Suppression of cell spreading by v-Crk requires Ras-MEK-MAP kinase signaling

We investigated the attachment and spreading of v-Crk-transformed cells, v-Crk3Y1, on fibronectin. Transfor-mation by v-Crk virtually suppressed the spreading, butnot the attachment, of cells on fibronectin. Thissuppression of cell spreading was not correlated withthe suppression of integrin a5 and b1 expression.However, the spreading of v-Crk3Y1 on fibronectin wasdramatically restored by either expression of dominant-negative Ras or treatment with manumycin A, a Rasfarnesyltransferase inhibitor. Moreover, both expressionof dominant-negative MEK1 and treatment of cells withU0126, a MEK1 inhibitor, restored the cell spreading ofv-Crk3Y1. In contrast, neither treatment withLY294002, a PI3K inhibitor, nor expression ofdominant-negative C3G showed no e•ect on cellspreading on fibronectin. Taken together, our resultssuggest that, among multiple signaling pathways acti-vated by v-Crk, the Ras-MEK1-MAP kinase cascadeplays a pivotal role in the suppression of cell spreadingon fibronectin, but C3G and the PI3 kinase do not.Oncogene (2001) 20, 5908–5912.Keywords: v-Crk; Ras; MAP kinase; cell spreading;integrinOne striking aspect of the phenotype of transformedcells is reduced adhesion to solid substrates. Manytransformed cells fail to spread as extensively as dotheir normal counterparts and grow independent ofsubstrate contact (Plantefaber and Hynes, 1989). Sincethis phenomenon appears to reflect the tumorigenicpotential of cells, extensive studies have been made. Inearlier studies, abnormalities in fibronectin (FN), amajor component of the extracellular matrix, and itsreceptor, integrin a5b1, have been identified. Whilemany of the transformed cells have reduced amountsof FN (Olden and Yamada, 1977; Adams et al., 1977),cells transformed with src or ras have dramaticallyreduced levels of integrin a5b1 and hence a diminishedability to adhere to FN (Plantefaber and Hynes,1989).Integrins are heteromeric transmembrane cell adhe-sion receptors comprising a-andb-subunits (Hynes,1992). From 16 di•erent a- and eight b-subunits, atleast 20 di•erent heterodimers are produced, givingthe variety of integrin function. The discovery ofintegrin as a signaling molecule (Hynes, 1992) lead tothe study of cell spreading as a bi-directionalsignaling event, ‘outside to in’ and ‘inside to out’.While ligand binding and integrin aggregation aresu†cient to recruit signaling molecules to the integrinclustered sites (Miyamoto et al., 1995), variousintracellular signaling can control focal adhesionformation (Clark and Brugge, 1995). For example,v-Src induces tyrosine phosphorylation of cellularproteins including FAK and p130