Molecular mechanisms underlying nucleocytoplasmic shuttling of actinin-4

Abstract

SummaryIn addition to its well-known role as a crosslinker of actin filaments at focal-adhesion sites, actinin-4 is known to be localized to the nucleus. In this study, we reveal the molecular mechanism underlying nuclear localization of actinin-4 and its novel interactions with transcriptional regulators. We found that actinin-4 is imported into the nucleus through the nuclear pore complex in an importinindependent manner and is exported by the chromosome region maintenance-1 (CRM1)-dependent pathway. Nuclear actinin-4 levels were significantly increased in the late G2 phase of the cell cycle and were decreased in the G1 phase, suggesting that active release from the actin cytoskeleton was responsible for increased nuclear actinin-4 in late G2. Nuclear actinin-4 was found to interact with the INO80 chromatin-remodeling complex. It also directs the expression of a subset of cell-cycle-related genes and interacts with the upstream-binding factor (UBF)-dependent rRNA transcriptional machinery in the M phase. These findings provide molecular mechanisms for both nucleocytoplasmic shuttling of proteins that do not contain a nuclear-localization signal and cell-cycle-dependent gene regulation that reflects morphological changes in the cytoskeleton.

Karyopherin-independent spontaneous transport of amphiphilic proteins through the nuclear pore

“…Semi-permeabilized cells were prepared by digitonin treatment, as previously described (Kumeta et al, 2010). 70 kDa fluorescein-and rhodamine B-conjugated dextran were purchased from Molecular Probes.…”

“…Nuclear translocation of b-catenin, primarily a focal adhesion component, is a constituent of the canonical Wnt signaling pathway, which is an indispensable step for asymmetric cell division during embryonic development. Such protein classes often contain amphiphilic helices and include: i) cytoskeletal proteins containing spectrin repeats (Kumeta et al, 2010); ii) nuclear shuttling/signaling molecules containing armadillo repeats (Yokoya et al, 1999); and iii) a group of proteins containing HEAT repeats, to which, interestingly, the karyopherins themselves belong. Recently, it was revealed through the use of chemically modified BSA that molecular surface hydrophobicity is sufficient to overcome the selectivity barrier of the NPC (Naim et al, 2009).…”

Intermolecular disulfide bonds among nucleoporins regulate karyopherin-dependent nuclear transport

“…The plasmids were introduced into HeLa cells with the transfection reagent, Effectene (Qiagen, Valencia, CA), according to the manufacturer's protocol. Recombinant proteins (importin a1, importin b1, Ran, and GFP) were expressed in E. coli cells (BL21-CodonPlus(DE3)-RIL, Agilent Technologies, Wilmington, DE) as affinitytagged fusion proteins (GST or His 66 ) and affinity purified as described previously (Kumeta et al, 2010;Yoshimura et al, 2006). As for GST-fused proteins, proteins were cleaved from GST by protease digestion, if necessary (Otsuka et al, 2008).…”

“…Cells were then fixed with 4% paraformaldehyde and immunostained with anti-Ran antibody and fluorescein isothiocyanate-conjugated anti-mouse IgG (Cappel Laboratories) as described in the previous report (Kumeta et al, 2010).…”

“…The transport assay was performed as described in previous reports (Kumeta et al, 2012;Kumeta et al, 2010), except that digitonin-treated cells were pre-incubated with or without DTT for 10 minutes at 37˚C before the addition of fluorescent proteins. For the transport assay of NLS cargo, the cells were incubated with 4 mM importin a, 4 mM importin b, 25 mM His 66 -RanGDP, 2.5 mM GST-NLS-GFP, and ATP regeneration system (1 mM ATP, 5 mM creatine phosphate and 200 U/ml creatine phosphokinase) at room temperature for 5 minutes.…”