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Receptor Clustering in Monocytes1
Departments of
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Internal Medicine and
Microbiology, Ohio State University, Columbus, OH 43210
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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R signal transduction in monocytes describe
a molecular cascade that begins upon clustering of FcR with the
phosphorylation of critical tyrosine residues in the cytoplasmic
domains of FcRIIa or the -chain subunit of FcRI and
FcRIIIa. The cascade engages several other tyrosine-phosphorylated
molecules, either enzymes or adapters, to manifest ultimately an array
of biological responses, including phagocytosis, cell killing,
secretion of a variety of inflammatory mediators, and activation.
Continuing to assess systematically the molecules participating in the
cascade, we have found that the SH2-containing 5'-inositol phosphatase
(SHIP) is phosphorylated on tyrosine early and transiently after FcR
clustering. This molecule in other systems, such as B cells and mast
cells, mediates an inhibitory signal. We find that clustering of either
FcRIIa or FcRI is effective in inducing SHIP phosphorylation,
that SHIP binds in vitro to a phosphorylated immunoreceptor
tyrosine-based activation motif, peptide from the cytoplasmic domain of
FcRIIa in activation-independent fashion, although SHIP binding
increases upon cell activation, and that FcRIIb and FcRIIc are
not responsible for the observed SHIP phosphorylation. These findings
prompt us to propose that SHIP inhibits FcR-mediated signal
transduction by engaging immunoreceptor tyrosine-based activation
motif-containing cytoplasmic domains of FcRIIa and
FcRI-associated -chain. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R), specifically those that produce a positive biological
response, conform to this schema (reviewed in 2). Thus, clustering
of these FcR by immune complexes or by anti-FcR Abs
stimulates Src family PTK activity and results in the phosphorylation
of ITAM tyrosines situated either in the cytoplasmic tail of FcRIIa
or in the -chain subunit of FcRI and FcRIIIa. These
phosphotyrosines along with the three amino acid residues immediately
downstream comprise docking sites for specific Src homology 2 (SH2)
domain-containing enzymes and adapter proteins, which, in turn,
propagate positive signals (3, 4). Critical to all downstream events is
the PTK Syk, which binds to phosphorylated ITAM (phospho-ITAM) residues
and becomes activated. A variety of subsequent major signaling
pathways, either downstream of Syk or dependent upon Syk, are then
activated or assembled. These subsequent pathways include one dependent
upon phospholipase C that generates the second messengers inositol
trisphosphate and diacylglycerol, one dependent upon Shc that cascades
via Grb2 and SOS to involve additional downstream elements of the Ras
activation pathway, and one dependent upon phosphatidylinositol
3-kinase (PI3K) that leads to phagocytosis and superoxide generation
(reviewed in 2). Recent progress suggests that the PI3K pathway to
phagocytosis requires recruitment of the p110 catalytic subunit of PI3K
to the plasma membrane and activation of members of the Rho family of
small GTPases (5, 6, 7).
While ITAM-associated receptors lead to a positive biological response,
a different group of receptors has recently been described that, when
clustered by Abs or ligand, act to inhibit cellular responses (8, 9).
Termed negative signaling receptors, these function somewhat like
positive signaling receptors by undergoing phosphorylation on
cytoplasmic tyrosine residues and subsequently recruiting SH2
domain-containing enzymes, but the enzymes are of a specific sort that
includes the phosphotyrosine phosphatases SHP-1 and -2 and the inositol
5-phosphatase, SHIP (9, 10, 11). The cytoplasmic tyrosine of negative
signaling receptors is situated in an immunoreceptor tyrosine-based
inhibitory motif (ITIM) (8, 9) that is structurally similar to the
activating ITAM. SH2 domain-containing phosphatases recruited to
phospho-ITIM of these receptors act to reverse biochemical events
elicited by positive signaling receptors and so reduce or inhibit the
biological responses. Included in this group of negative signaling
receptors are killer cell inhibitory receptors, pg49B1, PIR-B, CTLA4,
and others, including a single FcR (FcRIIb) (8, 9).
The difference in the primary sequence between the activating ITAM and the inhibiting ITIM appears minor. The ITAM contains the sequence YxxI/L-x (6, 7, 8, 9, 10, 11, 12)-YxxI/L, where x is any amino acid. The residues in the +1 and/or +2 position relative to the tyrosine of the ITAM are generally acidic and therefore form an optimal recognition sequence for phosphorylation by the Src family PTKs (12). Similarly, the ITIM is characterized by a YxxI/L, but is generally preceded by a small hydrophobic residue at the -2 position relative to the tyrosine. The residues in the +1 and/or +2 position are generally not acidic but, rather, are neutral (serine/threonine) or hydrophobic (leucine/alanine). These apparently minor differences nevertheless are sufficient to promote recruitment of a distinct set of SH2 domain-containing proteins: activating enzymes and adapters associate with the phospho-ITAM and inhibiting phosphatases associate with the phospho-ITIM (8, 9). The distinct protein associations probably account for the dichotomy of signaling function, such that ITAM-containing receptors promote only activation of cellular functions, while ITIM-containing receptors promote only inhibition of cellular functions.
Our studies here focus on signaling events emanating from clustered
FcRIIa and FcRI, the major FcR on monocytes. We report that
clustering of these receptors induces both tyrosine phosphorylation of
the inositol 5-phosphatase SHIP and its association with the adapter
protein Shc (13, 14). We show also that SHIP associates with
phosphopeptides corresponding to the ITAM of FcRIIa, and we note
that others have shown it to associate with the phospho-ITAM of
FcRI-associating -chain (15). These findings suggest that the
idea of a distinct dichotomy of positive and negative signaling
receptors (8, 9) may be an oversimplification; rather, some
ITAM-containing receptors may concomitantly induce both positive and
negative signaling events.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The human monocytic cell line U937 was maintained in RPMI
Complete which consisted of RPMI 1640 medium (Life Technologies,
Gaithersburg, MD) supplemented with 10% heat-inactivated FCS (HyClone,
Logan, UT), 2 mM L-glutamine, 100 µg/ml penicillin, and
100 µg/ml streptomycin. The THP-1 monocytic cell line, a gift from
Dr. Paul Guyre, Dartmouth University (Lebanon, NH), was grown in RPMI
Complete supplemented with 5.5 x 10-2 mM 2-ME. When
indicated, cells were treated with 150 U/ml recombinant human IFN-
(Genentech, San Francisco, CA) for 48–72 h. The human B cell line Raji
was maintained in RPMI 1640 supplemented with 20% heat-inactivated FCS
and 2 mM L-glutamine. PBMC were isolated from whole blood
of normal volunteers using Histopaque (Sigma, St. Louis, MO) according
to the instructions of the supplier.
Antibodies
Whole IgG and purified Fab of anti-FcRII mouse mAb IV3
(IgG2b), whole IgG anti-FcRI mouse mAb 197 (IgG2a), and whole
IgG and purified F(ab')2 of anti-FcRI mAb 32 and 22
(both IgG1) were supplied by Medarex (Annandale, NJ). The preparation
of anti-SHIP polyclonal Ab against SHIP amino acid residues
874–941 has been described previously (14). An
anti-phosphotyrosine mAb mixture consisted of Py20 (IgG2b),
purchased from Santa Cruz Biotechnology (Santa Cruz, CA); Py72.10.5,
obtained from Dr. Bart Sefton (The Salk Institute, La Jolla, CA) and
then purified from ascites; and 4G10 (IgG2b), purchased from Upstate
Biotechnology (Lake Placid, NY). These three mAb were used in a 30:30:1
ratio, respectively. AT10, a mouse anti-FcRII (mIgG1), was
obtained from Dr. Martin Glennie (University of Southhampton,
Southhampton, U.K.) (16). Rabbit anti-FcRIIa IgG Ab 260 (17) and
mouse anti-FcRIIb mAb II8D2 (18) were gifts, respectively, from
Dr. Jean-Luc Teillaud (Institut Curie, Paris, France) and Dr. Jurgen
Frey (University of Bielefeld, Bielefeld, Germany). MOPC141 (IgG2b) and
MOPC21 (IgG1) were purified from ascites fluid by ion exchange
chromatography after obtaining the cells from American Type Culture
Collection (Manassas, VA). Anti-Shc rabbit polyclonal was purchased
from Upstate Biotechnology. Goat F(ab')2 anti-mouse IgG
(GAM) was purchased from Pierce (Rockford, IL). HRP-conjugated
F(ab')2 of sheep anti-mouse and donkey anti-rabbit
secondary Abs were purchased from Amersham (Arlington Heights, IL).
Monocyte activation and immunoadsorption
For FcRII activation, U937 cells, THP1 cells, and PBMC were
washed twice and then suspended in either PBS or HBSS containing 10 mM
HEPES (pH 7.4) and 0.1% BSA (activation buffer) at 1 x
108 cells/ml. Cells (107/sample) were incubated
with mAb IV3 (3 µg/ml) at 4°C for 30 min and then at 37°C for 10
min, after which GAM was added (30 µg/ml) to initiate receptor
clustering and cellular activation. Activation proceeded for 3 min
unless indicated otherwise. The cells were then lysed for 20 min in
Triton lysis buffer (TLB; PBS, 10 mM HEPES, 10 mM EDTA, and 1% Triton
X-100, pH 7.4) that had been supplemented with 3 mM sodium
orthovanadate, 20 µg/ml aprotinin (Sigma, St. Louis, MO), 40 µg/ml
leupeptin (Calbiochem, La Jolla, CA), and 2 µg/ml pepstatin A (Sigma;
TLB+). Insoluble material was removed by centrifugation at
16,000 x g for 20 min, and the supernatant was
immunoadsorbed overnight at 4°C with specific Abs (anti-PY mAb
mixture, 1:33 dilution; rabbit anti-SHIP serum, 1:500; rabbit
anti-SHC Ab, 1 µg/ml) mixed with 25 µl of protein G or protein
A-Sepharose (Pharmacia, Piscataway, NJ). Following overnight
immunoadsorption, unbound proteins were removed with four washes of TLB
plus 1 mM sodium orthovanadate and were eluted as described below.
For the experiment shown in Fig. 4
, U937 and Raji cells (3 x
107) were lysed in TLB supplemented with enzyme inhibitors,
as described above, for 1 h on ice. The lysates were incubated
overnight at 4°C with GAM-Sepharose beads that had been preincubated
with 10 µg/ml IV3 Fab or a 1:100 dilution of AT10 ascites and then
washed free of unbound Ab. Samples were split equally among three gels
and were analyzed by Western blot.
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RI activation, U937 cells were first treated with IFN- for
48 h to increase expression of FcRI (19). U937 cells cultured
in IFN- or PBMC were washed and resuspended as described above, and
1 x 107 cells/sample were activated by receptor
clustering as previously described (20). Briefly, cells were incubated
with F(ab')2 of mAb 32 or 22 (20 or 50 µg/ml) or with
whole IgG of mAb 197, 32, or 22 (10 µg/ml) on ice for 20 min; unbound
Ab was removed quickly by centrifugation, and cells were equilibrated
to 37°C in prewarmed activation buffer for 5 min. This was followed
by incubation at 37°C with GAM (20 µg/ml) for 2 min. Lysis,
immunoadsorption, and elution were essentially as described above. This
modified protocol was also used for IV3 Fab/GAM activation of FcRII
in U937 cells and PBMC. Western blot analysis
After elution of adsorbed proteins from immobilized Abs by boiling for 1 min in Laemmli (21) sample buffer containing 5% 2-ME, the immunoadsorbed proteins along with Rainbow protein m.w. markers (Amersham), and Ab controls (Abs and Sepharose without lysate) were separated by SDS-PAGE and were electrophoretically transferred to Hybond-ECL membranes (Amersham). Membranes were incubated for either 1 h at room temperature or overnight at 4°C in 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.1% Tween-20 containing 5% BSA or nonfat dried milk. Blots were then incubated sequentially with the immunoblotting Ab and peroxidase-conjugated anti-mouse or anti-rabbit Ab for 1 h each at room temperature with four 15-min washes of 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.1% Tween-20 after each step. Bound Abs were then visualized by ECL (Amersham) and autophotography. Scanned images of the appropriate bands were quantified and compared using the public domain NIH Image program (developed at the U.S. National Institutes of Health and available on the internet at http://rsb.info.nih.gov/nih-image/).
Affinity adsorption with immobilized phospho-ITAM and ITIM peptides
U937 cells were washed and resuspended in activation buffer;
activation via FcRII/GAM cross-linking was accomplished essentially
as described above (3 µg/ml IV3 and 30 µg/ml GAM, activation for 2
min). Following lysis in TLB+ as described above, lysates
were supplemented with 2 mM PMSF (Sigma) and 1 µM biotinylated
peptide (phospho-ITAM, doubly phosphorylated ITAM of FcRIIa; ITAM,
unphosphorylated ITAM of FcRIIa; or phospho-ITIM, singly
phosphorylated ITIM of FcRIIb) was added (14, 22); this mixture was
incubated overnight at 4°C with rotation. The next day, 20 µl of
UltraLink Immobilized NeutrAvidin Plus beads (Pierce, Rockford, IL)
were added and allowed to mix at 4°C with rotation for 20 min. Beads
were then washed five times with TLB supplemented with 1 mM sodium
orthovanadate. Following this, samples were treated as described above
for Western blot analysis.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RIIa cross-linking in U937 induces phosphorylation of the
inositol 5-phosphatase SHIP
We incubated the human monocyte line U937 at 37°C with
anti-FcRII mAb IV3 and a secondary Ab, clustering the receptors
for 3 min, then lysed the cells, immunopurified tyrosine-phosphorylated
proteins by immunoadsorption, and analyzed for their presence by
Western analysis. In the absence of FcR clustering very few
phosphoproteins appeared, as noted in the first three lanes of Fig. 1. (The heavy and light chains of
immunoadsorbing mAb, the most prominent in all four lanes appearing at
about 50 kDa and just above 30 kDa, respectively, should be discounted
as procedural artifact.) Upon FcRII clustering, however, a variety
of proteins were tyrosine phosphorylated, as lane 4 of Fig. 1 shows. These proteins ranged in m.w. across the entire analyzed
spectrum, with several appearing dense and well defined. Some of these
have been identified in other studies. Among these several proteins was
a 145-kDa band consistent with the mobility of SHIP.
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shows, as indicated by an arrow in the
left margin, that a dense band representing tyrosine
phospho-SHIP appeared at 145 kDa only in the lane analyzing U937 cells
whose FcRII had been clustered and not in the lanes assessing
resting cells or adsorption with nonspecific rabbit serum. The same
experiment with PBMC, using IV3 Fab fragments and GAM cross-linking to
avoid potential interaction with other FcR, shows essentially the
same result in the upper right panel. The lower
panels of this figure were repeated probings of these same Western
blots with anti-SHIP Abs and showed that the amount of SHIP is
relatively constant from lane to lane and does not account for the
predominant single band seen in lanes 4 of the top
panels.
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and 2 with U937. Specifically, densitometric analysis of an
experiment using the Fig. 1 protocol showed the SHIP bands from THP1
and U937 cells to be 33- and 16-fold, respectively, greater than
background. Using the Fig. 2 protocol the SHIP bands were 26- and
18-fold greater than background from THP1 and U937 cells, respectively.
We considered the possibility that the anti-FcRIIa reagent mAb
IV3, being an intact IgG, was recruiting an unidentified IgG-binding
molecule into the cluster. Therefore, we employed Fab of mAb IV3 to
cluster FcRIIa and assessed SHIP phosphorylation in U937 cells by
Western analysis. As shown in Fig. 3, Fab
of mAb IV3, when clustered with an F(ab')2 secondary Ab,
were perfectly capable of stimulating a response that led to SHIP
phosphorylation. Thus, it would seem highly unlikely that IgG-binding
molecules other than FcRIIa, such as FcRIIb, FcRIIc, or
FcRI, were being recruited into the receptor cluster.
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RIIa mAb IV3 do not immunoadsorb FcRIIb
from U937 cells
To assess directly the possibility that mAb IV3 had affinity for
the ITIM-containing FcRIIb and was therefore capable of binding and
clustering FcRIIb in our experiments, we immunoadsorbed FcRII
from U937 lysates with Fab of mAb IV3 and F(ab')2 of GAM
and analyzed by Western blots with Abs specific for either the
FcRIIa (rabbit IgG Ab 260) or FcRIIb (mAb II8D2) isoforms. As
shown in Fig. 4 we found no evidence for
FcRIIb in mAb IV3 Fab immunoadsorbates of U937 cells
(lane 1, middle panel), but found a robust signal
indicating the presence of FcRIIa (lane 1, top
panel). The anti-FcRIIb blotting Ab identified FcRIIb
from lysates of Raji, a B cell line expressing FcRIIb,
immunoadsorbed with a pan-FcRII mAb AT10 (lane 4,
middle panel), but not from Raji lysates immunoadsorbed with mAb
IV3 Fab (lane 3, middle panel), corroborating the
inability of mAb IV3 to bind FcRIIb on Raji cells. It appears clear,
therefore, that mAb IV3 does not bind FcRIIb. We have not been able
to affirm whether the FcRIIb protein is actually expressed in U937
cells. Note that while a band of appropriate FcRII mobility is seen
in lane 2 of the middle panel of Fig. 4 assessing
the mAb AT10 immunoadsorbate by anti-FcRIIb Western analysis, a
band of similar mobility is seen when the same blot is probed with
murine IgG1 isotype control Ab (lane 2, bottom
panel). Thus, we found no FcRIIb protein expression in U937 as
we did in Raji cells. (The doublets near the bottom of the
middle and lower panels are unidentified
nonspecific bands related to the AT10 ascites.)
Kinetics of SHIP phosphorylation after FcRIIa clustering
To determine the rapidity and the duration of SHIP phosphorylation
after FcRIIa clustering, we repeated the experiment shown in Fig. 2
sampling at various times over the first 8 min following clustering,
assaying by Western blot with anti-SHIP and
anti-phosphotyrosine Abs. In the experiment shown in the top
panel of Fig. 5 we noted that SHIP
phosphorylation appeared very early, by 30 s, peaked at about 2
min, was diminishing at 4 min, and was near background levels by 8 min
after clustering. A repeat experiment showed somewhat faster kinetics.
Plotting the mean densitometric values of the two experiments in the
lower panel of Fig. 5 we saw that the major signal appeared
between 0.5 and 4 min. Reprobing with anti-SHIP indicated equal
recovery of SHIP over time (not shown).
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We attempted to determine whether SHIP associated with FcRIIa
either before or after clustering, but could find no evidence by
immunoadsorption with anti-FcRIIa Abs and Western analysis with
anti-SHIP Abs. However, knowing that the adaptor protein Shc
interacts with SHIP and that Shc is rapidly phosphorylated after
FcRIIa clustering (23), we assessed whether phospho-SHIP might be
found in U937 cell lysates by immunoadsorption with anti-Shc Abs.
The top panel of Fig. 6 shows
that anti-Shc immunoadsorbates contain a 145-kDa phosphoprotein
after, but not before, receptor clustering, and that this band is
identified by Western blotting with anti-SHIP Ab (Fig. 6, lower panel).
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Realizing that transient or weak binding of SHIP to the
cytoplasmic tail of FcRIIa may explain our inability to immunoadsorb
SHIP with anti-FcRIIa, we sought to test whether SHIP would bind
in vitro to phospho-ITAM peptides of FcRIIa. This was achieved by
adsorbing SHIP from U937 cell lysates, before and after FcRIIa
clustering, using immobilized FcRIIa ITAM peptide, phosphorylated at
both ITAM tyrosines. Detecting proteins eluted from immobilized ITAM by
Western analysis with anti-SHIP Abs (upper panel of Fig. 7), we found that while the
unphosphorylated FcRIIa ITAM bound no SHIP, phospho-ITAM bound SHIP
from lysates of cells before and after FcRIIa clustering,
considerably more after clustering than before. Likewise, Western blots
probed with anti-Shc (middle panel) showed that
phospho-ITAM adsorbed Shc from resting lysates and that the amount
bound was considerably enhanced by FcRIIa clustering. On the other
hand, the binding of Syk to phospho-ITAM (lower
panel, Western blot with anti-Syk) was not enhanced by
receptor clustering.
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RIIb phospho-ITIM in parallel
experiments (upper panel, Fig. 7). Moreover, SHIP binding to
the phospho-ITIM sequence appeared to be independent of FcRIIa
clustering. The phospho-ITIM showed affinity for Shc only after
receptor clustering but not for Syk (middle and lower
panels).
SHIP is tyrosine phosphorylated also after FcRI clustering
To assess whether clustering of the second class of FcR on
U937, i.e., FcRI, would result in SHIP phosphorylation we repeated
the experiments of Figs. 1 and 2 with three different anti-FcRI
Abs, namely, mAbs 32, 22, and 197. Intact IgG of all three mAbs when
cross-linked with secondary Ab resulted in SHIP phosphorylation, as
shown in the upper panel of Fig. 8, lanes 4, 7, and
8. This same result was seen using a source of normal cells,
namely, monocytes from PBMC after clustering FcRI with mAb 32 and
GAM (lower panel of Fig. 8). To eliminate the
possibility that other FcR capable of stimulating SHIP
phosphorylation, such as FcRII, were being recruited to the immune
complex by intact anti-FcRI Abs, we employed Fab of both 32 and
22 and found that they, too, were potent in stimulating SHIP
phosphorylation (lanes 2, 3, 5,
and 6 of upper panel of Fig. 8).
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RII was not inadvertently being clustered
along with FcRI was obtained from an alternative experiment in which
we blocked any putative association between FcRI and FcRII.
Specifically, we clustered FcRI of U937 cells with anti-FcRI
mAb 197, which binds FcRI as both Ab and ligand, being of the IgG2a
isotype. Simultaneously, we blocked incorporation of FcRII into the
receptor-ligand complexes with a great excess of Fab of
anti-FcRII mAb IV3. Phospho-SHIP was analyzed using the Fig. 8
protocol, and the SHIP band was quantified by densitometry. Adding as
much as 100 µg/ml IV3 Fab caused no greater inhibition (5%) of band
density than an equal amount of Fab of irrelevant specificity (12%).
These experiments indicate that the phosphorylation of SHIP seen after
FcRI clustering cannot be attributed to the incorporation of another
FcR, such as FcRII, into the cluster and implicate FcRI itself
as the trigger of SHIP phosphorylation in this experiment. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R, either
FcRIIa or FcRI, induces tyrosine phosphorylation of SHIP, an
inositol phosphatase that in all studies to date has been shown to
convey a negative cellular signal. It is known that the SHIP inositol
phosphatase specifically hydrolyzes 3-phosphorylated phosphoinositides,
products of the PI3K pathway, at the D5 position of the inositol ring
(24, 25). The enzyme can reverse signaling biochemistry in this way
(26, 27) or through its interaction with the Ras adapter protein Shc
(28, 29).
Experiments addressing a potential role for the inhibitory
(ITIM-containing) FcRIIb in our stimulation protocol were negative.
Specifically, all Abs used to cluster both target FcR were
F(ab')2, theoretically and experimentally unable to engage
FcRIIb by Fc interaction. Furthermore, the anti-FcRIIa mAb
IV3 used in our experiments did not immunoadsorb FcRIIb either from
lysates of U937 cells, where FcRIIb could not be detected, or from
lysates of the human B cell line Raji where we showed the expression of
FcRIIb. Thus, in our experiments the possibility that tyrosine
phosphorylation of SHIP is mediated by the negative signaling receptor,
FcRIIb, is very remote.
We have also considered whether the expression of the product of the
third FcRII gene, FcRIIc, might account for SHIP phosphorylation
upon FcRII clustering with mAb IV3. FcRIIc appears to be an
evolutionary hybrid of the other two FcRII receptors (30). It
contains an FcRIIb-identical sequence in the extracellular and
transmembrane domains and an FcRIIa-like sequence in the cytoplasmic
tail. Thus, it probably would not be recognized by mAb IV3 and would
therefore not participate in FcR clustering mediated by mAb IV3,
although were it to do so, it would almost certainly manifest an
FcRIIa-like effect, since its cytoplasmic tail expresses an ITAM.
Thus, SHIP phosphorylation in U937 cells appears solely due to
FcRIIa or FcRI clustering. Such clustering leads to the
phosphorylation of the associated ITAM, specifically the ITAM of the
tail of FcRIIa and the ITAM of the tail of FcRI-associated
-chain. By analogy with the tyrosine phosphorylation of SHIP in B
cells and mast cells (13, 14, 31), we suggest that SHIP associates via
its SH2 domain with a tyrosine-phosphorylated ITAM of FcRIIa and
FcRI-associated -chain. In support of this idea, we demonstrate
that SHIP is capable of binding in vitro to the phosphorylated, but not
to the unphosphorylated, ITAM of FcRIIa. Others have shown that the
SH2 domain of SHIP will engage the phosphorylated ITAM of the -chain
as well as the T cell Ag receptor-associated 
-chain, although in
these studies SHIP tyrosine phosphorylation was not induced in cells
expressing -chain and stimulated via Fc receptors for IgE (15).
It is important to point out that in our studies SHIP manifested
relatively high binding capacity for immobilized phospho-ITIM of
FcRIIb compared with phospho-ITAM of FcRIIa (Fig. 7), so
FcRIIb would have been an attractive candidate for mediating SHIP
phosphorylation after FcRIIa clustering were we to have shown its
presence in U937 or its immunoadsorption by the anti-FcR Abs we
have employed in this study. It also seems possible that monocytes may
express an unidentified, ITIM-containing receptor that is associated
with and phosphorylated by clustering FcRIIa or FcRI. A variety
of these proteins have been described recently in monocytes (32, 33, 34, 35),
although none has yet been found in complex with FcR.
Given our observation that the binding of SHIP to immobilized
phospho-ITAM is somewhat activation dependent (Fig. 7), it would also
seem possible that the putative interaction between SHIP and
phospho-ITAM is not direct, but is mediated by an adapter molecule
whose affinity for either SHIP or ITAM is dependent upon an
activation-mediated event, such as phosphorylation. One candidate for
such an intermediary adapter is Shc, which we show (Fig. 6) binds to
SHIP in an activation-dependent fashion, as have others (13, 36, 37).
Shc also binds in vitro the phosphorylated FcRIIa ITAM peptide in a
manner very similar to that of SHIP, i.e., greater amounts are adsorbed
from FcRIIa-activated lysates than from resting lysates, but Shc
does not bind the unphosphorylated peptide (Fig. 7). The increased
binding of Shc and SHIP to phospho-ITAM we detect upon FcRIIa
clustering is probably due to the additive effect of direct and
indirect recruitment of the two proteins, since SHIP and Shc interact
with each other as well as with the phospho-ITAM of FcRIIa. It is
also possible, however, that Shc recruitment to the phospho-ITAM brings
SHIP to the signaling complex and thereby promotes SHIP tyrosine
phosphorylation. Another possible adapter is Syk, which others have
shown manifests activation-dependent affinity for SHIP (36). Although
Syk binds well the immobilized, phosphorylated (but not the
unphosphorylated) ITAM peptide in vitro, such binding is not activation
dependent (Fig. 7). Nevertheless, perhaps the phosphorylation of Syk,
which previously we have shown occurs within 30 s after FcRIIa
clustering in platelets (22) and FcRI clustering in U937 (20),
enables it to serve as a bridge between SHIP and the phospho-ITAM, and
thereby to link SHIP with the FcR cluster. Immunoadsorbing from U937
lysates with Abs to both Syk and SHIP and analyzing by both Western
blots and in vitro kinase assays, we have been unable to copurify these
two molecules (not shown).
What function might SHIP subserve upon engaging FcR complexes is
speculative without additional data. At this point we would suggest two
possibilities. First, SHIP might promote a positive rather than a
negative signal by hydrolyzing phosphatidylinositol 3,4,5-trisphosphate
to phosphatidylinositol 3,4-bisphosphate. Phosphatidylinositol
3,4-bisphosphate has been shown to be an in vitro activator for the
proto-oncogene vav in the activation of Rac (38) and for the
Akt kinase (39); either of these proteins may promote downstream FcR
functions, including phagocytosis. Second, SHIP may down-regulate
FcR function as it does for the B cell receptor by blocking enzymes
dependent upon phosphatidylinositol 3,4,5-trisphosphate or
inositol-1,3,4,5-tetrakisphosphate and/or by binding Shc and blocking
induction of the Ras pathway (reviewed in 10). This latter
possibility is novel in that it stipulates that the ITAMs of monocyte
FcR are simultaneously triggering both positive and negative
signals. Such a situation is completely distinct from other
positive/negative signaling paradigms, such as the B cell FcRIIb and
the NK cell inhibitory receptors, in which different receptors promote
only one or the other type of response, but not both.
This latter possibility is somewhat like the situation described for
cytokine receptors, where positive and negative signaling concomitantly
are induced by cytokine engagement and are mediated by a single
receptor molecule. Indeed, SHIP was originally identified as a
phosphoprotein induced by stimulation of cells with IL-3 (24) or G-CSF
(25). Both the common ß-chain shared by receptors for IL-3, IL-5, and
GM-CSF (40) and the single G-CSF receptor molecule (41) encode
cytoplasmic tyrosines set in an optimal ITIM. Thus, these receptors,
which clearly induce positive signaling and promote cell growth, are
capable of recruiting negative signaling enzymes such as SHIP to
concomitantly induce a negative effect. Recent studies of the G-CSF
receptor indicate that positive and negative signals emanate from
different cytoplasmic domains of a single receptor molecule (42). Our
model of FcR-mediated SHIP phosphorylation would state that positive
and negative signals emanate not only from a single receptor molecule
but from a single cytoplasmic ITAM. Such a model would further imply
that control of signaling is modulated by the concentration and
affinity of SH2-bearing proteins for phosphotyrosines of receptor
cytoplasmic domains.
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Acknowledgments |
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Footnotes |
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2 D.L.M. and J.M.O. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Clark L. Anderson, 2054 Davis Research Center, 480 West Ninth Ave., Columbus, OH 43210. E-mail address:
4 Abbreviations used in this paper: ITAM, immunoreceptor tyrosine-mediated signal activation motif; PTK, protein tyrosine kinase; FcR, Fc receptors for IgG; SH2, Src homology 2; phospho-ITAM, phosphorylated ITAM; PI3K, phosphatidylinositol 3-kinase; SHIP, SH2-containing 5'-inositol phosphatase; ITIM, immunoreceptor tyrosine-based inhibitory motif; phospho-ITIM, phosphorylated ITIM; GAM, goat F(ab')2 anti-murine IgG; TLB, Triton lysis buffer; ECL, enhanced chemiluminescence.
Received for publication July 22, 1998. Accepted for publication March 17, 1999.
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