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 lin e 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 deat h process. To identify signaling molecules involved in this TNF-induced, re active oxygen intermediate-dependent cell death pathway, we performed a com parative study by two-dimensional gel electrophoresis of phosphoproteins fr om a mitochondria-enriched fraction derived from TNF-treated and control ce lls. TNF induced rapid and persistent phosphorylation of the phosphorylatio n-responsive regulator of the microtubule (MT) dynamics, oncoprotein 18 (Op 18). 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 promot es cell death and that Ser(16) and Ser(63) are the primary sites, This hype rphosphorylation of Op18 is known to completely turn off its NPT-destabiliz ing activity. As a result, TNF treatment of L929 cells induced elongated an d extremely tangled microtubules. These TNF-induced changes to the MT netwo rk were also observed in cells overexpressing wild type Op18 and, to a less er extent, in cells overexpressing the S25A/S38A mutant. No changes in the MT network were observed upon TNF treatment of cells overexpressing the S16 A/S63A mutant, and these cells were desensitized to TNF-induced cell death. These findings indicate that TNF-induced MT stabilization is mediated by h yperphosphorylation of Op18 and that this promotes cell death. The data sug gest that Op18 and the MT network play a functional role in transduction of the cell death signal to the mitochondria.