Nuclear Translocation of hARD1 Contributes to Proper Cell Cycle Progression

Arrest defective 1 (ARD1) is an acetyltransferase that is highly conserved across organisms, from yeasts to humans. The high homology and widespread expression of ARD1 across multiple species and tissues signify that it serves a fundamental role in cells. Human ARD1 (hARD1) has been suggested to be involved in diverse biological processes, and its role in cell proliferation and cancer development has been recently drawing attention. However, the subcellular localization of ARD1 and its relevance to cellular function remain largely unknown. Here, we have demonstrated that hARD1 is imported to the nuclei of proliferating cells, especially during S phase. Nuclear localization signal (NLS)-deleted hARD1 (hARD1ΔN), which can no longer access the nucleus, resulted in cell morphology changes and cellular growth impairment. Notably, hARD1ΔN-expressing cells showed alterations in the cell cycle and the expression levels of cell cycle regulators compared to hARD1 wild-type cells. Furthermore, these effects were rescued when the nuclear import of hARD1 was restored by exogenous NLS. Our results show that hARD1 nuclear translocation mediated by NLS is required for cell cycle progression, thereby contributing to proper cell proliferation.     

Nuclear sphingomyelin pathway in serum deprivation-induced apoptosis of embryonic hippocampal cells

Sphingomyelin (SM) cycle has been involved in the regulation of proliferation, differentiation, and apoptosis. Increases in ceramide have been found after a larger number of apoptotic stimuli including cytokines, cytotoxic drugs, and environmental stresses. Accumulating evidence suggest that the subcellular localization of ceramide generation is a critical factor in determining the cellular behavior. Since recently enzymes involved in ceramide metabolism such as sphingomyelinase, SM synthase, sphingosine kinase and ceramidase have been found in the nucleus of hepatocyte cells, we have studied first the presence and the physicochemical characteristics of SM metabolism enzymes in nuclei isolated from embryonic hippocampal cells (cell line HN9.10e). The activities of sphingomyelinase and SM-synthase have been assayed and the ceramide production evaluated at different times after serum deprivation in these neurones cultivated in serum-deficient medium. We report that both enzymes are present in the nucleus of embryonic hippocampal cells and differ from those present in the homogenate in optimum pH. After serum deprivation, that induces a time-dependent decrease in cell viability and increase of the cell percentage in G1 phase of the cell cycle, a nuclear sphingomyelinase activation together with SM-synthase inhibition and a consequent increase of nuclear ceramide pool have been demonstrated. No similar enzyme activity modifications in homogenate have been identified. The possible role of nuclear sphingomyelinase/sphingomyelin-synthase balance in serum deprivation-induced apoptosis in the embryonic hippocampal cell is discussed.     

Cell cycle-dependent expression and subcellular localization of fructose 1,6-bisphosphatase

Recently a gluconeogenic enzyme was discovered—fructose 1,6-bisphosphatase (FBPase)—that localizes in the nucleus of a proliferating cell, but its physiological role in this compartment remains unclear. Here, we demonstrate the link between nuclear localization of FBPase and the cell cycle progression. Results of our studies indicate that in human and mouse squamous cell lung cancer, as well as in the HL-1 cardiomyocytes, FBPase nuclear localization correlates with nuclear localization of S and G2 phase cyclins. Additionally, activity and expression of the enzyme depends on cell cycle stages. Identification of FBPase interacting partners with mass spectrometry reveals a set of nuclear proteins involved in cell cycle regulation, mRNA processing and in stabilization of genomic DNA structure. To our knowledge, this is the first experimental evidence that muscle FBPase is involved in cell cycle events.     

Dnd1的蛋白亚细胞定位及其对HeLa细胞增殖的抑制作用

小鼠睾丸生殖细胞瘤易感基因Dnd1编码的蛋白是一个在进化中保守的RNA结合蛋白.为探讨小鼠Dnd1的蛋白亚细胞定位和对细胞增殖的影响及其机制, 利用生物信息学技术, 采用组合的亚细胞定位分析软件对Dnd1进行真核生物亚细胞定位预测; 利用融合绿色荧光蛋白(green fluorescent protein, GFP)定位的方法, 通过构建pEGFP-Dnd1重组质粒, 将重组质粒pEGFP-Dnd1转染HeLa细胞和GC-1细胞, 在荧光显微镜下观察Dnd1的蛋白亚细胞定位; 用MTT法和流式细胞技术测定Dnd1过表达对HeLa细胞的增殖能力的影响和细胞周期的改变; 在HeLa细胞系中检测Dnd1对AP-1转录活性的影响. 结果表明: ① 生物信息学预测Dnd1主要在细胞核表达, 在细胞质中也有少量表达; 荧光显微镜下观察发现,Dnd1蛋白主要定位在细胞核, 在细胞质中也有少量分布; ② Dnd1基因在HeLa细胞系中的过表达抑制细胞增殖和诱导细胞周期G1期阻滞;③ Dnd1抑制AP-1的转录活性,从而抑制AP-1介导的转录是Dnd1抑制细胞增殖的可能机制.本研究初步明确了Dnd1的蛋白亚细胞定位及其对HeLa细胞的生长抑制作用, 这为进一步研究Dnd1基因的功能建立基础.     

Macromolecular crowding effect is critical for maintaining SIRT1's nuclear localization in cancer cells

SIRT1 is a principle class III histone deacetylase which exhibits versatile functions in stress response, development, and pathological processes including cancer. Although SIRT1 deacetylates a wide range of nuclear and cytoplasmic proteins, its subcellular localization in cancer cells has been controversial. In this study, we uncovered the inconsistent reports about SIRT1 subcellular localization is partially due to different analysis approaches. While immunofluorescence and live cell imaging reveal a predominant nuclear localization of SIRT1, conventional cell fractionation often results in a severe leaking of SIRT1 into the cytoplasm. Such a leakage is mainly caused by loss of cytoplasmic macromolecular crowding effect as well as hypotonic dwelling during the isolation of the nuclei. We also developed an improved cell fractionation procedure which maintains SIRT1 in its original subcellular localization. Analyzing a variety of human cancer cell lines using this approach and other methods demonstrate that SIRT1 predominantly localizes to the nucleus in cancer cells.     

Elevated nuclear localization of glycolytic enzyme TPI1 promotes lung adenocarcinoma and enhances chemoresistance

Increased glycolysis is a hallmark of tumor, which can provide tumor cells with energy and building blocks to promote cell proliferation. Recent studies have shown that not only the expression of glycolytic genes but also their subcellular localization undergoes a variety of changes to promote development of different types of tumors. In this study, we performed a comprehensive analysis of glycolysis and gluconeogenesis genes based on data from TCGA to identify those with significant tumor-promoting potential across 14 types of tumors. This analysis not only confirms genes that are known to be involved in tumorigenesis, but also reveals a significant correlation of triosephosphate isomerase 1 (TPI1) with poor prognosis, especially in lung adenocarcinoma (LUAD). TPI1 is a glycolytic enzyme that interconverts dihydroxyacetone phosphate (DHAP) to glyceraldehyde 3-phosphate (GAP). We confirm the upregulation of TPI1 expression in clinical LUAD samples and an inverse correlation with the overall patient survival. Knocking down of TPI1 in lung cancer cells significantly reduced cell migration, colony formation, and xenograft tumor growth. Surprisingly, we found that the oncogenic function of TPI1 depends on its translocation to cell nucleus rather than its catalytic activity. Significant accumulation of TPI1 in cell nucleus was observed in LUAD tumor tissues compared with the cytoplasm localization in adjacent normal tissues. Moreover, nuclear translocation of TPI1 is induced by extracellular stress (such as chemotherapy agents and peroxide), which facilitates the chemoresistance of cancer cells. Our study uncovers a novel function of the glycolytic enzyme TPI1 in the LUAD.Subject terms: Non-small-cell lung cancer, Cell biology     

Identification of subcellular targeting sequences of Cten reveals its role in cell proliferation

Spatial and temporal subcellular localization plays critical roles in regulating protein function. Cten (C-terminal tensin like) is a member of the tensin family. Cten recruits signaling molecules, such as DLC1, to focal adhesions, modulates homeostasis of receptor tyrosine kinases, including EGFR and c-Met, and promotes cell migration. These functions are likely controlled by Cten localization at focal adhesions and/or in the cytoplasm. In addition, Cten has been detected in the nucleus by which mechanism is unknown. To this end, we have examined the distribution of Cten in various cell lines, determined primary sequence requirements for its nuclear and focal adhesion localizations, and analyzed potential roles of nuclear Cten. Our results show that a proportion of Cten translocates to nuclei in cancer cell lines and that nuclear exporting of Cten is a CRM1-dependent process. A nuclear localization sequence and a nuclear export sequence are identified within Cten. In addition, like other tensins, Cten contains two independent focal adhesion binding sites. Although further expression of recombinant Cten showed no effect on cancer cell proliferation, silencing of Cten significantly reduced cell growth. Furthermore, expression of Cten mutants either with defective nuclear export sequence or tagged with SV40 nuclear localization sequence promoted cell growth. These results suggest that nuclear Cten contributes to cancer cell proliferation. Our findings identify a molecular mechanism for regulating Cten protein trafficking in mammalian cells and provide new insights into the dynamics of focal adhesion complexes in health and disease.     

The B56γ3 Regulatory Subunit of Protein Phosphatase 2A (PP2A) Regulates S Phase-specific Nuclear Accumulation of PP2A and the G1 to S Transition

Protein phosphatase 2A (PP2A) is a heterotrimeric enzyme consisting of a scaffold subunit (A), a catalytic subunit (C), and a variable regulatory subunit (B). The regulatory B subunits determine the substrate specificity and subcellular localization of the PP2A holoenzyme. Here, we demonstrate that the subcellular localization of the B56γ3 regulatory subunit is regulated in a cell cycle-specific manner. Notably, B56γ3 becomes enriched in the nucleus at the G₁/S border and in S phase. The S phase-specific nuclear enrichment of B56γ3 is accompanied by increases of nuclear A and C subunits and nuclear PP2A activity. Overexpression of B56γ3 promotes nuclear localization of the A and C subunits, whereas silencing both B56γ2 and B56γ3 blocks the S phase-specific increase in the nuclear localization and activity of PP2A. In NIH3T3 cells, B56γ3 overexpression reduces p27 phosphorylation at Thr-187, concomitantly elevates p27 protein levels, delays the G₁ to S transition, and retards cell proliferation. Consistently, knockdown of endogenous B56γ3 expression reduces p27 protein levels and increases cell proliferation in HeLa cells. These findings demonstrate that the dynamic nuclear distribution of the B56γ3 regulatory subunit controls nuclear PP2A activity, which regulates cell cycle controllers, such as p27, to restrain cell cycle progression, and may be responsible for the tumor suppressor function of PP2A.     

Glypican and Biglycan in the Nuclei of Neurons and Glioma Cells: Presence of Functional Nuclear Localization Signals and Dynamic Changes in Glypican During the Cell Cycle

We have investigated the expression patterns and subcellular localization in nervous tissue of glypican, a major glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan that is predominantly synthesized by neurons, and of biglycan, a small, leucine-rich chondroitin sulfate proteoglycan. By laser scanning confocal microscopy of rat central nervous tissue and C6 glioma cells, we found that a significant portion of the glypican and biglycan immunoreactivity colocalized with nuclear staining by propidium iodide and was also seen in isolated nuclei. In certain regions, staining was selective, insofar as glypican and biglycan immunoreactivity in the nucleus was seen predominantly in a subpopulation of large spinal cord neurons. The amino acid sequences of both proteoglycans contain potential nuclear localization signals, and these were demonstrated to be functional based on their ability to target β-galactosidase fusion proteins to the nuclei of transfected 293 cells. Nuclear localization of glypican β-galactosidase or Fc fusion proteins in transfected 293 cells and C6 glioma cells was greatly reduced or abolished after mutation of the basic amino acids or deletion of the sequence containing the nuclear localization signal, and no nuclear staining was seen in the case of heparan sulfate and chondroitin sulfate proteoglycans that do not possess a nuclear localization signal, such as syndecan-3 or decorin (which is closely related in structure to biglycan). Transfection of COS-1 cells with an epitope-tagged glypican cDNA demonstrated transport of the full-length proteoglycan to the nucleus, and there are also dynamic changes in the pattern of glypican immunoreactivity in the nucleus of C6 cells both during cell division and correlated with different phases of the cell cycle. Our data therefore suggest that in certain cells and central nervous system regions, glypican and biglycan may be involved in the regulation of cell division and survival by directly participating in nuclear processes.     

Growth factor regulation of cell growth and proliferation in the nervous system

This article discusses a novel intracrine mechanism of growth-factor action in the nervous system whereby fibroblast growth factor-2 (FGF-2) and its receptor accumulate in the cell nucleus and act as mediators in the control of cell growth and proliferation. In human and rat brain the levels and subcellular localization of FGF-2 differ between quiescent and reactive astrocytes. Quiescent cells express a low level of FGF-2, which is located predominantly within the cytoplasm. In reactive astrocytes, the expression of FGF-2 increases and the proteins are found in both the cytoplasm and nucleus. In glioma tumors, FGF-2 is overexpressed in the nuclei of neoplastic cells. Similar changes in FGF-2 expression and localization are found in vitro. The nuclear accumulation of FGF-2 reflects a transient activation of the FGF-2 gene by potentially novel transactivating factors interacting with an upstream regulatory promoter region. In parallel with FGF-2, the nuclei of astrocytes contain the high-affinity FGF-2 receptor, FGFR1. Nuclear FGFR1 is full length, retains kinase activity, and is localized within the nuclear interior in association with the nuclear matrix. Transfection of either FGF-2 or FGFR1 into cells that do not normally express these proteins results in their nuclear accumulation and concomitant increases in cell proliferation. A similar regulation of nuclear FGF-2 and FGFR1 is observed in neural crest-derived adrenal medullary cells and of FGF-2 in the nuclei of cerebellar neurons. Thus, the regulation of the nuclear content of FGF-2 and FGFR1 could serve as a novel mechanism controlling growth and proliferation of glial and neuronal cells.     

Phosphorylated BRCA1 is predominantly located in the nucleus and mitochondria

Multiple copies of the mitochondrial genome in eukaryotic cells are organized into protein-DNA complexes called nucleoids. Mitochondrial genome repair mechanisms have been reported, but they are less well characterized than their nuclear counterparts. To expand our knowledge of mitochondrial genome maintenance, we have studied the localization of the BRCA1 protein, known to be involved in nuclear repair pathways. Our confocal and immunoelectron microscopy results show that BRCA1 is present in mitochondria of several human cancer cell lines and in primary breast and nasal epithelial cells. BRCA1 localization in mitochondria frequently overlapped that of nucleoids. Small interfering RNA-mediated knockdown of BRCA1 in human cancer cells (confirmed by Western blot) results in decreased nuclear, cytoplasmic, and mitochondrial staining after immunofluorescence microscopy, establishing the specificity of the BRCA1 immunolabeling. Furthermore, using cell fractionation, dephosphorylation, and enzyme protection experiments, we show that a 220-kDa phosphorylated isoform of BRCA1 is enriched in mitochondrial and nuclear fractions but reduced in cytoplasmic subcellular fractions. Submitochondrial fractionation confirmed the presence of BRCA1 protein in isolated mitoplasts. Because phosphorylation of BRCA1 and subsequent changes in subcellular localization are known to follow DNA damage, our data support a universal role for BRCA1 in the maintenance of genome integrity in both mitochondria and nucleus.     

THE SUBCELLULAR LOCALIZATION OF CYTIDINE 5''-MONOPHOSPHO-N-ACETYLNEURAMINIC ACID SYNTHETASE IN CALF BRAIN

Abstract— The subcellular distribution of cytidine 5'-monophospho- N -acetylneuraminic acid synthetase, which is a key enzyme involved in the biosynthesis of sialo-glycoproteins and gangliosides, was studied in the frontal grey cortex and the corpus callosum of the calf brain. It appeared that the enzyme was highly concentrated in nuclear fractions, which were shown to be relatively pure as evaluated from morphological, enzymic and chemical data. The possibility that the nuclear localization of the enzyme was due to an artifact, as the result of adsorption onto the nuclear membrane or absorption into the nucleus during the homogenization of the tissue, was ruled out completely and so it appeared that cytidine 5'-monophospho- N -acetylneuraminic acid synthetase, at least for the greater part, is a true nuclear enzyme. The subnuclear localization of the enzyme was shown to be in the nuclear sap. Both neuronal and glial cell nuclei contained the enzyme, which makes it very likely that biosynthesis of sialo-glycoproteins and/or gangliosides occurs in neurons as well as in glial cells. The significance of the nuclear localization of the enzyme for the biosynthesis of sialic acid containing macromolecules and the possible regulatory role played by the nucleus in this process are discussed.     

Distinct subcellular localization patterns contribute to functional specificity of the Cln2 and Cln3 cyclins of Saccharomyces cerevisiae

The G(1) cyclins of budding yeast drive cell cycle initiation by different mechanisms, but the molecular basis of their specificity is unknown. Here we test the hypothesis that the functional specificity of G(1) cyclins is due to differential subcellular localization. As shown by indirect immunofluorescence and biochemical fractionation, Cln3p localization appears to be primarily nuclear, with the most obvious accumulation of Cln3p to the nuclei of large budded cells. In contrast, Cln2p localizes to the cytoplasm. We were able to shift localization patterns of truncated Cln3p by the addition of nuclear localization and nuclear export signals, and we found that nuclear localization drives a Cln3p-like functional profile, while cytoplasmic localization leads to a partial shift to a Cln2p-like functional profile. Therefore, forcing Cln3p into a Cln2p-like cytoplasmic localization pattern partially alters the functional specificity of Cln3p toward that of Cln2p. These results suggest that there are CLN-dependent cytoplasmic and nuclear events important for cell cycle initiation. This is the first indication of a cytoplasmic function for a cyclin-dependent kinase. The data presented here support the idea that cyclin function is regulated at the level of subcellular localization and that subcellular localization contributes to the functional specificity of Cln2p and Cln3p.     

Multi-lobulation of the nucleus in prolonged S phase by nuclear expression of Chk tyrosine kinase

Chk tyrosine kinase phosphorylates Src-family tyrosine kinases and suppresses their kinase activity. We recently showed that Chk localizes to the nucleus as well as the cytoplasm and inhibits cell proliferation. To investigate the role of nuclear Chk in proliferation, various Chk mutants were constructed and expressed. Nuclear localization of Chk-induced dynamic multi-lobulation of the nucleus and prolonged S phase of the cell cycle. The N-terminal domain of Chk and a portion of its kinase domain but not the kinase activity were responsible for induction of the multi-lobulation. Cell sorting analysis revealed that nuclear multi-lobulated cells were enriched in late S phase. Multi-lobulated nuclei were surrounded with lamin B1 that was particularly concentrated in concave regions of the nuclei. Furthermore, treatment with nocodazole or taxol disrupted multi-lobulation of the nucleus. These results suggest that nuclear multi-lobulation in late S phase, which is dependent on polymerization and depolymerization of microtubules, may be involved in nuclear Chk-induced inhibition of proliferation.     

Structure of human immunodeficiency virus type 1 Vpr(34-51) peptide in micelle containing aqueous solution.

Human immunodeficiency virus type 1 protein R (HIV-1 Vpr) promotes nuclear entry of viral nucleic acids in nondividing cells, causes G(2) cell cycle arrest and is involved in cellular differentiation and cell death. Vpr subcellular localization is as variable as its functions. It is known, that consistent with its role in nuclear transport, Vpr localizes to the nuclear envelope of human cells. Further, a reported ion channel activity of Vpr is clearly dependent on its localization in or at membranes. We focused our structural studies on the secondary structure of a peptide consisting of residues 34-51 of HIV-1 Vpr. This part of Vpr plays an important role in Vpr oligomerization, contributes to cell cycle arrest activity, and is essential for virion incorporation and binding to HHR23A, a protein involved in DNA repair. Employing NMR spectroscopy we found this part of Vpr to be almost completely alpha helical in the presence of micelles, as well as in trifluoroethanol containing and methanol/chloroform solvent. Our results provide structural data suggesting residues 34-51 of Vpr to contain an amphipathic, leucine-zipper-like alpha helix, which serves as a basis for oligomerization of Vpr and its interactions with cellular and viral factors involved in subcellular localization and virion incorporation of Vpr.     

Cells lacking IKKα show nuclear cyclin D1 overexpression and a neoplastic phenotype: role of IKKα as a tumor suppressor

The catalytic subunits of IκB kinase (IKK) complex, IKKα and IKKβ, are involved in activation of NF-κB and in mediating a variety of other biological functions. Though these proteins have a high-sequence homology, IKKα exhibits different functional characteristics as compared with IKKβ. Earlier, we have shown that cyclin D1 is overexpressed and predominantly localized in the nucleus of IKKα(-/-) cells, indicating that IKKα regulates turnover and subcellular distribution of cyclin D1, which is mediated by IKKα-induced phosphorylation of cyclin D1. Because cyclin D nuclear localization is implicated in tumor development, we examined whether the absence of IKKα leads to tumor development as well. In the current study, we show that IKKα plays a critical role in tumorigenesis. Though IKKα(-/-) MEF cells show a slower anchorage-dependent growth, they are clonogenic in soft agar. These cells are tumorigenic in nude mice. Microarray analysis of IKKα(-/-) cells indicates a differential expression of genes involved in proliferation and apoptosis. Furthermore, analysis of microarray data of human lung cancer cell lines revealed decreased IKKα RNA expression level as compared with cell lines derived from normal bronchial epithelium. These results suggest that IKKα may function as a tumor suppressor gene. Absence of IKKα may induce tumorigenicity by nuclear localization of cyclin D1 and modulating the expression of genes involved in neoplastic transformation.     

The presence of 19-kDa Bcl-2 in dividing cells

The 26-kDa bcl-2 gene product inhibits apoptosis and cell proliferation. Cleavage of Bcl-2 into a 22-kDa fragment inactivates its anti-apoptotic activity and is a key event in apoptosis. Here, and in recent work, we describe massive 19-kDa Bcl-2 immunoreactivity in non-apoptotic cells, suggesting a link with viability rather than cell death. Loss of 19 kDa Bcl-2 in adriamycin-induced apoptotic cells underlines this. G2/M-phase accumulation of cells by nocodazole-treatment also results in loss of 19 kDa Bcl-2. Next to its well-documented cytoplasmic localization, a substantial pool of Bcl-2 resides in nuclei. Hampered nuclear localization of Bcl-2 leads to a loss of cell cycle repression. This has led us to point at a pivotal role for nuclear Bcl-2 in cellular proliferation. In this report, cellular fractionation of bcl-2 transfected cells in various phases of the cell cycle reveals a constitutive cytoplasmic pool of 19 kDa Bcl-2. Nuclear 19-kDa Bcl-2 immunoreactivity is far more pronounced in rapidly dividing nuclei compared with more quiescent nuclear fractions. This implicates that ongoing cell proliferation involves cleavage of nuclear Bcl-2 with a 19-kDa fragment.     

Different effects of ZO-1, ZO-2 and ZO-3 silencing on kidney collecting duct principal cell proliferation and adhesion

Coordinated cell proliferation and ability to form intercellular seals are essential features of epithelial tissue function. Tight junctions (TJs) classically act as paracellular diffusion barriers. More recently, their role in regulating epithelial cell proliferation in conjunction with scaffolding zonula occludens (ZO) proteins has come to light. The kidney collecting duct (CD) is a model of tight epithelium that displays intense proliferation during embryogenesis followed by very low cell turnover in the adult kidney. Here, we examined the influence of each ZO protein (ZO-1, -2 and -3) on CD cell proliferation. We show that all 3 ZO proteins are strongly expressed in native CD and are present at both intercellular junctions and nuclei of cultured CD principal cells (mCCD_cl1). Suppression of either ZO-1 or ZO-2 resulted in increased G₀/G₁ retention in mCCD_cl1 cells. ZO-2 suppression decreased cyclin D1 abundance while ZO-1 suppression was accompanied by increased nuclear p21 localization, the depletion of which restored cell cycle progression. Contrary to ZO-1 and ZO-2, ZO-3 expression at intercellular junctions dramatically increased with cell density and relied on the presence of ZO-1. ZO-3 depletion did not affect cell cycle progression but increased cell detachment. This latter event partly relied on increased nuclear cyclin D1 abundance and was associated with altered β1-integrin subcellular distribution and decreased occludin expression at intercellular junctions. These data reveal diverging, but interconnected, roles for each ZO protein in mCCD_cl1 proliferation. While ZO-1 and ZO-2 participate in cell cycle progression, ZO-3 is an important component of cell adhesion.