INTRODUCTION

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Raf-1 is a critical component of an important transforming pathway emanating from oncogenic Ras, and its viral homologue, v-raf, is a potent oncogene on its own (1, 2). Raf-1 crucially participates in the regulation of a number of biological processes including cellular transformation, proliferation, differentiation, cell cycle progression, and apoptosis (3-8). Activated Raf phosphorylates and activates MEK-1¹ and MEK-2 (MAPK/ERK kinase), which in turn phosphorylate ERK1 and ERK2 (extracellular signal-regulated kinase). In many cells, this is sufficient to activate ERKs, which have a broad range of substrates, both in vitro and in vivo (9-11). Raf kinases and MEKs have a more restricted substrate specificity, with MEKs being the only unequivocally accepted substrates for Raf kinases and ERKs being the only known substrates for MEKs.

Several lines of evidence indicate that oncogenic forms of Raf mediate their biological effects primarily through the activation of MEK-1/2 and hence of ERK1/2. Activated MEK-1 can reproduce the transformation of 3T3 fibroblasts by activated Raf (12-14), whereas PD98059, a specific inhibitor of MEK activation (15, 16), or dominant negative ERK mutants can revert the transformed phenotype (17, 18). However, there is also evidence for different biological activities of the MEK/ERK and the Raf-1 signaling pathway. ERK activation does not invariably correlate with the transforming activity of Raf in all cell types (19). Activated Raf is able to induce differentiation of hippocampal H19-7 cells in the absence of pronounced activation of MEK and ERKs, while activated alleles of MEK and prolonged activation of ERKs fail to support differentiation (20).

Most of the experimental evidence for Raf-1 targeted signaling pathways stems from in vitro experiments and overexpression studies with activated or dominant negative mutants. These approaches are very powerful in defining the hierarchical order within signaling pathways and the interaction between individual components. However, since they focus on a single pathway, they are inherently biased and usually neither reveal the complexity of the cellular response nor allow a quantitative evaluation of this complexity. For instance, it is not clear which of the numerous ERK substrates (9, 11) are actually phosphorylated in response to Raf-1 activation and even less clear which participate in the biological response(s).

This prompted us to globally investigate Raf-1-induced phosphorylation(s) in intact cells to identify of the main in vivo targets. To activate Raf specifically, we generated cells harboring BXB-ER, a Raf-1 kinase domain-human estrogen receptor fusion protein, whose kinase activity is under the strict control of estrogen. Protein phosphorylations acutely induced in response to BXB-ER activation were analyzed by two-dimensional electrophoresis. The phosphorylated proteins were subsequently analyzed and identified by HPLC-coupled mass spectrometry. This approach circumvents artifacts such as relaxed substrate specificities of kinases in vitro or improper accessibility of the substrates. Since phosphorylations occur in intact cells, they represent physiological events likely to be involved in biological responses. This approach allowed us to detect several differentially phosphorylated proteins upon BXB-ER activation. Here, we describe the identification of stathmin as the most prominent immediate target of Raf activation in mouse fibroblasts. Stathmin destabilizes microtubules in living cells and its hyperphosphorylation is known to inhibit this destabilizing activity. BXB-ER activation immediately initiates the phosphorylation of stathmin on serine 25 and serine 38 via the activation of ERKs. This initial phosphorylation results in the appearance of stathmin forms phosphorylated to a higher stoichiometry, known to have an impaired microtubule-destabilizing activity. We further show that the appearance of hyperphosphorylated stathmin following BXB-ER activation correlates with the rearrangement of microtubular networks and the appearance of long bundled microtubules. These findings for the first time link activation of Raf kinase to specific changes in the cytoskeleton and identify stathmin as the responsible target.

	MATERIALS AND METHODS

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Cell Culture and Generation of Stable Lines-- Cells were grown in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum and 5% bovine serum in humidified atmosphere with 8% CO₂. BXB represents a Raf-1 mutant rendered transforming by deletion of amino acids 26-303 (1). The BXB-ER expression vector was constructed following a similar strategy as described by Samuels et al. (21). To facilitate detection of the BXB-ER fusion protein, an oligonucleotide encoding the epitope of the influenza virus hemagglutinin-specific 12CA5 antibody plus a stop codon was added to the 3'-end of the estrogen receptor portion. The construct was cloned into the pBABEpuro expression vector and transfected into NIH 3T3 cells (ATCC). Several of the more than 100 puromycin (Sigma)-resistant cell clones were tested for the expression of BXB-ER by immunoprecipitation with a Raf-1 antibody followed by Western blotting with the 12CA5 monoclonal antibody. Seven clones with equal expression levels were pooled to yield 3T3BXB-ER cells, which were cultured in the presence of 4 µg/ml puromycin.

Immunoprecipitation, Immunocomplex Kinase Assays, and Western Blotting-- Exponentially growing 3T3BXB-ER cells were stimulated with 5 µM estrogen with or without pretreatment for 30 min with 50 µM PD98059 (Life Technologies, Inc.) as indicated in the figure legend. For each time point, approximately 4 × 10⁶ cells were lysed exactly as described previously (22), adjusted to equal protein levels, and immunoprecipitated exactly as described previously (22) with the following antibodies: 12CA5 for BXB-ER, anti-MEK-1 sc436 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and a 1:1 mixture of anti-ERK1 sc436 and anti-ERK2 sc093 (Santa Cruz Biotechnology). Kinase assays were performed as described by Hafner et al. (22) using as substrates 50 ng of recombinant histidine-tagged MEK-1 (His-MEK1) and 150 ng of kinase-inactive recombinant ERK1 (His-ERK) (23) for BXB-ER kinase assays, 150 ng of His-ERK for MEK-1 kinase assays, and 2 µg of MBP (Life Technologies, Inc.) for ERK1/2 kinase assays. Kinase reactions were resolved by SDS-PAGE and Western blotted on polyvinylidene difluoride membranes (Millipore Corp.) as described (24). Western blots were routinely checked for equal amounts of immunoprecipitated kinases with a c-Raf-specific antibody (22) or with the antibodies used for immunoprecipitation prior to exposition on a phosphor imager (Fuji). Western blots were performed as described by Kolch et al. (24) using 10 mM CAPS (Sigma), 10% methanol, adjusted to pH 11 as blotting buffer and the ECL detection system (Amersham Pharmacia Biotech).

Metabolic Labeling-- Approximately 4 × 10⁶ cells in 10-cm culture dishes were washed twice in medium lacking either phosphate or methionine and cysteine and further incubated for 2 h in this medium. Cells were labeled with 250 µC/ml [³²P]orthophosphoric acid (Amersham Pharmacia Biotech) or [³⁵S]methionine/cysteine (ProMix, Amersham Pharmacia Biotech) for a total of 40 min (³²P label) or 3 h (³⁵S label). Where indicated in the figure legends, cells were treated during the last 30 min of the labeling period with 50 µM PD98059 or carrier (Me₂SO) and for the last 20 min with 5 µM estrogen or carrier (ethanol).

Two-dimensional Electrophoresis-- Two-dimensional electrophoresis was performed as described by Gorg et al. (25) with the modifications introduced by Bjellqvist and co-workers (26). Briefly, cells were trypsinized and washed twice with phosphate-buffered saline and once in 4 mM Na₂HPO₄, 0.75 mM KH₂PO₄, 70 mM NaCl, 1.5 mM KCl, before lysis in 100 µl of sample buffer (11 M urea, 4% CHAPS, 40 mM Tris, 1% dithioerythritol, 2.5 mM EDTA, 2.5 mM EGTA) per 10-µl cellular pellet, corresponding to approximately 2 × 10⁶ cells. For preparative purposes, 100 µl of sample buffer were used per 40 µl of cellular pellet, and urea was additionally added to a final concentration of 10 M. DNA was sheared using QIAshredders (QIAGEN) and removed by centrifugation at 100,000 × g for 50 min. Resolyte^TM, pH 4-8 (BDH), was added to the supernatant to a final concentration of 0.5%, and isoelectric focusing was performed using the Pharmacia dry strip kit (Amersham Pharmacia Biotech). 100-µl samples were applied to the acidic and basic part of Immobiline strips with a nonlinear gradient pH 3.5-10 (Amersham Pharmacia Biotech) using sample cup holders. Strips were reswollen in 10 M urea, 0.15% dithioerythritol, 2% CHAPS, 2.5 mM EDTA, EGTA, 1% Resolyte^TM, pH 4-8. A total of 100 kV-h was applied with several stepwise increases in voltage up to 3500 V. Second dimension was a standard SDS-PAGE using 12% gels (26). Apparent molecular weight and isoelectric point (pI) were determined by marker proteins (Bio-Rad, Sigma) separated on parallel processed gels.

Protein Analysis by HPLC-MS-- Proteins were cut out from the gels and digested with sequencing grade trypsin (Promega) as recommended by the manufacturer. Resulting peptides were dissolved in buffer A and separated on a 300-µM inner diameter/25-cm length µRPC C2/C18 column (LC PACKINGS) with a flow rate of 5 µl/min. The gradient was 0-50% B for 0-180 min, 50-100% B for 180-270 min (A: 1.5 mM ammonium acetate, 0.15% formic acid; B: 1.5 mM ammonium acetate, 0.15% formic acid, 70% acetonitril). The HPLC was coupled via an ion spray inlet to an API 100 quadrupole mass spectrometer (Perkin-Elmer). Masses were determined in 0.1 steps over the m/z (mass/charge) range from 400 to 1500 atomic mass units in the positive charge detection modus with an orifice voltage of 40 V.

Immunofluorescence-- Cells were seeded on Lab-Tak (Nunc) chamber slides 2 days prior to analysis. Indirect immunofluorescence was performed essentially as described (27) with some minor modifications. Cells were fixed in methanol at 20 °C. For blocking and all washes, phosphate-buffered saline containing 10% serum and 0.1% Triton X-100 was used. Cells were incubated with primary antibodies (anti-actin, anti--tubulin; Boehringer Mannheim) at a concentration of 1 µg/ml for 1 h at room temperature. Secondary antibody was used in a 1:50 dilution (fluorescein isothiocyanate-conjugated goat anti-mouse IgG; DAKO).

	RESULTS

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Analysis of Targets for Activated Raf-- The deletion of the regulatory domain of Raf-1 results in a constitutively activated kinase, BXB, with transforming properties (1, 28). The fusion of BXB to the hormone binding domain of the estrogen receptor renders the kinase activity hormone-dependent (21). Expression plasmids encoding such a fusion protein, termed BXB-ER, were stably introduced into NIH 3T3 cells. To avoid artifacts due to clonal variation, seven independent stable clones expressing equal levels of the fusion protein were pooled, and the resulting cells, 3T3BXB-ER, were used in all further experiments. These cells showed a robust induction of the BXB-ER kinase activity within minutes after the addition of estrogen, which was stable for several hours and slightly declined after 9 h but was still higher than in untreated proliferating cells (Fig. 1). The activity of MEK-1 and ERK1/2 followed the activity of BXB-ER throughout the time course. The rapid activation of MEK-1 and ERK1/2 could be reduced by adding the MEK inhibitor PD98059 (15, 16), whereas BXB-ER activity was not affected. BXB-ER activation resulted in the previously described effects such as morphological transformation (elongated shape, higher refractility) and block of the cell cycle in G₁ phase (6, 7). These effects were detectable within 6-9 h of hormone addition and fully established after 16-20 h (data not shown). The parental NIH 3T3 cells showed no detectable changes in any of the tested parameters in response to estrogen (data not shown).

View larger version (54K): [in this window] [in a new window]   Fig. 1.   Time course of BXB-ER, MEK-1, and ERK1/2 activation. Exponentially growing 3T3BXB-ER cells were stimulated with estrogen with or without prior addition of the MEK-1 inhibitor PD98059 and lysed at the indicated time points after hormone stimulation. Lysates were split in three equal aliquots, which were immunoprecipitated (IP) with the antibodies specific for the kinases indicated on the left. In vitro kinase assays with the appropriate substrates were performed as described under "Materials and Methods," with relevant substrates indicated on the right. Samples were resolved by SDS-PAGE and blotted. Shown are phosphor imager exposures of the blots from a representative of three independent experiments. MBP, myelin basic protein.

View larger version (33K): [in this window] [in a new window]   Fig. 2.   Detection of in vivo phosphorylated targets of BXB-ER. NIH 3T3 control cells and 3T3BXB-ER cells were serum-starved overnight, labeled with [32P]orthophosphoric acid for 20 min, and then treated with estrogen (+e.) or carrier for additional 20 min. Where indicated, PD98059 was administered 10 min before estrogen. Cell lysates were separated by two-dimensional electrophoresis (first dimension isoelectric focusing, anode on the right; second dimension SDS-PAGE). Gels were silver stained, dried and autoradiographed for 7 h. Shown are identical regions of representative autoradiographs from at least six independent experiments. The scheme shows the positions of reference proteins and Raf-regulated phosphoproteins RRPP2 (19 kDa/pI 5.4) and RRPP8 (19.5 kDa/pI 5.2). The positions of RRPP2 and RRPP8 are indicated by arrowheads.

Identification of RRPP2 as Stathmin-- To identify RRPP2, eight preparative two-dimensional gels were run from a total of 1 × 10⁸ serum-starved 3T3BXB-ER cells stimulated for 20 min with estrogen. A total of approximately 800 ng of Coomassie-stained RRPP2 was excised from the gels and digested with trypsin. The resulting peptides were separated by HPLC and injected on-line into an electrospray mass spectrometer to determine their exact masses. The peptide masses were used to search for matching peptide mass fingerprints in the European Bioinformatics Institute nonredundant protein data base using the PeptideSearch software. The search identified several stathmin sequences from different species, with Pr22 (mouse stathmin) being the best candidate. Mouse, rat, and human stathmins yielded 10-13 matching peptides, while the next best scores of other unrelated proteins were 5 matches (data not shown). The identification was verified by the analysis of metastable fragment ions. These derive from predictable fragmentations of the peptides during ionization and measurement (29). Since fragmentations preferentially proceed from either the N terminus (y-fragments) or the C terminus (b-fragments), they allow considerable sequence determination of peptides (30). Fig. 3 shows a representative metastable ion analysis of two such peptides. A series of observed masses could be exactly matched to the masses expected from the staggered y fragmentation of peptides corresponding to the stathmin sequence. Several other peptides were analyzed in the same way (data not shown) to prove unambiguously that RRPP2 is indeed stathmin.

View larger version (47K): [in this window] [in a new window]   Fig. 3.   Identification of RRPP2 as stathmin by HPLC-coupled ion spray MS. Tryptic peptides of RRPP2 were separated by HPLC, and their masses were determined by MS. RRPP2 was identified as stathmin by its peptide mass fingerprint as described under "Results." RRPP2 peptides matching to computed masses of tryptic stathmin peptides were further analyzed to verify the identification by deducing their primary sequence via the analysis of metastable fragment ions. The results of two selected peptides are shown. 10 single mass scans for each peptide were collectively analyzed. The predicted metastable fragment masses of the y-series peptides, expected from the computed stathmin sequence, were calculated by the BioTools software and compared with the actually measured masses. On the ordinate, masses are indicated as mass/charge ratios in atomic mass units for single charged peptides. Double charged peptides are marked with 2H+. Signal intensities are indicated on the abscissa in arbitrary instrument units.

Analysis of the Phosphorylation State of RRPP2/Stathmin-- Stathmin, also known as p19, Op18, prosolin, and oncoprotein 18, is a cytosolic phosphoprotein that can be phosphorylated on four different sites in vivo, namely Ser¹⁸, Ser²⁵, Ser³⁸, and Ser⁶³ (31, 32). Therefore, it was of interest to determine which sites are phosphorylated following BXB-ER activation. This was addressed by searching for phosphorylated tryptic peptides in the mass spectrum of RRPP2. We found the masses of the single phosphorylated tryptic peptides encompassing residues 15-27 and 29-40 containing the phosphorylation sites serine 18/25 and serine 38, respectively (Fig. 4). The phosphorylated peptides displayed the characteristic increase in mass due to the addition of a single phosphate group. No other phosphopeptides could be detected (data not shown).

View larger version (28K): [in this window] [in a new window]   Fig. 4.   Identification of RRPP2 as stathmin form with two phosphorylated peptides. The spectrum of the HPLC ion spray MS run of RRPP2 was searched for masses corresponding to unphosphorylated and phosphorylated tryptic stathmin peptides using BioMultiView software. The diagrams show masses corresponding to the 2H+ charged peptides from residues 15-27 (upper panel) and 29-40 (lower panel) derived from cumulatively analyzed mass scans. These were the only peptides from which phosphorylated forms could be detected. The elution time as well as the predicted sequence, phosphorylation state, and measured mass/charge ratio of each peptide is indicated. For clarity, only the 2H+ charged peptide masses are shown. Therefore, the typical increase of 80-Da observed in single charged phosphorylated peptides appears only as 40 Da in the 2H+ charged peptides.

View larger version (60K): [in this window] [in a new window]   Fig. 5.   Analysis of differentially phosphorylated stathmin forms by two-dimensional electrophoresis and Western blot. A, 3T3BXB-ER cells were metabolically labeled for 3 h with [35S]methionine/cysteine, lysed, and separated by two-dimensional electrophoresis. The gel was blotted and autoradiographed for 3 days (A, upper part). Subsequently, stathmin was visualized by immunostaining (A, middle part). Autoradiogram and Western blot were carefully aligned to correlate 35S and Western blot signals (A, lower part). B, cells were metabolically labeled for 3 h with [35S]methionine and stimulated with estrogen for the last 20 min. After lysis and two-dimensional separation, the gel was silver-stained, dried, and autoradiographed for 3 days. Stathmin forms were identified by aligning the autoradiograms of the blot shown in A and of the silver stained gel (B, upper part). The lower part of B shows the silver stain corresponding to the upper autoradiograph, with assignments of the stathmin forms derived from the Western blot and HPLC-MS analysis (RRPP2; compare Fig. 3). For better comparison, the stathmin N1 form is labeled by an asterisk.

Phosphorylation of Stathmin Is Further Increased at Later Time Points-- The combined experimental evidence suggests that in resting cells stathmin is mainly present in an unphosphorylated form N1 and to lesser extents in the single and double phosphorylated forms P1 and P2, respectively (Fig. 5). Activation of BXB-ER induces the rapid phosphorylation of stathmin by ERKs on serine 25 and serine 38. This results in a massive increase in the accumulation of RRPP2, the P2 form, within 20 min to a level severalfold higher than in unstimulated cells (Fig. 2).

View larger version (32K): [in this window] [in a new window]   Fig. 6.   Time course of stathmin hyperphosphorylation after BXB-ER activation. Exponentially growing NIH 3T3 control cells and 3T3BXB-ER cells were treated for the indicated times with estrogen. Lysates were separated by two-dimensional electrophoresis, blotted on polyvinylidene difluoride membranes, and probed with anti-stathmin antibody. As a further control, NIH 3T3 cells transformed by v-Raf from murine sarcoma virus 3611 were treated the same way. The nomenclature of the stathmin phosphoisomers according to Beretta and co-workers (32) is indicated.

Stathmin Hyperphosphorylation Correlates with Rearrangements of Microtubules-- Stathmin has recently been described as a regulator of microtubule dynamics, both in vitro and in vivo (36). Stathmin destabilizes microtubules and this destabilizing activity is reduced in vitro and in vivo by hyperphosphorylation of stathmin (37-41). Hyperphosphorylation of stathmin occurs in vivo prior to the onset of mitosis and is necessary to allow formation of stable microtubules to build up the mitotic spindle. However, rearrangement of the microtubular network also occurs in interphase cells in response to stimuli like serum, epidermal growth factor, insulin, and phorbol esters (42-45). Therefore, we analyzed whether the microtubules are also reorganized following BXB-ER activation.

View larger version (77K): [in this window] [in a new window]   Fig. 7.   Analysis of the rearrangement of microtubules after BXB-ER activation by immunofluorescence. Cells were grown on coverslips, treated as indicated, and stained by immunofluorescence with antibodies against tubulin or actin as described under "Materials and Methods." Pictures were taken at a 100-fold magnification. E, estrogen.

	DISCUSSION

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In the present study, we have identified stathmin as a prominent downstream target of the Raf/MEK/ERK pathway in living fibroblasts. Since the approach by which stathmin was identified was not biased and detected substrate phosphorylation in intact cells, stathmin is most likely a physiologically relevant downstream target of activated Raf.

Stathmin binds to heterodimeric tubulin and regulates microtubules in vitro and in vivo. Stathmin has been suggested to reduce the growth rate of microtubules (38, 46) or, alternatively, to enhance microtubule dynamics by increasing the frequency of catastrophes (i.e. the transition from growth to shrinkage) (36). There is consensus, however, that phosphorylation of stathmin inhibits its microtubule-destabilizing function. Whereas the overexpression of stathmin leads to the destabilization of microtubules in interphase cells, it does not affect the microtubules of mitotic spindles (39, 41). The loss of stathmin function correlates with the phosphorylation of serines 16, 25, 38, and 63 during mitosis (40, 41, 47). Overexpression of stathmin mutants, in which these phosphorylation sites were replaced by alanine in different combinations, results in mitotic arrest and endoreplication cycles, due to the lack of mitotic spindles. It is thus believed that the microtubule-destabilizing effect of endogenous stathmin must be suppressed by hyperphosphorylation in order to allow the formation of normal mitotic spindles and the undisturbed progression through mitosis (40, 41, 47, 48).

Stathmin phosphorylation is a hierarchical process requiring at least two different kinases (47). It can be phosphorylated by Cdc2 and ERKs on serines 25 and 38, by calcium/calmodulin-dependent kinase IV on serine 16 and by cAMP-dependent protein kinase on both serines 16 and 63 (34, 47, 49-53). The phosphorylation of serines 25 and 38 does not seem to affect stathmin function directly, but it seems to be a prerequisite for the phosphorylation of serines 16 and 63 by unknown kinases in mitotic cells (47). Phosphorylation of either serine 16 or serine 63 in combination with serines 25 and 38 inhibits stathmin activity (40). The phosphorylation of both serines 16 and 63 is sufficient to inhibit the microtubule-destabilizing activity in interphase or mitotic cells (37).

Our data suggest that in interphase fibroblasts, Raf-induced ERKs are the physiological kinases that phosphorylate stathmin on serines 25 and 38. Activation of Raf and hence ERKs results in an strong increase of the 2-fold phosphorylated stathmin form P2 (corresponding to spot RRPP2 in Fig. 2). Resembling the situation in mitotic cells, the P2 form is primed for further hyperphosphorylation, leading to the appearance of the multiply phosphorylated P3 form. Autoradiographically, P3 (corresponding to spot RRPP8 in Fig. 2) is already detectable 20 min after BXB-ER activation and further accumulates to become visible on Western blots between 3 and 9 h (Fig. 6). P3 is phosphorylated on serine 25/38 and on either serine 16 or serine 63 (32). As the kinases phosphorylating serine 16 or serine 63 in vivo remain to be identified, it is unclear whether they are also regulated by activated Raf. The accumulation of P3 after BXB-ER activation correlates with a rearrangement of the microtubular network (compare Figs. 6 and 7). Since stathmin acts stoichiometrically (38, 46), it is not surprising that a marked rearrangement of the microtubules only becomes visible when a clearly detectable fraction of stathmin is in the P3 form. The two candidate kinases for serine 16 or serine 63 phosphorylation, cAMP-dependent protein kinase (37) and calcium/calmodulin-dependent kinase IV (49), are unlikely to be responsible for the hyperphosphorylation of the P2 form in our cell system. Overexpression of calcium/calmodulin-dependent kinase IV or cAMP-dependent protein kinase results in an increase in microtubules without changes in the way they are organized (37, 49). In contrast, our data show that activated Raf induces the rearrangement of microtubules, resulting in the transformation of the tangled network to long bundled microtubules. This difference may not only arise from different stoichiometry and kinetics of stathmin phosphorylation, but also from different influences of each kinase on other regulators of microtubular organization, such as microtubule-associated proteins and microtubule-associated motor proteins (54, 55). Microtubule-associated proteins are good substrates for ERKs, and phosphorylation weakens their stabilizing effects on microtubules (56). Therefore, it is conceivable that ERKs activated by oncogenic Raf generate a specific phenotype of microtubule architecture.

Rearrangements of the microtubular network including a transient depolymerization are observed in response to various stimuli such as serum, epidermal growth factor, insulin, and phorbol esters, (42-45). It has been shown that transient microtubule depolymerization is not only necessary but may also be sufficient to initiate DNA synthesis in quiescent cells (57-59). In contrast, inhibition of microtubule depolymerization inhibits the mitogenic effects of thrombin and epidermal growth factor (59). Under our experimental conditions, BXB-ER induces the morphological transformation of the cells as well as a nearly complete block in the G₁ phase of the cell cycle (data not shown). This phenomenon was also observed by other investigators after strong activation of BXB-ER proteins and was attributed to the induction of the cyclin-dependent kinase inhibitor p21^waf/cip (5-7). In our cell system, robust stathmin hyperphosphorylation and the appearance of long bundled microtubules correlate with the cell cycle arrest. Therefore, it is tempting to speculate that the stabilization of microtubules contributes to the G₁ arrest induced by acutely activated Raf. This hypothesis is supported by the fact that NIH 3T3 cells transformed by stable overexpression of v-Raf derived from the murine sarcoma virus 3611 retrovirus show neither any significant alterations in their microtubular organization as compared with NIH 3T3 cells nor a growth arrest (data not shown). The involvement of microtubule stabilization in the Raf-induced growth arrest appears plausible considering that major ERK targets are linked with the microtubular compartment (56). ERKs reside within the microtubular network, and about half of the ERK activity induced by serum stimulation is associated with microtubules (60).

The regulation of microtubules by the Raf/MEK/ERK pathway also may have consequences for progression through mitosis. We and others have shown that endogenous Raf-1 as well as some transforming forms of Raf are activated during the G₂ and M phases of the cell cycle in a Ras-independent manner (61, 62). This could conceivably contribute to proper spindle formation due to stabilization of microtubules by stathmin hyperphosphorylation. Our preliminary results indicate that stathmin is indeed hyperphosphorylated within minutes upon BXB-ER activation in fibroblasts enriched at the S/G₂ border. Under these circumstances induction of the P3 form is stronger than in cycling or serum-starved cells (data not shown). This may indicate that the second kinase system responsible for phosphorylation of serine 16/63 is already active in later stages of the cell cycle (47).

In summary, our data identify stathmin as the protein whose phosphorylation exhibited the most pronounced increase in response to acute Raf activation. This suggests that stathmin is an important target of the Raf/MEK/ERK pathway and is fully concordant with an important role for stathmin phosphorylation in the reorganization of microtubules induced by activated Raf.