Nuclear localization and transactivation of SYS-1/β-catenin is the result of serial gene duplications and subfunctionalizations

β-catenin is a multifunctional protein capable of mediating cell adhesion via E-cadherin and transactivation of target genes of the canonical Wnt signaling pathway. The nematode, C. elegans contains four paralogs of β-catenin which are highly specific in their functions. Though similar in overall structure, the four β-catenins are functionally distinct, each regulating different aspects of development. Of the four, SYS-1 is a key player in Wnt dependent asymmetric cell division (ACD). In ACD, a polarized mother will give rise to a daughter with high nuclear SYS-1 and another with low nuclear SYS-1. Despite sequence dissimilarity, SYS-1 shares a close structural resemblance with human β-catenin where it retains an unstructured amino-terminus (NTD) and 12 armadillo repeats. Using existing genome sequence data from several nematodes species, we find that the four β-catenin paralogs result from 3 sequential gene duplications and neofunctionalizations during nematode evolution. SYS-1, however, lacks an unstructured carboxyl-terminus (CTD) that is essential for human β-catenin transactivation processes. This work supports the hypothesis that SYS-1 compensated for the lack of CTD by acquiring novel transactivation domains with cryptic nuclear localization signals in the NTD and the first four armadillo repeats, as shown by transactivation assays in worms and yeast. Furthermore, SYS-1 regulatory domains are not localized to the NTD as in canonical β-catenin and instead spans the entire length of the protein. Truncating SYS-1 abolishes the classical SYS-1 nuclear asymmetry, resulting in daughter cells with symmetrical SYS-1 truncation localization. A screen for SYS-1 physical interactors followed by in vivo cell fate and SYS-1 localization analyses suggest that proper SYS-1 nuclear export is facilitated by XPO-1, while an interaction with IMB-3, an importin β-like protein, suggests import mechanisms. Interestingly, XPO-1 is especially required for lowering SYS-1 in the Wnt-unsignaled nucleus, suggesting a distinct mechanism for regulating asymmetric nuclear SYS-1. In summary, we provide insights on the mechanism of β-catenin evolution within nematodes and inform SYS-1 transactivation and nuclear transport.

APC binds intermediate filaments and is required for their reorganization during cell migration

Intermediate filaments (IFs) are components of the cytoskeleton involved in most cellular functions, including cell migration. Primary astrocytes mainly express glial fibrillary acidic protein, vimentin, and nestin, which are essential for migration. In a wound-induced migration assay, IFs reorganized to form a polarized network that was coextensive with microtubules in cell protrusions. We found that the tumor suppressor adenomatous polyposis coli (APC) was required for microtubule interaction with IFs and for microtubule-dependent rearrangements of IFs during astrocyte migration. We also show that loss or truncation of APC correlated with the disorganization of the IF network in glioma and carcinoma cells. In migrating astrocytes, vimentin-associated APC colocalized with microtubules. APC directly bound polymerized vimentin via its armadillo repeats. This binding domain promoted vimentin polymerization in vitro and contributed to the elongation of IFs along microtubules. These results point to APC as a crucial regulator of IF organization and confirm its fundamental role in the coordinated regulation of cytoskeletons.

NF-κB Is Transported into the Nucleus by Importin α3 and Importin α4

NF-κB transcription factors are retained in the cytoplasm in an inactive form until they are activated and rapidly imported into the nucleus. We identified importin α3 and importin α4 as the main importin α isoforms mediating TNF-α-stimulated NF-κB p50/p65 heterodimer translocation into the nucleus. Importin α3 and α4 are close relatives in the human importin α family. We show that importin α3 isoform also mediates nuclear import of NF-κB p50 homodimer in nonstimulated cells. Importin α3 is shown to directly bind to previously characterized nuclear localization signals (NLSs) of NF-κB p50 and p65 proteins. Importin α molecules are known to have armadillo repeats that constitute the N-terminal and C-terminal NLS binding sites. We demonstrate by site-directed mutagenesis that NF-κB p50 binds to the N-terminal and p65 to the C-terminal NLS binding site of importin α3.In vitrocompetition experiments and analysis of cellular NF-κB suggest that NF-κB binds to importin α only when it is free of IκBα. The present study demonstrates that the nuclear import of NF-κB is a highly regulated process mediated by a subset of importin α molecules.