Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho- kinase)

Science

Volumn 273, Issue 5272, 1996, Pages 245-248

  Kimura, Kazushi ^a   Ito, Masaaki ^b   Amano, Mutsuki ^a   Chihara, Kazuyasu ^a   Fukata, Yuko ^a   Nakafuku, Masato ^a   Yamamori, Bunpei ^b   Feng, Jianhua ^b   Nakano, Takeshi ^b   Okawa, Katsuya ^c   Iwamatsu, Akihiro ^c   Kaibuchi, Kozo ^a  

^a NARA INSTITUTE OF SCIENCE AND TECHNOLOGY   (Japan)

^b MIE UNIVERSITY   (Japan)

^c KIRIN BREWERY CO LTD   (Japan)

Author keywords

[No Author keywords available]

Indexed keywords

MYOSIN; PHOSPHATASE; PHOSPHOTRANSFERASE;

ACTIN MYOSIN INTERACTION; ARTICLE; ENZYME INHIBITION; PRIORITY JOURNAL; PROTEIN PHOSPHORYLATION; REGULATORY MECHANISM; SMOOTH MUSCLE CONTRACTION;

EID: 9444242736     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.273.5272.245     Document Type: Article

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31. 2] containing 0.2 M NaCl, and bound proteins were eluted together with the GST fusion proteins by the addition of 0.825 ml of buffer A containing 10 mM glutathione.
32. Rabbit polyclonal antibodies to chicken MBS and to the 20-kD regulatory subunit were generated with the use of recombinant proteins (14). Antibodies to the 37-kD catalytic subunit were from Santa Cruz Biotechnology.
33. 32P-labeled MLC, and test samples] (14).
34. 35S]methionine-labeled products were mixed with glutathione-Sepharose 4B beads coated with GST-RhoA loaded with guanine nucleotides.
35. 8-lacZ] was transformed with plasmids encoding the LexA fusion proteins and plasmids encoding the Gal4p fusion proteins (15). The transformants were plated on synthetic medium lacking histidine. If the LexA fusion protein interacts with the Gal4p fusion protein, the transformant can express the HIS3 reporter gene and therefore grow in the absence of histidine.
36. V14 (10). After 40 hours, the cells were collected and homogenized. The cytosolic and particulate fractions were prepared and subjected to quantitative immunoblot analysis with antibodies (9E10) to the Myc epitope (10).
37. cMBS-N and cMBS-C were expressed as maltose-binding protein fusion proteins in Escherichia coli and purified (15).
38. 32P-labeled bands corresponding to MBS were visualized by autoradiography. Fold stimulation of MBS phosphorylation was determined with a Fuji image analyzer (BAS-2000).
39. Myosin phosphatase was purified from chicken gizzard (14).
40. 35S]ATP-γ-S (8 to 10 GBq/mmol) (28). After incubation for 6 min at 30°C, a portion of the mixture (40 μl) was subjected to SDS-PAGE and the remaining portion was assayed for MLC phosphatase activity (23).
41. v14-25 cell lines) under the control of IPTG [D. L. Wyborski and J. M. Short, Nucleic Acids Res. 19, 4647 (1991)].
42. 32P]orthophosphate. The cells were then lysed and MBS was immunoprecipitated. The washed immunoprecipitates were subjected to SDS-PAGE and auto-radiography. Fold stimulation of MBS phosphorylation was determined with a Fuji BAS-2000 image analyzer. Under similar conditions, with the exception that the cells were not labeled, cells were lysed and the lysates were subjected to immunoblot analysis with antibodies to MBS and RhoA.
43. IPTG-treated, serum-deprived NIH 3T3 cell lines (100-mm dishes) were treated with 10% (w/v) trichloroacetic acid. The resulting precipitates were subjected to glycerol-urea gel electrophoresis, and the relative amounts of nonphosphorylated and phosphorylated forms of MLC were determined by immunoblot analysis [D. A. Taylor and J. T. Stull, J. Biol. Chem. 263, 14456 (1988)]. NIH 3T3 cells were treated with 0.1 μM calyculin A for 10 min.
44. We thank M. Fujioka and J. Tanaka for preparing polyclonal antibodies to MBS and the 20-kD regulatory subunit of myosin phosphatase, Y. Hamajima for preparing NIH 3T3 cell lines, M. Inagaki for valuable discussions, and M. Nishimura for help in preparing the manuscript. Supported by grants-in-aid for scientific research and for cancer research from the Ministry of Education, Science, and Culture of Japan.