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  • Centrosomal targeting of tyrosine kinase activity does not enhance oncogenicity in chronic myeloproliferative disorders. 22015771

    Constitutive tyrosine kinase activation by reciprocal chromosomal translocation is a common pathogenetic mechanism in chronic myeloproliferative disorders. Since centrosomal proteins have been recurrently identified as translocation partners of tyrosine kinases FGFR1, JAK2, PDGFRα and PDGFRβ in these diseases, a role for the centrosome in oncogenic transformation has been hypothesized. In this study, we addressed the functional role of centrosomally targeted tyrosine kinase activity. First, centrosomal localization was not routinely found for all chimeric fusion proteins tested. Second, targeting of tyrosine kinases to the centrosome by creating artificial chimeric fusion kinases with the centrosomal targeting domain of AKAP450 failed to enhance the oncogenic transforming potential in both Ba/F3 and U2OS cells, although phospho-tyrosine-mediated signal transduction pathways were initiated at the centrosome. We conclude that the centrosomal localization of constitutively activated tyrosine kinases does not contribute to disease pathogenesis in chronic myeloproliferative disorders.
    Document Type:
    Reference
    Product Catalog Number:
    05-321X
    Product Catalog Name:
    Anti-Phosphotyrosine Antibody, clone 4G10®

  • A short linear motif in BNIP3L (NIX) mediates mitochondrial clearance in reticulocytes. 22906961

    Elimination of defective mitochondria is essential for the health of long-lived, postmitotic cells. To gain insight into this process, we examined programmed mitochondrial clearance in reticulocytes. BNIP3L is a mitochondrial outer membrane protein that is required for clearance. It has been suggested that BNIP3L functions by causing mitochondrial depolarization, activating autophagy, or engaging the autophagy machinery. Here we showed in mice that BNIP3L activity localizes to a small region in its cytoplasmic domain, the minimal essential region (MER). The MER is a novel sequence, which comprises three contiguous hydrophobic amino acid residues, and flanking charged residues. Mutation of the central leucine residue causes complete loss of BNIP3L activity, and prevents rescue of mitochondrial clearance. Structural bioinformatics analysis predicts that the BNIP3L cytoplasmic domain lacks stable tertiary structure, but that the MER forms an α-helix upon binding to another protein. These findings support an adaptor model of BNIP3L, centered on the MER.
    Document Type:
    Reference
    Product Catalog Number:
    06-536
    Product Catalog Name:
    Anti-Bak Antibody, NT

  • Naturally occurring germline and tumor-associated mutations within the ATP-binding motifs of PTEN lead to oxidative damage of DNA associated with decreased nuclear p53. 20926450

    Somatic and germline mutations in PTEN (phosphatase and tensin homolog deleted on chromosome 10) are found in sporadic cancers and Cowden syndrome patients, respectively. Recent identification of naturally occurring cancer and germline mutations within the ATP-binding motifs of PTEN (heretofore referred to as PTEN ATP-binding mutations) has revealed that these mutations disrupted the subcellular localization and tumor-suppressor activity of PTEN. However, very little is known about the underlying mechanisms of PTEN ATP-binding mutations in tumorigenesis. Here we show that these mutations impair PTEN's function both qualitatively and quantitatively. On the one hand, PTEN ATP-binding mutants lose their phosphatase activity and the effect of downregulation of cyclin D1. On the other, the mislocalized mutant PTEN results in a significantly decreased nuclear p53 protein level and transcriptional activity, enhanced production of reactive oxygen species, induction of Cu/Zn superoxide dismutase as well as dramatically increased DNA double-strand breaks (DSBs). When compared with wild-type PTEN, the ATP-binding mutant PTEN has reduced half-life in vitro and decreased protein expression levels in vivo. Our data, thus, reveal a novel mechanism of tumorigenesis in patients with germline or somatic mutations affecting PTEN ATP-binding motifs, i.e. qualitative and quantitative impairment of PTEN due to the loss of its phosphatase activity, and nuclear mislocalization, resulting in rapid PTEN protein degradation, suppression of p53-mediated transcriptional activity, loss of protection against oxidative stress as well as accumulation of spontaneous DNA DSBs.
    Document Type:
    Reference
    Product Catalog Number:
    05-636
    Product Catalog Name:
    Anti-phospho-Histone H2A.X (Ser139) Antibody, clone JBW301

  • Human SIRT1 associates with mitotic chromatin and contributes to chromosomal condensation. 21636977

    SIRT1 is a NAD-dependent deacetylase that participates in cellular controls of gene expression, metabolism, genomic stability and anti-aging. Here we report that SIRT1 levels rise in prometaphase leading to SIRT1 global association with mitotic chromatin until telophase. Moreover, SIRT1 contributes to chromosomal condensation by mediating chromosomal loading of histone H1 and the condensin I complex. Consistently, SIRT1 knockdown led to improper condensation and overall aberrant mitosis. Our data highlight new role for SIRT1 in maintenance of chromosome stability in mitosis and suggests how diminished SIRT1 activity during aging and tumorigenesis may lead to aneuploidy and genomic instability.
    Document Type:
    Reference
    Product Catalog Number:
    07-131
    Product Catalog Name:
    Anti-Sirt1(Sir2) Antibody

  • Domains of Tra1 important for activator recruitment and transcription coactivator functions of SAGA and NuA4 complexes. 21149579

    The Tra1 protein is a direct transcription activator target that is essential for coactivator function of both the SAGA and NuA4 histone acetyltransferase (HAT) complexes. The ∼400-kDa Saccharomyces cerevisiae Tra1 polypeptide and its human counterpart TRRAP contain 67 or 68 tandem α-helical HEAT and TPR protein repeats that extend from the N terminus to the conserved yet catalytically inactive phosphatidylinositol 3-kinase (PI3K) domain. We generated a series of mutations spanning the length of the protein and assayed for defects in transcription, coactivator recruitment, and histone acetylation at SAGA- and NuA4-dependent genes. Inviable TRA1 mutants all showed defects in SAGA and NuA4 complex stability, suggesting that similar surfaces of Tra1 mediate assembly of these two very different coactivator complexes. Nearly all of the viable Tra1 mutants showed transcription defects that fell into one of three classes: (i) defective recruitment to promoters, (ii) reduced stability of the SAGA and NuA4 HAT modules, or (iii) normal recruitment of Tra1-associated subunits but reduced HAT activity in vivo. Our results show that Tra1 recruitment at Gcn4-dependent and Rap1-dependent promoters requires the same regions of Tra1 and that separate regions of Tra1 contribute to the HAT activity and stability of the SAGA and NuA4 HAT modules.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • E-box-independent regulation of transcription and differentiation by MYC. 22020439

    MYC proto-oncogene is a key player in cell homeostasis that is commonly deregulated in human carcinogenesis(1). MYC can either activate or repress target genes by forming a complex with MAX (ref. 2). MYC also exerts MAX-independent functions that are not yet fully characterized(3). Cells possess an intrinsic pathway that can abrogate MYC-MAX dimerization and E-box interaction, by inducing phosphorylation of MYC in a PAK2-dependent manner at three residues located in its helix-loop-helix domain(4). Here we show that these carboxy-terminal phosphorylation events switch MYC from an oncogenic to a tumour-suppressive function. In undifferentiated cells, MYC-MAX is targeted to the promoters of retinoic-acid-responsive genes by its direct interaction with the retinoic acid receptor-? (RAR?). MYC-MAX cooperates with RAR? to repress genes required for differentiation, in an E-box-independent manner. Conversely, on C-terminal phosphorylation of MYC during differentiation, the complex switches from a repressive to an activating function, by releasing MAX and recruiting transcriptional co-activators. Phospho-MYC synergizes with retinoic acid to eliminate circulating leukaemic cells and to decrease the level of tumour invasion. Our results identify an E-box-independent mechanism for transcriptional regulation by MYC that unveils previously unknown functions for MYC in differentiation. These may be exploited to develop alternative targeted therapies.
    Document Type:
    Reference
    Product Catalog Number:
    06-598
    Product Catalog Name:
    Anti-acetyl-Histone H4 Antibody

  • Cyclin-dependent kinase-mediated phosphorylation of RBP1 and pRb promotes their dissociation to mediate release of the SAP30·mSin3·HDAC transcriptional repressor complex. 21148318

    Eukaryotic cell cycle progression is mediated by phosphorylation of protein substrates by cyclin-dependent kinases (CDKs). A critical substrate of CDKs is the product of the retinoblastoma tumor suppressor gene, pRb, which inhibits G(1)-S phase cell cycle progression by binding and repressing E2F transcription factors. CDK-mediated phosphorylation of pRb alleviates this inhibitory effect to promote G(1)-S phase cell cycle progression. pRb represses transcription by binding to the E2F transactivation domain and recruiting the mSin3·histone deacetylase (HDAC) transcriptional repressor complex via the retinoblastoma-binding protein 1 (RBP1). RBP1 binds to the pocket region of pRb via an LXCXE motif and to the SAP30 subunit of the mSin3·HDAC complex and, thus, acts as a bridging protein in this multisubunit complex. In the present study we identified RBP1 as a novel CDK substrate. RBP1 is phosphorylated by CDK2 on serines 864 and 1007, which are N- and C-terminal to the LXCXE motif, respectively. CDK2-mediated phosphorylation of RBP1 or pRb destabilizes their interaction in vitro, with concurrent phosphorylation of both proteins leading to their dissociation. Consistent with these findings, RBP1 phosphorylation is increased during progression from G(1) into S-phase, with a concurrent decrease in its association with pRb in MCF-7 breast cancer cells. These studies provide new mechanistic insights into CDK-mediated regulation of the pRb tumor suppressor during cell cycle progression, demonstrating that CDK-mediated phosphorylation of both RBP1 and pRb induces their dissociation to mediate release of the mSin3·HDAC transcriptional repressor complex from pRb to alleviate transcriptional repression of E2F.
    Document Type:
    Reference
    Product Catalog Number:
    06-875

  • The PAF1 complex differentially regulates cardiomyocyte specification. 21338598

    The specification of an appropriate number of cardiomyocytes from the lateral plate mesoderm requires a careful balance of both positive and negative regulatory signals. To identify new regulators of cardiac specification, we performed a phenotype-driven ENU mutagenesis forward genetic screen in zebrafish. In our genetic screen we identified a zebrafish ctr9 mutant with a dramatic reduction in myocardial cell number as well as later defects in primitive heart tube elongation and atrioventricular boundary patterning. Ctr9, together with Paf1, Cdc73, Rtf1 and Leo1, constitute the RNA polymerase II associated protein complex, PAF1. We demonstrate that the PAF1 complex (PAF1C) is structurally conserved among zebrafish and other metazoans and that loss of any one of the components of the PAF1C results in abnormal development of the atrioventricular boundary of the heart. However, Ctr9, Cdc73, Paf1 and Rtf1, but not Leo1, are required for the specification of an appropriate number of cardiomyocytes and elongation of the heart tube. Interestingly, loss of Rtf1 function produced the most severe defects, resulting in a nearly complete absence of cardiac precursors. Based on gene expression analyses and transplantation studies, we found that the PAF1C regulates the developmental potential of the lateral plate mesoderm and is required cell autonomously for the specification of cardiac precursors. Our findings demonstrate critical but differential requirements for PAF1C components in zebrafish cardiac specification and heart morphogenesis.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • The intersection of genetic and chemical genomic screens identifies GSK-3α as a target in human acute myeloid leukemia. 22326953

    Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults. Long-term survival of patients with AML has changed little over the past decade, necessitating the identification and validation of new AML targets. Integration of genomic approaches with small-molecule and genetically based high-throughput screening holds the promise of improved discovery of candidate targets for cancer therapy. Here, we identified a role for glycogen synthase kinase 3α (GSK-3α) in AML by performing 2 independent small-molecule library screens and an shRNA screen for perturbations that induced a differentiation expression signature in AML cells. GSK-3 is a serine-threonine kinase involved in diverse cellular processes, including differentiation, signal transduction, cell cycle regulation, and proliferation. We demonstrated that specific loss of GSK-3α induced differentiation in AML by multiple measurements, including induction of gene expression signatures, morphological changes, and cell surface markers consistent with myeloid maturation. GSK-3α-specific suppression also led to impaired growth and proliferation in vitro, induction of apoptosis, loss of colony formation in methylcellulose, and anti-AML activity in vivo. Although the role of GSK-3β has been well studied in cancer development, these studies support a role for GSK-3α in AML.
    Document Type:
    Reference
    Product Catalog Number:
    06-734
    Product Catalog Name:
    Anti-β-Catenin Antibody

  • Control of the RNA polymerase II phosphorylation state in promoter regions by CTD interaction domain-containing proteins RPRD1A and RPRD1B. 22231121

    RNA polymerase II (RNAP II) C-terminal domain (CTD) phosphorylation is important for various transcription-related processes. Here, we identify by affinity purification and mass spectrometry three previously uncharacterized human CTD-interaction domain (CID)-containing proteins, RPRD1A, RPRD1B and RPRD2, which co-purify with RNAP II and three other RNAP II-associated proteins, RPAP2, GRINL1A and RECQL5, but not with the Mediator complex. RPRD1A and RPRD1B can accompany RNAP II from promoter regions to 3'-untranslated regions during transcription in vivo, predominantly interact with phosphorylated RNAP II, and can reduce CTD S5- and S7-phosphorylated RNAP II at target gene promoters. Thus, the RPRD proteins are likely to have multiple important roles in transcription.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple