Identification of a putative target for Rho as the serine-threonine kinase protein kinase N

Science

Volumn 271, Issue 5249, 1996, Pages 648-650

  Amano, Mutsuki ^a   Mukai, Hideyuki ^b   Ono, Yoshitaka ^b   Chihara, Kazuyasu ^a   Matsui, Takeshi ^a   Hamajima, Yuko ^a   Okawa, Katsuya ^c   Iwamatsu, Akihiro ^c   Kaibuchi, Kozo ^a  

^a NARA INSTITUTE OF SCIENCE AND TECHNOLOGY   (Japan)

^b KOBE UNIVERSITY   (Japan)

^c KIRIN BREWERY CO LTD   (Japan)

Author keywords

[No Author keywords available]

Indexed keywords

BOTULINUM TOXIN; GUANOSINE TRIPHOSPHATASE; GUANOSINE TRIPHOSPHATE; LYSOPHOSPHATIDIC ACID; PROTEIN KINASE N;

ARTICLE; CATTLE; CELL STRAIN 3T3; CYTOSKELETON; ENZYME ACTIVATION; ENZYME PHOSPHORYLATION; NONHUMAN; PRIORITY JOURNAL;

BOS TAURUS;

EID: 0030035043     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.271.5249.648     Document Type: Article

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21. 2]. Solid ammonium sulfate was added to a final concentration of 40% saturation The precipitate was dissolved in 16 ml of buffer A, dialyzed against buffer A, and then passed over a 1-ml glutathione-Sepharose column. One-eighth of the fraction that passed through the column (2 ml) was loaded onto a 0 25-ml glutathione-Sepharose column containing 6 nmol of respective small GTPases preloaded with guanine nucleotides as indicated The columns were washed with 2.5 ml of buffer A and bound proteins were eluted with respective small GTPases by addition of 0 825 ml of buffer A containing 10 mM glutathione
22. The fraction that passed through the glutathione-Sepharose column (16 ml) was loaded onto a 1-ml glutathione-Sepharose column containing 24 nmol of GTP-γ-S-GST-RhoA p128 was eluted by addition of buffer A containing 0.2 M NaCl. The sample was dialyzed against buffer A and applied to a 0 3-ml DEAE-Sepharose column equilibrated with buffer A. The column was washed with 1.5 ml of buffer A containing 50 mM NaCl, and proteins were eluted with 1.5 ml of buffer A containing 75 mM NaCl. Fractions of 0.3 ml were collected, and portions (30 μl each) were subjected to SDS-PAGE followed by silver staining p128 appeared as a single peak in fractions 1 through 3.
23. 32P into the PKCα peptide was assessed by scintillation counting
24. A37, GDP-GST-Rac, GTP-γ-S-GST-Rac, GDP-GST-H-Ras, or GTP-γ-S-GST-H-Ras in 0.8 ml of buffer A. MBP-PKN was eluted three times by addition of 0.1 ml of buffer A containing 0.2 M NaCl and then three times by addition of 0.1 ml of buffer A containing 10 mM glutathione. Portions (30 μl each) of the first fraction of the glutathione eluate were subjected to SDS-PAGE followed by silver staining
25. 4, 5 mM NaF, leupeptin (2 5 μg/ml), 0.05% NP-40, and 0 05 M NaCl], and homogenized in a Dounce homogenizer The cytosol was subjected to immunoprecipitation by antibody to HA (12CA5).
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27. We thank M. Nakafuku, K. Umesono, and R Yu for discussion and critical reading of the manuscript and Y. Ohashi (Nihon Schering) for C3 exoenzyme Supported by grants-in-aid for scientific research and for cancer research from the Ministry of Education, Science, and Culture, Japan (1995).