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Identification of an Intracellular Protein that Specifically Interacts with Photoaffinity-Labeled Oncogenic P21 Protein

Lee, Grace ; Ronai, Zeev A. ; Pincus, Matthew R. ; Brandt-Rauf, Paul W. ; Murphy, Randall B. ; Delohery, Thomas M. ; Nishimura, Susumu ; Yamaizumi, Ziro ; Weinstein, I. Bernard

Proceedings of the National Academy of Sciences - PNAS, 1989-11, Vol.86 (22), p.8678-8682 [Periódico revisado por pares]

Washington, DC: National Academy of Sciences of the United States of America

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Título:
Identification of an Intracellular Protein that Specifically Interacts with Photoaffinity-Labeled Oncogenic P21 Protein
Autor: Lee, Grace ; Ronai, Zeev A. ; Pincus, Matthew R. ; Brandt-Rauf, Paul W. ; Murphy, Randall B. ; Delohery, Thomas M. ; Nishimura, Susumu ; Yamaizumi, Ziro ; Weinstein, I. Bernard
Assuntos: 3T3 cells ; 550201 - Biochemistry- Tracer Techniques ; Affinity Labels - metabolism ; AMINO ACIDS ; ANIMAL CELLS ; Animals ; Antibodies ; AZIDES ; Azides - metabolism ; BASIC BIOLOGICAL SCIENCES ; BETA DECAY RADIOISOTOPES ; BETA-MINUS DECAY RADIOISOTOPES ; BIOCHEMISTRY ; Biological and medical sciences ; BIOLOGICAL EFFECTS ; BIOLOGICAL RADIATION EFFECTS ; CARBOXYLIC ACIDS ; Cell fusion ; Cell physiology ; Cell transformation and carcinogenesis. Action of oncogenes and antioncogenes ; Cells, Cultured ; Cellular immunity ; CHEMICAL REACTIONS ; CHEMISTRY ; CONNECTIVE TISSUE CELLS ; CROSS-LINKING ; DAYS LIVING RADIOISOTOPES ; DNA ; DNA Replication ; DRUGS ; ELECTROMAGNETIC RADIATION ; ELECTROPHORESIS ; EVEN-ODD NUCLEI ; EXTREME ULTRAVIOLET RADIATION ; FIBROBLASTS ; Fluorescence ; Fundamental and applied biological sciences. Psychology ; Gels ; GENES ; Ha-ras ; IN VITRO ; ISOTOPES ; Kinetics ; LIGHT NUCLEI ; LIPOTROPIC FACTORS ; METHIONINE ; Methionine - metabolism ; Mice ; Molecular and cellular biology ; NIH 3T3 cells ; NITROGEN COMPOUNDS ; NUCLEI ; Oncogene Protein p21(ras) - metabolism ; ONCOGENES ; ORGANIC ACIDS ; ORGANIC COMPOUNDS ; ORGANIC SULFUR COMPOUNDS ; Pinocytosis ; POLYMERIZATION ; PRECIPITATION ; Product labeling ; Protein Binding ; Protein Biosynthesis ; protein p21 ; PROTEINS ; Proteins - isolation & purification ; Proteins - metabolism ; RADIATION EFFECTS ; RADIATIONS ; RADIOISOTOPES ; SEPARATION PROCESSES ; SOMATIC CELLS ; SULFUR 35 ; SULFUR ISOTOPES ; Sulfur Radioisotopes ; ULTRAVIOLET RADIATION ; Valine
É parte de: Proceedings of the National Academy of Sciences - PNAS, 1989-11, Vol.86 (22), p.8678-8682
Notas: ObjectType-Article-1
SourceType-Scholarly Journals-1
ObjectType-Feature-2
content type line 23
Descrição: An oncogenic 21-kDa (p21) protein (Harvey RAS protein with Val-12) has been covalently modified with a functional reagent that contains a photoactivatable aromatic azide group. This modified p21 protein has been introduced quantitatively into NIH 3T3 cells using an erythrocyte-mediated fusion technique. The introduced p21 protein was capable of inducing enhanced pinocytosis and DNA synthesis in the recipient cells. To identify the putative intracellular protein(s) that specifically interact with the modified p21 protein, the cells were pulsed with [35S]methionine at selected times after fusion and then UV-irradiated to activate the azide group. The resulting nitrene covalently binds to amino acid residues in adjacent proteins, thus linking the p21 protein to these proteins. The cells were then lysed, and the lysate was immunoprecipitated with the anti-p21 monoclonal antibody Y13-259. The immunoprecipitate was analyzed by SDS/PAGE to identify p21-protein complexes. By using this technique, we found that three protein complexes of 51, 64, and 82 kDa were labeled specifically and reproducibly. The most prominent band is the 64-kDa protein complex that shows a time-dependent rise and fall, peaking within a 5-hr period after introduction of the p21 protein into the cells. These studies provide evidence that in vitro the p21 protein becomes associated with a protein whose mass is about 43 kDa. We suggest that the formation of this complex may play a role in mediating early events involved with cell transformation induced by RAS oncogenes.
Editor: Washington, DC: National Academy of Sciences of the United States of America
Idioma: Inglês

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Identification of an Intracellular Protein that Specifically Interacts with Photoaffinity-Labeled Oncogenic P21 Protein

Lee, Grace ; Ronai, Zeev A. ; Pincus, Matthew R. ; Brandt-Rauf, Paul W. ; Murphy, Randall B. ; Delohery, Thomas M. ; Nishimura, Susumu ; Yamaizumi, Ziro ; Weinstein, I. Bernard

Washington, DC: National Academy of Sciences of the United States of America

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