Nuclear Matrix-associated Protein Research Articles

Nuclear Matrix-associated Protein SMAR1 Attenuated Acute Graft-versus-host Disease by Targeting JAK-STAT Signaling in CD4 + T Cells.

Acute graft-versus-host disease (aGVHD) mediated by alloreactive T cells remains a serious and life-threatening complication of allogeneic hematopoietic cell transplantation (allo-HCT). The contribution of the different CD4 + T helper cell subtypes to the pathogenesis and regulation of aGVHD is a central point in current research. The specialized effector subsets of T cells that differentiate from naive T cells into mature cells are closely related to scaffold/matrix-associated region-1-binding protein (SMAR1). However, the role of SMAR1 in aGVHD is unclear. Peripheral blood was collected from the patients with or without aGVHD after allo-HCT. The differences in CD4 + T cells transduced with the SMAR1 lentivirus vector and empty vector were analyzed. A humanized aGVHD mouse model was constructed to evaluate the function of SMAR1 in aGVHD. The expression of SMAR1 was significantly reduced in the CD4 + T cells from aGVHD patients and related to the occurrence of aGVHD. SMAR1 overexpression in human CD4 + T cells regulated CD4 + T-cell subsets differentiation and inflammatory cytokines secretion and inhibited the Janus kinase/signal transducer and activator of transcription pathway. Moreover, SMAR1 changed chromatin accessibility landscapes and affected the binding motifs of key transcription factors regulating T cells. Additionally, upregulation of SMAR1 expression in CD4 + T cells improved the survival and pathology in a humanized aGVHD mouse model. Our results showed that upregulation of SMAR1 regulated the CD4 + T-cell subpopulation and cytokines secretion and improved survival in a humanized aGVHD mouse model by alleviating inflammation. This study provides a promising therapeutic target for aGVHD.

YAP-Activated SATB2 Is a Coactivator of NRF2 That Amplifies Antioxidative Capacity and Promotes Tumor Progression in Renal Cell Carcinoma.

A YAP-SATB2-NRF2 regulatory axis amplifies antioxidative stress signaling and provides potential therapeutic targets to enhance response to chemotherapy in renal cell carcinoma.

HOXA9 Nucleates a Repressive Chromatin Complex with Nuclear Matrix-Associated Protein- Safb to Maintain Acute Myeloid Leukemia

HOXA9 Nucleates a Repressive Chromatin Complex with Nuclear Matrix-Associated Protein- Safb to Maintain Acute Myeloid Leukemia

Identification of DTL as Related Biomarker and Immune Infiltration Characteristics of Nasopharyngeal Carcinoma via Comprehensive Strategies.

PurposeAlthough considerable progress has been made in basic and clinical research on nasopharyngeal carcinoma (NPC), the biomarkers of the progression of NPC have not been fully studied and described. This study was designed to identify potential novel biomarkers for NPC using integrated analyses and explore the immune cell infiltration in this pathological process.MethodsFive GEO data sets were downloaded from gene expression omnibus database (GEO) and analysed to identify differentially expressed genes (DEGs), followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. The four algorithms were adopted for screening of novel and key biomarkers for NPC, including random forest (RF) machine learning algorithm, least absolute shrinkage and selection operator (LASSO) logistic regression, support vector machine-recursive feature elimination (SVM-RFE), and weighted gene co-expression network analysis (WGCNA). Lastly, CIBERSORT was used to assess the infiltration of immune cells in NPC, and the correlation between diagnostic markers and infiltrating immune cells was analyzed.ResultsHerein, we identified 46 DEGs, and enrichment analysis results showed that DEGs and several kinds of signaling pathways might be closely associated with the occurrence and progression of NPC. DTL was recognized as NPC-related biomarker. DTL, also known as retinoic acid-regulated nuclear matrix-associated protein (RAMP), or DNA replication factor 2 (CDT2), is reported to be correlated with the cell proliferation, cell cycle arrest and cell invasion in hepatocellular carcinoma, breast cancer and gastric cancer. Immune infiltration analysis demonstrated that macrophages M0, macrophages M1 and T cells CD4 memory activated were linked to pathogenesis of NPC.ConclusionIn summary, we adopted a comprehensive strategy to screen DTL as biomarkers related to NPC and explore the critical role of immune cell infiltration in NPC.

HNRNPU promotes the progression of hepatocellular carcinoma by enhancing CDK2 transcription

The nuclear matrix-associated protein Heterogeneous Nuclear Ribonucleoprotein U (HNRNPU), also known as SAF-A, is known to maintain active chromatin structure in mouse hepatocytes. However, the functional roles and molecular mechanisms of HNRNPU in the development of hepatocellular carcinoma (HCC) remain largely unknown. Herein, we found that HNRNPU was upregulated in HCC, and the proliferation of HCC cells was inhibited in vitro and in vivo upon HNRNPU knockdown. Moreover, the upregulation of HNRNPU was correlated with poor prognosis in HCC. Mechanistically, HNRNPU bound to the CDK2 gene locus, a key factor in cell cycle regulation, where it was enriched with H3K27 acetylation (H3K27ac), H3K9 acetylation (H3K9ac), and H3K4 mono-methylation (H3K4me1). Furthermore, HNRNPU knockdown reduced the levels of H3K27ac and H3K9ac at the binding site, where the levels of H3K27 tri-methylation (H3K27me3) were increased, eventually leading to the downregulation of CDK2. Collectively, our results provide a new mechanism whereby HNRNPU promotes HCC development by enhancing the transcription of CDK2.

Subcellular dynamics of estrogen-related receptors involved in transrepression through interactions with scaffold attachment factor B1.

Estrogen-related receptor (ERR), a member of the nuclear receptor superfamily, consists of three subtypes (α, β, γ) and has strong homology with estrogen receptor. No endogenous ligands have been identified for ERRs, but they play key roles in metabolic, hormonal, and developmental processes as transcription factors without ligand binding. Although subnuclear dynamics are essential for nuclear events including nuclear receptor-mediated transcriptional regulation, the dynamics of ERRs are poorly understood. Here, we report that ERRs show subcellular kinetic changes in response to diethylstilbestrol (DES), a synthetic estrogen that represses the transactivity of all three ERR subtypes, using live-cell imaging with fluorescent protein labeling. Upon DES treatment, all ERR subtypes formed discrete clusters in the nucleus, with ERRγ also displaying nuclear export. Fluorescence recovery after photobleaching analyses revealed significant reductions in the intranuclear mobility of DES-bound ERRα and ERRβ, and a slight reduction in the intranuclear mobility of DES-bound ERRγ. After DES treatment, colocalization of all ERR subtypes with scaffold attachment factor B1 (SAFB1), a nuclear matrix-associated protein, was observed in dot-like subnuclear clusters, suggesting interactions of the ERRs with the nuclear matrix. Consistently, co-immunoprecipitation analyses confirmed enhanced interactions between ERRs and SAFB1 in the presence of DES. SAFB1 was clarified to repress the transactivity of all ERR subtypes through the ERR-response element. These results demonstrate ligand-dependent cluster formation of ERRs in the nucleus that is closely associated with SAFB1-mediated transrepression. Taken together, the present findings provide a new understanding of the pathophysiology regulated by ERR/SAFB1 signaling pathways and their subcellular dynamics.

Deregulation of SATB2 in carcinogenesis with emphasis on miRNA-mediated control.

The special AT-rich DNA binding protein (SATB2) is a nuclear matrix-associated protein and an important transcription factor for biological development, gene regulation and chromatin remodeling. Aberrant regulation of SATB2 has been found to highly correlate with various types of cancers including lung, colon, prostate, breast, gastric and liver. Recent studies have revealed that a subset of small non-coding RNAs, termed microRNAs (miRNAs), are important regulators of SATB2 function. As post-transcriptional regulators, miRNAs have been found to have fundament importance maintaining normal cellular development. Evidence suggests that multiple miRNAs, including miR-31, miR-34, miR-182, miR-211, miR-599, are capable of regulating SATB2 in cancers of the lung, liver, colon and breast. This review examines the molecular functions of SATB2 and miRNAs in the text of cancer development and potential strategies for cancer therapy with a focus on systemic miRNA delivery.

The Cellular DNA Helicase ChlR1 Regulates Chromatin and Nuclear Matrix Attachment of the Human Papillomavirus 16 E2 Protein and High-Copy-Number Viral Genome Establishment.

ABSTRACTIn papillomavirus infections, the viral genome is established as a double-stranded DNA episome. To segregate the episomes into daughter cells during mitosis, they are tethered to cellular chromatin by the viral E2 protein. We previously demonstrated that the E2 proteins of diverse papillomavirus types, including bovine papillomavirus (BPV) and human papillomavirus 16 (HPV16), associate with the cellular DNA helicase ChlR1. This virus-host interaction is important for the tethering of BPV E2 to mitotic chromatin and the stable maintenance of BPV episomes. The role of the association between E2 and ChlR1 in the HPV16 life cycle is unresolved. Here we show that an HPV16 E2 Y131A mutant (E2Y131A) had significantly reduced binding to ChlR1 but retained transcriptional activation and viral origin-dependent replication functions. Subcellular fractionation of keratinocytes expressing E2Y131A showed a marked change in the localization of the protein. Compared to that of wild-type E2 (E2WT), the chromatin-bound pool of E2Y131A was decreased, concomitant with an increase in nuclear matrix-associated protein. Cell cycle synchronization indicated that the shift in subcellular localization of E2Y131A occurred in mid-S phase. A similar alteration between the subcellular pools of the E2WT protein occurred upon ChlR1 silencing. Notably, in an HPV16 life cycle model in primary human keratinocytes, mutant E2Y131A genomes were established as episomes, but at a markedly lower copy number than that of wild-type HPV16 genomes, and they were not maintained upon cell passage. Our studies indicate that ChlR1 is an important regulator of the chromatin association of E2 and of the establishment and maintenance of HPV16 episomes. IMPORTANCE Infections with high-risk human papillomaviruses (HPVs) are a major cause of anogenital and oropharyngeal cancers. During infection, the circular DNA genome of HPV persists within the nucleus, independently of the host cell chromatin. Persistence of infection is a risk factor for cancer development and is partly achieved by the attachment of viral DNA to cellular chromatin during cell division. The HPV E2 protein plays a critical role in this tethering by binding simultaneously to the viral genome and to chromatin during mitosis. We previously showed that the cellular DNA helicase ChlR1 is required for loading of the bovine papillomavirus E2 protein onto chromatin during DNA synthesis. Here we identify a mutation in HPV16 E2 that abrogates interaction with ChlR1, and we show that ChlR1 regulates the chromatin association of HPV16 E2 and that this virus-host interaction is essential for viral episome maintenance.

Epigenetic regulation of long noncoding RNA UCA1 by SATB1 in breast cancer.

Special AT-rich sequence binding protein 1 (SATB1) is a nuclear matrix-associated DNA-binding protein that functions as a chromatin organizer. SATB1 is highly expressed in aggressive breast cancer cells and promotes growth and metastasis by reprograming gene expression. Through genome-wide cross-examination of gene expression and histone methylation, we identified SATB1 target genes for which expression is associated with altered epigenetic marks. Among the identified genes, long noncoding RNA urothelial carcinoma-associated 1 (UCA1) was upregulated by SATB1 depletion. Upregulation of UCA1 coincided with increased H3K4 trimethylation (H3K4me3) levels and decreased H3K27 trimethylation (H3K27me3) levels. Our study showed that SATB1 binds to the upstream region of UCA1 in vivo, and that its promoter activity increases with SATB1 depletion. Furthermore, simultaneous depletion of SATB1 and UCA1 potentiated suppression of tumor growth and cell survival. Thus, SATB1 repressed the expression of oncogenic UCA1, suppressing growth and survival of breast cancer cells. [BMB Reports 2016; 49(10): 578-583]

FUS interacts with nuclear matrix-associated protein SAFB1 as well as Matrin3 to regulate splicing and ligand-mediated transcription.

FUS (Fused-in-Sarcoma) is a multifunctional DNA/RNA binding protein linked to familial amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD). Since FUS is localized mainly in the nucleus with nucleo-cytoplasmic shuttling, it is critical to understand physiological functions in the nucleus to clarify pathogenesis. Here we report a yeast two-hybrid screening identified FUS interaction with nuclear matrix-associated protein SAFB1 (scaffold attachment factor B1). FUS and SAFB1, abundant in chromatin-bound fraction, interact in a DNA-dependent manner. N-terminal SAP domain of SAFB1, a DNA-binding motif, was required for its localization to chromatin-bound fraction and splicing regulation. In addition, depletion of SAFB1 reduced FUS’s localization to chromatin-bound fraction and splicing activity, suggesting SAFB1 could tether FUS to chromatin compartment thorough N-terminal DNA-binding motif. FUS and SAFB1 also interact with Androgen Receptor (AR) regulating ligand-dependent transcription. Moreover, FUS interacts with another nuclear matrix-associated protein Matrin3, which is muted in a subset of familial ALS cases and reportedly interacts with TDP-43. Interestingly, ectopic ALS-linked FUS mutant sequestered endogenous Matrin3 and SAFB1 in the cytoplasmic aggregates. These findings indicate SAFB1 could be a FUS’s functional platform in chromatin compartment to regulate RNA splicing and ligand-dependent transcription and shed light on the etiological significance of nuclear matrix-associated proteins in ALS pathogenesis.

Cardiomyocyte--specific expression of the nuclear matrix protein, CIZ1, stimulates production of mono-nucleated cells with an extended window of proliferation in the postnatal mouse heart.

ABSTRACTMyocardial injury in mammals leads to heart failure through pathological cardiac remodelling that includes hypertrophy, fibrosis and ventricular dilatation. Central to this is inability of the mammalian cardiomyocyte to self-renew due to entering a quiescent state after birth. Modulation of the cardiomyocyte cell-cycle after injury is therefore a target mechanism to limit damage and potentiate repair and regeneration. Here, we show that cardiomyocyte-specific over-expression of the nuclear-matrix­-associated DNA replication protein, CIZ1, extends their window of proliferation during cardiac development, delaying onset of terminal differentiation without compromising function. CIZ1-expressing hearts are enlarged, but the cardiomyocytes are smaller with an overall increase in number, correlating with increased DNA replication after birth and retention of an increased proportion of mono-nucleated cardiomyocytes into adulthood. Furthermore, these CIZ1 induced changes in the heart reduce the impact of myocardial injury, identifying CIZ1 as a putative therapeutic target for cardiac repair.

Nuclear matrix-associated protein SMAR1 regulates alternative splicing via HDAC6-mediated deacetylation of Sam68

Pre-mRNA splicing is a complex regulatory nexus modulated by various trans-factors and their posttranslational modifications to create a dynamic transcriptome through alternative splicing. Signal-induced phosphorylation and dephosphorylation of trans-factors are known to regulate alternative splicing. However, the role of other posttranslational modifications, such as deacetylation/acetylation, methylation, and ubiquitination, that could modulate alternative splicing in either a signal-dependent or -independent manner remain enigmatic. Here, we demonstrate that Scaffold/matrix-associated region-binding protein 1 (SMAR1) negatively regulates alternative splicing through histone deacetylase 6 (HDAC6)-mediated deacetylation of RNA-binding protein Sam68 (Src-associated substrate during mitosis of 68 kDa). SMAR1 is enriched in nuclear splicing speckles and associates with the snRNAs that are involved in splice site recognition. ERK-MAPK pathway that regulates alternative splicing facilitates ERK-1/2-mediated phosphorylation of SMAR1 at threonines 345 and 360 and localizes SMAR1 to the cytoplasm, preventing its interaction with Sam68. We showed that endogenously, SMAR1 through HDAC6 maintains Sam68 in a deacetylated state. However, knockdown or ERK-mediated phosphorylation of SMAR1 releases the inhibitory SMAR1-HDAC6-Sam68 complex, facilitating Sam68 acetylation and alternative splicing. Furthermore, loss of heterozygosity at the Chr.16q24.3 locus in breast cancer cells, wherein the human homolog of SMAR1 (BANP) has been mapped, enhances Sam68 acetylation and CD44 variant exon inclusion. In addition, tail-vein injections in mice with human breast cancer MCF-7 cells depleted for SMAR1 showed increased CD44 variant exon inclusion and concomitant metastatic propensity, confirming the functional role of SMAR1 in regulation of alternative splicing. Thus, our results reveal the complex molecular mechanism underlying SMAR1-mediated signal-dependent and -independent regulation of alternative splicing via Sam68 deacetylation.

SMAR1 coordinates HDAC6-induced deacetylation of Ku70 and dictates cell fate upon irradiation.

Acetylation status of DNA end joining protein Ku70 dictates its function in DNA repair and Bax-mediated apoptosis. Despite the knowledge of HDACs and HATs that are reported to modulate the acetylation dynamics of Ku70, very little is known about proteins that critically coordinate these key modifications. Here, we demonstrate that nuclear matrix-associated protein scaffold/matrix-associated region-binding protein 1 (SMAR1) is a novel interacting partner of Ku70 and coordinates with HDAC6 to maintain Ku70 in a deacetylated state. Our studies revealed that knockdown of SMAR1 results in enhanced acetylation of Ku70, which leads to impaired recruitment of Ku70 in the chromatin fractions. Interestingly, ionizing radiation (IR) induces the expression of SMAR1 and its redistribution as distinct nuclear foci upon ATM-mediated phosphorylation at serine 370. Furthermore, SMAR1 regulates IR-induced G2/M cell cycle arrest by facilitating Chk2 phosphorylation. Alternatively, SMAR1 provides radioresistance by modulating the association of deacetylated Ku70 with Bax, abrogating the mitochondrial translocation of Bax. Thus, we provide mechanistic insights of SMAR1-mediated regulation of repair and apoptosis via a complex crosstalk involving Ku70, HDAC6 and Bax.

SAFB1 interacts with and suppresses the transcriptional activity of p53

A significant amount of nuclear p53 is found associated with the nuclear matrix in cells that were exposed to genotoxic stress. In this study we identified Scaffold attachment factor B1 (SAFB1), a nuclear matrix-associated protein that binds the scaffold or matrix attachment regions (S/MARs) of genomic DNA, as a novel p53-interacting protein. SAFB1 was able to associate with p53 through its C-terminal domain, while significant co-localization of the two proteins was observed in cells treated with 5-fluorouracil or mithramycin. Binding of p53 to SAFB1 had a significant functional outcome, since SAFB1 was shown to suppress p53-mediated reporter gene expression. These data suggest that nuclear matrix-associated proteins may play a critical role in regulating p53 localization and activity. Structured summaryp53physically interacts with SRPK1a: shown by two hybrid (view interaction)p53physically interacts with SRPK1a: shown by pull down (view interaction)p53physically interacts with SRPK1a: shown by anti bait coimmunoprecipitation (view interaction)p53physically interacts with SRPK1a: shown by anti tag coimmunoprecipitation (view interaction)SAFB1physically interacts with p53: shown by pull down (view interactions 1, 2)SAFB1physically interacts with p53: shown by anti bait coimmunoprecipitation (view interactions 1, 2)SAFB1 and p53colocalize: shown by fluorescence microscopy (view interaction)SAFB2physically interacts with p53: shown by pull down (view interaction)

Identification of retinoic acid-regulated nuclear matrix-associated protein as a novel regulator of gastric cancer.

Background: Retinoic acid-regulated nuclear matrix-associated protein (RAMP) is a WD40 repeat-containing protein that is involved in various biological functions, but little is known about its role in human cancer. This study aims to delineate the oncogenic role of RAMP in gastric carcinogenesis.Methods: RAMP expression was examined by real-time quantitative RT-PCR, immunohistochemistry and western blotting. Inhibition of RAMP expression was performed by siRNA-mediated knockdown. The functional effects of RAMP on cell kinetics were measured by cell viability assay, colony formation assay and flow cytometry. Cell lines stably expressing RAMP were established to investigate the oncogenic effects of RAMP in vitro.Results: Ramp was readily expressed in all seven gastric cancer cell lines and was significantly increased in human gastric cancer tissues when compared with their adjacent non-cancerous tissues (P<0.001). In keeping with this, expression of RAMP protein was higher in gastric cancer tissues compared with their adjacent non-cancerous tissues, whereas moderate protein expression were noted in intestinal metaplasia. Knockdown of RAMP in gastric cancer cells significantly reduced cell proliferation (P<0.01) and soft agar colony formation (P<0.001), but induced apoptosis and G2/M arrest. In additional, knockdown RAMP induced cell apoptosis is dependent on functional accumulation of p53 and p21 and induction of cleaved caspases-9, caspases-3 and PARP. Strikingly, overexpression of RAMP promoted anchorage-independent cell growth in soft agar.Conclusion: Our findings demonstrate that RAMP plays an oncogenic role in gastric carcinogenesis. Inhibition of RAMP may be a promising approach for gastric cancer therapy.

CK2β Is Expressed in Endometrial Carcinoma and Has a Role in Apoptosis Resistance and Cell Proliferation

Protein kinase CK2 (CK2) is a serine/threonine kinase that participates in important cellular processes. We have recently demonstrated that CK2 plays a role in resistance to TRAIL/Fas-induced apoptosis in endometrial carcinoma (EC) by regulating FLIP. Here, we assessed the immunohistochemical expression of CK2beta in EC and checked its role in cell proliferation and anchorage-independent cell growth. CK2beta immunostaining was assessed in two tissue microarrays, one constructed from paraffin-embedded blocks of 95 ECs and another from 70 samples of normal endometrium. CK2beta expression was correlated with histological type; grade and stage; cell proliferation (Ki-67) and apoptotic index; immunostaining for cyclin D1, PTEN, AKT, beta-catenin, and FLIP. Moreover, the Ishikawa EC cell line was subjected to down-regulation of CK2 by shRNA. CK2beta expression was frequent in EC (nuclear, 100%; cytoplasmic, 87.5%). The staining was more intense in EC than in normal endometrium (P = 0.000), and statistically correlated with AKT, PTEN, beta-catenin, and FLIP. In EC, CK2beta expression correlated with cell proliferation. Knock-down of CK2beta blocked colony formation of EC in soft agar, and also resulted in decreased expression of cyclin D1 and ERK phosphorylation. The results confirm that CK2beta is widely expressed in EC, and suggest a role in cell proliferation and anchorage-independent cell growth.

M1634 Overexpression of Retinoic Acid-Regulated Nuclear Matrix-Associated Protein (RAMP) Induces Cell Proliferation in Gastric Cancer: Potential Oncogenic Role of Ramp in Gastric Carcinogenesis

M1634 Overexpression of Retinoic Acid-Regulated Nuclear Matrix-Associated Protein (RAMP) Induces Cell Proliferation in Gastric Cancer: Potential Oncogenic Role of Ramp in Gastric Carcinogenesis

Subcellullar localization of tumor-associated antigen 3H11Ag

3H11Ag, a tumor-associated antigen defined by the monoclonal antibody 3H11 that specifically recognizes cancer cells in various tumor tissues, was successfully cloned recently, but its function is unknown. To explore the potential roles it plays in tumors, we analyzed its subcellular localization in the present study. By expressing 3H11Ag fused with fluorescent protein in COS-7 cells, we found that 3H11Ag localizes to both cytoplasm and nucleus, which was confirmed by subcellular fractionation. And sequentially extracting the nuclei of COS-7 cells transfected with 3H11Ag showed that it is a DNA- and nuclear matrix-associated protein. Moreover, by expressing a series of red fluorescent protein-tagged truncated forms of 3H11Ag, it was demonstrated that the 150 amino acid residues at its C-terminal are fully responsible for the subcellular localization. In addition, the results of the computational analysis of 3H11Ag were in accordance with those of the experimental analysis. All these data would be helpful to elucidate the functions of 3H11Ag.

Murine leukemia RL1 and sarcoma Meth A antigens recognized by cytotoxic T lymphocytes (CTL)

Peptide elution and expression cloning methods have been used to identify T cell-recognized antigens for which no molecular information is available. We identified a unique tumor antigen peptide pRL1a, IPGLPLSL that is recognized by CTL on BALB/c RL male 1 leukemia by peptide elution. The sequence of the peptide corresponded to the normally untranslated 5' region of akt. Cytotoxicity was generated in BALB/c spleen cells by in vivo and in vitro sensitization with pRL1a peptide in the form of multiple antigen peptide (MAP), but not the original form. pRL1a MAP immunization had a significant growth-inhibitory effect. pRL1a MAP was mostly internalized into the endosomal compartment of antigen-presenting cells, leaked to the cytosol, and degraded, and the pRL1a peptide produced was presented through the MHC class I pathway. In vivo depletion of CD4 T cells from tumor-inoculated BALB/c mice caused RL male 1 regression. Overexpression of the RLakt molecule seemed to induce CD4 immunoregulatory cells, which resulted in progressive RL male 1 growth in BALB/c mice. In vivo administration of anti-CD25 mAb (PC61) caused regression of RL male 1, suggesting that CD4(+) CD25(+) immunoregulatory cells were involved in the tumor growth. Recently, we improved the sensitivity and the efficacy of T cell antigen cloning from cDNA expression libraries by using large- and small-scale ELISPOT assays. Using the IFN-gamma ELISPOT method, we obtained a cDNA clone S35 of 937 bp recognized by AT-1 CTL on BALB/c Meth A sarcoma. S35 was a part of the retinoic acid-regulated nuclear matrix-associated protein (ramp). AT-1 CTL recognized the peptide LGAEAIFRL, which was derived from a newly created open reading frame due to the exon 14 extension.

In vitro modulation of the interaction between HA95 and LAP2beta by cAMP signaling.

The nuclear envelope mediates key functions by interacting with chromatin. We recently reported an interaction between the chromatin- and nuclear matrix-associated protein HA95 and the inner nuclear membrane integral protein LAP2beta, implicated in initiation of DNA replication (Martins et al. (2003) J. Cell Biol. 160, 177-188). Here, we show that in vitro, interaction between HA95 and LAP2beta is modulated by cAMP signaling via PKA. Exposure of an anti-HA95 immune precipitate from interphase HeLa cells to a mitotic extract promotes ATP-dependent release of LAP2beta from the HA95 complex. This coincides with Ser and Thr phosphorylation of HA95 and LAP2beta. Inhibition of PKA with PKI abolishes phosphorylation of HA95 and dissociation of LAP2beta from HA95, although LAPbeta remains phosphorylated. Antagonizing cAMP signaling in mitotic extract also abolishes the release of LAP2beta from HA95; however, disrupting PKA anchoring to A-kinase anchoring proteins has no effect. Inhibition of CDK activity in the extract greatly reduces LAP2beta phosphorylation but does not prevent LAP2beta release from HA95. Inhibition of PKC, MAP kinase, or CaM kinase II does not affect mitotic extract-induced dissociation of LAP2beta from HA95. PKA phosphorylates HA95 but not LAP2beta in vitro and elicits a release of LAP2beta from HA95. CDK1 or PKC phosphorylates LAP2beta within the HA95 complex, but neither kinase induces LAP2beta release. Our results indicate that in vitro, the interaction between HA95 and LAP2beta is influenced by a PKA-mediated phosphorylation of HA95 rather than by CDK1- or PKC-mediated phosphorylation of LAP2beta. This suggests an additional level of regulation of a chromatin-nuclear envelope interaction in dividing cells.