NUCKS1 is a highly modified, chromatin-associated protein involved in a diverse set of biological and pathophysiological processes.

The Nuclear Casein and Cyclin-dependent Kinase Substrate 1 (NUCKS1) protein is highly conserved in vertebrates, predominantly localized to the nucleus and one of the most heavily modified proteins in the human proteome. NUCKS1 expression is high in stem cells and the brain, developmentally regulated in mice and associated with several diverse malignancies in humans, including cancer, metabolic syndrome and Parkinson's disease. NUCKS1 function has been linked to modulating chromatin architecture and transcription, DNA repair and cell cycle regulation. In this review, we summarize and discuss the published information on NUCKS1 and highlight the questions that remain to be addressed to better understand the complex biology of this multifaceted protein.

Nondysplastic Ulcerative Colitis Has High Levels of the Homologous Recombination Repair Protein NUCKS1 and Low Levels of the DNA Damage Marker Gamma-H2AX.

Background: The colon and rectum are continuously exposed to oxidative stress that generates reactive oxygen species, which are a major cause of DNA double-strand breaks (DSB). Furthermore, chronic inflammatory diseases such as ulcerative colitis (UC) are characterized by an excess of reactive nitrogen species that can also lead to DNA double-strand breakage and genomic instability. We investigated the expression of the nuclear casein kinase and cyclin-dependent kinase substrate 1 (NUCKS1) protein in UC and sporadic colorectal cancer (CRC) due to its involvement in both DNA double-strand break repair and inflammatory signaling. Methods: NUCKS1 expression and expression of the DNA double-strand break marker gamma-H2AX (γH2AX) were assessed in formalin-fixed, paraffin-embedded UC and CRC patient biopsies using peroxidase immunohistochemistry. Expression levels for both proteins were evaluated together with previously published expression-level data for hTERT and TP53 proteins in the same material. Results: Nondysplastic UC lesions had 10-fold lower γH2AX expression and approximately 4-fold higher NUCKS1 expression compared with sporadic CRC, indicating minimal DNA DSB damage and heightened DNA DSB repair in these lesions, respectively. NUCKS1 expression in UC tended to decrease with increasing grades of dysplasia, whereas γH2AX, hTERT, and TP53 expression tended to increase with increasing grades of dysplasia. The highest γH2AX expression was seen in sporadic CRC, indicating considerable DNA DSB damage, whereas the highest NUCKS1 expression and hTERT expression were seen in nondysplastic UC. Conclusions: Overall, our data suggest that NUCKS1 may be involved in DNA DSB repair and/or inflammatory signaling in UC, but a more thorough investigation of both pathways in UC is warranted.

Identification of kinases phosphorylating 13 sites in the nuclear, DNA-binding protein NUCKS.

NUCKS is a vertebrate specific, nuclear and DNA-binding phospho protein. The protein is highly expressed in rapidly dividing cells, and is overexpressed in a number of cancer tissues. The phosphorylation of NUCKS is cell cycle and DNA-damage regulated, but little is known about the responsible kinases. By utilizing in vitro and in vivo phosphorylation assays using isolated NUCKS as well as synthetic NUCKS-derived peptides in combination with mass spectrometry, phosphopeptide mapping, phosphphoamino acid analyses, phosphospecific antibodies and the use of specific kinase inhibitors, we found that NUCKS is phosphorylated on 11 sites by CK2. At least 7 of the CK2 sites are phosphorylated in vivo. We also found that NUCKS is phosphorylated on two sites by ATM kinase and DNA-PK in vitro, and is phosphorylated in vivo by ATM kinase in γ-irradiated cells. All together, we identified three kinases phosphorylating 13 out of 39 in vivo phosphorylated sites in mammalian NUCKS. The identification of CK2 and PIKK kinases as kinases phosphorylating NUCKS in vivo provide further evidence for the involvement of NUCKS in cell cycle control and DNA repair.

Nucks1 synergizes with Trp53 to promote radiation lymphomagenesis in mice.

NUCKS1 is a 27 kD vertebrate-specific protein, with a role in the DNA damage response. Here, we show that after 4 Gy total-body X-irradiation, Trp53+/- Nucks1+/- mice more rapidly developed tumors, particularly thymic lymphoma (TL), than Trp53+/- mice. TLs in both cohorts showed loss of heterozygosity (LOH) of the Trp53+ allele in essentially all cases. In contrast, LOH of the Nucks1+ allele was rare. Nucks1 expression correlated well with Nucks1 gene dosage in normal thymi, but was increased in the majority of TLs from Trp53+/- Nucks1+/- mice, suggesting that elevated Nucks1 message may be associated with progression towards malignancy in vivo. Trp53+/- Nucks1+/- mice frequently succumbed to CD4- CD8- TLs harboring translocations involving Igh but not Tcra/d, indicating TLs in Trp53+/- Nucks1+/- mice mostly originated prior to the double positive stage and at earlier lineage than TLs in Trp53+/- mice. Monoclonal rearrangements at Tcrb were more prevalent in TLs from Trp53+/- Nucks1+/- mice, as was infiltration of primary TL cells to distant organs (liver, kidney and spleen). We propose that, in the context of Trp53 deficiency, wild type levels of Nucks1 are required to suppress radiation-induced TL, likely through the role of the NUCKS1 protein in the DNA damage response.

NUCKS1 is a novel RAD51AP1 paralog important for homologous recombination and genome stability.

NUCKS1 (nuclear casein kinase and cyclin-dependent kinase substrate 1) is a 27 kD chromosomal, vertebrate-specific protein, for which limited functional data exist. Here, we demonstrate that NUCKS1 shares extensive sequence homology with RAD51AP1 (RAD51 associated protein 1), suggesting that these two proteins are paralogs. Similar to the phenotypic effects of RAD51AP1 knockdown, we find that depletion of NUCKS1 in human cells impairs DNA repair by homologous recombination (HR) and chromosome stability. Depletion of NUCKS1 also results in greatly increased cellular sensitivity to mitomycin C (MMC), and in increased levels of spontaneous and MMC-induced chromatid breaks. NUCKS1 is critical to maintaining wild type HR capacity, and, as observed for a number of proteins involved in the HR pathway, functional loss of NUCKS1 leads to a slow down in DNA replication fork progression with a concomitant increase in the utilization of new replication origins. Interestingly, recombinant NUCKS1 shares the same DNA binding preference as RAD51AP1, but binds to DNA with reduced affinity when compared to RAD51AP1. Our results show that NUCKS1 is a chromatin-associated protein with a role in the DNA damage response and in HR, a DNA repair pathway critical for tumor suppression.

Dynamic expression of the vertebrate-specific protein Nucks during rodent embryonic development.

The nuclear casein kinase and cyclin-dependent kinase substrate 1 (NUCKS) is a highly phosphorylated nuclear protein that is overexpressed in many types of cancer. The flexibility of NUCKS and its extensive posttranslational modifications indicate that it is multifunctional, and its expression in most cell types suggests a housekeeping function. However, spatiotemporal expression of the Nucks protein during rodent development has not been reported. Thus, we investigated the expression of both the Nucks mRNA and protein during rat and mouse development by immunohistochemistry, in situ hybridization, Western immunoblotting, and reverse-transcription PCR analysis. We also used BLAST analysis against expressed sequence tag databases to determine whether a NUCKS homologue is expressed in invertebrate organisms. We found that Nucks expression increased during the initial stages of embryonic development, and then gradually decreased until birth in all tissues except the nervous tissue and muscle fibers. Interestingly, the expression of Nucks was very strong in migrating neural crest cells at E13.5 and ectoderm-derived tissues. In most tissues analyzed, the levels of Nucks correlated with the levels of Bax and activated caspase-3, which are indicative of apoptosis. Moreover, Nucks was upregulated very early during neuronal apoptosis in vitro. Expression analysis revealed that no transcript with close homology to the Nucks gene was present in invertebrates. The expression of Nucks in both proliferating and quiescent cells and its correlation with Bax levels and apoptosis strongly suggest that Nucks plays complex roles in cell homeostasis. Furthermore, the lack of homology in invertebrate organisms indicates a specific role for Nucks in vertebrate embryogenesis.

NUCKS overexpression in breast cancer.

BACKGROUND: NUCKS (Nuclear, Casein Kinase and Cyclin-dependent Kinase Substrate) is a nuclear, DNA-binding and highly phosphorylated protein. A number of reports show that NUCKS is highly expressed on the level of mRNA in several human cancers, including breast cancer. In this work, NUCKS expression on both RNA and protein levels was studied in breast tissue biopsies consisted of invasive carcinomas, intraductal proliferative lesions, benign epithelial proliferations and fibroadenomas, as well as in primary cultures derived from the above biopsies. Specifically, in order to evaluate the level of NUCKS protein in correlation with the histopathological features of breast disease, immunohistochemistry was employed on paraffin sections of breast biopsies of the above types. In addition, NUCKS expression was studied by means of Reverse Transcription PCR (RT-PCR), real-time PCR (qRT-PCR) and Western immunoblot analyses in the primary cell cultures developed from the same biopsies. RESULTS: The immunohistochemical Results showed intense NUCKS staining mostly in grade I and II breast carcinomas compared to normal tissues. Furthermore, NUCKS was moderate expressed in benign epithelial proliferations, such as adenosis and sclerosing adenosis, and highly expressed in intraductal lesions, specifically in ductal carcinomas in situ (DCIS). It is worth noting that all the fibroadenoma tissues examined were negative for NUCKS staining. RT-PCR and qRT-PCR showed an increase of NUCKS expression in cells derived from primary cultures of proliferative lesions and cancerous tissues compared to the ones derived from normal breast tissues and fibroadenomas. This increase was also confirmed by Western immunoblot analysis. Although NUCKS is a cell cycle related protein, its expression does not correlate with Ki67 expression, neither in tissue sections nor in primary cell cultures. CONCLUSION: The results show overexpression of the NUCKS protein in a number of non malignant breast lesions and cancerous tissues. In particular, the NUCKS overexpression in ADH and DCIS indicates a significant role of this protein in neoplastic progression.

Chromosomal changes in high- and low-invasive mouse lung adenocarcinoma cell strains derived from early passage mouse lung adenocarcinoma cell strains.

The incidence of adenocarcinoma of the lung is increasing in the United States, however, the difficulties in obtaining lung cancer families and representative samples of early to late stages of the disease have lead to the study of mouse models for lung cancer. We used Spectral Karyotyping (SKY), mapping with fluorescently labeled genomic clones (FISH), comparative genomic hybridization (CGH) arrays, gene expression arrays, Western immunoblot and real time polymerase chain reaction (PCR) to analyze nine pairs of high-invasive and low-invasive tumor cell strains derived from early passage mouse lung adenocarcinoma cells to detect molecular changes associated with tumor invasion. The duplication of chromosomes 1 and 15 and deletion of chromosome 8 were significantly associated with a high-invasive phenotype. The duplication of chromosome 1 at band C4 and E1/2-H1 were the most significant chromosomal changes in the high-invasive cell strains. Mapping with FISH and CGH array further narrowed the minimum region of duplication of chromosome 1 to 71-82 centimorgans (cM). Expression array analysis and confirmation by real time PCR demonstrated increased expression of COX-2, Translin (TB-RBP), DYRK3, NUCKS and Tubulin-alpha4 genes in the high-invasive cell strains. Elevated expression and copy number of these genes, which are involved in inflammation, cell movement, proliferation, inhibition of apoptosis and telomere elongation, were associated with an invasive phenotype. Similar linkage groups are altered in invasive human lung adenocarcinoma, implying that the mouse is a valid genetic model for the study of the progression of human lung adenocarcinoma.

Identification and characterization of two putative nuclear localization signals (NLS) in the DNA-binding protein NUCKS.

Immunofluorescence analyses show that the vertebrate specific and DNA-binding protein NUCKS is distributed throughout the cytoplasm in mitotic cells and targeted to the reforming nuclei in late telophase of the cell cycle. Computer analysis of the primary structure of NUCKS revealed the presence of two regions of highly charged, basic residues, which were identified as potential nuclear localization signals (NLSs). One of these signals (NLS1) is highly conserved between the species investigated, and fits to the description of being a classical bipartite NLS. The other amino acid motif (NLS2) is less conserved and does not constitute a classical bipartite NLS consensus sequence. We have shown that each of the two putative NLSs is capable of translocating green fluorescent protein (GFP) into the nucleus. The highly conserved NLS1 is monopartite, resembling the signals of c-Myc and RanBP3. Surprisingly, a natural occurring splice variant of NUCKS lacking 40 amino acids including NLS1, is not capable of translocating a corresponding NUCKS-GFP fusion protein into the nucleus, indicating that NLS1 is the main nuclear localization signal in NUCKS. This is also confirmed by site-directed mutagenesis of the full-length protein. By GFP-immunoprecipitation and GST-pull down experiments, we show that NUCKS binds to importin alpha3 and importin alpha5 in vitro, suggesting that the nuclear targeting of NUCKS follows a receptor-mediated and energy-dependent import mechanism.

Cytoplasmic retention of peroxide-activated ERK provides survival in primary cultures of rat hepatocytes.

Reactive oxygen species (ROS) are implicated in tissue damage causing primary hepatic dysfunction following ischemia/reperfusion injury and during inflammatory liver diseases. A potential role of extracellular signal-regulated kinase (ERK) as a mediator of survival signals during oxidative stress was investigated in primary cultures of hepatocytes exposed to ROS. Hydrogen peroxide (H(2)O(2)) induced a dose-dependent activation of ERK, which was dependent on MEK activation. The ERK activation pattern was transient compared with the ERK activation seen after stimulation with epidermal growth factor (EGF). Nuclear accumulation of ERK was found after EGF stimulation, but not after H(2)O(2) exposure. A slow import/rapid export mechanism was excluded through the use of leptomycin B, an inhibitor of nuclear export sequence-dependent nuclear export. Reduced survival of hepatocytes during ROS exposure was observed when ERK activation was inhibited. Ribosomal S6 kinase (RSK), a cytoplasmic ERK substrate involved in cell survival, was activated and located in the nucleus of H(2)O(2)-exposed hepatocytes. The activation was abolished when ERK was inhibited with U0126. In conclusion, our results indicate that activity of ERK in the cytoplasm is important for survival during oxidative stress in hepatocytes and that RSK is activated downstream of ERK. Supplementary material for this article can be found on the HEPATOLOGY website (http://www.interscience.wiley.com/jpages/0270-9139/suppmat/index.html).

Characterisation of the NUCKS gene on human chromosome 1q32.1 and the presence of a homologous gene in different species.

The NUCKS gene is located on human chromosome 1q32.1 and consists of seven exons and six introns. The gene lacks a TATA box but contains two Inr elements, two GC boxes, and one consensus-binding site for E2F-1. NUCKS is expressed in all human adult and foetal tissues investigated, and has all the features of being a housekeeping gene. Both data searches and Western immunoblotting experiments show that a homologous protein is present in fish, amphibians, and birds but not in insects and yeast, suggesting that NUCKS is a vertebrate specific gene. In all the species investigated, the protein contains several consensus phosphorylation sites for cyclin-dependent kinases and CK-2, and we have shown that the fish protein (like mammalian NUCKS) indeed is a substrate for CDK1 and CK-2 in vitro. The NUCKS protein is also conserved with respect to a DNA-binding domain previously characterised in mammals, and two putative bipartite nuclear localisation signals.

A putative DNA-binding domain in the NUCKS protein.

We have studied the DNA-binding properties of a NUCKS-derived, synthetic peptide containing an extended GRP motif. This peptide binds to random-sequence DNA, but did not bind preferentially to poly(dA-dT). A synthetic peptide with the same amino acid composition but with a random sequence did not bind to DNA, suggesting that the structure of the DNA-binding domain plays a pivotal role in the interaction with DNA. NMR and graphic modeling were employed to investigate the structure of the synthetic peptide. It was shown that the DNA-binding peptide constituted an alpha helix in phosphate buffer at pH 5.5. Docking results indicated an almost perfect fit for this small, helical peptide into the major groove of DNA with the possibility of four basic residues interacting with the phosphate backbone of DNA. One consensus site for phosphorylation by Cdk1 is located in the N-terminal end of the DNA-binding peptide. Upon phosphorylation of this site, the binding to DNA was completely prohibited. Immunofluorescence experiments showed that NUCKS was located in the nuclei in proliferating cells in interphase of the cell cycle, but was distributed throughout the cytoplasm in mitotic cells.

Nuclear receptor and apoptosis initiator NGFI-B is a substrate for kinase ERK2.

NGFI-B is an inducible orphan nuclear receptor that initiates apoptosis. Growth factors such as EGF activate the MAP kinase ERK, whose activity may determine if a cell survives or undergoes apoptosis. EGF stimulation of cells leads to phosphorylation of threonine in NGFI-B. Thr-142 of NGFI-B is comprised in a consensus MAP kinase site and was identified as a preferred substrate for ERK2 (but not ERK1) in vitro. These results suggest that NGFI-B may be a molecular target for ERK2 signals and thereby a substrate for crosstalk between a growth factor survival pathway and an inducible regulator of apoptosis.

UV induces tyrosine kinase-independent internalisation and endosome arrest of the EGF receptor.

We have compared the activation and trafficking of epidermal growth factor receptor (EGFR) induced by UV light and EGF. Tyrosine phosphorylation of EGFR was not detected in UV-exposed cells by immunoblotting of whole cell lysates or EGFR immunoprecipitates with antibodies specific for each of the five activated autophosphorylation sites of EGFR. In addition, EGFR of UV-irradiated cells did not demonstrate increased (32)P-incorporation. However, UV-exposed cells demonstrated a gel mobility shift of EGFR, which was not abolished by alkaline phosphatase treatment. UV-exposure did not induce dimerisation of EGFR. Furthermore, UV induced internalisation of EGFR without polyubiquitination or degradation. UV-exposed EGFR was transferred to early endosomes and arrested in transferrin-accessible endosomes close to the cell surface. Whereas inhibition of the EGFR tyrosine kinase effectively inhibited tyrosine phosphorylation and internalisation of EGF-activated EGFR, internalisation of UV-exposed EGFR was unaffected. UV induced neither relocalisation of Shc and Grb2 nor activation of Raf, but activation of MEK and MAPK was observed. Our work indicates that UV induces internalisation of EGFR independent of its phosphorylation or receptor tyrosine kinase activation, and altered EGFR trafficking compared with ligand-activated receptor. In addition, MAPK activation by UV does not appear to be mediated by EGFR activation.