Nuclear localization of ING3 is required to suppress melanoma cell migration, invasion and angiogenesis.

Inhibitor of growth family member 3 (ING3), a tumor suppressor, plays crucial roles in cell cycle regulation, apoptosis and transcription. Previous studies suggest important roles of nuclear ING3, however, the nuclear localization sequence (NLS) of ING3 is not defined and its biological functions remain to be elucidated. In this study, various ING3 mutants were generated to identify its NLS. The NLS of ING3 was determined as KKFK between 164 and 167 amino acids. More intriguingly, replacement of Lysine 164 residue of ING3 with alanine (K164A) resulted in retention of ING3 in the cytoplasm. Overexpression of ING3 led to inhibition of melanoma cell migration, invasion, and angiogenesis respectively, however, this inhibition was abrogated in cells with overexpression of ING3-K164A mutant. In conclusion, this study identified the NLS of ING3 and demonstrated the significance of ING3 nuclear localization for tumor suppressive functions of ING3, and future studies await to elucidate the role of ING3 (K164) post-modificaton in its nuclear transportation and cancer development.

Visualization of a blue light transmission area in living animals using light-induced nuclear translocation of fluorescent proteins.

Optical manipulations are widely used to analyze neuronal functions in vivo. Blue light is frequently used to activate channelrhodopsins or LOV domains, although the degrees of its absorption and scattering are higher than those of longer wavelength light. High spatial resolution of optical manipulation is easily achieved in vitro, while the light is unevenly scattered and absorbed in tissues due to many factors. It is difficult to spatially measure a blue light transmission area in vivo. Here, we propose a genetic method to visualize blue light transmission in the brain and other organs using light-induced nuclear translocation of fluorescent proteins with a LOV domain. A light-inducible nuclear localization signal (LINuS) consists of a LOV2 domain fused with a nuclear localization signal (NLS). We confirmed that blue light illumination induced reversible translocation of NES-tdTomato-LINuS from the cytosol to the nucleus within 30 min in HEK293 cells. By employing a PHP.eb capsid that can penetrate the blood-brain barrier, retro-orbital sinus injection of adeno-associated virus (AAV) vectors induced scattered expression of nuclear export signal (NES)-tdTomato-LINuS in the brain. We confirmed that 30-min transcranial blue light illumination induced nuclear translocation of NES-tdTomato-LINuS in the cortex, the hippocampus, and even the paraventricular nucleus of the thalamus. We also found that mice exposed to blue light in a shaved abdominal area exhibited a substantial increase in nuclear translocation in the ventral surface lobe of the liver. These results provide a simple way to obtain useful information on light transmission in tissues without any transgenic animals or skillful procedures.

Androgen receptor nuclear localization correlates with AR-V7 mRNA expression in circulating tumor cells (CTCs) from metastatic castration resistance prostate cancer patients.

Androgen receptor (AR) signaling drives prostate cancer (PC) progression and remains active upon transition to castration resistant prostate cancer (CRPC). Active AR signaling is achieved through the nuclear accumulation of AR following ligand binding and through expression of ligand-independent, constitutively active AR splice variants, such as AR-V7, which is the most commonly expressed variant in metastatic CRPC (mCRPC) patients. Most currently approved PC therapies aim to abrogate AR signaling and activity by inhibiting this ligand-mediated nuclear translocation. In a prospective multi-institutional clinical study, we recently showed that taxane based chemotherapy is also capable of impairing AR nuclear localization (ARNL) in circulating tumor cells (CTCs) from CRPC patients, whereas taxane induced decreases in ARNL were associated with response. Thus, quantitative assessment of ARNL in CTCs can be used to monitor therapeutic response in patients and help guide clinical decisions. Here, we describe the development and implementation of quantitative high throughput (QHT) image analysis algorithms to aid in CTC identification and quantitative assessment of percent ARNL (%ARNL). We applied this algorithm to fifteen CRPC patients at the start of taxane chemotherapy, quantified %ARNL in CTCs, and correlated with expression of AR-V7 mRNA (from CTCs enriched via negative, CD45+ depletion of peripheral blood) and with biochemical (prostate specific antigen; PSA) response to taxane chemotherapy. We found that CTCs from AR-V7 positive patients had higher baseline %ARNL compared to CTCs from AR-V7 negative patients, consistent with the constitutive nuclear localization of AR-V7. In addition, lower %ARNL in CTCs at baseline was associated with biochemical response to taxane chemotherapy. High inter- and intra-patient heterogeneity was also observed. As ARNL is required for active AR signaling, the QHT algorithms described herein can provide prognostic and/or predictive value in future clinical studies.

Molecular determinants of Drosophila immunophilin FKBP39 nuclear localization.

FK506-binding proteins (FKBPs) belong to a distinct class of immunophilins that interact with immunosuppressants. They use their peptidyl-prolyl isomerase (PPIase) activity to catalyze the cis-trans conversion of prolyl bonds in proteins during protein-folding events. FKBPs also act as a unique group of chaperones. The Drosophila melanogaster peptidyl-prolyl cis-trans isomerase FK506-binding protein of 39 kDa (FKBP39) is thought to act as a transcriptional modulator of gene expression in 20-hydroxyecdysone and juvenile hormone signal transduction. The aim of this study was to analyze the molecular determinants responsible for the subcellular distribution of an FKBP39-yellow fluorescent protein (YFP) fusion construct (YFP-FKBP39). We found that YFP-FKBP39 was predominantly nucleolar. To identify the nuclear localization signal (NLS), a series of YFP-tagged FKBP39 deletion mutants were prepared and examined in vivo. The identified NLS signal is located in a basic domain. Detailed mutagenesis studies revealed that residues K188 and K191 are crucial for the nuclear targeting of FKBP39 and its nucleoplasmin-like (NPL) domain contains the sequence that controls the nucleolar-specific translocation of the protein. These results show that FKBP39 possesses a specific NLS in close proximity to a putative helix-turn-helix (HTH) motif and FKBP39 may bind DNA in vivo and in vitro.

Efficient and dynamic nuclear localization of green fluorescent protein via RNA binding.

Classical nuclear localization signal (NLS) sequences have been used for artificial localization of green fluorescent protein (GFP) in the nucleus as a positioning marker or for measurement of the nuclear-cytoplasmic shuttling rate in living cells. However, the detailed mechanism of nuclear retention of GFP-NLS remains unclear. Here, we show that a candidate mechanism for the strong nuclear retention of GFP-NLS is via the RNA-binding ability of the NLS sequence. GFP tagged with a classical NLS derived from Simian virus 40 (GFP-NLS(SV40)) localized not only in the nucleoplasm, but also to the nucleolus, the nuclear subdomain in which ribosome biogenesis takes place. GFP-NLS(SV40) in the nucleolus was mobile, and intriguingly, the diffusion coefficient, which indicates the speed of diffusing molecules, was 1.5-fold slower than in the nucleoplasm. Fluorescence correlation spectroscopy (FCS) analysis showed that GFP-NLS(SV40) formed oligomers via RNA binding, the estimated molecular weight of which was larger than the limit for passive nuclear export into the cytoplasm. These findings suggest that the nuclear localization of GFP-NLS(SV40) likely results from oligomerization mediated via RNA binding. The analytical technique used here can be applied for elucidating the details of other nuclear localization mechanisms, including those of several types of nuclear proteins. In addition, GFP-NLS(SV40) can be used as an excellent marker for studying both the nucleoplasm and nucleolus in living cells.

Nuclear localization signal in a cancer-related transcriptional regulator protein NAC1.

Nucleus accumbens-associated protein 1 (NAC1) might have potential oncogenic properties and participate in regulatory networks for pluripotency. Although NAC1 is described as a transcriptional regulator, the nuclear import machinery of NAC1 remains unclear. We found, using a point mutant, that dimer formation was not committed to the nuclear localization of NAC1 and, using deletion mutants, that the amino-terminal half of NAC1 harbored a potential nuclear localization signal (NLS). Wild type, but not mutants of this region, alone was sufficient to drive the importation of green fluorescent protein (GFP) into the nucleus. Bimax1, a synthetic peptide that blocks the importin α/ß pathway, impaired nuclear localization of NAC1 in cells. We also used the binding properties of importin to demonstrate that this region is an NLS. Furthermore, the transcriptional regulator function of NAC1 was dependent on its nuclear localization activity in cells. Taken together, these results show that the region with a bipartite motif constitutes a functional nuclear import sequence in NAC1 that is independent of NAC1 dimer formation. The identification of an NAC1 NLS thus clarifies the mechanism through which NAC1 translocates to the nucleus to regulate the transcription of genes involved in oncogenicity and pluripotency.

Identification of a phosphorylation-dependent nuclear localization motif in interferon regulatory factor 2 binding protein 2.

BACKGROUND: Interferon regulatory factor 2 binding protein 2 (IRF2BP2) is a muscle-enriched transcription factor required to activate vascular endothelial growth factor-A (VEGFA) expression in muscle. IRF2BP2 is found in the nucleus of cardiac and skeletal muscle cells. During the process of skeletal muscle differentiation, some IRF2BP2 becomes relocated to the cytoplasm, although the functional significance of this relocation and the mechanisms that control nucleocytoplasmic localization of IRF2BP2 are not yet known. METHODOLOGY/PRINCIPAL FINDINGS: Here, by fusing IRF2BP2 to green fluorescent protein and testing a series of deletion and site-directed mutagenesis constructs, we mapped the nuclear localization signal (NLS) to an evolutionarily conserved sequence (354)ARKRKPSP(361) in IRF2BP2. This sequence corresponds to a classical nuclear localization motif bearing positively charged arginine and lysine residues. Substitution of arginine and lysine with negatively charged aspartic acid residues blocked nuclear localization. However, these residues were not sufficient because nuclear targeting of IRF2BP2 also required phosphorylation of serine 360 (S360). Many large-scale phosphopeptide proteomic studies had reported previously that serine 360 of IRF2BP2 is phosphorylated in numerous human cell types. Alanine substitution at this site abolished IRF2BP2 nuclear localization in C(2)C(12) myoblasts and CV1 cells. In contrast, substituting serine 360 with aspartic acid forced nuclear retention and prevented cytoplasmic redistribution in differentiated C(2)C(12) muscle cells. As for the effects of these mutations on VEGFA promoter activity, the S360A mutation interfered with VEGFA activation, as expected. Surprisingly, the S360D mutation also interfered with VEGFA activation, suggesting that this mutation, while enforcing nuclear entry, may disrupt an essential activation function of IRF2BP2. CONCLUSIONS/SIGNIFICANCE: Nuclear localization of IRF2BP2 depends on phosphorylation near a conserved NLS. Changes in phosphorylation status likely control nucleocytoplasmic localization of IRF2BP2 during muscle differentiation.

An in vivo imaging-based assay for detecting protein interactions over a wide range of binding affinities.

Identifying and characterizing protein interactions are fundamental steps toward understanding and modeling biological networks. Methods that detect protein interactions in intact cells rather than buffered solutions are likely more relevant to natural systems since molecular crowding events in the cytosol can influence the diffusion and reactivity of individual proteins. One in vivo, imaging-based method relies on the colocalization of two proteins of interest fused to DivIVA, a cell division protein from Bacillus subtilis, and green fluorescent protein (GFP). We have modified this imaging-based assay to facilitate rapid cloning by constructing new vectors encoding N- and C-terminal DivIVA or GFP molecular tag fusions based on site-specific recombination technology. The sensitivity of the assay was defined using a well-characterized protein interaction system involving the eukaryotic nuclear import receptor subunit, Importin alpha (Imp alpha), and variant nuclear localization signals (NLS) representing a range of binding affinities. These data demonstrate that the modified colocalization assay is sensitive enough to detect protein interactions with K(d) values that span over four orders of magnitude (1 nM to 15 microM). Lastly, this assay was used to confirm numerous protein interactions identified from mass spectrometry-based analyses of affinity isolates as part of an interactome mapping project in Rhodopseudomonas palustris.

Identification of a nuclear localization signal in suppressor of cytokine signaling 1.

Suppressor of cytokine signaling (SOCS) proteins are inducible feedback inhibitors of janus kinase and signal transducer and activators of transcription signaling pathways. In addition, SOCS1 has been identified to regulate stability of nuclear NF-kappaB subunits. However, details about the regulation of the nuclear pool of SOCS1 are unknown. Using different experimental approaches, we observed that SOCS1 but no further SOCS family members localized to the nucleus when expressed in various cell lines. Nuclear transport was confirmed for endogenous SOCS1 in macrophages stimulated with IFN-gamma. Sequence analysis revealed a bipartite nuclear localization signal (NLS) located between the src-homology 2 (SH2) domain and the SOCS box of SOCS1. Deletion of this region, introduction of a series of R/A point mutations, or substitution of this sequence with the respective region of SOCS3 resulted in loss of nuclear localization. Fusion of the SOCS1-NLS to cytokine-inducible SH2 region containing protein (CIS) resulted in nuclear localization of this otherwise cytoplasmic protein. SOCS1 mutants with loss of nuclear localization were still effective in suppressing IFN-alpha-mediated STAT1 tyrosine phosphorylation. However, they showed decreased inhibition of IFN-gamma-mediated induction of CD54. The results identify a hitherto unknown transport of SOCS1 into the nucleus which extends the spectrum of SOCS1 inhibitory activity.

DcpS scavenger decapping enzyme can modulate pre-mRNA splicing.

The human scavenger decapping enzyme, DcpS, functions to hydrolyze the resulting cap structure following cytoplasmic mRNA decay yet is, surprisingly, a nuclear protein by immunofluorescence. Here, we show that DcpS is a nucleocytoplasmic shuttling protein that contains separable nuclear import and Crm-1-dependent export signals. We postulated that the presence of DcpS in both cellular compartments and its ability to hydrolyze cap structure may impact other cellular events dependent on cap-binding proteins. An shRNA-engineered cell line with markedly diminished DcpS levels led to a corresponding reduction in cap-proximal intron splicing of a reporter minigene and endogenous genes. The impaired cap catabolism and resultant imbalanced cap concentrations were postulated to sequester the cap-binding complex (CBC) from its normal splicing function. In support of this explanation, DcpS efficiently displaced the nuclear cap-binding protein Cbp20 from cap structure, and complementation with Cbp20 reversed the reduced splicing, indicating that modulation of splicing by DcpS is mediated through Cbp20. Our studies demonstrate that the significance of DcpS extends beyond its well-characterized role in mRNA decay and involves a broader range of functions in RNA processing including nuclear pre-mRNA splicing.

Split pleckstrin homology domain-mediated cytoplasmic-nuclear localization of PI3-kinase enhancer GTPase.

Cytoplasm-nucleus shuttling of phosphoinositol 3-kinase enhancer (PIKE) is known to correlate directly with its cellular functions. However, the molecular mechanism governing this shuttling is not known. In this work, we demonstrate that PIKE is a new member of split pleckstrin homology (PH) domain-containing proteins. The structure solved in this work reveals that the PIKE PH domain is split into halves by a positively charged nuclear localization sequence. The PIKE PH domain binds to the head groups of di- and triphosphoinositides with similar affinities. Lipid membrane binding of the PIKE PH domain is further enhanced by the positively charged nuclear localization sequence, which is juxtaposed to the phosphoinositide head group-binding pocket of the domain. We demonstrate that the cytoplasmic-nuclear shuttling of PIKE is dynamically regulated by the balancing actions of the lipid-binding property of both the split PH domain and the nuclear targeting function of its nuclear localization sequence.

A functional nuclear localization signal in insulin-like growth factor binding protein-6 mediates its nuclear import.

IGF binding protein (IGFBP)-6 is a member of the IGFBP family that regulates the actions of IGFs. Although IGFBPs exert their functions extracellularly in an autocrine/paracrine manner, several members of the family, such as IGFBP-3 and -5, possess nuclear localization signals (NLS). To date, no NLS has been described for IGFBP-6, an IGFBP that binds preferentially to IGF-II. We report here that both exogenous and endogenous IGFBP-6 could be imported into the nuclei of rhabdomyosarcoma and HEK-293 cells. Nuclear import of IGFBP-6 was mediated by a NLS sequence that bears limited homology to those found in IGFBP-3 and -5. IGFBP-6 nuclear translocation was an active process that required importins. A peptide corresponding to the IGFBP-6 NLS bound preferentially to importin-alpha. A comprehensive peptide array study revealed that, in addition to positively charged residues such as Arg and Lys, amino acids, notably Gly and Pro, within the NLS, played an important part in binding to importins. Overexpression of wild-type IGFBP-6 increased apoptosis, and the addition of IGF-II did not negate this effect. Only the deletion of the NLS segment abolished the apoptosis effect. Taken together, these results suggest that IGFBP-6 is translocated to the nucleus with functional consequences and that different members of the IGFBP family have specific nuclear import mechanisms.

Analysis of a predicted nuclear localization signal: implications for the intracellular localization and function of the Saccharomyces cerevisiae RNA-binding protein Scp160.

Gene expression is controlled by RNA-binding proteins that modulate the synthesis, processing, transport and stability of various classes of RNA. Some RNA-binding proteins shuttle between the nucleus and cytoplasm and are thought to bind to RNA transcripts in the nucleus and remain bound during translocation to the cytoplasm. One RNA-binding protein that has been hypothesized to function in this manner is the Saccharomyces cerevisiae Scp160 protein. Although the steady-state localization of Scp160 is cytoplasmic, previous studies have identified putative nuclear localization (NLS) and nuclear export (NES) signals. The goal of this study was to test the hypothesis that Scp160 is a nucleocytoplasmic shuttling protein. We exploited a variety of yeast export mutants to capture any potential nuclear accumulation of Scp160 and found no evidence that Scp160 enters the nucleus. These localization studies were complemented by a mutational analysis of the predicted NLS. Results indicate that key basic residues within the predicted NLS of Scp160 can be altered without severely affecting Scp160 function. This finding has important implications for understanding the function of Scp160, which is likely limited to the cytoplasm. Additionally, our results provide strong evidence that the presence of a predicted nuclear localization signal within the sequence of a protein should not lead to the assumption that the protein enters the nucleus in the absence of additional experimental evidence.

Molecular determinants of nucleolar translocation of RNA helicase A.

RNA helicase A (RHA) is a member of the DEAH-box family of DNA/RNA helicases involved in multiple cellular processes and the life cycles of many viruses. The subcellular localization of RHA is dynamic despite its steady-state concentration in the nucleoplasm. We have previously shown that it shuttles rapidly between the nucleus and the cytoplasm by virtue of a bidirectional nuclear transport domain (NTD) located in its carboxyl terminus. Here, we investigate the molecular determinants for its translocation within the nucleus and, more specifically, its redistribution from the nucleoplasm to nucleolus or the perinucleolar region. We found that low temperature treatment, transcription inhibition or replication of hepatitis C virus caused the intranuclear redistribution of the protein, suggesting that RHA shuttles between the nucleolus and nucleoplasm and becomes trapped in the nucleolus or the perinucleolar region upon blockade of transport to the nucleoplasm. Both the NTD and ATPase activity were essential for RHA's transport to the nucleolus or perinucleolar region. One of the double-stranded RNA binding domains (dsRBD II) was also required for this nucleolar translocation (NoT) phenotype. RNA interference studies revealed that RHA is essential for survival of cultured hepatoma cells and the ATPase activity appears to be important for this critical role.

Different localization of Hsp105 family proteins in mammalian cells.

Hsp105alpha and Hsp105beta of the HSP105 family are alternatively spliced products derived from an hsp 105 gene transcript. Hsp105alpha is constitutively expressed and also induced by various stress, whereas Hsp105beta, lacking 44 amino acids from Hsp105alpha, is specifically expressed during mild heat shock. Although Hsp105alpha is shown to localize in the cytoplasm of mammalian cells, cellular localization of Hsp105beta is not known. In this study, we showed that Hsp105beta localized in the nucleus of cells in contrast to cytoplasmic Hsp105alpha, suggesting that these proteins function in different cellular compartments of cells. Using deletion and substitution mutants of Hsp105alpha and Hsp105beta, we revealed that these proteins had a functional nuclear localization signal (NLS) and a nuclear export signal (NES). Furthermore, Hsp105alpha accumulated in the nucleus of cells when treated with leptomycin B, a specific inhibitor of NES-dependent nuclear export. siRNA for importin beta, an essential component for NLS-dependent nuclear transport, inhibited the nuclear localization of Hsp105beta. Furthermore, the 44 amino acids sequence found in Hsp105alpha but not in Hsp105beta suppressed the NLS activity. Thus, the different localization of Hsp105alpha and Hsp105beta is suggested to be due to the suppressed NLS activity in Hsp105alpha.

Prothymosin alpha lacking the nuclear localization signal as an effective gene therapeutic strategy in collagen-induced arthritis.

Prothymosin alpha (ProT) is regulated by c-Myc, an oncoprotein overexpressed in synovium of rheumatoid arthritis, and is associated with cell proliferation. However, ProT also exerts immunomodulatory activities. The growth-promoting activity of ProT can be abolished by deleting its nuclear localization signal (NLS). In this study, we showed that AdProTDeltaNLS, an adenoviral vector encoding ProT lacking the NLS, did not enhance the proliferation of synovial fibroblasts. AdProTDeltaNLS treatment abolished the up-regulation of the MIP-1alpha promoter activity induced by TNF-alpha in synovial fibroblasts. AdProTDeltaNLS suppressed macrophage chemotaxis and reduced macrophage infiltration into the ankle joints in rats with collagen-induced arthritis (CIA). Neutralization test confirmed the involvement of MIP-1alpha in macrophage chemotaxis. Administration of AdProTDeltaNLS reduced the severity of CIA in the clinical, radiographic, and histological aspects. The levels of TNF-alpha (mean +/- SEM, 1261.9 +/- 107.9 vs 2880.1 +/- 561.4 pg/mg total protein; p < 0.05), IL-1beta (56.8 +/- 8.0 vs 109.2 +/- 4.9 pg/mg total protein; p < 0.01), and MIP-1alpha (41.7 +/- 3.6 vs 55.2 +/- 1.1 pg/mg total protein; p < 0.05) in the ankle joints were lower in the AdProTDeltaNLS-treated rats with CIA than those in their control counterparts. In the AdProTDeltaNLS-treated ankle joints, matrix metalloproteinase-9 expression was decreased by 40% and infiltrating macrophages reduced by 50%. Our results demonstrate that intra-articular delivery of AdProTDeltaNLS significantly ameliorated the clinical course of CIA in rats. This study is the first to suggest that ProT lacking the NLS may have therapeutic potential for the management of rheumatoid arthritis.

Multifocal two-photon laser scanning microscopy combined with photo-activatable GFP for in vivo monitoring of intracellular protein dynamics in real time.

We used multifocal two-photon laser scanning microscopy for local and selective protein activation and quantitative investigation of intracellular protein dynamics. The localized activation was realized with photo-activatable green-fluorescent-proteins (pa-GFP) and optical two-photon excitation in order to investigate the real-time intracellular dynamics in vivo. Such processes are of crucial importance for a deep understanding and modelling of regulatory and metabolic processes in living cells. Exemplarily, the intracellular dynamics of the Arabidopsis MYB transcription factor LHY/CCA1-like 1 (LCL1) that contains both a nuclear import and a nuclear export signal was quantitatively investigated. We used tobacco BY-2 protoplasts co-transfected with plasmids encoding photo-activatable green fluorescent protein (pa-GFP) fusion proteins and a red fluorescing transfection marker and measured the rapid nuclear export of pa-GFP-LCL1 after its photo-activation in the nucleus. In contrast, an export-negative mutant of LCL1 remained trapped inside the nucleus. We determined average time constants of 51 s and 125 s for the decrease of fluorescence in the nucleus due to active bi-directional nuclear transport of pa-GFP-LCL1 and diffusion of pa-GFP, respectively.

G2E3 is a nucleo-cytoplasmic shuttling protein with DNA damage responsive localization.

G2E3 was originally described as a G2/M-specific gene with DNA damage responsive expression. The presence of a conserved HECT domain within the carboxy-terminus of the protein indicated that it likely functions as a ubiquitin ligase or E3. Although HECT domains are known to function in this capacity for many proteins, we demonstrate that a portion of the HECT domain from G2E3 plays an important role in the dynamic subcellular localization of the protein. We have shown that G2E3 is a nucleo-cytoplasmic shuttling protein with nuclear export mediated by a novel nuclear export domain that functions independently of CRM1. In full-length G2E3, a separate region of the HECT domain suppresses the function of the NES. Additionally, G2E3 contains a nucleolar localization signal (NoLS) in its amino terminus. Localization of G2E3 to the nucleolus is a dynamic process, and the protein delocalizes from the nucleolus rapidly after DNA damage. Cell cycle phase-specific expression and highly regulated subcellular localization of G2E3 suggest a possible role in cell cycle regulation and the cellular response to DNA damage.

Tuberin nuclear localization can be regulated by phosphorylation of its carboxyl terminus.

Tuberin, the tuberous sclerosis 2 (TSC2) gene product, has been identified as a tumor suppressor protein genetically implicated in the pathology of tuberous sclerosis and the female-specific lung disease lymphangioleiomyomatosis. Tuberin and its predominant cytoplasmic binding partner hamartin have been shown to complex with a variety of intracellular signaling regulators and affect the processes of protein translation, cellular proliferation, cellular migration, and cellular transcription. In previous studies, we have presented evidence for tuberin binding to the calcium-dependent intracellular signaling protein calmodulin (CaM), overlap of tuberin CaM binding domain with a binding domain for estrogen receptor alpha, and the phosphorylation-associated nuclear localization of tuberin. In the study presented here, we expand our findings on the mechanism of tuberin nuclear localization to show that the CaM-estrogen receptor-alpha binding domain of tuberin can also serve as a tuberin nuclear localization sequence. Furthermore, we identify an Akt/p90 ribosomal S6 kinase-1 phosphorylation site within the carboxyl terminus of tuberin that can regulate tuberin nuclear localization and significantly affect the ability of tuberin to modulate estrogen genomic signaling events. These findings suggest a link between tuberin nuclear localization and a variety of intracellular signaling events that have direct implications with respect to the role of tuberin in the pathology of tuberous sclerosis and lymphangioleiomyomatosis.

Nuclear Ca2+/calmodulin-dependent protein kinase II in the murine heart.

Ca(2+) signaling through CaMKII is critical in regulating myocyte function with regard to excitation-contraction-relaxation cycles and excitation-transcription coupling. To investigate the role of nuclear CaMKII in cardiac function, transgenic mice were designed and generated to target the expression of a CaMKII inhibitory peptide, AIP (KKALRRQEAVDAL), to the nucleus. The transgenic construct consists of the murine alpha-myosin heavy chain promoter followed by the expression unit containing nucleotides encoding a four repeat concatemer of AIP (AIP(4)) and a nuclear localization signal (NLS). Western blot and immunohistochemical analyses demonstrate that AIP(4) is expressed only in the nucleus of cardiac myocytes of the transgenic mice (NLS-AIP(4)). The function of cytoplasmic CaMKII is not affected by the expression of AIP(4) in the nucleus. Inhibition of nuclear CaMKII activity resulted in reduced translocation of HDAC5 from nucleus to cytoplasm in NLS-AIP(4) mouse hearts. Loss of nuclear CaMKII activity causes NLS-AIP(4) mice to have smaller hearts than their nontransgenic littermates. Transcription factors including CREB and NFkappaB are not regulated by cardiac nuclear CaMKII. With physiological stresses such as pregnancy or aging (8 months), NLS-AIP(4) mice develop hypertrophy symptoms including enlarged atria, systemic edema, sedentariness, and morbidity. RT-PCR analyses revealed that the hypertrophic marker genes, such as ANF and beta-myosin heavy chain, were upregulated in pregnancy stressed mice. Our results suggest that absence of adequate Ca2+signaling through nuclear CaMKII regulated pathways leads to development of cardiac disease.