Wild‐type and specific mutant androgen receptor mediates transcription via 17β‐estradiol in sex hormone‐sensitive cancer cells

We previously encountered regulatory processes wherein dihydrotestosterone (DHT) exerted its inhibitory effect on parathyroid hormone‐related protein (PTHrP) gene repression through the estrogen receptor (ER)α, but not the androgen receptor (AR), in breast cancer MCF‐7 cells. Here, we investigated whether such aberrant ligand‐nuclear receptor (NR) interaction is present in prostate cancer LNCaP cells. First, we confirmed that LNCaP cells expressed large amounts of AR at negligible levels of ERα/β or progesterone receptor. Both suppression of PTHrP and activation of prostate‐specific antigen genes were observed after independent administration of 17β‐estradiol (E2), DHT, or R5020. Consistent with the notion that the LNCaP AR lost its ligand specificity due to a mutation (Thr‐Ala877), experiments with siRNA targeting the respective NR revealed that the AR monopolized the role of the mediator of shared hormone‐dependent regulation, which was invariably associated with nuclear translocation of this mutant AR. Microarray analysis of gene regulation by DHT, E2, or R5020 disclosed that more than half of the genes downstream of the AR (Thr‐Ala877) overlapped in the LNCaP cells. Of particular interest, we realized that the AR (wild‐type [wt]) and AR (Thr‐Ala877) were equally responsible for the E2‐AR interactions. Fluorescence microscopy experiments demonstrated that both EGFP‐AR (wt) and EGFP‐AR (Thr‐Ala877) were exclusively localized within the nucleus after E2 or DHT treatment. Furthermore, reporter assays revealed that some other cancer cells exhibited aberrant E2‐AR (wt) signaling similar to that in the LNCaP cells. We herein postulate the presence of entangled interactions between wt AR and E2 in certain hormone‐sensitive cancer cells. J. Cell. Physiol. 230: 1594–1606, 2015. © 2014 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.     

Decrease of GSK3β phosphorylation in the rat nucleus accumbens core enhances cocaine‐induced hyper‐locomotor activity

Glycogen synthase kinase 3β (GSK3β), which is abundantly present in the brain, is known to contribute to psychomotor stimulant‐induced locomotor behaviors. However, most studies have been focused in showing that GSK3β is able to attenuate psychomotor stimulants‐induced hyperactivity by increasing its phosphorylation levels in the nucleus accumbens (NAcc). So, here we examined in the opposite direction about the effects of decreased phosphorylation of GSK3β in the NAcc core on both basal and cocaine‐induced locomotor activity by a bilateral microinjection into this site of an artificially synthesized peptide, S9 (0.5 or 5.0 μg/μL), which contains sequences around N‐terminal serine 9 residue of GSK3β. We found that decreased levels of GSK3β phosphorylation in the NAcc core enhance cocaine‐induced hyper‐locomotor activity, while leaving basal locomotor activity unchanged. This is the first demonstration, to our knowledge, that the selective decrease of GSK3β phosphorylation levels in the NAcc core may contribute positively to cocaine‐induced locomotor activity, while this is not sufficient for the generation of locomotor behavior by itself without cocaine. Taken together, these findings importantly suggest that GSK3β may need other molecular targets which are co‐activated (or deactivated) by psychomotor stimulants like cocaine to contribute to generation of locomotor behaviors.     

The Mood‐Stabilizing Agent Valproate Inhibits the Activity of Glycogen Synthase Kinase‐3

Abstract : Valproic acid (VPA) is a potent broad‐spectrum anti‐epileptic with demonstrated efficacy in the treatment of bipolar affective disorder. It has previously been demonstrated that both VPA and lithium increase activator protein‐1 (AP‐1) DNA binding activity, but the mechanisms underlying these effects have not been elucidated. However, it is known that phosphorylation of c‐jun by glycogen synthase kinase (GSK)‐3β inhibits AP‐1 DNA binding activity, and lithium has recently been demonstrated to inhibit GSK‐3β. These results suggest that lithium may increase AP‐1 DNA binding activity by inhibiting GSK‐3β. In the present study, we sought to determine if VPA, like lithium, regulates GSK‐3. We have found that VPA concentration‐dependently inhibits both GSK‐3α and ‐3β, with significant effects observed at concentrations of VPA similar to those attained clinically. Incubation of intact human neuroblastoma SH‐SY5Y cells with VPA results in an increase in the subsequent in vitro recombinant GSK‐3β‐mediated ³²P incorporation into two putative GSK‐3 substrates (~85 and 200 kDa), compatible with inhibition of endogenous GSK‐3β by VPA. Consistent with GSK‐3β inhibition, incubation of SH‐SY5Y cells with VPA results in a significant time‐dependent increase in both cytosolic and nuclear β‐catenin levels. GSK‐3β plays a critical role in the CNS by regulating various cytoskeletal processes as well as long‐term nuclear events and is a common target for both lithium and VPA ; inhibition of GSK‐3β in the CNS may thus underlie some of the long‐term therapeutic effects of mood‐stabilizing agents.     

GSK‐3β activation index is a potential indicator for recurrent inflammation of chronic rhinosinusitis without nasal polyps

Chronic rhinosinusitis without nasal polyps (CRSsNP) is one of the most common otorhinolaryngologic diseases worldwide. However, the underlying mechanism remains unclear. In this study, the expression of glycogen synthase kinase 3 (GSK‐3) was quantitatively evaluated in patients with CRSsNP (n = 20) and healthy controls (n = 20). The mRNA levels of GSK‐3α and GSK‐3β were examined by qPCR, the immunoreactivities of GSK‐3β and nuclear factor‐κB (NF‐κB) were examined by immunohistochemistry (IHC) staining, and the protein levels of GSK‐3β, phospho‐GSK‐3β (p‐GSK‐3β, s9) and NF‐κB were examined using Western blot analysis. We found that GSK‐3 was highly expressed in both CRSsNP and control groups without significant difference in both GSK‐3β mRNA and protein levels. However, when compared with healthy control group, the GSK‐3β activation index, defined as the ratio of GSK‐3β over p‐GSK‐3β, was significantly decreased, whereas the NF‐κB protein abundance was significantly increased in CRSsNP group (P < 0.05). Strikingly, the GSK‐3β activation index, was highly correlated with NF‐κB protein level, as well as CT scores in CRSsNP group (P < 0.05). It was also highly correlated with the mRNA expressions of inflammation‐related genes, including T‐bet, IFN‐γ and IL‐4 in CRSsNP group (P < 0.05). Our findings suggest that GSK‐3β activation index, reflecting the inhibitory levels of GSK‐3β through phosphorylation, may be a potential indicator for recurrent inflammation of CRSsNP, and that the insufficient inhibitory phosphorylation of GSK‐3β may play a pivotal role in the pathogenesis of CRSsNP.     

GSK‐3β inhibition protects the rat heart from the lipopolysaccharide‐induced inflammation injury via suppressing FOXO3A activity

Sepsis‐induced cardiac dysfunction represents a main cause of death in intensive care units. Previous studies have indicated that GSK‐3β is involved in the modulation of sepsis. However, the signalling details of GSK‐3β regulation in endotoxin lipopolysaccharide (LPS)‐induced septic myocardial dysfunction are still unclear. Here, based on the rat septic myocardial injury model, we found that LPS could induce GSK‐3β phosphorylation at its active site (Y216) and up‐regulate FOXO3A level in primary cardiomyocytes. The FOXO3A expression was significantly reduced by GSK‐3β inhibitors and further reversed through β‐catenin knock‐down. This pharmacological inhibition of GSK‐3β attenuated the LPS‐induced cell injury via mediating β‐catenin signalling, which could be abolished by FOXO3A activation. In vivo, GSK‐3β suppression consistently improved cardiac function and relieved heart injury induced by LPS. In addition, the increase in inflammatory cytokines in LPS‐induced model was also blocked by inhibition of GSK‐3β, which curbed both ERK and NF‐κB pathways, and suppressed cardiomyocyte apoptosis via activating the AMP‐activated protein kinase (AMPK). Our results demonstrate that GSK‐3β inhibition attenuates myocardial injury induced by endotoxin that mediates the activation of FOXO3A, which suggests a potential target for the therapy of septic cardiac dysfunction.     

Identification of glycogen synthase kinase 3α as a therapeutic target in melanoma

Deregulated expression or activity of kinases can lead to melanomas, but often the particular kinase isoform causing the effect is not well established, making identification and validation of different isoforms regulating disease development especially important. To accomplish this objective, an siRNA screen was undertaken that which identified glycogen synthase kinase 3α (GSK3α) as an important melanoma growth regulator. Melanocytes and melanoma cell lines representing various stages of melanoma tumor progression expressed both GSK3α and GSK3β, but analysis of tumors in patients with melanoma showed elevated expression of GSK3α in 72% of samples, which was not observed for GSK3β. Furthermore, 80% of tumors in patients with melanoma expressed elevated levels of catalytically active phosphorylated GSK3α (pGSK3αY279), but not phosphorylated GSK3β (pGSK3βY216). siRNA‐mediated reduction in GSK3α protein levels reduced melanoma cell survival and proliferation, sensitized cells to apoptosis‐inducing agents and decreased xenografted tumor development by up to 56%. Mechanistically, inhibiting GSK3α expression using siRNA or the pharmacological agent AR‐A014418 arrested melanoma cells in the G0/G1 phase of the cell cycle and induced apoptotic death to retard tumorigenesis. Therefore, GSK3α is a key therapeutic target in melanoma.     

Nuclear export of ERK3 by a CRM1-dependent mechanism regulates its inhibitory action on cell cycle progression

Extracellular signal-regulated kinase 3 (ERK3) is an atypical member of the mitogen-activated protein kinase family of serine/threonine kinases. Little is known on the regulation of ERK3 function. Here, we report that ERK3 is constitutively localized in the cytoplasmic and nuclear compartments. In contrast to other mitogen-activated protein kinases, the cellular distribution of ERK3 remains unchanged in response to common mitogenic or stress stimuli and is independent of the enzymatic activity or phosphorylation of the kinase. The cytoplasmic localization of ERK3 is directed by a CRM1-dependent nuclear export mechanism. Treatment of cells with leptomycin B causes the nuclear accumulation of ERK3 in a high percentage of cells. Moreover, ectopic expression of CRM1 promotes the cytoplasmic relocalization of ERK3, whereas overexpression of snurportin 1, which binds CRM1 with high affinity, inhibits the nuclear export of ERK3. We also show that CRM1 binds to ERK3 in vitro. Importantly, we show that enforced localization of ERK3 in the nucleus or cytoplasm markedly attenuates the ability of the kinase to induce cell cycle arrest in fibroblasts. Our results suggest that nucleocytoplasmic shuttling of ERK3 is required for its negative regulatory effect on cell cycle progression.     

Characterization of an Importin α/β‐recognized nuclear localization signal in β‐dystroglycan

β‐dystroglycan (β‐DG) is a widely expressed transmembrane protein that plays important roles in connecting the extracellular matrix to the cytoskeleton, and thereby contributing to plasma membrane integrity and signal transduction. We previously observed nuclear localization of β‐DG in cultured cell lines, implying the existence of a nuclear targeting mechanism that directs it to the nucleus instead of the plasma membrane. In this study, we delineate the nuclear import pathway of β‐DG, characterizing a functional nuclear localization signal (NLS) in the β‐DG cytoplasmic domain, within amino acids 776–782. The NLS either alone or in the context of the whole β‐DG protein was able to target the heterologous GFP protein to the nucleus, with site‐directed mutagenesis indicating that amino acids R⁷⁷⁹ and K⁷⁸⁰ are critical for NLS functionality. The nuclear transport molecules Importin (Imp)α and Impβ bound with high affinity to the NLS of β‐DG and were found to be essential for NLS‐dependent nuclear import in an in vitro reconstituted nuclear transport assay; cotransfection experiments confirmed the dependence on Ran for nuclear accumulation. Intriguingly, experiments suggested that tyrosine phosphorylation of β‐DG may result in cytoplasmic retention, with Y⁸⁹² playing a key role. β‐DG thus follows a conventional Impα/β‐dependent nuclear import pathway, with important implications for its potential function in the nucleus. J. Cell. Biochem. 110: 706–717, 2010. © 2010 Wiley‐Liss, Inc.     

Nuclear exclusion of the androgen receptor by melatonin

Androgen receptors (AR) play a crucial role in androgen-mediated processes and prostate cancer progression. The pineal hormone melatonin attenuates the androgen-dependent growth of benign and cancer prostate epithelial cells in vitro and may reverse clinical resistance to androgen ablation therapy in patients progressing on gonadotropin releasing hormone (GnRH) analogue. Where along the AR cascade does melatonin act remains to be determined. The effects of melatonin on AR localization, level and activity were assessed using androgen-insensitive prostate carcinoma PC3 cells stably transfected with a wild-type AR-expressing vector (PC3-AR).AR was localized to the PC3-AR cell nucleus in the absence of dihydrotestosterone (DHT). Melatonin caused a robust exclusion of the AR from the cell nucleus to the cytoplasm. The nuclear export inhibitor, leptomycin B prevented this process. The exclusion was selective since melatonin had no such effect on the nuclear localization of estrogen receptors alpha (ERalpha) in these cells.Melatonin also caused nuclear exclusion of the AR in the presence of DHT. In addition, it attenuated androgen induced reporter gene activity in PC3 cells co-transfected with the human AR and AR reporter plasmids. Elevated androgen concentrations counteracted melatonin's effects. Melatonin did not decrease AR level or androgen binding in the cells.The nuclear localization of the AR is a hallmark of its cellular activity. These data point to AR nuclear exclusion as a possible mechanism to attenuate androgen responses in target tissues.     

β‐arrestin2/miR‐155/GSK3β regulates transition of 5′‐azacytizine‐induced Sca‐1‐positive cells to cardiomyocytes

Stem‐cell antigen 1–positive (Sca‐1+) cardiac stem cells (CSCs), a vital kind of CSCs in humans, promote cardiac repair in vivo and can differentiate to cardiomyocytes with 5′‐azacytizine treatment in vitro. However, the underlying molecular mechanisms are unknown. β‐arrestin2 is an important scaffold protein and highly expressed in the heart. To explore the function of β‐arrestin2 in Sca‐1+ CSC differentiation, we used β‐arrestin2–knockout mice and overexpression strategies. Real‐time PCR revealed that β‐arrestin2 promoted 5′‐azacytizine‐induced Sca‐1+ CSC differentiation in vitro. Because the microRNA 155 (miR‐155) may regulate β‐arrestin2 expression, we detected its role and relationship with β‐arrestin2 and glycogen synthase kinase 3 (GSK3β), another probable target of miR‐155. Real‐time PCR revealed that miR‐155, inhibited by β‐arrestin2, impaired 5′‐azacytizine‐induced Sca‐1+ CSC differentiation. On luciferase report assay, miR‐155 could inhibit the activity of β‐arrestin2 and GSK3β, which suggests a loop pathway between miR‐155 and β‐arrestin2. Furthermore, β‐arrestin2‐knockout inhibited the activity of GSK3β. Akt, the upstream inhibitor of GSK3β, was inhibited in β‐arrestin2‐Knockout mice, so the activity of GSK3β was regulated by β‐arrestin2 not Akt. We transplanted Sca‐1+ CSCs from β‐arrestin2‐knockout mice to mice with myocardial infarction and found similar protective functions as in wild‐type mice but impaired arterial elastance. Furthermore, low level of β‐arrestin2 agreed with decreased phosphorylation of AKT and increased phophorylation of GSK3β, similar to in vitro findings. The β‐arrestin2/miR‐155/GSK3β pathway may be a new mechanism with implications for treatment of heart disease.     

Glycogen synthase kinase 3β in the nucleus accumbens core mediates cocaine‐induced behavioral sensitization

Glycogen synthase kinase 3β (GSK‐3β) is a ubiquitous serine/threonine protein kinase involved in a number of signaling pathways. Previous studies have demonstrated a role for GSK‐3β in the synaptic plasticity underlying dopamine‐associated behaviors and diseases. Drug sensitization is produced by repeated exposure to the drug and is thought to reflect neuroadaptations that contribute to addiction. However, the role of GSK‐3β in cocaine‐induced behavior sensitization has not been examined. The present study investigated the effects of chronic cocaine exposure on GSK‐3β activity in the nucleus accumbens (NAc) and determined whether changes in GSK‐3β activity in the NAc are associated with cocaine‐induced locomotor sensitization. We also explored whether blockade of GSK‐3β activity in the NAc inhibits the initiation and expression of cocaine‐induced locomotor sensitization in rats using systemic or brain region‐specific administration of the GSK‐3β inhibitors lithium chloride (LiCl) and SB216763. GSK‐3β activity in the NAc core, but not NAc shell, increased after chronic cocaine (10 mg/kg, i.p.) administration. The initiation and expression of cocaine‐induced locomotor sensitization was attenuated by systemic administration of LiCl (100 mg/kg, i.p.) or direct infusion of SB216763 (1 ng/side) into the NAc core, but not NAc shell. Collectively, these results indicate that GSK‐3β activity in the NAc core, but not NAc shell, mediates the initiation and expression of cocaine‐induced locomotor sensitization, suggesting that GSK‐3β may be a potential target for the treatment of cocaine addiction.     

Rg1 protects H9C2 cells from high glucose‐/palmitate‐induced injury via activation of AKT/GSK‐3β/Nrf2 pathway

Our previous studies have assessed ginsenoside Rg1 (Rg1)‐mediated protection in a type 1 diabetes rat model. To uncover the mechanism through which Rg1 protects against cardiac injury induced by diabetes, we mimicked diabetic conditions by culturing H9C2 cells in high glucose/palmitate. Rg1 had no toxic effect, and it alleviated the high glucose/palmitate damage in a dose‐dependent manner, as indicated by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide assay and lactate dehydrogenase release to the culture medium. Rg1 prevented high glucose/palmitate‐induced cell apoptosis, assessed using cleaved caspase‐3 and terminal deoxynucleotidyl transferase dUTP nick end labelling staining. Rg1 also reduced high glucose‐/palmitate‐induced reactive oxygen species formation and increased intracellular antioxidant enzyme activity. We found that Rg1 activates protein kinase B (AKT)/glycogen synthase kinase‐3 (GSK‐3β) pathway and antioxidant nuclear factor erythroid 2‐related factor 2 (Nrf2) pathway, indicated by increased phosphorylation of AKT and GSK‐3β, and nuclear translocation of Nrf2. We used phosphatidylinositol‐3‐kinase inhibitor Ly294002 to block the activation of the AKT/GSK‐3β pathway and found that it partially reversed the protection by Rg1 and decreased Nrf2 pathway activation. The results suggest that Rg1 exerts a protective effect against high glucose and palmitate damage that is partially AKT/GSK‐3β/Nrf2‐mediated. Further studies are required to validate these findings using primary cardiomyocytes and animal models of diabetes.     

Hsp105α suppresses Adriamycin-induced cell death via nuclear localization signal-dependent nuclear accumulation

Heat shock protein 105 (Hsp105) is a molecular chaperone, and the isoforms Hsp105α and Hsp105β exhibit distinct functions with different subcellular localizations. Hsp105β localizes in the nucleus and induces the expression of the major heat shock protein Hsp70, whereas cytoplasmic Hsp105α is less effective in inducing Hsp70 expression. Hsp105 shuttles between the cytoplasm and the nucleus; the subcellular localization is governed by the relative activities of the nuclear localization signal (NLS) and nuclear export signal (NES). Here, we show that nuclear accumulation of Hsp105α but not Hsp105β is involved in Adriamycin (ADR) sensitivity. Knockdown of Hsp105α induces cell death at low ADR concentration, at which ADR is less effective in inducing cell death in the presence of Hsp105α. Of note, Hsp105 is localized in the nucleus under these conditions, even though Hsp105β is not expressed, indicating that Hsp105α accumulates in the nucleus in response to ADR treatment. The exogenously expressed Hsp105α but not its NLS mutant localizes in the nucleus of ADR-treated cells. In addition, the expression level of the nuclear export protein chromosomal maintenance 1 (CRM1) was decreased by ADR treatment of cells, and CRM1 knockdown caused nuclear accumulation of Hsp105α both in the presence and absence of ADR. These results indicating that Hsp105α accumulates in the nucleus in a manner dependent on the NLS activity via the suppression of nuclear export. Our findings suggest a role of nuclear Hsp105α in the sensitivity against DNA-damaging agents in tumor cells.     

Inhibition of glycogen synthase kinase-3β counteracts ligand-independent activity of the androgen receptor in castration resistant prostate cancer

In order to generate genomic signals, the androgen receptor (AR) has to be transported into the nucleus upon androgenic stimuli. However, there is evidence from in vitro experiments that in castration-resistant prostate cancer (CRPC) cells the AR is able to translocate into the nucleus in a ligand-independent manner. The recent finding that inhibition of the glycogen-synthase-kinase 3β (GSK-3β) induces a rapid nuclear export of the AR in androgen-stimulated prostate cancer cells prompted us to analyze the effects of a GSK-3β inhibition in the castration-resistant LNCaP sublines C4-2 and LNCaP-SSR. Both cell lines exhibit high levels of nuclear AR in the absence of androgenic stimuli. Exposure of these cells to the maleimide SB216763, a potent GSK-3β inhibitor, resulted in a rapid nuclear export of the AR even under androgen-deprived conditions. Moreover, the ability of C4-2 and LNCaP-SSR cells to grow in the absence of androgens was diminished after pharmacological inhibition of GSK-3β in vitro. The ability of SB216763 to modulate AR signalling and function in CRPC in vivo was additionally demonstrated in a modified chick chorioallantoic membrane xenograft assay after systemic delivery of SB216763. Our data suggest that inhibition of GSK-3β helps target the AR for export from the nucleus thereby diminishing the effects of mislocated AR in CRPC cells. Therefore, inhibition of GSK-3β could be an interesting new strategy for the treatment of CRPC.     

GSKIP,an inhibitor of GSK3β, mediates the N‐cadherin/β‐catenin pool in the differentiation of SH‐SY5Y cells

Emerging evidence has shown that GSK3β plays a pivotal role in regulating the specification of axons and dendrites. Our previous study has shown a novel GSK3β interaction protein (GSKIP) able to negatively regulate GSK3β in Wnt signaling pathway. To further characterize how GSKIP functions in neurons, human neuroblastoma SH‐SY5Y cells treated with retinoic acid (RA) to differentiate to neuron‐like cells was used as a model. Overexpression of GSKIP prevents neurite outgrowth in SH‐SY5Y cells. GSKIP may affect GSK3β activity on neurite outgrowth by inhibiting the specific phosphorylation of tau (ser396). GSKIP also increases β‐catenin in the nucleus and raises the level of cyclin D1 to promote cell‐cycle progression in SH‐SY5Y cells. Additionally, overexpression of GSKIP downregulates N‐cadherin expression, resulting in decreased recruitment of β‐catenin. Moreover, depletion of β‐catenin by small interfering RNA, neurite outgrowth is blocked in SH‐SY5Y cells. Altogether, we propose a model to show that GSKIP regulates the functional interplay of the GSK3β/β‐catenin, β‐catenin/cyclin D1, and β‐catenin/N‐cadherin pool during RA signaling in SH‐SY5Y cells. J. Cell. Biochem. 108: 1325–1336, 2009. © 2009 Wiley‐Liss, Inc.     

Transport of galectin-3 between the nucleus and cytoplasm. II. Identification of the signal for nuclear export

Galectin-3, a factor involved in the splicing of pre-mRNA, shuttles between the nucleus and the cytoplasm. Previous studies have shown that incubation of fibroblasts with leptomycin B resulted in the accumulation of galectin-3 in the nucleus, suggesting that the export of galectin-3 from the nucleus may be mediated by the CRM1 receptor. A candidate nuclear export signal fitting the consensus sequence recognized by CRM1 can be found between residues 240 and 255 of the murine galectin-3 sequence. This sequence was engineered into the pRev(1.4) reporter system, in which candidate sequences can be tested for nuclear export activity in terms of counteracting the nuclear localization signal present in the Rev(1.4) protein. Rev(1.4)-galectin-3(240-255) exhibited nuclear export activity that was sensitive to inhibition by leptomycin B. Site-directed mutagenesis of Leu247 and Ile249 in the galectin-3 nuclear export signal decreased nuclear export activity, consistent with the notion that these two positions correspond to the critical residues identified in the nuclear export signal of the cAMP-dependent protein kinase inhibitor. The nuclear export signal activity was also analyzed in the context of a full-length galectin-3 fusion protein; galectin-3(1-263; L247A) showed more nuclear localization than wild-type, implicating Leu247 as critical to the function of the nuclear export signal. These results indicate that residues 240-255 of the galectin-3 polypeptide contain a leucine-rich nuclear export signal that overlaps with the region (residues 252-258) identified as important for nuclear localization.