Peptide name : Nucleophosmin

Source/Organism : Human

Linear/Cyclic : Not found

Chirality : Not found

Peptide length: 294

C-terminal modification: Not found

N-terminal modification : Not found

Non-natural peptide information: None

Assay type : Antibody-based assay

Assay time : 48h

Activity : Not found

Cell line : HepG2

Cancer type : Not specified

Other activity : Not found

Amino acid composition bar chart :

Molecular mass : 32574.6569 Dalton

Aliphatic index : 0.626

Instability index : 51.3048

Hydrophobicity (GRAVY) : -0.969

Isoelectric point : 4.6389

Charge (pH 7) : -24.0053

Aromaticity : 0.044

Molar extinction coefficient (cysteine, cystine): (16960, 17085)

Hydrophobic/hydrophilic ratio : 0.69942196

hydrophobic moment : -0.009

Missing amino acid : None

Most occurring amino acid : D

Most occurring amino acid frequency : 34

Least occurring amino acid : W

Least occurring amino acid frequency : 2

Secondary structure fraction (Helix, Turn, Sheet): (0.3, 0.3, 0.2)

SMILES Notation: CC[C@H](C)[C@H](NC(=O)[C@H](C)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@@H](NC(=O)[C@H](CCSC)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@H](CS)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](NC(=O)[C@H](Cc1ccc(O)cc1)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@@H](NC(=O)[C@H](CCCCN)NC(=O)[C@@H]1CCCN1C(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)CNC(=O)CNC(=O)[C@H](CCCCN)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@@H](NC(=O)[C@H](CO)NC(=O)[C@H](C)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCSC)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@@H](NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H]1CCCN1C(=O)CNC(=O)[C@H](CCCCN)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](NC(=O)[C@H](CCCCN)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCCN)NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H]1CCCN1C(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCCCN)NC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](C)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@@H](NC(=O)[C@H](CO)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](NC(=O)[C@@H]1CCCN1C(=O)[C@H](C)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](C)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)CNC(=O)CNC(=O)CNC(=O)[C@@H]1CCCN1C(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](CCCCN)NC(=O)CNC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](C)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](Cc1c[nH]cn1)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](Cc1c[nH]cn1)NC(=O)[C@@H](NC(=O)[C@@H]1CCCN1C(=O)CNC(=O)[C@H](CO)NC(=O)CNC(=O)[C@H](CS)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@@H]1CCCN1C(=O)[C@@H]1CCCN1C(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](Cc1ccccc1)NC(=O)CNC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@@H]1CCCN1C(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](CO)NC(=O)[C@H](CCSC)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H](CCCCN)NC(=O)[C@@H](NC(=O)[C@@H]1CCCN1C(=O)[C@H](CO)NC(=O)CNC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](Cc1ccc(O)cc1)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCSC)NC(=O)[C@H](C)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](C)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H](Cc1c[nH]cn1)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)CNC(=O)[C@H](C)NC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](Cc1c[nH]cn1)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@@H](NC(=O)[C@H](CCCCN)NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@H](Cc1c[nH]cn1)NC(=O)[C@H](Cc1ccc(O)cc1)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](C)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CS)NC(=O)CNC(=O)[C@H](Cc1ccccc1)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](Cc1ccc(O)cc1)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H]1CCCN1C(=O)[C@H](CO)NC(=O)[C@H](CCSC)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCSC)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCSC)NC(=O)[C@H](CO)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)O)C(C)C)[C@@H](C)CC)C(C)C)[C@@H](C)CC)C(C)C)[C@@H](C)O)[C@@H](C)O)C(C)C)[C@@H](C)O)C(C)C)[C@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Paper title : New centromeric component CENP-W is an RNA-associated nuclear matrix protein that interacts with nucleophosmin/B23 protein.

Doi : https://doi.org/10.1074/jbc.M111.228411

Abstract : CENP-W was originally identified as a putative oncogene, cancer-upregulated gene 2 (CUG2) that was commonly up-regulated in many cancer tissues. Recently, CENP-W has also been identified as a new centromeric component that interacts with CENP-T. As a complex with CENP-T, CENP-W plays crucial roles in assembly of the functional kinetochore complex. In this study, the subnuclear localization of CENP-W was extensively analyzed using various approaches. We found that ectopically expressed CENP-W primarily accumulated in the nucleolus and remained substantially associated with the nucleolus in stable cells. The following fractionation study also showed that CENP-W is associated with RNA as well as DNA. Moreover, a considerable amount of CENP-W was found in the nuclear mesh-like structure, nuclear matrix, possibly indicating that CENP-W participates in diverse subnuclear activities. Finally, biochemical affinity binding analysis revealed that CENP-W specifically interacts with the nucleolar phosphoprotein, nucleophosmin (B23). Depletion of cellular B23 by siRNA treatment induced a dramatic decrease of CENP-W stability and severe mislocalization during prophase. Our data proposed that B23 may function in the assembly of the kinetochore complex by interacting with CENP-W during interphase.

Paper title : A quantitative atlas of mitotic phosphorylation.

Doi : https://doi.org/10.1073/pnas.0805139105

Abstract : The eukaryotic cell division cycle is characterized by a sequence of orderly and highly regulated events resulting in the duplication and separation of all cellular material into two newly formed daughter cells. Protein phosphorylation by cyclin-dependent kinases (CDKs) drives this cycle. To gain further insight into how phosphorylation regulates the cell cycle, we sought to identify proteins whose phosphorylation is cell cycle regulated. Using stable isotope labeling along with a two-step strategy for phosphopeptide enrichment and high mass accuracy mass spectrometry, we examined protein phosphorylation in a human cell line arrested in the G(1) and mitotic phases of the cell cycle. We report the identification of >14,000 different phosphorylation events, more than half of which, to our knowledge, have not been described in the literature, along with relative quantitative data for the majority of these sites. We observed >1,000 proteins with increased phosphorylation in mitosis including many known cell cycle regulators. The majority of sites on regulated phosphopeptides lie in [S/T]P motifs, the minimum required sequence for CDKs, suggesting that many of the proteins may be CDK substrates. Analysis of non-proline site-containing phosphopeptides identified two unique motifs that suggest there are at least two undiscovered mitotic kinases.

Paper title : Fusion of a kinase gene, ALK, to a nucleolar protein gene, NPM, in non-Hodgkin's lymphoma.

Doi : https://doi.org/10.1126/science.8122112

Abstract : The 2;5 chromosomal translocation occurs in most anaplastic large-cell non-Hodgkin's lymphomas arising from activated T lymphocytes. This rearrangement was shown to fuse the NPM nucleolar phosphoprotein gene on chromosome 5q35 to a previously unidentified protein tyrosine kinase gene, ALK, on chromosome 2p23. In the predicted hybrid protein, the amino terminus of nucleophosmin (NPM) is linked to the catalytic domain of anaplastic lymphoma kinase (ALK). Expressed in the small intestine, testis, and brain but not in normal lymphoid cells, ALK shows greatest sequence similarity to the insulin receptor subfamily of kinases. Unscheduled expression of the truncated ALK may contribute to malignant transformation in these lymphomas.

Paper title : The structure and functions of NPM1/Nucleophsmin/B23, a multifunctional nucleolar acidic protein.

Doi : https://doi.org/10.1093/jb/mvm222

Abstract : NPM1/Nucleophosmin/B23, also termed NO38 or numatrin, is an acidic nucleolar protein that plays multiple roles in cell growth and proliferation. In general, the expression level of B23 is proportional to the cell growth rate, suggesting that it plays a positive role(s) in cell growth and proliferation. It is important to note that the deletion of the B23 gene and expression of an aberrant type of this gene--caused by gene conversion via translocation or reading-frame shift via nucleotides insertion-have been observed in diverse haematopoietic malignancies. Thus, it is important to understand the function of B23 in the regulation of cell growth and proliferation. In addition, B23 has been reported to undergo a variety of post-translational modifications such as phosphorylation, ubiquitination, SUMOylation, acetylation and poly-(ADP-ribosyl)ation. In this review, the basic structure and functions of B23 as well as the regulation of these functions are summarized.

Paper title : Large-scale proteomics analysis of the human kinome.

Doi : https://doi.org/10.1074/mcp.M800588-MCP200

Abstract : Members of the human protein kinase superfamily are the major regulatory enzymes involved in the activity control of eukaryotic signal transduction pathways. As protein kinases reside at the nodes of phosphorylation-based signal transmission, comprehensive analysis of their cellular expression and site-specific phosphorylation can provide important insights into the architecture and functionality of signaling networks. However, in global proteome studies, low cellular abundance of protein kinases often results in rather minor peptide species that are occluded by a vast excess of peptides from other cellular proteins. These analytical limitations create a rationale for kinome-wide enrichment of protein kinases prior to mass spectrometry analysis. Here, we employed stable isotope labeling by amino acids in cell culture (SILAC) to compare the binding characteristics of three kinase-selective affinity resins by quantitative mass spectrometry. The evaluated pre-fractionation tools possessed pyrido[2,3-d]pyrimidine-based kinase inhibitors as immobilized capture ligands and retained considerable subsets of the human kinome. Based on these results, an affinity resin displaying the broadly selective kinase ligand VI16832 was employed to quantify the relative expression of more than 170 protein kinases across three different, SILAC-encoded cancer cell lines. These experiments demonstrated the feasibility of comparative kinome profiling in a compact experimental format. Interestingly, we found high levels of cytoplasmic and low levels of receptor tyrosine kinases in MV4-11 leukemia cells compared with the adherent cancer lines HCT116 and MDA-MB-435S. The VI16832 resin was further exploited to pre-fractionate kinases for targeted phosphoproteomics analysis, which revealed about 1200 distinct phosphorylation sites on more than 200 protein kinases. This hitherto largest survey of site-specific phosphorylation across the kinome significantly expands the basis for functional follow-up studies on protein kinase regulation. In conclusion, the straightforward experimental procedures described here enable different implementations of kinase-selective proteomics with considerable potential for future signal transduction and kinase drug target analysis.

Paper title : RRP1B targets PP1 to mammalian cell nucleoli and is associated with Pre-60S ribosomal subunits.

Doi : https://doi.org/10.1091/mbc.E10-04-0287

Abstract : A pool of protein phosphatase 1 (PP1) accumulates within nucleoli and accounts for a large fraction of the serine/threonine protein phosphatase activity in this subnuclear structure. Using a combination of fluorescence imaging with quantitative proteomics, we mapped the subnuclear localization of the three mammalian PP1 isoforms stably expressed as GFP-fusions in live cells and identified RRP1B as a novel nucleolar targeting subunit that shows a specificity for PP1β and PP1γ. RRP1B, one of two mammalian orthologues of the yeast Rrp1p protein, shows an RNAse-dependent localization to the granular component of the nucleolus and distributes in a similar manner throughout the cell cycle to proteins involved in later steps of rRNA processing. Quantitative proteomic analysis of complexes containing both RRP1B and PP1γ revealed enrichment of an overlapping subset of large (60S) ribosomal subunit proteins and pre-60S nonribosomal proteins involved in mid-late processing. Targeting of PP1 to this complex by RRP1B in mammalian cells is likely to contribute to modulation of ribosome biogenesis by mechanisms involving reversible phosphorylation events, thus playing a role in the rapid transduction of cellular signals that call for regulation of ribosome production in response to cellular stress and/or changes in growth conditions.

Paper title : Characterization of the transforming activity of p80, a hyperphosphorylated protein in a Ki-1 lymphoma cell line with chromosomal translocation t(2;5).

Doi : https://doi.org/10.1073/pnas.93.9.4181

Abstract : We have molecularly cloned a cDNA encoding a protein uniquely expressed and hyperphosphorylated at tyrosine residues in a Ki-1 lymphoma cell that contained chromosomal translocation t(2;5). The encoded protein p80 was shown to be generated by fusion of a protein-tyrosine kinase and a nucleolar protein B23/nucleophosmin (NPM). The coding sequence of this cDNA turned out to be virtually identical to that of the fusion cDNA for NPM-anaplastic lymphoma kinase (ALK) previously cloned from the transcript of the gene at the breakpoint of the same translocation. Overexpression of p80 in NIH 3T3 cells induced neoplastic transformation, suggesting that the p80 kinase is aberrantly activated. The normal form of p80 was predicted to be a receptor-type tyrosine kinase on the basis of its sequence similarity to the insulin receptor family of kinases. However, an immunofluorescence study using COS cells revealed that p80 was localized to the cytoplasm. Thus, subcellular translocation and activation of the tyrosine kinase presumably by its structural alteration would cause the malignant transformation. We also showed that a mutant p80 lacking the NPM portion was unable to transform NIH 3T3 cells. Thus, the NPM sequence is essential for the transforming activity, suggesting that the chromosomal translocation is responsible for the oncogenesis. Finally, Shc and insulin receptor substrate 1 (IRS-1) were tyrosine-phosphorylated and bound to p80 in p80-transformed cells. However, mutants of p80 that were defective for binding to and phosphorylation of Shc and insulin receptor substrate 1 could transform NIH 3T3 cells. Association of these mutants with GRB2 was still observed, suggesting that interaction of p80 with GRB2 but not with Shc or IRS-1 was relevant for cell transformation.

Paper title : Nucleolar targeting signal of Rex protein of human T-cell leukemia virus type I specifically binds to nucleolar shuttle protein B-23.

Doi : https://doi.org/Not available

Abstract : Rex protein, the post-transcriptional regulator of human T-cell leukemia virus type I, is located predominantly in the cell nucleolus and is associated with the cytoplasmic accumulation of unspliced and singly spliced viral mRNAs. The N-terminal 19-amino acid segment of Rex has been identified as the nucleolar targeting signal (NOS) and shown to be important for Rex function. To study the molecular interaction between the NOS region of Rex and its binding host protein(s) in the nucleolus, we chemically synthesized a functional NOS peptide (wild type) and mutant NOS peptides. Fluorescein isothiocyanate-conjugated functional NOS peptide was rapidly taken up by human cells and was transported to the nucleolus. Using affinity chromatography, we identified nucleolar protein B-23 as the major protein that binds to NOS. We also identified two highly acidic regions of B-23 (amino acids 120-132 and 161-188) as acceptor regions for NOS. Previous experiments have suggested that B-23 functions as a shuttle protein for the nucleolar transport of ribosomal components. Our results suggest that B-23 may also serve as a shuttle for the import of Rex from the cytoplasm to the nucleolus coupled to the export of viral mRNAs containing the Rex-responsive element.

Paper title : Polo-like kinase 2-dependent phosphorylation of NPM/B23 on serine 4 triggers centriole duplication.

Doi : https://doi.org/10.1371/journal.pone.0009849

Abstract : Duplication of the centrosome is well controlled during faithful cell division while deregulation of this process leads to supernumary centrosomes, chromosome missegregation and aneuploidy, a hallmark of many cancer cells. We previously reported that Polo-like kinase 2 (Plk2) is activated near the G1/S phase transition, and regulates the reproduction of centrosomes. In search for Plk2 interacting proteins we have identified NPM/B23 (Nucleophosmin) as a novel Plk2 binding partner. We find that Plk2 and NPM/B23 interact in vitro in a Polo-box dependent manner. An association between both proteins was also observed in vivo. Moreover, we show that Plk2 phosphorylates NPM/B23 on serine 4 in vivo in S-phase. Notably, expression of a non-phosphorylatable NPM/B23 S4A mutant interferes with centriole reduplication in S-phase arrested cells and leads to a dilution of centriole numbers in unperturbed U2OS cells. The corresponding phospho-mimicking mutants have the opposite effect and their expression leads to the accumulation of centrioles. These findings suggest that NPM/B23 is a direct target of Plk2 in the regulation of centriole duplication and that phosphorylation on serine 4 can trigger this process.

Paper title : Nucleophosmin (NPM1/B23) interacts with activating transcription factor 5 (ATF5) protein and promotes proteasome- and caspase-dependent ATF5 degradation in hepatocellular carcinoma cells.

Doi : https://doi.org/10.1074/jbc.M112.363622

Abstract : Nucleophosmin (NPM1/B23) and the activating transcription factor 5 (ATF5) are both known to subject to cell type-dependent regulation. NPM1 is expressed weakly in hepatocytes and highly expressed in hepatocellular carcinomas (HCC) with a clear correlation between enhanced NPM1 expression and increased tumor grading and poor prognosis, whereas in contrast, ATF5 is expressed abundantly in hepatocytes and down-regulated in HCC. Re-expression of ATF5 in HCC inhibits cell proliferation. We report here that using an unbiased approach, tandem affinity purification (TAP) followed with mass spectrometry (MS), we identified NPM1 as a novel ATF5-interacting protein. Unlike many other NPM1-interacting proteins that interact with the N-terminal oligomerization domain of NPM1, ATF5 binds via its basic leucine zipper to the C-terminal region of NPM1 where its nucleolar localization signal is located. NPM1 association with ATF5, whose staining patterns partially overlap in the nucleoli, promotes ATF5 protein degradation through proteasome-dependent and caspase-dependent pathways. NPM1-c, a mutant NPM1 that is defective in nucleolar localization, failed to stimulate ATF5 polyubiquitination and was unable to down-regulate ATF5. NPM1 interaction with ATF5 displaces HSP70, a known ATF5-interacting protein, from ATF5 protein complexes and antagonizes its role in stabilization of ATF5 protein. NPM1-promoted ATF5 down-regulation diminished ATF5-mediated repression of cAMP-responsive element-dependent gene transcription and abrogates ATF5-induced G(2)/M cell cycle blockade and inhibition of cell proliferation in HCC cells. Our study establishes a mechanistic link between elevated NPM1 expression and depressed ATF5 in HCC and suggests that regulation of ATF5 by NPM1 plays an important role in the proliferation and survival of HCC.

Paper title : Large-scale phosphoproteome analysis of human liver tissue by enrichment and fractionation of phosphopeptides with strong anion exchange chromatography.

Doi : https://doi.org/10.1002/pmic.200700884

Abstract : The mixture of phosphopeptides enriched from proteome samples are very complex. To reduce the complexity it is necessary to fractionate the phosphopeptides. However, conventional enrichment methods typically only enrich phosphopeptides but not fractionate phosphopeptides. In this study, the application of strong anion exchange (SAX) chromatography for enrichment and fractionation of phosphopeptides was presented. It was found that phosphopeptides were highly enriched by SAX and majority of unmodified peptides did not bind onto SAX. Compared with Fe(3+) immobilized metal affinity chromatography (Fe(3+)-IMAC), almost double phosphopeptides were identified from the same sample when only one fraction was generated by SAX. SAX and Fe(3+)-IMAC showed the complementarity in enrichment and identification of phosphopeptides. It was also demonstrated that SAX have the ability to fractionate phosphopeptides under gradient elution based on their different interaction with SAX adsorbent. SAX was further applied to enrich and fractionate phosphopeptides in tryptic digest of proteins extracted from human liver tissue adjacent to tumorous region for phosphoproteome profiling. This resulted in the highly confident identification of 274 phosphorylation sites from 305 unique phosphopeptides corresponding to 168 proteins at false discovery rate (FDR) of 0.96%.

Paper title : Nucleophosmin interacts with and inhibits the catalytic function of eukaryotic initiation factor 2 kinase PKR.

Doi : https://doi.org/10.1074/jbc.M301392200

Abstract : In normal cells the protein kinase PKR effects apoptosis in response to various extra and intracellular cues and can also function to suppress the neoplastic phenotype. Because most neoplastic cells are resistant to certain apoptotic cues, we reasoned that an early molecular event in carcinogenesis or leukemogenesis might be the inactivation of PKR by expression or activation of intracellular PKR inhibitors. Seeking novel PKR-modulating proteins we report here that nucleophosmin (NPM), a protein frequently overexpressed in a variety of human malignancies, binds to PKR, and inhibits its activation. Co-immunoprecipitation and in vitro binding experiments showed that NPM associated with PKR. Kinase assays demonstrated that recombinant NPM inhibited PKR activation in a dose-dependent manner. In addition, purified recombinant NPM was phosphorylated by activated PKR. Most importantly, overexpression of NPM suppressed PKR activity, enhanced protein synthesis, and inhibited apoptosis. Lymphoblasts from patients with Fanconi anemia (FA) expressed low levels of NPM, which correlated with high ground-state activation of PKR and cellular hypersensitivity to apoptotic cues, but enforced expression of NPM in these mutant cells reduced aberrant apoptotic responses. Inhibition of PKR by NPM may be one mechanism by which neoplastic clones evolve in sporadic malignancies and in neoplastic cells arising in the context of the cancer predisposition syndrome, Fanconi anemia.

Paper title : Proteomic characterization of the human centrosome by protein correlation profiling.

Doi : https://doi.org/10.1038/nature02166

Abstract : The centrosome is the major microtubule-organizing centre of animal cells and through its influence on the cytoskeleton is involved in cell shape, polarity and motility. It also has a crucial function in cell division because it determines the poles of the mitotic spindle that segregates duplicated chromosomes between dividing cells. Despite the importance of this organelle to cell biology and more than 100 years of study, many aspects of its function remain enigmatic and its structure and composition are still largely unknown. We performed a mass-spectrometry-based proteomic analysis of human centrosomes in the interphase of the cell cycle by quantitatively profiling hundreds of proteins across several centrifugation fractions. True centrosomal proteins were revealed by both correlation with already known centrosomal proteins and in vivo localization. We identified and validated 23 novel components and identified 41 likely candidates as well as the vast majority of the known centrosomal proteins in a large background of nonspecific proteins. Protein correlation profiling permits the analysis of any multiprotein complex that can be enriched by fractionation but not purified to homogeneity.

Paper title : Nucleolar protein B23/nucleophosmin regulates the vertebrate SUMO pathway through SENP3 and SENP5 proteases.

Doi : https://doi.org/10.1083/jcb.200807185

Abstract : Ubiquitin-like protein/sentrin-specific proteases (Ulp/SENPs) mediate both processing and deconjugation of small ubiquitin-like modifier proteins (SUMOs). Here, we show that Ulp/SENP family members SENP3 and SENP5 localize within the granular component of the nucleolus, a subnucleolar compartment that contains B23/nucleophosmin. B23/nucleophosmin is an abundant shuttling phosphoprotein, which plays important roles in ribosome biogenesis and which has been strongly implicated in hematopoietic malignancies. Moreover, we found that B23/nucleophosmin binds SENP3 and SENP5 in Xenopus laevis egg extracts and that it is essential for stable accumulation of SENP3 and SENP5 in mammalian tissue culture cells. After either codepletion of SENP3 and SENP5 or depletion of B23/nucleophosmin, we observed accumulation of SUMO proteins within nucleoli. Finally, depletion of these Ulp/SENPs causes defects in ribosome biogenesis reminiscent of phenotypes observed in the absence of B23/nucleophosmin. Together, these results suggest that regulation of SUMO deconjugation may be a major facet of B23/nucleophosmin function in vivo.

Paper title : Mapping of SUMO sites and analysis of SUMOylation changes induced by external stimuli.

Doi : https://doi.org/10.1073/pnas.1413825111

Abstract : SUMOylation is an essential ubiquitin-like modification involved in important biological processes in eukaryotic cells. Identification of small ubiquitin-related modifier (SUMO)-conjugated residues in proteins is critical for understanding the role of SUMOylation but remains experimentally challenging. We have set up a powerful and high-throughput method combining quantitative proteomics and peptide immunocapture to map SUMOylation sites and have analyzed changes in SUMOylation in response to stimuli. With this technique we identified 295 SUMO1 and 167 SUMO2 sites on endogenous substrates of human cells. We further used this strategy to characterize changes in SUMOylation induced by listeriolysin O, a bacterial toxin that impairs the host cell SUMOylation machinery, and identified several classes of host proteins specifically deSUMOylated in response to this toxin. Our approach constitutes an unprecedented tool, broadly applicable to various SUMO-regulated cellular processes in health and disease.

Paper title : The t(3;5)(q25.1;q34) of myelodysplastic syndrome and acute myeloid leukemia produces a novel fusion gene, NPM-MLF1.

Doi : https://doi.org/Not available

Abstract : A t(3;5)(q25.1;q34) chromosomal translocation associated with myelodysplastic syndrome and acute myeloid leukemia (AML) was found to rearrange part of the nucleophosmin (NPM) gene on chromosome 5 with sequences from a novel gene on chromosome 3. Chimeric transcripts expressed by these cells contain 5' NPM coding sequences fused in-frame to those of the new gene, which we named myelodysplasia/myeloid leukemia factor 1 (MLF1). RNA-based polymerase chain reaction analysis revealed identical NPM-MLF1 mRNA fusions in each of the three t(3;5)-positive cases of AML examined. The predicted MLF1 amino acid sequence lacked homology to previously characterized proteins and did not contain known functional motifs. Normal MLF1 transcripts were expressed in a variety of tissues, most abundantly in testis, ovary, skeletal muscle, heart, kidney and colon. Anti-MLF1 antibodies detected the wild-type 31 kDa protein in K562 and HEL erythroleukemia cell lines, but not in HL-60, U937 or KG-1 myeloid leukemia lines. By contrast, t(3;5)-positive leukemia cells expressed a 54 kDa NPM-MLF1 protein, but not normal MLF1. Immunostaining experiments indicated that MLF1 is normally located in the cytoplasm, whereas NPM-MLF1 is targeted to the nucleus, with highest levels in the nucleolus. The nuclear/nucleolar localization of NPM-MLF1 mirrors that of NPM, indicating that NPM trafficking signals direct MLF1 to an inappropriate cellular compartment in myeloid leukemia cells.

Paper title : Isolation and characterization of the human nucleophosmin/B23 (NPM) gene: identification of the YY1 binding site at the 5' enhancer region.

Doi : https://doi.org/10.1093/nar/25.6.1225

Abstract : NPM (nucleophosmin/B23) is a major nucleolar protein which is 20 times more abundant in tumor or proliferating cells than in normal resting cells. Recently, it was found that NPM gene is located at the breakpoints of the t(2:5), t(3:5) and t(5:17) chromosome translocation. To understand the human NPM gene's structure and regulation, four genomic clones were isolated from the human chromosome 5 library and their DNA sequences analyzed. The human NPM gene has 12 exons of sizes ranging from 58 to 358 bp. The chromosome breakpoint for t(2:5) and t(5:17) translocation is within intron 4 and the breakpoint for t(3:5) translocation is within intron 6. The initiation site is located 96 bp upstream from the ATG site. A typical TATA box (at -25 nt) and a GC box (at -65 nt) were identified in the promoter region. We identified two gel-shift bands (A and B) with DNA fragment E (-741/-250 nt) by EMSA. A DNA footprint was observed at (-371/-344 nt) with the nuclear extract. A double stranded DNA with the footprint sequence (-371/-344 nt) competed the formation of gel-shift bands A and B in EMSA suggesting that proteins A and B bind to the footprint region. We confirmed that protein A is transcription factor YY1. These results suggest that YY1 may play a role in NPM gene expression. This is the first report on human NPM gene structure and sequence.

Paper title : Modification of nucleolar protein B23 after exposure to ionizing radiation.

Doi : https://doi.org/Not available

Abstract : The responses of cells to ionizing radiation include induction and/or suppression of the expression of genes and proteins. In our investigations of alterations in cellular protein expression in response to ionizing radiation, we have used the techniques of two-dimensional polyacrylamide gel electrophoresis and silver staining. We compared the nuclear protein profiles of control and irradiated (6 Gy, 4 h postirradiation) radioresistant squamous carcinoma cells (SQ-20B) and observed an alteration in the expression of a 40 kDa protein: control nuclei express a protein isoform with pI values between 5.4-5.8, while irradiated nuclei express a more acidic variant with pI values between 5.2-5.5. Using the cyanogen bromide/O-phthalaldehyde method followed by microsequencing analysis, we obtained an internal amino acid sequence and identified the 40 kDa protein as nucleolar protein B23. Western blotting experiments confirmed the internal amino acid sequencing results and showed both species (control, 5.4-5.8, irradiated, 5.2-5.5) to be recognized by an anti-B23 monoclonal antibody. Radiolabeling of control and irradiated samples with [32P]NAD or [32P]orthophosphoric acid revealed the acidic species of B23 to be both ADP-ribosylated and phosphorylated. Therefore, exposure of SQ-20B cells to radiation results in the increase in expression of an ADP-ribosylated and phosphorylated species of B23.

Paper title : System-wide temporal characterization of the proteome and phosphoproteome of human embryonic stem cell differentiation.

Doi : https://doi.org/10.1126/scisignal.2001570

Abstract : To elucidate cellular events underlying the pluripotency of human embryonic stem cells (hESCs), we performed parallel quantitative proteomic and phosphoproteomic analyses of hESCs during differentiation initiated by a diacylglycerol analog or transfer to media that had not been conditioned by feeder cells. We profiled 6521 proteins and 23,522 phosphorylation sites, of which almost 50% displayed dynamic changes in phosphorylation status during 24 hours of differentiation. These data are a resource for studies of the events associated with the maintenance of hESC pluripotency and those accompanying their differentiation. From these data, we identified a core hESC phosphoproteome of sites with similar robust changes in response to the two distinct treatments. These sites exhibited distinct dynamic phosphorylation patterns, which were linked to known or predicted kinases on the basis of the matching sequence motif. In addition to identifying previously unknown phosphorylation sites on factors associated with differentiation, such as kinases and transcription factors, we observed dynamic phosphorylation of DNA methyltransferases (DNMTs). We found a specific interaction of DNMTs during early differentiation with the PAF1 (polymerase-associated factor 1) transcriptional elongation complex, which binds to promoters of the pluripotency and known DNMT target genes encoding OCT4 and NANOG, thereby providing a possible molecular link for the silencing of these genes during differentiation.

Paper title : Quantitative phosphoproteomic analysis of T cell receptor signaling reveals system-wide modulation of protein-protein interactions.

Doi : https://doi.org/10.1126/scisignal.2000007

Abstract : Protein phosphorylation events during T cell receptor (TCR) signaling control the formation of complexes among proteins proximal to the TCR, the activation of kinase cascades, and the activation of transcription factors; however, the mode and extent of the influence of phosphorylation in coordinating the diverse phenomena associated with T cell activation are unclear. Therefore, we used the human Jurkat T cell leukemia cell line as a model system and performed large-scale quantitative phosphoproteomic analyses of TCR signaling. We identified 10,665 unique phosphorylation sites, of which 696 showed TCR-responsive changes. In addition, we analyzed broad trends in phosphorylation data sets to uncover underlying mechanisms associated with T cell activation. We found that, upon stimulation of the TCR, phosphorylation events extensively targeted protein modules involved in all of the salient phenomena associated with T cell activation: patterning of surface proteins, endocytosis of the TCR, formation of the F-actin cup, inside-out activation of integrins, polarization of microtubules, production of cytokines, and alternative splicing of messenger RNA. Further, case-by-case analysis of TCR-responsive phosphorylation sites on proteins belonging to relevant functional modules together with network analysis allowed us to deduce that serine-threonine (S-T) phosphorylation modulated protein-protein interactions (PPIs) in a system-wide fashion. We also provide experimental support for this inference by showing that phosphorylation of tubulin on six distinct serine residues abrogated PPIs during the assembly of microtubules. We propose that modulation of PPIs by stimulus-dependent changes in S-T phosphorylation state is a widespread phenomenon applicable to many other signaling systems.

Paper title : Nucleotide sequence of a cDNA clone representing a third allele of human protein B23.

Doi : https://doi.org/Not available

Abstract : Not available

Paper title : An enzyme assisted RP-RPLC approach for in-depth analysis of human liver phosphoproteome.

Doi : https://doi.org/10.1016/j.jprot.2013.11.014

Abstract : UNLABELLED: Protein phosphorylation is one of the most common post-translational modifications. It plays key roles in regulating diverse biological processes of liver tissues. To better understand the role of protein phosphorylation in liver functions, it is essential to perform in-depth phosphoproteome analysis of human liver. Here, an enzyme assisted reversed-phase-reversed-phase liquid chromatography (RP-RPLC) approach with both RPLC separations operated with optimized acidic mobile phase was developed. High orthogonal separation was achieved by trypsin digestion of the Glu-C generated peptides in the fractions collected from the first RPLC separation. The phosphoproteome coverage was further improved by using two types of instruments, i.e. TripleTOF 5600 and LTQ Orbitrap Velos. A total of 22,446 phosphorylation sites, corresponding to 6526 nonredundant phosphoproteins were finally identified from normal human liver tissues. Of these sites, 15,229 sites were confidently localized with Ascore≥13. This dataset was the largest phosphoproteome dataset of human liver. It can be a public resource for the liver research community and holds promise for further biology studies. BIOLOGICAL SIGNIFICANCE: The enzyme assisted approach enabled the two RPLC separations operated both with optimized acidic mobile phases. The identifications from TripleTOF 5600 and Orbitrap Velos are highly complementary. The largest phosphoproteome dataset of human liver was generated.

Paper title : Lysine acetylation targets protein complexes and co-regulates major cellular functions.

Doi : https://doi.org/10.1126/science.1175371

Abstract : Lysine acetylation is a reversible posttranslational modification of proteins and plays a key role in regulating gene expression. Technological limitations have so far prevented a global analysis of lysine acetylation's cellular roles. We used high-resolution mass spectrometry to identify 3600 lysine acetylation sites on 1750 proteins and quantified acetylation changes in response to the deacetylase inhibitors suberoylanilide hydroxamic acid and MS-275. Lysine acetylation preferentially targets large macromolecular complexes involved in diverse cellular processes, such as chromatin remodeling, cell cycle, splicing, nuclear transport, and actin nucleation. Acetylation impaired phosphorylation-dependent interactions of 14-3-3 and regulated the yeast cyclin-dependent kinase Cdc28. Our data demonstrate that the regulatory scope of lysine acetylation is broad and comparable with that of other major posttranslational modifications.

Paper title : RPGR ORF15 isoform co-localizes with RPGRIP1 at centrioles and basal bodies and interacts with nucleophosmin.

Doi : https://doi.org/10.1093/hmg/ddi129

Abstract : The ORF15 isoform of RPGR (RPGR(ORF15)) and RPGR interacting protein 1 (RPGRIP1) are mutated in a variety of retinal dystrophies but their functions are poorly understood. Here, we show that in cultured mammalian cells both RPGR(ORF15) and RPGRIP1 localize to centrioles. These localizations are resistant to the microtubule destabilizing drug nocodazole and persist throughout the cell cycle. RPGR and RPGRIP1 also co-localize at basal bodies in cells with primary cilia. The C-terminal (C2) domain of RPGR(ORF15) (ORF15(C2)) is highly conserved across 13 mammalian species, suggesting that it is a functionally important domain. Using matrix-assisted laser desorption ionization time-of-flight mass spectrometry, we show that this domain interacts with a 40 kDa shuttling protein nucleophosmin (NPM). The RPGR(ORF15)-NPM interaction was confirmed by (i) yeast two-hybrid analyses; (ii) binding of both recombinant and native HeLa cell NPM to RPGR(ORF15) fusion proteins in vitro; (iii) co-immunoprecipitation of native NPM, RPGR(ORF15) and RPGRIP1 from bovine retinal extracts and of native HeLa cell NPM and transfected RPGR(ORF15) from cultured cells and (iv) co-localization of NPM and RPGR(ORF15) at metaphase centrosomes in cultured cells. NPM is a multifunctional protein chaperone that shuttles between the nucleoli and the cytoplasm and has been associated with licensing of centrosomal division. RPGR and RPGRIP1 join a growing number of centrosomal proteins involved in human disease.

Paper title : Lys-N and trypsin cover complementary parts of the phosphoproteome in a refined SCX-based approach.

Doi : https://doi.org/10.1021/ac9004309

Abstract : The analysis of proteome-wide phosphorylation events is still a major analytical challenge because of the enormous complexity of protein phosphorylation networks. In this work, we evaluate the complementarity of Lys-N, Lys-C, and trypsin with regard to their ability to contribute to the global analysis of the phosphoproteome. A refined version of low-pH strong cation exchange was used to efficiently separate N-terminally acetylated, phosphorylated, and nonmodified peptides. A total of 5036 nonredundant phosphopeptides could be identified with a false discovery rate of <1% from 1 mg of protein using a combination of the three enzymes. Our data revealed that the overlap between the phosphopeptide data sets generated with different proteases was marginal, whereas the overlap between two similarly generated tryptic data sets was found to be at least 4 times higher. In this way, the parallel use of Lys-N and trypsin enabled a 72% increase in the number of detected phosphopeptides as compared to trypsin alone, whereas a trypsin replicate experiment only led to a 25% increase. Thus, when focusing solely on the trypsin and Lys-N data, we identified 4671 nonredundant phosphopeptides. Further analysis of the detected sites showed that the Lys-N and trypsin data sets were enriched in significantly different phosphorylation motifs, further evidencing that multiprotease approaches are very valuable in phosphoproteome analyses.

Paper title : Nucleolar structure and function are regulated by the deubiquitylating enzyme USP36.

Doi : https://doi.org/10.1242/jcs.044461

Abstract : The nucleolus is a subnuclear compartment and the site of ribosome biogenesis. Previous studies have implicated protein ubiquitylation in nucleolar activity. Here we show that USP36, a deubiquitylating enzyme of unknown function, regulates nucleolar activity in mammalian cells. USP36 localized to nucleoli via the C-terminal region, which contains basic amino acid stretches. Dominant-negative inhibition of USP36 caused the accumulation of ubiquitin-protein conjugates in nucleoli, suggesting that nucleoli are the site of USP36 action. USP36 deubiquitylated the nucleolar proteins nucleophosmin/B23 and fibrillarin, and stabilized them by counteracting ubiquitylation-mediated proteasomal degradation. RNAi-mediated depletion of cellular USP36 resulted in reduced levels of rRNA transcription and processing, a less-developed nucleolar morphology and a slight reduction in the cytoplasmic ribosome level, which eventually led to a reduced rate of cell proliferation. We conclude that by deubiquitylating various nucleolar substrate proteins including nucleophosmin/B23 and fibrillarin, USP36 plays a crucial role in regulating the structure and function of nucleoli.

Paper title : Functional proteomic analysis of human nucleolus.

Doi : https://doi.org/10.1091/mbc.e02-05-0271

Abstract : The notion of a "plurifunctional" nucleolus is now well established. However, molecular mechanisms underlying the biological processes occurring within this nuclear domain remain only partially understood. As a first step in elucidating these mechanisms we have carried out a proteomic analysis to draw up a list of proteins present within nucleoli of HeLa cells. This analysis allowed the identification of 213 different nucleolar proteins. This catalog complements that of the 271 proteins obtained recently by others, giving a total of approximately 350 different nucleolar proteins. Functional classification of these proteins allowed outlining several biological processes taking place within nucleoli. Bioinformatic analyses permitted the assignment of hypothetical functions for 43 proteins for which no functional information is available. Notably, a role in ribosome biogenesis was proposed for 31 proteins. More generally, this functional classification reinforces the plurifunctional nature of nucleoli and provides convincing evidence that nucleoli may play a central role in the control of gene expression. Finally, this analysis supports the recent demonstration of a coupling of transcription and translation in higher eukaryotes.

Paper title : Nucleophosmin/B23 is a target of CDK2/cyclin E in centrosome duplication.

Doi : https://doi.org/10.1016/s0092-8674(00)00093-3

Abstract : In animal cells, duplication of centrosomes and DNA is coordinated. Since CDK2/cyclin E triggers initiation of both events, activation of CDK2/cyclin E is thought to link these two events. We identified nucleophosmin (NPM/B23) as a substrate of CDK2/cyclin E in centrosome duplication. NPM/B23 associates specifically with unduplicated centrosomes, and NPM/B23 dissociates from centrosomes by CDK2/cyclin E-mediated phosphorylation. An anti-NPM/B23 antibody, which blocks this phosphorylation, suppresses the initiation of centrosome duplication in vivo. Moreover, expression of a nonphosphorylatable mutant NPM/ B23 in cells effectively blocks centrosome duplication. Thus, NPM/B23 is a target of CDK2/cyclin E in the initiation of centrosome duplication.

Paper title : Substrate and functional diversity of lysine acetylation revealed by a proteomics survey.

Doi : https://doi.org/10.1016/j.molcel.2006.06.026

Abstract : Acetylation of proteins on lysine residues is a dynamic posttranslational modification that is known to play a key role in regulating transcription and other DNA-dependent nuclear processes. However, the extent of this modification in diverse cellular proteins remains largely unknown, presenting a major bottleneck for lysine-acetylation biology. Here we report the first proteomic survey of this modification, identifying 388 acetylation sites in 195 proteins among proteins derived from HeLa cells and mouse liver mitochondria. In addition to regulators of chromatin-based cellular processes, nonnuclear localized proteins with diverse functions were identified. Most strikingly, acetyllysine was found in more than 20% of mitochondrial proteins, including many longevity regulators and metabolism enzymes. Our study reveals previously unappreciated roles for lysine acetylation in the regulation of diverse cellular pathways outside of the nucleus. The combined data sets offer a rich source for further characterization of the contribution of this modification to cellular physiology and human diseases.

Paper title : Amino acid sequence of a specific antigenic peptide of protein B23.

Doi : https://doi.org/Not available

Abstract : A specific antigenic peptide was obtained from protein B23 (Mr/pI = 37,000/5.1) after 30 min of digestion with staphylococcal V8 protease (10 micrograms/ml/mg protein B23). The antigenic peptide was purified by DEAE-cellulose chromatography and high pressure liquid chromatography on a reverse-phase C18 column. The antigenic peptide contains 14.7 and 18.7 mol% of glutamic acid and lysine, respectively. Amino acid sequence analysis showed that the peptide has 68 amino acids and is located on the carboxyl-terminal sequence of protein B23. The sequence is Ser-Phe-Lys-Lys-Gln-Glu-Lys-Thr-Pro-Lys-Thr-Pro- Lys-Gly-Pro-Ser-Ser-Val-Glu-Asp-Ile-Lys-Ala-Lys-Met-Gln-Ala-Ser-Ile-Glu- Lys-Gly- Gly-Ser-Leu-Pro-Lys-Val-Glu-Ala-Lys-Phe-Ile-Asn-Tyr-Val-Lys-Asn-Cys-Phe- Arg-Met- Thr-Asp-Gln-Glu-Ala-Ile-Gln-Asp-Leu-Trp-Gln-Trp-Arg-Lys-Ser-Leu-Cooh. Extensive digestion of the antigenic peptide with V8 protease, trypsin, or chymotrypsin results in loss of the antigenic activity. Three cloned cDNAs (hpB1, hpB2, and hpB7) which code for the 82 amino acids at the COOH terminus of protein B23 and the 3' non-translating sequence were identified and characterized. All three clones have identical nucleotide sequences coding for the antigenic portion of the protein (68 amino acids at the COOH terminus), the stop codon, and the 3' non-translated region. However, mutation of 6 nucleotide bases of one clone (hpB2) caused changes in 4 amino acids in the sequence just preceding the immunoreactive region. The result suggests the presence of at least 2 immunologically similar but distinct proteins which are both recognized by the anti-B23 antibody.

Paper title : Sumoylation induced by the Arf tumor suppressor: a p53-independent function.

Doi : https://doi.org/10.1073/pnas.0502978102

Abstract : The mouse p19(Arf) protein has both p53-dependent and p53-independent tumor-suppressive activities. Arf triggers sumoylation of many cellular proteins, including Mdm2 and nucleophosmin (NPM/B23), with which p19(Arf) physically interacts in vivo, and this occurs equally well in cells expressing or lacking functional p53. In an Arf-null NIH 3T3 cell derivative (MT-Arf cells) engineered to reexpress an Arf transgene driven by a zinc-inducible metallothionein promoter, sumoylation of endogenous Mdm2 and NPM proteins was initiated as p19(Arf) was induced and was observed before p53-dependent cell cycle arrest. Predominately nucleoplasmic molecules visualized by immunofluorescence with antibodies to small ubiquitin-like modifier (SUMO) 1 localized to nucleoli as p19(Arf) accumulated there. Two Arf mutants, one of which binds to Mdm2 and NPM but is excluded from nucleoli and the other of which enters nucleoli but is handicapped in binding to Mdm2 and NPM, were defective in inducing sumoylation of these two target proteins and did not localize bulk sumoylated molecules to nucleoli. The CELO adenovirus protein, Gam1, which inhibits the SUMO activating enzyme (E1) and leads to down-regulation of the SUMO conjugating enzyme (E2/Ubc9), had no overt effect on the ability of p19(Arf) to activate p53 or the p53-responsive genes encoding Mdm2 and p21(Cip1), despite the fact that Arf-induced sumoylation of Mdm2 was blocked. Reduction of Ubc9 levels with short hairpin RNAs rendered similar results. We suggest that Arf's p53-independent effects on gene expression and tumor suppression might depend on Arf-induced sumoylation.

Paper title : The nucleolar phosphoprotein B23 interacts with hepatitis delta antigens and modulates the hepatitis delta virus RNA replication.

Doi : https://doi.org/10.1074/jbc.M010087200

Abstract : Hepatitis delta virus (HDV) encodes two isoforms of delta antigens (HDAgs). The small form of HDAg is required for HDV RNA replication, while the large form of HDAg inhibits the viral replication and is required for virion assembly. In this study, we found that the expression of B23, a nucleolar phosphoprotein involved in disparate functions including nuclear transport, cellular proliferation, and ribosome biogenesis, is up-regulated by these two HDAgs. Using in vivo and in vitro experimental approaches, we have demonstrated that both isoforms of HDAg can interact with B23 and their interaction domains were identified as the NH(2)-terminal fragment of each molecule encompassing the nuclear localization signal but not the coiled-coil region of HDAg. Sucrose gradient centrifugation analysis indicated that the majority of small HDAg, but a lesser amount of the large HDAg, co-sedimented with B23 and nucleolin in the large nuclear complex. Transient transfection experiments also indicated that introducing exogenous full-length B23, but not a mutated B23 defective in HDAg binding, enhanced HDV RNA replication. All together, our results reveal that HDAg has two distinct effects on nucleolar B23, up-regulation of its gene expression and the complex formation, which in turn regulates HDV RNA replication. Therefore, this work demonstrates the important role of nucleolar protein in regulating the HDV RNA replication through the complex formation with the key positive regulator being small HDAg.

Paper title : A two-dimensional gel database of human colon carcinoma proteins.

Doi : https://doi.org/10.1002/elps.1150180344

Abstract : The master two-dimensional gel database of human colon carcinoma cells currently lists cellular proteins from normal crypts and the colorectal cancer cell lines LIM 1863, LIM 1215 and LIM 1899 (Ward et al., Electrophoresis 1990, 11, 883-891; Ji et al., Electrophoresis 1994, 15, 391-405). Updated two-dimensional electrophoretic (2-DE) maps of cellular proteins from LIM 1215 cells, acquired under both nonreducing and reducing conditions, are presented. Fifteen cellular proteins are identified in the reducing 2-DE gel map, and seven in the nonreducing gel map, along with a tabular listing of their M(r)/pI loci and mode of identification. We also include our mass spectrometric based procedures for identifying 2-DE resolved proteins. This procedure relies on a combination of capillary column (0.10-0.32 mm internal diameter) reversed-phase HPLC peptide mapping of in-gel digested proteins, peptide mass fingerprinting, sequence analysis by either collision-induced dissociation or post-source-decay fragmentation, and protein identification using available database search algorithms. These data, and descriptions of the micro-techniques employed in this laboratory for identifying 2-DE resolved proteins can be accessed via the internet URL: http:(/)/www.ludwig.edu.au.

Paper title : Quantitative phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis.

Doi : https://doi.org/10.1126/scisignal.2000475

Abstract : Eukaryotic cells replicate by a complex series of evolutionarily conserved events that are tightly regulated at defined stages of the cell division cycle. Progression through this cycle involves a large number of dedicated protein complexes and signaling pathways, and deregulation of this process is implicated in tumorigenesis. We applied high-resolution mass spectrometry-based proteomics to investigate the proteome and phosphoproteome of the human cell cycle on a global scale and quantified 6027 proteins and 20,443 unique phosphorylation sites and their dynamics. Co-regulated proteins and phosphorylation sites were grouped according to their cell cycle kinetics and compared to publicly available messenger RNA microarray data. Most detected phosphorylation sites and more than 20% of all quantified proteins showed substantial regulation, mainly in mitotic cells. Kinase-motif analysis revealed global activation during S phase of the DNA damage response network, which was mediated by phosphorylation by ATM or ATR or DNA-dependent protein kinases. We determined site-specific stoichiometry of more than 5000 sites and found that most of the up-regulated sites phosphorylated by cyclin-dependent kinase 1 (CDK1) or CDK2 were almost fully phosphorylated in mitotic cells. In particular, nuclear proteins and proteins involved in regulating metabolic processes have high phosphorylation site occupancy in mitosis. This suggests that these proteins may be inactivated by phosphorylation in mitotic cells.

Paper title : Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype.

Doi : https://doi.org/10.1056/NEJMoa041974

Abstract : BACKGROUND: Nucleophosmin (NPM), a nucleocytoplasmic shuttling protein with prominent nucleolar localization, regulates the ARF-p53 tumor-suppressor pathway. Translocations involving the NPM gene cause cytoplasmic dislocation of the NPM protein. METHODS: We used immunohistochemical methods to study the subcellular localization of NPM in bone marrow-biopsy specimens from 591 patients with primary acute myelogenous leukemia (AML). We then correlated the presence of cytoplasmic NPM with clinical and biologic features of the disease. RESULTS: Cytoplasmic NPM was detected in 208 (35.2 percent) of the 591 specimens from patients with primary AML but not in 135 secondary AML specimens or in 980 hematopoietic or extrahematopoietic neoplasms other than AML. It was associated with a wide spectrum of morphologic subtypes of the disease, a normal karyotype, and responsiveness to induction chemotherapy, but not with recurrent genetic abnormalities. There was a high frequency of FLT3 internal tandem duplications and absence of CD34 and CD133 in AML specimens with a normal karyotype and cytoplasmic dislocation of NPM, but not in those in which the protein was restricted to the nucleus. AML specimens with cytoplasmic NPM carried mutations of the NPM gene that were predicted to alter the protein at its C-terminal; this mutant gene caused cytoplasmic localization of NPM in transfected cells. CONCLUSIONS: Cytoplasmic NPM is a characteristic feature of a large subgroup of patients with AML who have a normal karyotype, NPM gene mutations, and responsiveness to induction chemotherapy.

Paper title : Nucleophosmin phosphorylation by v-cyclin-CDK6 controls KSHV latency.

Doi : https://doi.org/10.1371/journal.ppat.1000818

Abstract : Nucleophosmin (NPM) is a multifunctional nuclear phosphoprotein and a histone chaperone implicated in chromatin organization and transcription control. Oncogenic Kaposi's sarcoma herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman disease (MCD). In the infected host cell KSHV displays two modes of infection, the latency and productive viral replication phases, involving extensive viral DNA replication and gene expression. A sustained balance between latency and reactivation to the productive infection state is essential for viral persistence and KSHV pathogenesis. Our study demonstrates that the KSHV v-cyclin and cellular CDK6 kinase phosphorylate NPM on threonine 199 (Thr199) in de novo and naturally KSHV-infected cells and that NPM is phosphorylated to the same site in primary KS tumors. Furthermore, v-cyclin-mediated phosphorylation of NPM engages the interaction between NPM and the latency-associated nuclear antigen LANA, a KSHV-encoded repressor of viral lytic replication. Strikingly, depletion of NPM in PEL cells leads to viral reactivation, and production of new infectious virus particles. Moreover, the phosphorylation of NPM negatively correlates with the level of spontaneous viral reactivation in PEL cells. This work demonstrates that NPM is a critical regulator of KSHV latency via functional interactions with v-cyclin and LANA.

Paper title : Nek2A kinase regulates the localization of numatrin to centrosome in mitosis.

Doi : https://doi.org/10.1016/j.febslet.2004.08.047

Abstract : Chromosome segregation in mitosis is orchestrated by the kinetochore and spindle microtubules stemming from two centrosomes. Our recent studies demonstrated the importance of Nek2A in faithful chromosome segregation during mitosis. Here, we report that Nek2A regulates the function of numatrin in mitosis. The biochemical interaction between Nek2A and numatrin in mitotic cells was revealed by a set of reciprocal immunoprecipitation experiments using Nek2A and numatrin antibodies, respectively. The interaction is validated by a pull-down assay using recombinant Nek2A and numatrin proteins. Moreover, our immunofluorescence studies demonstrate that numatrin becomes centrosome-associated as the cell enters into mitosis and depart from the centrosome after sister chromatid separation in anaphase. The co-localization of numatrin and Nek2A to the centrosome suggests their interaction with and involvement in centrosome function. Indeed, elimination of Nek2A kinase by siRNA diminished its association with the centrosome. Furthermore, we show that numatrin is phosphorylated by wild type but not kinase-death Nek2A. Our studies suggest that the Nek2A kinase cascade is essential for the localization of numatrin to the centrosome.

Paper title : The Nucleolar Protein GLTSCR2 Is an Upstream Negative Regulator of the Oncogenic Nucleophosmin-MYC Axis.

Doi : https://doi.org/10.1016/j.ajpath.2015.03.016

Abstract : The transcriptional factor MYC and the nucleophosphoprotein nucleophosmin (NPM) act in concert to regulate the proliferation of both normal and cancer cells. MYC directly interacts with NPM to form an NPM-MYC binary complex, which is recruited to the promoter of MYC target genes to induce the transcription of proteins required for transformation, thus forming an oncogenic NPM-MYC axis. However, the regulatory molecules and mechanisms that control the transcription of MYC target genes by NPM remain to be determined. Herein, we describe a novel function of the nucleolar protein glioblastoma tumor-suppressive candidate region gene 2 (GLTSCR2) in regulating the transcriptional activity of MYC through an NPM-dependent pathway in SK-BR3 breast cancer cells. GLTSCR2 bound to NPM weakly in the nucleolus, but the redistribution of GLTSCR2 to the nucleoplasm increased the binding affinity between the two proteins. Enhancing the GLTSCR2-NPM interaction competitively inhibited the formation of the NPM-MYC binary complex, resulting in a decrease in the recruitment of the NPM-MYC complex to the MYC target gene promoter. This process suppressed the transcriptional and transformational activities of MYC. Thus, our data demonstrated that GLTSCR2 was an upstream negative regulator of the NPM-MYC axis involved in controlling the transcriptional activity of MYC, thereby suggesting that GLTSCR2 may be a novel candidate molecule for suppressing the growth of cancer cells stimulated by MYC hyperactivation.

Paper title : The metastasis efficiency modifier ribosomal RNA processing 1 homolog B (RRP1B) is a chromatin-associated factor.

Doi : https://doi.org/10.1074/jbc.M109.023457

Abstract : There is accumulating evidence for a role of germ line variation in breast cancer metastasis. We have recently identified a novel metastasis susceptibility gene, Rrp1b (ribosomal RNA processing 1 homolog B). Overexpression of Rrp1b in a mouse mammary tumor cell line induces a gene expression signature that predicts survival in breast cancer. Here we extend the analysis of RRP1B function by demonstrating that the Rrp1b activation gene expression signature accurately predicted the outcome in three of four publicly available breast carcinoma gene expression data sets. In addition, we provide insights into the mechanism of RRP1B. Tandem affinity purification demonstrated that RRP1B physically interacts with many nucleosome binding factors, including histone H1X, poly(ADP-ribose) polymerase 1, TRIM28 (tripartite motif-containing 28), and CSDA (cold shock domain protein A). Co-immunofluorescence and co-immunoprecipitation confirmed these interactions and also interactions with heterochromatin protein-1alpha and acetyl-histone H4 lysine 5. Finally, we investigated the effects of ectopic expression of an RRP1B allelic variant previously associated with improved survival in breast cancer. Gene expression analyses demonstrate that, compared with ectopic expression of wild type RRP1B in HeLa cells, the variant RRP1B differentially modulates various transcription factors controlled by TRIM28 and CSDA. These data suggest that RRP1B, a tumor progression and metastasis susceptibility candidate gene, is potentially a dynamic modulator of transcription and chromatin structure.

Paper title : APE1/Ref-1 interacts with NPM1 within nucleoli and plays a role in the rRNA quality control process.

Doi : https://doi.org/10.1128/MCB.01337-08

Abstract : APE1/Ref-1 (hereafter, APE1), a DNA repair enzyme and a transcriptional coactivator, is a vital protein in mammals. Its role in controlling cell growth and the molecular mechanisms that fine-tune its different cellular functions are still not known. By an unbiased proteomic approach, we have identified and characterized several novel APE1 partners which, unexpectedly, include a number of proteins involved in ribosome biogenesis and RNA processing. In particular, a novel interaction between nucleophosmin (NPM1) and APE1 was characterized. We observed that the 33 N-terminal residues of APE1 are required for stable interaction with the NPM1 oligomerization domain. As a consequence of the interaction with NPM1 and RNA, APE1 is localized within the nucleolus and this localization depends on cell cycle and active rRNA transcription. NPM1 stimulates APE1 endonuclease activity on abasic double-stranded DNA (dsDNA) but decreases APE1 endonuclease activity on abasic single-stranded RNA (ssRNA) by masking the N-terminal region of APE1 required for stable RNA binding. In APE1-knocked-down cells, pre-rRNA synthesis and rRNA processing were not affected but inability to remove 8-hydroxyguanine-containing rRNA upon oxidative stress, impaired translation, lower intracellular protein content, and decreased cell growth rate were found. Our data demonstrate that APE1 affects cell growth by directly acting on RNA quality control mechanisms, thus affecting gene expression through posttranscriptional mechanisms.

Paper title : Interaction between ROCK II and nucleophosmin/B23 in the regulation of centrosome duplication.

Doi : https://doi.org/10.1128/MCB.01383-06

Abstract : Nucleophosmin (NPM)/B23 has been implicated in the regulation of centrosome duplication. NPM/B23 localizes between two centrioles in the unduplicated centrosome. Upon phosphorylation on Thr(199) by cyclin-dependent kinase 2 (CDK2)/cyclin E, the majority of centrosomal NPM/B23 dissociates from centrosomes, but some NPM/B23 phosphorylated on Thr(199) remains at centrosomes. It has been shown that Thr(199) phosphorylation of NPM/B23 is critical for the physical separation of the paired centrioles, an initial event of the centrosome duplication process. Here, we identified ROCK II kinase, an effector of Rho small GTPase, as a protein that localizes to centrosomes and physically interacts with NPM/B23. Expression of the constitutively active form of ROCK II promotes centrosome duplication, while down-regulation of ROCK II expression results in the suppression of centrosome duplication, especially delaying the initiation of centrosome duplication during the cell cycle. Moreover, ROCK II regulates centrosome duplication in its kinase and centrosome localization activity-dependent manner. We further found that ROCK II kinase activity is significantly enhanced by binding to NPM/B23 and that NPM/B23 acquires a higher binding affinity to ROCK II upon phosphorylation on Thr(199). Moreover, physical interaction between ROCK II and NPM/B23 in vivo occurs in association with CDK2/cyclin E activation and the emergence of Thr(199)-phosphorylated NPM/B23. All these findings point to ROCK II as the effector of the CDK2/cyclin E-NPM/B23 pathway in the regulation of centrosome duplication.

Paper title : Site-specific mapping of the human SUMO proteome reveals co-modification with phosphorylation.

Doi : https://doi.org/10.1038/nsmb.3366

Abstract : Small ubiquitin-like modifiers (SUMOs) are post-translational modifications (PTMs) that regulate nuclear cellular processes. Here we used an augmented K0-SUMO proteomics strategy to identify 40,765 SUMO acceptor sites and quantify their fractional contribution for 6,747 human proteins. Structural-predictive analyses revealed that lysines residing in disordered regions are preferentially targeted by SUMO, in notable contrast to other widespread lysine modifications. In our data set, we identified 807 SUMOylated peptides that were co-modified by phosphorylation, along with dozens of SUMOylated peptides that were co-modified by ubiquitylation, acetylation and methylation. Notably, 9% of the identified SUMOylome occurred proximal to phosphorylation, and numerous SUMOylation sites were found to be fully dependent on prior phosphorylation events. SUMO-proximal phosphorylation occurred primarily in a proline-directed manner, and inhibition of cyclin-dependent kinases dynamically affected co-modification. Collectively, we present a comprehensive analysis of the SUMOylated proteome, uncovering the structural preferences for SUMO and providing system-wide evidence for a remarkable degree of cross-talk between SUMOylation and other major PTMs.

Paper title : Function of homo- and hetero-oligomers of human nucleoplasmin/nucleophosmin family proteins NPM1, NPM2 and NPM3 during sperm chromatin remodeling.

Doi : https://doi.org/10.1093/nar/gks162

Abstract : Sperm chromatin remodeling after oocyte entry is the essential step that initiates embryogenesis. This reaction involves the removal of sperm-specific basic proteins and chromatin assembly with histones. In mammals, three nucleoplasmin/nucleophosmin (NPM) family proteins-NPM1, NPM2 and NPM3-expressed in oocytes are presumed to cooperatively regulate sperm chromatin remodeling. We characterized the sperm chromatin decondensation and nucleosome assembly activities of three human NPM proteins. NPM1 and NPM2 mediated nucleosome assembly independently of other NPM proteins, whereas the function of NPM3 was largely dependent on formation of a complex with NPM1. Maximal sperm chromatin remodeling activity of NPM2 required the inhibition of its non-specific nucleic acid-binding activity by phosphorylation. Furthermore, the oligomer formation with NPM1 elicited NPM3 nucleosome assembly and sperm chromatin decondensation activity. NPM3 also suppressed the RNA-binding activity of NPM1, which enhanced the nucleoplasm-nucleolus shuttling of NPM1 in somatic cell nuclei. Our results proposed a novel mechanism whereby three NPM proteins cooperatively regulate chromatin disassembly and assembly in the early embryo and in somatic cells.

Paper title : GLTSCR2 is an upstream negative regulator of nucleophosmin in cervical cancer.

Doi : https://doi.org/10.1111/jcmm.12474

Abstract : Nucleophosmin (NPM)/B23, a multifunctional nucleolar phosphoprotein, plays an important role in ribosome biogenesis, cell cycle regulation, apoptosis and cancer pathogenesis. The role of NPM in cells is determined by several factors, including total expression level, oligomerization or phosphorylation status, and subcellular localization. In the nucleolus, NPM participates in rRNA maturation to enhance ribosomal biogenesis. Consistent with this finding, NPM expression is increased in rapidly proliferating cells and many types of human cancers. In response to ribosomal stress, NPM is redistributed to the nucleoplasm, where it inactivates mouse double minute 2 homologue to stabilize p53 and inhibit cell cycle progression. These observations indicate that nucleolus-nucleoplasmic mobilization of NPM is one of the key molecular mechanisms that determine the role of NPM within the cell. However, the regulatory molecule(s) that control(s) NPM stability and subcellular localization, crucial to the pluripotency of intercellular NPM, remain(s) unidentified. In this study, we showed that nucleolar protein GLTSCR2/Pict-1 induced nucleoplasmic translocation and enhanced the degradation of NPM via the proteasomal polyubiquitination pathway. In addition, we showed that GLTSCR2 expression decreased the transforming activity of cells mediated by NPM and that the expression of NPM is reciprocally related to that of GLTSCR2 in cervical cancer tissue. In this study, we demonstrated that GLTSCR2 is an upstream negative regulator of NPM.

Paper title : Cytoplasmic nucleophosmin in myeloid sarcoma occurring 20 years after diagnosis of acute myeloid leukaemia.

Doi : https://doi.org/10.1016/S1470-2045(06)70661-1

Abstract : Not available

Paper title : Nucleolar retention of a translational C/EBPalpha isoform stimulates rDNA transcription and cell size.

Doi : https://doi.org/10.1038/emboj.2009.404

Abstract : The messenger RNA of the intronless CEBPA gene is translated into distinct protein isoforms through the usage of consecutive translation initiation sites. These translational isoforms have distinct functions in the regulation of differentiation and proliferation due to the presence of different N-terminal sequences. Here, we describe the function of an N-terminally extended protein isoform of CCAAT enhancer-binding protein alpha (C/EBPalpha) that is translated from an alternative non-AUG initiation codon. We show that a basic amino-acid motif within its N-terminus is required for nucleolar retention and for interaction with nucleophosmin (NPM). In the nucleoli, extended-C/EBPalpha occupies the ribosomal DNA (rDNA) promoter and associates with the Pol I-specific factors upstream-binding factor 1 (UBF-1) and SL1 to stimulate rRNA synthesis. Furthermore, during differentiation of HL-60 cells, endogenous expression of extended-C/EBPalpha is lost concomitantly with nucleolar C/EBPalpha immunostaining probably reflecting the reduced requirement for ribosome biogenesis in differentiated cells. Finally, overexpression of extended-C/EBPalpha induces an increase in cell size. Altogether, our results suggest that control of rRNA synthesis is a novel function of C/EBPalpha adding to its role as key regulator of cell growth and proliferation.

Paper title : The RNA binding activity of a ribosome biogenesis factor, nucleophosmin/B23, is modulated by phosphorylation with a cell cycle-dependent kinase and by association with its subtype.

Doi : https://doi.org/10.1091/mbc.02-03-0036

Abstract : Nucleophosmin/B23 is a nucleolar phosphoprotein. It has been shown that B23 binds to nucleic acids, digests RNA, and is localized in nucleolar granular components from which preribosomal particles are transported to cytoplasm. The intracellular localization of B23 is significantly changed during the cell cycle. Here, we have examined the cellular localization of B23 proteins and the effect of mitotic phosphorylation of B23.1 on its RNA binding activity. Two splicing variants of B23 proteins, termed B23.1 and B23.2, were complexed both in vivo and in vitro. The RNA binding activity of B23.1 was impaired by hetero-oligomer formation with B23.2. Both subtypes of B23 proteins were phosphorylated during mitosis by cyclin B/cdc2. The RNA binding activity of B23.1 was repressed through cyclin B/cdc2-mediated phosphorylation at specific sites in B23. Thus, the RNA binding activity of B23.1 is stringently modulated by its phosphorylation and subtype association. Interphase B23.1 was mainly localized in nucleoli, whereas B23.2 and mitotic B23.1, those of which were incapable of binding to RNA, were dispersed throughout the nucleoplasm and cytoplasm, respectively. These results suggest that nucleolar localization of B23.1 is mediated by its ability to associate with RNA.

Paper title : Crystal structure of human nucleophosmin-core reveals plasticity of the pentamer-pentamer interface.

Doi : https://doi.org/10.1002/prot.21504

Abstract : Not available

Paper title : B23/nucleophosmin serine 4 phosphorylation mediates mitotic functions of polo-like kinase 1.

Doi : https://doi.org/10.1074/jbc.M403264200

Abstract : Phosphoprotein profiling by Kinetworks trade mark analysis of M-phase-arrested HeLa cells by nocodazole treatment revealed that a novel mitosis-specific phosphorylation event occurred in the nucleolar protein B23/nucleophosmin at a conserved Ser-4 residue. Consistent with the resemblance of the Ser-4 phosphorylation site to the Polo-like kinase 1 (Plk1) consensus recognition sequence, inhibition of Plk1 by a kinase-defective mutation (K82M) abrogated B23 Ser-4 phosphorylation, whereas activation of Plk1 by a constitutively active mutation (T210D) enhanced its phosphorylation following in vivo transfection and in vitro phosphorylation assays. Depletion of endogenous Plk1 by RNA interference abolished B23 Ser-4 phosphorylation. The physical interaction of Plk1 and B23 was further demonstrated by their co-immunoprecipitation and glutathione S-transferase fusion protein pull-down assays. Interference of Ser-4 phosphorylation of B23 induced multiple mitotic defects in HeLa cells, including aberrant numbers of centrosomes, elongation and fragmentation of nuclei, and incomplete cytokinesis. The phenotypes of B23 mutants are reminiscent of a subset of those described previously in Plk1 mutants. Our findings provide insights into the biochemical mechanism underlying the role of Plk1 in mitosis regulation through the identification of Ser-4 in B23 as a major physiological substrate of Plk1.

Paper title : Methylation of ribosomal protein S10 by protein-arginine methyltransferase 5 regulates ribosome biogenesis.

Doi : https://doi.org/10.1074/jbc.M110.103911

Abstract : Modulation of ribosomal assembly is a fine tuning mechanism for cell number and organ size control. Many ribosomal proteins undergo post-translational modification, but their exact roles remain elusive. Here, we report that ribosomal protein s10 (RPS10) is a novel substrate of an oncoprotein, protein-arginine methyltransferase 5 (PRMT5). We show that PRMT5 interacts with RPS10 and catalyzes its methylation at the Arg(158) and Arg(160) residues. The methylation of RPS10 at Arg(158) and Arg(160) plays a role in the proper assembly of ribosomes, protein synthesis, and optimal cell proliferation. The RPS10-R158K/R160K mutant is not efficiently assembled into ribosomes and is unstable and prone to degradation by the proteasomal pathway. In nucleoli, RPS10 interacts with nucleophosmin/B23 and is predominantly concentrated in the granular component region, which is required for ribosome assembly. The RPS10 methylation mutant interacts weakly with nucleophosmin/B23 and fails to concentrate in the granular component region. Our results suggest that PRMT5 is likely to regulate cell proliferation through the methylation of ribosome proteins, and thus reveal a novel mechanism for PRMT5 in tumorigenesis.

Paper title : Amino acid sequence of protein B23 phosphorylation site.

Doi : https://doi.org/Not available

Abstract : A major phosphopeptide labeled in vivo, was identified in nucleolar protein B23 (Mr/pI = 37,000/5.1) after tryptic digestion. This peptide was purified by high performance liquid chromatography using reverse-phase (C8 and C18) columns. The phosphopeptide contains 20 amino acids including 1 phosphoserine, 7 glutamic acids, and 4 aspartic acids. The amino acid sequence is: His-Leu-Val-Ala-Val-Glu-Glu-Asp-Ala-Glu-Ser(P)-Glu-Asp-Glu-Asp- Glu-Glu-Asp-Val-Lys. This amino acid sequence is similar to that of nucleolar phosphoprotein C23 (8 consecutive amino acids were identical), and to the regulatory subunit (RII) of cAMP-dependent protein kinase (7 consecutive amino acids were identical, which is phosphorylated by casein kinase II (Carmichael, D.F., Geahlen, R.L., Allen, S.M., and Krebs, E.G. (1982) J. Biol. Chem 257, 10440-10445). The regions near these phosphorylation sites are enriched with glutamic and aspartic acids, suggesting that this acidic amino acid cluster may be essential for kinase recognition.

Paper title : Improved titanium dioxide enrichment of phosphopeptides from HeLa cells and high confident phosphopeptide identification by cross-validation of MS/MS and MS/MS/MS spectra.

Doi : https://doi.org/10.1021/pr070152u

Abstract : Enrichment is essential for phosphoproteome analysis because phosphorylated proteins are usually present in cells in low abundance. Recently, titanium dioxide (TiO2) has been demonstrated to enrich phosphopeptides from simple peptide mixtures with high specificity; however, the technology has not been optimized. In the present study, significant non-specific bindings were observed when proteome samples were applied to TiO2 columns. Column wash with an NH4Glu solution after loading peptide mixtures significantly increased the efficiency of TiO2 phosphopeptide enrichment with a recovery of up to 84%. Also, for proteome samples, more than a 2-fold increase in unique phosphopeptide identifications has been achieved. The use of NH4Glu for a TiO2 column wash does not significantly reduce the phosphopeptide recovery. A total of 858 phosphopeptides corresponding to 1034 distinct phosphosites has been identified from HeLa cells using the improved TiO2 enrichment procedure in combination with data-dependent neutral loss nano-RPLC-MS2-MS3 analysis. While 41 and 35% of the phosphopeptides were identified only by MS2 and MS3, respectively, 24% was identified by both MS2 and MS3. Cross-validation of the phosphopeptide assignment by MS2 and MS3 scans resulted in the highest confidence in identification (99.5%). Many phosphosites identified in this study appear to be novel, including sites from antigen Ki-67, nucleolar phosphoprotein p130, and Treacle protein. The study also indicates that evaluation of confidence levels for phosphopeptide identification via the reversed sequence database searching strategy might underestimate the false positive rate.

Paper title : Human histone chaperone nucleophosmin enhances acetylation-dependent chromatin transcription.

Doi : https://doi.org/10.1128/MCB.25.17.7534-7545.2005

Abstract : Histone chaperones are a group of proteins that aid in the dynamic chromatin organization during different cellular processes. Here, we report that the human histone chaperone nucleophosmin interacts with the core histones H3, H2B, and H4 but that this histone interaction is not sufficient to confer the chaperone activity. Significantly, nucleophosmin enhances the acetylation-dependent chromatin transcription and it becomes acetylated both in vitro and in vivo. Acetylation of nucleophosmin and the core histones was found to be essential for the enhancement of chromatin transcription. The acetylated NPM1 not only shows an increased affinity toward acetylated histones but also shows enhanced histone transfer ability. Presumably, nucleophosmin disrupts the nucleosomal structure in an acetylation-dependent manner, resulting in the transcriptional activation. These results establish nucleophosmin (NPM1) as a human histone chaperone that becomes acetylated, resulting in the enhancement of chromatin transcription.

Paper title : System-wide Analysis of SUMOylation Dynamics in Response to Replication Stress Reveals Novel Small Ubiquitin-like Modified Target Proteins and Acceptor Lysines Relevant for Genome Stability.

Doi : https://doi.org/10.1074/mcp.O114.044792

Abstract : Genotoxic agents can cause replication fork stalling in dividing cells because of DNA lesions, eventually leading to replication fork collapse when the damage is not repaired. Small Ubiquitin-like Modifiers (SUMOs) are known to counteract replication stress, nevertheless, only a small number of relevant SUMO target proteins are known. To address this, we have purified and identified SUMO-2 target proteins regulated by replication stress in human cells. The developed methodology enabled single step purification of His10-SUMO-2 conjugates under denaturing conditions with high yield and high purity. Following statistical analysis on five biological replicates, a total of 566 SUMO-2 targets were identified. After 2 h of hydroxyurea treatment, 10 proteins were up-regulated for SUMOylation and two proteins were down-regulated for SUMOylation, whereas after 24 h, 35 proteins were up-regulated for SUMOylation, and 13 proteins were down-regulated for SUMOylation. A site-specific approach was used to map over 1000 SUMO-2 acceptor lysines in target proteins. The methodology is generic and is widely applicable in the ubiquitin field. A large subset of these identified proteins function in one network that consists of interacting replication factors, transcriptional regulators, DNA damage response factors including MDC1, ATR-interacting protein ATRIP, the Bloom syndrome protein and the BLM-binding partner RMI1, the crossover junction endonuclease EME1, BRCA1, and CHAF1A. Furthermore, centromeric proteins and signal transducers were dynamically regulated by SUMOylation upon replication stress. Our results uncover a comprehensive network of SUMO target proteins dealing with replication damage and provide a framework for detailed understanding of the role of SUMOylation to counteract replication stress. Ultimately, our study reveals how a post-translational modification is able to orchestrate a large variety of different proteins to integrate different nuclear processes with the aim of dealing with the induced DNA damage.

Paper title : Serine ADP-Ribosylation Depends on HPF1.

Doi : https://doi.org/10.1016/j.molcel.2017.01.003

Abstract : ADP-ribosylation (ADPr) regulates important patho-physiological processes through its attachment to different amino acids in proteins. Recently, by precision mapping on all possible amino acid residues, we identified histone serine ADPr marks in the DNA damage response. However, the biochemical basis underlying this serine modification remained unknown. Here we report that serine ADPr is strictly dependent on histone PARylation factor 1 (HPF1), a recently identified regulator of PARP-1. Quantitative proteomics revealed that serine ADPr does not occur in cells lacking HPF1. Moreover, adding HPF1 to in vitro PARP-1/PARP-2 reactions is necessary and sufficient for serine-specific ADPr of histones and PARP-1 itself. Three endogenous serine ADPr sites are located on the PARP-1 automodification domain. Further identification of serine ADPr on HMG proteins and hundreds of other targets indicates that serine ADPr is a widespread modification. We propose that O-linked protein ADPr is the key signal in PARP-1/PARP-2-dependent processes that govern genome stability.

Paper title : Global, in vivo, and site-specific phosphorylation dynamics in signaling networks.

Doi : https://doi.org/10.1016/j.cell.2006.09.026

Abstract : Cell signaling mechanisms often transmit information via posttranslational protein modifications, most importantly reversible protein phosphorylation. Here we develop and apply a general mass spectrometric technology for identification and quantitation of phosphorylation sites as a function of stimulus, time, and subcellular location. We have detected 6,600 phosphorylation sites on 2,244 proteins and have determined their temporal dynamics after stimulating HeLa cells with epidermal growth factor (EGF) and recorded them in the Phosida database. Fourteen percent of phosphorylation sites are modulated at least 2-fold by EGF, and these were classified by their temporal profiles. Surprisingly, a majority of proteins contain multiple phosphorylation sites showing different kinetics, suggesting that they serve as platforms for integrating signals. In addition to protein kinase cascades, the targets of reversible phosphorylation include ubiquitin ligases, guanine nucleotide exchange factors, and at least 46 different transcriptional regulators. The dynamic phosphoproteome provides a missing link in a global, integrative view of cellular regulation.

Paper title : The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).

Doi : https://doi.org/10.1101/gr.2596504

Abstract : The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

Paper title : Initial characterization of the human central proteome.

Doi : https://doi.org/10.1186/1752-0509-5-17

Abstract : BACKGROUND: On the basis of large proteomics datasets measured from seven human cell lines we consider their intersection as an approximation of the human central proteome, which is the set of proteins ubiquitously expressed in all human cells. Composition and properties of the central proteome are investigated through bioinformatics analyses. RESULTS: We experimentally identify a central proteome comprising 1,124 proteins that are ubiquitously and abundantly expressed in human cells using state of the art mass spectrometry and protein identification bioinformatics. The main represented functions are proteostasis, primary metabolism and proliferation. We further characterize the central proteome considering gene structures, conservation, interaction networks, pathways, drug targets, and coordination of biological processes. Among other new findings, we show that the central proteome is encoded by exon-rich genes, indicating an increased regulatory flexibility through alternative splicing to adapt to multiple environments, and that the protein interaction network linking the central proteome is very efficient for synchronizing translation with other biological processes. Surprisingly, at least 10% of the central proteome has no or very limited functional annotation. CONCLUSIONS: Our data and analysis provide a new and deeper description of the human central proteome compared to previous results thereby extending and complementing our knowledge of commonly expressed human proteins. All the data are made publicly available to help other researchers who, for instance, need to compare or link focused datasets to a common background.

Paper title : Human protein factory for converting the transcriptome into an in vitro-expressed proteome,

Doi : https://doi.org/10.1038/nmeth.1273

Abstract : Appropriate resources and expression technology necessary for human proteomics on a whole-proteome scale are being developed. We prepared a foundation for simple and efficient production of human proteins using the versatile Gateway vector system. We generated 33,275 human Gateway entry clones for protein synthesis, developed mRNA expression protocols for them and improved the wheat germ cell-free protein synthesis system. We applied this protein expression system to the in vitro expression of 13,364 human proteins and assessed their biological activity in two functional categories. Of the 75 tested phosphatases, 58 (77%) showed biological activity. Several cytokines containing disulfide bonds were produced in an active form in a nonreducing wheat germ cell-free expression system. We also manufactured protein microarrays by direct printing of unpurified in vitro-synthesized proteins and demonstrated their utility. Our 'human protein factory' infrastructure includes the resources and expression technology for in vitro proteome research.

Paper title : Nucleophosmin serves as a rate-limiting nuclear export chaperone for the Mammalian ribosome.

Doi : https://doi.org/10.1128/MCB.01548-07

Abstract : Nucleophosmin (NPM) (B23) is an essential protein in mouse development and cell growth; however, it has been assigned numerous roles in very diverse cellular processes. Here, we present a unified mechanism for NPM's role in cell growth; NPM directs the nuclear export of both 40S and 60S ribosomal subunits. NPM interacts with rRNA and large and small ribosomal subunit proteins and also colocalizes with large and small ribosomal subunit proteins in the nucleolus, nucleus, and cytoplasm. The transduction of NPM shuttling-defective mutants or the loss of Npm1 inhibited the nuclear export of both the 40S and 60S ribosomal subunits, reduced the available pool of cytoplasmic polysomes, and diminished overall protein synthesis without affecting rRNA processing or ribosome assembly. While the inhibition of NPM shuttling can block cellular proliferation, the dramatic effects on ribosome export occur prior to cell cycle inhibition. Modest increases in NPM expression amplified the export of newly synthesized rRNAs, resulting in increased rates of protein synthesis and indicating that NPM is rate limiting in this pathway. These results support the idea that NPM-regulated ribosome export is a fundamental process in cell growth.

Paper title : Uncovering global SUMOylation signaling networks in a site-specific manner.

Doi : https://doi.org/10.1038/nsmb.2890

Abstract : SUMOylation is a reversible post-translational modification essential for genome stability. Using high-resolution MS, we have studied global SUMOylation in human cells in a site-specific manner, identifying a total of >4,300 SUMOylation sites in >1,600 proteins. To our knowledge, this is the first time that >1,000 SUMOylation sites have been identified under standard growth conditions. We quantitatively studied SUMOylation dynamics in response to SUMO protease inhibition, proteasome inhibition and heat shock. Many SUMOylated lysines have previously been reported to be ubiquitinated, acetylated or methylated, thus indicating cross-talk between SUMO and other post-translational modifications. We identified 70 phosphorylation and four acetylation events in proximity to SUMOylation sites, and we provide evidence for acetylation-dependent SUMOylation of endogenous histone H3. SUMOylation regulates target proteins involved in all nuclear processes including transcription, DNA repair, chromatin remodeling, precursor-mRNA splicing and ribosome assembly.

Paper title : Combining protein-based IMAC, peptide-based IMAC, and MudPIT for efficient phosphoproteomic analysis.

Doi : https://doi.org/10.1021/pr0705441

Abstract : Immobilized metal affinity chromatography (IMAC) is a common strategy used for the enrichment of phosphopeptides from digested protein mixtures. However, this strategy by itself is inefficient when analyzing complex protein mixtures. Here, we assess the effectiveness of using protein-based IMAC as a pre-enrichment step prior to peptide-based IMAC. Ultimately, we couple the two IMAC-based enrichments and MudPIT in a quantitative phosphoproteomic analysis of the epidermal growth factor pathway in mammalian cells identifying 4470 unique phosphopeptides containing 4729 phosphorylation sites.

Paper title : Characterization of the cDNA encoding human nucleophosmin and studies of its role in normal and abnormal growth.

Doi : https://doi.org/10.1021/bi00429a017

Abstract : A cDNA encoding human nucleophosmin (protein B23) was obtained by screening a human placental cDNA library in lambda gtll first with monoclonal antibody to rat nucleophosmin and then with confirmed partial cDNA of human nucleophosmin as probes. The cDNA had 1311 bp with a coding sequence encoding a protein of 294 amino acids. The identity of the cDNA was confirmed by the presence of encoded amino acid sequences identical with those determined by sequencing pure rat nucleophosmin (a total of 138 amino acids). The most striking feature of the sequence is an acidic cluster located in the middle of the molecule. The cluster consists of 26 Asp/Glu and 1 Phe and Ala. Comparison of human nucleophosmin and Xenopus nucleolar protein NO38 shows 64.3% sequence identity. The N-terminal 130 amino acids of human nucleophosmin also bear 50% identity with that of Xenopus nucleoplasmin. Northern blot analysis of rat liver total RNA with a partial nucleophosmin cDNA as probe demonstrated a homogeneous mRNA band of about 1.6 kb. Similar observations were made in hypertrophic rat liver and Novikoff hepatoma. However, the quantity of nucleophosmin mRNA is 50- and 5-fold higher in Novikoff hepatoma and hypertrophic rat liver, respectively, when compared with normal rat liver. Dot blot analysis also showed a nucleophosmin mRNA ratio of 64:5:1 in the three types of rat liver. When the protein levels were compared with Western blot immunoassays, Novikoff hepatoma showed 20 times more nucleophosmin, while only about 5 times more nucleophosmin was observed in hypertrophic rat liver than in unstimulated normal liver.

Paper title : Aurora-B regulates RNA methyltransferase NSUN2.

Doi : https://doi.org/10.1091/mbc.e06-11-1021

Abstract : Disassembly of the nucleolus during mitosis is driven by phosphorylation of nucleolar proteins. RNA processing stops until completion of nucleolar reformation in G(1) phase. Here, we describe the RNA methyltransferase NSUN2, a novel substrate of Aurora-B that contains an NOL1/NOP2/sun domain. NSUN2 was concentrated in the nucleolus during interphase and was distributed in the perichromosome and cytoplasm during mitosis. Aurora-B phosphorylated NSUN2 at Ser139. Nucleolar proteins NPM1/nucleophosmin/B23 and nucleolin/C23 were associated with NSUN2 during interphase. In mitotic cells, association between NPM1 and NSUN2 was inhibited, but NSUN2-S139A was constitutively associated with NPM1. The Aurora inhibitor Hesperadin induced association of NSUN2 with NPM1 even in mitosis, despite the silver staining nucleolar organizer region disassembly. In vitro methylation experiments revealed that the Aurora-B-phosphorylation and the phosphorylation-mimic mutation (S139E) suppressed methyltransferase activities of NSUN2. These results indicate that Aurora-B participates to regulate the assembly of nucleolar RNA-processing machinery and the RNA methyltransferase activity of NSUN2 via phosphorylation at Ser139 during mitosis.

Paper title : Complete sequencing and characterization of 21,243 full-length human cDNAs.

Doi : https://doi.org/10.1038/ng1285

Abstract : As a base for human transcriptome and functional genomics, we created the "full-length long Japan" (FLJ) collection of sequenced human cDNAs. We determined the entire sequence of 21,243 selected clones and found that 14,490 cDNAs (10,897 clusters) were unique to the FLJ collection. About half of them (5,416) seemed to be protein-coding. Of those, 1,999 clusters had not been predicted by computational methods. The distribution of GC content of nonpredicted cDNAs had a peak at approximately 58% compared with a peak at approximately 42%for predicted cDNAs. Thus, there seems to be a slight bias against GC-rich transcripts in current gene prediction procedures. The rest of the cDNAs unique to the FLJ collection (5,481) contained no obvious open reading frames (ORFs) and thus are candidate noncoding RNAs. About one-fourth of them (1,378) showed a clear pattern of splicing. The distribution of GC content of noncoding cDNAs was narrow and had a peak at approximately 42%, relatively low compared with that of protein-coding cDNAs.

Paper title : The t(5;17) variant of acute promyelocytic leukemia expresses a nucleophosmin-retinoic acid receptor fusion.

Doi : https://doi.org/Not available

Abstract : We have studied an acute promyelocytic leukemia (APL) patient with a variant t(5;17)(q32;q12). This translocation fuses the gene for the nucleolar phosphoprotein nucleophosmin (NPM) to the retinoic acid receptor alpha (RARA). Two alternatively spliced transcripts are expressed, which differ in 129 bases immediately upstream of the RARA sequence. The NPM sequences contained in the shorter NPM-RAR cDNA are identical to the NPM sequences contained in the NPM-ALK fusion gene expressed in t(2;5) lymphomas. The RARA sequences are the same as the RARA sequences found in the PML-RAR and PLZF-RAR fusion seen in t(15;17) and t(11;17) APL, respectively. Both NPM-RAR transcripts fuse NPM and RARA sequence in the same reading frame, to generate translation products of 57 kD and 62 kD. Both NPM-RAR proteins are expressed in the patient's leukemic cells, along with wild-type RARA derived from the uninvolved allele. In transcriptional assays using a retinoic acid response element reporter construct, both NPM-RAR fusion proteins act as retinoic acid-dependent transcriptional activators. This case defines a third class of APL rearrangements, all of which generate fusion proteins of RARA.

Paper title : Toward a comprehensive characterization of a human cancer cell phosphoproteome.

Doi : https://doi.org/10.1021/pr300630k

Abstract : Mass spectrometry (MS)-based phosphoproteomics has achieved extraordinary success in qualitative and quantitative analysis of cellular protein phosphorylation. Considering that an estimated level of phosphorylation in a cell is placed at well above 100,000 sites, there is still much room for improvement. Here, we attempt to extend the depth of phosphoproteome coverage while maintaining realistic aspirations in terms of available material, robustness, and instrument running time. We developed three strategies, where each provided a different balance between these three key parameters. The first strategy simply used enrichment by Ti(4+)-IMAC followed by reversed chromatography LC-MS (termed 1D). The second strategy incorporated an additional fractionation step through the use of HILIC (2D). Finally, a third strategy was designed employing first an SCX fractionation, followed by Ti(4+)-IMAC enrichment and additional fractionation by HILIC (3D). A preliminary evaluation was performed on the HeLa cell line. Detecting 3700 phosphopeptides in about 2 h, the 1D strategy was found to be the most sensitive but limited in comprehensivity, mainly due to issues with complexity and dynamic range. Overall, the best balance was achieved using the 2D based strategy, identifying close to 17,000 phosphopeptides with less than 1 mg of material in about 48 h. Subsequently, we confirmed the findings with the K562 cell sample. When sufficient material was available, the 3D strategy increased phosphoproteome allowing over 22,000 unique phosphopeptides to be identified. Unfortunately, the 3D strategy required more time and over 1 mg of material before it started to outperform 2D. Ultimately, combining all strategies, we were able to identify over 16,000 and nearly 24,000 unique phosphorylation sites from the cancer cell lines HeLa and K562, respectively. In summary, we demonstrate the need to carry out extensive fractionation for deep mining of the phosphoproteome and provide a guide for appropriate strategies depending on sample amount and/or analysis time.

Paper title : ABH2 couples regulation of ribosomal DNA transcription with DNA alkylation repair.

Doi : https://doi.org/10.1016/j.celrep.2013.07.027

Abstract : Transcription has been linked to DNA damage. How the most highly transcribed mammalian ribosomal (rDNA) genes maintain genome integrity in the absence of transcription-coupled DNA damage repair is poorly understood. Here, we report that ABH2/ALKBH2, a DNA alkylation repair enzyme, is highly enriched in the nucleolus. ABH2 interacts with DNA repair proteins Ku70 and Ku80 as well as nucleolar proteins nucleolin, nucleophosmin 1, and upstream binding factor (UBF). ABH2 associates with and promotes rDNA transcription through its DNA repair activity. ABH2 knockdown impairs rDNA transcription and leads to increased single-stranded and double-stranded DNA breaks that are more pronounced in the rDNA genes, whereas ABH2 overexpression protects cells from methyl-methanesulfonate-induced DNA damage and inhibition of rDNA transcription. In response to massive alkylation damage, ABH2 rapidly redistributes from the nucleolus to nucleoplasm. Our study thus reveals a critical role of ABH2 in maintaining rDNA gene integrity and transcription and provides insight into the ABH2 DNA repair function.

Paper title : DDX31 regulates the p53-HDM2 pathway and rRNA gene transcription through its interaction with NPM1 in renal cell carcinomas.

Doi : https://doi.org/10.1158/0008-5472.CAN-12-1645

Abstract : Studies of renal cell carcinoma (RCC) have led to the development of new molecular-targeted drugs but its oncogenic origins remain poorly understood. Here, we report the identification and critical roles in renal carcinogenesis for DDX31, a novel nucleolar protein upregulated in the vast majority of human RCC. Immunohistochemical overexpression of DDX31 was an independent prognostic factor for patients with RCC. RNA interference (RNAi)-mediated attenuation of DDX31 in RCC cells significantly suppressed outgrowth, whereas ectopic DDX31 overexpression in human 293 kidney cells drove their proliferation. Endogenous DDX31 interacted and colocalized with nucleophosmin (NPM1) in the nucleoli of RCC cells, and attenuation of DDX31 or NPM1 expression decreased pre-ribosomal RNA biogenesis. Notably, in DDX31-attenuated cells, NPM1 was translocated from nucleoli to the nucleoplasm or cytoplasm where it bound to HDM2. As a result, HDM2 binding to p53 was reduced, causing p53 stablization with concomitant G(1) phase cell-cycle arrest and apoptosis. Taken together, our findings define a mechanism through which control of the DDX31-NPM1 complex is likely to play critical roles in renal carcinogenesis.

Paper title : Acetylcholinesterase associates differently with its anchoring proteins ColQ and PRiMA.

Doi : https://doi.org/10.1074/jbc.M801364200

Abstract : Acetylcholinesterase tetramers are inserted in the basal lamina of neuromuscular junctions or anchored in cell membranes through the interaction of four C-terminal t peptides with proline-rich attachment domains (PRADs) of cholinesterase-associated collagen Q (ColQ) or of the transmembrane protein PRiMA (proline-rich membrane anchor). ColQ and PRiMA differ in the length of their proline-rich motifs (10 and 15 residues, respectively). ColQ has two cysteines upstream of the PRAD, which are disulfide-linked to two AChE(T) subunits ("heavy" dimer), and the other two subunits are disulfide-linked together ("light" dimer). In contrast, PRiMA has four cysteines upstream of the PRAD. We examined whether these cysteines could be linked to AChE(T) subunits in complexes formed with PRiMA in transfected COS cells and in the mammalian brain. For comparison, we studied complexes formed with N-terminal fragments of ColQ, N-terminal fragments of PRiMA, and chimeras in which the upstream regions containing the cysteines were exchanged. We also compared the effect of mutations in the t peptides on their association with the two PRADs. We report that the two PRADs differ in their interaction with AChE(T) subunits; in complexes formed with the PRAD of PRiMA, we observed light dimers, but very few heavy dimers, even though such dimers were formed with the PQ chimera in which the N-terminal region of PRiMA was associated with the PRAD of ColQ. Complexes with PQ or with PRiMA contained heavy components, which migrated abnormally in SDS-PAGE but probably resulted from disulfide bonding of four AChE(T) subunits with the four upstream cysteines of the associated protein.

Paper title : BRCA2 and nucleophosmin coregulate centrosome amplification and form a complex with the Rho effector kinase ROCK2.

Doi : https://doi.org/10.1158/0008-5472.CAN-10-0030

Abstract : BRCA2 germline mutations account for the majority of heredity breast and ovarian cancer. Besides its role in DNA damage repair, BRCA2 also plays an important role in cytokinesis, transcription regulation, and cancer cell proliferation. Recently, we reported that BRCA2 localizes to centrosomes as well as nuclei and the dysfunction of BRCA2 in a centrosome causes abnormalities in cell division. Here, we identified a nucleolar phosphoprotein, nucleophosmin (NPM), as a novel BRCA2-associated protein. We also detected the binding of BRCA2 to ROCK2, an effector of Rho small GTPase. Because it is known that ROCK2 binds to NPM at centrosomes, these 3 proteins may form a complex. NPM-binding region was within amino acids 639-1,000 of BRCA2. Exogenous expression of this BRCA2 region resulted in aberrant centrosome amplification and a high frequency of multinucleated cells. Our results suggested that a complex consisting of BRCA2, NPM, and ROCK2 maintains the numerical integrity of centrosomes and accurate cell division and that dysfunction of this regulation might be involved in the tumorigenesis of breast cancer.

Paper title : N-terminome analysis of the human mitochondrial proteome.

Doi : https://doi.org/10.1002/pmic.201400617

Abstract : The high throughput characterization of protein N-termini is becoming an emerging challenge in the proteomics and proteogenomics fields. The present study describes the free N-terminome analysis of human mitochondria-enriched samples using trimethoxyphenyl phosphonium (TMPP) labelling approaches. Owing to the extent of protein import and cleavage for mitochondrial proteins, determining the new N-termini generated after translocation/processing events for mitochondrial proteins is crucial to understand the transformation of precursors to mature proteins. The doublet N-terminal oriented proteomics (dN-TOP) strategy based on a double light/heavy TMPP labelling has been optimized in order to improve and automate the workflow for efficient, fast and reliable high throughput N-terminome analysis. A total of 2714 proteins were identified and 897 N-terminal peptides were characterized (424 N-α-acetylated and 473 TMPP-labelled peptides). These results allowed the precise identification of the N-terminus of 693 unique proteins corresponding to 26% of all identified proteins. Overall, 120 already annotated processing cleavage sites were confirmed while 302 new cleavage sites were characterized. The accumulation of experimental evidence of mature N-termini should allow increasing the knowledge of processing mechanisms and consequently also enhance cleavage sites prediction algorithms. Complete datasets have been deposited to the ProteomeXchange Consortium with identifiers PXD001521, PXD001522 and PXD001523 (http://proteomecentral.proteomexchange.org/dataset/PXD001521, http://proteomecentral.proteomexchange.org/dataset/PXD0001522 and http://proteomecentral.proteomexchange.org/dataset/PXD001523, respectively).

Paper title : Isolation and characterization of a molecular cDNA clone of a human mRNA from interferon-treated cells encoding nucleolar protein B23, numatrin.

Doi : https://doi.org/10.1016/0006-291x(89)91699-9

Abstract : A cDNA clone encoding human nucleolar phosphoprotein B23, numatrin, was isolated from a library prepared with mRNA from human U cells. The complete nucleotide sequence was determined; it revealed a single open reading frame of 294 amino acids that included four in-frame AUG codons as potential sites of translation initiation. Comparison of the human B23 nucleotide sequence with the rat and mouse B23 sequences revealed 91% homology (hum:rat, and hum:mus) in the coding region; the predicted B23 proteins displayed 94% amino acid identity. Northern gel blot analysis revealed a single B23 mRNA species of approximately 1.5 kb. The level of B23 mRNA in U cells was not detectably altered by treatment with either alpha or gamma interferon. Southern gel blot analysis revealed polymorphism within the human B23 gene structure, and suggested the presence of multiple B23 genes and/or extensive splicing of B23 RNA transcripts.

Paper title : SUMO-2 Orchestrates Chromatin Modifiers in Response to DNA Damage.

Doi : https://doi.org/10.1016/j.celrep.2015.02.033

Abstract : Small ubiquitin-like modifiers play critical roles in the DNA damage response (DDR). To increase our understanding of SUMOylation in the mammalian DDR, we employed a quantitative proteomics approach in order to identify dynamically regulated SUMO-2 conjugates and modification sites upon treatment with the DNA damaging agent methyl methanesulfonate (MMS). We have uncovered a dynamic set of 20 upregulated and 33 downregulated SUMO-2 conjugates, and 755 SUMO-2 sites, of which 362 were dynamic in response to MMS. In contrast to yeast, where a response is centered on homologous recombination, we identified dynamically SUMOylated interaction networks of chromatin modifiers, transcription factors, DNA repair factors, and nuclear body components. SUMOylated chromatin modifiers include JARID1B/KDM5B, JARID1C/KDM5C, p300, CBP, PARP1, SetDB1, and MBD1. Whereas SUMOylated JARID1B was ubiquitylated by the SUMO-targeted ubiquitin ligase RNF4 and degraded by the proteasome in response to DNA damage, JARID1C was SUMOylated and recruited to the chromatin to demethylate histone H3K4.

Paper title : The role of nucleophosmin in centrosome duplication.

Doi : https://doi.org/10.1038/sj.onc.1205708

Abstract : In higher animal cells, duplication of centrosomes is triggered by CDK2/cyclin E-mediated phosphorylation. Nucleophosmin (NPM)/B23, a multifunctional protein, has recently been identified as one of the substrates of CDK2/cyclin E in centrosome duplication. Centrosome-bound NPM/B23 dissociates from centrosome upon phosphorylation by CDK2/cyclin E, which in turn triggers initiation of centriole duplication. Duplicated centrosomes remain free of NPM/B23 till mitosis. When the nuclear membrane breaks down during mitosis, NPM/B23 re-localizes to centrosomes. Upon cytokinesis, each daughter cell receives one centrosome bound by NPM/B23, which again dissociates from the centrosome upon exposure to CDK2/cyclin E at mid-late G1 phase of the next cell cycle. Thus, NPM/B23 would constitute one of the licensing systems for centrosome duplication, ensuring the coordination of centrosome and DNA duplication, which limiting duplication once per cell cycle.

Paper title : The nucleotide sequence of a human cDNA encoding the highly conserved nucleolar phosphoprotein B23.

Doi : https://doi.org/10.1016/0006-291x(89)92100-1

Abstract : A cDNA clone containing the complete coding sequence for the human nucleolar phosphoprotein B23 was isolated from a Burkitt's lymphoma cDNA library by immunoscreening with human autoantibodies. The B23 clone contained a 1.3 kb cDNA insert encoding a polypeptide of 294 amino acids with a predicted molecular mass of 32,539 daltons. The deduced B23 amino acid sequence contained 2 acidic domains rich in aspartic and glutamic acid, a feature shared by a number of nuclear and nucleolar proteins. The human B23 amino acid sequence showed 98% homology with rat B23 and 68% homology with the Xenopus laevis nucleolar phosphoprotein, NO38 showing that the primary structure of B23 is highly conserved among these species.

Paper title : Kinase-selective enrichment enables quantitative phosphoproteomics of the kinome across the cell cycle.

Doi : https://doi.org/10.1016/j.molcel.2008.07.007

Abstract : Protein kinases are pivotal regulators of cell signaling that modulate each other's functions and activities through site-specific phosphorylation events. These key regulatory modifications have not been studied comprehensively, because low cellular abundance of kinases has resulted in their underrepresentation in previous phosphoproteome studies. Here, we combine kinase-selective affinity purification with quantitative mass spectrometry to analyze the cell-cycle regulation of protein kinases. This proteomics approach enabled us to quantify 219 protein kinases from S and M phase-arrested human cancer cells. We identified more than 1000 phosphorylation sites on protein kinases. Intriguingly, half of all kinase phosphopeptides were upregulated in mitosis. Our data reveal numerous unknown M phase-induced phosphorylation sites on kinases with established mitotic functions. We also find potential phosphorylation networks involving many protein kinases not previously implicated in mitotic progression. These results provide a vastly extended knowledge base for functional studies on kinases and their regulation through site-specific phosphorylation.

Paper title : The nucleolar SUMO-specific protease SENP3 reverses SUMO modification of nucleophosmin and is required for rRNA processing.

Doi : https://doi.org/10.1038/embor.2008.3

Abstract : The ubiquitin-like SUMO system functions by a cyclic process of modification and demodification, and recent data suggest that the nucleolus is a site of sumoylation-desumoylation cycles. For example, the tumour suppressor ARF stimulates sumoylation of nucleolar proteins. Here, we show that the nucleolar SUMO-specific protease SENP3 is associated with nucleophosmin (NPM1), a crucial factor in ribosome biogenesis. SENP3 catalyses desumoylation of NPM1-SUMO2 conjugates in vitro and counteracts ARF-induced modification of NPM1 by SUMO2 in vivo. Intriguingly, depletion of SENP3 by short interfering RNA interferes with nucleolar ribosomal RNA processing and inhibits the conversion of the 32S rRNA species to the 28S form, thus phenocopying the processing defect observed on depletion of NPM1. Moreover, mimicking constitutive modification of NPM1 by SUMO2 interferes with 28S rRNA maturation. These results define SENP3 as an essential factor for ribosome biogenesis and suggest that deconjugation of SUMO2 from NPM1 by SENP3 is critically involved in 28S rRNA maturation.

Paper title : Comparative large scale characterization of plant versus mammal proteins reveals similar and idiosyncratic N-α-acetylation features.

Doi : https://doi.org/10.1074/mcp.M111.015131

Abstract : N-terminal modifications play a major role in the fate of proteins in terms of activity, stability, or subcellular compartmentalization. Such modifications remain poorly described and badly characterized in proteomic studies, and only a few comparison studies among organisms have been made available so far. Recent advances in the field now allow the enrichment and selection of N-terminal peptides in the course of proteome-wide mass spectrometry analyses. These targeted approaches unravel as a result the extent and nature of the protein N-terminal modifications. Here, we aimed at studying such modifications in the model plant Arabidopsis thaliana to compare these results with those obtained from a human sample analyzed in parallel. We applied large scale analysis to compile robust conclusions on both data sets. Our data show strong convergence of the characterized modifications especially for protein N-terminal methionine excision, co-translational N-α-acetylation, or N-myristoylation between animal and plant kingdoms. Because of the convergence of both the substrates and the N-α-acetylation machinery, it was possible to identify the N-acetyltransferases involved in such modifications for a small number of model plants. Finally, a high proportion of nuclear-encoded chloroplast proteins feature post-translational N-α-acetylation of the mature protein after removal of the transit peptide. Unlike animals, plants feature in a dedicated pathway for post-translational acetylation of organelle-targeted proteins. The corresponding machinery is yet to be discovered.