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Published online 27 December 2000. doi:10.1083/jcb.151.7.1549
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© The Rockefeller University Press, 0021-9525/2000/12/1549/ $5.00
The Journal of Cell Biology, Volume 151, Number 7, December 25, 2000 1549-1560

Original Article

Activated R-Ras, Rac1, PI 3-Kinase and PKC{epsilon} Can Each Restore Cell Spreading Inhibited by Isolated Integrin ß1 Cytoplasmic Domains

Allison L. Berriera, Anthony M. Mastrangeloa, Julian Downwardb, Mark Ginsbergc, and Susan E. LaFlammea
a Center for Cell Biology and Cancer Research, Albany Medical College, Albany, New York 12208
b Imperial Cancer Research Fund, London WC2A 3PX, United Kingdom
c Department of Vascular Biology, The Scripps Research Institute, La Jolla, California 92037

Correspondence to: Susan E. LaFlamme, Center for Cell Biology & Cancer Research, Mail Code 165, Albany Medical College, 47 New Scotland Avenue, Albany, NY 12208. Tel:(518) 262-6256 Fax:(518) 262-5669 E-mail:laflams{at}mail.amc.edu.

Attachment of many cell types to extracellular matrix proteins triggers cell spreading, a process that strengthens cell adhesion and is a prerequisite for many adhesion-dependent processes including cell migration, survival, and proliferation. Cell spreading requires integrins with intact ß cytoplasmic domains, presumably to connect integrins with the actin cytoskeleton and to activate signaling pathways that promote cell spreading. Several signaling proteins are known to regulate cell spreading, including R-Ras, PI 3-kinase, PKC{epsilon} and Rac1; however, it is not known whether they do so through a mechanism involving integrin ß cytoplasmic domains. To study the mechanisms whereby cell spreading is regulated by integrin ß cytoplasmic domains, we inhibited cell spreading on collagen I or fibrinogen by expressing tac-ß1, a dominant-negative inhibitor of integrin function, and examined whether cell spreading could be restored by the coexpression of either V38R-Ras, p110{alpha}-CAAX, myr-PKC{epsilon}, or L61Rac1. Each of these activated signaling proteins was able to restore cell spreading as assayed by an increase in the area of cells expressing tac-ß1. R-Ras and Rac1 rescued cell spreading in a GTP-dependent manner, whereas PKC{epsilon} required an intact kinase domain. Importantly, each of these signaling proteins required intact ß cytoplasmic domains on the integrins mediating adhesion in order to restore cell spreading. In addition, the rescue of cell spreading by V38R-Ras was inhibited by LY294002, suggesting that PI 3-kinase activity is required for V38R-Ras to restore cell spreading. In contrast, L61Rac1 and myr-PKC{epsilon} each increased cell spreading independent of PI 3-kinase activity. Additionally, the dominant-negative mutant of Rac1, N17Rac1, abrogated cell spreading and inhibited the ability of p110{alpha}-CAAX and myr-PKC{epsilon} to increase cell spreading. These studies suggest that R-Ras, PI 3-kinase, Rac1 and PKC{epsilon} require the function of integrin ß cytoplasmic domains to regulate cell spreading and that Rac1 is downstream of PI 3-kinase and PKC{epsilon} in a pathway involving integrin ß cytoplasmic domain function in cell spreading.

Key Words: cell spreading, integrin, signaling, Rac1, R-Ras


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