Originally published In Press as doi:10.1074/jbc.M003163200 on August 15, 2000
J. Biol. Chem., Vol. 275, Issue 44, 34701-34709, November 3, 2000
Involvement of Gangliosides in
Glycosylphosphatidylinositol-anchored Neuronal Cell Adhesion Molecule
TAG-1 Signaling in Lipid Rafts*
Kohji
Kasahara
§,
Kazutada
Watanabe¶,
Kosei
Takeuchi¶
,
Harumi
Kaneko¶,
Atsuhiko
Oohira**,
Tadashi
Yamamoto, and
Yutaka
Sanai
From the Department of Biochemical Cell Research, The
Tokyo Metropolitan Institute of Medical Science, Tokyo Metropolitan
Organization for Medical Research, Honkomagome, Bunkyo-ku,
Tokyo 113-8613, the ¶ Department of Cell Recognition, Tokyo
Metropolitan Institute of Gerontology, Itabashi-ku, Tokyo 173-0015, the ** Department of Perinatology and Neuroglycoscience, Institute for
Developmental Research, Kasugai, Aichi 480-0392, and the
Department of Oncology, Institute of
Medical Science, University of Tokyo, Minato-ku,
Tokyo 108-0071, Japan
Received for publication, April 13, 2000, and in revised form, August 1, 2000
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ABSTRACT |
The association of ganglioside GD3 with
TAG-1, a glycosylphosphatidylinositol-anchored neuronal cell adhesion
molecule, was examined by coimmunoprecipitation experiments.
Previously, we have shown that the anti-ganglioside GD3 antibody (R24)
immunoprecipitated the Src family kinase Lyn from the rat cerebellum,
and R24 treatment of primary cerebellar cultures induced Lyn activation
and rapid tyrosine phosphorylation of an 80-kDa protein (p80).
We now report that R24 coimmunoprecipitates a 135-kDa protein
(p135) from primary cerebellar cultures. Treatment with
phosphatidylinositol-specific phospholipase C revealed that p135 was
glycosylphosphatidylinositol-anchored to the membrane. It was
identified as TAG-1 by sequential immunoprecipitation with an
anti-TAG-1 antibody. Antibody-mediated cross-linking of TAG-1 induced
Lyn activation and rapid tyrosine phosphorylation of p80. Selective
inhibitor for Src family kinases reduced the tyrosine phosphorylation
of p80. Sucrose density gradient analysis revealed that the TAG-1 and
tyrosine-phosphorylated p80 in cerebellar cultures were present in the
lipid raft fraction. These data show that TAG-1 transduces signals via
Lyn to p80 in the lipid rafts of the cerebellum. Furthermore,
degradation of cell-surface glycosphingolipids by endoglycoceramidase
induced an alteration of TAG-1 distribution on an OptiPrep gradient and
reduced the TAG-1-mediated Lyn activation and tyrosine phosphorylation
of p80. These observations suggest that glycosphingolipids are involved
in TAG-1-mediated signaling in lipid rafts.
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INTRODUCTION |
Gangliosides, sialic acid-containing glycosphingolipids
(GSLs),1 are found in the
outer leaflet of the plasma membrane of all vertebrate cells and are
thought to play functional roles in cellular interactions and the
control of cell proliferation (1-4). In the nervous system, where
gangliosides are particularly abundant, the species and amounts of
gangliosides undergo profound changes during development, suggesting
that they may play fundamental roles in this process (5). The
accumulation of gangliosides within the neurons in ganglioside storage
diseases results in extensive neurite outgrowth (6). Exogenously
administered gangliosides accelerate the regeneration of neurons in the
central nervous system in vivo after lesioning (7). The
addition of exogenous gangliosides to primary cultures of neurons and
neuroblastoma cells in vitro stimulates cellular
differentiation with concomitant neurite sprouting and extension
(8-10). Glucosylceramide synthesis, the first glycosylation step of
GSL synthesis, is required for axonal growth in hippocampal neurons
(11) and for embryonic development (12). Transfection of the
ganglioside GD32 synthase
cDNA into neuroblastoma cells induces their cholinergic differentiation and neurite sprouting (13). Finally, mice lacking complex gangliosides exhibit axonal degeneration (14). These data show
that gangliosides are involved in neural cell differentiation and brain
development. However, the molecular mechanisms underlying the
ganglioside-dependent neural functions remain obscure.
GSLs are known to exist in clusters and form microdomains containing
cholesterol at the surface of the plasma membrane. These microdomains
are variously referred to as lipid rafts, detergent-resistant membranes, detergent-insoluble glycosphingolipid-enriched domains, or
caveolae membranes (15-20). The GSL microdomains have been implicated in signal transduction because a variety of signaling molecules, such
as the Src family kinases, are associated with them. However, the
precise functions of GSLs in the microdomains remain to be explored.
We investigated the association of gangliosides with specific proteins
in the central nervous system by coimmunoprecipitation experiments
using an anti-ganglioside antibody. We previously demonstrated that
anti-ganglioside GD3 antibody (R24) coimmunoprecipitates the Src family
kinase Lyn from the rat cerebellum (21). R24 treatment of primary
cerebellar cultures induced Lyn activation and rapid tyrosine
phosphorylation of an 80-kDa protein (p80). Furthermore, sucrose
density gradient analysis showed that Lyn, in both cerebella and CHO
cells transfected with Lyn cDNA, was detected in the lipid raft
fraction. R24 immunoprecipitated caveolin, a caveolae marker protein,
from CHO cells transfected with GD3 synthase cDNA (22). These
observations suggest that GSL may regulate the functions of Lyn in
lipid rafts.
In the present study, we attempted to identify the cell-surface
molecules involved in Lyn signaling because Lyn is a nonreceptor-type kinase, and we found that R24 coimmunoprecipitates TAG-1, a
glycosylphosphatidylinositol (GPI)-anchored neuronal cell adhesion
molecule and that the antibody-mediated cross-linking of TAG-1 induced
Lyn activation and rapid tyrosine phosphorylation of p80. Furthermore,
we investigated the roles of GSLs in TAG-1 signaling.
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EXPERIMENTAL PROCEDURES |
Materials--
Endoglycoceramidase II and activator II (EGCase
II ACT) and phosphatidylinositol-specific phospholipase C (PI-PLC) were
purchased from Takara Biomedicals (Osaka, Japan) and Funakoshi (Tokyo,
Japan), respectively. OptiPrep was purchased from Nycomed Pharma (Oslo, Norway). Triton X-100 and EGTA were purchased from Sigma. PP1, PP2, and
PP3 were purchased from Alexis Biochemicals (San Diego, CA) and
Calbiochem, respectively. The 3.1C12 mouse monoclonal IgG for rat TAG-1
was obtained from the Developmental Studies Hybridoma Bank developed
under the auspices of the NICHD, National Institutes of Health, and
maintained by the Department of Biological Sciences, University of
Iowa. The mouse IgG3 anti-ganglioside GD3 monoclonal antibody, R24,
anti-Lyn (Lyn8) monoclonal antibody, and anti-NCAM polyclonal antibody
were obtained from Signet Laboratories (Dedham, MA), Wako Chemicals
(Osaka, Japan), and Affinity Research Products (Mamhead, UK),
respectively. The anti-caveolin polyclonal antibody, horseradish
peroxidase-conjugated anti-phosphotyrosine antibody (PY20), and
fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG
antibody were purchased from Transduction Laboratories (Lexington, KY)
and Zymed Laboratories Inc. (San Francisco, CA),
respectively. Anti-rat N-methyl-D-aspartate
(NMDA) receptor, subunit 2C (amino acids 25-130, extracellular
domain), polyclonal rabbit IgG, and anti-integrin 
5
(cytoplasmic domain) polyclonal rabbit IgG were obtained from Molecular
Probes (Eugene, OR) and Chemicon International (Temecula, CA),
respectively. The anti-T-cadherin polyclonal antibody (amino acids
1-20) was a gift from Dr. Eichmann (University of California, San
Diego). Phosphacan was purified as described by Maeda et al.
(23).
Primary Culture--
Cerebellar granule cell cultures, 98%
pure, were prepared from the cerebella of 6-day-old rats according to
the method of Levi et al. (24) with some modifications.
Briefly, the cerebella were incubated in 1% trypsin or 0.4 mg/ml
dispase for 30 min at room temperature and triturated with a Pasteur
pipette in 0.05% DNase until no tissue aggregates remained. Then
2.5 × 106 cells were suspended in basal Eagle's
medium containing 25 mM KCl and 5% fetal bovine serum and
plated onto 35-mm poly-D-lysine-coated plastic dishes. To
avoid the proliferation of glial cells, 100 µM cytosine
arabinoside was added 18 h after the seeding.
Antibodies--
Anti-TAG-1 serum was generated against
recombinant proteins expressed in Escherichia coli using the
pET vector system from the rat TAG-1 encoding immunoglobulin domains
I-II (amino acids 31-143). Based on the sequence information of the
rat TAG-1 cDNA, two degenerate oligonucleotides were synthesized.
The sequences of the 5' and 3' primers were
5'-TCATATGCAGGGAACCCCAGCTACCTTTGG-3' and
5'-TGAGGAGCCAGCGGTAGGACAAACC-3'. For PCR amplification, first strand
cDNA was synthesized from the total RNA of rat brain. Thirty cycles
(94 °C for 10 s, 60 °C for 30 s, and 72 °C for
45 s) were run. The PCR product, around 400 base pairs in length,
was subcloned into the pCR II vector, and the
NdeI-BamHI fragment was inserted into the pET-3b
vector (Novagen). The protein was expressed in E. coli
BL21(DE3)pLysS after induction with
isopropyl-1-thio--D-galactopyranoside and then partially
purified as inclusion bodies. After SDS-PAGE, the recombinant protein
was eluted electrophoretically from the gel. Aliquots containing about
1 mg of the protein were emulsified with complete or incomplete
Freund's adjuvant and used to immunize rabbits.
Anti-contactin/F3 serum was generated against recombinant proteins
expressed in E. coli using the pET vector system from rat contactin/F3 encoding immunoglobulin domains I-II (amino acids 22-255). Based on the sequence information of rat contactin/F3 cDNA, two degenerate oligonucleotides were synthesized. The
sequences of the 5' and 3' primers were
5'-ATACATATGTTTACATGGCACAGAAGATAT-3' and
5'-AGATCTGAATTCATGGTGTATATGTCCTTG-3'. For PCR amplification, first
strand cDNA was synthesized from the total RNA of rat brain. Thirty
cycles (94 °C for 10 s, 55 °C for 30 s, and 72 °C
for 45 s) were run. The PCR product, around 680 base pairs in
length, was subcloned into the pCRII vector, and the
NdeI-BamHI fragment was inserted into the pET-3b
vector. Aliquots containing about 1 mg of the protein were emulsified
with complete or incomplete Freund's adjuvant and used to immunize rabbits.
Metabolic Labeling and Immunoprecipitation--
Primary
cerebellar cultures were labeled with the
EXPRE35S35S Protein Labeling Mix (PerkinElmer
Life Sciences) for 3 h in a methionine-/cysteine-depleted medium,
at a final concentration of 0.1 mCi/ml. The cells were solubilized in
lysis buffer (1% Triton X-100, 50 mM Tris-HCl (pH 7.4),
150 mM NaCl, 1 mM
Na3VO4, 1 mM EGTA, 1 mM
phenylmethylsulfonyl fluoride) at 4 °C for 20 min. The postnuclear
supernatants were collected after centrifugation at 14,000 rpm for 3 min. Aliquots (0.5 ml, 750 µg of protein) of the supernatants were
precleared with protein G-Sepharose (7.5 µl) and then incubated with
the anti-GD3 antibody, R24 (2.5 µg), for 1 h and precipitated
with protein G-Sepharose (7.5 µl). Following immunoprecipitation, the beads were washed three times with lysis buffer, and after the addition
of the Laemmli sample buffer, the samples were subjected to SDS-PAGE
(10% acrylamide) followed by autoradiography. In the sequential
immunoprecipitation experiment, the immunoprecipitates were boiled for
5 min in lysis buffer with 1% SDS, diluted 10-fold with lysis buffer,
and then reimmunoprecipitated with anti-GPI-anchored protein antibodies.
Cell-surface Labeling and PI-PLC Treatment--
Primary
cerebellar cells, 5 × 106, were washed with PBS. To
the cells in 0.5 ml of PBS were added 100 µl of lactoperoxidase (1 units/ml in PBS) and 500 µCi of Na125I at room
temperature. The reaction was started by the addition of 1 µl of
freshly prepared 0.0015% H2O2. The diluted
H2O2 was added at 1-min intervals over 4 min.
To terminate the reaction, an equal volume of 1 mg/ml tyrosine in PBS
was added.
For PI-PLC treatment, the surface-labeled cells were washed with PBS
and incubated in a medium containing 2.5 units/ml PI-PLC from
Bacillus thuringiensis for 1 h at 37 °C.
Antibody-mediated Cross-linking of TAG-1 and Measurement of Lyn
Activity in Primary Cerebellar Cultures--
Cerebellar cultures of 3 days in vitro in a 35-mm dish were incubated with 20 µg/ml
anti-TAG-1 monoclonal antibody (3.1C12) on ice for 30 min in BSA medium
(culture medium without fetal bovine serum containing 1% BSA and 20 mM HEPES (pH 7.4)). After washing with cold BSA medium, the
cells were incubated with 50 µg/ml anti-mouse IgG affinity-purified
polyclonal antibody (secondary antibody) in BSA medium at 37 °C for
the indicated times on the dish. After washing with ice-cold PBS,
lysates were prepared in 1% Triton X-100, 50 mM Tris-HCl
(pH 7.4), 150 mM NaCl, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride at 4 °C. After
centrifugation at 14,000 rpm for 3 min, the supernatants were incubated
with the anti-Lyn antibody and precipitated with protein G-Sepharose. The immunoprecipitates were incubated with kinase buffer containing 10 µM ATP and 5 µCi of [
-32P]ATP (3,000 Ci/mmol). Kinase activity was measured by in vitro autophosphorylation.
For cross-linking of ganglioside GD3, cells were incubated with 20 µg/ml R24 on ice for 30 min in BSA medium. After washing with cold
BSA medium, the cells were incubated with 50 µg/ml anti-mouse IgG
affinity-purified polyclonal antibody in BSA medium at 37 °C for 3 min.
For cross-linking of NMDA receptors, cells were incubated with 20 µg/ml anti-NMDA receptor antibody on ice for 30 min in BSA medium.
After washing with cold BSA medium, the cells were incubated with 50 µg/ml anti-rabbit IgG affinity-purified polyclonal antibody (secondary antibody) in BSA medium at 37 °C for 3 min.
Alternatively, cells were incubated with 20 µg/ml anti-NMDA receptor
antibody in BSA medium at 37 °C for 3 min.
For mock cross-linking of TAG-1 or ganglioside GD3, cells were
incubated with 20 µg/ml normal mouse IgG on ice for 30 min in BSA
medium. After washing with cold BSA medium, the cells were incubated
with 50 µg/ml anti-mouse IgG affinity-purified polyclonal antibody in
BSA medium at 37 °C for 3 min. For mock cross-linking of NMDA
receptors, cells were incubated with 20 µg/ml normal rabbit IgG on
ice for 30 min in BSA medium. After washing with cold BSA medium, the
cells were incubated with 50 µg/ml anti-rabbit IgG affinity-purified
polyclonal antibody (secondary antibody) in BSA medium at 37 °C for
3 min.
Phosphacan-mediated Stimulation and Detection of Tyrosine
Phosphorylation of Proteins in Primary Cerebellar Cultures--
Two
methods were used for the phosphacan-mediated stimulation. For method
1, the cerebellar cultures were incubated with 10 µg/ml phosphacan in
BSA medium at 37 °C for 3 min. For method 2, the cerebellar cultures
were incubated with 10 µg/ml phosphacan in BSA medium on ice for 30 min. After washing with cold BSA medium, the cells were incubated with
a 10-fold concentrated culture supernatant of hybridoma 6B4
(anti-phosphacan) (25) at 37 °C for 3 min. As a negative control
experiment, the cells were incubated with the concentrated 6B4
supernatant at 37 °C for 3 min without phosphacan pretreatment.
For the detection of tyrosine phosphorylation of proteins, cells were
lysed with lysis buffer and subjected to SDS-PAGE and immunoblotting
with the anti-phosphotyrosine antibody. Tyrosine phosphorylation of p80
was quantified by densitometry.
Protein Kinase Activity Coprecipitated with Anti-TAG-1 Antibody
or R24--
Membrane fractions were prepared from newborn rat
cerebella. The cerebella were homogenized in ice-cold buffer A (0.32 M sucrose, 1 mM Tris-HCl (pH 7.4) and 0.1 mM EDTA) using a Teflon motor-driven glass homogenizer. The
homogenate was centrifuged at 900 × g for 1 min. The
supernatant was centrifuged at 11,500 × g for 3 min. The resulting pellet of the rat brain or the centrifuged cultured cells
was solubilized in lysis buffer (1% Triton X-100, 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM
Na3VO4, 1 mM EGTA, 1 mM
phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin and 5 µg/ml
pepstatin A) at 4 °C for 20 min. The supernatants were collected
after centrifugation at 14,000 rpm for 3 min. Aliquots (0.5 ml, 750 µg of protein) of the supernatants were precleared with protein
G-Sepharose (7.5 µl), then incubated with anti-TAG-1 antibody or R24
(2.5 µg) for 1 h, and precipitated with protein G-Sepharose (7.5 µl). Following immunoprecipitation, the beads were washed three times
with lysis buffer, once with kinase buffer (30 mM HEPES (pH
7.5), 10 mM MgCl2, and 2 mM
MnCl2), and then resuspended in 20 µl of kinase buffer. The reaction was started by the addition of 5 µCi of
[-32P]ATP (3,000 Ci/mmol, PerkinElmer Life Sciences),
and the samples were incubated for 10 min at room temperature.
Phosphorylation was stopped by the addition of the Laemmli sample
buffer, and the samples were subjected to SDS-PAGE followed by
autoradiography. We used normal mouse IgG, anti-NMDA receptor antibody,
and anti-integrin 5 antibody as negative controls.
Density Gradient Analysis--
TAG-1 distribution on a density
gradient was investigated by three different methods. Sucrose gradient
analysis with sodium carbonate was performed according to a method
described previously (26). Rat primary cerebellar cultures in a 35-mm
dish were homogenized using a Teflon glass homogenizer and sonicated in
2 ml of 500 mM sodium carbonate at pH 11.0. The sucrose
content in the homogenate was then adjusted to 45% by the addition of
90% sucrose in MBS (25 mM MES (pH 6.5), 0.15 M
NaCl). A linear sucrose gradient (5-35%) in 6 ml of MBS containing
250 mM sodium carbonate was layered over the lysate. The
gradients were centrifuged for 16-20 h at 39,000 rpm at 4 °C in a
Hitachi RPS40T rotor. 10 × 1-ml fractions were collected from the
top of the gradient.
Sucrose gradient analysis with Triton X-100 was performed according to
a method described previously (27). The cells were homogenized using a
Teflon glass homogenizer in 2 ml of TNE/Triton X-100 buffer (1% Triton
X-100, 25 mM Tris-HCl (pH 7.5), 150 mM NaCl,
and 1 mM EGTA). The sucrose content in the homogenate was then adjusted to 40% by the addition of 80% sucrose. A linear sucrose
gradient (5-30%) in 6 ml of TNE without Triton X-100 was layered over
the lysate. The gradients were centrifuged for 16-20 h at 39,000 rpm
at 4 °C in a Hitachi RPS40T rotor. 10 × 1 ml fractions were
collected from the top of the gradient.
OptiPrep gradient analysis was performed according to a previously
described method with some modifications (28). The cells were
homogenized using a Teflon glass homogenizer and a sonicator in 2 ml of
buffer A (0.25 M sucrose, 1 mM EDTA, and 20 mM Tricine (pH 7.8)). The OptiPrep content in the
homogenate was then adjusted to 25% by the addition of 50% OptiPrep
in buffer A. A linear OptiPrep gradient (10-20%) in 6 ml of buffer A
was layered over the lysate. The gradients were centrifuged for 16-20
h at 39,000 rpm at 4 °C in a Hitachi RPS40T rotor. 10 × 1 ml
fractions were collected from the top of the gradient.
After trichloroacetic acid precipitation of each fraction, TAG-1 was
detected by immunoblotting using the anti-TAG-1 polyclonal antibody.
Caveolin in CHO cells was used as a marker protein of the lipid raft
fraction, because caveolin was not detected in rat cerebellar cultures.
After CHO cells in a 35-mm dish were subjected to density gradient
analysis, caveolin in each fraction was detected by immunoblotting using an anti-caveolin polyclonal antibody.
Distribution of tyrosine phosphorylation of p80 was investigated by
sucrose density gradient with Triton X-100. After antibody-mediated cross-linking of TAG-1 or GD3 in cerebellar cultures in five 35-mm dishes, the Triton X-100 extracts were subjected to density gradient analysis. For the detection of the tyrosine phosphorylation of proteins, each fraction was subjected to SDS-PAGE and immunoblotting with the anti-phosphotyrosine antibody.
EGCase Treatment--
EGCase II ACT (1 units/ml EGCase II, 0.5 mM activator II) was diluted with the culture medium and
applied to the culture at 2-3 days in vitro at a final
concentration of 2 to 60 milliunits/ml (EGCase) and 1-30
µM (activator). To investigate the reversibility of the
EGCase effect, the cells were washed and cultured with the conditioned
medium after the EGCase treatment. Heat-inactivated EGCase was prepared
by boiling for 5 min. The colorimetric assay of cell viability was
performed using MTT according to the manufacturer's instructions
(Chemicon International, Temecula, CA).
We investigated the EGCase effect on the following: 1) cell-surface
ganglioside GD3 expression; 2) Lyn activation by TAG-1 cross-linking;
3) tyrosine phosphorylation of p80 by TAG-1 or GD3 cross-linking; 4)
cell morphology; 5) cell viability; and 6) TAG-1 distribution on
density gradient.
Flow Cytometry--
After treatment with EGCase, the rat primary
cerebellar cultures were harvested. The cells were first treated with
R24, then with FITC-conjugated anti-mouse IgG antibody, and analyzed on a FACSCalibur (Becton-Dickinson).
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RESULTS |
Anti-ganglioside GD3 Antibody Coprecipitates p135--
The
immunoprecipitate obtained with the anti-GD3 antibody (R24) from the
Triton X-100 extract of rat primary cerebellar cultures metabolically
labeled with [35S] methionine was analyzed by
autoradiography. R24 coimmunoprecipitated two proteins of 135 and 16 kDa, as revealed by SDS-PAGE (Fig. 1).
This observation suggests that ganglioside GD3 associates with these
two proteins.
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Fig. 1.
Anti-ganglioside GD3 antibody
(R24)-precipitated protein from primary cerebellar cultures labeled
with 35S. Primary cerebellar cultures metabolically
labeled with 35S were solubilized in lysis buffer. The
supernatants were immunoprecipitated with R24, and the
immunoprecipitates were subjected to SDS-PAGE and autoradiography.
Lane 1, precipitate with control mouse IgG3; lane
2, precipitate with R24. Arrow indicates p135.
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Identification of p135 as the GPI-anchored Neuronal Cell Adhesion
Molecule, TAG-1--
125I surface labeling and
immunoprecipitation with R24 were performed to investigate whether or
not the 135-kDa protein (p135) was a surface protein. p135, but not the
16-kDa protein, was also coprecipitated with R24 from the
surface-labeled cells (Fig.
2A). The appearance of another
smaller band was possibly due to the degradation of p135 by trypsin
during cell culture, since the cell surface was labeled 12 h after
trypsinization.
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Fig. 2.
p135 is surface-labeled and released by
PI-PLC treatment of primary cerebellar cultures. A, primary
cerebellar cultures whose p135 was surface-labeled with
125I were solubilized in lysis buffer. The supernatants
were immunoprecipitated with R24, and the immunoprecipitates were
subjected to SDS-PAGE and autoradiography. Lane 1 is same as
lane 2 of Fig. 1. Lane 2, precipitate with
control mouse IgG3; lane 3, precipitate with R24.
B, the cultures with surface-labeled p135 were treated with
PI-PLC. Lane 1, immunoprecipitates with R24; lane
2, cell lysate; lane 3, culture supernatant.
Right lane, PI-PLC-treated cells, left lane,
untreated cells. Proteins were visualized by autoradiography.
Arrow indicates p135.
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p135 was not coprecipitated with R24 after treatment of the
surface-labeled cells with PI-PLC (Fig. 2B, lane 1). PI-PLC
treatment released p135 from the cell surface (Fig. 2B, lane
2) into the culture medium (Fig. 2B, lane 3),
indicating that p135 is a GPI-anchored protein.
p135 was identified as the GPI-anchored neuronal cell adhesion
molecule, TAG-1, by sequential immunoprecipitation with R24 and the
anti-TAG-1 antibody. Immunoprecipitation with R24 was performed using
cerebellar cultures metabolically labeled with [35S]methionine, after which the immune complexes were
disrupted by boiling in SDS-containing buffer (Fig.
3, lane 5) and subjected to
second immunoprecipitation with antibodies specific for GPI-anchored neuronal cell adhesion molecules, i.e. TAG-1, contactin/F3,
NCAM, and T-cadherin. The anti-TAG-1 polyclonal antibody, but not
anti-contactin/F3, anti-NCAM, anti-T-cadherin antibodies, specifically
precipitated p135 in the re-immunoprecipitation experiment (Fig. 3,
lane 1).
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Fig. 3.
Identification of p135 as the neuronal cell
adhesion molecule, TAG-1. The R24 precipitates from the cerebellar
cultures labeled with 35S (lane 5) were eluted
by boiling in 1% SDS. After 10-fold dilution with lysis buffer,
re-immunoprecipitation was performed with anti-TAG-1 (lane
1), anti-contactin/F3 (lane 2), anti-NCAM (lane
3), and anti-T-cadherin (lane 4) polyclonal antibody,
respectively. The immunoprecipitates were subjected to SDS-PAGE and
autoradiography. Arrow indicates p135.
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Antibody-mediated Cross-linking of TAG-1 Activates Lyn Signaling in
Lipid Rafts--
To examine the possibility of TAG-1-mediated signal
transduction via Lyn, we measured the Lyn activity in cerebellar
cultures after antibody-mediated cross-linking of TAG-1. Triton X-100
extracts were prepared from cerebellar cultures, which were
sequentially treated with the anti-TAG-1 monoclonal antibody for 30 min
on ice and secondary antibody for the indicated time at 37 °C.
Immunoprecipitation was carried out with the anti-Lyn antibody. Kinase
activity was measured by in vitro autophosphorylation.
Treatment with the secondary antibody resulted in a rapid (within 1 min) and significant (3.5-fold) increase in Lyn activity (Fig.
4, lanes 1-5).
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Fig. 4.
Lyn activation induced by antibody-mediated
cross-linking of TAG-1 in rat primary cerebellar cultures. After
antibody-mediated cross-linking of TAG-1 or NMDA receptors for the
indicated times, lysates were prepared and immunoprecipitated with
anti-Lyn monoclonal antibody. Kinase activity was measured by in
vitro auto-phosphorylation. Cross-linking of TAG-1 was allowed to
proceed for 1 (lane 2), 3 (lane 3), 7 (lane
4), and 15 min (lane 5). Mock cross-linking using
normal mouse IgG was allowed for 1 min (lane 1).
Cross-linking of NMDA receptors was allowed to proceed for 3 min
(lane 7). Mock cross-linking using normal rabbit IgG was
allowed for 3 min (lane 6). Arrows indicate
p53Lyn and p56Lyn.
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The possible increase in tyrosine phosphorylation of cellular proteins
as a result of antibody-mediated cross-linking of TAG-1 was
investigated. After SDS-PAGE, phosphotyrosines were detected by
anti-phosphotyrosine immunoblotting. Treatment with the secondary antibody induced tyrosine phosphorylation of several proteins, including the prominent phosphorylation of p80 (Fig.
5A). The phosphorylation
peaked at 2 min and returned to the control level at 20 min. Treatment
with the anti-TAG-1 monoclonal antibody only at 37 °C, even at 100 µg/ml, resulted in no change in tyrosine phosphorylation of p80 (data
not shown). p80 was also phosphorylated by antibody-mediated
cross-linking of ganglioside GD3 (Fig. 5B). Mock
cross-linking with normal mouse IgG and antibody-mediated cross-linking
of NMDA receptor did not induce increase in Lyn activity (Fig. 4,
lanes 6 and 7) and protein tyrosine
phosphorylation (Fig. 5D). Phosphacan, a neural
tissue-specific chondroitin sulfate proteoglycan, is known to be one of
the high affinity ligands of TAG-1 (29). Sequential treatment of
cerebellar cultures with phosphacan and anti-phosphacan antibody also
induced tyrosine phosphorylation of p80 (Fig. 5C).
Furthermore, PP1 and PP2, selective inhibitor for Src family kinases,
significantly reduced tyrosine phosphorylation of p80 by
antibody-mediated cross-linking of TAG-1 (Fig.
6, lanes 3-6). However, the
inactive compound PP3 did not reduce (Fig. 6, lanes 7 and
8). These observations suggest that TAG-1 transduces signals
via Lyn to p80 in the rat cerebellum.
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Fig. 5.
Tyrosine phosphorylation of p80 induced by
antibody-mediated cross-linking of TAG-1 or by phosphacan-mediated
stimulation. A, time-dependent tyrosine
phosphorylation of the protein. After antibody mediated cross-linking
of TAG-1 for the indicated times, the lysates were subjected to
SDS-PAGE and immunoblotted with anti-phosphotyrosine antibody.
Lane 1, 0 min; lane 2, 1 min; lane 3,
2 min; lane 4, 4 min; lane 5, 6 min; lane
6, 8 min; lane 7, 10 min; lane 8, 12 min;
lane 9, 14 min; lane 10, 16 min. B,
tyrosine phosphorylation of p80 induced by antibody-mediated
cross-linking of ganglioside GD3. Mock cross-linking was allowed for 3 min (lane 1), TAG-1 cross-linking for 3 min (lane
2), and GD3 cross-linking for 3 min (lane 3).
C, tyrosine phosphorylation of p80 by phosphacan-mediated
stimulation. Untreated cells (lane 1), phosphacan-treated
cells (lane 2), cells sequentially treated with phosphacan
and anti-phosphacan antibody (lane 3), and cells treated
with anti-phosphacan antibody alone (lane 4) are shown.
D, tyrosine phosphorylation of proteins induced by
antibody-mediated cross-linking of NMDA receptors. Untreated cells
(lane 1), cells treated with anti-NMDA receptor antibody for
3 min (lane 2), cells sequentially treated with anti-NMDA
receptor, and anti-rabbit IgG secondary antibody for 3 min (lane
3) are shown. Arrow indicates p80.
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Fig. 6.
Effect of selective inhibitors for Src family
kinases on tyrosine phosphorylation of p80. Tyrosine
phosphorylation of p80 induced by antibody-mediated cross-linking of
TAG-1 for 3 min after 10 min pretreatment with inhibitors (lane
3-8). Mock cross-linking was allowed for 3 min (lane
1) and TAG-1 cross-linking for 3 min (lane 2) without
the pretreatment. Pretreatment with 1 µM PP1 (lane
3), 10 µM PP1 (lane 4), 1 µM PP2 (lane 5), 10 µM PP2
(lane 6), 1 µM PP3 (lane 7), 10 µM PP3 (lane 8) is shown. Tyrosine
phosphorylation of p80 was visualized by anti-phosphotyrosine
immunoblotting.
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Previously, we found that R24 treatment of primary cerebellar cultures
induced Lyn activation and tyrosine phosphorylation of p80 and that Lyn
was present in the lipid raft fraction of sucrose density gradient
analysis (21). Therefore, we investigated whether tyrosine
phosphorylation of p80 was detected in the lipid raft fraction.
Antibody-mediated cross-linking of TAG-1 or GD3 induced tyrosine
phosphorylation of p80 in low density lipid raft fractions 3-5 on
sucrose density gradient (Fig. 7,
B and C). TAG-1 was also detected in the lipid
raft fraction by immunoblotting (Fig. 7D). In contrast,
tyrosine phosphorylation of about 110-kDa protein, but not p80, and
most cellular proteins were detected in high density sample fractions
7-10 (Fig. 7, A-C and E). These observations
suggest that TAG-1 transduces signal via Lyn in lipid rafts of rat
cerebellum and that antibody-mediated cross-linking of ganglioside GD3
can mimic the TAG-1 signaling.
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Fig. 7.
Tyrosine phosphorylation of p80 in lipid
rafts. After antibody-mediated cross-linking of TAG-1 or GD3 in
cerebellar cultures, the Triton X-100 extracts were subjected to
sucrose density gradient analysis. For the detection of tyrosine
phosphorylation of proteins, each fraction was subjected to SDS-PAGE
and immunoblotting with anti-phosphotyrosine antibody. A,
mock cross-linking; B, TAG-1 cross-linking; C,
ganglioside GD3 cross-linking. TAG-1 was detected by immunoblotting
(D). The cellular proteins of D were stained with
Ponceau S (E). Arrow indicates p80.
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Association of Protein Kinase Activity with TAG-1 and Ganglioside
GD3--
Previously we found that R24 coprecipitated protein kinase
activity from the rat cerebellum (21). Therefore, we investigated whether the protein kinase activity was precipitated or not by the
anti-TAG-1 monoclonal antibody (Fig.
8A). Proteins of 80, 56, 53, and 40 kDa were phosphorylated as revealed by an immune complex kinase
assay using R24 from postnatal days 5-21. In addition, a 46-kDa
protein was phosphorylated as revealed by an immune complex kinase
assay using R24 from postnatal days 5-14. Proteins of 56 and 53 kDa
were identified as two isoforms of the Src family kinase Lyn by
reimmunoprecipitation with the anti-Lyn antibody (Fig. 8B,
lane 3). The 46-kDa protein was also detected by the immune complex kinase assay using the anti-TAG-1 antibody from postnatal days
5-14 at the same level as that of R24. However, the phosphorylation of
Lyn and the 80- and 40-kDa proteins was weak in contrast with that by
R24. This indicates that association with TAG-1 of Lyn and the 40-kDa
and 80-kDa proteins is weak or unstable in comparison to their
association with ganglioside GD3. The anti-NMDA receptor antibody and
anti-integrin 5 antibody also precipitated protein kinase activity. However, Lyn and the 80- and 40-kDa proteins were not
detected by the immune complex kinase assay (Fig. 8C, lanes
3 and 4). Furthermore, normal mouse IgG precipitated no protein kinase activity (Fig. 8C, lane 1). Therefore, the
association of Lyn and the 80- and 40-kDa proteins with GD3 and TAG-1
is specific. Furthermore, the level of phosphorylation of Lyn and the
40- and 80-kDa proteins coprecipitated with anti-TAG-1 antibody
gradually decreased from postnatal days 5-21 in contrast with the
constant level of phosphorylation of proteins coprecipitated with R24. TAG-1 and Lyn are expressed in cerebellum from newborn to adult rats
(30, 31). Consistent with this, the protein levels of TAG-1 and Lyn
were not changed from postnatal days 5-21 (data not shown). These
observations suggest a possibility that the association of TAG-1 with
Lyn may be transient during the early stage of cerebellar
development.
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Fig. 8.
Protein kinase activity coprecipitated with
anti-TAG-1 antibody or R24 from cerebellum. A, the membrane
fractions of newborn rat cerebella (P5-P21) were solubilized in 1%
Triton X-100 lysis buffer. The supernatants were immunoprecipitated
with anti-TAG-1 monoclonal antibody (lane T) or R24
(lane G), and the immunoprecipitates were subjected to an
in vitro kinase assay and SDS-PAGE. Phosphorylation was
visualized by autoradiography. P5 (lane 1), P10 (lane
2), P14 (lane 3), P17 (lane 4), and P21
(lane 5). B, identification of the 56- and 53-kDa
proteins as the Src family kinase Lyn. R24 precipitates from the rat
cerebella (P7) were subjected to an in vitro kinase reaction
(lane 1) and eluted by boiling in 1% SDS. After 10-fold
dilution with lysis buffer, reimmunoprecipitation was allowed to
proceed with normal mouse IgG (lane 2) and anti-Lyn antibody
(lane 3). C, protein kinase activity from the rat
cerebella (P5) coprecipitated with normal rabbit IgG (lane
1), anti-Lyn (lane 2), anti-NMDA receptor (lane
3), and anti-integrin 5 antibody (lane
4).
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Density Gradient Analysis of TAG-1--
Although GPI-anchored
proteins associate with lipid rafts during signal transduction, there
is some debate as to whether GPI-anchored proteins associate with lipid
rafts under the steady state (32, 33). Therefore, three different
methods of density gradient analysis were performed to investigate
whether TAG-1 associates with lipid rafts in cerebellar cultures.
Distribution of caveolin in lipid raft fractions for each method is
shown in Fig. 9 (upper panels). Most of the TAG-1 was present in the lipid raft fractions 3-5 in sucrose gradients with sodium carbonate (pH 11) and sucrose gradient with Triton X-100 (Fig. 9, A and B,
lower panesl). However, most of the TAG-1 was absent in
lipid raft fractions 1-6 in OptiPrep gradients (without detergent,
neutral pH) (Fig. 9C). The discrepancy may be explained by a
previous finding that the GPI-anchored protein was concentrated in
lipid rafts after detergent treatment (34). Therefore, results of Fig.
8 and Fig. 9 suggest that TAG-1 is associated with lipid rafts, but the
association may be unstable. However, two membrane subdomains,
ganglioside-microdomains and caveolin-enriched microdomains, seem to
exist among lipid rafts (35). Therefore, we cannot deny the possibility
that discrepancy of distribution between TAG-1 and caveolin on OptiPrep
gradient may be due to separation of the subdomains.
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Fig. 9.
Density gradient analysis of TAG-1 in primary
cerebellar cultures. The cultures in a 35-mm dish were homogenized
in each buffer, and linear density gradients were formed over them. Ten
fractions were collected from top to bottom after
centrifugation. TAG-1 of the cerebellar cultures was visualized in each
fraction by immunoblotting (lower panel). A,
sodium carbonate (pH 11) and sucrose gradient 5-35%; B,
1% Triton X-100 and sucrose gradient 5-30%; C, Tricine
(pH 7.8) and OptiPrep gradient 10-20%. Caveolin of CHO cells was
visualized by immunoblotting (upper panel).
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Effect of GSL Hydrolysis by EGCase on TAG-1 Signaling--
In the
present study, anti-GSL antibody coimmunoprecipitated TAG-1, and
antibody-mediated cross-linking of GSL mimicked TAG-1 signaling in
lipid rafts. What is the role of GSL in TAG-1 signaling? EGCase is an
enzyme that specifically hydrolyzes the linkage between carbohydrates
and ceramides in GSLs (36). With the aid of an activator protein, it is
capable of releasing the carbohydrate moiety of GSLs on the cell
surface without affecting other lipids and glycoproteins (37, 38).
Treatment of cerebellar cultures with EGCase and an activator protein
greatly reduced the cell-surface ganglioside GD3 content (Fig.
10A) and R24-mediated
tyrosine phosphorylation of p80 (Fig. 10C, lane 4).
Interestingly, EGCase treatment reduced TAG-1-mediated Lyn activation
(Fig. 10B, lanes 3 and 4) and tyrosine
phosphorylation of p80 (Fig. 10C, lane 5) with a concomitant
neurite retraction (Fig. 11D,
panel b). The EGCase effect on TAG-1-mediated tyrosine phosphorylation of p80 was dose- and time-dependent (Fig.
11, A and B) and reversible (Fig.
11C). Hydrolysis of cell-surface GSL by EGCase induces an
increase in GSL synthesis de novo for maintaining the
cell-surface GSL contents (36). Consistent with this, GD3 expression
was restored by removal of EGCase (Fig. 11E, panel c). TAG-1-mediated tyrosine phosphorylation of p80 and neurites are also
restored following the removal of EGCase (Fig. 11C, panel c,
and D, panel c). EGCase treatment did not decrease cell
viability as determined by the MTT method under this condition. No
change in TAG-1-mediated tyrosine phosphorylation of p80 was observed following treatment with heat-inactivated EGCase (data not shown). Therefore, the inhibition of TAG-1-mediated signaling is probably due
to the degradation of cell-surface GSLs. Furthermore, EGCase treatment
altered TAG-1 distribution on the OptiPrep gradient to a low density
fraction (Fig. 12C).
Similarly, although slightly, the alteration was also observed in
sucrose density gradients. TAG-1 was additionally detected in fraction
2 with sodium carbonate (Fig. 12A) and in fraction 4 with
Triton X-100 (Fig. 12B) after EGCase treatment. This
suggests that degradation of cell-surface GSLs may affect the
properties of lipid rafts. Taken together, these results indicate that
GSLs are involved in TAG-1 signaling in lipid rafts.
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Fig. 10.
Effect of EGCase treatment on TAG-1
signaling. A, flow cytometric analysis of GD3 expression.
After treatment with 50 milliunits/ml of EGCase at 37 °C for 6 h, the cells were stained with R24, followed by that with
FITC-conjugated anti-mouse IgG antibody. Untreated cells (thick
line) and EGCase-treated cells (thin line). Cells
stained with FITC-conjugated anti-mouse IgG antibody alone (dot
line) are indicated as the control. B, effect of EGCase
on Lyn activation induced by TAG-1 cross-linking. Lyn activity was
measured by in vitro autophosphorylation after mock
cross-linking (lane 1) and TAG-1 cross-linking (lane
2) for 1 min of untreated cells, or after mock cross-linking
(lane 3) and TAG-1 cross-linking (lane 4) for 1 min of EGCase-treated cells. C, effect of EGCase on tyrosine
phosphorylation of p80 induced by the antibody-mediated cross-linking
of TAG-1 or the ganglioside GD3. Mock cross-linking (lane
1), GD3 cross-linking (lanes 2 and 4), TAG-1
cross-linking (lanes 3 and 5); untreated cells
(lanes 1-3), EGCase-treated cells (lanes 4 and
5). Arrow indicates tyrosine phosphorylation of
p80.
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Fig. 11.
Dose- and time-dependent and
reversible effect of EGCase on TAG-1 signaling and cell morphology.
A-C, measurement of tyrosine phosphorylation of p80.
D, cell morphology; E, cell-surface GD3
expression. A, dose-dependent effect of EGCase
treatment for 6 h on TAG-1-mediated tyrosine phosphorylation of
p80. The concentrations of EGCase and the activator are 2 milliunits/ml
and 1 µM (lane 2), 5 milliunits/ml and 2.5 µM (lane 3), 10 milliunits/ml and 5 µM (lane 4), and 60 milliunits/ml and 30 µM (lane 5), respectively; untreated cells
(lane 1). Relative percentage of tyrosine phosphorylation of
p80 by TAG-1 cross-linking in untreated cells is indicated.
B, time-dependent effect of EGCase treatment on
TAG-1-mediated tyrosine phosphorylation of p80. The concentrations of
EGCase and the activator are 50 milliunits/ml and 25 µM,
respectively. Untreated cells (lane 1), and cells treated
for 2 (lane 2), 4 (lane 3), and 6 h
(lane 4). C-E, reversible effect of treatment
with 50 milliunits/ml EGCase. C, TAG-1-mediated tyrosine
phosphorylation of p80, D, time course of changes of cell
morphology; E, flow cytometric analysis of GD3 expression;
the dotted lines are indicated as the control. Untreated
cells (a), EGCase-treated cells for 5 h (b),
and cells 12 h after the removal of EGCase (c).
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Fig. 12.
Effect of EGCase treatment on TAG-1
distribution in a density gradient. Sodium carbonate (pH 11) and
sucrose gradient 5-35% (A), 1% Triton X-100 and sucrose
gradient 5-30% (B), Tricine (pH 7.8) and OptiPrep gradient
10-20% (C). Untreated cells (upper panel) and
EGCase-treated cells (lower panel) were subjected to density
gradient analysis. TAG-1 in each fraction was visualized by
immunoblotting.
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DISCUSSION |
TAG-1 Signaling via Src Family Kinase Lyn in Rat
Cerebellum--
In our study, we have shown the association of
ganglioside GD3 with GPI-anchored neuronal cell adhesion molecule,
TAG-1, and the Src family kinase Lyn by a coimmunoprecipitation technique.
Several GPI-anchored proteins have been implicated in transmembrane
signaling via Src family tyrosine kinases (39, 40). In leukocytes,
antibodies against a variety of GPI-anchored proteins coimmunoprecipitate Src family kinases. Antibody-mediated cross-linking of GPI-anchored proteins induces transient tyrosine phosphorylation of
several substrates and, concomitantly, cell activation (40). Some
groups have reported coprecipitation of Src family kinases with
GPI-anchored proteins in the nervous system. Src family kinase Fyn is
coprecipitated with Thy-1 in the chick brain (41), with contactin/F3 in
the mouse cerebellum (42) and with NCAM120 in oligodendrocytes (43).
Antibody-mediated cross-linking of F11(chicken contactin/F3) on
embryonic chick neuronal cells induces Fyn activation (44).
Antibody-mediated cross-linking of contactin/F3 on oligodendrocytes induces Fyn activation (43). In the present study, we found that Lyn
can interact with TAG-1 in the rat cerebellum and that antibody-mediated cross-linking of TAG-1 induced Lyn activation in rat
primary cerebellar cultures. These observations show that TAG-1
transduces signals via Lyn in the rat cerebellum.
The main site of TAG-1 signaling via Lyn in the cerebellum is probably
the granule cells. Indeed, both TAG-1 and Lyn are predominantly expressed in the cerebellar granule cells, as has been shown by in situ hybridization and immunohistochemical studies (30,
31, 45-47). During development, cerebellar granule cells in the
external granular layer undergo proliferation, axon outgrowth,
migration to the internal granular layer, and formation of synaptic
connections (48). Regulation of tyrosine phosphorylation through Src
family kinases is critical in the control of neural development (49). For example, Fyn-deficient mice exhibit impaired myelination
(50). It has been shown that Src family kinases are involved in the intracellular signaling pathway for responses by the neural cell adhesion molecules of the immunoglobulin superfamily (51). TAG-1, a
GPI-anchored neuronal cell adhesion molecule of the immunoglobulin superfamily, is involved in neurite outgrowth, pathfinding, and fasciculation (52). Therefore, TAG-1 signaling via Lyn might play a
role in the development of cerebellar granule cells.
Isolation of Lipid Rafts by Anti-GSL Antibody--
GPI-anchored
proteins are restricted to the outer leaflet of the lipid bilayer and
are integrated into the membrane by GPI anchors. These lipid anchors
have no direct contact with the cytoplasm. Src family kinases anchor
onto the inner leaflet via N-terminal lipid modification,
palmitoylation, and myristoylation. How do GPI-anchored proteins
transduce signals via Src family kinases? This is probably possible via
the lipid rafts that are rich in GSLs, cholesterol, GPI-anchored
proteins, and signaling molecules such as Src family kinases (53, 54).
The basic forces driving lipid raft formation are considered to be
lipid interactions, which are weak and transient (16, 55). The
saturated acyl chains and high acyl chain-melting temperature of GSLs
mediate GSL clustering in combination with cholesterol, which has the properties of a "liquid-ordered phase" with restricted fluidity (16). In contrast, most phospholipids have unsaturated acyl chains, low
melting temperature, and the properties of a "liquid phase" with
high fluidity. Lipid rafts may exist as phase-separated domains in the
membrane. Interactions between the carbohydrates of GSLs by hydrogen
bonds is also assumed to contribute to raft formation (19). The linkage
of GPI-anchored proteins and Src family kinases to saturated acyl
chains is considered to facilitate targeting to lipid rafts. The lipid
composition renders lipid rafts and their constituent proteins
resistant to solubilization with non-ionic detergents. Thus, lipid
rafts can be isolated as low density membrane fractions in sucrose
density gradients with detergents. Lipid rafts can be also isolated in
OptiPrep gradients without detergents or by immunoprecipitation with
the anti-caveolin antibody (17). We found that the anti-GSL antibody
coprecipitates TAG-1, Lyn, and caveolin, suggesting that
immunoprecipitation with this antibody is an immunoabsorption method
for isolating lipid rafts. Consistent with this idea, other anti-GSL
antibodies such as anti-ganglioside GM3, anti-ganglioside 
GalGD1b,
and anti-globotriaosylceramide Gb3 can immunoprecipitate Src family
kinases and GPI-anchored proteins (56-59).
Role of GSL in GPI-anchored Protein Signaling in Lipid
Rafts--
A number of studies have examined the influence of
cholesterol on the physical properties and signaling of lipid rafts by (i) cholesterol depletion using cyclodextrin or by oxidation to cholestenone, (ii) inhibition of cholesterol biosynthesis by compactin or lovastatin, and (iii) using cholesterol-binding agents, such as
filipin or nystatin. Cholesterol depletion results in an increase in
solubility of GPI-anchored proteins in non-ionic detergents and
perturbation of raft-mediated signaling (16, 17, 60-64).
In contrast to cholesterol, only a few studies have examined the
influence of GSL, another core lipid of lipid rafts, on the physical
properties and signaling in lipid rafts (65, 66). Inhibition of
sphingolipid biosynthesis results in an increase in solubility of
GPI-anchored proteins in non-ionic detergents (62). Single particle
tracking of GPI-anchored proteins and GSL in native membranes showed
their transient confinement in patches, which is assumed to represent
lipid rafts in vivo (67). The size of the confining domain
for the GPI-anchored protein is reduced by treatment with inhibitors of
GSL biosynthesis (68). These observations suggest that GSLs can
regulate the physical properties of lipid rafts.
What is the role of GSL in raft-mediated signaling? In the present
study, we showed that GSL signaling and GPI-anchored protein signaling
have many properties in common. (i) Both the anti-ganglioside GD3
antibody (R24) and anti-TAG-1 antibody (3.1C12) coprecipitate protein
kinase activity. Proteins of 80 and 40 kDa, and 56/53 kDa:Lyn are
phosphorylated as revealed by immune complex kinase assays. (ii)
Cross-linking mediated by both R24 and 3.1C12 induces Lyn activation.
(iii) Cross-linking mediated by both R24 and 3.1C12 induces rapid
tyrosine phosphorylation of p80 in lipid rafts. It was reported that
both the anti-ganglioside GalGD1b antibody and anti-Thy-1 antibody
coprecipitate Lyn, as well as phosphoproteins of 80 and 40 kDa.
Cross-linking mediated by these antibodies also induces Lyn activation
in lipid rafts, rapid tyrosine phosphorylation of proteins including an
80-kDa protein, and calcium flux in rat basophilic leukemia RBL-2H3
cells (58, 69-72). The 80-kDa phosphoprotein as revealed by the immune
complex kinase assays is identical to the 80-kDa protein phosphorylated
in RBL-2H3 cells by antibody cross-linking (58). Treatment with the
anti-GSL antibody can mimic GPI-anchored protein signaling, suggesting
that GPI-anchored proteins and GSLs transduce signals into the cell
using the same signaling pathway. Several groups also reported that
antibodies against various GPI-anchored proteins and GSLs coprecipitate
protein kinase activity. Proteins of 80 and 40 kDa and 60-53-kDa:Src
family kinases are phosphorylated at the tyrosine residue as indicated by the immune complex kinase assays (40, 41, 58, 73-75). GPI-anchored
ephrin-A5 also induces tyrosine phosphorylation of an 80-kDa protein in
the lipid raft fraction by binding its receptor, and the
phosphorylation was abrogated in the presence of a selective inhibitor
against Src family kinases and in Src family kinase-deficient mice
(76). These observations suggest that p80 may be a membrane protein in
lipid rafts and a putative substrate for Src family kinases. Thus, Src
family kinases, p80 and 40-kDa protein are sets of signaling components
in lipid rafts for both GPI-anchored proteins and GSLs.
We used the GSL-degrading enzyme, EGCase, to investigate the function
of GSLs in lipid rafts. EGCase specifically hydrolyzes cell-surface
GSLs of intact cells with the assistance of its activator protein
(36-38). The application of EGCase to living cells provides a novel
approach to determining the physiological roles of endogenous GSLs. In
the present study, we showed that EGCase treatment reduced cell-surface
GSL and impairs TAG-1-dependent signaling via Lyn. GD3
expression and TAG-1-mediated tyrosine phosphorylation of p80 was
restored by removal of EGCase. The inhibition of TAG-1-mediated signaling by EGCase is not due to degradation of TAG-1 by protease contaminants in EGCase, because TAG-1 protein levels were not changed
following EGCase treatment (Fig. 12). This is also not due to its
cytotoxic effects, because EGCase treatment did not decrease cell
viability and the EGCase effect was reversible. Therefore, the
inhibition of TAG-1-mediated signaling is probably due to the
degradation of GSLs and that cell-surface GSLs are probably necessary
for TAG-1 signaling in lipid rafts. We also found that EGCase treatment
induces neurite retraction. However, the molecular mechanism underlying
this remains unknown.
What is the mechanism underlying inhibition of TAG-1-mediated signaling
by EGCase? We found that enzymatic removal of the carbohydrate moiety
from GSLs by EGCase alters the TAG-1 distribution on an OptiPrep
gradient. This suggests that carbohydrate moiety of GSLs can affect the
interaction of TAG-1 with lipid rafts or the physical properties of
lipid rafts. A recent report showed that exogenously administered
ganglioside, which was incorporated in lipid raft fraction, displaced
GPI-anchored proteins from lipid rafts and increased their detergent
solubility (77, 78). This finding suggests that GSL can affect
distribution of GPI-anchored proteins in lipid rafts. Taken together,
our results suggest that GSLs are involved in GPI-anchored protein
signaling in lipid rafts. Further experiments are required to clarify
the role of GSLs in signal transduction by GPI-anchored proteins.
| |
ACKNOWLEDGEMENTS |
We are indebted to Dr. Makoto Ito (Kyushu
University) for technical assistance with EGCase treatment. We thank
Dr. Vance Lemmon (Case Western Reserve University) and Dr. Hiroaki Aso
(Tokyo Metropolitan Institute of Gerontology) for the helpful
discussion. We also thank Dr. Youichi Tajima and Dr. Yumiko Watanabe
(Department of Biochemical Cell Research) for the technical assistance
with flow cytometry. We are grateful to Dr. Yoshitaka Nagai
(Mitsubishi Kasei Institute of Life Sciences, Tokyo) for
encouraging this work.
| |
FOOTNOTES |
*
This work was supported by Grants-in-aid 11121237 (to
K. K.) and 10178102 (to Y. S.) for Scientific Research on Priority
Areas, by Grant-in-aid 10780376 (to K. K.) for Encouragement of Young Scientists, by Grant-in-aid 10480173 (to Y. S.) for Scientific Research (B) from the Ministry of Education, Science, Sports and Culture of Japan, and by the Yamada Science Foundation.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.
§
To whom correspondence should be addressed: Dept. of
Biochemical Cell Research, The Tokyo Metropolitan Institute of Medical Science, Tokyo Metropolitan Organization for Medical Research, 3-18-22, Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan. Tel.: 81-3-3823-2101, ext. 5235; Fax: 81-3-3828-6663; E-mail: kasahara@rinshoken.or.jp.
Present address: Dept. of Biological Science, Graduate School
of Science, Nagoya University, Nagoya 464-8602, Japan.
Published, JBC Papers in Press, August 15, 2000, DOI 10.1074/jbc.M003163200
2
The nomenclature for gangliosides follows the
system of Svennerholm (79).
| |
ABBREVIATIONS |
The abbreviations used are:
GSL, glycosphingolipid;
GPI, glycosylphosphatidylinositol;
PI-PLC, phosphatidylinositol-specific phospholipase C;
NMDA, N-methyl-D-aspartate;
FITC, fluorescein
isothiocyanate;
EGCase, endoglycoceramidase;
MTT, [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide];
PAGE, polyacrylamide gel electrophoresis;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine;
MES, 4-morpholineethanesulfonic acid;
PBS, phosphate-buffered saline;
BSA, bovine serum albumin;
CHO, Chinese hamster ovary;
PCR, polymerase chain
reaction.
| |
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TOP
ABSTRACT
INTRODUCTION
