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Departments of
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Biochemistry and Molecular Biology and
Physiology and Biophysics, University of Calgary, Calgary, Alberta, Canada
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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subunits (4, 5, 11, 12, 13). Recently, the palmitoylated
adaptor protein, linker for activation of T cells (LAT), which couples
TCR activation to the phospholipase C
(PLC) and Ras signaling
pathways, was found to localize to lipid rafts along with
phosphorylated PLC and phosphatidylinositol 3-kinase (PI-3K),
suggesting a role for these domains in TCR-mediated signaling
(14, 15). This is supported by the observation that
disruption of rafts by cholesterol depletion inhibits calcium
mobilization (14, 16). Similar data was reported for T
cells grown in the presence of lovastatin, which lowers cellular
cholesterol by preventing sterol synthesis (17). The densely packed, liquid-ordered environment of rafts excludes most integral membrane proteins; however, a few transmembrane proteins translocate into lipid rafts with stimulation. These include the high-affinity IgE receptor in mast cells (18, 19), components of the TCR (14, 20), and CD20, a regulator of cell cycle progression in B lymphocytes (21). We show here that this unusual property is shared by the BCR, in agreement with a recent report by Cheng et al. (22). Stimulation of the BCR results in rapid translocation of a subset of BCRs to lipid rafts and induces the appearance of tyrosine-phosphorylated substrates within the rafts. Surprisingly, disruption of rafts by cholesterol sequestration enhanced BCR-mediated calcium mobilization, suggesting that these domains may play a role in down-regulating intracellular calcium release. Consistent with this, the Src homology 2 (SH2) domain-containing inositol 5-phosphatase (SHIP) transiently translocated to lipid rafts within the first minute of BCR stimulation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Ramos, Raji, and Cess B cells were maintained by culture in RPMI 1640, 10% FBS. Primary B cells were isolated from tonsils obtained from the Alberta Children’s Hospital, essentially as described (23). Briefly, cells released from the tonsil tissue were depleted of T lymphocytes using SRBC pretreated with 2-aminoethylisothiouronium bromide (Sigma, St. Louis, MO), leaving >95% B lymphocytes. Dense (resting) B cells were then separated on Percoll density gradients.
Goat anti-human IgM F(ab')2 (Jackson
ImmunoResearch, West Grove, PA) was used to stimulate the BCR.
Monoclonal Abs 4G7 (CD19), 1F5 (CD20), G29.5 (CD21), HD39 (CD22), G28.1
(CD37), G28.5 (CD40), 9.4 (CD45), HIDE (MHC class I), and HB10a (MHC
class II) were provided by Drs. J. Ledbetter and E. Clark (University
of Washington, Seattle, WA). Anti-CD23 was purchased from Immunotech
(Westbrook, ME), and anti-CD32 was obtained from Serotec (Oxford,
U.K.). Anti-phosphotyrosine mAb 4G10 and anti-mitogen-activated
protein kinase (MAPK)/extracellular regulated kinase (ERK) were
obtained from Upstate Biotechnology (Lake Placid, NY),
anti-phosphoMAPK was obtained from New England Biolabs (Beverly,
MA), anti-PKC
and anti-paxillin were obtained from
Transduction Laboratories (Lexington, KY), anti-actin was obtained
from Boehringer Mannheim (Mannheim, Germany), anti-ezrin was
obtained from Neomarkers (Fermont, CA),
anti-Gi was obtained from Oncogene
Research Products (Cambridge, MA), and anti-tubulin was obtained
from Sigma. Anti-Ig was provided by Dr. Linda Matsuuchi, and SHIP
Abs were provided by Drs. L. Rohrscheider (Seattle, WA) and A.
Veillette (Montreal, Quebec, Canada). Cell-surface labeling was
performed with sulfo-NHS-LC-biotin (Pierce, Rockford, IL).
Methyl-ß-cyclodextrin (MBC) was purchased from Sigma.
Sample preparation
F(ab')2 anti-IgM (1
µg/106 cells) was added to cells at 37°C in
an equal volume of warm RPMI 1640 to ensure efficient sample mixing.
Cell stimulation was stopped by the addition of an equal volume of
ice-cold 2x lysis buffer (2% Triton X-100 (Pierce) in 20 mM Tris, pH
7.5, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 2 mM PMSF, 10 mM EDTA,
2 mM Na3VO4), and samples
were immediately placed on ice for 15 min. Sucrose density gradient
centrifugation was performed essentially as described
(21). Lysates from 108 cells were
mixed with an equal volume of 80% sucrose in lysis buffer,
overlayered with a discontinuous sucrose density gradient (4
ml 30% sucrose, then 6 ml 5% sucrose), and centrifuged at 37,000
rpm for 3 h. To confirm the location of lipid rafts on the
gradients, 1-ml fractions were collected from the top to the bottom of
the gradients, and the insoluble high-density pellets were washed four
times and dissolved in 1 ml SDS sample buffer. Then, 10 µl of each
fraction was mixed with an equal volume of SDS sample buffer and probed
by immunoblot for the presence of Gi, a dually
acylated heterotrimeric G protein subunit known to reside in rafts
(13, 24). Gi was exclusively
detected in fraction 6, which included the 5%/30% interface where
flocculent material was visible (Fig. 1
).
The majority of Gi was pelletable as shown by
dilution of fraction 6, recentrifugation, and dissolving the pelleted
material in 100 µl SDS sample buffer (Fig. 1, lane 6*).
Tubulin and paxillin, predominantly cytosolic proteins, were detected
primarily in fractions 11 and 12 (Fig. 1). Actin and ezrin, proteins
associated with the cortical cytoskeleton, were detected in the
high-density insoluble pellet, as well as in fractions 6, 11, and
12.
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10 ml morpholineethanesulfonic acid-buffered
saline, and centrifuged at 37,000 rpm for 1 h to pellet and
concentrate the raft-associated proteins. The pellets were solubilized
in 100 µl SDS sample buffer. Second, a 1-ml sample of the soluble
lysate was collected from the bottom of the tube (corresponding to
fraction 12 in Fig. 1
) and mixed with 0.33 ml of 4x SDS sample buffer.
Third, the insoluble high-density pellet was washed four times and
dissolved in 0.5 ml SDS sample buffer. Except where otherwise noted, 10
µl of each sample was loaded onto gels, thus representing
107, 7 x 105, and
2 x 106 cell equivalents from the
low-density insoluble fraction, the soluble fraction, and the
high-density insoluble pellet, respectively.
For experiments reported in Table I and
Fig. 6, whole-cell lysates were cleared of insoluble material by
centrifugation at 14,000 x g for 15 min at 4°C. For
experiments in Table I, the insoluble pellet was washed and solublized
in SDS sample buffer as described (21) and contains both
the high- and low-density insoluble fractions. Proteins were separated
on 10% polyacrylamide gels and transferred to Immobilon P (Millipore,
Bedford, MA) for immunoblotting.
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Cells were incubated for 20 min with 20 µM fluo-3AM (Molecular Probes, Eugene, OR), washed, resuspended in 20 mM HEPES buffer, pH 7.4, containing 150 mM NaCl, 1.5 mM CaCl2, 3 mM KCl, 10 mM glucose, and 250 µM sulfinpyrazone (Sigma), and placed in a cuvette with magnetic stirrer in a SLM-Aminco series 2 luminescence spectrometer (SLM Instruments, Urbana, IL). Cells were excited at 480 nm (8 nm bandwidth), and emission was measured at 530 nm (8 nm bandwidth). A 495-nm long-pass filter was placed in front of the emission monochromator. Minimum and maximum fluorescence values for each experiment were obtained using EDTA (5 mM) and either A23187 (10 µM) or saponin (0.02%), respectively.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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We initially sought to determine whether engagement of the BCR
induces the redistribution of integral membrane surface proteins to
lipid rafts. To that end, rafts were isolated by sucrose density
gradient fractionation from Ramos B cells that had been surface labeled
with biotin and stimulated with F(ab')2
anti-IgM for 15 min before lysis (Fig. 2A). Detection of labeled
proteins by avidin-HRP blotting revealed very few surface-labeled
proteins in low-density fractions from unstimulated cells, even on
overexposed films (Fig. 2A, lane 1). However, BCR
stimulation induced the appearance in the low-density fraction of two
prominent bands migrating at 80 and 25 kDa (Fig. 2A,
lane 2) that were simultaneously lost from the
detergent-soluble fraction (Fig. 2A, lanes 3 and
4). Similar results were obtained using dense tonsil B cells
(Fig. 2B, and data not shown). Because the sizes of these
proteins correspond to those of the heavy and light chains of the IgM
BCR, biotin-labeled F(ab')2 anti-IgM was used
to stimulate Ramos B cells to determine whether receptor-bound Ab could
be detected in the low-density insoluble fraction. The cells were
stimulated with biotin-labeled F(ab')2
anti-IgM for 15 min, washed, lysed, and fractionated on sucrose
density gradients. A total of 106 cell
equivalents from each fraction was analyzed by Western blotting with
avidin-HRP (Fig. 2C). The intensity of the bands in each
lane was measured by densitometry using a Fluor-S Max MultiImaging
system (Bio-Rad, Richmond, CA). The data indicated that only 16% of
engaged receptors remained in the soluble fraction after 15 min
stimulation, while 13% partitioned into the low-density insoluble
fraction. The majority of receptors were found in the high-density
insoluble pellet, consistent with previous reports of BCR translocation
to the cytoskeleton (25, 26, 27).
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A). The mature IgM heavy
chain (Fig. 3A, upper bands in all panels) was selectively
and rapidly mobilized to lipid rafts (upper panels) and
simultaneously lost from the detergent soluble fraction (middle
panels). Receptor translocation to rafts was detected at the
earliest time point tested (7 s) and peaked in the first 5 min of
stimulation. Densitometry analysis of the upper bands in the top
panels indicated that at 15 min there was 1.6-fold less BCR than
at 5 min. Because we estimated 13% of engaged receptors in rafts at 15
min, this indicates that 20% of BCRs are present in rafts 5 min
after stimulation. Interestingly, translocation to the high-density
insoluble pellet (Fig. 3A, bottom panels)
involved both mature and immature (lower bands in all panels) forms of
the receptor and, in contrast to raft-associated receptors, continued
to increase over the 15-min period, accounting for the steady loss of
receptors from the soluble fraction. Some irregularity in the protein
bands in the pellets reflects the difficulty in solublizing them for
analysis.
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/Igß components that facilitate
signal transmission. Because it was recently reported that a proportion
of BCRs dissociate from Ig/Igß after stimulation
(28), we sought to determine whether Ig/Igß
translocated to rafts with the BCR. Surface-labeled proteins
corresponding to Ig and Igß were not detected in lipid rafts after
receptor stimulation (Fig. 2); however, we found that Ig and Igß
were only weakly labeled with biotin in comparison with the heavy and
light chains of the BCR and may not have been detectable in lipid rafts
by avidin blotting (data not shown). Immunoblotting with anti-Ig
demonstrated that Ig was transiently recruited to lipid rafts with
similar kinetics as the IgM component of the BCR (Fig. 3B).
Similar data were obtained for Igß (data not shown). Therefore, in
agreement with the data of Cheng et al. (22), it is likely
that the intact BCR/Ig/Igß complex translocates to lipid
rafts.
We have previously reported that Ab-mediated ligation of CD20, an
integral membrane protein expressed on B cells, induces its
redistribution to lipid rafts (21). To determine whether
raft localization of surface proteins was a common consequence of Ab
cross-linking, the detergent solubility of several other integral
membrane proteins expressed in B cells, namely CD19, CD21, CD22, CD23,
CD32, CD37, CD40, CD45, and MHC class I and II, was examined after Ab
binding (Table I). Cells were exposed to specific Ab for 15 min at
37°C, unbound Ab was washed away, and receptor-bound Ab was tracked
by immunoblotting with anti-mouse IgG-HRP. In addition to CD20 and
the BCR, CD32 and CD45 were partly insoluble in Triton X-100, but all
other proteins were completely soluble (Table I). To assess the
subcellular localization of insoluble CD32 and CD45, low- and
high-density detergent-insoluble proteins were isolated by sucrose
density gradient centrifugation from cells pretreated with
receptor-specific Abs. The insoluble components of both CD32 and CD45
were found exclusively in the high-density insoluble pellet, in
contrast to CD20 and the BCR. This indicates that translocation to
lipid rafts is not an inevitable outcome of Ab-mediated cross-linking
of cell-surface proteins. Indeed, CD20 translocation to lipid rafts can
be induced with highly purified Fab, indicating that cross-linking is
not required (M. J. Polyak, H. Li, R. J. Petrie, and J. Deans,
manuscript in preparation). It remains to be determined whether Fab of
anti-IgM or monovalent Ag can induce BCR redistribution to lipid
rafts.
Phosphorylation of tyrosine kinase substrates in lipid rafts
The earliest BCR-mediated signaling event known to occur is the
activation of src family tyrosine kinases (1, 2, 3). Evidence
of tyrosine kinase activation in lipid rafts was sought by
phosphotyrosine immunoblotting of raft proteins after 15-min BCR
stimulation of Ramos B cells (Fig. 4A). Substrates in the soluble
fraction and in the high-density insoluble pellets were simultaneously
assessed for comparison with the low-density fraction. Heavily
phosphorylated proteins at 55–60 kDa and 75–80 kDa were observed
in lipid rafts from unstimulated samples and obscured any BCR-mediated
signals that might otherwise be evident in that region of the membrane.
The 55- to 60-kDa proteins are likely to include src family kinases,
but the identities of the 75- to 80-kDa proteins are not known.
Increased tyrosine phosphorylation was observed on several substrates
(arrows in Fig. 4A), indicating either the movement into
rafts of phosphorylated proteins or the phosphorylation of resident
proteins within rafts by activated kinases. A similar pattern of
tyrosine-phosphorylated substrates was observed in lipid rafts isolated
from dense tonsil B cells stimulated for 2 min with
F(ab')2 anti-IgM (Fig. 4B). The
difference in relative intensities of some of the bands observed in
BCR-stimulated tonsil B cells compared with those in Ramos B cells can
be attributed to the difference in stimulation time, because kinetic
differences were noted in the phosphorylation of some of the
raft-associated proteins. Peak detection of a protein migrating at
29 kDa, barely visible at the 15-min time point shown in Fig. 4A, occurred at 15 s, in contrast to the more delayed
phosphorylation kinetics of substrates migrating between 35 and 50 kDa
(data not shown). While the identity of the phosphorylated proteins has
not yet been determined, the data suggest that a subset of BCR signals
is relayed to lipid rafts after receptor engagement. It remains to be
determined whether these signals are initiated from outside the rafts
or are a direct consequence of BCR translocation into this compartment.
It is established that dually acylated src family tyrosine kinases are
highly enriched in low-density membranes, but because CD45 tyrosine
phosphatase is excluded from rafts (29) (Table I), the
mechanism of their activation is unknown. However, src family kinases
can be hyperactivated in the absence of CD45 (30, 31),
perhaps through accumulated phosphorylation on the positive
regulatory tyrosine residue (32), or via high-affinity
interactions with the SH3 domain (33).
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The rapid redistribution of BCRs and the appearance of
tyrosine-phosphorylated proteins in lipid rafts after receptor
cross-linking, suggests a role for rafts in early signaling events.
MBC, a small cyclic oligosaccharide with a hydrophobic core that
selectively extracts cholesterol into its interior cavity (34, 35), was used to test the effect of raft disruption on
BCR-mediated signaling. Unlike other compounds that extract
cholesterol, MBC does so without binding or inserting into the cell
membrane (36, 37, 38). Preliminary experiments indicated that
pretreatment of Ramos B cells with 10 mM MBC for 10 min at 37°C would
disrupt rafts without compromising cell viability as measured by trypan
blue exclusion. Based on the work of others, these conditions are
expected to extract 30–35% of membrane cholesterol (36).
As shown in Fig. 5, pretreatment of Ramos
cells for 10 min with 10 mM MBC prevented translocation of mature BCRs
to the low-density insoluble fraction, confirming the disruption of
lipid rafts with these conditions. Fig. 5 also shows that translocation
of BCRs to the high-density insoluble pellet occurs regardless of MBC
treatment, suggesting that cytoskeletal attachment of BCRs may occur
independently of rafts. Lower exposures did not reveal any difference
in the representation of either upper or lower bands in the
high-density insoluble fraction after receptor stimulation in the
presence and absence of MBC pretreatment.
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(PKC) and MAPK/ERK as downstream indicators of
activated signaling pathways. Recently, we reported that PKC is
activated and tyrosine phosphorylated in response to BCR cross-linking
(39). However, disruption of lipid rafts did not inhibit
BCR-mediated tyrosine phosphorylation of PKC (Fig. 6A), indicating that raft
integrity is not required for tyrosine kinase activation. Tyrosine
phosphorylation of PKC is dependent not only on src family tyrosine
kinase activity, but also on the activity of PI-3K and PLC
(39). Therefore, tyrosine phosphorylation of PKC after
raft disruption also suggests that activation of PI-3K and PLC can
occur independent of raft integrity. One of the major signaling
pathways downstream of BCR-mediated tyrosine kinase activation leads to
activation of MAPK/ERK. ERK activation, as measured by detection of its
phosphorylated form, was also not inhibited by raft disruption (Fig. 6B), even when suboptimal doses of anti-IgM were used to
stimulate the BCR (data not shown). Others have shown that ERK is
hyperactivated by the epidermal growth factor receptor in
cholesterol-depleted fibroblasts (40). Although we did not
measure ERK activity per se, the detection of similar levels of
phosphorylated ERK in MBC-treated and untreated BCR-stimulated B cells
indicates that MAPK can occur independently of lipid rafts. Raft disruption enhances intracellular Ca2+ concentration [Ca2+]i mobilization
To investigate a potential role for lipid rafts in signaling
pathways leading to intracellular calcium elevation, Ramos cells were
loaded with fluo-3 and pretreated with MBC before BCR stimulation. MBC
pretreatment caused a marked enhancement in the initial calcium burst
induced by BCR stimulation (Fig. 7A, left panel).
The enhanced calcium response in MBC-treated cells was resistant to
chelation of extracellular calcium (Fig. 7A, right
panel), suggesting that it was due to greater release from
internal stores. MBC-treated and untreated Ramos B cells had identical
levels of basal calcium and internal calcium stores as shown in Fig. 7A (right panel inset). The higher levels of
[Ca2+]i observed in
BCR-stimulated MBC-treated Ramos cells was confirmed to be due to
enhanced release from intracellular stores, because, compared with
untreated BCR-stimulated cells, there was less calcium available for
subsequent release by A23187 (data not shown).
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B,
left panel). Compared with Ramos cells, tonsil B cells
showed a more sustained elevation in
[Ca2+]i, presumably due
to calcium influx. It is interesting that in MBC-treated tonsil B cells
there was a more rapid decline in
[Ca2+]i following the
initial peak, perhaps indicating some dependence of the calcium
channels on lipid microdomains. In both untreated and MBC-treated
primary B cells, the height of the calcium peak was lower in the
absence of extracellular calcium because of the loss of the component
of the peak that is due to calcium influx (Fig. 7B,
right panel). In primary B cells, as in Ramos, MBC enhanced
calcium release even in the absence of extracellular calcium. However,
whereas in Ramos cells the enhancing effect of cholesterol depletion
appears to be completely independent of extracellular calcium, in
primary B cells the enhancing effect is not as great as in the presence
of calcium, again consistent with the possibility that calcium influx
is partially dependent on lipid microdomains.
Others have reported that MBC inhibits the TCR-mediated calcium flux in
T lymphocytes (14). This suggests that T and B lymphocytes
may differ with respect to the role of lipid microdomains in Ag
receptor signaling. The requirement for lipid rafts in TCR-mediated
calcium mobilization can be understood in light of the fact that the
adaptor protein LAT, which couples the TCR to PLC activation
(41, 42), is palmitoylated and selectively localized to
rafts (15, 43). No B cell homologue of LAT has been
described; however, an unrelated adaptor protein, BLNK, couples the BCR
to PLC activation (44, 45, 46, 47). We have looked for, and not
detected, BLNK in lipid rafts either before or after BCR activation
(data not shown), supporting the interpretation that, in contrast to
the situation in T cells, signals leading to PLC activation in B
cells do not absolutely require the integrity of lipid rafts.
Transient accumulation of SHIP in rafts following BCR stimulation
The enhanced calcium flux observed after raft disruption could be
due to the release of sequestered phosphatidylinositol 4,5-biphosphate
(48, 49), src family tyrosine kinases, or other factors
involved in the activation of PLC in B cells. However, if this were
the case, one might expect to see an enhanced calcium flux in other
cell types in contrast to the inhibitory effects reported.
Alternatively, lipid microdomains may compartmentalize factors involved
in calcium down-regulation. SHIP is known to participate in calcium
regulation in B cells by dephosphorylating phosphatidylinositol
3,4,5-trisphosphate, a membrane target for the pleckstrin homology
domain of Btk tyrosine kinase (50, 51, 52). The involvement of
SHIP in regulating BCR-mediated calcium flux has been most studied in
models of immune complex-mediated down-regulation of the response, in
which SHIP is recruited to FcRIIB and terminates calcium influx
(53). It is important to note that in the studies reported
here FcRIIB is not involved because F(ab')2
anti-IgM was always used for BCR stimulation. However, SHIP is also
involved in regulating BCR signals in the absence of FcRIIB
engagement (54, 55). Evidence indicates that it is the
early, inositol 1,4,5-triphosphate-dependent phase of calcium flux that
is primarily regulated by SHIP (55). Therefore, we
examined low-density insoluble fractions of Ramos lysates for the
presence of SHIP before and after BCR stimulation and found that SHIP
was detectable at very low levels in rafts before stimulation, but
rapidly accumulated there upon BCR cross-linking (Fig. 8A). Phosphotyrosine blots of
raft fractions revealed a phosphorylated protein that comigrated with
SHIP (Fig. 8B), suggesting that raft-associated SHIP is
tyrosine phosphorylated. The mechanism of SHIP recruitment to rafts is
not yet known but could conceivably involve phosphotyrosyl-based
docking interactions.
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C). Interestingly, comparison
of SHIP and PI-3K in the same samples revealed that the kinetics of
SHIP accumulation appeared to be slightly faster than that of PI-3K
(Fig. 8C). The amount of SHIP and PI-3K detected in rafts
was a small proportion of the total cellular SHIP and PI-3K, as
indicated by the relative intensities of the bands in lanes
1–6 with that in lane 7; however, compared with PI-3K,
a higher proportion of total cellular SHIP was detected in rafts.
Recruitment of signaling molecules to rafts is selective because, as
mentioned earlier, we did not detect BLNK. Similarly, PKC, which is
rapidly activated and tyrosine phosphorylated in Ramos cells upon BCR
stimulation (39), was not detected in lipid rafts before
or after receptor stimulation (data not shown). Our preliminary data indicate that 42–43% of total phosphatidylinositol phosphates in unstimulated cells are found in the low-density insoluble fraction. When lipids were extracted from either soluble or low-density insoluble fractions and analyzed by TLC, we found that phosphatidylinositol trisphosphate accumulated predominantly in the low-density insoluble fraction after BCR stimulation and was rapidly dephosphorylated (data not shown). Because TLC of extracted lipids does not discriminate between different isoforms of phosphatidylinositol phosphates, the complexity of the phosphatidylinositol bisphosphate content of lipid rafts after receptor stimulation was not revealed by these preliminary experiments, and further detailed work needs to be done to determine the activity of SHIP in lipid microdomains.
Lipid rafts isolated from Ramos B cells exclude >99% of total
surface-labeled proteins (data not shown). In this report, we have
demonstrated that, in both Ramos cells and in primary human B cells, a
subset of engaged BCRs translocates into lipid rafts. This is an
unusual property among B cell integral membrane proteins that has
previously been ascribed only to CD20. In contrast to CD20, which
translocates into lipid rafts but is never detected in the high-density
insoluble pellet, the BCR is rapidly translocated to both compartments.
Although rafts contain both actin and ezrin, whether and how lipid
rafts are tethered to the cytoskeleton is presently unknown.
Cytoskeletal attachment of the BCR did not appear to be dependent on
prior residency in lipid rafts (Fig. 5), and inhibition of actin
polymerization with cytochalasin D did not prevent translocation of
receptors into rafts (data not shown).
Phosphorylation of tyrosine kinase substrates in rafts after BCR
engagement, as shown in both Ramos and primary B cells, indicates that
selected early signaling events are targeted to lipid rafts. Although
the integrity of lipid rafts does not appear to be essential for the
activation of signaling pathways leading to phosphorylation of PKC
or ERK, it is nevertheless possible that signaling events involved in
these pathways normally occur in rafts. Further, signaling pathways
other than those tested here may have a requirement for intact raft
microdomains. Whether or not rafts provide an essential conduit for
receptor-mediated signaling pathways, it seems likely that signaling
events occurring in rafts play a role in modulating the outcome of BCR
signaling. Our preliminary data indicates that turnover of inositol
phosphates occurs primarily in the raft compartment, and the rapid
kinetics of SHIP recruitment to rafts offers a possible explanation for
the observed enhancement of calcium mobilization in MBC-treated B
cells. Characterization of the changes in protein and lipid content of
rafts after BCR engagement will help to clarify the physiological role
of raft microdomains in B cells.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Julie Deans, Department of Biochemistry and Molecular Biology, University of Calgary, Health Sciences Center, 3330 Hospital Drive NW, Calgary, Alberta, T2N 4N, Canada.
3 Abbreviations used in this paper: BCR, B cell receptor; MAPK, mitogen-activated protein kinase; ERK, extracellular regulated kinase; MBC, methyl-ß-cylcodextrin; PKC, protein kinase C ; PI-3K, phosphatidylinositol 3-kinase; PLC, phospholipase C ; SH, Src homology; SHIP, SH2-domain containing inositol phosphatase; LAT, linker for activation of T cells; [Ca2+]i, intracellular Ca2+ concentration.
Received for publication December 22, 1999. Accepted for publication May 9, 2000.
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 J. H. Kim, J. A. Rutan, and B. J. Vilen The transmembrane tyrosine of {micro}-heavy chain is required for BCR destabilization and entry of antigen into clathrin-coated vesicles Int. Immunol., December 1, 2007; 19(12): 1403 - 1412. [Abstract] [Full Text] [PDF]
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N. W. Kin and V. M. Sanders CD86 Regulates IgG1 Production via a CD19-Dependent Mechanism J. Immunol., August 1, 2007; 179(3): 1516 - 1523. [Abstract] [Full Text] [PDF]
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S. Itoh, C. Susuki, K. Takeshita, K. Nagata, and T. Tsuji Redistribution of P-selectin glycoprotein ligand-1 (PSGL-1) in chemokine-treated neutrophils: a role of lipid microdomains J. Leukoc. Biol., June 1, 2007; 81(6): 1414 - 1421. [Abstract] [Full Text] [PDF]
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 H. Mayer, S. Duit, C. Hauser, W. J. Schneider, and J. Nimpf Reconstitution of the Reelin Signaling Pathway in Fibroblasts Demonstrates that Dab1 Phosphorylation Is Independent of Receptor Localization in Lipid Rafts Mol. Cell. Biol., January 1, 2006; 26(1): 19 - 27. [Abstract] [Full Text] [PDF]
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W. Xiao, H. Nishimoto, H. Hong, J. Kitaura, S. Nunomura, M. Maeda-Yamamoto, Y. Kawakami, C. A. Lowell, C. Ra, and T. Kawakami Positive and Negative Regulation of Mast Cell Activation by Lyn via the Fc{epsilon}RI J. Immunol., November 15, 2005; 175(10): 6885 - 6892. [Abstract] [Full Text] [PDF]
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 S. Hao and A. August Actin Depolymerization Transduces the Strength of B-Cell Receptor Stimulation Mol. Biol. Cell, May 1, 2005; 16(5): 2275 - 2284. [Abstract] [Full Text] [PDF]
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D. Mielenz, C. Vettermann, M. Hampel, C. Lang, A. Avramidou, M. Karas, and H.-M. Jack Lipid Rafts Associate with Intracellular B Cell Receptors and Exhibit a B Cell Stage-Specific Protein Composition J. Immunol., March 15, 2005; 174(6): 3508 - 3517. [Abstract] [Full Text] [PDF]
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A. Cherukuri, R. H. Carter, S. Brooks, W. Bornmann, R. Finn, C. S. Dowd, and S. K. Pierce B Cell Signaling Is Regulated by Induced Palmitoylation of CD81 J. Biol. Chem., July 23, 2004; 279(30): 31973 - 31982. [Abstract] [Full Text] [PDF]
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A. Ehrhardt, M. D. David, G. R. A. Ehrhardt, and J. W. Schrader Distinct Mechanisms Determine the Patterns of Differential Activation of H-Ras, N-Ras, K-Ras 4B, and M-Ras by Receptors for Growth Factors or Antigen Mol. Cell. Biol., July 15, 2004; 24(14): 6311 - 6323. [Abstract] [Full Text] [PDF]
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P. Munoz, M.-d.-C. Navarro, E. J. Pavon, J. Salmeron, F. Malavasi, J. Sancho, and M. Zubiaur CD38 Signaling in T Cells Is Initiated within a Subset of Membrane Rafts Containing Lck and the CD3-{zeta} Subunit of the T Cell Antigen Receptor J. Biol. Chem., December 12, 2003; 278(50): 50791 - 50802. [Abstract] [Full Text] [PDF]
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H. Li, L. M. Ayer, J. Lytton, and J. P. Deans Store-operated Cation Entry Mediated by CD20 in Membrane Rafts J. Biol. Chem., October 24, 2003; 278(43): 42427 - 42434. [Abstract] [Full Text] [PDF]
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 K.-W. Ong, A. D. Wilson, T. R. Hirst, and A. J. Morgan The B Subunit of Escherichia coli Heat-Labile Enterotoxin Enhances CD8+ Cytotoxic-T-Lymphocyte Killing of Epstein-Barr Virus-Infected Cell Lines J. Virol., April 1, 2003; 77(7): 4298 - 4305. [Abstract] [Full Text] [PDF]
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T. Baba, N. Fusaki, N. Shinya, A. Iwamatsu, and N. Hozumi Actin Tyrosine Dephosphorylation by the Src Homology 1-Containing Protein Tyrosine Phosphatase Is Essential for Actin Depolymerization After Membrane IgM Cross-Linking J. Immunol., April 1, 2003; 170(7): 3762 - 3768. [Abstract] [Full Text] [PDF]
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N. Gupta and A. L. DeFranco Visualizing Lipid Raft Dynamics and Early Signaling Events during Antigen Receptor-mediated B-Lymphocyte Activation Mol. Biol. Cell, February 1, 2003; 14(2): 432 - 444. [Abstract] [Full Text] [PDF]
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M. A. Putnam, A. E. Moquin, M. Merrihew, C. Outcalt, E. Sorge, A. Caballero, T. A. Gondre-Lewis, and J. R. Drake Lipid Raft-Independent B Cell Receptor-Mediated Antigen Internalization and Intracellular Trafficking J. Immunol., January 15, 2003; 170(2): 905 - 912. [Abstract] [Full Text] [PDF]
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R. Galandrini, I. Tassi, G. Mattia, L. Lenti, M. Piccoli, L. Frati, and A. Santoni SH2-containing inositol phosphatase (SHIP-1) transiently translocates to raft domains and modulates CD16-mediated cytotoxicity in human NK cells Blood, December 15, 2002; 100(13): 4581 - 4589. [Abstract] [Full Text] [PDF]
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R. J. Petrie and J. P. Deans Colocalization of the B Cell Receptor and CD20 Followed by Activation-Dependent Dissociation in Distinct Lipid Rafts J. Immunol., September 15, 2002; 169(6): 2886 - 2891. [Abstract] [Full Text] [PDF]
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A. D. Stuart, H. E. Eustace, T. A. McKee, and T. D. K. Brown A Novel Cell Entry Pathway for a DAF-Using Human Enterovirus Is Dependent on Lipid Rafts J. Virol., August 12, 2002; 76(18): 9307 - 9322. [Abstract] [Full Text] [PDF]
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 E. B. BABIYCHUK, K. MONASTYRSKAYA, F. C. BURKHARD, S. WRAY, and A. DRAEGER Modulating signaling events in smooth muscle: cleavage of annexin 2 abolishes its binding to lipid rafts FASEB J, August 1, 2002; 16(10): 1177 - 1184. [Abstract] [Full Text] [PDF]
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B. J. Vilen, K. M. Burke, M. Sleater, and J. C. Cambier Transmodulation of BCR Signaling by Transduction- Incompetent Antigen Receptors: Implications for Impaired Signaling in Anergic B Cells J. Immunol., May 1, 2002; 168(9): 4344 - 4351. [Abstract] [Full Text] [PDF]
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M. J. Polyak and J. P. Deans Alanine-170 and proline-172 are critical determinants for extracellular CD20 epitopes; heterogeneity in the fine specificity of CD20 monoclonal antibodies is defined by additional requirements imposed by both amino acid sequence and quaternary structure Blood, May 1, 2002; 99(9): 3256 - 3262. [Abstract] [Full Text] [PDF]
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F. Barabe, E. Rollet-Labelle, C. Gilbert, M. J. G. Fernandes, S. N. Naccache, and P. H. Naccache Early Events in the Activation of Fc{gamma}RIIA in Human Neutrophils: Stimulated Insolubilization, Translocation to Detergent-Resistant Domains, and Degradation of Fc{gamma}RIIA J. Immunol., April 15, 2002; 168(8): 4042 - 4049. [Abstract] [Full Text] [PDF]
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F. Barabe, G. Pare, M. J. G. Fernandes, S. G. Bourgoin, and P. H. Naccache Cholesterol-modulating Agents Selectively Inhibit Calcium Influx Induced by Chemoattractants in Human Neutrophils J. Biol. Chem., April 12, 2002; 277(16): 13473 - 13478. [Abstract] [Full Text] [PDF]
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V. Ayllon, A. Fleischer, X. Cayla, A. Garcia, and A. Rebollo Segregation of Bad from Lipid Rafts Is Implicated in the Induction of Apoptosis J. Immunol., April 1, 2002; 168(7): 3387 - 3393. [Abstract] [Full Text] [PDF]
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I. Halova, L. Draberova, and P. Draber A novel lipid raft-associated glycoprotein, TEC-21, activates rat basophilic leukemia cells independently of the type 1 Fc{varepsilon} receptor Int. Immunol., February 1, 2002; 14(2): 213 - 223. [Abstract] [Full Text] [PDF]
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M. J. Aman, A.-C. Tosello-Trampont, and K. Ravichandran Fcgamma RIIB1/SHIP-mediated Inhibitory Signaling in B Cells Involves Lipid Rafts J. Biol. Chem., November 30, 2001; 276(49): 46371 - 46378. [Abstract] [Full Text] [PDF]
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R. Li, Y. Liu, and S. Ladisch Enhancement of Epidermal Growth Factor Signaling and Activation of Src Kinase by Gangliosides J. Biol. Chem., November 9, 2001; 276(46): 42782 - 42792. [Abstract] [Full Text] [PDF]
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M. Dykstra, A. Cherukuri, and S. K. Pierce Rafts and synapses in the spatial organization of immune cell signaling receptors J. Leukoc. Biol., November 1, 2001; 70(5): 699 - 707. [Abstract] [Full Text] [PDF]
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R. J. Benschop, E. Brandl, A. C. Chan, and J. C. Cambier Unique Signaling Properties of B Cell Antigen Receptor in Mature and Immature B Cells: Implications for Tolerance and Activation J. Immunol., October 15, 2001; 167(8): 4172 - 4179. [Abstract] [Full Text] [PDF]
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T. Suzuki, N. Kiyokawa, T. Taguchi, T. Sekino, Y. U. Katagiri, and J. Fujimoto CD24 Induces Apoptosis in Human B Cells Via the Glycolipid-Enriched Membrane Domains/Rafts-Mediated Signaling System J. Immunol., May 1, 2001; 166(9): 5567 - 5577. [Abstract] [Full Text] [PDF]
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 M. Higuchi, K. M. Izumi, and E. Kieff Epstein-Barr virus latent-infection membrane proteins are palmitoylated and raft-associated: Protein 1 binds to the cytoskeleton through TNF receptor cytoplasmic factors PNAS, April 10, 2001; 98(8): 4675 - 4680. [Abstract] [Full Text] [PDF]
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P. C. Cheng, B. K. Brown, W. Song, and S. K. Pierce Translocation of the B Cell Antigen Receptor into Lipid Rafts Reveals a Novel Step in Signaling J. Immunol., March 15, 2001; 166(6): 3693 - 3701. [Abstract] [Full Text] [PDF]
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J. E. Damen, M. D. Ware, J. Kalesnikoff, M. R. Hughes, and G. Krystal SHIP's C-terminus is essential for its hydrolysis of PIP3 and inhibition of mast cell degranulation Blood, March 1, 2001; 97(5): 1343 - 1351. [Abstract] [Full Text] [PDF]
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M. Zubiaur, O. Fernandez, E. Ferrero, J. Salmeron, B. Malissen, F. Malavasi, and J. Sancho CD38 Is Associated with Lipid Rafts and upon Receptor Stimulation Leads to Akt/Protein Kinase B and Erk Activation in the Absence of the CD3-zeta Immune Receptor Tyrosine-based Activation Motifs J. Biol. Chem., January 4, 2002; 277(1): 13 - 22. [Abstract] [Full Text] [PDF]
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