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 Ser16 and Ser63are 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.

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 TNFinduced 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.
Tumor necrosis factor (TNF) 1 is a pleiotropic cytokine, orig-inally described for its ability to cause hemorrhagic necrosis of certain tumors in vivo (1). In addition to its anti-tumor and anti-malignant cell effects, TNF has been reported to influence mitogenesis, differentiation, and immunoregulation of various cell types.
The activities of TNF are mediated through two cell surface receptors, namely TNF-R55 (CD120a) and TNF-R75 (CD120b), which are expressed by most cell types. TNF's effects are mediated primarily through TNF-R55. Upon activation of the receptor, adaptor proteins such as TRADD and TRAF are recruited and bind to the intracellular part of the clustered receptor (for a review, see Ref. 2). These receptor-associated molecules that initiate signaling events are largely specific to the TNF/nerve growth factor receptor family. However, the downstream signaling molecules are not unique to the TNF system but also mediate effects of other inducers. Downstream signaling molecules in the TNF system identified so far include caspases, phospholipases, the three mitogen-activated protein (MAP) kinases, and the NF-B activation cascade.
In the mouse fibrosarcoma cell line L929 used in this study, TNF induces cytotoxicity and also has gene-inducing activity, some of which is mediated through NF-B activation. It has been shown that these activities are uncoupled in L929 cells. Specifically, SB203580, an inhibitor of the p38 MAP kinase, which is involved in TNF-induced cytokine expression, completely inhibits TNF-induced synthesis of IL-6, while TNFinduced cytotoxicity is unaffected (3). Furthermore, stable overexpression of a transdominant negative form of IB-␣ results in the inability to activate NF-B and inducibly express interleukin-6 but has no effect on TNF-induced cell death (4).
TNF-induced cell death in L929 cells is characterized by a necrosis-like phenotype and does not involve DNA fragmentation (Ref. 5; for a review, see Ref. 6). It is independent of caspase activation and cytochrome c release but is dependent on mitochondria (7,8) and is accompanied by increased production of reactive oxygen intermediates in the mitochondria that are essential to the death process (9,10). Furthermore, the mitochondria translocate from a dispersed distribution to a perinuclear cluster (11); functional implications of this mitochondrial translocation remain unclear.
While much effort has been directed at the molecular mechanism of the caspase-dependent cell death pathway, relatively little is known about the TNF-induced reactive oxygen intermediate-dependent cell death pathway. To identify molecules involved in the latter, we performed a comparative study of the phosphoproteins from TNF-treated and control cells by twodimensional gel electrophoresis. It is known that upon activation of the TNF receptor, several kinases/phosphatases are activated (12,13). However, most of the changes in phosphorylation occur very rapidly (2-15 min) upon binding of TNF to its receptors, and most of them are transient and related to the gene-inductive activities of TNF.
To identify molecules that are involved in the cytotoxic process downstream of the receptor-proximal events, we studied lysates from cells that had been stimulated with TNF for 1.5 h.
To date, three proteins with reproducible and large increases in phosphorylation upon TNF treatment have been detected; in this study we examined one of those proteins, oncoprotein 18 (Op18, stathmin). Op18 is a conserved cytoplasmic phosphoprotein that is highly expressed in a wide variety of cancers, including a subset of leukemias and breast carcinomas (14 -16). Its high abundance seems to be necessary for the maintenance of the transformed phenotype (17). Op18 is a phosphorylationresponsive regulator of microtubule (MT) dynamics that increases the catastrophe rate of MTs (depolymerization or shrinkage phase of individual MTs) in a dose-dependent manner (18,19).
Op18 can be phosphorylated in vivo on four Ser residues (Ser 16 , Ser 25 , Ser 38 , and Ser 63 ) (20) and is a target for both cell cycle and cell surface receptor-coupled kinase systems (for a review, see Ref. 21). Phosphorylation on all four Ser residues completely turns off its MT-destabilizing activity, leading to increased levels of MT polymer and stabilized MTs in vivo (18,(22)(23)(24). This multisite phosphorylation occurs during mitosis and is essential for formation of the mitotic spindle and thus progression through the cell cycle. However, the actual role of Op18 in mitosis could be rather passive, and the only thing that may be required during mitosis is switching off its microtubuledestabilizing activity.
The high stoichiometry of Op18 phosphorylation by several receptor-regulated kinase systems suggests that the primary role for Op18 may be to regulate the MT system in response to external signals in interphase cells. In this paper, we describe for the first time a functional role for Op18 in TNF-induced cell death.

Reagents
Murine TNF was produced in Escherichia coli and purified to at least 99% homogeneity in our laboratory. It had a specific activity of 1.9 ϫ 10 8 IU/mg of protein (National Institute for Biological Standards and Control, Potters Bar, UK), contained 4 ng of endotoxin/mg of protein, and was used at 1000 IU/ml. Recombinant human interferon-␣, which is also active on murine cells, was a generous gift from Dr. C. Weissmann (University of Zü rich); it had a specific antiviral activity of 7.9 ϫ 10 7 units/mg, as determined on murine cells in a L929 cell/vesicular stomatitis virus assay and was used at 1000 units/ml. Propidium iodide (PI) and cycloheximide (CHX) (all from Sigma) were used at concentrations of 30 M and 50 g/ml, respectively. The polyclonal anti-Op18 antiserum was a generous gift from Dr. M. Gullberg (University of Umeå, Umeå, Sweden).

Plasmids
The cDNAs encoding WT Op18 and phosphorylation site-deficient mutants were generous gifts from Dr. M. Gullberg (University of Umeå). Plasmid pSP64Mx, containing the murine Mx promoter, was donated by Dr. C. Weissmann (25). Plasmid pSV2neo contains the bacterial neomycin gene used as a selection marker in transfection experiments (26).

Construction of Expression Plasmids
Inserts encoding the mutants and WT Op18 were isolated as XhoI/ SmaI fragments from the Bluescript pBS plasmid and cloned as blunted fragments into the blunted BamHI site of the pSP64Mx expression vector.

Measurement of TNF-induced Cell Death by Flow Cytometry
Cell death in L929 and L929-derived cell lines was induced by the addition of TNF (1000 IU/ml) to the cell suspension. Cell death was measured by quantifying PI-positive cells by FACS (FACSCalibur, Becton Dickinson, San Jose, CA). The PI dye was excited with an argon-ion laser at 488 nm; PI fluorescence was measured above 590 nm using a long pass filter. Routinely, 3,000 cells were analyzed. Cell death is expressed as the percentage of PI-positive cells in the total cell population.

Radiolabeling of Cells and Preparation of the Subcellular Protein Fractions
L929 cells were plated 48 h prior to the experiment. 32 P labeling was carried out as described in Ref. 13. TNF treatments (1000 IU/ml, 1.5 h) were done in the presence of CHX, to synchronize cell death. To simplify the two-dimensional phosphoprotein pattern and subsequent computerassisted analysis, we prepared two subcellular protein fractions. The cytosolic protein fraction, containing soluble cytoplasmic molecules and molecules derived from single-membrane organelles, was obtained as the supernatant from digitonin (0.03%)-permeabilized cells. After rinsing once with excess phosphate-buffered saline buffer, the remaining cell fraction was lysed in a CHAPS (2%)-containing buffer as described in Ref. 13. This lysate was then centrifuged (20,000 ϫ g), and the supernatant was used as the organelle fraction; it is enriched for mitochondrial and cytoskeleton-derived proteins. The three protein spots detected with increased phosphorylation upon TNF treatment were present in both subcellular fractions.

Two-dimensional Gel Electrophoresis
Isoelectric Focusing-Isoelectric focusing was carried out on 18-cm IPG strips, pH 4 -7 (Amersham Pharmacia Biotech), according to the manufacturer's instructions. Protein samples were precipitated with ethanol and redissolved in lysis buffer.
Western Blotting-Proteins were separated by SDS-polyacrylamide gel electrophoresis (14%) and transferred to a polyvinylidene difluoride membrane (Hybond-P, Amersham Pharmacia Biotech). The blots were incubated with the desired antibody, followed by ECL-based detection (Amersham Pharmacia Biotech).

Amino Acid Sequence Analysis by Nanoelectrospray Mass Spectrometry
Amino acid sequence analysis of peptides derived from an in situ digest of a Coomassie Blue-stained two-dimensional gel protein spot was performed as described previously (27).

Immunofluorescence Microscopy
L929 cells were grown on glass coverslips for 48 h. Following TNF treatment, cells were fixed with methanol at Ϫ20°C. MTs were stained with a rat anti-tubulin hybridoma YOL1/34 supernatant (Harlan Sera-Lab, Crawley Down, UK) and detected with a fluorescein isothiocyanate-conjugated goat anti-rat IgG antibody (Harlan Sera-Lab). Coverslips were mounted in VectaShield and analyzed using a Zeiss Axiophot microscope. Fig. 1A shows the autoradiogram of the two-dimensional gels from TNF-treated and control samples. The protein spot with increased phosphorylation identified as Op18 is indicated by the arrow. It was identified by nanoelectrospray mass spectrometry analysis of tryptic peptides derived from an in situ digest of the excised protein spot (data not shown). The identity of the spot was further confirmed by Western blotting using a polyclonal antibody against Op18 (Fig. 1B). As shown, TNF treatment increased the amount of the mono-and diphosphorylated forms of Op18, which correspond with the ␣1, ␣2, and ␣ 1 1 forms, according to the pattern described in Ref. 20 mobility on SDS gels, probably due to the close proximity of the two phosphoserine residues). Increased phosphorylation of Op18 was observed after 10 min of TNF treatment (data not shown). However, the increase in phosphorylation was more pronounced after 1.5 h of TNF treatment. These data indicate that TNF-induced phosphorylation of Op18 is an early but long lasting event.

TNF Induces Increased Phosphorylation of Op18 -
TNF-induced Phosphorylation of Op18 Is Not Affected by the p38 MAP Kinase Inhibitor SB203580 -As already mentioned TNF can induce cytotoxicity and has gene-inducing activity in L929 cells. These two activities are uncoupled, and only the gene-inducing pathway is sensitive to the p38 MAP kinase inhibitor SB203580 (3). To determine which pathway the TNFinduced phosphorylation of Op18 was part of, we examined whether TNF-induced phosphorylation of Op18 was inhibited by SB203580. None of the TNF-induced phosphoisoforms was affected by the inhibitor (data not shown), indicating that phosphorylation of Op18 is independent of gene activation.

TNF-induced Hyperphosphorylation of Op18 Promotes Cell
Death-To investigate the role of Op18 and its phosphorylation in TNF-induced cell death, we overexpressed WT Op18 and the phosphorylation site-deficient mutants Op18 S25A/S38A and Op18 S16A/S63A in L929 cells. We chose an inducible expression vector for two reasons. First, it has been shown that overexpression of phosphorylation site-deficient mutants of Op18 causes a block during mitosis (18). A second and more important reason is the large clonal variability in sensitivity to TNF-induced cell killing, ranging in L929 subclones from almost resistant to complete cell killing in 3 h. 2 Because of the latter, it is unwise to compare TNF sensitivity among different clones. However, use of an inducible expression vector circumvents this clonal variability, because TNF sensitivity can be compared in the same clone upon induced expression of the protein.
Murine fibrosarcoma L929 cells were transfected with pSP64MxOp18 WT and phosphorylation site-deficient mutants using the calcium phosphate procedure. In this expression vector, Op18 is under the control of the murine Mx promoter that is inducible by type I interferon (IFN). Although the murine Mx promotor has been reported to result in tightly controlled heterologous gene expression in VERO cells (28), we found quite leaky expression in our system.
After a preliminary screen of G418-resistant clones, clones were further selected according to the following criteria: low leak expression, strong induced expression, and little or no cell toxicity upon induced expression of the protein. As a result, we obtained five clones expressing WT Op18 and only two clones per mutant with a low level of leak expression that was well tolerated by the cell and strongly induced expression of the proteins.
For induction, cells were incubated with 1000 IU/ml human interferon-␣ for 16 h prior to TNF treatment. Levels of the ectopically expressed proteins in the Op18 clones in the noninduced and induced states and the endogenous levels of Op18 in the control clones (Neo) are shown in Fig. 2, A and B. Essentially, all Op18 clones had detectable leak expression but also a strong induced expression.
TNF-induced cell death in the different clones was measured as a function of time by flow cytometry, using PI uptake as the parameter for the number of dead cells (5). For practical reasons, the experiments were performed in two sets. In the first set, we measured the effect of induced expression of WT Op18 on TNF-induced cell death. In a second set of experiments, we measured the effect of induced expression of the phosphorylation site-deficient mutants on TNF-induced cell death in comparison with clones overexpressing WT Op18 and control clones. The experiments were repeated several times, and similar results were obtained each time. Representative experiments are shown in Fig. 2. C and D show the percentage of cell death after 5 h of TNF treatment of the different clones in noninduced and induced conditions. The same results are presented in E and F, respectively, as the percentage increase in cell death in induced over noninduced conditions. As Fig. 2 clearly indicates, induced overexpression of WT Op18 caused a significant increase (average of 40 -50%) in TNF-induced cell death compared with the same clones in the noninduced state. A slight increase (average of 6%) in cell death was also observed in control clones (Neo), due to human interferon-␣ treatment of the cells. An increase (average of 34%) in cell death was also observed in the clones overexpressing the Op18 S25A/ S38A mutant. On the contrary, no increase, or even a decrease (average of Ϫ2%), in cell death was observed in the clones 2 K. Vancompernolle, unpublished observations. FIG. 1. TNF-induced increase in phosphorylation of Op18. A, autoradiogram of the two-dimensional gels of an organelle fraction from TNF-treated (TNF ϩ CHX; 1.5 h) and control (CHX) cells. CHX was used to synchronize cell death. The protein spot that was identified by mass spectrometry as Op18 is indicated by an arrow and corresponds presumably to the ␣2 form in B. The phosphoprotein spot indicated by the arrowhead, which is also increased in TNF-treated cells, corresponds presumably to the ␣1 form in B. B, Western blotting with a polyclonal anti-Op18 antibody on an organelle fraction from TNFtreated and control L929 cells. Note a marked increase of the double phosphorylated forms ␣2 and ␣ 1 1 in the TNF-treated sample. Nomenclature of the phosphoforms is according to the pattern described in Ref. 20. The ␣0 and ␤0 forms are unphosphorylated Op18, and the ␣1 is monophosphorylated. The ␤0 isoform is derived from the same mRNA as the ␣0 form but has an as yet unknown post-translational modification. IEF, isoelectric focusing. overexpressing the Op18 S16A/S63A mutant. This decrease would be even more pronounced if the slight increase in cell death in the human interferon-␣-treated control clones were taken into consideration.
To verify whether there was a correlation between the phosphorylation status of the ectopically expressed proteins and the effect on TNF-induced cell death, we examined the two-dimensional gel pattern of the phosphoisoforms of WT Op18 and mutants in TNF-treated and control cells (Fig. 3). Based on the two-dimensional pattern of the phosphoisoforms described in Ref. 20, TNF induces phosphorylation on all four Ser residues (Ser 16 , Ser 25 , Ser 38 , and Ser 63 ) of WT Op18, with a marked increase in the ␣2 and ␣3 phosphoforms. In Fig. 3, the triple (␣ 1 2 ) and quadruple (␣ 2 2 ) phosphorylated forms are evident. The triple and quadruple phosphorylated forms were not detected in parental cells (Fig. 1B), probably because they were below the limit of detection. This hyperphosphorylation on all four Ser residues leads to maximum inactivation of the MT destabilizing activity of Op18 (23,22,29). The ␤0 isoform was also apparent upon TNF treatment; this isoform is derived from the same mRNA as the ␣0 form but has an as yet unknown posttranslational modification (30). In the clone overexpressing the Ser 25 /Ser 38 mutant, TNF still increased the amount of monoand diphosphorylated forms and thus represents phosphoryla-

FIG. 2. Effect of induced overexpression of WT Op18 and phosphorylation site-deficient mutants on TNF-induced cell death in L929 cells.
A and B, expression levels (exogenous plus endogenous) of WT Op18 and mutants in the selected clones in the noninduced (ni) and induced (i) condition. Note that basically all Op18 clones have some leak expression. Endogenous levels of Op18 are hardly detectable in the control (Neo) clones (the same amount of total protein was loaded as for the Op18 clones). C and D, percentage of cell death after 5 h of TNF treatment of the different clones in the noninduced (white bars) and induced condition (black bars). E and F, percentage increase in cell death of the induced condition over the noninduced condition for the same experiment as represented in C and D, respectively. tion on Ser 16 and Ser 63 . However, we cannot exclude the possibility that the increase is derived from the endogenous protein, since it has been shown that during mitosis dual phosphorylation of Ser 25 and Ser 38 is required for phosphorylation of Ser 16 and Ser 63 (23). However, this might be different in interphase cells. Strikingly, upon TNF treatment, no change in the phosphorylation pattern was observed in the clone overexpressing the S16A/S63A mutant.
Taken together, these results clearly indicate that TNFinduced hyperphosphorylation of Op18 promotes cell death, and phosphorylation on Ser 16 and Ser 63 seems to be a prerequisite for this. These data correlate well with the down-regulation of the MT-destabilizing activity of Op18 by phosphorylation described in the literature. Specifically, phosphorylation of either Ser 16 or Ser 63 in combination with Ser 25 and Ser 38 inhibits the MT-destabilizing activity of Op18 (23), while phosphorylation of both Ser 16 and 63 is sufficient to inhibit the MT destabilizing activity of Op18 in interphase and mitotic cells (24).
TNF-induced Microtubule Stabilization Is Mediated by Hyperphosphorylated Op18 -Given the function of Op18 as a regulator of MT dynamics, we tested whether TNF actually induced changes in the organization and polymerization status of the MTs in L929 cells. As shown by immunofluorescence microscopy in Fig. 4, parental L929 cells have a fine and not very dense network of MT filaments dispersed throughout the cell, excluding only the nucleus. After only 10 min of TNF treatment, at which point increased phosphorylation of Op18 was already detectable, the MT network had changed. Specifically, there was an overall increase in the immunofluorescence signal, showing a denser microtubule network. Even more pronounced changes in the organization of the MTs were evident after 1.5-3 h of TNF treatment (Fig. 4). A strong increase in the immunofluorescence signal was observed and a very dense and extremely tangled network of MTs was present. Additionally, the MTs were considerably more elongated and extended toward the rim of the cell. In addition, TNF-treated cells had a more spread phenotype (before they started to die), in comparison with control cells.
To investigate whether these TNF-induced changes in the MT network were mediated by phosphorylation of Op18, we examined the organization of the MT network in stable L929 clones that inducibly overexpressed WT Op18 and the phosphorylation site-deficient mutants Op18 S25A/S38A and Op18 S16A/S63A. Overexpression of WT Op18 and phosphorylation site-deficient mutants resulted in depolymerization of MTs, in agreement with previous reports (18,22). In Fig. 5 (left panels), it can be seen that there are few microtubules present in the untreated cells (control) and that they are not homogeneous in thickness. Furthermore, the overall fluorescence signal was weak, indicating low amounts of MT polymer. TNF treatment of cells overexpressing WT Op18 resulted in the appearance of a dense network of elongated MTs. Additionally, the MTs appeared thicker than those in TNF-treated parental cells (Fig.  4). A TNF-induced increase in MTs was also observed in cells overexpressing the Op18 S25A/S38A mutant, although to a lesser extent than in cells overexpressing the WT protein (Fig.  5). However, the MTs were not as thick as in the TNF-treated cells overexpressing WT Op18. On the contrary, no change in the MT network could be detected upon TNF treatment of cells overexpressing the Op18 S16A/S63A mutant. It is noteworthy that the degree of tangling of MTs observed after TNF treatment is less pronounced in cells overexpressing WT Op18 than in parental cells. This may be because untreated cells overexpressing WT Op18 have lower levels of MT polymer than parental cells. In this case, higher levels of phosphorylated Op18 would be required to cause the same morphological change in the MT network observed after TNF treatment of parental cells. It is also possible that the TNF-induced changes in the MT network may be caused not only by phosphorylated Op18 but also by other regulators of MT dynamics.
Taking the data together, we draw the conclusion that TNF induces the stabilization of MTs, mediated by hyperphosphorylation of Op18. This MT stabilization is responsible for promotion of TNF-induced cell death. DISCUSSION In this study, we have identified Op18 as a protein that becomes rapidly and persistently phosphorylated upon TNF treatment of the fibrosarcoma cell line L929. TNF-induced cell death in these cells is independent of caspase activation and cytochrome c release but is dependent on mitochondria (7,8) and is accompanied by increased production of reactive oxygen ). TNF also induced an increase of the mono-and diphosphorylated forms in cells overexpressing the Op18 S25A/S38A mutant. However, no change in phosphorylation status could be detected upon TNF treatment of cells overexpressing the Op18 S16A/S63A mutant. intermediates in the mitochondria (9,10). TNF treatment of the L929 cells also resulted in morphological changes in the MT network. A dense network of more elongated and extremely tangled MTs appeared and extended toward the rim of the cell.
The role of Op18 and its phosphorylation in TNF-induced cell death was further investigated by induced overexpression of the WT protein and phosphorylation site-deficient mutants Op18 S25A/S38A and Op18 S16A/S63A in L929 cells. Induced overexpression of WT Op18 significantly increased TNF-induced cell death. TNF treatment of these cells induced hyperphosphorylation of Op18 (on all four Ser residues), which is known to turn off its MT-destabilizing activity. Accordingly, TNF treatment also resulted in more elongated MTs and increased levels of MT polymer. The effect of induced overexpression of the phosphorylation site-deficient mutant Op18 S25A/ S38A on TNF-mediated cell death was not significantly different from that of WT Op18. TNF still induced phosphorylation in this mutant, with an increase of the mono-and diphosphorylated forms. Furthermore, increased levels of MT polymer were observed upon TNF treatment of the cells, but to a lesser extent than cells overexpressing WT Op18. On the contrary, induced overexpression of the phosphorylation site-deficient mutant Op18 S16A/S63A significantly decreased, but did not block, TNF-induced cell death. No increase in phosphorylation in this mutant was observed, nor could we detect any change in the MT network after TNF treatment of the cells. These results indicate that promotion of TNF-induced cell death by phosphorylated Op18 is directly correlated with increased levels and stabilization of the MTs.
These data correlate well with previous reports in the literature. It has been shown that phosphorylation of either Ser 16 or Ser 63 in combination with Ser 25 and Ser 38 inhibits the activity of Op18 (23) and that dual phosphorylation on Ser 16 and Ser 63 by protein kinase A is necessary and sufficient to switch off the MT-destabilizing activity of Op18 in intact cells (24). Phosphorylation on Ser 25 and Ser 38 are of only minor importance in regulating the activity of Op18 (23); this may explain why overexpression of the Op18 S25A/S38A mutant still has a cell death-promoting role, since its MT-destabilizing activity still can be turned off.
On the contrary, overexpression of the Op18 S16A/S63A mutant does not promote and may even decrease TNF-induced cell death, because its MT-destabilizing activity cannot be turned off. There are several possible explanations as to why TNF-induced cell death is not completely blocked by the latter. First, endogenous WT Op18 is still present, and thus the mutant protein has to compete with the WT protein. Second, TNF can also modulate the activity of other MT regulatory proteins that may all contribute to cell death. Furthermore, the MT system is not the only event involved in TNF-induced cell death in L929 cells. 3 This suggests that TNF-induced cell death is the result of a concerted series of events.
In summary, we conclude that TNF-induced hyperphosphorylation of Op18 promotes cell death and that phosphorylation on Ser 16 and Ser 63 is necessary for this. Furthermore, the TNF-induced changes in the MT network (MT stabilization) can be attributed to the TNF-induced phosphorylation of Op18, and MT stabilization is in fact responsible for the TNF-induced cell death-promoting activity of phosphorylated Op18.
Analysis of the phosphorylation status of the ectopically expressed proteins after TNF treatment also reveals that the hierarchical order of Op18 phosphorylation is different from that in mitosis. During mitosis, dual phosphorylation on Ser 25 and Ser 38 is required for phosphorylation on Ser 16 and Ser 63 (23). However, in TNF-treated cells, no change in phosphorylation of the Op18 S16A/S63A mutant could be detected, while there was an increase in the mono-and diphosphorylated forms of the Op18 S25A/S38A mutant. Thus, during TNF-induced cell death, phosphorylation on Ser 16 and/or Ser 63 is required before phosphorylation on Ser 25 and/or Ser 38 can occur. Therefore, the hierarchical order of Op18 phosphorylation might determine a differential way of regulating MT-destabilizing activity of Op18 during cell proliferation and cell death.
The TNF-induced multisite phosphorylation of Op18 also indicates that several kinases become activated. It is tempting to speculate that protein kinase A is one of the likely candidates, since it has been shown that protein kinase A is activated by TNF (31), and that induced expression of the catalytic subunit of protein kinase A can switch off the activity of Op18 in vivo by phosphorylation on Ser 16 and Ser 63 (24). However, several other kinases can also phosphorylate Op18 (21,32), and the kinases involved in TNF-triggered phosphorylation of Op18 remain to be determined.
Op18 and the MT network seem not only to play a role in TNF-induced cell death, but it has been reported that activation of p53 in cells also appears to target MT regulatory proteins. Notably, Op18 and MAP4 are down-regulated following p53 induction in, among others, MCF-7 cells (33). Furthermore, it has been shown that restoration of p53 function in these cells sensitizes cells that are TNF-resistant (because of the presence of mutated p53) to the cytotoxic action of TNF (34). Downregulation of Op18 would also result in MT stabilization, since it has been shown that microinjection of neutralizing anti-Op18 antibodies into newt long cells results in an increase of MT polymer and an associated decrease in catastrophe frequency 3 K. Vancompernolle, unpublished data.
FIG. 5. TNF-induced microtubule stabilization is mediated by phosphorylated Op18. The MT network was analyzed by immunofluorescence microscopy in TNF-treated (3 h) and control cells after induced overexpression of WT Op18 and the phosphorylation site-deficient mutants, Op18 S25A/S38A and Op18 S16A/S63A. TNF induced a strong increase in MT polymer in cells overexpressing WT Op18, and the MTs were long and thick. An increase in MT polymer after TNF treatment was also observed in cells overexpressing the Op18 S25A/ S38A mutant but to a lesser extent than WT Op18. On the contrary, no change in the MT network could be detected after TNF treatment of cells overexpressing the Op18 S16A/S63A mutant. (35). This may mean that Op18 is not only targeted by TNF via the kinase pathway but also transcriptionally, via p53.
The question of whether Op18 phosphorylation and MT stabilization directly or indirectly contribute to the TNF-induced mitochondria-and reactive oxygen intermediate-dependent cell death pathway remains unresolved. Currently, more and more data are emerging that indicate that microtubules play a role in signal transduction (reviewed in Ref. 36). MTs, together with MAPs and/or motor proteins, could contribute to the transmission of signals from the cell membrane to downstream targets in the cytoplasm. Three models have been proposed for this: 1) MT sequestering and delivery, 2) MT delivery, in which motor proteins are involved, and 3) MT scaffolding of signaling molecules (36). Thus, it could be that the role of Op18 in TNF signaling to cell death is indirect and that the only function of phosphorylated Op18 is to stabilize the MTs. This would mean that a primary signaling cascade is initiated starting from the TNF receptor and leading to MT stabilization that in turn may contribute to and promote the transmission of the death signal to the mitochondria. This primary signaling cascade presumably involves not only Op18; TNF might also modulate the activity of other MT regulatory molecules. The exact mechanism of signal transfer from the MTs to the mitochondria remains to be determined.
However, a more direct role of Op18 in the signal transfer to the mitochondria is not excluded. It is interesting to note that Op18 is differentially expressed in the brain of mitochondrial Mn-superoxide dismutase-deficient mice (37). This Mn-superoxide dismutase deficiency causes oxidative stress and mitochondrial dysfunction, two phenomena also occurring in TNFinduced cell death in L929 cells. As a result, it could be that Op18, in addition to regulating MT dynamics, has an additional, as yet unidentified cellular function.