We investigated intracellular signaling events involved in fibronectin-accelerated TNF-alpha-mediated PMN apoptosis by means of 2-D gel electrophoresis and western blotting. Proteins were sequenced with electrospray ionization mass spectrometry. Apoptosis was quantitated by flow cytometry. We detected a cluster of acidic, high molecular-weight proteins that were only tyrosine phosphorylated when TNF-alpha-treated PMN interacted with fibronectin. Sequence analysis revealed that one of these proteins was Ly-GDI, a regulator of Rho GTPases. Fibronectin increased the TNF-alpha-induced Ly-GDI cleavage, yielding a 23-kD fragment. At 8 h, intact Ly-GDI was decreased to 33% on fibronectin, compared with 69% on PolyHema (P<0.05). Inhibition of tyrosine phosphorylation prevented phosphorylation of Ly-GDI, fibronectin-accelerated Ly-GDI cleavage, and fibronectin-accelerated apoptosis in TNF-alpha-treated PMN. We found that Ly-GDI cleavage was dependent on caspase-3 activation and that caspase-3 inhibition decreased apoptosis. We conclude that tyrosine phosphorylation of Ly-GDI, followed by increased caspase-3-mediated Ly-GDI cleavage, is a signaling event associated with accelerated TNF-alpha-mediated apoptosis on fibronectin.