Reduced ubiquitin-dependent degradation of c-Jun after phosphorylation by map kinases

Science

Volumn 275, Issue 5298, 1997, Pages 400-402

  Musti, Anna Maria ^a,b   Treier, Mathias ^a,c   Bohmann, Dirk ^a  

^a EUROPEAN MOLECULAR BIOLOGY LABORATORY   (Germany)

^b CNR   (Italy)

^c UNIVERSITY OF CALIFORNIA   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

COMPLEMENTARY DNA; MITOGEN ACTIVATED PROTEIN KINASE; MONOCLONAL ANTIBODY; ONCOPROTEIN; POLYCLONAL ANTIBODY; TRANSCRIPTION FACTOR; UBIQUITIN;

ANIMAL CELL; ARTICLE; ENZYME ACTIVATION; ENZYME PHOSPHORYLATION; GENE EXPRESSION REGULATION; NONHUMAN; PRIORITY JOURNAL; PROTEIN PHOSPHORYLATION; PROTO ONCOGENE; SIGNAL TRANSDUCTION; TRANSCRIPTION REGULATION;

3T3 CELLS; ANIMALS; CA(2+)-CALMODULIN DEPENDENT PROTEIN KINASE; CDC42 GTP-BINDING PROTEIN, SACCHAROMYCES CEREVISIAE; CELL CYCLE PROTEINS; GENE EXPRESSION REGULATION; GTP-BINDING PROTEINS; JNK MITOGEN-ACTIVATED PROTEIN KINASES; MICE; MITOGEN-ACTIVATED PROTEIN KINASES; PHOSPHORYLATION; PROTO-ONCOGENE PROTEINS C-JUN; SIGNAL TRANSDUCTION; TRANSFECTION; UBIQUITINS;

EID: 0031023756     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.275.5298.400     Document Type: Article

References (21)

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18. 6-tagged or HA-tagged c-Jun expression vectors with essentially identical results (compare Figs. 1 and 2).
19. -) cell transfections, purification of Jun-ubiquitin conjugates, and immunoblot analysis were as described (6).
20. 35S-cysteine per milliliter of medium, followed by incubation in a medium that contained 2 mM each of unlabeled methionine and cysteine for 0, 90, 180, or 270 min. The cells that had been transfected with the JNK1 expression vector were treated with 10 mM anisomycin during the labeling and chase periods to induce kinase activity. Cells were lysed in RIPA buffer [10 mM tris (pH 7.5), 45 mM β-glycerophosphate, 50 mM NaF, 5 mM sodium molybdate, 0.1% SDS, 1 mM EDTA, 1% NP-40, 0.5% deoxycholate] supplemented with 1 mM phenylmethylsulfonyl fluoride and 10 mg each of leupeptin, aprotinin, and pepstatin per milliliter of buffer. Samples containing equal amounts of acid-insoluble radioactivity were incubated with protein G-and protein A-agarose (Oncogene Science) and then incubated with the monoclonal antibody 12CA5 to HA. Precipitates were collected on protein G-and protein A-agarose, washed once with buffer A [10 mM tris (pH 7.5), 45 mM β-glycerophosphate, 50 mM NaF, 5 mM sodium molybdate, 0.1% SDS, 1 mM EDTA, 0.5% NP-40], once with buffer B [10 mM tris (pH 7.5), 410 mM NaCI, 45 mM β-glycerophosphate, 0.1% SDS, 1 mM EDTA, 0.5% NP-40], once with 10 mM tris (pH 7.5), and analyzed by SDS-polyacrylamide gel electrophoresis (PAGE; 10% gel) and autoradiography.
21. We thank S. Gutkind for plasmid vectors, L. Staszewski for technical assistance, and I. Mattaj, S. Cohen, T. Graf, A. Isaksson, L. Kockel, A. Papavassilou, and C. Ovitt for comments on the manuscript. Supported by a grant from the CNR to A.M.M.