Hyperosmotic Stress Signaling to the Nucleus Disrupts the Ran Gradient and the Production of RanGTP

Abstract

The RanGTP gradient depends on nucleocytoplasmic shuttling of Ran and its nucleotide exchange in the nucleus. Here we show that hyperosmotic stress signaling induced by sorbitol disrupts the Ran protein gradient and reduces the production of RanGTP. Ran gradient disruption is rapid and is followed by early (10 -20 min) and late (30 -60 min) phases of recovery. Results from SB203580 and siRNA experiments suggest the stress kinase p38 is important for Ran gradient recovery. NTF2 and Mog1, which are transport factors that regulate the nuclear localization of Ran, showed kinetics of delocalization and recovery similar to Ran. Microinjection of a nuclear localization signal reporter protein revealed that sorbitol stress decreases the rate of nuclear import. Sorbitol stress also slowed RCC1 mobility in the nucleus, which is predicted to reduce RCC1 dissociation from chromatin and RanGTP production. This was tested using a FRET biosensor that registers nuclear RanGTP levels, which were reduced in response to sorbitol stress. Although sorbitol alters nucleotide levels, we show that inverting the GTP/GDP ratio in cells is not sufficient to disrupt the Ran gradient. Thus, the Ran system is a target of hyperosmotic stress signaling, and cells use protein localization-based mechanisms as part of a rapid stress response. INTRODUCTIONCells subjected to UV radiation or oxidative, mechanical, or aniso-osmotic stress undergo both short-and long-term adaptation. Hyperosmotic stress induces rapid dehydration that drives an increase in intracellular salt concentration and a decrease in cell volume, placing physical strain on both the cytoskeleton and the plasma membrane (Lang et al., 1998). In response to these changes, cells rapidly initiate a stress signaling cascade and attempt to restore iso-osmolarity through transport of inorganic ions and initiate an accompanying reorganization of the actin cytoskeleton (Haussinger, 1996;Di Ciano et al., 2002). Short-term recovery of cell volume is facilitated by increasing intracellular concentrations of inorganic ions; however, elevated levels of intracellular salts can be detrimental to protein structure and function (Yancey et al., 1982;Russo et al., 2003). Long-term adaptation to hyperosmotic stress is achieved through signal transduction pathways that communicate with the metabolic and transcriptional machinery, resulting in the production or accumulation of organic osmolytes that increase intracellular osmolarity without adversely affecting protein structure or function (O'Neill, 1999).One of the most thoroughly studied stress kinases is the mitogen-activated protein kinase (MAPK) p38. p38 and its yeast homologue Hog1p are activated in response to a wide variety of adverse environmental conditions. These include osmotic, UV, and mechanical stress. Cytokines, such as interleukins and tumor necrosis factor ␣ are also known to activate p38 (Ono and Han, 2000). Hog1p and p38 are both phosphorylated on a threonine, glycine, tyrosine (TGY) motif within the kinase activation loop (Thr 180 an...

Inhibition of active nuclear transport is an intrinsic trigger of programmed cell death in trypanosomatids

“…26 Conversely, in response to external stimuli triggering apoptosis, a change in the RanGTP/RanGDP gradient is observed, leading to the inhibition of active nuclear transport. 7,9 In this study, using trypanosomatid protozoa, we have (i) identified Ran and several of its main partners in this divergent cell model, (ii) specified their subcellular localisation at the NPCs and (iii) characterized the apoptotic phenotypes induced by the depletion of their expression.…”

“…6 Besides, intracellular redistribution of nucleocytoplasmic transport factors is an early feature of apoptosis, which precedes caspase activation, 1,7 and which has been observed in response to cellular stresses including UV irradiation, oxidative stress and heat-shock 8 and hyperosmotic stress. 9 However, it is our opinion that all these reports do not allow inferring any firm conclusions on the link between the inhibition of the nucleocytoplasmic transport and apoptosis, in view of the diverse and essential functions of the proteins studied. In particular, the small, evolutionarily conserved, eukaryotic GTPase of the Ras family, Ran together with its partners, were first discovered to be essential in nucleocytoplasmic transport.…”

Disruption of the Ran System by Cysteine Oxidation of the Nucleotide Exchange Factor RCC1

“…Ran undergoes rapid shuttling between the nucleus and the cytoplasm, and formation of the Ran protein gradient requires its nucleus-localized nucleotide exchange factor RCC1 (4,11,12,46). tsBN2 cells, developed by the Nishimoto laboratory, harbor a temperature-sensitive allele of RCC1 and display a significant reduction in the Ran N/C distribution when cells are shifted to the nonpermissive temperature (13-15, 47, 48).…”

“…Quantitative analysis of IF microscopy images (ratios of nuclear intensity to cytoplasmic intensity [N/C ratios; N/C ratios ϭ mean fluorescence of nucleus/mean fluorescence of cytoplasm]) was done as described previously (11,13), with ImageJ (National Institutes of Health). Both the nuclear and cytoplasmic regions of each cell were selected, and the mean IF was determined by ImageJ software.…”

“…Thus, Ran undergoes import, nucleotide exchange, export, and nucleotide hydrolysis. These reactions generate two nuclear/cytoplasmic (N/C) Ran gradients, a Ran protein gradient (ϳ3:1) and a RanGTP gradient (ϳ100:1) (11,12). Disruption of the Ran protein gradient by depletion of NTF2 reduces Ran GTPdependent import (13,14), and inhibition of GTP loading with a temperature-sensitive allele of RCC1 disrupts the Ran protein gradient (15).…”