Nuclear import of NLS- RARα is mediated by importin α/ß.

The promyelocytic leukemia-retinoic acid receptor α (PML/RARα) is hypothesized to play a vital role in the pathogenesis of acute promyelocytic leukemia (APL). A previous study has demonstrated that PML/RARα is cleaved by neutrophil elastase (NE) in early myeloid cells, which leads to an increase in the nuclear localization signal (NLS) in RARα and in the incidence of APL. In this study, we explored the effects of NLS-RARα on acute myeloid leukemia (AML) cells and studied the mechanism of its localization. LV-NLS-RARα recombinant lentivirus and negative control LV-NC lentivirus were transfected into HL-60 cells and U937 cells while mutant NLS-RARα were transfected into U937 cells, and all groups were treated with 1α, 25-dihydroxyvitamin D3(1,25D3). The results showed that NLS-RARα was located mainly in the nucleus while mutant NLS-RARα was located in the cytoplasm. Overexpression of NLS-RARα downregulated the expression of CD11b, CD11c, CD14, and three forms of CEBPß compared to the overexpression of NC and mutant NLS-RARα. It was speculated that the abnormal localization of NLS-RARα was mediated via importin-α/ß in the pathogenesis of APL. By producing point mutations in the two NLSs in NLS-RARα, we showed that the nuclear import of NLS-RARα was mainly dependent on the NLS of the RARα portion. Subsequently, we found that importin-α1 (KPNA2)/importin-ß1 (KPNB1) participates in the nuclear transport of NLS-RARα. Taken together, abnormal localization of NLS-RARα blocks the differentiation of APL cells, and nuclear localization of NLS-RARα depends on NLS of the RARα portion and is mediated via binding with importin-α/ß.

Nuclear-cytoplasmic Shuttling in Chronic Myeloid Leukemia: Implications in Leukemia Maintenance and Therapy.

Nuclear-cytoplasmic shuttling is a highly regulated and complex process, which involves both proteins and nucleic acids. Changes in cellular compartmentalization of various proteins, including oncogenes and tumor suppressors, affect cellular behavior, promoting or inhibiting proliferation, apoptosis and sensitivity to therapies. In this review, we will recapitulate the role of various shuttling components in Chronic Myeloid Leukemia and we will provide insights on the potential role of shuttling proteins as therapeutic targets.

Identification of a putative nuclear localization signal in the tumor suppressor maspin sheds light on its nuclear import regulation.

The tumor suppressor activity of maspin (mammary serine protease inhibitor) has been associated with its nuclear localization. In this study we explore the regulation of maspin nuclear translocation. An in vitro nuclear import assay suggested that maspin can passively enter the nucleus. However, in silico analysis identified a putative maspin nuclear localization signal (NLS), which was able to mediate the nuclear translocation of a chimeric protein containing this NLS fused to five green fluorescent protein molecules in tandem (5GFP). Dominant-negative Ran-GTPase mutants RanQ69L or RanT24N suppressed this process. Unexpectedly, the full-length maspin fused to 5GFP failed to enter the nucleus. As maspin's putative NLS is partially hidden in its three-dimensional structure, we suggest that maspin nuclear transport could be conformationally regulated. Our results suggest that maspin nuclear translocation involves both passive and active mechanisms.

Nuclear localization and phosphorylation modulate pathological effects of alpha-synuclein.

Alpha-synuclein (aSyn) is a central player in Parkinson's disease (PD) but the precise molecular mechanisms underlying its pathogenicity remain unclear. It has recently been suggested that nuclear aSyn may modulate gene expression, possibly via interactions with DNA. However, the biological behavior of aSyn in the nucleus and the factors affecting its transcriptional role are not known. Here, we investigated the mechanisms underlying aSyn-mediated transcription deregulation by assessing its effects in the nucleus and the impact of phosphorylation in these dynamics. We found that aSyn induced severe transcriptional deregulation, including the downregulation of important cell cycle-related genes. Importantly, transcriptional deregulation was concomitant with reduced binding of aSyn to DNA. By forcing the nuclear presence of aSyn in the nucleus (aSyn-NLS), we found the accumulation of high molecular weight aSyn species altered gene expression and reduced toxicity when compared with the wild-type or exclusively cytosolic protein. Interestingly, nuclear localization of aSyn, and the effect on gene expression and cytotoxicity, was also modulated by phosphorylation on serine 129. Thus, we hypothesize that the role of aSyn on gene expression and, ultimately, toxicity, may be modulated by the phosphorylation status and nuclear presence of different aSyn species. Our findings shed new light onto the subcellular dynamics of aSyn and unveil an intricate interplay between subcellular location, phosphorylation and toxicity, opening novel avenues for the design of future strategies for therapeutic intervention in PD and other synucleinopathies.

Nuclear localization signal targeting to macronucleus and micronucleus in binucleated ciliate Tetrahymena thermophila.

Ciliated protozoa possess two morphologically and functionally distinct nuclei: a macronucleus (MAC) and a micronucleus (MIC). The MAC is transcriptionally active and functions in all cellular events. The MIC is transcriptionally inactive during cell growth, but functions in meiotic events to produce progeny nuclei. Thus, these two nuclei must be distinguished by the nuclear proteins required for their distinct functions during cellular events such as cell proliferation and meiosis. To understand the mechanism of the nuclear transport specific to either MAC or MIC, we identified specific nuclear localization signals (NLSs) in two MAC- and MIC-specific nuclear proteins, macronuclear histone H1 and micronuclear linker histone-like protein (Mlh1), respectively. By expressing GFP-fused fragments of these proteins in Tetrahymena thermophila cells, two distinct regions in macronuclear histone H1 protein were assigned as independent MAC-specific NLSs and two distinct regions in Mlh1 protein were assigned as independent MIC-specific NLSs. These NLSs contain several essential lysine residues responsible for the MAC- and MIC-specific nuclear transport, but neither contains any consensus sequence with known monopartite or bipartite NLSs in other model organisms. Our findings contribute to understanding how specific nuclear targeting is achieved to perform distinct nuclear functions in binucleated ciliates.

The role of nuclear NS1 protein in highly pathogenic H5N1 influenza viruses.

The non-structural protein (NS1) of influenza A viruses (IAV) performs multiple functions during viral infection. NS1 contains two nuclear localization signals (NLS): NLS1 and NLS2. The NS1 protein is located predominantly in the nucleus during the early stages of infection and subsequently exported to the cytoplasm. A nonsense mutation that results in a large deletion in the carboxy-terminal region of the NS1 protein that contains the NLS2 domain was found in some IAV subtypes, including highly pathogenic avian influenza (HPAI) H7N9 and H5N1 viruses. We introduced different mutations into the NLS domains of NS1 proteins in various strains of IAV, and demonstrated that mutation of the NLS2 region in the NS1 protein of HPAI H5N1 viruses severely affects its nuclear localization pattern. H5N1 viruses expressing NS1 protein that is unable to localize to the nucleus are less potent in antagonizing cellular antiviral responses than viruses expressing wild-type NS1. However, no significant difference was observed with respect to viral replication and pathogenesis. In contrast, the replication and antiviral defenses of H1N1 viruses are greatly attenuated when nuclear localization of the NS1 protein is blocked. Our data reveals a novel functional plasticity for NS1 proteins among different IAV subtypes.

The truncated NLR protein TIR-NBS13 is a MOS6/IMPORTIN-α3 interaction partner required for plant immunity.

Importin-α proteins mediate the translocation of nuclear localization signal (NLS)-containing proteins from the cytoplasm into the nucleus through nuclear pore complexes (NPCs). Genetically, Arabidopsis IMPORTIN-α3/MOS6 (MODIFIER OF SNC1, 6) is required for basal plant immunity and constitutive disease resistance activated in autoimmune mutant snc1 (suppressor of npr1-1, constitutive 1), suggesting that MOS6 plays a role in the nuclear import of proteins involved in plant defense signaling. Here, we sought to identify and characterize defense-regulatory cargo proteins and interaction partners of MOS6. We conducted both in silico database analyses and affinity purification of functional epitope-tagged MOS6 from pathogen-challenged stable transgenic plants coupled with mass spectrometry. We show that among the 13 candidate MOS6 interactors we selected for further functional characterization, the TIR-NBS-type protein TN13 is required for resistance against Pseudomonas syringae pv. tomato (Pst) DC3000 lacking the type-III effector proteins AvrPto and AvrPtoB. When expressed transiently in N. benthamiana leaves, TN13 co-immunoprecipitates with MOS6, but not with its closest homolog IMPORTIN-α6, and localizes to the endoplasmic reticulum (ER), consistent with a predicted N-terminal transmembrane domain in TN13. Our work uncovered the truncated NLR protein TN13 as a component of plant innate immunity that selectively binds to MOS6/IMPORTIN-α3 in planta. We speculate that the release of TN13 from the ER membrane in response to pathogen stimulus, and its subsequent nuclear translocation, is important for plant defense signal transduction.

The Effector Domain of MARCKS Is a Nuclear Localization Signal that Regulates Cellular PIP2 Levels and Nuclear PIP2 Localization.

Translocation to the nucleus of diacylglycerol kinase (DGK)- ζ is dependent on a sequence homologous to the effector domain of Myristoylated Alanine Rich C-Kinase Substrate (MARCKS). These data would suggest that MARCKS could also localize to the nucleus. A single report demonstrated immunofluorescence staining of MARCKS in the nucleus; however, further experimental evidence confirming the specific domain responsible for this localization has not been reported. Here, we report that MARCKS is present in the nucleus in GBM cell lines. We then over-expressed wild-type MARCKS (WT) and MARCKS with the effector domain deleted (ΔED), both tagged with V5-epitope in a GBM cell line with low endogenous MARCKS expression (U87). We found that MARCKS-WT localized to the nucleus, while the MARCKS construct without the effector domain remained in the cytoplasm. We also found that over-expression of MARCKS-WT resulted in a significant increase in total cellular phosphatidyl-inositol (4,5) bisphosphate (PIP2) levels, consistent with prior evidence that MARCKS can regulate PIP2 levels. We also found increased staining for PIP2 in the nucleus with MARCKS-WT over-expression compared to MARCKS ΔED by immunofluorescence. Interestingly, we observed MARCKS and PIP2 co-localization in the nucleus. Lastly, we found changes in gene expression when MARCKS was not present in the nucleus (MARCKS ΔED). These data indicate that the MARCKS effector domain can function as a nuclear localization signal and that this sequence is critical for the ability of MARCKS to regulate PIP2 levels, nuclear localization, and gene expression. These data suggests a novel role for MARCKS in regulating nuclear functions such as gene expression.

A Doubletime Nuclear Localization Signal Mediates an Interaction with Bride of Doubletime to Promote Circadian Function.

Doubletime (DBT) has an essential circadian role in Drosophila melanogaster because it phosphorylates Period (PER). To determine if DBT antagonism can produce distinct effects in the cytosol and nucleus, forms of a dominant negative DBT(K/R) with these 2 alternative localizations were produced. DBT has a putative nuclear localization signal (NLS), and mutation of this signal confers cytosolic localization of DBT in the lateral neurons of Drosophila clock cells in the brain. By contrast, addition of a strong NLS domain (e.g., SV40 NLS) to DBT's C terminus leads to more nuclear localization. Expression of DBT(K/R) with the mutated NLS (DBT(K/R) NLS(-)) using a timGAL4 driver does not alter the circadian period of locomotor activity, and the daily oscillations of PER detected by immunoblot and immunofluorescence persist, like those of wild-type flies. By contrast, expression of DBT(K/R) with the strong NLS (DBT(K/R) stNLS) using the timGAL4 driver lengthens period more strongly than DBT(K/R), with damped oscillations of PER phosphorylation and localization. Both DBT(K/R) and DBT(WT) without the NLS fail to interact with Bride of Doubletime (BDBT) protein, which is related to FK506-binding proteins and shown to interact with DBT to enhance its circadian function. This result suggests that the DBT(K/R) NLS(-) has lost its dominant negative property because it does not form normal clock protein complexes. DBT(WT) proteins with the same changes (NLS(-) and stNLS) also produce equivalent changes in localization that do not produce opposite period phenotypes. Additionally, a DBT(K/R) protein with both the stNLS and NLS(-) mutation does not affect circadian period, although it is nuclear, demonstrating that the lack of a dominant negative for the DBT(K/R) NLS(-) is not due to failure to localize to nuclei. Finally, bdbt RNAi increases the cytosolic localization of DBT(K/R) but not of DBT(WT), suggesting a role for BDBT in DBT kinase-dependent nuclear localization of DBT.

Role of a nuclear localization signal on the minor capsid proteins VP2 and VP3 in BKPyV nuclear entry.

BK Polyomavirus (BKPyV) is a ubiquitous nonenveloped human virus that can cause severe disease in immunocompromised populations. After internalization into renal proximal tubule epithelial cells, BKPyV traffics through the ER and enters the cytosol. However, it is unclear how the virus enters the nucleus. In this study, we elucidate a role for the nuclear localization signal located on the minor capsid proteins VP2 and VP3 during infection. Site-directed mutagenesis of a single lysine in the basic region of the C-terminus of the minor capsid proteins abrogated their nuclear localization, and the analogous genomic mutation reduced infectivity. Additionally, through use of the inhibitor ivermectin and knockdown of importin ß1, we found that the importin α/ß pathway is involved during infection. Overall these data are the first to show the significance of the NLS of the BKPyV minor capsid proteins during infection in a natural host cell.

Functional analysis of the C-terminal region of human adenovirus E1A reveals a misidentified nuclear localization signal.

The immortalizing function of the human adenovirus 5 E1A oncoprotein requires efficient localization to the nucleus. In 1987, a consensus monopartite nuclear localization sequence (NLS) was identified at the C-terminus of E1A. Since that time, various experiments have suggested that other regions of E1A influence nuclear import. In addition, a novel bipartite NLS was recently predicted at the C-terminal region of E1A in silico. In this study, we used immunofluorescence microscopy and co-immunoprecipitation analysis with importin-α to verify that full nuclear localization of E1A requires the well characterized NLS spanning residues 285-289, as well as a second basic patch situated between residues 258 and 263 ((258)RVGGRRQAVECIEDLLNEPGQPLDLSCKRPRP(289)). Thus, the originally described NLS located at the C-terminus of E1A is actually a bipartite signal, which had been misidentified in the existing literature as a monopartite signal, altering our understanding of one of the oldest documented NLSs.

Nuclear import of human histone lysine-specific demethylase LSD1.

Upregulation and nuclear retention of the human histone demethylase LSD1 are correlated with aggressiveness and poor outcome of several cancer types, but the molecular mechanism of LSD1 nuclear import remains unclear. Here, we found that the N-terminal flexible region of LSD1 contains a nuclear localization signal (NLS), (112)RRKRAK(117). Mutation or deletion of the NLS completely abolished the nuclear import of LSD1, suggesting the motif is a bona fide NLS. More importantly, our GST pull-down assay showed that LSD1 physically interacts with three proteins of importin α family. In addition, our data suggest that the nuclear localization of LSD1 via the NLS is not a cell-type specific event. Thus, these findings demonstrate for the first time that the NLS motif within the N-terminal flexible domain of LSD1 is critical for its nuclear localization via interaction with importin α proteins.

The R35 residue of the influenza A virus NS1 protein has minimal effects on nuclear localization but alters virus replication through disrupting protein dimerization.

The influenza A virus NS1 protein has a nuclear localization sequence (NLS) in the amino terminal region. This NLS overlaps sequences that are important for RNA binding as well as protein dimerization. To assess the significance of the NS1 NLS on influenza virus replication, the NLS amino acids were individually mutated to alanines and recombinant viruses encoding these mutations were rescued. Viruses containing NS1 proteins with mutations at R37, R38 and K41 displayed minimal changes in replication or NS1 protein nuclear localization. Recombinant viruses encoding NS1 R35A were not recovered but viruses containing second site mutations at position D39 in addition to the R35A mutation were isolated. The mutations at position 39 were shown to partially restore NS1 protein dimerization but had minimal effects on nuclear localization. These data indicate that the amino acids in the NS1 NLS region play a more important role in protein dimerization compared to nuclear localization.

Identification of a functional nuclear localization signal within the human USP22 protein.

Ubiquitin-specific processing enzyme 22 (USP22), a member of the deubiquitinase family, is over-expressed in most human cancers and has been implicated in tumorigenesis. Because it is an enzymatic subunit of the human SAGA transcriptional cofactor, USP22 deubiquitylates histone H2A and H2B in the nucleus, thus participating in gene regulation and cell-cycle progression. However, the mechanisms regulating its nuclear translocation have not yet been elucidated. It was here demonstrated that USP22 is imported into the nucleus through a mechanism mediated by nuclear localization signal (NLS). The bipartite NLS sequence KRELELLKHNPKRRKIT (aa152-168), was identified as the functional NLS for its nuclear localization. Furthermore, a short cluster of basic amino acid residues KRRK within this bipartite NLS plays the primary role in nuclear localization and is evolutionarily conserved in USP22 homologues. In the present study, a functional NLS and the minimal sequences required for the active targeting of USP22 to the nucleus were identified. These findings may provide a molecular basis for the mechanism underlying USP22 nuclear trafficking and function.

Transportin-1 and Transportin-2: protein nuclear import and beyond.

Nearly 20 years after its identification as a new ß-karyopherin mediating the nuclear import of the RNA-binding protein hnRNP A1, Transportin-1 is still commonly overlooked in comparison with its best known cousin, Importin-ß. Transportin-1 is nonetheless a considerable player in nucleo-cytoplasmic transport. Over the past few years, significant progress has been made in the characterization of the nuclear localization signals (NLSs) that Transportin-1 recognizes, thereby providing the molecular basis of its diversified repertoire of cargoes. The recent discovery that mutations in the Transportin-dependent NLS of FUS cause mislocalization of this protein and result in amyotrophic lateral sclerosis illustrates the importance of Transportin-dependent import for human health. Besides, new functions of Transportin-1 are emerging in processes other than nuclear import. Here, we summarize what is known about Transportin-1 and the related ß-karyopherin Transportin-2.

Targeting nuclear import shuttles, importins/karyopherins alpha by a peptide mimicking the NFκB1/p50 nuclear localization sequence.

BACKGROUND: We recently reported that a bifunctional nuclear transport modifier (NTM), cSN50.1 peptide, reduced atherosclerosis, plasma cholesterol, triglycerides, and glucose along with liver fat and inflammatory markers, in a murine model of familial hypercholesterolemia. We determined that cSN50.1 improved lipid homeostasis by modulating nuclear transport of sterol regulatory element-binding proteins through interaction with importin ß. Previous studies established that cSN50.1 and related NTMs also modulate nuclear transport of proinflammatory transcription factors mediated by binding of their nuclear localization sequences (NLSs) to importins/karyopherins α. However, selectivity and specificity of NTMs for importins/karyopherins α were undetermined. METHODS AND RESULTS: We analyzed interaction of the NTM hydrophilic module, N50 peptide, derived from the NLS of NFκB1/p50, with endogenous human importins/karyopherins α to determine the mechanism of NTM modulation of importin α-mediated nuclear transport. We show that N50 peptide forms stable complexes with multiple importins/karyopherins α. However, only interaction with importin α5 (Imp α5) displayed specific, high-affinity binding. The 2:1 stoichiometry of the N50-Imp α5 interaction (KD1 = 73 nmol/L, KD2 = 140 nmol/L) indicated occupancy of both major and minor NLS binding pockets. Utilizing in silico 3-dimensional (3-D) docking models and comparative structural analysis, we identified a structural component of the Imp α5 major NLS binding pocket that may stabilize N50 binding. Imp α5 also displayed rapid stimulus-induced turnover, which could influence its availability for nuclear transport during the inflammatory response. CONCLUSIONS: These results provide direct evidence that N50 peptide selectively targets Imp α5, encouraging further refinement of NLS-derived peptides as new tools to modulate inflammatory disorders.

Nuclear Respiratory Factor 2ß (NRF-2ß) recruits NRF-2α to the nucleus by binding to importin-α:ß via an unusual monopartite-type nuclear localization signal.

Nuclear respiratory factor 2 (NRF-2) is a mammalian transcription factor composed of two distinct and unrelated proteins: NRF-2α, which binds to DNA through its Ets domain, and NRF-2ß, which contains the transcription activation domain. The activity of NRF-2 in neurons is regulated by nuclear localization; however, the mechanism by which NRF-2 is imported into the nucleus remains unknown. By using in vitro nuclear import assays and immuno-cytofluorescence, we dissect the nuclear import pathways of NRF-2. We show that both NRF-2α and NRF-2ß contain intrinsic nuclear localization signals (NLSs): the Ets domain within NRF-2α and the NLS within NRF-2ß (amino acids 311/321: EEPPAKRQCIE) that is recognized by importin-α:ß. When NRF-2α and NRF-2ß form a complex, the nuclear import of NRF-2αß becomes strictly dependent on the NLS within NRF-2ß. Therefore, the nuclear import mechanism of NRF-2 is unique among Ets factors. The NRF-2ß NLS contains only two lysine/arginine residues, unlike other known importin-α:ß-dependent NLSs. Using ELISA-based binding assays, we show that it is bound by importin-α in almost the same manner and with similar affinity to that of the classical monopartite NLSs, such as c-myc and SV40 T-antigen NLSs. However, the part of the tryptophan array of importin-α that is essential for the recognition of classical monopartite NLSs by generating apolar pockets for the P3 and the P5 lysine/arginine side chains is not required for the recognition of the NRF-2ß NLS. We conclude that the NRF-2ß NLS is an unusual but is, nevertheless, a bona fide monopartite-type NLS.

RecQ5 protein translocation into the nucleus by a nuclear localization signal.

RecQ5, a member of the RecQ helicase family, maintains genome stability via participation in many DNA metabolic processes including DNA repair, DNA resolution, and RNA transcription, processes occurring in the nucleus. Previously, we reported that RecQ5 and Rad51, also involved in DNA repair, become co-localized in nuclei when co-expressed in cultured cells. Nuclear localization of RecQ5 appears to be important for cellular function along with Rad51. However, little is known about the nuclear localization of RecQ5. Here, we generated enhanced green fluorescent protein (EGFP)-tagged RecQ5 transgenic flies and analyzed localization of this protein in early embryos by live imaging. In syncytial embryos, RecQ5 was localized synchronously in interphase nuclei, and spread repeatedly over the embryos in mitosis. Thus, RecQ5 was transported into nuclei at the early interphase. Furthermore, we examined the subcellular localization of a series of truncated forms of Drosophila RecQ5 in cultured cells to determine the nuclear localization signal (NLS). Entire coding or deleted RecQ5 sequences of various sizes were ligated into EGFP vectors, which were then used to transfect cultured Drosophila cells. The region responsible for nuclear localization of Drosophila RecQ5 contained a short stretch of positively charged basic amino acids, 2 of which were particularly important for the nuclear localization. This stretch was sufficient for nuclear localization when fused with EGFP. Although the NLS of Drosophila RecQ5 was distinct from that of human RECQL5 in terms of position and amino acid sequence, this fly RecQ5 protein was translocated into the nucleus by an NLS.

Genetic disruption of Abl nuclear import reduces renal apoptosis in a mouse model of cisplatin-induced nephrotoxicity.

DNA damage activates nuclear Abl tyrosine kinase to stimulate intrinsic apoptosis in cancer cell lines and mouse embryonic stem cells. To examine the in vivo function of nuclear Abl in apoptosis, we generated Abl-µNLS (µ, mutated in nuclear localization signals) mice. We show here that cisplatin-induced apoptosis is defective in the renal proximal tubule cells (RPTC) from the Abl(µ/µ) mice. When injected with cisplatin, we found similar levels of platinum in the Abl(+/+) and the Abl(µ/µ) kidneys, as well as similar initial inductions of p53 and PUMAα expression. However, the accumulation of p53 and PUMAα could not be sustained in the Abl(µ/µ) kidneys, leading to reductions in renal apoptosis and tubule damage. Co-treatment of cisplatin with the Abl kinase inhibitor, imatinib, reduced the accumulation of p53 and PUMAα in the Abl(+/+) but not in the Abl(µ/µ) kidneys. The residual apoptosis in the Abl(µ/µ) mice was not further reduced in the Abl(µ/µ); p53(-/-) double-mutant mice, suggesting that nuclear Abl and p53 are epistatic to each other in this apoptosis response. Although apoptosis and tubule damage were reduced, cisplatin-induced increases in phospho-Stat-1 and blood urea nitrogen were similar between the Abl(+/+) and the Abl(µ/µ) kidneys, indicating that RPTC apoptosis is not the only factor in cisplatin-induced nephrotoxicity. These results provide in vivo evidence for the pro-apoptotic function of Abl, and show that its nuclear localization and tyrosine kinase activity are both required for the sustained expression of p53 and PUMAα in cisplatin-induced renal apoptosis.

Nonmyofilament-associated troponin T3 nuclear and nucleolar localization sequence and leucine zipper domain mediate muscle cell apoptosis.

Troponin T (TnT) plays a major role in striated muscle contraction. We recently demonstrated that the fast skeletal muscle TnT3 isoform is localized in the muscle nucleus, and either its full-length or COOH-terminus leads to muscle cell apoptosis. Here, we further explored the mechanism by which it enters the nucleus and promotes cytotoxicity. Amino acid truncation and substitution showed that its COOH-terminus contains a dominant nuclear/nucleolar localization sequence (KLKRQK) and the basic lysine and arginine residues might play an important role in the nuclear retention and nucleolar enrichment of KLKRQK-DsRed fusion proteins. Deleting this domain or substituting lysine and arginine residues (KLAAQK) resulted in a dramatic loss of TnT3 nuclear and nucleolar localization. In contrast, the GATAKGKVGGRWK domain-DsRed construct localized exclusively in the cytoplasm, indicating that a nuclear exporting sequence is possibly localized in this region. Additionally, we identified a classical DNA-binding leucine zipper domain (LZD) which is conserved among TnT isoforms and species. Deletion of LZD or KLKRQK sequence significantly reduced cell apoptosis compared to full-length TnT3. We conclude that TnT3 contains both a nuclear localization signal and a DNA-binding domain, which may mediate nuclear/nucleolar signaling and muscle cell apoptosis.