Tumor necrosis factor-induced microtubule stabilization mediated by hyperphosphorylated oncoprotein 18 promotes cell death.
Tumor necrosis factor (TNF)-induced cell death in the fibrosarcoma cell line L929 occurs independently of caspase activation and cytochrome c release. However, it is dependent on mitochondria and is characterized by increased production of reactive oxygen intermediates that are essential to the death process. To identify signaling molecules involved in this TNF-induced, reactive oxygen intermediate-dependent cell death pathway, we performed a comparative study by two-dimensional gel electrophoresis of phosphoproteins from a mitochondria-enriched fraction derived from TNF-treated and control cells. TNF induced rapid and persistent phosphorylation of the phosphorylation-responsive regulator of the microtubule (MT) dynamics, oncoprotein 18 (Op18). By using induced overexpression of wild type Op18 and phosphorylation site-deficient mutants S25A/S38A and S16A/S63A in L929 cells, we show that TNF-induced phosphorylation on each of the four Ser residues of Op18 promotes cell death and that Ser(16) and Ser(63) are the primary sites. This hyperphosphorylation of Op18 is known to completely turn off its MT-destabilizing activity. As a result, TNF treatment of L929 cells induced elongated and extremely tangled microtubules. These TNF-induced changes to the MT network were also observed in cells overexpressing wild type Op18 and, to a lesser extent, in cells overexpressing the S25A/S38A mutant. No changes in the MT network were observed upon TNF treatment of cells overexpressing the S16A/S63A mutant, and these cells were desensitized to TNF-induced cell death. These findings indicate that TNF-induced MT stabilization is mediated by hyperphosphorylation of Op18 and that this promotes cell death. The data suggest that Op18 and the MT network play a functional role in transduction of the cell death signal to the mitochondria.
Department of Molecular Biology, Ghent University and Flanders Interuniversity Institute for Biotechnology, K. L. Ledeganckstraat 35, B-9000 Ghent, Belgium. vancompernolle@dmb.rug.ac.be
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Vancompernolle
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Boonefaes
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Mann
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Fiers
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Grooten
D000818:Animals; D016923:Cell Death / Q000187:drug effects; D007093:Imidazoles / Q000494:pharmacology; D051379:Mice; D008868:Microtubule Proteins; D008870:Microtubules / Q000187:drug effects* / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000502:physiology; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011725:Pyridines / Q000494:pharmacology; D050777:Stathmin; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D048051:p38 Mitogen-Activated Protein Kinases
D007093:Imidazoles; D008868:Microtubule Proteins; D010750:Phosphoproteins; D011725:Pyridines; D050777:Stathmin; C493994:Stmn1 protein, mouse; D014409:Tumor Necrosis Factor-alpha; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; C093642:SB 203580