From the Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853
Received for publication, November 8, 2002
, and in revised form, March 14, 2003.
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ABSTRACT |
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INTRODUCTION |
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One current hypothesis focuses on the interaction of Lyn kinase with
FcRI. Metzger and colleagues
(3,
4) described evidence that a
small percentage of Lyn is constitutively associated with Fc
RI on
RBL-2H3 mast cells but is unable to phosphorylate these receptors in the
absence of aggregation. In the transphosphorylation model proposed in these
studies, Lyn can only phosphorylate an adjacent Fc
RI following
aggregation of two or more receptors. Subsequent studies from the Metzger
laboratory showed that the
subunit of Fc
RI is capable of weak
interactions with Lyn in the absence of phosphorylation
(5,
6). However, other studies
showed that signaling occurs via human Fc
RI in the absence of the
subunit (7,
8) and that
plays an
amplifying role in Fc
RI signaling
(9,
10).
An alternative model focuses on the ordered lipid environment of lipid
rafts that are proposed to facilitate the productive interaction between
aggregated FcRI and Lyn
(11,
12). Lipid rafts are enriched
in cholesterol, sphingolipids, and glycerophospholipids with saturated acyl
chains and can be isolated due to their insolubility in nonionic detergents at
4 °C (13,
14). A large percentage of
cellular Lyn fractionates with lipid rafts following sucrose gradient analysis
of Triton X-100-lysed RBL mast cells
(11,
15). Cholesterol depletion by
methyl-
-cyclodextrin reversibly inhibits antigen-stimulated tyrosine
phosphorylation of Fc
RI and in parallel causes reversible loss of both
Lyn and cross-linked Fc
RI from lipid rafts
(16). Thus, either the
association of Lyn or Fc
RI or both with rafts is important for
initiating this process.
Previous studies that focused on the relatively low abundance of proteins
in isolated lipid rafts suggested that localization of signaling proteins in
rafts serves to concentrate them and thereby promote signaling
(17,
18). However, it is now clear
that these ordered regions of the plasma membrane constitute a large
percentage of its lipid and surface area
(19,
20). To gain more insight
about the mechanism by which lipid rafts facilitate functional coupling
between cross-linked FcRI and Lyn, we investigated the role of membrane
environment on the kinase activity of Lyn. We find that Lyn solubilized from
RBL mast cells by Triton X-100 represents a subset of Lyn with reduced kinase
activity, and this subset largely fractionates with nonraft proteins in
sucrose gradients. Lyn isolated with lipid rafts has a substantially higher
kinase activity than Lyn from nonraft fractions, and this increase in activity
correlates with greater tyrosine phosphorylation of raft-associated Lyn in its
kinase domain. Cross-linking of Fc
RI does not increase the overall
kinase activity of Lyn in these cells but rather promotes functional coupling
by causing co-compartmentalization of these receptors with the active form of
Lyn in the raft environment.
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EXPERIMENTAL PROCEDURES |
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Sucrose Gradient FractionationRBL-2H3 cells were lysed at 3 x 107 cells/ml in 0.25% Triton X-100 in lysis buffer for 10 min on ice. The lysates were then brought to 40% sucrose by a 1:1 (v/v) dilution with an 80% sucrose stock solution, and 1 ml of this solution was applied to the bottom of an 11 x 60-mm centrifuge tube (Beckman Instruments, Inc., Palo Alto, CA). 2 ml of 30% sucrose and then 1 ml of 5% sucrose were layered above the lysate. Samples were ultracentrifuged as previously described for 1218 h at 250,000 x g (15). After ultracentrifugation, samples were fractionated to obtain lipid raft and nonlipid raft fractions. These fractions were diluted 2-fold with lysis buffer containing RIPA for subsequent immunoprecipitation of Lyn.
Lyn Immunoprecipitation and in Vitro Kinase AssayLyn was
immunoprecipitated from 0.51 ml of detergent extracts by incubation
with 2 µg anti-Lyn mouse monoclonal antibody H6 (Santa Cruz Biotechnology,
Inc., Santa Cruz, CA) for 12 h on ice and then rotated for 45 min with
35 µl of ImmunoPure Immobilized Protein A (Pierce) at 4 °C.
Immunoprecipitates were washed twice with lysis buffer without detergent and
then once with kinase assay buffer (20 mM Tris, pH 7.6, 10
mM MgCl2, and 1 mM
Na3VO4). After washing, Lyn immunoprecipitates were
subjected to in vitro kinase assays by adding 200 µl of kinase
assay buffer containing either 1 mM ATP, no ATP, or 1 mM
ATP and 100 µg of dephosphorylated -casein (Sigma) as an exogenous
substrate. Samples were then incubated at either 37 °C for samples without
-casein, or 30 °C for samples with
-casein, for 15 min.
Reactions were quenched by the addition of 50 µlof5x nonreducing SDS
sample buffer (15) followed
immediately by boiling.
ImmunoblottingSamples were separated by electrophoresis on 10% acrylamide SDS gels under nonreducing conditions, and then electrophoresed proteins were transferred to an Immobilon P membrane (Millipore Corp., Bedford, MA) with a Panther semidry electroblotter (Owl Separation Systems, Inc., Portsmouth, NH). Anti-phosphotyrosine blotting was performed using 0.1 µg/ml 4G10 mouse monoclonal antibody conjugated to horseradish peroxidase (Upstream Biotechnology, Inc., Lake Placid, NY) diluted in a 1:1 (v/v) mixture of Tris-buffered saline solution (TBST) (50 mM Tris, pH 7.6, 150 mM NaCl2, and 0.1% (v/v) Tween 20) and StabilZyme SELECT (SurModics, Eden Prairie, MN). Blots were then stripped by incubation with 0.2 M NaOH for 530 min and quenched with TBST. Anti-Lyn blotting was performed on the stripped blots using 0.2 µg/ml anti-Lyn rabbit polyclonal antibody 44 (Santa Cruz Biotechnology) and a horseradish peroxidase-labeled antirabbit Ig secondary antibody (Amersham Biosciences), both in a solution of 0.8% bovine serum albumin in TBST. All immunoblots were developed as previously described (15).
Quantitation of Lyn Basal Phosphorylation and Specific
Activity Western blots were scanned from film with an Epson
Expression 1600 digital scanner (Epson, Long Beach, CA), and density was
determined using UnScanit software (Silk Scientific, Orem, UT); intensities of
multiple samples on a single blot were normalized to a single sample in each
blot. Lyn basal phosphorylation is defined as the amount of phosphorylation on
immunopurified Lyn after an in vitro kinase incubation in the absence
of ATP. Lyn specific activity is defined as the amount of phosphorylation on
immunopurified Lyn following an in vitro kinase assay incubation in
the presence of ATP minus the basal phosphorylation of a parallel sample.
Values were calculated by dividing the normalized intensity of tyrosine
phosphorylation on Lyn from the 4G10 blot by the normalized intensity of Lyn
from the reprobed anti-Lyn blot in the same lane from nonsaturated film
following Western blotting. Alternatively, specific activity was determined as
the intensity of phosphorylated -casein following an in vitro
kinase assay incubation in the presence of ATP and
-casein in the
kinase assay buffer, divided by the amount of Lyn in the same lane.
Phosphopeptide Mapping of LynLyn was immunopurified using 35 µl of anti-Lyn conjugated to agarose beads (Santa Cruz Biotechnology) and 12 x 108 cell equivalents of RBL-2H3 cells lysed in the RIPA detergent buffer as above. Some Lyn samples were subjected to autophosphorylation in the in vitro kinase assay prior to elution and mapping. Lyn was eluted from the anti-Lyn beads by incubation with 0.1 M glycine HCl, pH 2.5. Next, the sample was dried using vacuum centrifugation (Thermo Savant, Holbrook, NY) and exchanged to 70% formic acid containing 100 mg/ml cyanogen bromide (CNBr) (ICN Biomedicals, Inc., Aurora, OH) to digest the protein overnight at room temperature in the dark. The following day, 500 µl of H2O was added to digests, and this mixture was then evaporated to dryness in the vacuum centrifuge. This wash step was repeated three more times, and dried samples were solubilized in 3050 µl of 1x sample buffer (125 mM Tris, pH 6.8, 4% SDS, 10% glycerol, 5% 2-mercaptoethanol, and 0.2 mg/ml bromphenol blue). Half of the sample was run on a 16.5% acrylamide Tricine gel (Bio-Rad) and then transferred to an Immobilon PSQ membrane (Millipore Corp.) by semidry transfer. Membranes were subsequently blocked for 4 h with 4% bovine serum albumin in TBST and then probed with anti-phosphotyrosine 4G10, washed, and detected by chemiluminescence as described above.
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RESULTS |
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To investigate further the effects of the two different lysis conditions on Lyn specific activity, we performed additional control experiments. To test whether RIPA enhances Lyn kinase activity, we washed Triton X-100-solubilized, immunoprecipitated Lyn with RIPA prior to the in vitro kinase reaction. We found no difference in kinase activity of Lyn with and without the RIPA wash, indicating no differential effects of detergents on Lyn kinase activity.2 Also, because of the large differences in Lyn recovery from RIPA and Triton X-100 PNS, we verified that results obtained were independent of the concentration of Lyn under the range of concentrations used for the in vitro kinase assay (Fig. 1C, inset).
The Triton X-100 Postnuclear Supernatant Is Depleted of Raft-associated LynThe insolubility of a substantial percentage of cellular Lyn following RBL-2H3 lysis in 0.5% Triton X-100 led us to examine the distribution of solubilized Lyn in the Triton X-100 PNS by sucrose gradient analysis. Fig. 2 compares the distribution of this Lyn, obtained as PNS (Fig. 1A), with the distribution of Lyn from cells lysed in the same concentration of Triton X-100 but directly loaded onto a sucrose gradient without precentrifugation. The representative blots shown in Fig. 2 illustrate that the Triton X-100 PNS is significantly depleted of lipid raft-associated Lyn; 4% of the Lyn from the PNS fractionates in the lipid raft region of the gradient, whereas 45% of the total cellular Lyn is found in the raft fraction under these conditions. Similar results were observed for lysis in 1% Triton X-100, indicating that Lyn solubility is not limited by the amount of detergent under these conditions.2 As observed previously (25), the alternatively spliced 53- and 56-kDa forms of Lyn are more highly resolved in the nonraft fractions in Fig. 2. These results, taken together, indicate that Triton X-100 lysis yields a PNS that is selectively depleted of lipid raft-associated Lyn.
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Raft-associated Lyn Has Enhanced Kinase ActivityThe results
of Fig. 2 suggest that the
difference in specific activity between Lyn from Triton X-100 and RIPA PNSs
could be due to different amounts of raft-associated Lyn. This interpretation
is consistent with studies by Ilangumaran et al.
(22) and Kabouridis et
al. (23) indicating that
the Src family kinases Lck and Fyn are differentially regulated by their lipid
environment. To investigate directly whether the lipid raft environment
affects Lyn activity, we isolated Lyn from either lipid raft or nonraft
fractions obtained from sucrose gradient separation of RBL cells lysed by
Triton X-100. A high concentration of cells was used in these experiments to
permit sufficient recovery of Lyn from gradient fractions for
immunoprecipitation and in vitro kinase analysis. The ratio of
detergent to cells chosen is similar to that used in previous studies to
preserve the interactions between cross-linked IgE-FcRI complexes and
lipid rafts (11,
15). Under these conditions,
uncross-linked IgE-Fc
RI was fully solubilized, fractionating with the
nonlipid raft components, indicating complete plasma membrane
lysis.2 The sucrose
gradient distribution of Lyn is shown in
Fig. 3A and is similar
to that in Fig. 2 and in
previous experiments carried out under these conditions
(11).
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Lipid raft-associated Lyn was recovered from the interface between the 5 and 30% sucrose layers (fraction 2; Fig. 2), and nonraft Lyn was recovered from the soluble lysate in the 40% sucrose fraction (fraction 5; Fig. 2). Both fractions were then diluted into RIPA buffer, and Lyn was immunoprecipitated and analyzed as in Fig. 1. Representative Western blots from these experiments are shown in Fig. 3B. Consistent with results in Fig. 1C, we find that Lyn isolated from lipid rafts has a 3.6-fold higher specific activity than Lyn isolated from nonraft fractions (Fig. 3C, shaded bars). Also, the levels of basal phosphorylation are 6-fold higher for Lyn from the detergent-resistant lipid raft environment compared with solubilized Lyn (Fig. 3C, open bars). Thus, lipid raft-associated Lyn has substantially more kinase activity than Lyn from a more disordered membrane environment in this autophosphorylation assay. Furthermore, this higher specific activity correlates with higher basal phosphorylation of Lyn.
To test the validity of conclusions based on in vitro Lyn
autophosphosphorylation, we performed in vitro kinase assays using
dephosphorylated -casein as an exogenous substrate. We first determined
that dephosphorylated
-casein is specifically phosphorylated by Lyn as
indicated by a lack of phosphorylation in mock immunoprecipitations done
without the Lyn
antibody.2 Activity
was detected by quantitative Western blot analysis of the
-casein band
with anti-phosphotyrosine and calculated per amount of Lyn detected by
reprobing the blot as for the autophosphorylation assay. A representative
Western blot is shown in Fig.
4A, and Fig.
4B summarizes the relative specific activities for Lyn
from lipid raft and nonraft fractions. Consistent with the autophosphorylation
results obtained in Figs. 1 and
3, raft-associated Lyn has a
4.7-fold higher specific activity toward
-casein than
nonraft-associated Lyn.
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Specific Activity of Lyn Does Not Increase in Stimulated
CellsIn previous studies, the total Lyn kinase activity associated
with FcRI was shown to increase following antigen stimulation, but the
specific activity of receptor-associated Lyn toward an exogenous substrate was
found to be unchanged (4,
24). To determine whether the
specific activity of total cellular Lyn is altered by antigen stimulation,
IgE-sensitized RBL-2H3 cells were stimulated with an optimal dose of antigen
(0.9 µg/ml denitrophenylated-bovine serum albumin) for various times. Lyn
was then immunoprecipitated from RIPA-lysed cells, and in vitro
kinase activity was determined with
-casein as an exogenous substrate.
The results are summarized in Table
I. Consistent with previous results from Metzger and colleagues
(4,
24), there is little change in
Lyn specific kinase activity following stimulation for 2 min at 37 °C.
Interestingly, Lyn specific activity was found to decrease by 60% after 5 min
of stimulation (Table I); this
may be related to the decline in Fc
RI tyrosine phosphorylation observed
at later times of antigen stimulation
(25) (see
"Discussion"). These results indicate that Fc
RI
cross-linking does not cause detectable activation of Lyn kinase per
se. Rather, Lyn kinase activity in resting cells is sufficient for
stimulated Fc
RI phosphorylation that results from cross-linking by
antigen. We conclude that the increase in Fc
RI phosphorylation commonly
observed after cross-linking results from changes in Fc
RI proximity to
active Lyn.
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Tyrosine 397 Phosphorylation Is the Predominant Detectable Site of Tyrosine Phosphorylation on Lyn from Unstimulated RBL CellsResults summarized in Figs. 1, 3, and 4 indicate that the higher specific activity of Lyn isolated from a lipid raft environment correlates with higher basal tyrosine phosphorylation. This higher specific activity is preserved following solubilization of Lyn away from the ordered lipids of the raft membranes by RIPA. Thus, it is likely that a covalent modification of Lyn, such as phosphorylation, is responsible for the higher activity state. Lyn, like other Src family kinases, has two major sites of tyrosine phosphorylation that regulate activity: one in the active site at residue 397 (Lyn A notation) and a second at the C terminus at residue 508 (26). Phosphorylation of Tyr397 has been reported to increase the specific activity of Lyn by 17-fold (27), whereas phosphorylation of the C-terminal regulatory site, Tyr508, reduces Lyn activity, similar to other Src family members (28).
To investigate the relative abundance of tyrosine phosphorylation at these
sites both before and after the in vitro kinase assay, we performed
CNBr peptide mapping. CNBr cleavage of Lyn is predicted to yield separate
fragments containing the active site and C-terminal site tyrosines with sizes
of 8.2 and 4.1 kDa, respectively (Fig.
5). For these experiments, RIPA-solubilized Lyn was
immunoprecipitated and subjected to an in vitro kinase incubation,
with or without ATP, as in previous experiments, followed by treatment with
CNBr. As shown in Fig. 5, basal
Lyn phosphorylation is readily detectable in an 8-kDa fragment,
consistent with the size expected for the minimal peptide containing
Tyr397. Much less is detected in the
4-kDa peptide that is the
size of the C-terminal fragment, similar to previous results in B-cells
(29). After the in
vitro kinase incubation with ATP, Lyn autophosphorylation is again
preferentially found in the
8-kDa fragment, similar to trends observed in
two previous studies (27,
44). Incomplete digestion of
Lyn by CNBr makes it difficult to determine whether the 4.1-kDa C-terminal
fragment is produced as efficiently as the 8.2-kDa fragment containing
Tyr397, but the results indicate that phosphorylation at the active
site loop contributes significantly, if not predominantly, to basal tyrosine
phosphorylation of Lyn in RBL cells. Furthermore, the relative enhancements of
phosphorylation following the in vitro kinase assay in this and in
Figs. 1 and
3 show that only a small
fraction of Lyn is phosphorylated in the basal state, suggesting that Lyn
activity is regulated primarily by phosphorylation at Tyr397, and
that Tyr508 phosphorylation plays a lesser role in the regulation
of Lyn kinase activity in unstimulated RBL-2H3 cells.
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Tyrosine 397 Phosphorylation Is Responsible for Increased Kinase
Activity of Raft-associated LynLipid raft-associated Lyn has a
higher level of basal phosphorylation than nonraft Lyn
(Fig. 3), and RIPA-solubilized
Lyn appears to be phosphorylated primarily at Tyr397
(Fig. 5). We hypothesized that
lipid raft-associated Lyn has a higher level of tyrosine 397 phosphorylation
than nonraft Lyn in RBL cells, and this leads to its higher specific activity.
We were unable to isolate sufficient Lyn from sucrose gradient fractions to
directly evaluate CNBr digests of basally phosphorylated Lyn from lipid rafts.
However, a prediction of our hypothesis is that the enhanced kinase activity
of raft-associated Lyn should be selectively reduced by the actions of
tyrosine phosphatases during cell lysis. To test this prediction, we compared
the specific activities of lipid raft-associated and nonraft Lyn obtained from
cells lysed either in the presence (as in previous experiments) or in the
absence of the tyrosine phosphatase inhibitors NaVO4 and
-glycerophosphate.
As shown in Fig. 6, lipid raft-associated Lyn isolated in the absence of phosphatase inhibitors (shaded bars) has a markedly decreased specific activity compared with Lyn isolated in the presence of phosphatase inhibitors (white bars). In fact, raft-associated Lyn isolated in the absence of phosphatase inhibitors has a kinase activity similar to that of Lyn isolated from nonraft environments. Thus, dephosphorylation of Lyn during cell lysis in the absence of tyrosine phosphatase inhibitors substantially reduces kinase activity of Lyn from lipid rafts, suggesting that this Lyn derives its higher specific activity from enhanced basal phosphorylation that is preserved during lysis in the presence of phosphatase inhibitors. Taken together with the phosphopeptide mapping results, these data imply that raft-associated Lyn is more active because it has a higher amount of phosphorylation at Tyr397. Conversely, the specific activity of soluble, nonlipid raft, Lyn is slightly increased when isolated from cells lysed in the absence of phosphatase inhibitors (Fig. 6). This suggests that the specific activity of Lyn outside the lipid raft environment may be negatively regulated by phosphorylation at Tyr508.
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DISCUSSION |
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Our present results demonstrate that the lipid raft environment regulates
the specific activity of Lyn kinase. Specifically, Lyn from a lipid raft
environment has a substantially higher in vitro kinase activity than
nonlipid raft-associated Lyn (Figs.
1,
3, and
4). Furthermore, this activity
measured with autophosphorylation or -casein does not increase
following antigen stimulation. Phosphopeptide mapping of Lyn indicates that
Tyr397, the active site tyrosine, is a major site for tyrosine
phosphorylation in unstimulated cells. This basal phosphorylation at
Tyr397 is found primarily on Lyn isolated from lipid rafts, and
dephosphorylation by omission of tyrosine phosphatase inhibitors during cell
lysis reduces raft-associated Lyn kinase activity to levels similar to that of
Lyn isolated from more fluid regions of the membrane. We conclude that
cocompartmentalization of active Lyn with Fc
RI in lipid rafts after
antigen cross-linking results in increased receptor phosphorylation and
thereby initiation of intracellular signaling.
Our initial experiments used different detergents to solubilize Lyn for immunoprecipitation and in vitro kinase analysis, and we found that Triton X-100 under standard lysis conditions resulted in recovery of less than half of the total cellular Lyn in the PNS. Octyl glucoside yielded much better recoveries (data not shown), and the best recoveries of Lyn kinase were with the RIPA mixture of Triton X-100, deoxycholate, and SDS. Furthermore, the specific activity of Lyn solubilized by Triton X-100 was found to be substantially less than that from RIPA-lysed cells, and sucrose gradient analysis showed that the Triton X-100 PNS was almost completely depleted of lipid raft-associated Lyn (Fig. 2). Because RIPA and octyl glucoside solubilize lipid rafts (30) (data not shown), sucrose gradient fractionation is not useful for cells lysed in these detergents.
These initial experiments led us to compare the recovery and distribution of Lyn using standard sucrose gradient analyses of Triton X-100-lysed cells. In the absence of a clarification step to obtain a PNS, nearly half of the Lyn is recovered in the lipid raft floating fractions. The remainder is either in mixed micelles in the middle of the gradient or at the bottom of the gradient, where cytoskeletal and other large, detergent-insoluble structures are found. These results, together with sucrose gradient analysis of the PNS, indicate that most Lyn associated with lipid rafts pellets in the centrifugation step that immediately follows cell lysis with Triton X-100 in a standard immunoprecipitation protocol, possibly because of association of lipid rafts with the cytoskeleton at the time of cell lysis. During overnight centrifugation in the sucrose gradients, this interaction is lost, allowing Lyn and most raft-associated components to float at low densities. Previous studies showed that clustered lipid rafts on intact cells are major sites for attachment of the actin cytoskeleton to the plasma membrane (25, 31), and other studies indicated that Triton X-100-resistant rafts exist as a continuous network supported by the cellular cytoskeleton immediately following cell lysis (32, 33). Taken together, our results indicate that Lyn kinase activity from Triton X-100 PNS represents a subset of its total cellular activity that is depleted of lipid raft-associated Lyn.
Using RIPA solubilization to analyze total cellular Lyn, we find that
antigen does not cause a detectable increase in total Lyn tyrosine kinase
activity after 2 min of stimulation but rather results in a decrease in this
activity at subsequent time points (Table
I). Honda et al.
(34) reported that a rapid,
transient increase in Lyn kinase activity, which preceded maximal
phosphorylation of Syk and other phosphotyrosine substrates, occurs at
13 min in wild type RBL-2H3 cells. Because they solubilized cells in a
mixture of Triton X-100 and deoxycholate, it is unclear whether they analyzed
a subset depleted in lipid raft-associated Lyn. Recent results by Ohtake
et al. (35) indicate
that, upon stimulation via FcRI, Csk is recruited to lipid rafts via its
binding to Cbp/PAG, and this results in a decrease in Fc
RI tyrosine
phosphorylation. The decrease in specific activity of Lyn we observe following
antigen stimulation may result from the C-terminal phosphorylation of
raft-associated Lyn by recruited Csk, and this may contribute to the
time-dependent decrease in stimulated Fc
RI phosphorylation that occurs.
We previously showed that inhibition of stimulated actin polymerization by
cytochalasin causes transient Fc
RI phosphorylation to become more
sustained in response to antigen
(25). Thus, it will be
interesting to investigate whether actin polymerization regulates Lyn kinase
activity under stimulating conditions and whether Csk is involved in this
process.
Together, our results indicate that tyrosine phosphorylation of the active
site is a major determinant of Lyn kinase activity, both before and
immediately after antigen stimulation. Our results are consistent with a role
for Csk-dependent C-terminal phosphorylation as a mechanism to down-regulate
Lyn kinase activity at longer times of stimulation. A previous study in
RBL-2H3 mast cells indicated that overexpression of membrane-associated Csk
can increase negative regulation of Lyn by enhancing C-terminal
phosphorylation (28).
Expression of mutated Lyn missing its C-terminal Tyr in these cells caused
elevated basal phosphorylation of FcRI and a stimulated increase by
antigen. These results indicated that C-terminal phosphorylation of Lyn
contributes to its regulation but is not the primary determinant of
antigen-stimulated Fc
RI phosphorylation. For Src, the prototype of this
family, it is clear that C-terminal phosphorylation plays a dominant role in
regulating its basal activity in mammalian cells
(36), but this kinase exhibits
only a very modest 2-fold increase in activity due to phosphorylation of its
active site loop (37). In
contrast, the large, 17-fold increase in Lyn kinase activity that results from
Tyr397 phosphorylation makes this phosphorylation site much more
important in determining the overall regulation of Lyn kinase activity
(27).
Based on our results, we propose a model for signal initiation in which Lyn
in an ordered lipid raft environment is protected from dephosphorylation at
its active site. In this environment, Lyn would have little contact with
transmembrane tyrosine phosphatases, such as CD45, which is excluded from
lipid rafts (38). CD45 itself
is not expressed detectably on most RBL-2H3 cells
(39), but it represents a
large family of receptor-like transmembrane phosphatases, one or more of which
might serve a negative regulatory role on Lyn kinase activity. In our model,
cross-linking of IgE-FcRI by antigen coalesces small, dynamic lipid raft
domains containing active Lyn into larger more stable domains containing
active Lyn and cross-linked Fc
RI. Within this environment, active Lyn
phosphorylates ITAM sequences on Fc
RI
and
subunits to
initiate the signaling cascade. The kinetics of this process parallels lipid
raft association of Fc
RI determined by sucrose gradient analysis
(11,
25). Proteins in these
coalesced lipid rafts domains would thus be protected from transmembrane
phosphatases, which are excluded and prevented from dephosphorylating Lyn or
Fc
RI.
Metzger and colleagues (40,
45) have shown that FcRI
and other substrates of Lyn or Syk kinase are not protected from
dephosphorylation when ongoing stimulation is halted by monovalent hapten.
Based on these and related results, they argue that lipid rafts fail to
protect from the action of tyrosine phosphatases. However, in breaking up
cross-links, monovalent hapten reverses lateral immobilization of cross-linked
receptors (41), and this
probably reflects dispersal of clustered lipid rafts. In another recent study,
Kovarova et al. (42)
indicated that Lyn with a single acyl chain failed to fractionate detectably
with lipid rafts, but antigen-stimulated phosphorylation of Fc
RI was
preserved with this construct. Lyn with a single saturated acyl chain would
still be expected to associate significantly with an ordered lipid
environment, but its interaction energy would be reduced, and thus it would
probably be less able to withstand the perturbation of Triton X-100
solubilization (43).
Furthermore, the construct tested in these experiments contained a green
fluorescent protein domain attached to its C terminus, so it is unclear
whether this construct can be negatively regulated by C-terminal
phosphorylation, which may contribute to the negative regulation of wild type
Lyn in the fluid membrane environment (Fig.
6).
It is not yet clear whether the phosphatase(s) responsible for FcRI
dephosphorylation that occurs after cross-linking are also effective in
dephosphorylating the active site phosphotyrosine of Lyn; these may represent
distinct molecular species having differential interactions with lipid rafts.
Testing this model critically will require identification and
co-reconstitution of these phosphatase(s) with Fc
RI and Lyn under
conditions in which antigen-stimulated Fc
RI phosphorylation can be
observed. Our working model predicts that stimulated Fc
RI
phosphorylation depends on co-segregation of cross-linked receptor and Lyn
from a phosphatase that is excluded from lipid rafts. The role of lipid rafts
to coordinate interactions between active Lyn and cross-linked Fc
RI is
central to regulating the initial events of signal transduction in RBL-2H3
mast cells.
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FOOTNOTES |
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To whom correspondence should be addressed: Dept. of Chemistry and Chemical
Biology, Cornell University, Baker Laboratory, Ithaca, NY 14850. Tel.:
607-255-4095; Fax: 607-255-4137; E-mail:
bab13{at}cornell.edu.
1 The abbreviations used are: RIPA, radioimmune precipitation assay; PNS,
postnuclear supernatant; Tricine,
N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
2 R. M. Young, D. Holowka, and B. Baird, unpublished results.
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ACKNOWLEDGMENTS |
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REFERENCES |
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