Biomedical subjects

Blagoy Blagoev

Publications and source records attributed to Blagoy Blagoev.

17 recordsLinked to original sources

PubMed2006

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

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.

Binding Sites↗

PubMed2006

Regulation of ubiquitin-binding proteins by monoubiquitination.

Proteins containing ubiquitin-binding domains (UBDs) interact with ubiquitinated targets and regulate diverse biological processes, including endocytosis, signal transduction, transcription and DNA repair. Many of the UBD-containing proteins are also themselves monoubiquitinated, but the functional role and the mechanisms that underlie this modification are less well understood. Here, we demonstrate that monoubiquitination of the endocytic proteins Sts1, Sts2, Eps15 and Hrs results in intramolecular interactions between ubiquitin and their UBDs, thereby preventing them from binding in trans to ubiquitinated targets. Permanent monoubiquitination of these proteins, mimicked by the fusion of ubiquitin to their carboxyl termini, impairs their ability to regulate trafficking of ubiquitinated receptors. Moreover, we mapped the in vivo monoubiquitination site in Sts2 and demonstrated that its mutation enhances the Sts2-mediated effects of epidermal-growth-factor-receptor downregulation. We propose that monoubiquitination of ubiquitin-binding proteins inhibits their capacity to bind to and control the functions of ubiquitinated targets in vivo.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Actin homolog MreB and RNA polymerase interact and are both required for chromosome segregation in Escherichia coli.

The actin-like MreB cytoskeletal protein and RNA polymerase (RNAP) have both been suggested to provide the force for chromosome segregation. Here, we identify MreB and RNAP as in vivo interaction partners. The interaction was confirmed using in vitro purified components. We also present convincing evidence that MreB and RNAP are both required for chromosome segregation in Escherichia coli. MreB is required for origin and bulk DNA segregation, whereas RNAP is required for bulk DNA, terminus, and possibly also for origin segregation. Furthermore, flow cytometric analyses show that MreB depletion and inactivation of RNAP confer virtually identical and highly unusual chromosome segregation defects. Thus, our results raise the possibility that the MreB-RNAP interaction is functionally important for chromosome segregation.

Actins↗

PubMed2006

Quantitative proteomics to study mitogen-activated protein kinases.

In the last several years, the impact of mass spectrometry (MS)-based proteomics on cell signaling research has increased dramatically. This development has been driven both by better instrumentation and by the progression of proteomics from mainly qualitative measurements towards quantitative analyses. In this regard, Stable Isotope Labeling by Amino acids in Cell culture (SILAC) has established itself as one of the most popular and useful quantitative proteomic methodologies to study signaling networks. SILAC relies on the metabolic incorporation of non-radioactive heavy isotopes in the whole proteome of desired cell line, making all proteins from these cells easily distinguishable in the mass spectrometers from the proteins originating from control cells. The procedure does not involve any chemical derivatization steps and, importantly, allows mixing of the two cell populations for combined additional sample manipulation, thus leading to highly reliable results with minimal errors. In this chapter, we describe in detail the SILAC labeling procedure and explain how to design SILAC experiments to examine the level and duration of phosphorylation of endogenous MAP kinases and their substrates in cell culture systems.

Amino Acids↗

PubMed2005

Specificity in Toll-like receptor signalling through distinct effector functions of TRAF3 and TRAF6.

Toll-like receptors (TLRs) are activated by pathogen-associated molecular patterns to induce innate immune responses and production of pro-inflammatory cytokines, interferons and anti-inflammatory cytokines. TLRs activate downstream effectors through adaptors that contain Toll/interleukin-1 receptor (TIR) domains, but the mechanisms accounting for diversification of TLR effector functions are unclear. To dissect biochemically TLR signalling, we established a system for isolating signalling complexes assembled by dimerized adaptors. Using MyD88 as a prototypical adaptor, we identified TNF receptor-associated factor 3 (TRAF3) as a new component of TIR signalling complexes that is recruited along with TRAF6. Using myeloid cells from TRAF3- and TRAF6-deficient mice, we show that TRAF3 is essential for the induction of type I interferons (IFN) and the anti-inflammatory cytokine interleukin-10 (IL-10), but is dispensable for expression of pro-inflammatory cytokines. In fact, TRAF3-deficient cells overproduce pro-inflammatory cytokines owing to defective IL-10 production. Despite their structural similarity, the functions of TRAF3 and TRAF6 are largely distinct. TRAF3 is also recruited to the adaptor TRIF (Toll/IL-1 receptor domain-containing adaptor-inducing IFN-beta) and is required for marshalling the protein kinase TBK1 (also called NAK) into TIR signalling complexes, thereby explaining its unique role in activation of the IFN response.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Mechanism of divergent growth factor effects in mesenchymal stem cell differentiation.

Closely related signals often lead to very different cellular outcomes. We found that the differentiation of human mesenchymal stem cells into bone-forming cells is stimulated by epidermal growth factor (EGF) but not platelet-derived growth factor (PDGF). We used mass spectrometry-based proteomics to comprehensively compare proteins that were tyrosine phosphorylated in response to EGF and PDGF and their associated partners. More than 90% of these signaling proteins were used by both ligands, whereas the phosphatidylinositol 3-kinase (PI3K) pathway was exclusively activated by PDGF, implicating it as a possible control point. Indeed, chemical inhibition of PI3K in PDGF-stimulated cells removed the differential effect of the two growth factors, bestowing full differentiation effect onto PDGF. Thus, quantitative proteomics can directly compare entire signaling networks and discover critical differences capable of changing cell fate.

Cell Differentiation↗

PubMed2004

Mouse embryonic fibroblasts derived from Odin deficient mice display a hyperproliiferative phenotype.

Odin is a recently identified cytosolic phosphotyrosine binding (PTB) domain containing negative regulatory protein, that was discovered on the basis of its ability to undergo tyrosine phosphorylation upon stimulation by epidermal growth factor in HeLa cells. The protein was originally obtained as a KIAA clone (KIAA 0229) from the Kazusa DNA Research Institute which maintains the HUGE protein database--a database devoted to the analysis of long cDNA clones encoding large proteins (>50 kDa). Odin has been demonstrated to cause downregulation of c-Fos promoter activity and to inhibit PDGF-induced mitogenesis in cell lines. To further investigate the role of Odin in growth factor receptor signaling and to elucidate its biological function in vivo, we have generated mice deficient in Odin by gene targeting. Odin-deficient mice do not display any obvious phenotype, and histological examination of the kidney, lung and liver does not show any major abnormalities as compared to wild-type controls. However, mouse embryonic fibroblasts (MEFs) generated from Odin-deficient mice exhibit a hyperproliferative phenotype compared to wild-type-derived MEFs, consistent with its role as a negative regulator of growth factor receptor signaling. Our results confirm that although Odin expression in mice is not essential for any major developmental pathway, it could play a significant functional role to negatively regulate growth factor receptor signaling pathways.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Temporal analysis of phosphotyrosine-dependent signaling networks by quantitative proteomics.

To study the global dynamics of phosphotyrosine-based signaling events in early growth factor stimulation, we developed a mass spectrometric method that converts temporal changes to differences in peptide isotopic abundance. The proteomes of three cell populations were metabolically encoded with different stable isotopic forms of arginine. Each population was stimulated by epidermal growth factor for a different length of time, and tyrosine-phosphorylated proteins and closely associated binders were affinity purified. Arginine-containing peptides occurred in three forms, which were quantified; we then combined two experiments to generate five-point dynamic profiles. We identified 81 signaling proteins, including virtually all known epidermal growth factor receptor substrates, 31 novel effectors and the time course of their activation upon epidermal growth factor stimulation. Global activation profiles provide an informative perspective on cell signaling and will be crucial to modeling signaling networks in a systems biology approach.

Epidermal Growth Factor↗

PubMed2003

A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling.

Mass spectrometry-based proteomics can reveal protein-protein interactions on a large scale, but it has been difficult to separate background binding from functionally important interactions and still preserve weak binders. To investigate the epidermal growth factor receptor (EGFR) pathway, we employ stable isotopic amino acids in cell culture (SILAC) to differentially label proteins in EGF-stimulated versus unstimulated cells. Combined cell lysates were affinity-purified over the SH2 domain of the adapter protein Grb2 (GST-SH2 fusion protein) that specifically binds phosphorylated EGFR and Src homologous and collagen (Shc) protein. We identified 228 proteins, of which 28 were selectively enriched upon stimulation. EGFR and Shc, which interact directly with the bait, had large differential ratios. Many signaling molecules specifically formed complexes with the activated EGFR-Shc, as did plectin, epiplakin, cytokeratin networks, histone H3, the glycosylphosphatidylinositol (GPI)-anchored molecule CD59, and two novel proteins. SILAC combined with modification-based affinity purification is a useful approach to detect specific and functional protein-protein interactions.

Epidermal Growth Factor↗

PubMed2002

Cloning of a novel phosphotyrosine binding domain containing molecule, Odin, involved in signaling by receptor tyrosine kinases.

We have used a proteomic approach using mass spectrometry to identify signaling molecules involved in receptor tyrosine kinase signaling pathways. Using affinity purification by anti-phosphotyrosine antibodies to enrich for tyrosine phosphorylated proteins, we have identified a novel signaling molecule in the epidermal growth factor receptor signaling pathway. This molecule, designated Odin, contains several ankyrin repeats, two sterile alpha motifs and a phosphotyrosine binding domain and is ubiquitously expressed. Using antibodies against endogenous Odin, we show that it undergoes tyrosine phosphorylation upon addition of growth factors such as EGF or PDGF but not by cytokines such as IL-3 or erythropoietin. Immunofluorescence experiments as well as Western blot analysis on subcellular fractions demonstrated that Odin is localized to the cytoplasm both before and after growth factor treatment. Deletion analysis showed that the phosphotyrosine binding domain of Odin is not required for its tyrosine phosphorylation. Overexpression of Odin, but not an unrelated adapter protein, Grb2, inhibited EGF-induced activation of c-Fos promoter. Microinjection of wild-type or a mutant version lacking the PTB domain into NIH3T3 fibroblasts inhibited PDGF-induced mitogenesis. Taken together, our results indicate that Odin may play a negative role in growth factor receptor signaling pathways.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Inhibition of adipocyte differentiation by resistin-like molecule alpha. Biochemical characterization of its oligomeric nature.

A novel family of cysteine-rich secreted proteins with unique tissue distribution has recently been identified. One of the members, resistin (for "resistance to insulin"), also called FIZZ3, was identified in a screen for molecules that are down-regulated in mature adipocytes upon administration of thiazolidinediones. The prototypical member of this family was originally identified from bronchoalveolar lavage fluid of inflamed lungs and designated FIZZ1 ("found in inflammatory zone"). This molecule was also found to be highly expressed in adipose tissue and was named resistin-like molecule alpha (RELMalpha). Here we demonstrate that RELMalpha inhibits the differentiation of 3T3-L1 preadipocytes into adipocytes. RELMalpha has no effect on proliferation of 3T3-L1 preadipocytes. Pretreatment of 3T3-L1 preadipocytes with RELMalpha does not affect insulin- or platelet-derived growth factor-induced mitogenesis. IRS-1 phosphorylation and glucose transport stimulated by insulin in mature adipocytes were also unaffected by RELMalpha. We show that RELMalpha forms disulfide-linked homooligomers based on results from electrophoresis under reducing and nonreducing conditions, coimmunoprecipitation experiments as well as by mass spectrometry. In addition, RELMalpha is able to form heterooligomers with resistin but not RELMbeta. Since RELMalpha is expressed by adipose tissue and it is a secreted factor, our findings suggest that RELMalpha may be involved in the control of the adipogenesis as well as in the process of muscle differentiation.

3T3 Cells↗

PubMed2002

A rapid screening method to monitor expression of recombinant proteins from various prokaryotic and eukaryotic expression systems using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry.

Rapid methods using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry to monitor recombinant protein expression from various prokaryotic and eukaryotic cell culture systems were devised. Intracellular as well as secreted proteins from both induced and constitutive expression systems were measured and monitored from whole cells and growth media, thus providing an alternative to time-consuming traditional methods for screening and monitoring of protein expression. The methods described here involve minimal processing of samples and are therefore relevant to high-throughput screening applications.

Animals↗

PubMed2002

Cloning and characterization of PAK5, a novel member of mammalian p21-activated kinase-II subfamily that is predominantly expressed in brain.

The p21-activated kinase (PAK) family of protein kinases has recently attracted considerable attention as an effector of Rho family of small G proteins and as an upstream regulator of MAPK signalling pathways during cellular events such as re-arrangement of the cytoskeleton and apoptosis. We have cloned a novel human PAK family kinase that has been designated as PAK5. PAK5 contains a CDC42/Rac1 interactive binding (CRIB) motif at the N-terminus and a Ste20-like kinase domain at the C-terminus. PAK5 is structurally most related to PAK4 and PAK6 to make up the PAK-II subfamily. We have shown that PAK5 preferentially binds to CDC42 in the presence of GTP and that CRIB motif is essential for this interaction. PAK5 is a functional protein kinase but unlike PAK-I family kinases (PAK1, 2, and 3), the kinase activity of PAK5 does not seem to require the binding of CDC42. Overexpression of PAK5 activates the JNK kinase pathway but not p38 or ERK pathways. PAK5 transcript is predominantly expressed in brain as revealed by Northern blot and in situ hybridization. The expression pattern of PAK5 is distinct from that of PAK4 and PAK6, suggesting a functional division among PAK-II subfamily kinases based on differential tissue distribution.

Amino Acid Motifs↗

PubMed2002

Cloning of rat thymic stromal lymphopoietin receptor (TSLPR) and characterization of genomic structure of murine Tslpr gene.

Thymic stromal derived lymphopoietin receptor (TSLPR) is a novel receptor subunit that is related in sequence to the interleukin (IL)-2 receptor common gamma chain. TSLPR forms a heterodimeric complex with the IL-7 receptor alpha chain to form the receptor for thymic stromal derived lymphopoietin, a cytokine involved in B- and T-cell function. We have cloned the TSLP receptor from rat and find that the WSXWX motif commonly found in extracellular domains of cytokine receptors is conserved as a W(T/S)XV(T/A) motif among TSLP receptors from mouse, rat and human. As in the mouse, TSLP receptor is widely expressed in rats suggesting that TSLPR may have roles in signaling outside the hematopoietic system. A zooblot analysis revealed that TSLPR is expressed in all vertebrate species examined. The absence of TSLPR in Saccharomyces cerevisiae, Drosophila melanogaster and Caenorhabditis elegans genomes is similar to the expression of several other cytokine receptors that have been characterized thus far. We have also characterized the genomic structure of the murine Tslpr gene which shows that in addition to primary sequence homology, it shares a common genomic organization of coding exons with the murine IL-2 receptor common gamma chain (Il2rg). Use of an alternative splice acceptor site leads to two alternatively spliced transcript variants of murine TSLPR, both of which are functional receptors. Finally, using linkage analysis, we mapped the murine Tslpr gene to mouse chromosome 5 between the Ecm2 and Pxn genes.

Alternative Splicing↗

PubMed2002

A proteomic approach for identification of secreted proteins during the differentiation of 3T3-L1 preadipocytes to adipocytes.

We have undertaken a systematic proteomic approach to purify and identify secreted factors that are differentially expressed in preadipocytes versus adipocytes. Using one-dimensional gel electrophoresis combined with nanoelectrospray tandem mass spectrometry, proteins that were specifically secreted by 3T3-L1 preadipocytes or adipocytes were identified. In addition to a number of previously reported molecules that are up- or down-regulated during this differentiation process (adipsin, adipocyte complement-related protein 30 kDa, complement C3, and fibronectin), we identified four secreted molecules that have not been shown previously to be expressed differentially during the process of adipogenesis. Pigment epithelium-derived factor, a soluble molecule with potent antiangiogenic properties, was found to be highly secreted by preadipocytes but not adipocytes. Conversely, we found hippocampal cholinergic neurostimulating peptide, neutrophil gelatinase-associated lipocalin, and haptoglobin to be expressed highly by mature adipocytes. We also used liquid chromatography-based separation followed by automated tandem mass spectrometry to identify proteins secreted by mature adipocytes. Several additional secreted proteins including resistin, secreted acidic cysteine-rich glycoprotein/osteonectin, stromal cell-derived factor-1, cystatin C, gelsolin, and matrix metalloprotease-2 were identified by this method. To our knowledge, this is the first study to identify several novel secreted proteins by adipocytes by a proteomic approach using mass spectrometry.

3T3 Cells↗

PubMed2002

Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.

Quantitative proteomics has traditionally been performed by two-dimensional gel electrophoresis, but recently, mass spectrometric methods based on stable isotope quantitation have shown great promise for the simultaneous and automated identification and quantitation of complex protein mixtures. Here we describe a method, termed SILAC, for stable isotope labeling by amino acids in cell culture, for the in vivo incorporation of specific amino acids into all mammalian proteins. Mammalian cell lines are grown in media lacking a standard essential amino acid but supplemented with a non-radioactive, isotopically labeled form of that amino acid, in this case deuterated leucine (Leu-d3). We find that growth of cells maintained in these media is no different from growth in normal media as evidenced by cell morphology, doubling time, and ability to differentiate. Complete incorporation of Leu-d3 occurred after five doublings in the cell lines and proteins studied. Protein populations from experimental and control samples are mixed directly after harvesting, and mass spectrometric identification is straightforward as every leucine-containing peptide incorporates either all normal leucine or all Leu-d3. We have applied this technique to the relative quantitation of changes in protein expression during the process of muscle cell differentiation. Proteins that were found to be up-regulated during this process include glyceraldehyde-3-phosphate dehydrogenase, fibronectin, and pyruvate kinase M2. SILAC is a simple, inexpensive, and accurate procedure that can be used as a quantitative proteomic approach in any cell culture system.

3T3 Cells↗

PubMed2001

Cloning of MASK, a novel member of the mammalian germinal center kinase III subfamily, with apoptosis-inducing properties.

We have cloned a novel human GCK family kinase that has been designated as MASK (Mst3 and SOK1-related kinase). MASK is widely expressed and encodes a protein of 416 amino acid residues, with an N-terminal kinase domain and a unique C-terminal region. Like other GCK-III subfamily kinases, MASK does not activate any mitogen-activated protein kinase pathways. Wild type MASK, but not a form lacking the C terminus, exhibits homophilic binding in the yeast two-hybrid system and in coimmunoprecipitation experiments. Additionally, deletion of this C-terminal region of MASK leads to an increased kinase activity toward itself as well as toward an exogenous substrate, myelin basic protein. A potential caspase 3 cleavage site (DESDS) is present in the C-terminal region of MASK, and we show that MASK is cleaved in vitro by caspase 3. Finally, wild type and C-terminally truncated forms of MASK can both induce apoptosis upon overexpression in mammalian cells that is abrogated by CrmA, suggesting involvement of MASK in the apoptotic machinery in mammalian cells.

Amino Acid Motifs↗