Nucleocytoplasmic shuttling by nucleoporins Nup153 and Nup214 and CRM1-dependent nuclear export control the subcellular distribution of latent Stat1

Abstract

Interferon stimulation of cells leads to the tyrosine phosphorylation of latent Stat1 and subsequent transient accumulation in the nucleus that requires canonical transport factors. However, the mechanisms that control the predominantly cytoplasmic localization in unstimulated cells have not been resolved. We uncovered that constitutive energy- and transport factor-independent nucleocytoplasmic shuttling is a property of unphosphorylated Stat1, Stat3, and Stat5. The NH2- and COOH-terminal Stat domains are generally dispensable, whereas alkylation of a single cysteine residue blocked cytokine-independent nuclear translocation and thus implicated the linker domain into the cycling of Stat1. It is revealed that constitutive nucleocytoplasmic shuttling of Stat1 is mediated by direct interactions with the FG repeat regions of nucleoporin 153 and nucleoporin 214 of the nuclear pore. Concurrent active nuclear export by CRM1 created a nucleocytoplasmic Stat1 concentration gradient that is significantly reduced by the blocking of energy-requiring translocation mechanisms or the specific inactivation of CRM1. Thus, we propose that two independent translocation pathways cooperate to determine the steady-state distribution of Stat1.

Microinjected antibodies interfere with protein nucleocytoplasmic shuttling by distinct molecular mechanisms

“…In addition, certain large proteins can enter the nucleus, independent of metabolic energy and transport factors via direct interactions with constituents of the nuclear pore (6), and an increasing number of proteins, e.g. STAT1 and importin-b are known to use both pathways consecutively or in parallel (7,8). Thus, there is a growing need for reliable methods to detect and analyze nucleocytoplasmic shuttling regardless of the transport mechanism.…”

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

“…The background expression of hCD2 reflects amounts of Stat1 in the nucleus in the absence of ligand stimulation. In this regard, it has been shown that Stat1 shuttles between the cytoplasm and the nucleus even in the absence of ligand stimulation [38,39]. In contrast, significant upregulation of hCD2 was observed 7 hr after IFNγ stimulation.…”

“…Studies by two other groups later showed that both Stat3 and Stat5B have functional NES [33,37]. Nuclear export, which occurs via exportin-dependent or -independent mechanisms, is an important process by which cytoplasmic accumulation of latent Stats occur [30,38,39].…”

“…In addition to cytokine-induced nuclear translocation, constitutive shuttling was detected for Stat1, Stat3, and Stat5 [30,33,38], leading to the observation that unphosphorylated Stats can enter the nucleus either through importin-dependent or -independent pathways [30,38]. 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.…”

“…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].…”