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RAPID COMMUNICATION
Protein-Tyrosine Kinase ~
7 in FcyRI
2
~ Receptor
~
~ Signaling
By Donald L. Durden and Yen bou Liu
In this report we show that y-interferon (IFN) induces the
expressionof
the nonreceptor protein tyrosinekinase,
~ 7 2 * ~and
‘ , that cross-linking the FcyRl receptorin IFN-differentiated U937 cells (U9371F cells) resultsin the activation of
syk kinase. We show that syk is tyrosine phosphorylated
(It-fold increase) after FcyRl cross-linking. In vitro kinase
assays demonstrate that the specific kinase activity of syk
increased eightfold after FcyRl cross-linking. The activation
of signal transduction through the FcyRI receptor, as measured by the respiratory burst, is associated with the tyrosine phosphorylation and catalytic activationof the syk kinase. We show that syk coprecipitateswith the y subunit
of the FcyRI, FcyRly. The
data suggest that ~
7 is involved
2
~
in signal transductionthrough the FcyRl receptor, involving
the FcyRly subunit.
0 1994 by The American Society of Hematology.
0
amount of the information continues to accumulate concerning Fc receptor signaling, the interaction between the tyrosine kinase and serinekhreonine kinase cascades and other
downstream pathways, including the G protein-linked paths,
remain obscure.
The y subunit, initially described as a subunit of the FccRI
receptor, is now known to be part of the FcyRI and FcyRIII
re~eptor.’.’~”~
The gene for the FccRIy and TCRL chains
belong to the same family and are both located on human
and mouse chromosome number l.’.’ Amino acid sequence
homology andtheir patterns of exodintron organization suggest that they likely arose through gene duplication.’.’Takai
et a l l 4 recently reported that the targeted deletion of y subunit
gene in mice results in a disruption of FcyRIII and FcyRI
function. Other receptor subunits also have sequence homology to these proteins, including the Igcw and Igp subunits of
the surface Ig receptor on B cells (BCR).2.3.15
The ARHI
containing subunits serve to connect cell-surface receptors
to intracellular effectors of signal transduction.* The y and
6 are generally expressed as homodimeric subunits of Fc
receptors or part of the TCWCD3 complex, respectively.
The y and subunits show a striking degree of sequence
homology and have the same protein t~pology.’.~
It is interesting to note that the similarity of structure and function,
which is observed between the y and L homodimers, parallels certain observed similarities between signal transduction
between the Fc receptors and the TCWCD3 cornplex.l6For
example, both signaling pathways are regulated by the receptor protein tyrosine phosphatase, CD45.’6-2’ FcR and TCR
ST involves members of the src family of protein tyrosine
kinase and another group of nonreceptor kinase, the sykl
ZAWO
Study of the TCR signaling has suggested that during Tcell receptor activation, the src kinase, Zck, is activated and
phosphorylates a second nonreceptor PTK, ZAP70.’6.23,24
The
phosphorylation of ZAP70 results in the activation of this
kinase.” Chan et aIz3 usedheterologous expression in COS7
cells to show that the noncovalent association of ZAP70
with the 6 subunit sequence required the cotransfection of
the Zck or fyn kinase. Hence, the model now put forward by
Weiss and Littmani6 suggests that the src kinases are upstream from the syWZAP7O family of kinases in ST. Less is
known about the role of syk in myeloid signal relay. It has
been shown that FccRI cross-linking results in the tyrosine
phosphorylation of the syk protein,” and that syk is noncovalently associated with the y subunit of FceRLZ5More re-
VER THE PAST several years, much has been learned
about the structure and function of the Fc receptor
family.”3 FcRs are defined by their specificity for the various
Ig isotypes; eg, Fc receptors for IgG are referred to as FcyRs
and Fc receptors for IgE as FURS.’ Three groups of FcyRs
have been historically designated FcyRI,FcyRII,
and
FcyRI1I.l Biologically, FcRs have a wide cellular distribution and carry out diverse cellular functions in hematopoietic
cells. This diversity of function relates to a number of divergent intracytoplasmic and transmembrane sequences within
the FcyR family superimposed on a largely conserved backbone amino acid sequence, making this family of receptors
particularly interesting in terms of protein
Signal transduction (ST) through the Fc receptors for Ig
involves the tyrosine phosphorylation of cell protein^."^ In
mast cells, cross-linking FceRI stimulates tyrosine phosphorylation of cell proteins, including the y subunit! Eiseman
and Bolens reported that the FceRI is associated with two
src-family protein tyrosine kinases (PTKs), Zyn and yes, and
upon FCEreceptor activation these PTKs are activated, as
evidenced by an increase in their kinase activity. Huang et
al’ have reported that cross-linking the FcyRII receptors on
platelets and a human erythroleukemia cell line results in
the tyrosine phosphorylation of multiple proteins, including
FcyRII itself. Others have reported that cross-linking of
FcyRI or FcyRII in U937 cells resulted in the rapid hydrolysis of PtdIns-4,5-P2, resulting from the tyrosine phosphorylation and activation of PLC-y l .9 Although a considerable
From the Neil Bogart Memorial Laboratories, the Department of
Pediatrics, the Division of Hematology-Oncology, Childrens Hospital Los Angeles, University of Southern California, School of Medicine, L o s Angeles, CA.
Submitted March 28, 1994; accepted June 28, 1994.
Supported inpart by a grant to the NeilBogart Memorial Laboratories by the T.J. Martell Foundation for Leukemia, Cancer and
AIDS Research.
Address reprint requests to Donald L. Durden, MD, PhD, Department of Pediatrics, Division of Hematology-Oncology, Mailstop #57,
Childrens Hospital L o s Angeles, 4650 Sunset Blvd, Los Angeles, CA
9002 7.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 I994 by The American Society of Hematology.
0006-4971/94/8407-09$3.00/0
2102
Blood, Vol 84, No 7 (October l ) , 1994 pp 2102-2108
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FCyRl SIGNAL TRANSDUCTION
cently, work in rat macrophages demonstrated that FcyRII
mediated phagocytosis, and FcyRII cross-linking was associated with the tyrosine phosphorylation of syk kinase.26
However, it has not been shown the syk kinase activity increases after FcyRI receptor activation. We and others have
used U937 cells differentiated in y interferon (IFN), termed
U937IF cells, to study macrophage activation and the fundamental features of signal transduction through the FcyRI
receptor.lO.l 127.28 Our data show that FcyRI cross-linking of
U937IF cells results in an increase in the specific activity of
~72"' kinase and that the syk protein is tyrosine phosphorylated in response to FcyRI cross-linking. Our results show
that syk expression is induced by IFN and correlates with
the capacity of U937 cells to signal through the FcyRI receptor to the respiratory burst'' and that syk is noncovalently
associated with the FcyRIlyARHI, suggesting that FcyRY
yARHI signal transduction involves the nonreceptor protein
tyrosine kinase, p72"yk.
MATERIALS AND METHODS
Cells. The U937 cell line was obtained from American Type
Culture Collection (ATCC; Rockville, MD) and cultured in RPMI
1640 + 10% fetal bovine serum (FBS).U937IF cells were prepared
by culturing U937 cells inRPMI 1640 + 10%FBS + 250 U/mL
human recombinant IFN for different time intervals. The U937IF
cells were maintained at a concentration of 5 X lo5 cells/mL and
the medium was replaced with fresh tissue culture medium + IFN
every 2 days, as described."
Reagents and chemicals. Cytochrome c, superoxide dismutase,
and sodium orthovanadate were obtained from Sigma (St Louis,
MO). The FcyR specific antibodies were obtained from Medarex,
Inc (West Lebanon, NH). The monoclonal antibodies (MoAbs) 32
and 197 are Fab; fragments and are specific for the FcyRI receptor.
The cross-linking antibody was a rabbit antimouse Fab; fragment
purchased from Cappel Labs (Durham, NC). The enhanced chemiluminescence, ECL kit was purchased from Amersham Corp (Arlington Heights, IL).
Anti-y subunit antibodies. Antipeptide antisera against the y
subunit was generated by immunizing rabbits with a peptide representing the extreme C-terminal sequence of the y subunit conjugated
to keyhole limpet hemocyanin (KLH), as described (ie, NQETYETLKHEKPPQ)."." Another anti-y antibody (4D8) was generously
provided by Dr J. Kochan (Hoffman-LaRoche, Inc, Nutley, NJ).zs~z9
Stimulation of lJ937IF cells. We prepared whole-cell lysates of
U937 cells after different incubation times in250 U/mL of IFN.
Proteins in these lysates were resolved by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and blotted with
anti-syk antisera. For FcyRI cross-linking experiments, U937IF (day
4 of IF'N) cells were collected and washed twice in cold Hanks'
buffered saline solution (HBSS) and adjusted to a concentration of
10 X 107/mL. MoAbs against the FcyRI receptor were usedto
activate U937IF cells. Cells, 1 X IO', in 1.0 mL of HBSS were
incubated on ice for 30 minutes with 2 pg/mL of the primary MoAb
(FcyRI, 32.2). We then added the secondary rabbit-antimouse antibody (Fab;) at a concentration of 10 pglmL and placed the cells at
37°C for different times. The addition of the secondary antibody at
37°C was considered the start of stimulation. Stimulated cells were
cooled rapidly by the addition of an equal volume of cold HBSS.
The cells were then centrifuged at 500g in a cold room for 30
seconds andthe supernate quickly aspirated. We then added 600
pL of the extraction buffer (EB) lysis buffer and performed anti-y
immunoprecipitation, as described below.
2103
Low-stringency immunoprecipitation procedure. For immunoprecipitations designed to preserve noncovalent interactions between
effector molecules involved in FcyRIST, we used a Triton X-100
(Sigma) extraction buffer (EB buffer). Cell lysates were prepared in
a lysis buffer containing 1 % Triton X-100, 10 mmoVL Tris pH 7.6,
50 mmoVLNaCI, 0.1% bovine serum albumin (BSA), 1 mmol/L
phenylmethyl sulfonyl fluoride, 1 % aprotinin, 5 mmoVL EDTA, 50
mmollL NaF, 0.1% 2-mercaptoethanol, 5 pmoVL PAO, and 100
pmoVL vanadate (EB lysis buffer). Lysates were cleared by centrifugation at 15,000g for 45 minutes at 4°C.To precipitate the y subunit,
we added 5 pL of the 5927.2 or 4D8 MoAb to clarified cell lysates.
To precipitate the syk protein, we added 3 pL of the polyclonal antisyk antisera to these lysates. After 2 hours at O'C, 50 p L of a 10%
solution of formalin-fixed Staphylococcus aureus was added to the
5927 immunoprecipitation (IP) and anti-syk IP and 50 pL protein G
Sepharose (Pharmacia, Piscataway, NJ) was added to the 4D8 IP.
The IPS were further incubated for an hour at 0°C. The resultant
immune complexes were washed several times in EB lysis buffer.
We then added 25 pL of lx sample buffer and heated these samples
at 98°C for 5 minutes or for the in vitro kinase assay, we washed
the anti-syk IP several times with PAN (20 mmol/L PIPES, pH 7.0,
0.1 mol& NaCI, 20 pg/mL aprotinin) before performing the kinase
assay.
Respiratoryburst. U937IF cells (2 x 10' cells) (day 4 of IFN
differentiation) were incubated on ice withthe primary antibody,
anti-FcyRI, (Fab; fragments, 2 pg/mL)(aCD64, MoAb 32 from
Medarex, Inc) for 30 minutes. We then added 80 pmol/L cytochrome
c to the reaction mixture, followed by the addition of rabbit-antimouse (Fab; fragments, 10 pg/mL) at 37°C. The addition of the
cross-linking antibody was considered the time zero for stimulation.
The reduction of cytochrome c was quantitated by measuring the
change in absorbance at 550 nm wavelength over time. The generation of superoxide anions by U937IF cells was measured as the
superoxide dismutase inhibitable reduction of fenicytochrome c":
measured atthe 550-nm wavelength in a microtiter plate reader
(Molecular Devises Inc, Menlo Park, CA), using air-oxidized and
dithionite-reduced cytochrome c as standards. The final reaction mixture contained 2 X 10' U937IF cells and 80 p m o K femcytochrome
c, in a volume of 250 pL of HBSS. One half of the wells received
superoxide dismutase (SOD) (25 pg/mL). After the addition of rabbit-antimouse Fab; antibody, the plates were incubated at 37°C and
agitated. Serial spectrophotometric determinations were recorded to
construct a kinetic curve for the production of superoxide. Maximum
reduction of fenicytochrome c (25 nmol) was achieved by adding
5 pL of freshly prepared sodium dithionite. Superoxide production
is expressed as nanomoles of superoxide dismutase inhibitable cytochrome c reduction per 2 X IO6 cells.
Electrophoresis and immunoblotfing. Immunoprecipitates and
whole-cell lysates were resolved on 10% or 20% acrylamide, 0.193%
bisacrylamide gels by SDS-PAGE, as described." Proteins were
transferred to nitrocellulose filters (1 mA-h/cm*) usinga dry transfer
system (Ellard Inc, Seattle, WA), as described." The blot was incubated with block (10 mmol/L Tris-HCI, pH 7.5, 150 mmol/L NaCI;
1% BSA; 1% ovalbumin) for 1 hour at room temperature and then
incubated with specific anti-y, anti-syk, or anti-PY antisera for 2
hours at room temperature with continuous agitation. We used the
anti-y antisera (5927) at a concentration of 1:2,OOOsolution in block.
The anti-syk antisera, obtained from Dr Joseph Bolen (Bristol-Meyers Squibb Research Institute, Princeton, NJ), was used at a dilution
of 1: 1,ooO in 3% dry milk in rinse solution (10 mmoVL Tris-HC1,
pH 7.5, 150 mmoVL NaCI). The anti-PY antibody (4G10) was used
at 2 pg/mL in block. After four washes in rinse solution (10 mmol/
L Tris-HCI, pH 7.5, 150 mmoVL NaCI), the membranes were incu-
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DURDENANDLIU
2104
bated at room temperature for 1 hour with secondary antibody. Secondary antibody was horseradish peroxidase-conjugated antirabbit
or antimouse antibody provided in the enhanced chemiluminescence,
ECL kit from Amersham Corp. Proteins were visualized using the
ECL detection system.
Protein tyrosine kinase assay. We used a rabbit anti-syk polyclonal antibody provided by Dr Joseph Bolen, and commercially
prepared anti-svk antisera (no. 12583) from Upstate Biotechnology
(Lake Placid, NY) to IP syk from resting U937IF cells or U9371F
cells stimulated with FcyRl cross-linking. The PTK activity of svk
was determined using an in vitro kinase assay, as described." Immunoprecipitates were incubated in a solution containing 20 mmol/L
PIPES, pH 7.0; I O mmol/L MnCI2; S uCi [Y'~P]ATP (3,000 Cil
mmol); and 20 pglmL aprotinin in a final volume of I O pL. After
I O minutes at 30"C, the reactions were terminated by adding I O pL
of 2x reducing sample buffer and heated at 98°C for S minutes."
The syk protein wasresolved by 10% SDS-PAGE,visualized by
autoradiography, and quantitated using a phosphorimaging system
supplied with the Phosphor-analyst software (Bio-Rad GS 250 Molecular Imager, Hercules, CA).
aeyk IP
E
asvk 1P
RESULTS
The protein tyrosine kinase, p 7 F k , is activated afer
FcyRI cross-linking. Our results show that syk immunoprecipitated from FcyRI-stimulated U937IF cells is catalytically activated (eightfold activation) (Fig IA, lanes 1 through
3). Anti-syk immunoblots show that an equal amount of syk
protein was immunoprecipitated from the resting and FcyRIactivated U937IF cells (Fig IA, lanes 5 through 8). Therefore, the specific activity of syk kinase is markedly activated
after FcyRI cross-linking. In contrast, stimulation with
FcyRI antibody alone did not activate the syk kinase (Fig
IA, lane 2). Preimmune rabbit antisera do not IP syk or other
associated tyrosine phosphorylated proteins from FcyRI activated U937IF cells, and when we perform in vitro kinase
assays on these IPSwe do not observe the syk kinase or other
phosphoproteins (data not shown). These results support the
conclusion that the activation of syk is specific for conditions
in which we IP syk and is not explained based on a nonspecific IgG binding other molecules, including FcyRs.
syk is tyrosine phosphorylated after FcyRI cross-linking.
We immunoprecipitated the syk kinase from resting or stimulated U937IF cells and performed anti-PY immunoblot analysis. FcyRI cross-linking results in the tyrosine phosphorylation of the syk protein ( 1 2-fold increase in phosphotyrosine)
(Fig IB, lanes 1 and3). Incubation of U937IF cells with
phorbol myristic acid (PMA), calcium ionophore, formyl
met-leu-phe, FcyRI primary antibody alone, or FcyRII
cross-linking did not result in the tyrosine phosphorylation
of syk (data not shown, Fig IB, lane 2).
FcyRl cross-linking results in the activation of the respiratory burst. We performed experiments quantitating the
respiratory burst (RB) in U937IF cells under conditions of
FcyRI cross-linking. U937IF cells stimulated with primary
antibody (aFcyRI, Fab; 32.2) or secondary antirabbit Fab;
are shown in Fig 2.The results are similar to previous reports
showing that the activation of the respiratory burst in U937IF
cells requires FcyRI cross-linking.".2RPrimary and secondary antibody alone do not induce the phosphorylation of y
or the activation of the RB in these cells. In other experi-
uPY blot
Fig 1. The protein tyrosine kinase p72** is activated and tyrosine
phosphorylatedafter FcyRl activation. (AI Anti-syk immunoprecipitates from 4 x lo6 U9371Fcells were assayedfor in vitro kinase
activity, as described in Materials and Methods. Proteins were resolved by 10% SDS-PAGE, transferred to nitrocellulose, and blotted
with anti-syk antisera. The autophosphorylatedsyk kinasewas visualized usingautoradiography (lanes 1 through 41. We probed the
same blot with anti-syk antisera and observed that the phosphorylated bandssuperimposed
withthe
syk-immunoreactivebands
(lanes 5 through 8 ) . The lanes correspond to resting U9371Fcells,
lanes 1 and 5; U9371F cellsstimulated with Fab; 32.2 MoAb antibody
alone, lanes 2 and 6; cells stimulated with crFcyRl cross-linking for1
minute, lanes 3 and 7; cells stimulated with crFcyRIfor 5 minutes,
lanes 7 and 8, respectively, as described in Materials and Methods.
(B) We probed a parallel set of anti-syk immunoprecipitatesfrom 4
x lo6 U9371F cells with antiphosphotyrosine antibody (46101(lanes
1 through 41. The lanes correspond to resting U9371Fcells, lane 1;
U9371F stimulated with Fab;, 32.2 MoAb alone, lane 2; U9371F cells
stimulated with FcyRl cross-linking for1 and 5 minutes at 37°C lanes
3 and 4, respectively.
ments, we have shown that FcyRI cross-linking is required
for the phosphorylation of the y subunit.'" The kinetics of
the RB after FcyRI activation shows that a measurable burst
begins 1 minute after FcyRI cross-linking. The activation of
syk kinases and the tyrosine phosphorylation of syk occur
by 1 minute and quickly returnto baseline (Fig IA). The
respiratory burst response parallels the observed tyrosine
phosphorylation of syk (Fig IB).
IFN induces the expression of ~ 7 2 kinase
" ~ in U937 cells.
Treatment of U937 cells with 250 U/mL of IFN for 4 days
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FCyRl SIGNAL TRANSDUCTION
2105
results in an eightfold increase in the level of syk expression
(Fig 3, lanes 1 through 4). syk expression is increased at 4
days of IFN differentiation, a time when y subunit expression is also increased 8- to IO-fold, and this correlates with
the expression of phosphorylated isoform of the y subunit,
y l , and the capacity to signal through the FcyRI receptor to
the respiratory burst."
syk coprecipitates with the y subunit of FcyRI receptor.
We used the 4D8 antibody to immunoprecipitate the y subunit from resting and FcyRI-stimulated U937IF cells (Fig
4). We have previously described the mobility shift that
occurs in the y subunit after FcyRI cross-linking in U937IF
cells.I0 This mobility shift is associated with FcyRI activa-
IFN treatment
0
1
2
3
4
2
3
4
c-syk
a s y k imrnunoblot
Fig 3. IFN induces the expression of oyk. Anti-oyk Western blot
performed onwhole-cell lysates of U937 cells at different stages of
IFN differentiation. Lysates were prepared from U937 cells incubated
with IFN for different times (250 UlmL). Proteins were resolved by
SDS-PAGE (20% gels1 under reducing conditions and blotted with
the anti-syk antisera. Lanes 1 through 4 show levels of syk protein
expressed after 0,2,3, and 4 days of incubation in IFN, respectively.
+
,
primary + secondary
+nostimulation
"
I
C primary antibody
V secondary antibody
tion and the serine and tyrosine phosphorylation of the y
subunit.'0.'' In Fig 4C, we observed this mobility shift ( y o
to 7'). confirming that the y subunit was activated in these
cells after FcyRI cross-linking. We immunoprecipitated the
y subunit, using the 4D8 MoAb, and probed the 4D8 immunoprecipitate with antiphosphotyrosine (4G10). FcyRI
cross-linking results in tyrosine phosphorylation of a 72-kD
protein, as shown by antiphosphotyrosine immunoblotting
(Fig 4B, lanes 4 and 5). We then stripped the blot and reprobed with anti-syk antibody. The tyrosine-phosphorylated
72-kD protein that coprecipitates with the y subunit protein
reacts with anti-syk antibody (Fig 4A, lanes 4 and 5). We
observed an equivalent amount of sykprotein in the y subunit
IPS of resting and stimulated U937IF cells (Fig 4A, lanes 2
through 5). Isotype control MoAb does not IP the y subunit,
syk or a tyrosine phosphorylated 72-kD molecule (data not
shown). The isotype control IPS probed with anti-syk and
antiphosphotyrosine are identical to the results of our sham
IP (Fig 4, A and B, lane 1).
DISCUSSION
20
40
60
time (min)
Fig 2. FcyRl cross-linking results in the activation of the respiratory burst. Cross-linking of FcyRl induces the respiratory burst in
U9371F cells. The kinetics of the respiratory burst was quantitated
by measuring superoxide production as the superoxide dismutase
inhibitable reduction of ferricytochrome c, as described in Materials
and Methods. Data are included for theeffects of primary antibody
and secondary antibody alone and for the effects of primary and
secondary antibody cross-linking on the respiratory burst (see legend). Bars represent the SEM.
The high-affinity Fc receptor for IgG (FcyRI), the highaffinity Fc receptor for IgE (FccRI), and the T-cell receptor
(TCR) signal through a conserved stretch of amino acids
termed the antigen receptor homology I domain (ARHI),"2
the tyrosine activation motif (TAM)? or the antigen recognition activation motif (ARAM).'" ST through the ARHI domain involves the tyrosine phosphorylation of conserved tyrosine residues within the ARHI sequence (DExxYxxL
(X)68YxxL).'6Investigators have suggested that the tyrosine
phosphorylation of ARHI occurs through the activation of
nonreceptor protein tyrosine kinases,'".2' but the specific kinases responsible have not been clearly identified. The y
subunit of the FcyRI and FccRI as well as the 6 subunit of
the TCWCD3 contain ARHI s e q ~ e n c e s . ~ ~ 'Our
~ ~ ~data
'~~"
show that FcyRI activation results in the tyrosine phosphorylation of syk and the catalytic activation of syk kinase activity
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DURDEN AND LIU
2106
A.
1
408 anti y IP
2 3 4
5
4D8 antl TIP
1. %lm
4b8 anti y IP
I
W
“1
anti-y blot
_ ~ ~.
_ .”
.
. .
-
a PY blot
Fig 4. syk coprecipitates with the y subunit of FcyfN receptor. (A)
Anti-y immunoprecipitates (using 4D8
the MoAb) from4 x 10’ U9371F
cells were resolved by 10% SDS-PAGE, transferred t o nitrocellulose,
and blotted with anti-syk antisera (lanes 1 through 5). The lanes
correspond t o resting U9371F cells, lanes 2;U9371F cells stimulated
with FcyRl alone, lane 3; U9371F stimulated with Fcyfil cross-linking
for 1 and 5 minutes, lanes 4 and 5, respectively. Lane 1 represents a
sham IP performed with anti-y antisera and no cell lysate. (B)Antiy immunoprecipitates (using the 4D8 MoAbl from 4 x lo6 U9371F
cells were resolved by 10% SDS-PAGE, transferred t o nitrocellulose
and blotted for antiphosphotyrosine(4610) (lanes 1 through 5). The
lanes correspond t o resting U9371F cells, lane 2; U9371F cells stimulated with Fcyfil alone, lane 3;U9371F stimulated with FcyfiI crosslinking for1 and 5 minutes, lanes 4 and 5, respectively. Lane 1 represents a sham IP performed with anti-y antisera and no cell lysate.
Panels (AI and (B)represent the same blot first probed with antiphosphotyrosine then stripped and reprobed with anti-syk antisera. The
tyrosine phosphorylatedp72 superimposed with thesyk immunoreactive bands. (ClThis represents the lower portion of a 4D8 antiy immunoprecipitate (20% gel) probed with another anti-y (5927)
antisera. The lanes are of resting U9371F cells, lane 1; or U9371F stimulated with Fcyfil cross-linking for 1 minute, lane 2. We show the
position of the yo and y’ bands corresponding to thenonphosphoryy subunit, respectively, as prelated and phosphorylated form of the
viously described.” This blot isa separate representative experiment
( l 0 experiments performed).
(Fig 1). We demonstrate that syk expression is increased
on day 4 of I F N differentiation (Fig 3). On day 4 of I F N
differentiation, we have observed the appearance of a hyperphosphorylated form of the y subunit, termed y ’ , which
correlates with the capacity of U9371F cells to transmit a
signal through FcyRI.’” The observation of IFN-induced expression of syk may suggest a role of syk kinase in FcyRI
ST. syk is constitutively associated with the FcyRIlyARHI
receptor complex in U937IF cells (Fig 4).This suggests a
role for p7Tykin the y I F N priming of signal transduction
through the FcyRI receptor FcyRI. Signal transduction, as
measured by the quantitation of the respiratory burst, is associated with the activation of syk kinase activity (Fig 2). Currently, it is unclear whether syk kinase or FcyRIy are directly
involved in the downstream activation of the burst response
or other myeloid responses. Overexpression or antisense experiments may answer this question.
The characterization of nonreceptor protein kinases has
shown a considerable degree of diversity. There are now
eight families of nonreceptor protein tyrosine kinases.” The
syWZAP7O family have nowbeen
implicated inFceRI,
FcyR, and TCR signal transduction, respectively.z~~z’~z~~~’
Experiments performed in T cells have shown that ZAWO is
associated with the 6 subunit of the TCR/CD3 complex,
under condition of TCR activation.23The p7Tykkinase has
also been implicated in ST in B cells and in platelets.36
Benhamou et aI2’ showed that syk is tyrosine phosphorylated
and noncovalently associated with the y subunit of the FccRI
receptor after receptor activation. Agarwal et al.” reported
in vitro kinase data showing that syk kinase is activated in
HL60 cells stimulated with human IgG, followed by crosslinking with rabbit-antihuman IgG (Fab;). In these experiments, HL60 cells were activated in the presence of 50 pmol/
L sodium orthovanadate, a concentration previously shown
to inhibit FcyRI signal transduction and phosphorylation of
the FcyRIy subunit.’’ The pleiotropic effects of vanadate
make the interpretation of these experiments more difficult.”
While this manuscript was in preparation, other investigators
have shown that syk kinase is activated after FcyRI and
FcyRII acti~ation.’~
We have shown that syk is constitutively
associated with the FcyRIy subunit and undergoes catalytic
activation and tyrosine phosphorylation in vivo after FcyRI
cross-linking (8-fold induction of autophosphorylation and
12-fold increase in tyrosine phosphorylation). The increased
syk kinase activity likely occurs through the autophosphorylation of syk; however, it is possible that some other kinase
may coprecipitate with syk. Preimmune antisera does not
precipitate syk from U937IF cells stimulated with FcyRI
cross-linking and these IPS do not contain the syk kinase
activity (data not shown). Experiments performed with preimmune antisera argue against the nonspecific association
of other kinases and suggest that the activation of syk after
FcyRI stimulation is specific. The constitutive association
of nonphosphorylated syk and FcyRIy in I F N primed U937
cells is consistent with the recent observations of Iwashima
et al,’9 who showed that the tyrosine phosphorylation and
tyrosine kinase activity of ZAP70 kinase isnot required
for its association with the 6 subunit. They used synthetic
phosphopeptides to show that tyrosine phosphorylation of
the two tandem tyrosine residues in 6 sequence was required
for the association of ZAP70.39The binding of ZAP70 to
6 did not require the tyrosine phosphorylation or catalytic
activation of ZAWO, but did require the presence ofboth
tandem SH2 domain^.'^ More recently, Duplay et aI4’
showed that ZAWO and lck are noncovalently associated in
T cells. Our results are similar to Hutchcroft et a14’in that
nonphosphorylated syk is constitutively associated with the
FcyRIy (Fig 4A, lanes 2 through 5). The localization of the
syk kinase to the FcyRI receptor complex likely involves
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FCrRl SIGNAL TRANSDUCTION
the interaction of the two tandem SH2 domains of syk with
the tyrosine-phosphorylated tandem yARHI motifs of
FcyRIy. Our results suggest a role for syk in signal relay
through the FcyRUyARHI receptor complex and suggest
a role for the sywFcyRIy association in IFN priming of
macrophages. Our previous data may argue that the constitutive association of syk with FcyRIy is not mediated through
an increase in the tyrosine phosphorylation of the yARHI
motifs in resting U9371F cells." Alternatively, a very low
level of tyrosine phosphorylation may be required to drive
this association or that this association occurs through some
other molecular mechanism. The further study of the role of
syk kinase in FcyRI ST will likely clarify these questions
and contribute to the general understanding of how this nonreceptor protein tyrosine kinase functions in macrophage
activation andin interferon priming of macrophage functions.
ACKNOWLEDGMENT
We thank Dr Joseph Bolen for generously providing the polyclonal anti-syk antisera and Dr J. Kochan for providing us with the
anti-y subunit antibody (4D8). We thank J. Blanchard for secretarial
support.
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1994 84: 2102-2108
Protein-tyrosine kinase p72syk in Fc gamma RI receptor signaling
DL Durden and YB Liu
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