TNF-alpha-mediated neutrophil apoptosis involves Ly-GDI, a Rho GTPase regulator

We investigated intracellular signaling events involved in fibronectin-acce lerated TNF-alpha-mediated PMN apoptosis by means of 2-D gel electrophoresi s and western blotting. Proteins were sequenced with electrospray ionizatio n mass spectrometry. Apoptosis was quantitated by flow cytometry. We detect ed a cluster of acidic, high molecular- weight proteins that were only tyro sine phosphorylated when TNF-alpha-treated PMN interacted with fibronectin. Sequence analysis revealed that one of these proteins was Ly-GDI a regulat or of Rho GTPases. Fibronectin increased the TNF-alpha-induced Ly-GDI cleav age, 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 tyros ine phosphorylation prevented phosphorylation of Ly-GDI, fibronectin-accele rated Ly-GDI cleavage, and fibronectin-accelerated apoptosis hi TNF-alpha-t reated PMN. We found that Ly-GDI cleavage was dependent on caspase-3 activa tion and that caspase-3 inhibition decreased apoptosis. We conclude that ty rosine phosphorylation of Ly-GDI, follow ed by increased caspase-3-mediated Ly-GDI cleavage, is a signaling event associated with accelerated TNF-alph a-mediated apoptosis on fibronectin.