Activated R-Ras, Rac1, Pi 3-Kinase and Pkcε Can Each Restore Cell Spreading Inhibited by Isolated Integrin β1 Cytoplasmic Domains

Abstract

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, PKCε 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, p110α-CAAX, myr-PKCε, 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 PKCε 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-PKCε 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 p110α-CAAX and myr-PKCε to increase cell spreading. These studies suggest that R-Ras, PI 3-kinase, Rac1 and PKCε require the function of integrin β cytoplasmic domains to regulate cell spreading and that Rac1 is downstream of PI 3-kinase and PKCε in a pathway involving integrin β cytoplasmic domain function in cell spreading.