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Molecular and Cellular Biology, August 2001, p. 5437-5446, Vol. 21, No. 16
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.16.5437-5446.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Inhibition of the Motility and Growth of B16F10 Mouse Melanoma Cells by Dominant Negative Mutants of Dok-1

Tetsuya Hosooka,1 Tetsuya Noguchi,1,* Hiroshi Nagai,2 Tatsuya Horikawa,2 Takashi Matozaki,3 Masamitsu Ichihashi,2 and Masato Kasuga1

Second Department of Internal Medicine1 and Department of Dermatology,2 Kobe University School of Medicine, Chuo-ku, Kobe 650-0017, and Biosignal Research Center, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512,3 Japan

Received 10 April 2001/Accepted 23 May 2001

Dok-1 (p62Dok) is a multiple-site docking protein that acts downstream of receptor and nonreceptor tyrosine kinases. Although it has been proposed to contribute to the control of cell growth and migration through association with the Ras GTPase-activating protein and the adapter protein Nck, the role of Dok-1 remains largely unknown. The functions of Dok-1 have now been investigated by the generation of two different COOH-terminal truncation mutants of this protein: one (DokPH+PTB) containing the pleckstrin homology and phosphotyrosine-binding domains, and the other (DokPH) composed only of the pleckstrin homology domain. Both of these mutant proteins were shown to act in a dominant negative manner. Overexpression of each of the mutants in highly metastatic B16F10 mouse melanoma cells thus both inhibited the tyrosine phosphorylation of endogenous Dok-1 induced by cell adhesion as well as reduced the association of the endogenous protein with cellular membranes and the cytoskeleton. Overexpression of DokPH+PTB in these cells also markedly reduced both the rates of cell spreading, migration, and growth as well as the extent of Ras activation. The effects of DokPH on these processes were less pronounced than were those of DokPH+PTB, indicating the importance of the phosphotyrosine-binding domain. These results suggest that at least in B16F10 cells, Dok-1 positively regulates not only cell spreading and migration but also cell growth and Ras activity.

* Corresponding author. Mailing address: Second Department of Internal Medicine, Kobe University School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. Phone: 81-78-382-5861. Fax: 81-78-382-2080. E-mail: noguchi{at}med.kobe-u.ac.jp.

Molecular and Cellular Biology, August 2001, p. 5437-5446, Vol. 21, No. 16
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.16.5437-5446.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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