From the Geneva Biomedical Research Institute, Glaxo
Wellcome Research and Development S.A., 1228 Plan-les-Ouates,
Switzerland, ¶ Glaxo Inc., Research Triangle Park,
North Carolina 27709, and ** Institut de Biologie Cellulaire
et de Morphologie, University of Lausanne, 1005 Lausanne, Switzerland
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
SCG10 is a neuron-specific, membrane-associated protein that is highly concentrated in growth cones of developing neurons. Previous studies have suggested that it is a regulator of microtubule dynamics and that it may influence microtubule polymerization in growth cones. Here, we demonstrate that in vivo, SCG10 exists in both phosphorylated and unphosphorylated forms. By two-dimensional gel electrophoresis, two phosphoisoforms were detected in neonatal rat brain. Using in vitro phosphorylated recombinant protein, four phosphorylation sites were identified in the SCG10 sequence. Ser-50 and Ser-97 were the target sites for protein kinase A, Ser-62 and Ser-73 for mitogen-activated protein kinase and Ser-73 for cyclin-dependent kinase. We also show that overexpression of SCG10 induces a disruption of the microtubule network in COS-7 cells. By expressing different phosphorylation site mutants, we have dissected the roles of the individual phosphorylation sites in regulating its microtubule-destabilizing activity. We show that nonphosphorylatable mutants have increased activity, whereas mutants in which phosphorylation is mimicked by serine-to-aspartate substitutions have decreased activity. These data suggest that the microtubule-destabilizing activity of SCG10 is regulated by phosphorylation, and that SCG10 may link signal transduction of growth or guidance cues involving serine/threonine protein kinases to alterations of microtubule dynamics in the growth cone.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
SCG10 is a growth-associated protein abundant in the growth cones of developing neurons (1-3). The gene encoding SCG10 is a member of the stathmin gene family (4). Both stathmin and SCG10 are microtubule (MT)1-destabilizing factors (5-7). In in vitro assays of MT assembly, these molecules inhibit microtubule polymerization and induce depolymerization. Unlike stathmin, which is a cytosolic protein and expressed in most tissues (8), SCG10 is a membrane-associated and neuron-specific protein (1, 9). The expression of SCG10 is developmentally regulated, with high levels in embryonic and postnatal nervous system (1, 2). In the adult, its expression persists in several brain regions that are associated with synaptic plasticity (10), and up-regulation of SCG10 has been found following lesion experiments (11). Overexpression of SCG10 in a neuronal cell line was found to enhance neurite outgrowth (7). Moreover, SCG10 is highly concentrated in the central domain of growth cones (3) where the distal ends of MTs are in a dynamic state of growth and shrinkage (12-14). Thus, SCG10 may be a regulator of MT dynamic instability during neurite outgrowth and structural plasticity. While the role of MTs in growth cone motility and neurite elongation is well established (15-18), little is known about their regulation in response to the environment and the signaling pathways involved. An understanding of phosphorylation and dephosphorylation events regulating the activity of SCG10 may lead to important insights into the intracellular mechanisms that modulate growth cone motility.
We have previously shown that recombinant SCG10 is an in vitro target for the serine/threonine protein kinases PKA, MAP kinase, and cyclin-dependent kinase (CDK) p34cdc2 (19). Both MAP kinase and PKA are present in growth cones and associated with microtubules (20). The CDK p34cdc2 is not expressed in neurons, but another member of the CDK family, CDK5/p25, which is highly homologous to p34cdc2, has been identified in neurons (21, 22) and is localized in growth cones (23).
Here, we demonstrate that SCG10 is phosphorylated in vivo and we have identified four phosphorylation sites in the recombinant protein using liquid chromatography/electrospray ionization mass spectrometry. To further elucidate the molecular mechanism of SCG10 function, we have analyzed the effect of phosphorylation on the activity of the protein in intact cells. Our findings suggest that SCG10 is a phosphoprotein in developing brain and that its MT-destabilizing activity is regulated by phosphorylation.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Antibodies--
A rabbit antibody directed against SCG10
(anti-SCG10-BR) was generated by injecting 80 µg of recombinant,
NH2-terminal truncated SCG10 that had been phosphorylated
with PKA, MAP kinase, and p34cdc2 (19) in complete Freund's
adjuvant. One month after the first injection, the animal was boosted
every 2 weeks with 40 µg of antigen in incomplete Freund's adjuvant.
Five days after the third boost, the animal was deeply anesthetized
with Nembutal and bled by cardiac puncture. The anti-SCG10 serum was
tested for specificity in Western blots. For immunofluorescence
experiments, the previously described rabbit polyclonal antibody
directed against SCG10 (9) and a mouse monoclonal antibody for
-tubulin (clone B-5-1-2, Sigma) were used.
Tissue Preparation, Immunoblots, and
Dephosphorylation--
Wistar rats of different ages (birth, postnatal
days (P) 0, 5, 10, 15, and 20) and adult rats (3 months) were prepared
for biochemistry as described earlier (24). Animals were deeply anesthetized (40 mg of sodium pentobarbital per kg body weight) and
decapitated. The brains were removed, immediately frozen in liquid
nitrogen, and kept at 80 °C until use. Proteins (50 µg/slot) were separated on 5-20% gradient SDS-polyacrylamide gel
electrophoresis and either stained with Coomassie Blue or transferred
to nitrocellulose sheets. Western blots were incubated with
anti-SCG10-BR antiserum (1:3000) followed by a peroxidase-conjugated
secondary antibody and developed with 4-chloro-1-naphthol (24). For
dephosphorylation, the brain of a P5 animal was homogenized in
dephosphorylation buffer (50 mM Tris/HCl, pH 8.2, 135 mM NaCl, 0.1 mM EDTA, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 30 µg/ml each of leupeptin, antipain, and pepstatin, and 15 µg/ml E64), and incubated with and
without 0.2 IU alkaline phosphatase (Boehringer Mannheim, molecular
grade)/µg of protein overnight at 37 °C (25, 26). The reaction was
stopped by adding electrophoresis sample buffer and boiling. Control
homogenates were incubated with 1 mM 4-nitrophenyl phosphate and 50 mM sodium fluoride to inhibit phosphatase
activity. Samples were separated on SDS-PAGE, blotted onto
nitrocellulose, and immunostained.
Two-dimensional Gel Electrophoresis and Blots--
Because SCG10
could not be resolved by regular isoelectrofocusing, brain homogenates
and phosphorylated forms of the recombinant NH2-terminal
truncated SCG10 were separated in the first dimension by a
nonequilibrium pH gradient (NEPHGE) (27). Protein samples were
dissolved in sample buffer (9.5 M urea, 2% CHAPS, 1% DTT, 0.8% ampholine, pH 3-10, 1.2% ampholine, pH 7-9, and 0.4%
ampholine, pH 9-11) at 10 µg of protein/µl. Each gel was loaded
with 50 µg of brain homogenate, or 1 and 0.3 µg of recombinant
SCG10 for Coomassie Blue staining and Western blot, respectively. The
upper buffer chamber contained 0.1 M
H3PO4, the lower 0.02 M NaOH. The polarity was the reverse of that used for isoelectrofocusing gels, with
the cathode (+) at the top and the anode () at the bottom. After
application of the sample, the proteins were separated for 2 h at
800 V, 4 °C. The gels were incubated for 30 min with equilibration buffer (3% SDS, 1 mM EDTA, 10% glycerol, 0.2%
2-mercaptoethanol, 125 mM Tris-HCl, pH 6.8), and separated
on a 5-20% acrylamide gradient (or 15% SDS-PAGE). Proteins were
visualized by Coomassie Blue staining or after transfer to
nitrocellulose by immunostaining.
In Vitro Phosphorylation--
Recombinant SCG10 was purified as
described earlier and stored in 20 mM Tris-HCl, 0.2 mM DTT, pH 7,5 at 80 °C (19, 28). The purified protein
was over 98% pure on reverse phase-HPLC and showed apparent
homogeneity on SDS-PAGE. For the mass spectrometry studies, full-length
SCG10 (19) was used, whereas for the two-dimensional gel analysis,
NH2-terminal truncated SCG10 (28) was used. For each
phosphorylation assay, 250 pmol (5 µg) of the protein were used in a
total reaction volume of 50 µl in the buffers described below. MAP
kinase (p44mpk, Upstate Biotechnology Inc., Lake Placid, NY):
15 ng MAP kinase in 15 mM MOPS, pH 7.0, 10 mM
MgCl2, 0.5 mM EGTA, 50 mM NaF, and 1 mM DTT. cAMP-dependent protein kinase,
catalytic subunit (PKA, Boehringer Mannheim, Germany): 5 milliunits of
PKA in 20 mM MOPS, pH 7.0, 10 mM
MgCl2, 0.5 mM EGTA, and 1 mM DTT.
p34cdc2 kinase (Promega, Madison, WI): 10 units
pp34cdc2 in 20 mM MOPS, pH 7.0, 10 mM
MgCl2, 0.5 mM EGTA, 50 mM NaF, and 0.5 mM DTT. All reaction mixtures contained 0.2 mM ATP. The samples were incubated at 35 °C for 60 min.
The reactions were stopped by addition of 20 mM EDTA and
the samples were stored at
80 °C until analyzed.
Electrospray Ionization-Mass Spectrometry-- Electrospray ionization-mass spectrometry (MS) was performed using an API-III triple quadrupole mass spectrometer (PE-Sciex, Concord, Ontario, Canada) equipped with an HP1090 microbore HPLC ternary pump system (Hewlett Packard, Palo Alto, CA). Separations were carried out using a PorosTM R2/H 300 mm × 10-cm capillary perfusion column (LC Packings, San Francisco, CA). Buffer A was 0.05% trifluoroacetic acid in H2O. Buffer B was 0.035% trifluoroacetic acid in 90/10 acetonitrile/H2O. The column flow rate was 50 µl/min and was achieved using a pre-column flow split. An aliquot of purified SCG10, corresponding to 50-100 pmol, was loaded onto the capillary perfusion column and eluted using a gradient of 15% buffer B to 65% buffer B in 5 min. The mass spectrometer was scanned from m/z 825 and m/z 1125 Da every 3 s during the gradient HPLC separation. The resolution of the mass spectrometer unit was up to m/z 2200 (20% valley definition) as determined from the infusion of a (poly)propylene glycol standard calibrant solution (supplied by PE-Sciex). Mass spectra were acquired using a 0.2-Da step size (permitting 5 data points/Da) and a 2.0-ms dwell time per step.
Enzymatic Digestion and LC/MS Characterization-- Aliquots of protein samples, corresponding to 5-25 pmol, were injected onto an immobilized trypsin perfusion column (PerSeptive Biosystems, Framingham, MA) and digested on-column (total on-column digestion time of 2 min). Peptide fragments were trapped onto a reversed phase C18 column and eluted into the ion source of the mass spectrometer as described previously (29). Tryptic peptides were separated on a 15 cm × 300-µm capillary C18 column (LC Packings) operated at 5 µl/min using a linear gradient of 1 to 21% buffer B in 5 min and 21 to 41% buffer B in 15 min. Electrospray mass spectra were acquired by scanning the mass spectrometer from 300-1800 Da in 3 s using a 0.5-Da step and a 1.0-ms dwell time.
Alternatively, an aliquot of SCG10 corresponding to 25 pmol, was purified by reversed phase perfusion column HPLC and subjected to digestion with endoprotease Glu-C (Boehringer Mannheim, Germany) in NH4HCO3 at pH 8.0 for 12 h and at an enzyme:substrate ratio of 1:50 to confirm the sites of phosphorylation identified from the tryptic digestions.Phosphopeptide Identification--
Phosphopeptides were
identified from the tryptic and Glu-C digests using the stepped orifice
voltage technique, previously reported by Huddleston et al.
(30) and Ding et al. (31). An aliquot of protein digest
(5-25 pmol) was separated on the capillary C18 column and analyzed by
electrospray ionization-MS. Peptides were ionized in the negative ion
mode, and phosphopeptides were identified based on their ability to
form a prominent PO3 ion, indicative
of phosphorylation, at m/z 79.
LC/MS/MS to Identify Phosphorylation Sites-- Tryptic phosphopeptides identified by negative ion stepped orifice potential experiments were subjected to on-line LC/MS/MS. These peptides were separated using the same column and gradient as described above. LC/MS/MS spectra of phosphopeptides were acquired by scanning the mass spectrometer from 50 Da to the mass of the precursor (M + H)+ ion in 3 s. The collision gas was set to 3 × 1014 collision gas thickness units. The mass spectrometer resolution was set to 1000 (full-width, half-maximum).
The size of some of the tryptic fragments precluded their ability to be characterized fully by LC/MS/MS. Consequently, some of these larger tryptic fragments were subjected to digestion with Glu-C. These peptides were characterized by LC/MS/MS in a manner identical to the tryptic peptides, described above.Generation of SCG10 Mutants-- The rat SCG10 cDNA was a generous gift of Dr. N. Mori (Kyoto, Japan). To replace serine with alanine or aspartate at positions 50, 62, 73, and 97 in the SCG10 sequence, site-directed mutagenesis was performed according to polymerase chain reaction procedures as described previously (32). All mutants were subcloned into the BamHI and XbaI sites of the pcDNA3 expression vector (Invitrogen) and constructs were confirmed by DNA sequencing.
Expression of SCG10 Mutants in COS-7 Cells and Measurement of
MT-depolymerizing Activity--
COS-7 cells were cultured and
electroporated as described previously (9) and plated in 100-mm Petri
dishes containing 15-mm coverslips. After 48 h, cells were fixed
for 20 min in 4% formaldehyde in phosphate-buffered saline (PBS) and
washed 3 times with PBS. Cells were incubated for 2 h at room
temperature with a rabbit antiserum to SCG10 (9) and mouse monoclonal
antibodies to -tubulin (clone B5-1-2, Sigma) in PBS containing 10%
normal goat serum (Sigma), 0.3% Triton X-100, and 2% bovine serum
albumin (Sigma). Following three washes with PBS, cells were incubated
for 30 min at room temperature with fluorescein-conjugated goat
anti-mouse and Cy3-conjugated goat anti-rabbit antibodies (Jackson
Laboratories, Bar Harbor, ME). Cells were washed three times with PBS,
and coverslips were mounted using Vectashield mounting medium (Vector).
Using a fluorescent microscope (Zeiss Axioscop), cells expressing high levels of SCG10, i.e. where SCG10 staining was not
restricted to the Golgi area, were assessed for their microtubule
content. Cells were considered as cells containing polymerized MT if
they contained more than 50 intact MTs. In three independent
experiments, 50 cells were scored, and the percentage of cells
containing polymerized MTs was calculated. As controls, we analyzed
untransfected cells and cells that were transfected with an inactive
SCG10 construct where the first 99 amino acids were deleted. In both
cases 100% of the interphase cells showed a dense MT network. To
compare the activity of SCG10 in transfected cells with that of a
soluble form of SCG10 missing the membrane-binding domain (9), 100 transfected cells from three independent experiments were counted regardless of their level of expression, and the percentage of cells
containing polymerized MTs was determined as described above.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
SCG10 Phosphoisoforms in Rat Brain-- An anti-SCG10 serum, raised against in vitro phosphorylated recombinant SCG10 recognized two major bands in Western blots of postnatal brain extracts, one at 22 kDa and one at 25 kDa (Fig. 1A). As expected, due to the developmental change of SCG10 protein in rat brain (2), the levels of expression decreased after postnatal day 5 and became undetectable in the adult (Fig. 1A). Following alkaline phosphatase treatment of P5 brain extract, the band at 25 kDa was no longer detectable, indicating that it corresponds to one or several phosphorylated forms of SCG10 (Fig. 1B). Two-dimensional gel electrophoresis revealed three isoforms of SCG10 in postnatal brain extract (Fig. 1C). The electrophoretic mobility of these isoforms was compared with that of in vitro phosphorylated, NH2-terminal truncated SCG10 (28) (as described under "Experimental Procedures") (Fig. 1, D-F). A mixture of nonphosphorylated SCG10 and SCG10 that was phosphorylated by CDK or by a combination of CDK, MAP kinase, and PKA generated a SCG10 pattern similar to that found in brain extracts (Fig. 1G). These results indicate that postnatal rat brain contains unphosphorylated as well as two different phosphoisoforms of SCG10.
|
Phosphorylation Sites in SCG10-- For determination of the phosphorylation sites, recombinant SCG10 was in vitro phosphorylated by PKA, MAP kinase, and CDK as described under "Experimental Procedures." The phosphorylated samples were first analyzed by ion-spray mass spectrometry to determine the number of phosphorylation sites. Ion-spray mass spectrometry gave a mass of 20,624 ± 1.4 for unphosphorylated SCG10, which is in agreement with the calculated molecular mass (20,624 Da). After phosphorylation with PKA, the main molecular mass was found to be 20,803, which corresponds to diphosphorylated SCG10. MAP kinase phosphorylation gave both mono- and diphosphorylated SCG10 with molecular masses of 20,715 and 20,803, respectively. However, after 120 min of incubation with the kinase, the protein was entirely diphosphorylated (Fig. 2A). Samples phosphorylated with CDK showed mainly monophosphorylated protein with a molecular mass of 20,715. To map the sites of phosphorylation, the samples were digested with either trypsin or Glu-C, and the peptides were separated on reverse phase-HPLC and sequenced with tandem mass spectrometry (MS/MS).
|
|
|
|
Regulation of SCG10 Activity--
SCG10 inhibits the assembly of
microtubules and induces their disassembly in vitro (7). To
study the effect of SCG10 on microtubules in intact cells and to
identify the role of phosphorylation in its activity, we have
transiently transfected COS-7 cells, which do not express endogenous
SCG10. We assessed the effects of expression of wild-type SCG10 on the
MT array by immunofluorescence staining using an anti--tubulin
antibody. Then, we tested mutants in which the serines, individually or
in combination, were mutated to alanine or aspartate to prevent or
mimic phosphorylation, respectively (Fig. 4C). In cells
expressing low levels of wild-type SCG10 in which SCG10 staining was
observed in the area of the Golgi apparatus, as previously shown (9),
no obvious abnormalities could be observed. These cells were similar to
the controls where 100% of the interphase cells showed a dense MT
network. However, cells expressing high levels of SCG10, where the
protein was also abundant in the cytoplasm (Fig.
5B), showed a dramatic,
sometimes complete, disappearance of the microtubule network (Fig. 5,
A and B). When we compared the activity of
wild-type SCG10 with that of a construct that is missing the
NH2-terminal membrane-binding domain and thus expressed as
a cytosolic protein (9), we found that SCG10 is less active in the
membrane-bound form. While 43.7 ± 1.2% of cells transfected with
full-length SCG10, only 20.3 ± 0.9% of cells transfected with
soluble SCG10 contained polymerized MTs (p < 0.001).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
SCG10, a growth cone-enriched MT-destabilizing protein, has been recently characterized as an in vitro substrate for various serine/threonine kinases including PKA, MAP kinase, and CDK (19). We have found that SCG10 is phosphorylated in vivo in developing rat brain. The in vivo isoforms correspond to unphosphorylated, monophosphorylated, and multiphosphorylated forms. More detailed studies will be required to reveal the specific phosphorylation states of this protein in cells and tissues under a variety of physiological conditions.
In this work, the sites of SCG10 phosphorylated by PKA, MAP kinase, and CDK have been identified. Our results, based upon tryptic peptide mapping followed by LC/MS and MS/MS sequencing, are summarized in Fig. 4A, where they are compared with the sites that have been reported for the related protein stathmin (33-35). The PKA phosphorylation sites present in SCG10 are conserved to the in vitro and in vivo phosphorylation sites known for stathmin. The sites for MAP kinase phosphorylation were identified as Ser-62 and Ser-73 of SCG10. Both sites contain a proline residue C-terminal to the serine consistent with the serine (threonine)-proline specificity of this kinase (Fig. 4B). In contrast to stathmin, where the major site for MAP kinase was Ser-25, no preferred phosphorylation site was found for SCG10. Two sites (Ser-25 and Ser-38) are phosphorylated by CDK in stathmin, whereas we found only one major site in SCG10 that was phosphorylated by this kinase (Ser-73). Since the kinase used for these experiments is not expressed in neurons, the neuronal cdc2-like kinase CDK5/p25, which was also found to efficiently phosphorylate SCG10 (data not shown), may be the physiologically relevant kinase. Our results suggest that the function of SCG10 is regulated by multiple protein kinases and that the kinases PKA, MAP kinase, and CDK5/p25, all three of which are present in growth cones (20, 23), are good candidates to phosphorylate SCG10 in vivo.
To reveal physiological functions of SCG10 phosphorylation, we assessed whether phosphorylation at the identified sites had an effect on the MT-destabilizing activity of the protein. We found that overexpression of wild-type SCG10 in COS-7 cells caused disruption of the MT network consistent with its microtubule-depolymerizing effect in vitro (7). A similar activity was recently reported for the cytosolic protein stathmin upon transfection (6) or microinjection of recombinant protein (36) into cells. However, SCG10 was significantly less active than a truncated cytosolic form of the protein, and its effect was observed mainly in highly overexpressing cells, where SCG10 localization was not restricted to the area of the Golgi complex (9) but also found in the cytoplasm. This may indicate that Golgi-associated SCG10 is either not very active or, more likely, not in a subcellular compartment where it can induce depolymerization of interphase microtubules. It is not known yet whether SCG10 functions while it is bound to organelles in neuronal growth cones (3) or whether it has to be released from membranes.
By expressing a series of phosphorylation site mutants, we showed that the MT-destabilizing effect of SCG10 could be modulated. While the nonphosphorylatable mutant showed higher activity than the wild-type protein, the activity of the mutant in which phosphorylation on all four sites was mimicked by an aspartate residue was greatly reduced. These data suggest that the nonphosphorylated state of SCG10 represents the most active form of the protein. Observations of stathmin in transfected or microinjected cells (6, 36) as well as in an in vitro assay of MT assembly (37, 38) suggest a similar mechanism of regulation of the activity of the two proteins. However, for stathmin it has been reported that alanine substitution on only two of the four serines (Ser-25 and Ser-38) increased its MT-destabilizing activity to nearly the same extend as mutating all four sites. This was not the case for SCG10, where the S50A,S62A,S73A,S97A mutant was significantly more active than any single or double alanine substitutions. Interestingly, these two serine residues in stathmin (Ser-25 and Ser-38) are not precisely conserved in the SCG10 sequence (Fig. 4B) and exhibit differences between stathmin and SCG10 in their phosphorylation by MAP kinase and CDK (Fig. 4A).
We also tested the effect of mutation of phosphorylation sites in SCG10 by introducing aspartate residues to replace each of the four phosphorylatable serines, both individually and in various combinations. Of the single mutants, only S73D and S97D were statistically different from wild-type, but not from each other. Also the double mutants were not statistically different from each other, though they were different from the S50D,S62D,S73D,S97D mutant. Whether the decreased activity of aspartate mutants is caused by a reduced binding to tubulin as is the case for in vitro phosphorylated stathmin (37, 38) needs to be determined.
In summary, our results strongly suggest that the activity of SCG10 is controlled by phosphorylation and that its activity can be down-regulated to different extents by multiple phosphorylations. Therefore, SCG10 may be a key factor that links growth or guidance cues to the local control of MT assembly in growth cones. Fine tuning of its activity, possibly by several signal transduction pathways that act in concert, may be involved in regulation of the dynamics of MTs required for growth cone advance and turning.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank all members of the neurobiology group at Geneva Biomedical Research Institute for their helpful discussion. We are grateful to R. Golsteyn for experiments on SCG10 phosphorylation with neuronal CDK. We also thank A. Bernard, H. Blasey, J. Y. Bonnefoy, N. Gullu, C. Hebert, S. Herren, P. Graber, S. Montessuit, R. Porchet, L. Potier, and E. Sebille for their help at various stages of this work. We thank S. Catsicas and J.K. Staple for many helpful comments and critical reading the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported in part by the National Science Foundation of Switzerland, Grants 31-43137.95 (to B. M. R.) and 3100-050948.97 (to G.G.).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.
§ Present address: Serono Pharmaceutical Research Institute S. A., 1228 Plan-les-Ouates, Switzerland.
Present address: CombiChem, Inc., San Diego, CA 92121.
To whom correspondences should be addressed: IBCM,
Université de Lausanne, Rue du Bugnon 9, 1005 Lausanne,
Switzerland. Tel.: 41-21-692 5100; Fax: 41-21-692 5105.
1 The abbreviations used are: MT, microtubule; PKA, cAMP-dependent protein kinase; MAP, mitogen-activated protein; CDK (p34cdc2 and CDK5/p25), cyclin-dependent protein kinase; PAGE, polyacrylamide gel electrophoresis; NEPHGE, nonequilibrium pH gradient electrophoresis; HPLC, high performance liquid chromatography; MS, mass spectrometry; MS/MS tandem MS; LC, liquid chromatography; PBS, phosphate-buffered saline; DTT, dithiothreitol; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|