Dissecting the Signaling Events That Impact Classical Nuclear Import and Target Nuclear Transport Factors

Abstract

BackgroundSignaling through MEK→ERK1/2 and PI3 kinases is implicated in many aspects of cell physiology, including the survival of oxidant exposure. Oxidants play a role in numerous physiological and pathophysiological processes, many of which rely on transport in and out of the nucleus. However, how oxidative stress impacts nuclear trafficking is not well defined.Methodology/Principal FindingsTo better understand the effect of stress on nucleocytoplasmic trafficking, we exposed cells to the oxidant diethyl maleate. This treatment activated MEK→ERK1/2 as well as PI3 kinase→Akt cascades and triggered the inhibition of classical nuclear import. To define the molecular mechanisms that regulate nuclear transport, we examined whether MEK and PI3 kinase signaling affected the localization of key transport factors. Using recently developed tools for image acquisition and analysis, the subcellular distributions of importin-α, CAS, and nucleoporins Nup153 and Nup88 were quantified in different cellular compartments. These studies identified specific profiles for the localization of transport factors in the nucleus and cytoplasm, and at the nuclear envelope. Our results demonstrate that MEK and PI3 kinase signaling as well as oxidative stress control nuclear trafficking and the localization of transport components. Furthermore, stress not only induced changes in transport factor distribution, but also upregulated post-translational modification of transport factors. Our results are consistent with the idea that the phosphorylation of importin-α, CAS, Nup153, and Nup88, and the O-GlcNAc modification of Nup153 increase when cells are exposed to oxidant.Conclusions/SignificanceOur studies defined the complex regulation of classical nuclear import and identified key transport factors that are targeted by stress, MEK, and PI3 kinase signaling.

Role of the ubiquitin-like protein Urm1 as a noncanonical lysine-directed protein modifier

“…It remains equally enigmatic why components of its own pathway, as well as CAS and possibly other members of the nuclear translocation machinery, are modified by Urm1. Nuclear translocation is halted on exposure to oxidants (39). Urmylation of CAS could contribute to an arrest in nuclear transport (e.g., by decreasing the pool of cytosolic importin α).…”

Intermolecular disulfide bonds among nucleoporins regulate karyopherin-dependent nuclear transport

“…We performed similar immunoblot analyses with other ROS, including menadione, an oxidative agent like H 2 O 2 , and diethylmaleate, a sulfhydryl-reactive agent that has been shown to affect nucleocytoplasmic transport (Crampton et al, 2009;Kodiha et al, 2009;Kodiha et al, 2008;Matsuura and Stewart, 2004;Sato et al, 1993). These reagents also increased the intracellular ROS level as measured by the fluorescent probe (Fig.…”

“…This implies that oxidative stress regulates the efflux via mechanisms other than S-S bond formation. Previous studies have demonstrated that Nups 214, 153, 98, 88 and 62 are phosphorylated and/or glycosylated by oxidative stress (Crampton et al, 2009;Kodiha et al, 2009). Other studies have reported that the Nup214/Nup88 complex and Nup98 are involved in export but not in import (Hutten and Kehlenbach, 2006;Oka et al, 2010).…”

“…The importin a/b pathway is the best known route for transporting nuclear localization signal (NLS)-containing proteins from the cytoplasm to the nucleoplasm (Mattaj and Englmeier, 1998). It has been reported that oxidative stress inhibits macromolecular transport through the NPC (Crampton et al, 2009;Kodiha et al, 2004;Kodiha et al, 2009;Kodiha et al, 2008;Miyamoto et al, 2004;Patel et al, 2012;Stochaj et al, 2000;Yasuda et al, 2006). Oxidative stress influences the intracellular distribution of Ran and transport mediators, which in turn affects the intracellular localization of the cargo proteins (Kodiha et al, 2004;Kodiha et al, 2008;Yasuda et al, 2006).…”

“…Oxidative stress influences the intracellular distribution of Ran and transport mediators, which in turn affects the intracellular localization of the cargo proteins (Kodiha et al, 2004;Kodiha et al, 2008;Yasuda et al, 2006). Oxidative stress also alters the modifications of Nups (phosphorylation and glycosylation), which might affect the passage of proteins through the NPC (Crampton et al, 2009;Kodiha et al, 2009).…”