Jerzy Ostrowski

Members of the poly (rC) binding protein family stimulate the activity of the c-myc internal ribosome entry segment in vitro and in vivo

The 5′ untranslated region of the proto-oncogene c-myc contains an internal ribosome entry segment and c-Myc translation can be initiated by cap-independent as well as cap-dependent mechanisms. In contrast to the process of cap-dependent initiation, the trans-acting factor requirements for cellular internal ribosome entry are poorly understood. Here, we show that members of the poly (rC) binding protein family, poly (rC) binding protein 1 (PCBP1), poly (rC) binding protein 2 (PCBP2) and hnRNPK were able to activate the IRES in vitro up to threefold when added in combination with upstream of N-ras and unr-interacting protein. The interactions of PCBP1, PCBP2 and hnRNPK with c-myc-IRES-RNA were shown to be specific by ultraviolet crosslinking analysis and electrophoretic mobility shift assays, while immunoprecipitation of the three proteins using specific antibodies followed by reverse transcriptase–polymerase chain reaction showed that they were able to bind c-myc mRNA. c-myc–IRES-mediated translation from the reporter vector was stimulated by cotransfection of plasmids encoding PCBP1, PCBP2 and hnRNPK. Interestingly, the mutated version of the c-myc IRES that is prevalent in patients with multiple myeloma bound hnRNPK more efficiently in vitro and was stimulated by hnRNPK to a greater extent in vivo.

Diverse molecular interactions of the hnRNP K protein

© 1997 Federation of European Biochemical Societies.

The K Protein Domain That Recruits the Interleukin 1-responsive K Protein Kinase Lies Adjacent to a Cluster of c-Src and Vav SH3-binding Sites IMPLICATIONS THAT K PROTEIN ACTS AS A DOCKING PLATFORM

The heterogeneous ribonucleoprotein particle (hnRNP) K protein interacts with multiple molecular partners including DNA, RNA, serine/threonine, and tyrosine kinases and the product of the proto-oncogene, Vav. The K protein is phosphorylated in vivo and in vitro on serine/threonine residues by an interleukin 1 (IL-1)-responsive kinase with which it forms a complex. In this study we set out to map the K protein domains that bind kinases. We demonstrate that the K protein contains a cluster of at least three SH3-binding sites (P1, PPGRGGRPMPPSRR, amino acids 265-278; P2, PRRGPPPPPPGRG, 285-297; and P3, RARNLPLPPPPPPRGG, 303-318) and that each one of these sites is capable of selectively engaging c-Src and Vav SH3 domains but not SH3 domains of Abl, p85 phosphatidylinositol 3-kinase, Grb-2, and Csk. We demonstrate that the K protein domain that recruits and is phosphorylated in an RNA-dependent manner by the IL-1-responsive kinase, designated KPK for K protein kinase, is contained within the 338-425-amino acid stretch and thus is contiguous but does not include the cluster of the SH3-binding sites. K protein and KPK co-immunoprecipitate from cell extracts with either c-Src or Vav, suggesting that K protein-KPK-c-Src and K protein-KPK-Vav complexes exist in vivo. Furthermore, in the context of K protein, c-Src can reactivate KPK in vitro. The succession of kinase-binding sites contained within the K protein that allow it to form multienzyme complexes and facilitate kinase cross-talk suggest that K protein may serve as a docking platform that promotes molecular interactions occurring during signal transduction.

Regulated interaction of protein kinase Cdelta with the heterogeneous nuclear ribonucleoprotein K protein.

The heterogeneous nuclear ribonucleoprotein (hnRNP) K protein recruits a diversity of molecular partners that are involved in signal transduction, transcription, RNA processing, and translation. K protein is phosphorylated in vivo andin vitro by inducible kinase(s) and contains several potential sites for protein kinase C (PKC) phosphorylation. In this study we show that K protein is phosphorylated in vitro by PKCδ and by other PKCs. Deletion analysis and site-directed mutagenesis revealed that Ser302 is a major K protein site phosphorylated by PKCδ in vitro. This residue is located in the middle of a short amino acid fragment that divides the two clusters of SH3-binding domains. Mutation of Ser302decreased the level of phosphorylation of exogenously expressed K protein in phorbol 12-myristate 13-acetate-treated COS cells, suggesting that Ser302 is also a site for PKC-mediated phosphorylation in vivo. In vitro, PKCδ binds K protein via the highly interactive KI domain, an interaction that is blocked by poly(C) RNA. Mutation of Ser302 did not alter the K protein-PKCδ interaction in vitro, suggesting that phosphorylation of this residue alone is not sufficient to alter this interaction. Instead, binding of PKCδ to K protein in vitro and in vivo was greatly increased by K protein phosphorylation on tyrosine residues. The ability of PKCδ to bind and phosphorylate K protein may serve not only to alter the activity of K protein itself, but K protein may also bridge PKCδ to other K protein molecular partners and thus facilitate molecular cross-talk. The regulated nature of the PKCδ-K protein interaction may serve to meet cellular needs at sites of active transcription, RNA processing and translation in response to changing extracellular environment.

Zik1, a Transcriptional Repressor That Interacts with the Heterogeneous Nuclear Ribonucleoprotein Particle K Protein

The heterogeneous nuclear ribonucleoprotein particle (hnRNP) K protein is comprised of multiple modular domains that serve to engage a diverse group of molecular partners including DNA, RNA, the product of the proto-oncogene vav, and tyrosine and serine/threonine kinases. To identify additional K protein molecular partners and to further understand its function, we used a fragment of K protein as a bait in the yeast two-hybrid screen. The deduced primary structure of one of the positive clones revealed a novel zinc finger protein, hereby denoted as Zik1. In addition to the nine contiguous zinc fingers in the C terminus, Zik1 contains a KRAB-A domain thought to be involved in transcriptional repression. Zik1 and K protein bound in vitro and co-immunoprecipitated from cell extracts indicating that in vivo their interaction is direct. Expression of Gal4 DNA-binding domain-Zik1 fusion protein repressed a gene promoter bearing Gal4-binding elements, indicating that from cognate DNA elements Zik1 is a transcriptional repressor. The known diverse nature of K protein molecular interactions and now the identification of a K protein partner that is a transcriptional repressor lends support to the notion that K protein is a remarkably versatile molecule that may be acting as a docking platform to facilitate communication among molecules involved in signal transduction and gene expression.

Integrating proteomic and transcriptomic high-throughput surveys for search of new biomarkers of colon tumors

To the search of new colon tumor biomarkers in the transition from normal colon (NC) mucosa to adenoma (AD) and adenocarcinoma (AC), we integrated microarray data with the results of a high-throughput proteomic workflow. In proteomic study, we used a modified isoelectric focusing protocol on strips with an immobilized pH gradient to separate peptides labeled with iTRAQ (isobaric tags for relative and absolute quantitation) tags followed by liquid chromatography–tandem mass spectrometry analysis. Gene expression measurements were done using Affymetrix GeneChip HG-U133plus2 microarrays and quantitative reverse transcriptase PCR (q-RT-PCR). We identified 3,886 proteins with at least two peptides. Of them, 1,061 proteins were differentially expressed [FC ≥ 1.5; FDR ≤ 0.01] in two pair-wise comparisons: AD vs. NC and AC vs. AD while 15 and 23 proteins were progressively up-regulated and down-regulated in the NC/AD/AC sequence, respectively. The quantitative proteomic information was subsequently correlated with microarray data. For a collection of genes with the same direction of changes of both mRNA and protein levels, we obtained 785/853/795 genes in AD vs. NC/AC vs. NC/AC vs. AD comparison, respectively. Further evaluation of sequentially altered gene expression by q-RT-PCR on individual samples of 24 NCs, 42 ADs, and 26 ACs confirmed progressive expression of six genes: biglycan, calumenin, collagen type XII, alpha 1 (COL12A1), monoamine oxidase A (MAOA), ectonucleoside triphosphate diphosphohydrolase 5 (ENTPD5), and MOCO sulphurase C-terminal domain-containing 2 (MOSC2). Among them, three continuously down-regulated (MAOA, ENTPD5, and MOSC2) and one continuously overexpressed (COL12A1) are reported, to our best knowledge, for the first time in a connection to colon cancer onset.

Quantitative detection for low levels of Helicobacter pylori infection in experimentally infected mice by real-time PCR.

Accurate diagnosis of Helicobacter pylori infection is important in both clinical practice and clinical research. Molecular methods are highly specific and sensitive, and various PCR-based tests have been developed to detect H. pylori in gastric biopsy specimens. We optimized a sensitive and specific quantitative SYBR Green I real-time PCR assay for detection of H. pylori based on amplification of the fragment of a 26-kDa Helicobacter species-specific antigen gene that allows for detection of 5 bacterial cells per PCR sample. Under the assay conditions, SYBR Green I real-time PCR is highly reproducible with a precise log-linear relation in the range of six orders of magnitude of bacterial DNA concentrations. For accurate comparison of H. pylori infection in different tissue samples, the amount of total host DNA in each sample is normalized by TaqMan real-time PCR of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) pseudogenes. The developed method was validated in prophilactically immunized and experimentally infected mice and revealed a level of H. pylori gastric colonisation that was below the limit of detection for a rapid urease test. This new method established for a quantitative analysis of H. pylori in the hosts stomach may be useful in experimental studies evaluating new anti-H. pylori drugs and vaccines.

Molecular defense mechanisms of Barrett’s metaplasia estimated by an integrative genomics

Barrett’s esophagus is characterized by the replacement of squamous epithelium with specialized intestinal metaplastic mucosa. The exact mechanisms of initiation and development of Barrett’s metaplasia remain unknown, but a hypothesis of “successful adaptation” against noxious reflux components has been proposed. To search for the repertoire of adaptation mechanisms of Barrett’s metaplasia, we employed high-throughput functional genomic and proteomic methods that defined the molecular background of metaplastic mucosa resistance to reflux. Transcriptional profiling was established for 23 pairs of esophageal squamous epithelium and Barrett’s metaplasia tissue samples using Affymetrix U133A 2.0 GeneChips and validated by quantitative real-time polymerase chain reaction. Differences in protein composition were assessed by electrophoretic and mass-spectrometry-based methods. Among 2,822 genes differentially expressed between Barrett’s metaplasia and squamous epithelium, we observed significantly overexpressed metaplastic mucosa genes that encode cytokines and growth factors, constituents of extracellular matrix, basement membrane and tight junctions, and proteins involved in prostaglandin and phosphoinositol metabolism, nitric oxide production, and bioenergetics. Their expression likely reflects defense and repair responses of metaplastic mucosa, whereas overexpression of genes encoding heat shock proteins and several protein kinases in squamous epithelium may reflect lower resistance of normal esophageal epithelium than Barrett’s metaplasia to reflux components. Despite the methodological and interpretative difficulties in data analyses discussed in this paper, our studies confirm that Barrett’s metaplasia may be regarded as a specific microevolution allowing for accumulation of mucosal morphological and physiological changes that better protect against reflux injury.

Cooperative binding of the hnRNP K three KH domains to mRNA targets.

The heterogeneous nuclear ribonucleoprotein (hnRNP) K homology (KH) domain is an evolutionarily conserved module that binds short ribonucleotide sequences. KH domains most often are present in multiple copies per protein. In vitro studies of hnRNP K and other KH domain bearing proteins have yielded conflicting results regarding the relative contribution of each KH domain to the binding of target RNAs. To assess this RNA‐binding we used full‐length hnRNP K, its fragments and the yeast ortholog as baits in the yeast three‐hybrid system. The results demonstrate that in this heterologous in vivo system, the three KH domains bind RNA synergistically and that a single KH domain, in comparison, binds RNA weakly.

Comprehensive Analysis of the Palindromic Motif TCTCGCGAGA: A Regulatory Element of the HNRNPK Promoter

Definitive identification of promoters, their cis-regulatory motifs, and their trans-acting proteins requires experimental analysis. To define the HNRNPK promoter and its cognate DNA–protein interactions, we performed a comprehensive study combining experimental approaches, including luciferase reporter gene assays, chromatin immunoprecipitations (ChIP), electrophoretic mobility shift assays (EMSA), and mass spectrometry (MS). We discovered that out of the four potential HNRNPK promoters tested, the one containing the palindromic motif TCTCGCGAGA exhibited the highest activity in a reporter system assay. Although further EMSA and MS analyses, performed to uncover the identity of the palindrome-binding transcription factor, did identify a complex of DNA-binding proteins, neither method unambiguously identified the pertinent direct trans-acting protein(s). ChIP revealed similar chromatin states at the promoters with the palindromic motif and at housekeeping gene promoters. A ChIP survey showed significantly higher recruitment of PARP1, a protein identified by MS as ubiquitously attached to DNA probes, within heterochromatin sites. Computational analyses indicated that this palindrome displays features that mark nucleosome boundaries, causing the surrounding DNA landscape to be constitutively open. Our strategy of diverse approaches facilitated the direct characterization of various molecular properties of HNRNPK promoter bearing the palindromic motif TCTCGCGAGA, despite the obstacles that accompany in vitro methods.