Cytoskeletal Rearrangements and Transcriptional Activation of c-fos Serum Response Element by Rho-kinase*
- Kazuyasu Chihara,
- Mutsuki Amano,
- Nao Nakamura,
- Takeo Yano‡,
- Masao Shibata‡,
- Toshiya Tokui§,
- Hisashi Ichikawa§,
- Reiko Ikebe§,
- Mitsuo Ikebe§ and
- Kozo Kaibuchi¶
- From the Division of Signal Transduction, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma 630-01, the‡Medical and Biological Laboratories, Ina 396, Japan, and the §Department of Physiology, University of Massachusetts Medical Center, Worcester, Massachusetts 01655
Abstract
The small GTPase Rho is implicated in cytoskeletal rearrangements including stress fiber and focal adhesion formation and in the transcriptional activation of c-fosserum response element. In vitro, Rho-kinase, which is activated by Rho, phosphorylates not only myosin light chain (MLC) (thereby activating myosin ATPase) but also myosin phosphatase, thus inactivating myosin phosphatase. Rho-kinase is involved in the formation of stress fibers and focal adhesions in fibroblasts. Here we show that the expression of constitutively active Rho-kinase increased the level of MLC phosphorylation. The activity of Rho-kinase was necessary for maintaining the vinculin-containing focal adhesions, whereas organized actin stress fibers were not necessary for this. The microinjection of constitutively active Rho-kinase into fibroblasts induced the formation of focal adhesions to some extent under the conditions where organized actin stress fibers were disrupted. The expression of constitutively active Rho-kinase also stimulated the transcriptional activity of c-fos serum response element. These results suggest that Rho-kinase has distinct roles in divergent pathways downstream of Rho, which include MLC phosphorylation leading to stress fiber formation, focal adhesion formation, and gene expression.
Footnotes
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↵* This study was supported by grants-in-aid for scientific research and for cancer research from the Ministry of Education, Science, and Culture of Japan (1996), by grants from the Mitsubishi Foundation and Kirin Brewery Company Limited, and by National Institutes of Health Grants HL37117, HL47530, and AR41653.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵¶ To whom correspondence should be addressed. Tel.: 81-7437-2-5440; Fax: 81-7437-2-5449; E-mail:kaibuchi{at}bs.aist-nara.ac.jp.
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↵1 The abbreviations used are: SRE, serum response element; SRF, serum response factor; MBS, myosin-binding subunit; MLC, myosin light chain; GST, glutathione S-transferase; CAT, catalytic domain; C3, C3 transferase; HA, hemagglutinin; tk, thymidine kinase; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; Tricine,N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; GTPγS, guanosine 5′-(3-O-thio)triphosphate; GFAP, glial fibrillary acidic protein; KD, kinase dead.
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↵2 Yano, T., Shibata, M., Tokui, T., Ichikawa, H., Ikebe, R., and Ikebe, M., unpublished data.
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- Received April 21, 1997.
- Revision received June 16, 1997.
