| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Receptor-Mediated Phagocytosis1
*
Department of Allergy and Rheumatology, Faculty of Medicine, University of Tokyo, Tokyo, Japan;
Division of Protein Metabolism, Institute for Protein Research, Osaka University, Osaka, Japan; and
Department of Oncology, Institute of Medical Science, University of Tokyo, Tokyo, Japan
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
receptors (FcRs) is essential for macrophage functions including
phagocytosis. Although the initial step is ascribed to Src family
tyrosine kinases, the role of individual kinases in phagocytosis
signaling is still to be determined. In reconstitution experiments, we
first showed that expression in the RAW 264.7 cell line of C-terminal
Src kinase (Csk) inhibited and that of a membrane-anchored,
gain-of-function Csk abolished the FcR-mediated signaling that leads
to phagocytosis in a kinase-dependent manner. We next tested
reconstruction of the signaling in the membrane-anchored,
gain-of-function Csk-expressing cells by introducing Src family kinases
the C-terminal negative regulatory sequence of which was replaced with
a c-myc epitope. Those constructs derived from Lyn and Hck (a-Lyn and
a-Hck) that associated with detergent-resistant membranes successfully
reconstructed FcR-mediated Syk activation, filamentous actin
rearrangement, and phagocytosis. In contrast, c-Src-derived construct
(a-Src), that was excluded from detergent-resistant membranes, could
not restore the series of phagocytosis signaling. Tyrosine
phosphorylation of Vav and c-Cbl was restored in common by a-Lyn,
a-Hck, and a-Src, but FcRIIB tyrosine phosphorylation, which is
implicated in negative signaling, was reconstituted solely by a-Lyn and
a-Hck. These findings suggest that Src family kinases are
differentially involved in FcR-signaling and that selective kinases
including Lyn and Hck are able to fully transduce phagocytotic
signaling. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Rs3), namely
FcRI, FcRIIs, and FcRIIIs (1, 2, 3, 4, 5, 6). In murine
macrophages, FcRI, FcRIIB, and FcRIIIA are expressed (3, 5, 7). Of these, FcRI and FcRIIIA are composed of an a
subunit possessing IgG-binding sites and a dimer of the subunit
that contains a cell activation motif referred to as immunoreceptor
tyrosine-based activation motifs (ITAMs) (5, 8, 9, 10, 11, 12). ITAM
tyrosines in the subunit are phosphorylated after the clustering of
FcRI and FcRIIIA, and the tyrosine-phosphorylated ITAM creates
sites for the assembly of Src homology 2 (SH2) domains, including that
of Syk tyrosine kinase. (13, 14, 15, 16, 17, 18). Requirement of a
subunit for FcR-mediated phagocytosis was unequivocally established
by the targeted disruption of the subunit gene (19).
The pivotal roles of Syk in connecting the early signaling to
phagocytosis have also been shown by various approaches (4, 20, 21). FcRIIB does not associate with the subunit, but it
possesses a motif defined as immunoreceptor tyrosine-based inhibition
motif (ITIMs) (22, 23). Coligation of FcRIIB
with cell-activating Ag or Fc receptors has been shown to
result in the down-regulation of inflammatory functions,
presumably through the action of SH2-containing inositol 5'-phosphatase
recruited to phosphorylated ITIM (5). Recent findings that
FcRIIB-/- murine
macrophages exhibit enhanced phagocytosis of IgG-coated particles are
consistent with the idea (24).
The initial event of the phosphorylation of ITAM tyrosines is presumed
to be catalyzed by Src family tyrosine kinases. This notion was first
supported by pharmacological findings that herbimycin A, a tyrosine
kinase inhibitor relatively specific for Src-family kinases, potently
suppressed Fc receptor-mediated functions (25, 26). In
addition, Src family members were found to physically associate with
resting Fc receptors, and their catalytic activity was shown to be
augmented by the receptor aggregation (27, 28, 29, 30, 31). To further
dissect the roles of Src family kinases in FcR functions, targeted
disruption of single or multiple Src family genes have been conducted
(21, 32, 33, 34). Recently, Crowley et al. (21)
found that FcR-mediated phagocytosis is delayed but preserved in
Lyn-/-Hck-/-Fgr-/-
macrophages. One of our laboratories (35) showed that mast
cell degranulation mediated by Fc
RI, a closely related member to
FcRs, is not diminished in
Lyn-/- mast cells. These
observations provided important information that Src family kinases
possess overlapping roles in these FcR-mediated functions, but their
significance is still left to be determined.
As an alternative approach, C-terminal Src kinase (Csk) has been
utilized to suppress the activity of Src family kinases
(36, 37, 38, 39, 40, 41). Src family kinases are assumed to be in an
equilibrium between C-terminal tyrosine phosphorylated ("inactive")
and dephosphorylated ("partially active") states and Csk shift the
balance to the former by phosphorylating the negative regulatory
tyrosine (42). We have previously shown that expression of
membrane-anchored Csk mutant (mCsk) effectively down-regulates mast
cell functions including FcRI-mediated degranulation and
integrin-mediated cell motility and that coexpression of selective Src
family members lacking C-terminal negative regulatory tyrosine (termed
a-Src kinases) could rescue these functions (37, 38). In
the current study, we utilized the reconstitution strategy to
investigate the roles of individual Src family kinase in
FcR-mediated phagocytosis. We observed that Csk suppressed and mCsk
almost abolished FcR-mediated Syk activation, filamentous actin
(F-actin) assembly, and phagocytosis, but a kinase-defective mCsk
(mCsk(-)) did not. Reconstitution experiments revealed that the
impaired signaling cascade by mCsk was successfully restored by the
coexpression of a-Lyn or by that of a-Hck but not by that of a-Src.
These results strongly suggested that Src family kinases are required
for FcR-mediated phagocytosis and that the function is catalyzed by
selective Src family members including Lyn and Hck in macrophages.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
All the culture media and Geneticin were purchased from Life Tech Oriental (Osaka, Japan). FCS was from Equitec Bio (Ingram, TX). Puromycin and HRP-conjugated cholera toxin B were from Sigma (St. Louis, MO). Rhodamine-conjugated phalloidin, fluorescein-conjugated Escherichia coli particles and fluorescein-conjugated zymosan particles were from Molecular Probes (Eugene, OR). Protein G-Sepharose was from Pharmacia-LKB (Uppsala, Sweden). SRBCs and rabbit anti-SRBC IgG were from InterCell Technologies (Hopewell, NJ). M-280 supraparamagnetic tosyl-activated beads and M-280 supraparamagnetic beads precoated with sheep anti-rabbit IgG (referred to as IgG-beads) were purchased from Dynal (Lake Success, NY).
Antibodies
Hybridoma producing 2.4G2, a rat anti-mouse FcRII/III
mAb, was obtained from the American Type Culture Collection (Manassas,
VA). Cultured supernatants of the hybridoma were applied to protein
G-Sepharose to purify 2.4G2 mAb. FITC-conjugated 2.4G2 mAb was
purchased from PharMingen (San Diego, CA). Anti-phosphotyrosine mAb,
4G10, was from ICN Biochemicals (Costa Mesa, CA). Polyclonal Abs
against c-myc, Syk, c-Cbl, Vav, and Csk were from Santa Cruz
Biotechnology (Santa Cruz, CA). Rabbit polyclonal anti-mouse
FcRIIB Ab was a generous gift from Dr. Toshiyuki Takai (Tohoku
University, Sendai, Japan).
Preparation of RAW 264.7 cell lines expressing Csk and its mutants
RAW 264.7 cells were maintained in DMEM supplemented with 10% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C in humidified 5% CO2 as described (37). cDNAs for rat Csk, a membrane-anchoring Csk mutant (mCsk) possessing myristoylation signal from rat c-Src, and its kinase-defective form (mCsk(-)) were described previously (37). These cDNAs were subcloned into an expression vector, pCXN2 harboring neomycin-resistant gene (43), and introduced into RAW 264.7 cells by electroporation. Geneticin-resistant cell lines were screened by immunoblotting with anti-Csk Ab, and independent cell lines expressing Csk, mCsk, or mCsk(-) were established.
Preparation of RAW 264.7 cell lines expressing mCsk in combination with mutated Src family kinases
To create c-myc-tagged Src family kinases lacking C-terminal negative regulatory tyrosine, C-terminal amino acids of rat c-Src (aa 527–536), human Lyn A (aa 505–512), and human p59hck (aa 498–505), corresponding to one another, were deleted and replaced with a c-myc epitope sequence (TSVDEQKLISEEDLN) as described (38). The resultant cDNAs, termed a-Src, a-Lyn, and a-Hck, respectively, were subcloned into an expression vector, pCAGGS (43), and stably introduced into mCsk expressing cells with the aid of a puromycin-resistant vector as described (38). Puromycin-resistant clones were screened by immunoblotting with anti-c-myc Ab and with anti-Csk Ab, and independent cell lines expressing a-Src, a-Lyn, or a-Hck in combination with mCsk were created.
Flow cytometry
To evaluate surface expression of FcRIIIA/IIB, cells were
harvested by brief trypsinization, as described (38), and
stained with 5 µg/ml of FITC-conjugated 2.4G2 mAb or an
isotype-matched control Ab in PBS supplemented with 2% horse serum and
0.01% NaN3. Cells were washed three times with
PBS, and fluorescence intensity was measured by EPICS XL flow cytometer
(Beckman Coulter, Fullerton, CA).
Rosetting assay
SRBCs (1%) were opsonized with rabbit anti-SRBC Ab for 30 min at 37°C at a subagglutinating concentration. Adherent RAW cells in ice-cold assay medium (DMEM containing 0.1% BSA and 10 mM HEPES-NaOH, pH 7.4) were loaded with precooled SRBCs and incubated for 60 min on ice to prevent SRBC internalization. Cells were washed twice with PBS, fixed with PBS containing 4% formaldehyde, and observed with light microscopy. Positive rosetting was defined as RAW cells binding three or more IgG-opsonized SRBCs.
Phagocytosis assay
M-280 tosyl-activated beads were covalently conjugated with
2.4G2 mAb (referred to as 2.4G2-beads) according to the manufacturer’s
instructions. FcR-mediated phagocytosis was assayed by using the
2.4G2 beads or with sheep IgG-coated M-280 beads (IgG-beads). Cells
were cultured on eight-well Falcon CultureSlides (Becton Dickinson,
Franklin Lakes, NJ) overnight and incubated in the assay medium for
3 h for serum starvation. Then, cells were loaded either with
IgG-beads or with 2.4G2-beads and incubated for 20 min at 4°C to
allow beads to attach to the cell surface. Excess beads were removed by
one gentle washing with ice-cold assay medium, and phagocytosis was
initiated by incubating the cells at 37°C. After 1 h of
incubation, beads outside the cells were stained with FITC-conjugated
anti-IgG at 20°C for 5 min and fixed with PBS containing 4%
formaldehyde. FITC-staining was observed and photographed by
fluorescence microscopy as described (38). Beads within
the cells were simultaneously photographed using visible light.
Phagocytosis index was calculated as the number of ingested beads per
100 cells.
Phagocytosis of Escherichia coli, nonopsonized zymosan, and complement-opsonized zymosan (COZ) was performed by using fluorescein-conjugated particles as described previously (44). For complement-opsonization, zymosan particles were incubated for 1 h at 37°C in fresh FCS as described (26). Nonopsonized zymosan particles were added to adherent RAW cells and allowed to be ingested for 1 h at 37°C in assay medium. In the case of COZ phagocytosis, cells were pretreated with 200 nM PMA for 15 min at 37°C to elicit CD11b/CD18 receptor activation (26) and loaded with COZ for 1 h at 37°C. To examine E. coli phagocytosis, cells were loaded with E. coli in assay medium supplemented with 2 mM EDTA and 10% FCS for 1 h at 37°C to evaluate the CD14-dependent phagocytotic pathway (45). After the incubations, adherent cells were washed three times with PBS, and particles remaining on the outer cell surface were removed by treating cells with trypsin/EDTA for 1.5 h at 37°C, as described (44). The quantities of internalized fluorescent particles were determined by flow cytometry or visualized with fluorescence microscopy.
FcR clustering by 2.4G2 mAb and cell lysis
Cells (1 x 107) in 6-well plastic plates were serum starved overnight in assay medium. Cells were washed once with ice-cold assay medium, equilibrated with assay medium at 4°C, and reacted with 10 µg/ml 2.4G2 mAb for 30 min. After two washes with ice-cold assay medium, reaction was initiated by addition of assay medium at 37°C containing 30 µg/ml rabbit anti-rat IgG. Cells were incubated for the indicated periods at 37°C, medium was aspirated, and cells were solubilized with Nonidet P-40 lysis buffer (20 mM Tris-HCl (pH 7.4), 1% Nonidet P-40, 0.1% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, 20 mM ß-glycerophosphate, 10 µg/ml aprotinin, 5 µg/ml leupeptin, and 0.2 mM PMSF). Insoluble materials were removed by centrifugation at 12,000 rpm for 10 min, and the supernatant was used as total cell lysate.
Immunoprecipitation and immunoblotting
In FcR stimulation experiments, total cell lysates were first
incubated with 15 µl protein G-Sepharose beads alone (50% slurry)
for 1 h at 4°C under continuous rotation to absorb 2.4G2 mAb-
and rabbit anti-rat IgG-bound FcRIIB and FcRIIIA. Samples
were centrifuged at 12,000 rpm at 4°C for 1 min, and the supernatant
was saved. Beads were washed three times with 500 µl Nonidet P-40
lysis buffer, and bound materials were eluted with boiling 2% SDS
sample buffer. The eluted proteins were used as 2.4G2 mAb
immunoprecipitates. The saved supernatants were incubated first with
various first Abs and then with 15 µl suspension of protein
G-Sepharose beads for 1 h at 4°C under continuous rotation. The
beads were washed, and bounded materials were eluted as described
above.
Immunoprecipitated proteins or the total cell lysate was subjected to SDS-PAGE, and separated proteins were electrotransferred onto a polyvinylidene fluoride membrane (Millipore, Bedford, MA). Proteins were probed with first Abs and then reacted with HRP-conjugated second Abs. Signals were detected by chemiluminescence (ECL Western Blotting System, Amersham, Arlington Heights, IL), as described (37).
In vitro kinase assay
In vitro kinase assay of Syk and the mutated Src family kinases
was performed as described previously (37, 46). Syk was
immunoprecipitated using anti-Syk polyclonal Ab as described above.
Beads were washed three times with Nonidet P-40 lysis buffer, washed
three times with kinase buffer (50 mM HEPES-NaOH (pH 7.6), 10 mM
MnCl2, 2 mM MgCl2, 10 µM
Na3VO4, 1 mM 4-nitrophenyl
phosphate), and resuspended in kinase buffer. Reaction was initiated by
the addition of 2 µM ATP and 10 µCi
[-32P]ATP, proceeded for 10 min at 30°C.
a-Src, a-Lyn, and a-Hck were immunoprecipitated with anti-c-myc Ab.
Beads were washed twice with Nonidet P-40 lysis buffer and once with
kinase buffer. Reaction was initiated by the addition of 1 µg
enolase, 2 µM ATP, and 10 µCi [-32P]ATP
and proceeded for 1 min at 25°C. Under the reaction conditions,
[-32P]ATP incorporation into enolase was
linear with time (37). Reactions were terminated by the
addition of 2% SDS sample buffer. After boiling, samples were
centrifuged, and the supernatants were subjected to SDS-PAGE. Gels were
dried and subjected to phosphorimager analysis using a Fuji BAS 2000
image analyzer (Fujifilm Medical, Tokyo, Japan).
Detection of F-actin accumulation
To prepare a 2.4G2 mAb-coated surface, LAB-TEK chamber slides (Permanox, Nunc, Naperville, IL) were filled overnight with or without 10 µg/ml 2.4G2 mAb in carbonate buffer, pH 9.6, at 4°C. Wells were washed once with PBS, blocked with 20 mg/ml fatty acid-free BSA in PBS for 1 h at 37°C, and again washed twice with PBS and once with DMEM. RAW cells in assay medium were seeded onto the chamber slides and allowed to adhere to the 2.4G2 mAb-coated surface at 37°C for indicated periods. Accumulation of F-actin at the attachment sites was detected by staining with rhodamine-conjugated phalloidin and observed by fluorescence microscopy as described (38).
Sucrose density gradient centrifugation
Protein association with detergent-resistant membranes (DRMs) was analyzed by solubilizing cells with low concentration Triton X-100 followed by ultracentrifugation of cell lysates on sucrose density gradients according to the method of Field et al. (47). In brief, 4 x 106/ml cell suspension was solubilized with 0.05% Triton X-100, cell lysate was layered onto 80 to 10% discontinuous sucrose gradients prepared in a Hitachi 13 PA tube (1.5 x 9.6 cm), and centrifuged at 35,000 rpm at 4°C for 18 h (47). Aliquots (1 ml) of the gradients were collected, proteins were extracted following the methods of Wessel and Flugge (48), and the sample was subjected to Western blotting as described above.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
R-mediated
phagocytosis
As an approach to investigation of the roles of Src family kinases
in FcR-mediated phagocytosis, we utilized Csk, mCsk
(36, 37, 38), and mCsk(-), which functions as a dominant
negative molecule (37). These Csk-based molecules were
transfected into the RAW 264.7 macrophage cell line, and multiple
independent clones stably expressing each of them were established. A
representative immunoblot with anti-Csk Ab of RAW cells
overexpressing Csk, mCsk, or mCsk(-) is shown in Fig. 1
.
|
RII/III mAb,
2.4G2. Cells were loaded with the beads and allowed to internalize them
for 60 min at 37°C. As seen in Fig. 2A, 2.4G2-coated beads
remaining on the cell surface were positively stained with
FITC-conjugated anti-IgG (bright green particles), and internalized
beads were observed as dark yellow particles. RAW 264.7 cells (WT-RAW)
efficiently phagocytosed 2.4G2-beads. Csk-overexpressing cells ingested
the beads less efficiently, and mCsk-overexpressing cells were
virtually unable to ingest them. mCsk(-)-overexpressing cells
phagocytosed the beads almost comparably with WT-RAW. Phagocytosis
index (number of ingested beads per 100 cells) of the cell lines is
shown as a bar graph in Fig. 2B. Csk and mCsk significantly
suppressed the FcR-mediated phagocytosis, and the effects were
kinase dependent. Surface expression of FcRIIB/FcRIIIA, as
analyzed by fluorescent 2.4G2 mAb staining, and IgG-opsonized SRBC
rosetting on the cells were not significantly altered by the expression
of Csk or the mutated Csks (not shown). Therefore, those inhibitory
effects of Csk and mCsk could not be ascribed to decreased density or
affinity of FcRIIB and IIIA, but most likely to impaired
postreceptor signaling pathway.
|
R-IgG contact sites, which is an early and obligatory step for
FcR-mediated phagocytosis (49, 50). Cells were allowed
to adhere onto 2.4G2-coated surfaces, and F-actin accumulation was
detected by staining with rhodamine-phalloidin. As seen in Fig. 2
C, a ring-like F-actin accumulation developed at ventral
surfaces of cells in WT-RAW within 5 min. Such structures were not
observed in Csk- or mCsk-overexpressing cells within 60
min, although those structures were clearly observed in
mCsk(-)-overexpressing cells within 5 min (Fig. 2C). These
findings were consistent with Csk-and mCsk-mediated inhibition of
phagocytosis and indicated that Csk inhibits FcR-mediated
phagocytosis at a step before F-actin rearrangement. Differential inhibitory effects of Csk on the phagocytosis of E. coli, nonopsonized zymosan, and COZ
We next compared the effects of Csk-derived molecules on other
phagocytosis pathways. Nonopsonized zymosan and COZ were shown to be
internalized mainly via receptors for mannose-fucose and ß-glucan and
via CD11b/CD18 integrin, respectively (6, 51, 52). Under
the current experimental conditions, E. coli was shown to be
ingested via the CD14-dependent pathway (45). Those
particles conjugated with fluorescein were loaded onto RAW cell lines,
and phagocytosis was evaluated by flow cytometry. As seen in Fig. 3, WT cells efficiently ingested these
three materials. Csk and mCsk were found to exert differential effects
on these phagocytotic activities: phagocytosis of nonopsonized zymosan
was most severely affected by their expressions, while E.
coli- and COZ phagocytosis was only marginally influenced. As
compared with control WT cells, 97.7 and 89.8% of mCsk cells ingested
E. coli, and COZ, respectively, whereas only 27.8% of mCsk
cells phagocytosed nonopsonized zymosan. These findings suggested that
increased Csk activity resulted in the suppression of selective
phagocytotic receptor functions.
|
RII/III-mediated Syk activation
Because Syk plays pivotal roles in FcR-mediated phagocytosis
(20, 21), we next evaluated the effects of Csk and mCsk on
Syk activation. FcRIIB and IIIA were aggregated with 2.4G2 and
second Ab at 37°C, and cells were lysed 3 min after the receptor
clustering. Syk was immunoprecipitated from the lysates and subjected
to in vitro kinase assay or to immunoblotting. As seen in Fig. 4, top (IVK), Syk activity, as
assessed by autophosphorylation, was clearly increased after FcRIIB
and FcIIIA clustering in WT-RAW. Csk inhibited and mCsk more
potently inhibited clustering-induced Syk activation. In mCsk(-)
cells, Syk activation was almost preserved. As seen in Fig. 4, middle (pY blot), Syk was tyrosine phosphorylated under
basal conditions in WT cells, and FcRIIB and FcIIIA clustering
resulted in enhanced Syk tyrosine phosphorylation. As indicated by an
asterisk, tyrosine-phosphorylated proteins of 
20 kDa, presumably
corresponding to the subunit (53), were
coimmunoprecipitated with Syk, and the signals were increased by
FcRII/III clustering. Csk expression suppressed basal and
clustering-induced Syk tyrosine phosphorylation and decreased the
signals of 20 kDa tyrosine phosphorylation. mCsk expression almost
abolished tyrosine phosphorylation of Syk and associated 20 kDa
tyrosine phosphorylation. In contrast, mCsk(-) expression did not
appreciably inhibit these signals. These observation indicated that Csk
and mCsk inhibited FcRII/III-mediated activation of Syk in a
stepwise manner and that the effects were kinase dependent.
|
The above findings that Csk and mCsk inhibited
FcRII/III-mediated Syk activation, actin cytoskeleton
reorganization, and phagocytosis suggested that Src family kinases play
pivotal roles in the signaling pathway. To further confirm the
requirement of Src family kinases and to investigate the roles of
individual Src family kinase for the functions, we created Src family
kinases in which C-terminal sequences containing negative regulatory
tyrosine were replaced with c-myc epitope (Fig. 5A). These partially active
constructs, termed a-Src kinases, were reconstituted in mCsk cells and
tested for the ability to reconstruct FcRII/III-mediated signaling.
In this study, we focused on Lyn, Hck, and c-Src. Among the Src family
members, Lyn and Hck are expressed predominantly in hemopoietic cells,
whereas c-Src is ubiquitously expressed. RAW 264.7 cells express these
molecules (not shown). a-Lyn and a-Src, in which the corresponding
C-terminal amino acid sequences (aa 505–512 for Lyn A
(p56lyn), aa 527–536 for c-Src
(p60c-src)) were replaced with a c-myc
epitope tag sequence were described previously (38).
a-Hck, in which aa 498–505 were replaced with c-myc epitope, was
created as described in Materials and Methods (Fig. 5A). Puromycin-resistant vector alone or in combination with
each a-Src kinase construct was transfected into mCsk cells, and
multiple mCsk cell lines coexpressing comparable levels of a-Lyn,
a-Hck, or a-Src were obtained. Representative immunoblots of the clones
with anti-c-myc Ab and with anti-Csk Ab are shown in Fig. 5B. Expression of these a-Src kinases did not affect surface
expression of FcRIIB and FcRIIIA, as assessed by fluorescent
2.4G2 staining (not shown).
|
C, all the constructs efficiently phosphorylated enolase to
roughly comparable levels, although activity of a-Src exceeded those of
a-Lyn and a-Hck. We could not find the reason for the difference, but
it might be ascribed to different intrinsic kinase activities of c-Src,
Lyn, and Hck.
FcR-mediated phagocytosis and F-actin rearrangement were
reconstructed by a-Lyn or by a-Hck, but not by a-Src
We compared FcRII/III-mediated phagocytosis in the cells
expressing mCsk or mCsk with a-Src kinases. Cells were loaded with
2.4G2-beads or sheep IgG-beads for 60 min at 37°C, and beads
internalized in the cells were observed and counted as described above.
As seen in Fig. 6A, vector
control cells (puro/mCsk cells) failed to ingest 2.4G2-beads, as
expected. Intriguingly, expression of a-Lyn or that of a-Hck in mCsk
cells clearly reconstructed the ability to ingest 2.4G2-beads (Fig. 6A). In contrast, a-Src expression did not efficiently
restore the phagocytosis. As seen in the bar graph of the calculated
phagocytosis index (Fig. 6B), expression of a-Lyn or that of
a-Hck significantly increased phagocytosis index above vector control,
to almost comparable levels in WT cells, but expression of a-Src did
not. FcR-mediated F-actin assembly was also examined by using
2.4G2-coated surface. The ring-like structures of accumulated F-actin
at the contact sites were clearly observed in a-Lyn/mCsk cells and in
a-Hck/mCsk cells, but these structures were barely detectable in
a-Src/mCsk cells (Fig. 6C).
|
RII/III-mediated Syk activation was restored by a-Lyn or by
a-Hck but not by a-Src
To investigate the mechanisms underlying the differential
restoration of phagocytosis by a-Src kinases, we next examined
FcRII/III clustering-induced Syk activation. As seen in Fig. 7, top, Syk
autophosphorylation activity in puro/mCsk control cells was almost
undetectable, as seen in parent mCsk cells (Syk ip IVK). In
a-Lyn/mCsk cells and in a-Hck/mCsk cells, receptor clustering resulted
in clear increase in the kinase activity (Fig. 7). In contrast,
receptor clustering did not induce detectable Syk activation in
a-Src/mCsk cells (Fig. 7). As seen in Fig. 7, middle (pY
blot), Syk tyrosine phosphorylation was almost undetectable before and
after FcRII/III clustering in puro/mCsk cells. In a-Lyn/mCsk and
a-Hck/mCsk cells, basal Syk tyrosine phosphorylation was clearly
observed, and FcRII/III clustering resulted in intense Syk tyrosine
phosphorylation. In addition, the 20 kDa signal, presumably
corresponding to the tyrosine-phosphorylated subunit, was
coimmunoprecipitated with Syk under basal conditions, and the intensity
of the signals was increased after the receptor clustering. In
a-Src/mCsk cells, Syk tyrosine phosphorylation under resting conditions
was barely detectable, and it was increased to a lesser extent than
those in a-Lyn/mCsk and a-Hck/mCsk cells after the receptor clustering.
Therefore, Syk activation was successfully reconstructed by a-Lyn or
a-Hck. a-Src weakly tyrosine phosphorylate Syk upon clustering but
could not restore Syk activation.
|
RII/III clustering
The above findings indicated that a-Lyn and a-Hck almost fully
reconstructed FcRII/III-mediated signaling leading to phagocytosis,
whereas a-Src could only weakly transmit Syk tyrosine
phosphorylation. To further explore their differential abilities to
transduce FcRIIIA/IIB signaling, we surveyed basal and
clustering-induced tyrosine phosphorylation of signaling molecules
including c-Cbl, Vav, and FcRIIB. Vav transmits positive signals
through GTP/GDP exchange of Rac, which leads to JNK activation and the
regulation of actin cytoskeleton (54, 55). c-Cbl and
FcRIIB seem to function as negative regulators, through the
down-regulation of Syk activity and/or the degradation of Syk molecule
via ubiquitination (56, 57), and by the recruitment of SH2
containing inositol phosphatase, respectively (58, 59). As
seen in Fig. 8A (c-Cbl ip),
all three a-Src kinases commonly increased basal and clustering induced
c-Cbl tyrosine phosphorylation above control levels (puro/mCsk cells).
In a-Hck/mCsk cells, clustering-induced signal appeared weak, but
significantly reduced c-Cbl recovery after clustering (see c-Cbl blot)
suggested that it was presumably due to a-Hck association with
detergent-insoluble cytoskeletal fraction (60, 61). They
also in common augmented basal and clustering-induced Vav tyrosine
phosphorylation (Fig. 8A, Vav ip). The smaller clustering
effects in a-Lyn/mCsk and a-Hck/mCsk cells than that in a-Src/mCsk
cells presumably reflected intense constitutive tyrosine
phosphorylation. In contrast to c-Cbl and Vav tyrosine phosphorylation,
FcRIIB tyrosine phosphorylation was reconstructed only by a-Lyn and
a-Hck, but not by a-Src (Fig. 8B, pY blot); a-Lyn and a-Hck
expression resulted in detectable basal FcRIIB tyrosine
phosphorylation and intense FcRIIB tyrosine phosphorylation after
clustering, whereas a-Src expression did not exert such effects.
|
Recent studies have revealed that localization of Src family
kinases at specialized low density membrane domain, called DRMs or
sphingolipid-cholesterol rafts (62), is critical for Ag
receptor signal transduction (47, 63, 64, 65, 66, 67). We thus
examined the association of a-Lyn, a-Hck, and a-Src with DRMs by
sucrose density gradient centrifugation (47, 65). a-Src
kinases were detected with anti-c-myc Ab, and GM1 ganglioside, a
marker of DRMs (62), with cholera toxin B. As seen in Fig. 9, a-Lyn and a-Hck distributions
exhibited separated peaks at high density (fractions 2–6) and low
density (fractions 8 and 9) fractions, and the latter colocalized with
GM1 (a marker of DRMs). In contrast, a-Src was recovered mainly from
high density fractions (fractions 2–7), and its association with DRMs
was minimal. These findings revealed that a-Lyn and a-Hck were in part
associated with DRMs and that a-Src was almost excluded from
DRMs.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
R-mediated signaling. Several lines of evidence indicated that
the earliest event after FcR clustering is phosphorylation of ITAM
tyrosines in the subunit and that this modification is prerequisite
for transducing downstream signaling (16, 68, 69).
Impaired Syk activation in FcR-stimulated
Lyn-/-Hck-/-Fgr-/-
murine macrophages further evidenced the close proximity of Src
family kinases to FcRs (21). However, the findings that
FcR-mediated phagocytosis is delayed but preserved in the
Lyn-/-Hck-/-Fgr-/-
cells still left the roles of Src family kinases to be determined.
Furthermore, the specificity of Src family kinases in transducing
phagocytosis signaling has not been elucidated. As an approach to
complementing the knowledge obtained from gene targeting studies, we
designed reconstitution experiments using membrane-anchored Csk that
functions as a gain-of-function molecule (37).
Through the analysis of RAW 264.7 murine macrophage cell line
overexpressing Csk, mCsk, or mCsk(-), it was first observed that
FcR-mediated phagocytosis was inhibited in Csk-expressing cells and
almost abolished in mCsk-expressing cells under our experimental
conditions, and that the inhibitory effects of mCsk was kinase
dependent (see Fig. 2, A and B). Several lines of
evidence indicate that Syk tyrosine kinase is indispensable for
FcR-mediated phagocytosis:
Syk-/- murine macrophages
are incapable of FcR-mediated phagocytosis (21), and
clustering of a FcR-Syk chimera is sufficient to induce phagocytosis
when expressed in COS cells (20). It is also established
that F-actin assembly around phagosomes is an early and obligatory
process in the phagocytosis (49, 50). We observed that
these early signals were also inhibited by Csk and by mCsk in a
stepwise manner and that the inhibitory effects were kinase dependent.
These findings strongly indicate that Src family kinases are
indispensable upstream regulators for FcRII/III-mediated
phagocytosis, and they confirm the idea that Src family kinases lies
upstream of Syk activation (21). It might be also possible
that Src family kinases regulate common downstream signaling (e.g.,
actin polymerization) and that the inhibitory effects of mCsk are not
specific for selective receptors. This hypothesis seems unlikely
because mCsk exerted differential effects on nonopsonized zymosan, COZ,
and E. coli phagocytosis. Marginal effects of mCsk on COZ
and E. coli internalization are consistent with previous
reports showing that herbimycin A did not affect COZ phagocytosis
(26) and that CD14-mediated signaling was essentially
normal in
Lyn-/-Hck-/-Fgr-/-
triple knockout macrophages (70). Mechanisms of
nonopsonized zymosan, presumably mediated by receptors for
mannose-fucose and ß-glucan (6, 51, 52), have not been
fully elucidated (6), but our findings suggested essential
roles of Src family kinases in these pathways.
The prominent inhibitory effects of mCsk on the phagocytosis of
IgG-coated beads are apparently contradictory to the data from
Lyn-/-Hck-/-Fgr-/-
primary macrophages challenged with IgG-coated SRBC (21).
The delayed kinetics of phagocytosis in the triple knockout macrophages
is consistent with our data indicating positive regulatory roles of Src
family kinases. However, net phagocytosis was unchanged in the triple
knockout cells after long incubation periods
(21), whereas it clearly decreased in mCsk-expressing RAW
cells. One possible explanation for the discrepancy is that
FcR-mediated phagocytosis in
Lyn-/-Hck-/-Fgr-/-
primary macrophages was compensated by other Src family kinases
including Yrk, as suggested by the authors (70), and that
mCsk blocked the activity of the residual kinases. Alternatively, it
might be ascribed to different carriers, SRBC in the study by Crowley
et al. and artificial beads in this study. Although quantitative data
were not available, Lowell et al. (32) showed that uptake
of IgG-coated beads was reduced but that IgG-SRBC phagocytosis was
normal in
Hck-/-Fgr-/-
double knockout macrophages, findings suggestive of the influence of
carrier particles. RBCs express several ligands for phagocytotic
receptors including scavenger receptors and asialoglycoprotein
receptor, especially at senescent or physically damaged
states (6, 71, 72, 73, 74). Therefore, it may also be
possible that the slow phagocytosis of IgG-SRBC in
Lyn-/-Hck-/-Fgr-/-
cells is catalyzed via ligands on SRBC surfaces.
The clear suppression of FcR-mediated phagocytosis in
mCsk-overexpressing cells led us to test the abilities of individual
Src family kinase to reconstruct the functions by coexpression
procedures. Basal tyrosine phosphorylation of Syk and 20 kDa
protein(s) corresponding to subunit were decreased in Csk cells and
almost vanished in mCsk cells, but not in mCsk(-) cells (Fig. 4, pY
blot). Similar observations were made in
Lyn-/- mast cells
(34) and in
Lyn-/-Hck-/-Fgr-/-
primary macrophages (21). These findings strongly
indicated that under basal conditions, Src family kinases are partly in
an active (C-terminal tyrosine dephosphorylated) conformation, and the
mCsk stronly reduced the probability for the kinases to take the active
conformation. We thus tested the effects of the coexpression of
C-terminal tyrosine-deleted a-Src kinases in mCsk cells on
FcRII/III-mediated phagocytotic signaling.
Reconstitution experiments showed that FcR-mediated phagocytosis was
restored by a-Lyn or by a-Hck, but not by a-Src. The successful
reconstruction of the phagocytosis by the two hemopoietic Src family
kinases, together with Csk-mediated inhibition of the phagocytosis,
further strengthened the idea that Src family kinases are indispensable
for FcR-mediated phagocytosis. In addition, the differential
abilities of a-Src kinases strongly suggested that FcR-mediated
phagocytosis is catalyzed by selective Src family members. The
inability of a-Src to restore the phagocytosis was not due to low
expression level of the kinase (see Fig. 5B), or to
defective catalytic activity of a-Src construct (Fig. 5C).
Preserved surface expression of FcRII/III and IgG-opsonized SRBC
rosetting in a-Src/mCsk cells excluded the possibility that a-Src
expression altered ligand-FcR binding. We thus concluded that a-Src
could not drive postreceptor signaling. Consistent with the notion,
clustering-mediated activation of Syk and accumulation of F-actin on a
2.4G2-coated surface were reconstructed by a-Lyn and by a-Hck, but not
by a-Src. The inability of a-Src to restore FcR-mediated
phagocytosis is most likely ascribed to its inability to induce
clustering-mediated Syk activation. Although it should be taken into
account that these a-Src kinases are not in normal equilibrium, the
above findings strongly suggest that FcR-mediated phagocytosis could
be driven by selective Src family members including Lyn and Hck. As an
alternative explanation, it may be possible that a-Src kinases
conversely down-regulated mCsk activity or displaced it from a proper
position and that FcR signaling was initiated by endogenous Src
family kinases, but not by a-Src kinases. These possibilities should be
examined by further studies. However, it was noted that
FcRIIIA/IIB-clustering induced physical association of the receptor
complexes with a-Lyn, but not with a-Src (H. Kono and Z.-i. Honda,
unpublished observation). These observations suggest that a-Lyn
positively participates in the signal initiation rather than
nonspecifically down-regulating mCsk functions.
The mechanisms underlying the specificity should be elucidated by
future studies, but one potential clue is that N-terminal
palmitoylation sites were found in Lyn and Hck but not in c-Src
(75). It has become increasingly clear that palmitoylation
of Src family kinases is required for kinases to associate with
functional membrane subdomains variously called detergent-resistant
membranes (DRMs) or sphingolipid-cholesterol rafts (62).
Association of Src family kinases with DRMs seems to be essential for
kinases to transduce signaling originated from aggregated TCR
(63, 64). Recently, we provided evidence that early
FcRI signaling is catalyzed by Lyn and Fyn, but not by c-Src, and
that creation of palmitoylation site in c-Src rendered it competent to
transmit FcRI signaling (76). In the current study, we
confirmed that a-Lyn and a-Hck partially associated with DRMs, whereas
a-Src did not. The differential localization may in part explain the
specificity of Src family kinases in FcRIIIA/IIB-mediated
phagocytosis.
Although a-Src could not restore FcRIIIA/IIB-mediated phagocytosis,
a-Src did not seem to be completely separated from the
FcRIIIA/IIB-signaling pathway. Expression of a-Src up-regulated
clustering-induced tyrosine phosphorylation of c-Cbl and Vav. Syk
tyrosine phosphorylation was also weakly up-regulated, but Syk
activation was not detectable in a-Src/mCsk cells, suggesting that
activation loop tyrosine was not efficiently phosphorylated. Concerning
the roles of c-Src for other Fc receptor functions, physical
association of c-Src with Fcµ receptor and Fcµ- and
FcRI-mediated c-Src activation have been noted (27, 77). These data including ours argue for involvement of a-Src in
Fc-receptor signaling, albeit that it could transduce merely abortive
signaling. Considering that the Fc receptor 
subunit localizes at
the outside of DRMs before clustering (47), it might be
possible that FcR signaling could be initiated at the outside of DRMs,
but that sufficient amplification of signaling to induce biological
functions (i.e., phagocytosis) could be catalyzed only by
DRM-associated Src family kinases.
Intriguingly, FcRIIB tyrosine phosphorylation was not increased by
a-Src expression, while it was intensely augmented by a-Lyn and a-Hck
expression. The striking specificity is of interest, because FcRIIB
is implicated in negative signaling in a variety of Ag and Fc receptors
(5, 22, 23, 59, 78, 79). One of our laboratory showed that
tyrosine phosphorylation of FcRIIB ITIM after coligation with
surface IgM is almost exclusively catalyzed by Lyn in murine B cells
(34). The current findings revealed through a different
approach that FcRIIB could be tyrosine phosphorylated by
DRM-associated Lyn and Hck. We are now investigating the mechanisms how
FcRIIB tyrosine phosphorylation is catalyzed solely by
DRM-associated kinases.
Because distributions and functions of Src family kinases are highly
overlapping, it is crucial to define selective Src family kinases in
inflammatory signaling pathways. The current study provided further
evidence that Src family kinases are important upstream regulators of
FcR-mediated phagocytosis signaling, and for the first time
suggested that the phagocytosis signaling could be driven by Lyn and by
Hck, but only incompletely by c-Src. To further define the roles of
other Src family kinases and to elucidate the submolecular structures
responsible for the FcR functions are the next problems that should
be investigated.
Since the submission of the paper, Fitzer-Attas et al. (80) showed that phagocytosis of IgG-coated SRBC in Lyn-/-Hck-/-Fgr-/- primary macrophages was not only delayed but also substantially decreased. These findings are consistent with our observations that mCsk almost abolished phagocytosis of IgG-coated beads in RAW cells.
|
Acknowledgments |
|---|
RIIB Ab and H. Ota-Ichijo and M. Saka for
excellent technical assistance. |
Footnotes |
|---|
2 Address correspondence and reprint requests to Zen-ichiro Honda, Department of Allergy and Rheumatology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan.
3 Abbreviations used in this paper: FcR, Fc receptor; ITAM, immunoreceptor tyrosine-based activation motif; ITIM, immunoreceptor tyrosine-based inhibition motif; Csk, C-terminal Src kinase; mCsk, membrane-anchored C-terminal Src kinase; mCsk(-), kinase-defective, membrane-anchored C-terminal Src kinase; F-actin, filamentous actin; COZ, complement-opsonized zymosan; DRM, detergent-resistant membrane; SH2, Src homology 2; WT, wild type; WT-RAW, wild-type RAW 264.7 cells.
Received for publication November 8, 1999. Accepted for publication April 12, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
receptor classes on human leukocytes. J. Exp. Med. 171:1333.RIIa (CD32) introduced into mouse fibroblasts mediates phagocytosis of sensitized erythrocytes. Blood 79:1651. receptor mediated phagocytosis. Blood 86:4389. subunit are required for a phagocytic signal mediated by Fc gamma RIIIA. J. Clin. Invest. 92:2073.
RIIIA transmits a phagocytic signal that requires the cytoplasmic domain of its subunit. J. Clin. Invest. 92:1967.
receptor, FcRIIIA: sequence requirements and interaction with protein-tyrosine kinases. Proc. Natl. Acad. Sci. USA 92:7381.RI) results in phosphorylation of its associated gamma-chain. J. Biol. Chem. 269:12111. subunit of Fc receptors, p72syk, and paxillin during Fc receptor-mediated phagocytosis in macrophages. J. Biol. Chem. 269:3897. receptors on macrophages. J. Exp. Med. 186:1027. RIIB modulates B-cell receptor signalling. Nature 369:340.[Medline]
RII in monocytic THP-1 cells. J. Biol. Chem. 269:8878. RIIIA (CD16) in natural killer cells. J. Exp. Med. 177:1475. RIIIA activates the receptor-associated protein tyrosine kinase Syk and induces phosphorylation of multiple proteins including p95Vav and p62/GAP-associated protein. J. Immunol. 152:5429.[Abstract]
RI to the Rac1-JNK pathway. J. Biol. Chem. 272:10751.)RIIB. Nature 383:263.[Medline]
RII-deficient mice. Nature 379:346.[Medline]
RI with detergent-resistant membranes. J. Biol. Chem. 274:1753.-chain in tyrosine kinase activation via murine Fc gamma RIII. EMBO J. 11:2747.[Medline]
RI chain as a function of the receptor context. J. Exp. Med. 181:247. signaling. Mol. Cell. Biol. 20:1759.
- and FcRI -chains and with src family protein tyrosine kinases. J. Immunol. 157:1485.[Abstract]
RIIB, regulates negatively BCR-, TCR-, and FcR-dependent cell activation. Immunity 3:635.[Medline]
receptor-mediated phagocytosis in macrophages lacking the Src family tyrosine kinases Hck, Fgr, and Lyn. J. Exp. Med. 191:669.This article has been cited by other articles:
|
|
 |
|
  P. Haberzettl, R. P. F. Schins, D. Hohr, V. Wilhelmi, P. J. A. Borm, and C. Albrecht Impact of the Fc{gamma}II-receptor on quartz uptake and inflammatory response by alveolar macrophages Am J Physiol Lung Cell Mol Physiol, June 1, 2008; 294(6): L1137 - L1148. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Cougoule, S. Hoshino, A. Dart, J. Lim, and E. Caron Dissociation of Recruitment and Activation of the Small G-protein Rac during Fc{gamma} Receptor-mediated Phagocytosis J. Biol. Chem., March 31, 2006; 281(13): 8756 - 8764. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. Sanjuan, N. Rao, K.-T. A. Lai, Y. Gu, S. Sun, A. Fuchs, W.-P. Fung-Leung, M. Colonna, and L. Karlsson CpG-induced tyrosine phosphorylation occurs via a TLR9-independent mechanism and is required for cytokine secretion. J. Cell Biol., March 27, 2006; 172(7): 1057 - 1068. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Sobota, A. Strzelecka-Kiliszek, E. Gladkowska, K. Yoshida, K. Mrozinska, and K. Kwiatkowska Binding of IgG-Opsonized Particles to Fc{gamma}R Is an Active Stage of Phagocytosis That Involves Receptor Clustering and Phosphorylation J. Immunol., October 1, 2005; 175(7): 4450 - 4457. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Pertosa, S. Simone, M. Soccio, D. Marrone, L. Gesualdo, F. P. Schena, and G. Grandaliano Coagulation Cascade Activation Causes CC Chemokine Receptor-2 Gene Expression and Mononuclear Cell Activation in Hemodialysis Patients J. Am. Soc. Nephrol., August 1, 2005; 16(8): 2477 - 2486. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Okazawa, S.-i. Motegi, N. Ohyama, H. Ohnishi, T. Tomizawa, Y. Kaneko, P.-A. Oldenborg, O. Ishikawa, and T. Matozaki Negative Regulation of Phagocytosis in Macrophages by the CD47-SHPS-1 System J. Immunol., February 15, 2005; 174(4): 2004 - 2011. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. A. Swanson and A. D. Hoppe The coordination of signaling during Fc receptor-mediated phagocytosis J. Leukoc. Biol., December 1, 2004; 76(6): 1093 - 1103. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z.-Y. Huang, S. Hunter, M.-K. Kim, P. Chien, R. G. Worth, Z. K. Indik, and A. D. Schreiber The monocyte Fc{gamma} receptors Fc{gamma}RI/{gamma} and Fc{gamma}RIIA differ in their interaction with Syk and with Src-related tyrosine kinases J. Leukoc. Biol., August 1, 2004; 76(2): 491 - 499. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Song, S. Tanaka, D. Cox, and S. C. Lee Fc{gamma} receptor signaling in primary human microglia: differential roles of PI-3K and Ras/ERK MAPK pathways in phagocytosis and chemokine induction J. Leukoc. Biol., June 1, 2004; 75(6): 1147 - 1155. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. G. Khadaroo, A. Kapus, K. A. Powers, M. I. Cybulsky, J. C. Marshall, and O. D. Rotstein Oxidative Stress Reprograms Lipopolysaccharide Signaling via Src Kinase-dependent Pathway in RAW 264.7 Macrophage Cell Line J. Biol. Chem., November 28, 2003; 278(48): 47834 - 47841. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Strzelecka-Kiliszek, K. Kwiatkowska, and A. Sobota Lyn and Syk Kinases Are Sequentially Engaged in Phagocytosis Mediated by Fc{gamma}R J. Immunol., December 15, 2002; 169(12): 6787 - 6794. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P.-Y. Berclaz, Y. Shibata, J. A. Whitsett, and B. C. Trapnell GM-CSF, via PU.1, regulates alveolar macrophage Fcgamma R-mediated phagocytosis and the IL-18/IFN-gamma -mediated molecular connection between innate and adaptive immunity in the lung Blood, December 1, 2002; 100(12): 4193 - 4200. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. O. C. Cory, R. Garg, R. Cramer, and A. J. Ridley Phosphorylation of Tyrosine 291 Enhances the Ability of WASp to Stimulate Actin Polymerization and Filopodium Formation J. Biol. Chem., November 15, 2002; 277(47): 45115 - 45121. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. C. Edberg, H. Qin, A. W. Gibson, A. M. F. Yee, P. B. Redecha, Z. K. Indik, A. D. Schreiber, and R. P. Kimberly The CY Domain of the Fcgamma RIa alpha -Chain (CD64) Alters gamma -Chain Tyrosine-based Signaling and Phagocytosis J. Biol. Chem., October 18, 2002; 277(43): 41287 - 41293. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Mahalingam and B. A. Lidbury Suppression of lipopolysaccharide-induced antiviral transcription factor (STAT-1 and NF-kappa B) complexes by antibody-dependent enhancement of macrophage infection by Ross River virus PNAS, October 15, 2002; 99(21): 13819 - 13824. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. P. Scott, F. Zappacosta, E. Y. Kim, R. S. Annan, and W. T. Miller Identification of Novel SH3 Domain Ligands for the Src Family Kinase Hck. WISKOTT-ALDRICH SYNDROME PROTEIN (WASP), WASP-INTERACTING PROTEIN (WIP), AND ELMO1 J. Biol. Chem., July 26, 2002; 277(31): 28238 - 28246. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Kono, T. Suzuki, K. Yamamoto, M. Okada, T. Yamamoto, and Z.-i. Honda Spatial Raft Coalescence Represents an Initial Step in Fc{gamma}R Signaling J. Immunol., July 1, 2002; 169(1): 193 - 203. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Carreno, E. Caron, C. Cougoule, L. J. Emorine, and I. Maridonneau-Parini p59Hck Isoform Induces F-actin Reorganization to Form Protrusions of the Plasma Membrane in a Cdc42- and Rac-dependent Manner J. Biol. Chem., May 31, 2002; 277(23): 21007 - 21016. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Liu, K. Aupperle, and R. Terkeltaub Src family protein tyrosine kinase signaling mediates monosodium urate crystal-induced IL-8 expression by monocytic THP-1 cells J. Leukoc. Biol., December 1, 2001; 70(6): 961 - 968. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Majeed, E. Caveggion, C. A. Lowell, and G. Berton Role of Src kinases and Syk in Fc{gamma} receptor-mediated phagocytosis and phagosome-lysosome fusion J. Leukoc. Biol., November 1, 2001; 70(5): 801 - 811. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. G. Marshall, J. W. Booth, V. Stambolic, T. Mak, T. Balla, A. D. Schreiber, T. Meyer, and S. Grinstein Restricted Accumulation of Phosphatidylinositol 3-Kinase Products in a Plasmalemmal Subdomain during Fc{gamma} Receptor-mediated Phagocytosis J. Cell Biol., June 18, 2001; 153(7): 1369 - 1380. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
