Activation of β1 Integrin Signaling Stimulates Tyrosine Phosphorylation of p190 RhoGAP and Membrane-protrusive Activities at Invadopodia

doi:10.1074/jbc.273.1.9

Activation of β1 Integrin Signaling Stimulates Tyrosine Phosphorylation of p190^RhoGAP and Membrane-protrusive Activities at Invadopodia*

From the ^‡Lombardi Cancer Center & Department of Cell Biology, Georgetown University Medical Center, Washington, D. C. 20007 and the ^¶Craniofacial Developmental Biology and Regeneration Branch, NIDR, National Institutes of Health, Bethesda, Maryland 20892

Abstract

The ligation of available α6β1 integrin in adherent LOX melanoma cells by laminin G peptides and integrin stimulatory antibodies induced cell invasiveness, independent of adhesion activity of integrins that were pre-bound to extracellular matrix (Nakahara, H., Nomizu, M., Akiyama, S. K., Yamada, Y., Yeh, Y., and Chen, W.-T. (1996) J. Biol. Chem. 271, 27221–27224). Here, we show that this induced invasion involves an increase in tyrosine phosphorylation of a 190-kDa GTPase-activating protein for Rho family members (p190^RhoGAP; p190) and membrane-protrusive activities at invadopodia. This tyrosine phosphorylation does not occur when the adherent cells are treated with non-activating antibody against β1 integrin, control laminin peptides, or tyrosine kinase inhibitors genistein and herbimycin A. Although p190 and F-actin co-distribute in all cell cortex extensions, tyrosine-phosphorylated proteins including p190 appear to associate with F-actin specifically in invadopodia. In addition, the localized matrix degradation and membrane-protrusive activities were blocked by treatment of LOX cells with tyrosine kinase inhibitors as well as microinjection of antibodies directed against p190 but not by non-perturbing antibodies or control buffers. We suggest that activation of the α6β1 integrin signaling regulates the tyrosine phosphorylation state of p190 which in turn connects downstream signaling pathways through Rho family GTPases to actin cytoskeleton in invadopodia, thus promoting membrane-protrusive and degradative activities necessary for cell invasion.

Footnotes

↵* This work was supported by United States Public Health Service Grants R01 CA-39077 and HL-33711 (to W.-T. C.) and CA-61273 (to S. C. M.) and in part by United States Public Health Service Grant 2P30-CA-51008 from the Lombardi Cancer Center Microscopy & Imaging Shared Resource.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.
↵§ Present address: The First Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Osaka University, Osaka, 565 Japan.
↵‖ To whom correspondence should be addressed: Lombardi Cancer Center & Department of Cell Biology, TRB E415 Georgetown University, 3970 Reservoir Rd. N.W., Washington, D. C. 20007. Tel.: 202-687-1769; Fax: 202-687-3300; E-mail: chenw{at}gunet.georgetown.edu.
↵1 The abbreviations used are: ECM, extracellular matrix; FAK, focal adhesion kinase; GAP, GTPase activating protein; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; SH2, Src homology 2 domains; YPP, tyrosine-phosphorylated proteins.
- Received October 17, 1997.
- Revision received November 11, 1997.
The American Society for Biochemistry and Molecular Biology, Inc.