Extracellular signal-dependent nuclear import of Stat1 is mediated by nuclear pore-targeting complex formation with NPI-1, but not Rch1

Abstract

In response to interferon-γ (IFN-γ), Stat1 is tyrosine phosphorylated and translocates to the nucleus where it activates transcription. In this study, we identified factors which mediate the nuclear import of Stat1. Tyrosine-phosphorylated Stat1 associated with the β subunit (a 97 kDa component) of the nuclear poretargeting complex via the NPI-1 family, but not the Rch1 family, of α subunit (a 58 kDa component) as a result of IFN-γ stimulation. Antibodies against NPI-1 or β subunit consistently inhibited the IFN-γ-dependent nuclear import of Stat1 in living cells, although antibodies reactive to Rch1 had no effect. Solution binding assays with deletion mutants of NPI-1 showed that the Stat1-binding domain of NPI-1 was located in the carboxy-terminal region, which is clearly distinct from the SV40 large T antigen nuclear localization signal (NLS)-binding region. These results indicate that the extracellular signal-dependent nuclear transport of Stat1 is mediated by NPI-1, but not Rch1, in conjunction with β subunit, and that these factors participate in, not only constitutive, but also the conditional nuclear import of proteins.

Microinjected antibodies interfere with protein nucleocytoplasmic shuttling by distinct molecular mechanisms

“…However, microinjection of this antibody not only inhibited the carrier-independent shuttling of unphosphorylated STAT1 in unstimulated cells, but also prevented the nuclear translocation of activated STAT1 in IFN-c-treated cells, which requires metabolic energy as well as the transport factors importin-a and importin-b (16,30,31). Accordingly, and contrary to the unphosphorylated STAT1 (Fig.…”

“…2A-2D) interferon-c expectedly had no influence on the distribution of either antibody. However, IFN-c treatment of cells triggers the association of both STAT1 and importin-b with the karyopherin importin-a, which affords their nuclear import via a GTPase-dependent mechanism (3,30,31). Surprisingly, while the translocating antibody accumulated in the nucleus, it nonetheless prevented the concurrent nuclear accumulation of STAT1 (Fig.…”

Cytokine-induced nuclear translocation of signaling proteins and their analysis using the inducible translocation trap system

“…In this regard, it has been previously suggested that the nuclear import of activated Stat1 is mediated by NLS interaction with importin α5 [24,25]. Sekimoto et al showed that activated Stat1 associates with importin α5 and that its inhibition impairs nuclear translocation of Stat1 [24]. They also showed that dimerization is required for interaction with importin α5.…”

“…Both groups showed that this particular arginine/lysine-rich region mediates interaction with importin α, particularly importin α5 [22,23]. In this regard, it has been previously suggested that the nuclear import of activated Stat1 is mediated by NLS interaction with importin α5 [24,25]. Sekimoto et al showed that activated Stat1 associates with importin α5 and that its inhibition impairs nuclear translocation of Stat1 [24].…”

“…Although non-phosphorylated Stat1 constitutively shuttles between the nucleus and the cytoplasm, nuclear import of the phosphorylated dimers does not use the same mechanism as the unphosphorylated monomer. It has been shown that unphosphorylated Stat1 does not use importins for its nuclear translocation but mediated by direct contact with NPC [38] while the phosphorylated dimers (homodimer or heterodimer with Stat2) nuclear entry is facilitated by importin α5 [22][23][24]. It was also reported that dimerization, which is mediated by tyrosinephosphorylation of Stat1, is required for interaction with importins [24].…”