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J. Biol. Chem., Vol. 278, Issue 33, 31419-31425, August 15, 2003
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Activity by Glycosphingolipids*
From the Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan 48109-0676
Received for publication, April 10, 2003 , and in revised form, May 23, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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1. In the present study, the depletion of endogenous
glucosylceramide by D-t-EtDO-P4 in cultured ECV304 cells
induced autophosphorylation of Src kinase at tyrosine residue 418 within the
catalytic loop and dephosphorylation of Src kinase at tyrosine residues 529
within the carboxyl-terminal regulatory region. Phosphotransferase activities
of Src kinase were also induced in the glucosylceramide-depleted cells. c-Src
kinase activity and phosphorylations at Src Tyr-418 and epidermal growth
factor (EGF) receptor Tyr-1068 were significantly enhanced by bradykinin in
response to 100 nM D-t-EtDO-P4 compared with control
cells. The phosphorylation and dephosphorylation on Tyr-418 and Tyr-529
residues of c-Src were reversed by treatment of
4-amino-5-(4-chlorophenyl)-7-t-butyl(pyrazolo)[3,4-d]pyrimidine
(PP2), an inhibitor of Src kinase, in control cells. Glucosylceramide-depleted
cells resisted treatment with PP2, and both phosphorylation of Tyr-418 and
dephosphorylation of Tyr-529 induced by depletion of glucosylceramide were
maintained. Compared with untreated cells, tyrosine phosphorylation of
phospholipase C-1 was enhanced by EGF stimulation in
glucosylceramide-depleted cells, associated with enhanced tyrosine
phosphorylation of the EGF receptor at Tyr-1068 and Tyr-1086 stimulated by
EGF. The Src inhibitor, PP2, significantly blocked EGF-induced tyrosine
phosphorylation of phospholipase C-1 in control cells, whereas in
glucosylceramide-depleted cells, suppression of Src kinase activity by PP2
toward EGF-induced tyrosine phosphorylation of phospholipase C-1 was
less significant. Thus the activation of Src kinase by depletion of
glucosylceramide-based glycosphingolipids in cultured ECV304 cells is a
critical up-stream event in the activation of phospholipase C-1. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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In previously reported work, glycosphingolipids were shown to regulate
phospholipase C-1 activity within lipid rafts
(11). Specifically, the
depletion of glucosylceramide and other glucosylceramide-based
glycosphingolipids in ECV304 cells was marked by increased phospholipase C
activity and inositol trisphosphate formation. The increase in phospholipase
activity was the result of phosphorylation and not expression of the lipase.
The increase in phosphorylation and activity was observed under both basal
conditions and in response to stimulation with bradykinin. ECV304 cells were
originally characterized as a human endothelial cell line but have
characteristics that are more consistent with those of a bladder carcinoma
cell line. ECV304 cells were used in this study, because they demonstrate a
high rate of turnover of glycosphingolipids and have many lipid
raft-associated signaling molecules, including bradykinin and
EGF1 receptors, c-Src,
and phospholipase C-1
(11).
The mechanism responsible for the glycosphingolipid-dependent change in
phospholipase C activity remains to be elucidated. Indeed, phospholipase
C-1 is phosphorylated and activated in response to a receptor tyrosine
kinase such as EGF and recruited to the inner membrane during cell signaling.
Glycosphingolipids, on the other hand, are present in the outer leaflet. A
direct interaction between phospholipase C-1 and a
glucosylceramide-based glycolipid is unlikely to account for these
observations. In the present study, ECV304 cells were employed to further
investigate the basis for the observed increase in phospholipase C-1
activity in the setting of glycosphingolipid depletion.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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1 antibody was from BD Pharmingen (San Diego, CA),
and [-32P]ATP was from PerkinElmer Life Sciences. Protein
A-agarose, EGF, and bradykinin were purchased from Sigma Chemical Co. (St.
Louis, MO). D-t-EtDO-P4 and
D-e-EtDO-P4 enantiomers were synthesized and isolated as
previously described (12). Cell Culture—Human ECV304 cells were routinely maintained in Medium 199 (M199) supplemented with 10% (v/v) newborn calf serum, 2 mM L-glutamine, 4.5 g/liter D-glucose, 100 µg/ml streptomycin, and 100 units/ml penicillin. For lipid extraction, cells were plated onto 150-mm dishes. For immunoprecipitation, Western blotting analysis, and Src kinase activity measurements, the cells were plated onto 100-mm dishes. The cells were grown for 2 days to 85–90% confluence prior to treatment with glucosylceramide synthase inhibitors. The medium containing 10% serum was removed and replaced with serum-free M199 medium with or without the glucosylceramide synthase inhibitors. Stock solutions of D-t-EtDO-P4 and D-e-EtDO-P4 (4 mM) were made in 100% Me2SO and diluted with 250 volumes of serum-free M199 medium just prior to use. Equivalent Me2SO concentrations served as vehicle controls. All cells were maintained at 37 °C in a humidified 5% CO2 atmosphere. The normal morphology of cells and the total protein content of cultured cells were unaffected by treatment with up to 3 µM D-t-EtDO-P4 for 48 h.
Immunoprecipitation and Immunoblotting—Serum-starved ECV304 cells in the presence or absence of D-t-EtDO-P4 or D-e-EtDO-P4 were exposed to either bradykinin, EGF, PP2, or AG1478 at various concentrations and time intervals. The exposures were stopped by aspiration of the incubation medium. Cells were then rinsed twice with ice-cold phosphate-buffered saline containing 1 mM Na3VO4 and lysed in 1 ml of ice-cold Tris-Triton lysis buffer (25 mM Tris-HCl (pH 7.4), 1% Triton X-100, 10% glycerol, 20 mM NaF, 2 mM EDTA, 2 mM Na3VO4, 137 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 20 µg/ml aprotinin, and 1 µg/ml pepstatin). Insoluble debris was removed by centrifugation at 21,000 x g for 20 min at 4 °C. Protein concentrations were determined by use of the bicinchoninic acid assay using bovine serum albumin as a standard. Immunoprecipitation and immunoblots were performed as previously described (13). Briefly, equal amounts of protein from each cell lysate were incubated with selected antibodies at 4 °C for 2 h or subjected to direct Western blotting. Immunoprecipitates were absorbed by protein A-agarose beads and washed with ice-cold washing buffer (25 mM Tris-HCl (pH 7.4), 0.1% Triton X-100, 1 mM Na3VO4, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 µg/ml pepstatin). The immunoprecipitates or whole cell extracts were resolved by SDS-PAGE using a 4–13% gradient and transferred to a nylon membrane. The blots were blocked overnight at 4 °C in 5% skimmed milk in TBS buffer (20 mM Tris-HCl (pH 7.6), 150 mM NaCl) and probed with selected antibodies diluted in TBS containing 1% skimmed milk. The immunoreactive bands were detected with the ECL-plus system (PerkinElmer Life Sciences).
Src Dephosphorylation, Phosphorylation, and in Vitro Kinase
Assays—The measurement of Src dephosphorylation was performed by
immunoprecipitation combined with immunoblotting. The clarified cell lysates
(200 µg of protein) were incubated with a monoclonal anti-human Src
antibody (2 µg/200 µg of lysate protein) for 2 h at 4 °C. The
anti-Src immunocomplexes were then immobilized on protein A-agarose beads. The
recovered samples were washed four times with washing buffer, solubilized in
SDS sample buffer, electrophoresed, and immunoblotted with a rabbit polyclonal
anti-phosphotyrosine antibody that specifically recognizes the
carboxyl-terminal (TSTEPQpYQPGENL) sequence of human c-Src, corresponding to
the sequence surrounding the Tyr-529 residue. Alternatively, equal amounts of
total cellular lysate protein (30 µg) were directly subjected to Western
blotting and probed with a polyclonal phosphospecific anti-human Src (pY529)
antibody (0.25 µg/ml). The phosphotransferase activity of Src kinase was
assayed in parallel by incubation of cell lysates (500 µg of protein) with
a mouse antibody against human Src kinase (4 µg/ml). The Src
immunoprecipitates were washed three times in ice-cold washing buffer (50
mM Tris-HCl (pH 7.2), 10 mM MgCl2, 2
mM EGTA, 100 µM Na3VO4, and 1
mM dithiothreitol). The washed immunocomplexes were then incubated
in 30 µl of kinase reaction buffer (50 mM Tris-HCl (pH 7.2), 10
mM MgCl2, 10 mM MnCl2, 2
mM EGTA, 100 µM Na3VO4, and 1
mM dithiothreitol) supplemented with 150 µM synthetic
peptide (KVEKIGEGTYGVVYK) as exogenous substrate. The kinase reactions were
initiated by the addition of 8 µM [-32P]ATP (8
µCi) in 10 µl of Src Mn/ATP mixture (50 mM Tris-HCl (pH 7.2),
25 mM MnCl2, 100 µM ATP, 25 mM
-glycerol phosphate, 2 mM EGTA, 100 µM
Na3VO4, and 1 mM dithiothreitol). The assays
were run at 30 °C for 10 min with agitation and terminated by the addition
of 30 µl of 50% acetic acid (v/v). The samples were centrifuged, and the
supernatants were spotted onto P81 ion exchange chromatography paper. The
paper squares were washed five times for 5 min each in 0.75% phosphoric acid,
once in acetone, and then air dried. The phosphotransferase activity of human
Src was measured as specific 32P incorporation into the substrate
(KVEKIGEGT(32pY)GVVYK) by liquid scintillation counting.
Alternatively, rabbit muscle enolase was used as an exogenous substrate. The
enolase phosphorylation by Src kinase was assessed by the incubation of whole
cellular lysate (500 µg of protein) with a mouse monoclonal anti-human Src
antibody (4 µg/500 µg of protein) as described above. The precipitated
immunocomplexes were incubated with 30 µl of kinase reaction buffer with
2.5 µg of acid-denatured enolase and 8 µM
[-32P]ATP (8 µCi) at 30 °C for 10 min. The assays
were terminated by the addition of 2x SDS loading buffer. The products
were resolved on a 4–13% gradient SDS-PAGE. The acrylamide gel was
dried, and the 32P-labeled enolase was visualized by
autoradiography.
Lipid Extraction and Analysis—ECV304 cells that were treated or untreated with glucosylceramide synthase inhibitors were washed with ice-cold phosphate-buffered saline, fixed with ice-cold methanol, and harvested by scraping. Chloroform was then added to yield a theoretical ratio of chloroform:methanol:water at 1:2:0.8 (v/v). The mixture of cells, chloroform, and water was sonicated for 15 min in a bath sonicator and centrifuged at 2200 x g for 30 min. The resultant supernatants were partitioned into aqueous and organic phases by the addition of chloroform and water to create a ratio of chloroform:methanol:water (2:1:0.8, v/v). The lower organic phases were carefully extracted, washed with 0.9% NaCl, and evaporated under a stream of nitrogen gas. The residues were redissolved into a chloroform/methanol solution (2:1, v/v). Total cellular phospholipid was measured with a lipid phosphate assay. For the analysis of glucosylceramide, equal amounts of total lipid phosphate (150 nmol) from each sample were subjected to base hydrolysis by incubation with 2 ml of chloroform and 1 ml of 0.21 N NaOH in 100% methanol at 37 °C for 1 h. The incubation was terminated by the addition of 0.8 ml of 0.3 M acetic acid. Glucosylceramide was extracted from the lower organic phase, dried under a stream of nitrogen gas, and analyzed by high performance thin layer chromatography with a solvent system consisting of chloroform:methanol:water (65/25/4, v/v), and detected by charring with 8% cupric sulfate in 8% phosphoric acid. The levels of glucosylceramide were quantified by densitometric scanning using Image version 1.62 (National Institutes of Health) and compared with authentic standards run in parallel on the same plates.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The possibility that activation of c-Src kinase, a non-receptor
protein-tyrosine kinase, may mediate the tyrosine phosphorylation of
phospholipase C-1 observed with glucosylceramide depletion of ECV304
cells was studied. The phosphotransferase activity of c-Src kinase was
measured as the capacity of c-Src immunoprecipitated from cell lysates to
phosphorylate a c-Src-specific substrate peptide (KVEKIGEGTYGVVYK)
(Fig. 2). The activities of Src
kinase from immunoprecipitates of cells treated with
D-e-EtDO-P4 showed no difference compared with untreated
control cells. In contrast, Src kinase activity from immunoprecipitates of
D-t-EtDO-P4-treated cells exhibited a dose-dependent
increase. The maximal increase in the Src kinase activity was observed in the
cells exposed to 100 nM D-t-EtDO-P4. An increase of
D-t-EtDO-P4 concentrations over 100 nM,
however, led to a decrease in the Src kinase activities to basal levels. The
basal levels of kinase activities were progressively lower as the cells were
maintained in serum-free medium. However, the peak increase in kinase activity
was not significantly different between cells grown for 24, 36, and 48 h in
the absence of serum. In addition, the direct addition of either
D-t-EtDO-P4 or D-e-EtDO-P4 to the
assay mixture was without effect on Src kinase activity.
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The coupling of bradykinin stimulation of ECV304 cells to Src kinase activation was next studied. More specifically, the effects of bradykinin on the dephosphorylation of Src kinase at tyrosine residue 529 (Tyr-529), the inhibitory site within the C-terminal regulatory region, and the phosphorylation of c-Src kinase at tyrosine residue 418 (Tyr-418), the activation site, within the catalytic loop was evaluated in control cells and in cells treated with 100 nM D-t-EtDO-P4 (Fig. 3A). Western blot analysis revealed that the depletion of glucosylceramide by D-t-EtDO-P4 (100 nM) in cultured ECV304 cells induced autodephosphorylation of c-Src at the Tyr-529 residue and autophosphorylation of c-Src at the Tyr-418 residue. Upon stimulation with bradykinin, a rapid and transient dephosphorylation of Src at Tyr-529 and phosphorylation of Src at Tyr-418 was observed in control cells. These changes were dependent on the dose and time of bradykinin exposure. In glucosylceramide-depleted cells, both Tyr-529 dephosphorylation and Tyr-418 phosphorylation of c-Src kinase were significantly enhanced and markedly extended. The total cellular expression of c-Src was no different in the absence or presence of the glucosylceramide synthase inhibitor, bradykinin, or both.
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The phosphotransferase activities of Src kinase were assayed in vitro in parallel experiments (Fig. 3B). Bradykinin induced an increase in the Src kinase activity in both control and in cells treated with 100 nM D-t-EtDO-P4. The basal and stimulated Src kinase activities were higher in the inhibitor-treated cells, and the stimulated kinase activity displayed a peak activity at 1 min versus 30 s. The pattern of Src kinase activation was similar in the control and D-t-EtDO-P4-treated cells.
The association between glucosylceramide depletion and Src kinase activity was studied further with the c-Src kinase inhibitor PP2 (Fig. 4). Serum-starved cells incubated with or without 100 nM D-t-EtDO-P4 for 24 h were exposed to 0.1, 0.5, and 1.0 µM PP2 for another 12 h (Fig. 4A). The phosphorylation status of c-Src kinase at Tyr-529 residue was detected by immunoprecipitation with a monoclonal anti-human Src antibody, followed by immunoblot analysis using a polyclonal antibody recognizing the c-Src carboxyl-terminal sequence (TSTEPQpYQPGENL) surrounding the Tyr-529 residue. In the case of controls, cells exposed to a relatively low dose of PP2 (0.1 µM) for 12 h maximally restored phosphorylation of c-Src kinase at the carboxyl-terminal inhibitory tyrosine residue, Tyr-529. However, glucosylceramide-depleted cells were observed to be insensitive to PP2 treatment. The dose-dependent phosphorylation of human c-Src at Tyr-529 by PP2 was shifted rightward in D-t-EtDO-P4-treated cells. The phosphorylation in D-t-EtDO-P4-treated cells was observed in those cells exposed to a relatively high dose of PP2 (1.0 µM) for 12 h. By contrast, the phosphorylation of c-Src kinase at Tyr-418 residue (the activation site) within the catalytic loop was gradually decreased in control and in D-e-EtDO-P4-treated cells with even greater PP2 concentrations (5, 10, and 15 µM for 2 h) (Fig. 4B). Under the same experimental conditions, D-t-EtDO-P4-treated cells were resistant to the inhibitory effect of PP2 on phosphorylation of c-Src kinase at Tyr-418. This resistance was still observed at the highest concentration of PP2 employed in this study. The activities of Src kinase were assayed in vitro in parallel experiments using rabbit muscle enolase as an exogenous substrate (Fig. 4C). The measured change in c-Src kinase activities paralleled those predicted by the change in phosphorylation status under the identical experimental conditions. D-e-EtDO-P4-treated cells demonstrated no change in c-Src kinase activity with the rabbit enolase substrate (Fig. 4D).
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Bradykinin stimulation of phospholipase C-1 activity may be mediated
via activation of the EGF receptor. Therefore, the effect of glucosylceramide
depletion on bradykinin-stimulated phosphorylation of the EGF receptor was
next evaluated. The cells were deprived of serum, and growth factors for 48 h
and total cell lysates were subjected to Western blot analysis using two
phosphospecific antibodies: anti-human EGF receptor (pY1068) and anti-human
EGF receptor (pY1173) (Fig. 5).
Under control conditions, no background phosphorylation at either Tyr-1068 or
Tyr-1173 was observed. However, in glucosylceramide-depleted cells, tyrosine
phosphorylation of the EGF receptor was detected at Tyr-1068 but not at
Tyr-1173. In control cells, bradykinin induction phosphorylated the EGF
receptor at carboxyl-terminal Tyr-1068 and Tyr-1173 residues in a dose- and
time-dependent manner. The pretreatment of ECV304 cells with
D-t-EtDO-P4 resulted in the enhanced phosphorylation of
the Tyr-1068 residue but impaired phosphorylation of the Tyr-1173 residue. No
changes in the total cellular expression of the EGF receptor were observed
under these conditions.
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Phospholipase C-1 is a substrate for the EGF receptor tyrosine
kinase. Therefore, the phosphorylation of phospholipase C-1 was studied
in response to stimulation with EGF in the presence and absence of
D-t-EtDO-P4 treatment
(Fig. 6, A and
B). EGF stimulation induced a time- and
concentration-dependent increase in phospholipase C-1 phosphorylation.
In the presence of D-t-EtDO-P4, the degree of
phosphorylation, the rapidity of the response, and the duration of the
phosphorylation were increased.
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In the presence of the c-Src inhibitor, PP2, the tyrosine phosphorylation
of phospholipase C-1 in response to EGF was attenuated
(Fig. 7A). This
attenuation was reversed by glucosylceramide depletion with
D-t-EtDO-P4 (Fig.
7B). These results suggest that Src kinase functionally
contributes to the tyrosine phosphorylation of phospholipase C-1 during
cell stimulation with both EGF and bradykinin.
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Because differences in the sensitivity of carboxyl-terminal residues of the EGF receptor to glucosylceramide depletion were observed in response to bradykinin, tyrosine phosphorylation was also studied in response to EGF stimulation (Fig. 8). Whole cell lysates prepared from control cells and cells pretreated with D-t-EtDO-P4 cells that were then simulated with EGF were analyzed by Western blot employing four different phosphospecific antibodies. These antibodies included anti-human EGF receptors pY1068, pY1086, pY1148, and pY1173. Tyrosine phosphorylation in response to EGF was observed at each residue. In each case phosphorylation was inhibited with AG1478. In glucosylceramide-depleted cells, the EGF receptor tyrosine phosphorylation was significantly elevated in response to EGF stimulation at Tyr-1068 and to a lesser extent at Tyr-1086. Similar effects from glycolipid depletion were not observed at Tyr-1148 or Tyr-1173. D-t-EtDO-P4 partially reversed the sensitivity to inhibition of phosphorylation at Tyr-1068 and Tyr-1086 but had no effect at Tyr-1148 and Tyr-1173.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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1. These changes occurred in the absence of secondary increases in
ceramide levels. Increased phospholipase C-1 phosphorylation and
inositol 1,4,5-trisphosphate formation were observed in the absence of agonist
stimulation, but these changes were enhanced in the presence of bradykinin.
Glycosphingolipid depletion did not alter the expression of any isoform of
phospholipase C, nor did it change the expression of other raft-associated
proteins, including Ras, c-Raf-1, eNOS, and annexin II. Finally, the changes
in phospholipase C-1 phosphorylation and inositol 1,4,5-trisphosphate
formation were consistently increased as a function of
D-t-EtDO-P4 concentration and time following bradykinin
stimulation (11). Both changes
were maximal at 100 nM D-t-EtDO-P4 and at 2 min following
2 µM bradykinin exposure.
Although these observations were consistent with a possible direct
interaction between a glucosylceramide-based glycolipid and phospholipase
C-1, such an interaction would be unlikely given the known localization
of the glycolipids and the phospholipase within cells. The former is typically
localized to the outer plasma membrane leaflet, and the latter is recruited to
the inner plasma membrane following agonist stimulation. Phospholipase
C-1 stimulation can result from activation of G protein-coupled
receptors by agonists such as bradykinin or may result from stimulation of
receptor tyrosine kinases such as the EGF receptor. In the current model of G
protein-coupled receptor activation, phospholipase C-1 phosphorylation
is secondary to the activation of the receptor tyrosine kinase via Src kinase
(17). For this reason, the
role of Src kinase in the glycosphingolipid-dependent change in phospholipase
C activity was evaluated.
Src kinase activity was increased following exposure to
D-t-EtDO-P4. The increase in kinase activity was secondary
to glycosphingolipid depletion, because the inactive erythro
enantiomer, D-e-EtDO-P4, was without effect. The change in
kinase activity was biphasic with the maximal effect observed at100
nM inhibitor, independent of time of exposure. This concentration
response paralleled that previously observed for phospholipase C-1
phosphorylation and inositol 1,4,5-trisphosphate formation. The basis for the
biphasic change in Src kinase activity is not obvious from the present data.
Although the previous study demonstrated that no change in ceramide content is
observed at the inhibitor concentrations employed, other
glucosylceramide-based glycosphingolipids may demonstrate a different
concentration-dependent relationship. An understanding of the relationship
between these glycosphingolipids and Src kinase activity will require further
study (11).
The increase in Src kinase activity was accompanied by a decrease in phosphorylation of Tyr-529 and an increase at Tyr-418. These changes are consistent with a current model of Src kinase activation in which the phosphorylation of Tyr-529 is associated with the closed inactive state and phosphorylation of Tyr-418 is associated with the active state of the kinase. These changes in phosphorylation were observed in the absence of agonist stimulation and were enhanced as a function of bradykinin concentration and time following agonist exposure. The Src kinase inhibitor PP2 enhanced Tyr-529 and inhibited Tyr-418 phosphorylation as well as Src kinase activity. These effects were mitigated by D-t-EtDO-P4 but unaltered by the inactive enantiomer D-e-EtDO-P4.
The role of glycosphingolipid depletion in mediating EGF-stimulated
phospholipase C-1 phosphorylation was subsequently studied.
Phosphorylation was increased in a time- and concentration-dependent manner in
response to EGF. The phosphorylation was increased in the presence of
D-t-EtDO-P4 but blocked by the Src kinase inhibitor PP2.
The transactivation of the EGF receptor in response to bradykinin was manifest
by an increase in phosphorylation of Tyr-1068 and Tyr-1173. Glycolipid
depletion by D-t-EtDO-P4, however, increased Tyr-1068
phosphorylation but decreased Tyr-1173 phosphorylation. Stimulation by EGF
induced autophosphorylation of EGF receptor at Tyr-1068, Tyr-1086, Tyr-1148,
and Tyr-1173. However, D-t-EtDO-P4 was primarily
associated with an increase in autophosphorylation only at Tyr-1068 and
Tyr-1086. This observation was somewhat surprising, because phospholipase
C-1 activation has been tied to phosphorylation of Tyr-1173
(18). Clearly, Tyr-1173
phosphorylation alone is not sufficient to explain activation of phospholipase
C-1.
The enhanced activation of phospholipase C-1 in the setting of
glycosphingolipid depletion is perhaps best explained by changes in EGF
receptor activity. In the current model of G protein receptor-coupled
activation of this growth factor receptor, Src kinase serves as a
transactivator for the EGF receptor. The current study presents evidence in
support of enhanced Src kinase activity both by direct measurements of the
kinase and the secondary changes in Tyr-529 and Tyr-418. These changes appear
to occur in addition to but independent of direct interactions with the EGF
receptor. Several studies have now implicated the association of most every
non-receptor tyrosine kinase with glycosphingolipids. These include c-Src
(19), Lyk, Fyn
(20), Lyn
(21,
22), Yes
(23), Cbl
(24), FAK
(19), and Csk
(25). These studies have
either localized these tyrosine kinases in raft fractions by density gradient
separations or have co-immunoprecipitated these kinases with
anti-glycosphingolipid antibodies. Signal transduction of non-receptor
tyrosine kinases has also been demonstrated in association with several
glycosphingolipids. This includes the activation of c-Src by exogenous GM3
(25), the activation of Lyn by
anti-ganglioside GD3 antibody
(22), activation of Lck with
exogenous GM1 (26), the
activation of Lyn with anti-
GalGD1b antibody
(27), and the activation of
Yes by the globotriaosylceramide binding toxin shiga toxin
(23). The mechanism
responsible for the regulation of non-receptor tyrosine kinases by
glycosphingolipids is not understood. In general glycosphingolipids affect the
physical properties of lipid rafts and thus may serve to activate
raft-associated enzymes by allowing molecules to physically associate, perhaps
by coalescence. Alternatively, they may function to exclude molecules that
negatively regulate tyrosine kinase activity, such as Csk, from associating
with the non-receptor tyrosine kinase
(25). It also remains to be
determined whether glycosphingolipids regulate these tyrosine kinases directly
or do so through adaptor molecules.
Glycosphingolipids and gangliosides in particular have been recognized as
independent regulators of growth factor receptors as well. The increase in
phosphorylation of phospholipase C-1 in the presence of the Src kinase
inhibitor PP2 and D-t-EtDO-P4 are consistent with a role
for glycosphingolipids in mediating the changes in phospholipase C activity
independent of changes in Src activity. Three models have been proposed for a
direct regulation of growth factor receptors by gangliosides
(28). These models include a
direct interaction of the growth factor with the ganglioside, the regulation
of receptor dimerization by ganglioside, and the modulation of receptor
activation state and subcellular localization by ganglioside. The use of
specific inhibitors of glucosylceramide synthase provides an alternative
method for evaluating these possibilities. Although the purpose of the present
study was not to distinguish among these various models, the data suggest that
ganglioside ligand interactions and dimerization alone are insufficient to
explain the results. The former is unlikely because changes in EGF receptor
phosphorylation and phospholipase C-1 phosphorylation were observed in
the absence of EGF. The latter model is unlikely, because the effects of
D-t-EtDO-P4 treatment were specific for particular Tyr
residues. This would be unlikely if receptor dimerization were specifically
enhanced.
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FOOTNOTES |
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To whom correspondence should be addressed: Nephrology Division, Dept. of
Internal Medicine, University of Michigan, Box 0676, Rm. 1560 MSRB II, 1150
West Medical Center Drive, Ann Arbor, MI 48109-0676. Tel.: 734-763-0992; Fax:
734-763-0982; E-mail:
jshayman{at}umich.edu.
1 The abbreviations used are: EGF, epidermal growth factor;
D-e-EtDO-P4,
D-erythro-ethylendioxyphenyl-2-palmitoylamino-3-pyrrolidinopropanol;
D-t-EtDO-P4,
D-threo-ethylendioxyphenyl-2-palmitoylamino-3-pyrrolidinopropanol;
PP2,
4-amino-5-(4-chlorophenyl)-7-t-butyl(pyrazolo)[3,4-d]pyrimidine;
GM1, N-acetylneuraminylgangliotetraosylceramide; GM3,
N-acetylneuraminyllactosylceramide.
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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K. Mitsuzuka, K. Handa, M. Satoh, Y. Arai, and S. Hakomori A Specific Microdomain ("Glycosynapse 3") Controls Phenotypic Conversion and Reversion of Bladder Cancer Cells through GM3-mediated Interaction of {alpha}3{beta}1 Integrin with CD9 J. Biol. Chem., October 21, 2005; 280(42): 35545 - 35553. [Abstract] [Full Text] [PDF]
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 L. Shu, H. S. Murphy, L. Cooling, and J. A. Shayman An In Vitro Model of Fabry Disease J. Am. Soc. Nephrol., September 1, 2005; 16(9): 2636 - 2645. [Abstract] [Full Text] [PDF]
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M. S. Toledo, E. Suzuki, K. Handa, and S. Hakomori Effect of Ganglioside and Tetraspanins in Microdomains on Interaction of Integrins with Fibroblast Growth Factor Receptor J. Biol. Chem., April 22, 2005; 280(16): 16227 - 16234. [Abstract] [Full Text] [PDF]
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J. J. Rahn, Q. Shen, B. K. Mah, and J. C. Hugh MUC1 Initiates a Calcium Signal after Ligation by Intercellular Adhesion Molecule-1 J. Biol. Chem., July 9, 2004; 279(28): 29386 - 29390. [Abstract] [Full Text] [PDF]
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J. Haendeler, G. Yin, Y. Hojo, Y. Saito, M. Melaragno, C. Yan, V. K. Sharma, M. Heller, R. Aebersold, and B. C. Berk GIT1 Mediates Src-dependent Activation of Phospholipase C{gamma} by Angiotensin II and Epidermal Growth Factor J. Biol. Chem., December 12, 2003; 278(50): 49936 - 49944. [Abstract] [Full Text] [PDF]
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