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  • PHOSPHORYLATION  (4)
  • PROTEINS  (4)
  • 1
    Keywords: CELLS ; EXPRESSION ; carcinoma ; CELL ; Germany ; human ; GENE ; GENE-EXPRESSION ; GENES ; PROTEIN ; PROTEINS ; MOLECULES ; COMPLEX ; COMPLEXES ; KERATINOCYTES ; BOVINE PAPILLOMAVIRUS ; DOWN-REGULATION ; GROWTH-FACTOR RECEPTOR ; papillomavirus ; MOLECULE ; MHC ; TRANSPORT ; IDENTIFICATION ; LESIONS ; gene expression ; DESIGN ; DIFFERENCE ; PLASMA ; MEMBRANE ; STRESS ; SPECTROMETRY ; human papillomavirus ; TYPE-16 ; MASS-SPECTROMETRY ; SURFACE ; CLASS-I ; EPITHELIAL-CELL LINE ; GOLGI-APPARATUS ; HUMAN-PAPILLOMAVIRUS ; MHC class I ; MHC CLASS-I ; CARCINOMAS ; DIFFERENTIAL EXPRESSION ; MEMBRANE PROTEIN ; HaCaT ; MEMBRANE-PROTEIN ; GEL-ELECTROPHORESIS ; MASSES ; E5 PROTEIN ; CERVICAL NEOPLASIA
    Abstract: Membrane proteins differentially expressed in human papillomavirus type 16 (HPV-16) E5-transfected HaCaT cells have been identified. Membrane proteins were isolated and separated by two-dimensional gel electrophoresis. Spots showing quantitative differences between E5-transfected and control cells were extracted and the proteins were identified by nanoelectrospray ionization mass spectrometry. A total of 24 spots was analysed. Among the proteins showing differential expression, a decreased amount of calnexin and increased expression of hsp70, proteins both involved in maturation and transport of MHC class I complexes to the plasma membrane, were noticed. These findings correlate with the decreased surface expression of MHC class I molecules described in E5-expressing cells, HPV-positive cervical lesions and cervical carcinomas. These results stress the value of the proteomic approach, as used here in the experimental design, which allows the correlation of changes in host gene expression with biological functions of viral genes
    Type of Publication: Journal article published
    PubMed ID: 15166425
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  • 2
    Keywords: CELLS ; EXPRESSION ; proliferation ; tumor ; carcinoma ; KINASE ; DISEASE ; PROTEIN ; DIFFERENTIATION ; TUMORS ; CONTRAST ; ACTIVATED PROTEIN-KINASE ; PHOSPHORYLATION ; BREAST ; IN-SITU ; LESIONS ; immunohistochemistry ; MALIGNANCIES ; PATTERNS ; EPITHELIAL-CELLS ; CARCINOMAS ; INTERCELLULAR COMMUNICATION ; MALIGNANCY ; MOLECULAR-BASIS ; BASAL LAMINA ; breast carcinoma ; connexin43 ; GAP JUNCTIONAL COMMUNICATION ; gap junctions
    Abstract: We applied an antiserum (SA226P) specifically recognizing the phosphorylated form of connexin43 (P-Cx43) to human breast samples including normal breast samples, with fibrocystic disease (FCD), fibroadenomas (FA), in situ and infiltrating carcinomas of all major types, and miscellaneous extramarnmary tumors. The findings were compared with those obtained with commercial antisera recognizing all Cx43 forms (pan-Cx43). A subset of samples was stained for Her2-neu and p44/42 to mitogen-activated protein kinase. Paraffin step sections were used. Immunoblots were performed on frozen samples of a representative subset of cases. In the normal breast, FCD, and FA, SA226P stained strongly and extensively most myoepithelial cells (MECs); luminal cells remained unstained. In proliferative FCD and some cellular FA, SA226P stained MEC and the capillary endothelium (CE). In ductal and lobular in situ carcinomas, SA226P reacted strongly and diffusely with the remaining MEC, the CE, and the transformed luminal cells. SA226P stained all infiltrating carcinomas except the tubular variant. In all breast carcinomas, the CE within and adjacent to tumors and some myofibroblasts stained with SA226P. By contrast, pan-Cx43 stained weakly and sporadically the MEC and rare samples of invasive carcinomas. Notably, Mab p44/42 reacted in parallel with the samples stained with SA226P, whereas reactions with Her2 were negative. Immunoblot findings paralleled those obtained immunohistochemically. We conclude that P-Cx43, restricted to MEC in the normal breast, is up-regulated in the same cells in hyperplasias and dysplasias and FA and is strongly up-regulated in invasive carcinomas. Notably, in some proliferative FCD and in most in situ and infiltrating carcinomas, P-Cx43 is strongly expressed in CE within and adjacent to the lesions but not away from them. These findings were paralleled by the strong nuclear reactions noted with Mab p44/42. These phenomena, although not exclusive to malignancy, are particularly conspicuous in breast carcinomas and seemingly reflect active proliferation associated with abnormal gap junctional intercellular communication. (c) 2005 Elsevier Inc. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 15948121
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  • 3
    Keywords: CANCER ; CELLS ; EXPRESSION ; carcinoma ; Germany ; SITE ; GENES ; PROTEIN ; PROTEINS ; TISSUE ; CARCINOGENESIS ; PHOSPHORYLATION ; antibodies ; MOUSE ; LESIONS ; PROGRESSION ; immunohistochemistry ; CERVIX ; CELL-LINE ; REGION ; REGIONS ; CARCINOMAS ; INTERCELLULAR COMMUNICATION ; STRATIFIED EPITHELIUM ; JUNCTION ; premalignant ; gap junction ; cell communication
    Abstract: Connexins are proteins that form the connexons, gap junction structures, which allow cells to communicate. Phosphorylation of connexins has been found to impair this communication. Using an antibody specifically recognizing the S279/S282-phosphorylated form of connexin43 (Cx43) for immunohistochemistry, we have analysed Cx43 phosphorylation in normal epithelium, CIN III lesions, and carcinomas of the cervix. We found that in normal epithelium the basal layer was devoid of staining and most of the protein was localized in stratum spinosum and stratum granulosum. In pre-malignant CIN-III lesions Cx43 was strongly phosphorylated, but the basal layer was still negative. In squamous carcinomas, the cells were intensely stained. In these tumours, sites of strong staining were adjacent to less stained regions, suggesting that the tumours are intrinsically heterogeneous. Immunoblotting of proteins extracted from carcinomas with the specific antibody showed the classical pattern of multiple reacting bands, with the appearance of low migrating forms of the protein. Our results suggest that increased S279/S282 phosphorylation of Cx43 is the result of altered tissue structure rather than of cell malignization. (c) 2005 Elsevier Ireland Ltd. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 15958277
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  • 4
    Keywords: KINASE ; PHOSPHORYLATION ; UBIQUITINATION ; LIVING CELLS ; MAP KINASES ; INTERCELLULAR COMMUNICATION ; connexin43 ; GAP-JUNCTION PROTEIN ; TURNOVER ; EGF ; V-SRC
    Abstract: Connexins are the structural units of gap junctions, structures allowing interchanging of information between the adjacent cells. Connexin43 (Cx43) is the most abundant gap junction protein. Cx43 can be degraded by lysosome- and proteasome-mediated processes upon internalisation of the entire structure. Only little is known about the role of phosphorylation during the gap junction degradation. In Cx43, a protein containing 14 amino acids susceptible to be phosphorylated, amino acids S279 and S282 are phosphorylated upon epidermal growth factor (EGF) treatment by erk1/2 MAP kinases. Here, we show that the wild-type Cx43 protein, as well as HeLa cells expressing the mutated Cx43 proteins S279A, S282A and S279A/S282A, is mainly located at the plasma membrane. However, the protein stability is not altered in the isolated single mutants, whereas the double mutant S279A/S282A is strongly degradation impaired upon EGF treatment. This effect is not due to the decreased Cx43 internalisation, but seems to be related to a reduced ubiquitination.
    Type of Publication: Journal article published
    PubMed ID: 24399133
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  • 5
    Keywords: APOPTOSIS ; CANCER ; CELLS ; EXPRESSION ; proliferation ; CELL ; Germany ; human ; INFORMATION ; GENE ; GENOME ; PROTEIN ; PROTEINS ; transcription ; DIFFERENTIATION ; TISSUE ; MICE ; DNA ; TISSUES ; SKIN ; BINDING ; BIOLOGY ; MOLECULAR-BIOLOGY ; papillomavirus ; TARGET ; virus ; NO ; TRANSGENIC MICE ; PROMOTER ; PROMOTERS ; cervical cancer ; CERVICAL-CANCER ; REGION ; human papillomavirus ; HIGH-RISK ; HPV ; transactivation ; ONCOGENE ; HUMAN-PAPILLOMAVIRUS ; CERVICAL-CARCINOMA ; NETHERLANDS ; REPRESSION ; molecular biology ; molecular ; RE ; PATTERN ; LEVEL ; analysis ; methods ; EVENTS ; LOSSES ; UBIQUITIN ; CANCERS ; microbiology ; E2 PROTEIN ; HUMAN PAPILLOMAVIRUSES ; animal model ; host ; viral ; UPSTREAM ; UBIQUITIN-C ; biotechnology ; E2 ; READING FRAMES ; E6 PROMOTER ; PAPILLOMAVIRUS DNA-REPLICATION ; ubiquitin C promoter
    Abstract: The E2 early protein of human papillomaviruses (HPV) has been found associated with the mitotic spindle therefore being implicated in the partition of the replicated viral DNA to daughter cells. In addition, E2 proteins bind to the upstream regulatory region of the virus and to cellular promoters modulating thereby cellular transcription and differentiation. In many cervical cancers, the E2 reading frame is interrupted upon incorporation of the viral genome into the host DNA. This results in the loss of the E2 mediated transcriptional repression and uncontrolled expression of the viral oncogenes. All these results have been obtained in transfected cells but no information is available on the E2 effects in the context of the entire organism. Transgenic mice were generated expressing the E2 protein of HPV11 under the control of the Ubiquitin C promoter. E2 mRNA is present in all mice tissues analysed and the E2 protein expressed in the skin (the target tissue of HPV11) was shown by Western blotting, albeit at a very low level. Analysis of the transgenic mice shows no major histological changes in the skin or all other tissues investigated. These data indicate that in transgenic mice the human papillomavirus type 11 E2 does not grossly modulate cellular proliferation or differentiation events
    Type of Publication: Journal article published
    PubMed ID: 17701441
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  • 6
    Keywords: PEPTIDE ; CELLS ; EXPRESSION ; Germany ; human ; KINASE ; MICROSCOPY ; NEW-YORK ; PROTEIN ; PROTEINS ; cell line ; TISSUE ; FAMILY ; ACTIVATED PROTEIN-KINASE ; PHOSPHORYLATION ; treatment ; cell culture ; ACIDS ; antibodies ; antibody ; MAP KINASE ; DISRUPTION ; PLASMA ; MEMBRANE ; STRESS ; LOCALIZATION ; CHROMATOGRAPHY ; AMINO-ACIDS ; PHORBOL-ESTER ; serine ; CHANNEL ; GAP-JUNCTIONAL COMMUNICATION ; growth factors ; hyperosmosis ; INTERCELLULAR COMMUNICATION ; LIVER EPITHELIAL-CELLS ; plasma membranes ; sorbitol ; threonine phosphorylation
    Abstract: We have developed polyclonal antibodies (SA226P) to a peptide of the human connexin43 (Cx43) protein between amino acids 271 and 288 containing phosphorylated S279 and S282. Antibodies specific for the phosphorylated form of the peptide were isolated by double immunoaffinity chromatography and were characterised using proteins of the cell line WB-F344, known to contain large amounts of Cx43. SA226P recognises specifically the slowest migrating Cx43 band in immunoblots of proteins isolated from untreated cells. In immunofluorescence experiments SA226P scarcely stains the plasma membrane in untreated cells in contrast to a commercial antibody recognising all isoforms of the Cx43 protein. EGF or stress treatment of the cells results in a rapid increase in the phosphorylated forms of Cx43 as revealed by immunoblotting. Immunofluorescence experiments reveal that both phosphorylated and non-phosphorylated Cx43 could be found at the plasma membrane. Whether phosphorylation of S279/S282 takes place before or after incorporation of Cx43 into the membranes is so far unknown. More interestingly, confocal microscopy using our antibodies and a commercial antibody recognising all isoforms of Cx43 shows the coexistence of differentially phosphorylated forms of the protein at the plasma membrane. Our results indicate that MAP kinases erk1/2 are mainly responsible for this phosphorylation, as already published. Nevertheless, treatment of the cells with anisomycin, known to activate stress kinase p38 but not erk1/2, also results in a weak but reproducible Cx43 phosphorylation
    Type of Publication: Journal article published
    PubMed ID: 12483281
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