BackgroundClassical nuclear localization signal (NLS) dependent nuclear import is carried out by a heterodimer of importin α and importin β. NLS cargo is recognized by importin α, which is bound by importin β. Importin β mediates translocation of the complex through the central channel of the nuclear pore, and upon reaching the nucleus, RanGTP binding to importin β triggers disassembly of the complex. To date, six importin α family members, encoded by separate genes, have been described in humans.ResultsWe sequenced and characterized a seventh member of the importin α family of transport factors, karyopherin α 7 (KPNA7), which is most closely related to KPNA2. The domain of KPNA7 that binds Importin β (IBB) is divergent, and shows stronger binding to importin β than the IBB domains from of other importin α family members. With regard to NLS recognition, KPNA7 binds to the retinoblastoma (RB) NLS to a similar degree as KPNA2, but it fails to bind the SV40-NLS and the human nucleoplasmin (NPM) NLS. KPNA7 shows a predominantly nuclear distribution under steady state conditions, which contrasts with KPNA2 which is primarily cytoplasmic.ConclusionKPNA7 is a novel importin α family member in humans that belongs to the importin α2 subfamily. KPNA7 shows different subcellular localization and NLS binding characteristics compared to other members of the importin α family. These properties suggest that KPNA7 could be specialized for interactions with select NLS-containing proteins, potentially impacting developmental regulation.

The mutant form of lamin A responsible for the premature aging disease Hutchinson-Gilford progeria syndrome (termed progerin) acts as a dominant negative protein that changes the structure of the nuclear lamina. How the perturbation of the nuclear lamina in progeria is transduced into cellular changes is undefined. Using patient fibroblasts and a variety of cell-based assays, we determined that progerin expression in Hutchinson-Gilford progeria syndrome inhibits the nucleocytoplasmic transport of several factors with key roles in nuclear function. We found that progerin reduces the nuclear/cytoplasmic concentration of the Ran GTPase and inhibits the nuclear localization of Ubc9, the sole E2 for SUMOylation, and of TPR, the nucleoporin that forms the basket on the nuclear side of the nuclear pore complex. Forcing the nuclear localization of Ubc9 in progerin-expressing cells rescues the Ran gradient and TPR import, indicating that these pathways are linked. Reducing nuclear SUMOylation decreases the nuclear mobility of the Ran nucleotide exchange factor RCC1 in vivo, and the addition of SUMO E1 and E2 promotes the dissociation of RCC1 and Ran from chromatin in vitro. Our data suggest that the cellular effects of progerin are transduced, at least in part, through reduced function of the Ran GTPase and SUMOylation pathways.The nuclear lamina provides an architectural framework that defines the size, shape, and physical properties of the nucleus (29). A critical function of the nuclear lamina is to provide a scaffold for chromatin attachment, and there is a growing body of evidence linking the nuclear lamina to the regulation of gene expression and chromosome positioning within interphase cells (49). The nuclear periphery, including the region proximal to the lamina, is rich in heterochromatin and provides a nuclear subcompartment that promotes transcriptional silencing (19). The mechanisms responsible for transcriptional silencing associated with the lamina appear to involve epigenetic regulation and modulation of the higherorder chromatin structure (2). Other functions of the lamina include roles in DNA replication and apoptosis (22,29). The principal components of the lamina are lamin A/C and lamin B, which are encoded by the LMNA and LMNB genes, respectively (22, 29). More than 300 mutations in LMNA have been described (http://www.umd.be/LMNA/) and have been linked to 12 diseases collectively known as laminopathies. These diseases include dilated cardiomyopathy with conduction defects (DCM-CD), familial partial lipodystrophy (FPLD), atypical Werner's syndrome, Emery-Dreifuss muscular dystrophy (EDMD), and Hutchinson-Gilford progeria syndrome (HGPS) (9, 70, 77).The nuclear lamina also provides a scaffold for organizing nuclear pore complexes (NPCs) within the nuclear membrane (1). NPCs span the nuclear lamina and both the inner and outer nuclear membranes and serve as conduits for nuclear import and export (73). Nucleoporins that comprise the NPC are organized into subcomplexes that disassemble and reassemble duri...

Summary G proteins and their associated receptors process information from a variety of environmental stimuli to induce appropriate cellular responses. Generally speaking, each cell in a population responds within defined limits despite large variation in the expression of protein signaling components. Therefore we postulated that noise suppression is encoded within the signaling system. Using the yeast mating pathway as a model we evaluated the ability of a regulator of G protein signaling (RGS) protein to suppress noise. We found that the RGS protein Sst2 limits variability in transcription and morphogenesis in response to pheromone stimulation. While signal suppression is a result of both the GAP (GTPase accelerating) and receptor binding functions of Sst2, noise suppression requires only the GAP activity. Taken together our findings reveal a hitherto overlooked role of RGS proteins as noise suppressors, and demonstrate an ability to uncouple signal and noise in a prototypical stimulus-response pathway.

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...

Summary Background Septins are well known to form a boundary between mother and daughter cells in mitosis, but their role in other morphogenic states is poorly understood. Results Using microfluidics and live cell microscopy, coupled with new computational methods for image analysis, we investigated septin function during pheromone-dependent chemotropic growth in yeast. We show that septins colocalize with the regulator of G-protein signaling (RGS) Sst2, a GTPase-activating protein that dampens pheromone receptor signaling. We show further that the septin structure surrounds the polar cap, ensuring that cell growth is directed toward the source of pheromone. When RGS activity is abrogated, septins are partially disorganized. Under these circumstances the polar cap travels toward septin structures and away from sites of exocytosis, resulting in a loss of gradient tracking. Conclusion Septin organization is dependent on RGS protein activity. When assembled correctly, septins promote turning of the polar cap and proper tracking of a pheromone gradient.

We describe a mechanism for protein phosphatase 2A (PP2A) targeting to the androgen receptor (AR) and provide insight into the more general issue of kinase and phosphatase interactions with AR. Simian virus 40 (SV40) small t antigen (ST) binding to N-terminal HEAT repeats in the PP2A A subunit induces structural changes transduced to C-terminal HEAT repeats. This enables the C-terminal HEAT repeats in the PP2A A subunit, including HEAT repeat 13, to discriminate between androgen-and androgen antagonist-induced AR conformations. The PP2A-AR interaction was used to show that an AR mutant in prostate cancer cells (T877A) is activated by multiple ligands without acquiring the same conformation as that induced by androgen. The correlation between androgen binding to AR and increased phosphorylation of the activation function 1 (AF-1) region implies that changes in AR conformation or chaperone composition are causal to kinase access to phosphorylation sites. However, AF-1 phosphorylation sites are kinase accessible prior to androgen binding. This suggests that androgens can enhance the phosphorylation state of AR either by negatively regulating the ability of the ligand-binding domain to bind phosphatases or by inducing an AR conformation that is resistant to phosphatase action. SV40 ST subverts this mechanism by promoting the direct transfer of PP2A onto androgen-bound AR, resulting in multisite dephosphorylation.The nuclear receptor superfamily of transcription factors directs the expression of genes whose products regulate diverse biological pathways. The domain organization of nuclear receptors is conserved and includes an N-terminal activation function 1 (AF-1) region, a central DNA binding domain (DBD), and a C-terminal ligand-binding domain (LBD). Ligand binding to the LBD initiates a series of changes in nuclear receptor structure, chaperone composition, localization, transactivation potential, and protein half-life (t 1/2 ) (10,28,36,44). Understanding how ligand binding elicits these changes is fundamental to understanding nuclear receptor regulation and activity. Defining how ligands control nuclear receptor activity should also provide insight into certain disease mechanisms and aid in the design of drugs such as selective androgen receptor modulators and selective estrogen receptor modulators (4, 27).In addition to ligand binding, nuclear receptors can be regulated by signal transduction pathways. Kinases including those controlled by growth factor-dependent pathways act directly or indirectly on a variety of nuclear receptors (37). Kinases reported to regulate androgen receptor (AR)-dependent transcription include the mitogen-activated protein kinases (p42/ 44, p38, and Jun N-terminal protein kinase), protein kinase A, and protein kinase C (8). The mitogen-activated protein kinases p38 and Jun N-terminal protein kinase also regulate the nucleocytoplasmic distribution of AR (15). Determining exactly how kinases regulate nuclear receptor transcription activity has been challenging because of cross talk bet...

The androgen receptor (AR) has critical functions as a transcription factor in both normal and cancer cells, but the specific mechanisms that regulate its nuclear localization are not well defined. We found that an AR mutation commonly reported in prostate cancer generates an androgen-independent gain of function for nuclear import. The substitution, Thr877Ala, is within the ligand-binding domain, but the nuclear import gain of function is mediated by the bipartite nuclear localization signal (NLS) spanning the DNA-binding domain (DBD) and hinge region. Bipartite NLS activity depends on the structure provided by the DBD, and protein interactions with the bipartite NLS are repressed by the hinge region. The bipartite NLS is recognized by importin 7, a nuclear import receptor for several proteins. Importin 7 binding to AR, however, inhibits import by shielding the bipartite NLS. Androgen binding relieves the inhibition by inducing a switch that promotes exchange of importin 7 for karyopherin alpha import receptors. Importin 7 contributes to the regulation of AR import by restraining import until androgen is detected in the cytoplasm. N uclear import of proteins is mediated by cis-acting nuclear localization signals (NLSs) that usually contain either one or two clusters of basic amino acids (1). Import signals similar to the monopartite NLS in simian virus 40 (SV40) (PKKKRRV) and the bipartite NLS in nucleoplasmin (KRPAATKKAGQAKKKK) have been identified in hundreds of proteins and provide the specificity for import through interactions with nuclear import receptors (1). Three steps common to all NLS and transport receptor-mediated pathways are (i) receptor recognition of the NLS, (ii) translocation of the NLS-receptor complex through the nuclear pore complex (NPC), and (iii) dissociation of the NLS-receptor complex in the nucleoplasm (2). After release of the NLS-containing protein into the nucleoplasm, import receptors are exported to the cytoplasm for a new round of protein import.The import receptors that recognize NLSs in proteins are phylogenetically conserved. Within a species, import receptors are either members of the importin ␤ superfamily, which also includes structurally related export receptors (3) or the importin-␣ family of import receptors (4). The latter are termed karyopherin ␣ (KPNA) proteins. Importin ␤ superfamily members that mediate import are 95-to 125-kDa polypeptides composed of HEAT repeats, which were first recognized in Huntingtin, elongation factor 3, the PR65/A subunit of protein phosphatase 2A, and the lipid kinase TOR and can bind directly to NLSs. Importin ␤ proteins then undergo transient binding to nucleoporins during translocation through the NPC (5). Certain HEAT repeats within importin ␤ family members form a domain that can bind RanGTP, an interaction that promotes NLS cargo release from these receptors within the nucleus (6). KPNA proteins are encoded by seven genes in humans (7). They are polypeptides of ϳ60 kDa in size that are composed of Armadillo (ARM) repeats, which bin...

The sequestration of DNA within the membrane-bound nucleus is a defining characteristic of eukaryotic cells. Replication and transcription are therefore restricted to the nucleus, however, the regulation of these events relies on cytoplasmic processes including protein synthesis and signal transduction pathways. Because a variety of cellular activities depend on nuclear transport, researchers from diverse fields have found it useful to examine the nuclear localization of proteins of interest. Here we present some important technical considerations for studying nuclear and cytoplasmic localization, and provide guidance for quantifying protein levels using fluorescence microscopy and ImageJ software. We include discussion of the use of regions of interest and image segmentation for quantification of protein localization. Nucleocytoplasmic transport is fundamentally important for controlling protein levels and activity in the nucleus or cytoplasm, and quantitative analysis can provide insight into how biological output is achieved.