Activated R-Ras, Rac1, PI 3-kinase and PKC is an element of can each restore cell spreading inhibited by isolated integrin beta 1 cytoplasmic domains

Attachment of many cell types to extracellular matrix proteins triggers cel l 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 beta cytop lasmic domains, presumably to connect integrins with the actin cytoskeleton and to activate signaling pathways that promote cell spreading. Several si gnaling 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 beta cytoplasmic domains. To study the mechanisms whereby cell spreading is regulated by integrin beta cytopla smic domains, we inhibited cell spreading on collagen I or fibrinogen by ex pressing tac-beta1, a dominant-negative inhibitor of integrin function, and examined whether cell spreading could be restored by the coexpression of e ither V38R-Ras, p110 alpha -CAAX, myr-PKC epsilon, or L61Rac1. Each of thes e activated signaling proteins was able to restore cell spreading as assaye d by an increase in the area of cells expressing tac-beta1. R-Ras and Rac1 rescued cell spreading in a GTP-dependent manner, whereas PKC epsilon requi red an intact kinase domain. Importantly, each of these signaling proteins required intact beta cytoplasmic domains on the integrins mediating adhesio n in order to restore cell spreading. In addition, the rescue of cell sprea ding by V38R-Ras was inhibited by LY294002, suggesting that PI 3-kinase act ivity is required for V38R-Ras to restore cell spreading. In contrast, L61R ac1 and myr-PKC epsilon each increased cell spreading independent of PI 3-k inase activity. Additionally, the dominant-negative mutant of Rac1, N17Rac1 , abrogated cell spreading and inhibited the ability of p110 alpha -CAAX an d 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 bet a cytoplasmic domains to regulate cell spreading and that Rac1 is downstrea m of PI 3-kinase and PKC epsilon in a pathway involving integrin beta cytop lasmic domain function in cell spreading.