TNF-alpha-mediated neutrophil apoptosis involves Ly-GDI, a Rho GTPase regulator.
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.
Kettritz
R
R
Franz Volhard Clinic and Max Delbrück Center for Molecular Medicine, Medical Faculty of the Charité, Humboldt University of Berlin, Germany.
Xu
Y X
YX
Faass
B
B
Klein
J B
JB
Müller
E C
EC
Otto
A
A
Busjahn
A
A
Luft
F C
FC
Haller
H
H
D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology*; D002478:Cells, Cultured; D020730:Guanine Nucleotide Dissociation Inhibitors; D006801:Humans; D009504:Neutrophils / Q000473:pathology* / Q000502:physiology*; D011506:Proteins / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D025521:Tumor Suppressor Proteins; D020741:rho GTP-Binding Proteins / Q000502:physiology; D062949:rho Guanine Nucleotide Dissociation Inhibitor beta; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors
C569658:ARHGDIB protein, human; D020730:Guanine Nucleotide Dissociation Inhibitors; D011506:Proteins; D014409:Tumor Necrosis Factor-alpha; D025521:Tumor Suppressor Proteins; D062949:rho Guanine Nucleotide Dissociation Inhibitor beta; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; D020741:rho GTP-Binding Proteins