From the Medical Research Council Laboratory
for Molecular Cell Biology, Cancer Research Campaign Oncogene
and Signal Transduction Group, and the
Department of Biochemistry, University
College London, Gower Street, London WC1E 6BT, United Kingdom, the
¶ Department of Biochemistry and Medical Protein Research,
Flanders Interuniversity Institute for Biotechnology, Ghent University,
B-900 Belgium, and ** INSERM U440, IFM, 17 rue du Fer
à Moulin, 75005 Paris, France
Received for publication, September 12, 2000, and in revised form, October 27, 2000
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ABSTRACT |
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We have identified a rapid protein
phosphorylation event at residue serine 16 of stathmin using
two-dimensional gel electrophoresis coupled to matrix-assisted laser
desorption/ionization mass spectrometry in combination with post-source
decay analysis, which is induced by the epidermal growth factor
receptor. Phosphorylation is specifically mediated by the small
GTPases Rac and Cdc42 and their common downstream target, the
serine/threonine kinase p65PAK. Both GTPases have previously
been shown to regulate the dynamics of actin polymerization. Because
stathmin destabilizes microtubules, and this process is inhibited by
phosphorylation at residue 16, Rac and Cdc42 can potentially regulate
both F-actin and microtubule dynamics.
Members of the Rho GTPase family, Rho, Rac, and Cdc42, control the
assembly of filamentous actin structures in all mammalian cells (1).
Their ability to link extracellular signals to the reorganization of
the actin cytoskeleton suggests that they are likely to be important
regulators of actin-driven cell processes, and Rac, for example, is
crucial for growth cone guidance and cell migration both in tissue
culture cells and in vivo in Drosophila and
Caenorhabditis elegans (2-4). We report here that
activation of Rac and to a lesser extent Cdc42 by
EGF1 leads to the
phosphorylation of stathmin at residue 16. Phosphorylation at this site
has been shown to inhibit stathmin-induced destabilization of
microtubules, and our results suggest, therefore, that Rac and Cdc42
can regulate the dynamics of both the actin and the microtubule
cytoskeletons (5, 6).
Reagents, Antibodies, and Plasmids--
Human recombinant EGF
was from Collaborative Biomedical Products. Toxins B1470 and B10463
were gifts from C. von Eichel-Streiber (Inst. for Microbial Medicine,
Mainz, Germany). CNF-1 was a gift from G. Schmidt (University of
Freiburg, Germany). Polyclonal antisera directed against stathmin and
stathmin that has been phosphorylated at serines 16, 25, or 38 were
described previously (7). Monoclonal phosphospecific anti-ERK1,2
antibody was from Sigma. The PAK1-(83-149) autoinhibitory fragment
cloned into pRK5Myc was derived from murine PAK1 (8). All other
expression plasmids encoded Myc-tagged proteins and have been described
previously (7, 9).
Cell Culture and Transfections--
HEp-2 cells were cultured in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum. 1 × 105 cells per well were seeded into
12-well dishes and serum-starved 24 h later. For preparative
purposes, cells were seeded at 1 × 106 cells per
10-cm dish and were serum-starved 2 days later for another 20 h
prior to lysis. For [32P] or
[33P]orthophosphate labeling, cells were incubated in
phosphate-free medium in the presence of 12.5 µ Ci/ml
32Pi for preparative or 25 µ Ci/ml
32Pi or 100 µ Ci/ml 33Pi
for analytical purposes for 3 h prior to lysis. For transfection experiments, HEp-2 cells were seeded at 2 × 105 per
well into 6-well dishes 20 h before transfection. Cells were incubated for 4 h in 1.0 ml of serum-free medium containing 6 µl
of LipofectAMINE (Life Technologies, Inc.) and 1.5 to 1.7 µg of total
DNA per well. The transfection mixture was supplemented with 1 ml of
medium containing 20% fetal bovine serum, and cells were lysed 20 h later.
Cell Lysis, Gel Electrophoresis, and Immunoblotting--
Cell
lysis was performed in 50 mM Tris, pH 7.5, 100 mM dithiothreitol, 0.3% SDS, 5 mM sodium
pyrophosphate, 150 units/ml benzonase plus additives (1 mM
EDTA, 2 mM MgCl2, 1 mM sodium
fluoride, 1 mM orthovanadate, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride). For
immunoprecipitations, lysis buffer contained 50 mM Hepes pH
7.5, 150 mM NaCl, 0.4% Triton X-100, 10 mM
sodium pyrophosphate, 10% glycerol plus additives. Both buffers
solubilized stathmin to the same extent. For two-dimensional gel
electrophoresis, isoelectric focusing was performed using linear 11-cm
immobilized pH 4-7 gradient drystrips on an IPGphor (Amersham
Pharmacia Biotech). For preparative purposes, the final focusing step
was at 8000 V for 4 h.
Protein Purification--
Fourteen 10-cm dishes of serum-starved
and 32Pi-labeled HEp-2 cells were stimulated
with 100 ng/ml EGF for 7 min and lysed (350 µl per dish). Lysates
were precleared, supplemented with 80 µl of 20% SDS and 120 µl of
87% glycerol per ml, and the sample containing about 30 mg of total
protein was loaded on the Model 491 Prep Cell (Bio-Rad). Preparative
gel electrophoresis was performed utilizing a 11% separating gel.
Aliquots containing proteins in the 19,000- to 23,000-dalton
range were precipitated and analyzed by two-dimensional gel
electrophoresis and autoradiography of silver-stained gels (10). The
21,000-dalton phosphoprotein of interest, which was found in two
fractions, was dried to one-tenth of its original volume, precipitated,
and subjected to preparative two-dimensional gel electrophoresis.
MALDI-MS Analysis--
The protein spot of interest was
visualized by Coomassie staining and excised, and the gel fragment was
washed in water. The gel piece was allowed to shrink in 100 µl of
acetonitrile/water (1:1) (both Baker HPLC Analyzed, Mallinckrodt Baker
B.V., Deventer, The Netherlands) for 30 min and dried in a centrifugal
vacuum concentrator. 10 µl of 50 mM ammonium bicarbonate
buffer containing 0.05 µg of sequencing grade modified trypsin
(Promega) was added followed by ammonium bicarbonate buffer to submerge
the gel piece. Digestion proceeded overnight at 37 °C and was
stopped by acidification. The supernatant was removed from the gel
pieces and a fraction (10%) was purified/concentrated on added Poros®
50 R2 beads (Roche Molecular Biochemicals GmbH, Mannheim, Germany) and
used for direct MALDI-MS peptide mass fingerprint analysis (11). The
remainder of the digestion mixture was separated on a RP-HPLC column;
eluting peptides were automatically collected on Poros R2 beads and
used for MALDI-PSD measurements (12). Measurements were performed on a
Bruker Reflex III MALDI-TOF-MS (Bruker Daltonik GmbH, Bremen, Germany)
operating in the reflectron mode. RP-HPLC fractions were first scanned
in the reflectron mode, and candidate peptides were selected and their
PSD spectra recorded (12). The information present in the PSD spectra
was used by the SEQUEST algorithm (13) to identify the protein in a
public nonredundant protein database.
Rac interacts with numerous cellular targets including at least
three families of Ser/Thr kinases, p65PAK, MLK, and p70S6kinase. To
identify phosphorylation events controlled by Rac, metabolically labeled HEp-2 cells were pretreated with either clostridial toxin B1470, which inactivates Rac, R-ras, Rap1, Ral and, to a lesser extent,
Cdc42, or with toxin B10463, which inactivates Rho, Rac, and Cdc42 (14,
15). Cells were then stimulated with EGF, a known activator of Rac, for
7 min and cell lysates were analyzed by two-dimensional gel
electrophoresis. The increase in intensity of only 2 of about 20 EGF-stimulated phosphoproteins was blocked by both toxins (Fig.
1A). One was isolated from a
preparative two-dimensional gel and subjected to MALDI-MS post-source
decay analysis. The excised protein was identified as stathmin.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
EGF-induced and
GTPase-dependent phosphorylation of stathmin.
A, HEp-2 cells were serum-starved for 18 h and labeled
with 100 µCi/ml 33Pi for 3 h. Cells were
pretreated with 160 ng/ml toxin B1470 or 2 µg/ml toxin B10463 for
3 h prior to stimulation with 100 ng/ml EGF for 5 min. After cell
lysis and two-dimensional gel electrophoresis, phosphoproteins were
visualized by autoradiography. B, HEp-2 cells were
serum-starved for 18 h and labeled with 25 µCi/ml
32Pi for 3 h before EGF stimulation and cell
lysis. After two-dimensional gel electrophoresis and transfer to
nitrocellulose membrane, phosphoproteins were visualized by
autoradiography (upper panels) and protein by anti-stathmin
antiserum C (lower panels). A and B,
the position of the phosphoprotein purified for MALDI-MS analysis is
indicated by arrows. Molecular mass and apparent pH are
indicated on the left side and on the bottom of
each panel, respectively. C, electrophoretic
mobilities of unphosphorylated (N) and the various
mono-(P1), di-(P2), and triphosphorylated
(P3) stathmin phosphoforms on two-dimensional gels are shown
(16). D, cell extracts (see B) were immunoblotted
with antisera specific for stathmin phosphorylated at serines 16, 25, or 38. Molecular mass is on the left and the positions of
unphosphorylated stathmin and its mono-, di-, and triphosphorylated
phosphoforms are shown on the bottom of each
panel.
As shown in Fig. 1B, EGF treatment resulted in the appearance of two new forms of stathmin and a significant increase in the intensity of at least one other form. Stathmin has been shown to be phosphorylated on four distinct serines: 16, 25, 38, and 63, resulting in a variety of migration patterns on two-dimensional gels (Fig. 1C and Refs. 7 and 16). To identify which residues are phosphorylated in response to EGF, antibodies specific for three phosphorylated forms (serines 16, 25, and 38) were used on Western blots (7). As seen in Fig. 1D, EGF induces a dramatic increase in phosphorylation at Ser-16 and Ser-25. Ser-38 is phosphorylated in control cells and does not increase significantly upon EGF addition. The lack of a significant change in the P3 isoform at 19 kDa (Refs. 25, 38, 63 and Fig. 1D) coupled with the strong increase in the P3 isoform at 23 kDa (16, 25, 38) suggests that very little phosphorylation is induced at Ser-63 by EGF.
To identify which phosphorylation event is mediated by Rho GTPases,
phosphospecific antibodies were used to analyze Western blots of
one-dimensional gels of lysates from cells treated with the two
clostridial toxins. As can be seen in Fig.
2A, 160 ng/ml toxin B1470
results in a complete inhibition of EGF-induced phosphorylation of
Ser-16 although at even 10-fold higher concentrations, the toxin does
not inhibit EGF-induced phosphorylation at Ser-25. Ser-25 has been
reported to be phosphorylated by ERK MAP kinases and the EGF-stimulated
activation of ERK1 and ERK2 was not changed by toxin treatment (Fig.
2A, bottom left and Ref. 17). Toxin B10463 gave
similar results (data not shown). The basal levels of phosphorylated
Ser-38 were unaffected by both toxins. Cytotoxic necrotizing factor 1 (CNF1) from Escherichia coli activates all members of the
Rho family and when added to HEp-2 cells results in increased
phosphorylation of stathmin only on Ser-16 (Fig. 2B and Ref.
18). We conclude that EGF-induced phosphorylation at Ser-16 of stathmin
is mediated by Rac and/or Cdc42.
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To confirm that Rac and Cdc42 can induce phosphorylation at Ser-16,
HEp-2 cells were transfected with a Myc-tagged stathmin expression
vector either alone or with dominant-negative Rac (N17Rac1) or Cdc42
(N17Cdc42) and treated 24 h later with EGF. Inhibition of Rac
almost completely prevented phosphorylation on Ser-16, whereas
inhibition of Cdc42 inhibited phosphorylation by around 50% (Fig.
3A). Next, stathmin was
cotransfected with constitutively activated GTPases. L61Rac and
L61Cdc42, but not L63Rho, induced phosphorylation specifically at
Ser-16 (Fig. 3B).
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Three isoforms (1, 2, and 3) of the Ser/Thr kinase p65PAK interact
directly with Rac and Cdc42 leading to a variety of cellular effects
(8, 19). To determine whether p65PAK mediates Ser-16 phosphorylation,
HEp-2 cells were transfected with stathmin along with an autoinhibitory
fragment (residues 83-149) derived from p65PAK (8). As seen in Fig.
4, A and B,
inhibition of p65PAK completely prevented EGF-, L61Rac-, and
L61Cdc42-induced phosphorylation on Ser-16.
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In conclusion, we have show that the addition of EGF to HEp-2 cells leads to a Rac/Cdc42 and p65PAK-dependent phosphorylation of stathmin at Ser-16. Whether p65PAK directly phosphorylates stathmin or whether it activates another downstream kinase is not currently known. Two kinases have been reported to phosphorylate stathmin at Ser-16, the cAMP-dependent kinase A and the Ca2+/calmodulin-dependent kinase isoforms, CaMK IV/Gr and CaMKII (5, 16, 20). The cAMP-dependent kinase A is unlikely to be involved because it preferentially phosphorylates Ser-63, and pretreatment of HEp-2 cells with EGTA to block Ca2+ influx had no effect, suggesting that CaM kinases are not involved (data not shown).
Stathmin plays an important role in controlling microtubule dynamic
either by sequestering /
tubulin heterodimers or by increasing
catastrophe frequency at the plus ends of microtubules or both
(21-23). As a result, stathmin causes destabilization of growing
microtubules and phosphorylation at Ser-16 appears to block this
activity (5, 6). Rac and Cdc42 regulate actin polymerization and form
membrane protrusions at the leading edge of migrating cells and
neuronal growth cones. The results described here suggest, therefore,
that Rac and Cdc42 might control both F-actin and microtubule dynamics
in localized regions associated with cell protrusions (24, 25).
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ACKNOWLEDGEMENTS |
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We thank Drs. C. von Eichel-Streiber, G. Schmidt, and K. Aktories for their kind gifts of bacterial toxins. We also thank Karen Knox for stathmin DNA isolation, P. Curmi and O. Gavet for stathmin mutant constructions, V. Manceau for stathmin phosphospecific antisera, and Richard Lamb and Sandrine Etienne- Manneville for stimulating discussions.
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FOOTNOTES |
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* This work was supported in part by the Cancer Research Campaign, United Kingdom (to A. H. and H. D). The work in Ghent was supported by Grant IUAP P4/23 from the Interuniversity Attraction Poles of the Prime Minister's Office.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of an EMBO long term fellowship. Present address: Axxima Pharmaceuticals AG, Am Klopferspitz 19, 82152 Martinsried, Germany.
Postdoctoral Fellow of the Fund for Scientific Research,
Flanders, Belgium (F. W. O.-Vlaanderen).
§§ To whom correspondence should be addressed. E-mail: alan. hall@ucl.ac.uk.
Published, JBC Papers in Press, October 31, 2000, DOI 10.1074/jbc.C000635200
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ABBREVIATIONS |
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The abbreviations used are: EGF, epidermal growth factor; MALDI-TOF-MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; PSD, post-source decay analysis; MAP, mitogen-activated protein; RP-HPLC, reversed phase-high performance liquid chromatography; ERK, extracellular receptor kinase.
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