNA binding homeodomain itself, which is composed of three helices (1, 2). The "paired type" subgroup of the superfamily is characterized by a set of highly conserved residues. There are 26 members of this subgroup in man. The aristaless-related homeobox (ARX) 4 protein, a paired-type homeodomain containing protein, is mutated in multiple human conditions (3-5). ARX is expressed most strongly in the brain (6) and is important for development of the forebrain, pancreas, and testis (7). Arx proteins are highly conserved (Fig. 1) and contain 562 amino acids (564 amino acids in mouse) with four poly(A) (alanine) tracts of variable length, a paired-type homeodomain, and a conserved "aristaless" domain (8). Endogenous Arx was found in the nucleus of some types of nerve cells (9, 10). Because Arx has a molecular mass larger than 60 kDa, its nuclear import is likely to be signal-dependent. Three putative basic NLSs in Arx have been proposed (8, 11): NLS1 from aa 82 to 89, BC1 from aa 327 to 334, and BC2 from aa 381 to 388 (Fig. 1). It is not clear whether or how these putative NLSs function in the nuclear localization of Arx.As for other trinucleotide-repeat-containing genes (12, 13), the first poly(A) tract can expand in Arx. Expansion of this tract or loss of the 3Ј aristaless domain in Arx are both associated with infantile spasms syndrome and mental retardation (14, 15). Moreover, expansion of the first poly(A) tract of Arx results in for...

SYNOPSIS ARHI (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 the transcriptional 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, β1, α1, α3, α5, α6, α7 as well as mutant α1). 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 α1 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. In nuclear import assays, addition of ARHI blocked nuclear localization of phosphorylated Stat3. ARHI protein also 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. By GST-pull down assays, we found that ARHI could compete for Ran-impotin 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.

Homeodomain proteins are crucial transcription factors for cell differentiation, cell proliferation and organ development. Interestingly, their homeodomain signature structure is important for both their DNA-binding and their nucleocytoplasmic trafficking. The accurate nucleocytoplasmic distribution of these proteins is essential for their functions. We summarize information on a) the roles of karyopherins for import and export of homeoproteins, b) the regulation of their nuclear transport during development, and c) the corresponding complexity of homeoprotein nucleocytoplasmic transport signals.

Nucleocytoplasmic transport of transcription factors is essential in eukaryotes. We previously reported the presence of two functional NLSs in the homeodomain protein, aristaless-related homeobox (Arx) protein, which is a key transcriptional repressor of LMO1, SHOX2, and PAX4 during development. NLS2, that overlaps the homeodomain, is recognized directly by multiple importin βs, but not by importin αs. In this study, we found that the N-terminal NLS1 of Arx is targeted by multiple importin α proteins, including importin α3 and α5. Both in vivo and in vitro assays demonstrated that nuclear import of Arx via NLS1 is mediated by the importin α/β pathway. Mutagenesis analysis indicated that two basic amino acids, (84)K and (87)R, are essential to the function of NLS1, and that their mutation prevents interactions of Arx with importin αs. Interestingly, inhibition of nuclear import of Arx via NLS1 clearly attenuates its ability of transcriptional repression, suggesting that nuclear import of Arx via NLS1 contributes to its transcriptional function.