Activated Raf Induces the Hyperphosphorylation of Stathmin and the Reorganization of the Microtubule Network
1998; Elsevier BV; Volume: 273; Issue: 35 Linguagem: Inglês
10.1016/s0021-9258(18)48797-2
ISSN1083-351X
AutoresJosip Lovrić, Sascha Dammeier, Arnd Kieser, Harald Mischak, Walter Kölch,
Tópico(s)Microtubule and mitosis dynamics
ResumoRaf kinases are regulators of cellular proliferation, transformation, differentiation, and apoptosis. To identify downstream targets of Raf-1 in vivo, we used NIH 3T3 fibroblasts expressing a Raf-1 kinase domain-estrogen receptor fusion protein (BXB-ER), whose activity can be acutely regulated by estrogen. Proteins differentially phosphorylated 20 min after BXB-ER activation in living cells were displayed by two-dimensional electrophoresis. The protein with the most prominent newly induced phosphorylation was identified as stathmin, a phosphorylation-sensitive regulator of microtubule dynamics. Stathmin is rapidly phosphorylated on two ERK phosphorylation sites (serines 25 and 38) upon BXB-ER activation. The mitogen-activated protein kinase/extracellular signal-regulated kinase-kinase (MEK) inhibitor PD98059 abolished this phosphorylation, demonstrating that stathmin is targeted by BXB-ER via the MEK/ERK pathway. Prolonged BXB-ER activation resulted in the accumulation of a stathmin phosphoisomer with impaired microtubule-destabilizing activity. The appearance of this phosphoisomer after BXB-ER activation correlated with rearrangements in the microtubule network, resulting in the formation of long bundled microtubules extending toward the rim of the cells. Our results identify stathmin as a main target of the Raf/MEK/ERK kinase cascadein vivo and strongly suggest that ERK-mediated stathmin phosphorylation plays an important role for the microtubule reorganization induced by acute activation of Raf-1. Raf kinases are regulators of cellular proliferation, transformation, differentiation, and apoptosis. To identify downstream targets of Raf-1 in vivo, we used NIH 3T3 fibroblasts expressing a Raf-1 kinase domain-estrogen receptor fusion protein (BXB-ER), whose activity can be acutely regulated by estrogen. Proteins differentially phosphorylated 20 min after BXB-ER activation in living cells were displayed by two-dimensional electrophoresis. The protein with the most prominent newly induced phosphorylation was identified as stathmin, a phosphorylation-sensitive regulator of microtubule dynamics. Stathmin is rapidly phosphorylated on two ERK phosphorylation sites (serines 25 and 38) upon BXB-ER activation. The mitogen-activated protein kinase/extracellular signal-regulated kinase-kinase (MEK) inhibitor PD98059 abolished this phosphorylation, demonstrating that stathmin is targeted by BXB-ER via the MEK/ERK pathway. Prolonged BXB-ER activation resulted in the accumulation of a stathmin phosphoisomer with impaired microtubule-destabilizing activity. The appearance of this phosphoisomer after BXB-ER activation correlated with rearrangements in the microtubule network, resulting in the formation of long bundled microtubules extending toward the rim of the cells. Our results identify stathmin as a main target of the Raf/MEK/ERK kinase cascadein vivo and strongly suggest that ERK-mediated stathmin phosphorylation plays an important role for the microtubule reorganization induced by acute activation of Raf-1. 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 (1Heidecker G. Huleihel M. Cleveland J.L. Kolch W. Beck T.W. Lloyd P. Pawson T. Rapp U.R. Moll. Cell Biol. 1990; 10: 2503-2512Crossref PubMed Scopus (206) Google Scholar, 2Avruch J. Zhang X.F. Kyriakis J.M. Trends Biochem. Sci. 1994; 19: 279-283Abstract Full Text PDF PubMed Scopus (541) Google Scholar). Raf-1 crucially participates in the regulation of a number of biological processes including cellular transformation, proliferation, differentiation, cell cycle progression, and apoptosis (3Kolch W. Heidecker G. Lloyd P. Rapp U.R. Nature. 1991; 349: 426-428Crossref PubMed Scopus (354) Google Scholar, 4Samuels M.L. McMahon M. Mol. Cell. Biol. 1994; 14: 7855-7866Crossref PubMed Google Scholar, 5Pumiglia K.M. Decker S.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 448-452Crossref PubMed Scopus (328) Google Scholar, 6Sewing A. Wiseman B. Lloyd A.C. Land H. Mol. Cell. Biol. 1997; 17: 5588-5597Crossref PubMed Scopus (419) Google Scholar, 7Woods D. Parry D. Cherwinski H. Bosch E. Lees E. McMahon M. Mol. Cell. Biol. 1997; 17: 5598-5611Crossref PubMed Scopus (574) Google Scholar, 8Weissinger E.M. Eissner G. Grammer C. Fackler S. Haefner B. Yoon L.S. Lu K.S. Bazarov A. Sedivy J.M. Mischak H. Kolch W. Mol. Cell. Biol. 1997; 17: 3229-3241Crossref PubMed Scopus (59) Google Scholar). Activated Raf phosphorylates and activates MEK-1 1The abbreviations used are: MEKmitogen-activated protein kinase/extracellular signal-regulated kinase-kinaseERKextracellular signal regulated kinasePAGEpolyacrylamide gel electrophoresisHPLChigh performance liquid chromatographyMSmass spectrometryCAPS3-(cyclohexylamino)-1-propanesulfonic acidCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.1The abbreviations used are: MEKmitogen-activated protein kinase/extracellular signal-regulated kinase-kinaseERKextracellular signal regulated kinasePAGEpolyacrylamide gel electrophoresisHPLChigh performance liquid chromatographyMSmass spectrometryCAPS3-(cyclohexylamino)-1-propanesulfonic acidCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. 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 (9Robinson M.J. Cobb M.H. Curr. Opin. Cell Biol. 1997; 9: 180-186Crossref PubMed Scopus (2281) Google Scholar, 10Cobb M.H. Xu S. Hepler J.E. Hutchison M. Frost J. Robbins D.J. Cell. Mol. Biol. Res. 1994; 40: 253-256PubMed Google Scholar, 11Crews C.M. Alessandrini A. Erikson R.L. Cell Growth Differ. 1992; 3: 135-142PubMed Google Scholar). 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. mitogen-activated protein kinase/extracellular signal-regulated kinase-kinase extracellular signal regulated kinase polyacrylamide gel electrophoresis high performance liquid chromatography mass spectrometry 3-(cyclohexylamino)-1-propanesulfonic acid 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. mitogen-activated protein kinase/extracellular signal-regulated kinase-kinase extracellular signal regulated kinase polyacrylamide gel electrophoresis high performance liquid chromatography mass spectrometry 3-(cyclohexylamino)-1-propanesulfonic acid 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. 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 (12Brunet A. Pages G. Pouyssegur J. Oncogene. 1994; 9: 3379-3387PubMed Google Scholar, 13Cowley S. Paterson H. Kemp P. Marshall C.J. Cell. 1994; 77: 841-852Abstract Full Text PDF PubMed Scopus (1852) Google Scholar, 14Mansour S.J. Matten W.T. Hermann A.S. Candia J.M. Rong S. Fukasawa K. Vande Woude G.F. Ahn N.G. Science. 1994; 265: 966-970Crossref PubMed Scopus (1259) Google Scholar), whereas PD98059, a specific inhibitor of MEK activation (15Alessi D.R. Cuenda A. Cohen P. Dudley D.T. Saltiel A.R. J. Biol. Chem. 1995; 270: 27489-27494Abstract Full Text Full Text PDF PubMed Scopus (3252) Google Scholar, 16Dudley D.T. Pang L. Decker S.J. Bridges A.J. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7686-7689Crossref PubMed Scopus (2589) Google Scholar), or dominant negative ERK mutants can revert the transformed phenotype (17Kortenjann M. Thomae O. Shaw P.E. Mol. Cell. Biol. 1994; 14: 4815-4824Crossref PubMed Scopus (169) Google Scholar, 18Weyman C.M. Ramocki M.B. Taparowsky E.J. Wolfman A. Oncogene. 1997; 14: 697-704Crossref PubMed Scopus (44) Google Scholar). 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 (19Gallego C. Gupta S.K. Heasley L.E. Qian N.X. Johnson G.L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7355-7359Crossref PubMed Scopus (92) Google Scholar). 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 (20Kuo W.L. Abe M. Rhee J. Eves E.M. McCarthy S.A. Yan M. Templeton D.J. McMahon M. Rosner M.R. Mol. Cell. Biol. 1996; 16: 1458-1470Crossref PubMed Scopus (79) Google Scholar). 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 (9Robinson M.J. Cobb M.H. Curr. Opin. Cell Biol. 1997; 9: 180-186Crossref PubMed Scopus (2281) Google Scholar, 11Crews C.M. Alessandrini A. Erikson R.L. Cell Growth Differ. 1992; 3: 135-142PubMed Google Scholar) 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 vitroor 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. 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 (1Heidecker G. Huleihel M. Cleveland J.L. Kolch W. Beck T.W. Lloyd P. Pawson T. Rapp U.R. Moll. Cell Biol. 1990; 10: 2503-2512Crossref PubMed Scopus (206) Google Scholar). The BXB-ER expression vector was constructed following a similar strategy as described by Samuelset al. (21Samuels M.L. Weber M.J. Bishop J.M. McMahon M. Mol. Cell. Biol. 1993; 13: 6241-6252Crossref PubMed Scopus (322) Google Scholar). 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. 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 (22Hafner S. Adler H.S. Mischak H. Janosch P. Heidecker G. Wolfman A. Pippig S. Lohse M. Ueffing M. Kolch W. Mol. Cell. Biol. 1994; 14: 6696-6703Crossref PubMed Scopus (292) Google Scholar), adjusted to equal protein levels, and immunoprecipitated exactly as described previously (22Hafner S. Adler H.S. Mischak H. Janosch P. Heidecker G. Wolfman A. Pippig S. Lohse M. Ueffing M. Kolch W. Mol. Cell. Biol. 1994; 14: 6696-6703Crossref PubMed Scopus (292) Google Scholar) 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. (22Hafner S. Adler H.S. Mischak H. Janosch P. Heidecker G. Wolfman A. Pippig S. Lohse M. Ueffing M. Kolch W. Mol. Cell. Biol. 1994; 14: 6696-6703Crossref PubMed Scopus (292) Google Scholar) using as substrates 50 ng of recombinant histidine-tagged MEK-1 (His-MEK1) and 150 ng of kinase-inactive recombinant ERK1 (His-ERK) (23Gardner A.M. Vaillancourt R.R. Johnson G.L. J. Biol. Chem. 1993; 268: 17896-17901Abstract Full Text PDF PubMed Google Scholar) 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 (24Kolch W. Weissinger E. Mischak H. Troppmair J. Showalter S.D. Lloyd P. Heidecker G. Rapp U.R. Oncogene. 1990; 5: 713-720PubMed Google Scholar). Western blots were routinely checked for equal amounts of immunoprecipitated kinases with a c-Raf-specific antibody (22Hafner S. Adler H.S. Mischak H. Janosch P. Heidecker G. Wolfman A. Pippig S. Lohse M. Ueffing M. Kolch W. Mol. Cell. Biol. 1994; 14: 6696-6703Crossref PubMed Scopus (292) Google Scholar) 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. (24Kolch W. Weissinger E. Mischak H. Troppmair J. Showalter S.D. Lloyd P. Heidecker G. Rapp U.R. Oncogene. 1990; 5: 713-720PubMed Google Scholar) using 10 mm CAPS (Sigma), 10% methanol, adjusted to pH 11 as blotting buffer and the ECL detection system (Amersham Pharmacia Biotech). Approximately 4 × 106cells 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 was performed as described by Gorg et al. (25Gorg A. Postel W. Domscheit A. Gunther S. Electrophoresis. 1988; 9: 681-692Crossref PubMed Scopus (103) Google Scholar) with the modifications introduced by Bjellqvist and co-workers (26Bjellqvist B. Pasquali C. Ravier F. Sanchez J.C. Hochstrasser D. Electrophoresis. 1993; 14: 1357-1365Crossref PubMed Scopus (367) Google Scholar). Briefly, cells were trypsinized and washed twice with phosphate-buffered saline and once in 4 mmNa2HPO4, 0.75 mmKH2PO4, 70 mm NaCl, 1.5 mm KCl, before lysis in 100 μl of sample buffer (11m 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. ResolyteTM, 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% ResolyteTM, 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 (26Bjellqvist B. Pasquali C. Ravier F. Sanchez J.C. Hochstrasser D. Electrophoresis. 1993; 14: 1357-1365Crossref PubMed Scopus (367) Google Scholar). Apparent molecular weight and isoelectric point (pI) were determined by marker proteins (Bio-Rad, Sigma) separated on parallel processed gels. 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. Cells were seeded on Lab-Tak (Nunc) chamber slides 2 days prior to analysis. Indirect immunofluorescence was performed essentially as described (27Goodnight J.A. Mischak H. Kolch W. Mushinski J.F. J. Biol. Chem. 1995; 270: 9991-10001Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar) 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-β-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). The deletion of the regulatory domain of Raf-1 results in a constitutively activated kinase, BXB, with transforming properties (1Heidecker G. Huleihel M. Cleveland J.L. Kolch W. Beck T.W. Lloyd P. Pawson T. Rapp U.R. Moll. Cell Biol. 1990; 10: 2503-2512Crossref PubMed Scopus (206) Google Scholar, 28Bruder J.T. Heidecker G. Rapp U.R. Genes Dev. 1992; 6: 545-556Crossref PubMed Scopus (396) Google Scholar). The fusion of BXB to the hormone binding domain of the estrogen receptor renders the kinase activity hormone-dependent (21Samuels M.L. Weber M.J. Bishop J.M. McMahon M. Mol. Cell. Biol. 1993; 13: 6241-6252Crossref PubMed Scopus (322) Google Scholar). 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 (15Alessi D.R. Cuenda A. Cohen P. Dudley D.T. Saltiel A.R. J. Biol. Chem. 1995; 270: 27489-27494Abstract Full Text Full Text PDF PubMed Scopus (3252) Google Scholar, 16Dudley D.T. Pang L. Decker S.J. Bridges A.J. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7686-7689Crossref PubMed Scopus (2589) Google Scholar), 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 (6Sewing A. Wiseman B. Lloyd A.C. Land H. Mol. Cell. Biol. 1997; 17: 5588-5597Crossref PubMed Scopus (419) Google Scholar, 7Woods D. Parry D. Cherwinski H. Bosch E. Lees E. McMahon M. Mol. Cell. Biol. 1997; 17: 5598-5611Crossref PubMed Scopus (574) Google Scholar). 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). To detect targets of activated Raf, NIH 3T3 and 3T3BXB-ER cells were serum-starved overnight and metabolically labeled with [32P]orthophosphoric acid for 20 min prior to the addition of estrogen and harvested after an additional 20 min. Lysates were separated by two-dimensional electrophoresis, and phosphoprotein patterns were analyzed. From the nearly 2000 proteins detected by silver stain, more than 300 were phosphorylated to a readily detectable level within the short labeling period. Upon stimulation of BXB-ER only eight phosphoproteins showed reproducible increases in their intensities, ranging from 2- to 8-fold. In control experiments, additional assays were performed in which the MEK activation was inhibited by PD98059 prior to hormone addition. In these assays, only one phosphoprotein showed MEK-independent alterations after BXB-ER activation (data not shown). Hormone addition did not change the phosphoprotein patterns of NIH 3T3 control cells, demonstrating the specificity of the system. In this study, we will focus on the analysis of the Raf-regulated phosphoproteins RRPP2 and RRPP8, while the other BXB-ER targets are still under investigation. Fig. 2 shows the results from one representative out of six independent experiments for two of the BXB-ER-regulated phosphoproteins designated RRPP2 and RRPP8. The phosphorylation of RRPP2 was increased by a factor of 8 within 20 min of BXB-ER activation in both serum-starved and exponentially growing cells (Fig. 2 and data not shown). RRPP8 could not be detected in NIH 3T3 cells and was exclusively observed in estrogen-stimulated 3T3BXB-ER cells. Since PD98059 completely blocked the BXB-ER induced hyperphosphorylation of RRPP2 and RRPP8, we conclude that they are not phosphorylated by BXB-ER directly, but rather as a result of the activation of MEKs or ERKs (compare Fig. 1). RRPP2 showed the strongest increase in intensity of all proteins analyzed and was therefore chosen for further analysis. Protein staining indicated that the amount of RRPP2 was less than 0.001% of the total cellular protein, while the RRPP8 protein was below the detection limit (data not shown). 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 (29Nguyen D.N. Becker G.W. Riggin R.M. J. Chromatogr. A. 1995; 705: 21-45Crossref PubMed Scopus (98) Google Scholar). Since fragmentations preferentially proceed from either the N terminus (y-fragments) or the C terminus (b-fragments), they allow considerable sequence determination of peptides (30Siuzdak G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11290-11297Crossref PubMed Scopus (236) Google Scholar). Fig. 3shows 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. 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 (31Belmont L. Mitchison T. Deacon H.W. Trends Biochem. Sci. 1996; 21: 197-198Abstract Full Text PDF PubMed Scopus (61) Google Scholar, 32Beretta L. Dobransky T. Sobel A. J. Biol. Chem. 1993; 268: 20076-20084Abstract Full Text PDF PubMed Google Scholar). 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). To distinguish which of the possible in vivo phosphorylation sites on peptide 15–27 are actually phosphorylated, we analyzed the metastable fragment ions of the peptide. We could not find a single y-fragment ion mass that would correspond to the phosphorylation of serine 16 but found eight y-fragment ion masses that could only be generated when serine 25 was phosphorylated (TableI). These data clearly show that serine 25 and serine 38 of stathmin are phosphorylated in RRPP2.Table IIdentification of serine 25 as the phosphorylated residue in peptide 16–27Shown are all expected y-series fragments that allow the discrimination between the phosphorylation of either serine 16 or serine 25. Fragment ion masses actually found in the mass scans of the phosphorylated peptide 16–27 are in boldface type.* Found as (2H+) ion.** Found as y9 (−17) ion due to the loss of ammonia from the carboxy-terminal arginine (29Nguyen D.N. Becker G.W. Riggin R.M. J. Chromatogr. A. 1995; 705: 21-45Crossref PubMed Scopus (98) Google Scholar). Open table in a new tab Shown are all expected y-series fragments that allow the discrimination between the phosphorylation of either serine 16 or serine 25. Fragment ion masses actually found in the mass scans of the phosphorylated peptide 16–27 are in boldface type. * Found as (2H+) ion. ** Found as y9 (−17) ion due to the loss of ammonia from the carboxy-terminal arginine (29Nguyen D.N. Becker G.W. Riggin R.M. J. Chromatogr. A. 1995; 705: 21-45Crossref PubMed Scopus (98) Google Scholar). To confirm and extend our mass spectrometrical analysis of the stathmin phosphorylation, several experiments with a stathmin-specific antibody were performed. Two-dimensional gels of 3T3BXB-ER cells metabolically labeled with [35S]methionine/cysteine were blotted and probed with an anti-stathmin antibody (33Koppel J. Boutterin M.C. Doye V. Peyro-Saint-Paul H. Sobel A. J. Biol. Chem. 1990; 265: 3703-3707Abstract Full Text PDF PubMed Google Scholar). The signals from the anti-stathmin antibody were overlaid and carefully aligned with the autoradiograph of the same blot (Fig.5 A). Three forms of stathmin could be detected by the stathmin antibody and assigned to [35S]methionine/cysteine-labeled proteins. The observed migration pattern of stathmin in the two-dimensional gel is similar to the pattern described previously (34Luo X.N. Mookerjee B. Ferrari A. Mistry S. Atweh G.F. J. Biol. Chem. 1994; 269: 10312-10318Abstract Full Text PDF PubMed Google Scholar). It is characteristic for several protein forms distinguished by the extent of phosphorylation. The least phosphorylated form occupies the most basic position and displays the lowest apparent molecular weight. Each phosphorylation causes a shift to the acidic part of the gel and reduces the electrophoretic mobility in the SDS-PAGE, resulting in an increased apparent molecular weight. In addition, the Western blot was aligned with a gel also prepared from [35S]methio
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