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© The Rockefeller University Press,
0021-9525/2000//1549 $5.00
The Journal of Cell Biology, Volume 151, Number 7,
, 2000 1549-1560
Original Article |
Activated R-Ras, Rac1, Pi 3-Kinase and Pkc
Can Each Restore Cell Spreading Inhibited by Isolated Integrin β1 Cytoplasmic Domains
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 β 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
and Rac1; however, it is not known whether they do so through a mechanism involving integrin β cytoplasmic domains. To study the mechanisms whereby cell spreading is regulated by integrin β cytoplasmic domains, we inhibited cell spreading on collagen I or fibrinogen by expressing tac-β1, a dominant-negative inhibitor of integrin function, and examined whether cell spreading could be restored by the coexpression of either V38R-Ras, p110
-CAAX, myr-PKC, 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-β1. R-Ras and Rac1 rescued cell spreading in a GTP-dependent manner, whereas PKC required an intact kinase domain. Importantly, each of these signaling proteins required intact β 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 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-CAAX and myr-PKC to increase cell spreading. These studies suggest that R-Ras, PI 3-kinase, Rac1 and PKC require the function of integrin β cytoplasmic domains to regulate cell spreading and that Rac1 is downstream of PI 3-kinase and PKC in a pathway involving integrin β cytoplasmic domain function in cell spreading.
Key Words: cell spreading • integrin • signaling • Rac1 • R-Ras
© 2000 The Rockefeller University Press
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