Supplementary Table 1 from Quantitative Phosphoproteomics Reveals a Cluster of Tyrosine Kinases That Mediates Src Invasive Activity in Advanced Colon Carcinoma Cells

Supplementary Table 2 from Quantitative Phosphoproteomics Reveals a Cluster of Tyrosine Kinases That Mediates Src Invasive Activity in Advanced Colon Carcinoma Cells

Résumé Patrick Jouin œuvre sur des projets d’hôtels, de mobilier urbain ou d’automobiles, en se fondant dans les marques pour lesquelles il travaille. Il cherche leurs codes par la discussion et l’émotion, un travail collectif et personnel, et des recherches techniques pointues.

Abstract The non-receptor spleen tyrosine kinase Syk has mainly been studied in hematopoietic cells in which it plays a key role in the immune-response signalling. We previously demonstrated that Syk is also present in mammary epithelial cells and that its expression is lost during malignant progression of breast cancer cell lines. Clinical studies confirmed loss of Syk expression, not only in breast tumors but also in other carcinomas and melanoma. Xenotransplantation animal experiments evidenced that Syk acts as a tumor and metastasis suppressor in breast cancer and melanoma. The mechanisms by which Syk exerts its anti-oncogenic activity remain, however, unrevealed. We previously demonstrated that Syk localizes to the centrosomes and negatively affects cell division. The centrosomal Syk substrates and possible effectors of its anti-oncogenic activity remain, nevertheless, unknown. In this study, we demonstrate that Syk may negatively affect cell cycle progression through the phosphorylation of the Cdk1 kinase, a key protein involved in the control of mitosis. Using immunofluorescence, we observed that endogenous Syk and Cdk1 colocalize at the centrosomes and that transient DsRed-Syk transfection induces pY15-Cdk1-positive epitopes at the centrosomes. In vitro kinase assays demonstrate that Syk can phosphorylate Cdk1 in the presence of different cyclins. Mass spectrometry allowed identification of four different Cdk1 tyrosine residues phosphorylated by Syk, amongst which the Y15 is known to negatively affect mitotic entry when phosphorylated by the Wee1 kinase. Transient overexpression of DsRed-Syk or GFP-Cdk1 carrying phospho-mimicking mutations resulted in a cell cycle block as observed by FACS analysis and led to a senescence-like growth arrest. Finally, we demonstrate that DNA damage-inducing agents induce Syk activation and Cdk1 tyrosine phosphorylation in a Syk-dependent manner, as evidenced by the use of a Syk-targeting shRNA. In conclusion, our results reveal that Syk overexpression or Syk activation by genotoxic agents induce a cell cycle arrest through the phosphorylation of Cdk1. These novel aspects of Syk function will contribute to a better understanding of its onco-suppressive activity and, more generally, of the DNA damage-induced signalling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2959. doi:10.1158/1538-7445.AM2011-2959

You have accessJournal of Urology1 Apr 2009SERUM PRO-MATRIX METALLOPROTEINASE-7 (PRO-MMP-7) AS A PREDICTIVE MARKER FOR EARLY PROGRESSION AFTER NEPHRECTOMY IN RENAL CELL CARCINOMA Pierre Bigot, Patricia Fergelot, Nathalie Rioux-Leclercq, Gaiane Sarkissian, Karim Bensalah, Pierre-Jean Lamy, Stéphane Culine, Rodolphe Thuret, Patrick Jouin, Bruno Darbouret, and Jean-Jacques Patard Pierre BigotPierre Bigot More articles by this author , Patricia FergelotPatricia Fergelot More articles by this author , Nathalie Rioux-LeclercqNathalie Rioux-Leclercq More articles by this author , Gaiane SarkissianGaiane Sarkissian More articles by this author , Karim BensalahKarim Bensalah More articles by this author , Pierre-Jean LamyPierre-Jean Lamy More articles by this author , Stéphane CulineStéphane Culine More articles by this author , Rodolphe ThuretRodolphe Thuret More articles by this author , Patrick JouinPatrick Jouin More articles by this author , Bruno DarbouretBruno Darbouret More articles by this author , and Jean-Jacques PatardJean-Jacques Patard More articles by this author View All Author Informationhttps://doi.org/10.1016/S0022-5347(09)60312-8AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail "SERUM PRO-MATRIX METALLOPROTEINASE-7 (PRO-MMP-7) AS A PREDICTIVE MARKER FOR EARLY PROGRESSION AFTER NEPHRECTOMY IN RENAL CELL CARCINOMA." The Journal of Urology, 181(4S), p. 109 © 2009 by American Urological AssociationFiguresReferencesRelatedDetails Volume 181Issue 4SApril 2009Page: 109 Advertisement Copyright & Permissions© 2009 by American Urological AssociationMetricsAuthor Information Pierre Bigot More articles by this author Patricia Fergelot More articles by this author Nathalie Rioux-Leclercq More articles by this author Gaiane Sarkissian More articles by this author Karim Bensalah More articles by this author Pierre-Jean Lamy More articles by this author Stéphane Culine More articles by this author Rodolphe Thuret More articles by this author Patrick Jouin More articles by this author Bruno Darbouret More articles by this author Jean-Jacques Patard More articles by this author Expand All Advertisement PDF downloadLoading ...

Mosquito-transmitted pathogens pass through the insect's midgut (MG) and salivary gland (SG). What occurs in these organs in response to a blood meal is poorly understood, but identifying the physiological differences between sugar-fed and blood-fed (BF) mosquitoes could shed light on factors important in pathogens transmission. We compared differential protein expression in the MGs and SGs of female Aedes aegypti mosquitoes after a sugar- or blood-based diet. No difference was observed in the MG protein expression levels but certain SG proteins were highly expressed only in BF mosquitoes. In sugar-fed mosquitoes, housekeeping proteins were highly expressed (especially those related to energy metabolism) and actin was up-regulated. The immunofluorescence assay shows that there is no disruption of the SG cytoskeletal after the blood meal. We have generated for the first time the 2-DE profiles of immunogenic Ae. aegypti SG BF-related proteins. These new data could contribute to the understanding of the physiological processes that appear during the blood meal.

Depletion of major blood proteins is one of the most promising approaches to access low abundant biomarkers using proteomics. Immunocapture columns often used for this purpose exist in different formats depending on the number of major proteins removed. In this article, we compared the relative interest of depleting either one (albumin), six (albumin, IgG, IgA, transferrin, α1-antitrypsin, and haptoglobin), twelve (the previous six and apo A-I and -II, orosomucoid, α2-macroglobulin, fibrinogen, IgM) or twenty blood proteins (the previous twelve and IgD, ceruloplasmin, apo B, complement C1q, C3, C4, plasminogen, and prealbumin). Such study raises interesting issues related to the reproducibility, practicability, specificity of the immunocapture, and to the impact of removing not only the selected molecules, but also associated peptides and proteins. Depleted sera were here analysed using different proteomic approaches, including two dimensional electrophoresis and SELDI–TOF. Altogether, our results clearly confirmed the interest of depleting major blood proteins for the proteomic detection of low abundant components. However, we observed that increasing the number of depleted proteins from twelve to twenty had a limited beneficial impact and might increase drawbacks in removing associated peptides and proteins. This conclusion is however related to the technologies that we have used, and we believe that it is necessary to adapt the immunocapture to the analytical method employed, and to the ratio between wanted and unwanted proteins removed.

Animal trypanosomosis is a major constraint to livestock productivity in the tropics and has a significant impact on the life of millions of people globally (mainly in Africa, South America and south–east Asia). In Africa, the disease in livestock is caused mainly by Trypanosoma congolense, Trypanosoma vivax, Trypanosoma evansi and Trypanosoma brucei brucei. The extracellular position of trypanosomes in the bloodstream of their host requires consideration of both the parasite and its naturally excreted–secreted factors (secretome) in the course of pathophysiological processes. We therefore developed and standardised a method to produce purified proteomes and secretomes of African trypanosomes. In this study, two strains of T. congolense exhibiting opposite properties of both virulence and pathogenicity were further investigated through their secretome expression and its involvement in host–parasite interactions. We used a combined proteomic approach (one-dimensional SDS–PAGE and two-dimensional differential in-gel electrophoresis coupled to mass spectrometry) to characterise the whole and differentially expressed protein contents of secretomes. The molecular identification of differentially expressed trypanosome molecules and their correlation with either the virulence process or pathogenicity are discussed with regard to their potential as new diagnostic or therapeutic tools against animal trypanosomosis.

ABSTRACT The non-receptor tyrosine kinase Src is freque ntly overexpressed and/or activated in human colon carcinoma (CRC) and its increased activity has been associated with a poor clinical outcome. Src has been then implicated in growth and invasion of these can cer cells by still not well known mechanisms. Here we addressed thes e Src oncogenic signaling using quantitative phosphoproteomics. Src overexpression increased gr owth and invasiveness of the metastatic SW620 CRC cells. Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) in combination with LC-MS/MS allowed the iden tification of 136 proteins which exhibited a significant increased and/or association with tyrosine phosphorylation upon Src expression. These mainly include signaling, cytoskeleton and vesicular-associated pr oteins. Interestingly, Src also phosphorylated a cluster of tyrosine kinases, ie the receptors Met and EphA2, the cytoplasmic tyrosine kinase Fa k and pseudo-tyrosine kinase Sg K223, which were required for its invasive activity. Similar re sults were obtained with the me tastatic Colo205 CRC cells that exhibit high endogenous Sr c activity. We concluded that Src uses a tyrosine kinases network to promote its invasive activity in CRC and this implicates a reverse signaling via tyrosine kinase receptors. Targeting thes e tyrosine kinases may be of si gnificant therapeutic value in hal-00367875, version 1 - 12 Mar 2009 this cancer.

Abstract The nonreceptor tyrosine kinase Src is frequently overexpressed and/or activated in human colorectal carcinoma (CRC), and its increased activity has been associated with a poor clinical outcome. Src has been implicated in growth and invasion of these cancer cells by still not well-known mechanisms. Here, we addressed Src oncogenic signaling using quantitative phosphoproteomics. Src overexpression increased growth and invasiveness of metastatic SW620 CRC cells. Stable isotope labeling with amino acids in cell culture in combination with liquid chromatography tandem mass spectrometry allowed the identification of 136 proteins which exhibited a significant increase in and/or association with tyrosine phosphorylation upon Src expression. These mainly include signaling, cytoskeleton, and vesicular-associated proteins. Interestingly, Src also phosphorylated a cluster of tyrosine kinases, i.e., the receptors Met and EphA2, the cytoplasmic tyrosine kinase Fak, and pseudo-tyrosine kinase SgK223, which were required for its invasive activity. Similar results were obtained with metastatic Colo205 CRC cells that exhibit high endogenous Src activity. We concluded that Src uses a tyrosine kinases network to promote its invasive activity in CRC and this implicates a reverse signaling via tyrosine kinase receptors. Targeting these tyrosine kinases may be of significant therapeutic value in this cancer. [Cancer Res 2009;69(6):2279–86]

The spleen tyrosine kinase Syk has predominantly been studied in hematopoietic cells in which it is involved in immunoreceptor-mediated signaling. Recently, Syk expression was evidenced in numerous nonhematopoietic cells and shown to be involved in tumor formation and progression. The Syk downstream signaling effectors in nonhematopoietic cells remain, however, to be uncovered, and were investigated using MS-based quantitative phosphoproteomics. Two strategies, based on the inhibition of the Syk catalytic activity and on the loss of Syk expression were employed to identify phosphotyrosine-dependent complexes. Quantitative measurements were obtained on 350 proteins purified with phosphotyrosine affinity columns using the SILAC method. Forty-one proteins are dependent on both Syk expression and catalytic activity and were selected as signaling effectors. They are involved in a variety of biological processes such as signal transduction, cell–cell adhesion and cell polarization. We investigated the functional involvement of Syk in cell–cell adhesion and demonstrated the phosphorylation of E-cadherin and α-catenin. In addition, Syk is localized at cell–cell contacts, and Syk-mediated phosphorylation of E-cadherin seems to be important for the proper localization of p120-catenin at adherens junctions. Identification of the biochemical pathways regulated by Syk in human cancer cells will help to uncover its role in tumor formation and progression.

Human cerebrospinal fluid (CSF) proteome is actively investigated to identify relevant biomarkers and therapeutic targets for neurological disorders. Approximately 80% of CSF proteome originate from plasma, yielding a high dynamic range in CSF protein concentration and precluding identification of potential biomarkers originating from CNS cells. Here, we have adapted the most complete multiaffinity depletion method available to remove 20 abundant plasma proteins from a CSF pool originating from patients with various cognitive disorders. We identified 622 unique CSF proteins in immunodepleted plus retained fractions versus 299 in native CSF, including 22 proteins hitherto not identified in CSF. Parallel analysis of neuronal secretome identified 34 major proteins secreted by cultured cortical neurons (cell adhesion molecules, proteins involved in neurite outgrowth and axonal guidance, modulators of synaptic transmission, proteases and protease inhibitors) of which 76% were detected with a high confidence in immunodepleted CSF versus 50% in native CSF. Moreover, a majority of proteins previously identified as secretory products of choroid plexus cells or astrocytes were detected in immunodepleted CSF. Hence, removal of 20 major plasma proteins from CSF improves detection of brain cell-derived proteins in CSF and should facilitate identification of relevant biomarkers in CSF proteome profiling analyses.

BACKGROUND:No validated renal cell carcinoma (RCC) marker is known for detection of asymptomatic disease in selected populations or for prognostic purposes or treatment monitoring. We identified immunogenic proteins as tumor markers for RCC by combining conventional proteome analysis with serological screening, and we investigated the diagnostic clinical value of such markers in serum.METHODS:We studied the immunogenic protein expression profile of CAL 54, a human RCC cell line, by 2-dimensional electrophoresis combined with immunoblotting using sera from healthy donors compared with RCC patients. We developed a homogeneous, fluorescent, dual-monoclonal immunoassay for metalloproteinase 7 (MMP-7) and used it to measure MMP-7 in sera from 30 healthy donors, 30 RCC patients, and 40 control patients.RESULTS:Pro-MMP-7 (29 kDa; pI 7.7) in the CAL 54 cell line secretome was an immunogenic protein reactive with RCC patient sera but not with control sera. The concentrations of pro-MMP-7 were increased (P <0.0001) in sera of RCC patients (median 7.56 microg/L; range 3.12-30.5 microg/L) compared with healthy controls (median 2.13 microg/L; range 0.17-3.5 microg/L). Serum pro-MMP-7 had a sensitivity of 93% (95% CI 78%-99%) at a specificity of 75% (59%-87%) for RCC in the samples tested.CONCLUSION:Proteomics technology combined with serology led to the identification of serum pro-MMP-7 as a marker of RCC and represents a powerful tool in searching for candidate proteins as biomarkers.