(Received for publication, May 3, 1995; and in revised form, June 16, 1995)
From the
One of the primary responses observed following antigen-induced
cross-linking in mast cells is an increase in the phosphorylation of
certain cellular proteins on tyrosine residues. Stimulation of
protein-tyrosine kinase activity appears to be necessary for induction
of downstream responses such as degranulation. The role of nonreceptor
protein-tyrosine kinases in the signal transduction pathway initiated
by FcRI engagement in an interleukin-3-dependent mast cell line
has been examined. The results presented here show that the enzymatic
activity of Lyn is increased within seconds of receptor engagement. Syk
activity also undergoes a rapid and transient increase, reaching a peak
at approximately 30 s. Similarly, the activity of Fer, representing a
third class of nontransmembrane protein-tyrosine kinase increases as
well, with its activity peak reached at 1 min poststimulation. The
enzymatic activities of Syk and Fer were found to correspond to
anti-phosphotyrosine antibody reactivity. Phosphorylation of tyrosine
residues of the
and
chains of Fc
RI increased
concomitant with increased protein-tyrosine kinase activity. These
results indicate that at least three classes of nontransmembrane
protein-tyrosine kinases are involved in mast cell FceRI signaling and
that the activation of these classes of enzymes is temporally
regulated.
Mast cells are the primary effectors in immediate type
hypersensitivity reactions. Upon exposure to allergen, mast cells are
induced to rapidly secrete preformed mediators from cytoplasmic
granules. Mast cells bind IgE avidly via specific, high affinity Fc
receptors, termed FcRI. Fc
RI are heterotetrameric complexes
comprising an IgE-binding
subunit, a
subunit, and two
subunits(1, 2) . The cytoplasmic domains of the
and
subunits each contain two tyrosine residues, which are
located within a conserved consensus sequence termed the immunoreceptor
tyrosine activation motif (ITAM) (
)or antigen recognition
activation motif(3, 4, 5, 6) .
Phosphorylation of this pair of conserved tyrosines is required for
successful signal transduction in mast cells (7) and other
hemopoietic cell types(8, 9) . Cell activation follows
cross-linking of surface-bound IgE molecules by multivalent
allergen(10) .
Phosphorylation of a select set of cellular
proteins on tyrosine residues is a hallmark of the activation of cells
through engagement of multichain immune recognition receptors (MIRR)
and is one of the earliest detectable events following stimulation of
mast cells through
FcRI(11, 12, 13, 14, 15) .
Protein tyrosine phosphorylation following engagement of the Fc
RI
is thought to be mediated by the nontransmembrane protein-tyrosine
kinases Lyn and Syk. Lyn, a member of the Src family of
protein-tyrosine kinases, is constitutively associated with
Fc
RI(15), binding to the unactivated
subunit(7) .
Syk is a 72-kDa protein-tyrosine kinase (16) that, along with
p70
(Zap)(9) , defines a family of
cytoplasmic enzymes distinguished by the presence of tandem SH2 domains
located amino-terminal to the catalytic domain. Syk becomes activated
upon ligation of both B-cell (17) and mast cell MIRRs (7, 18) and associates with phosphorylated ITAMs
present within the cytoplasmic sequences of the MIRRs. In mast cells,
Syk binds to the phosphorylated ITAMs of Fc
RI
(19, 20, 21, 22) .
Fer is a ubiquitously expressed 94-kDa cytoplasmic protein-tyrosine kinase related to Fes(23) . At present, little is known about the involvement of Fer in cellular signaling pathways.
Temporal
differences in the activation of nonreceptor protein-tyrosine kinases
following MIRR engagement have been reported in B-cell and T-cell
lines(17, 24) . The purpose of this study was to gain
a further understanding of mast cell stimulation by examining the
timing and order of activation of nontransmembrane protein-tyrosine
kinases following FcRI engagement. We find that Lyn and Syk are
activated sequentially and that Fer also undergoes transient
activation.
Figure 1:
Time course of protein tyrosine
phosphorylation (A) and Ca flux (B)
in PT18 cells following IgE/DNP stimulation. Cells were sensitized with
DNP-specific IgE and washed as described under ``Materials and
Methods.'' A, the cells were then lysed as a function of
the indicated times following addition of DNP-HSA, and cellular
proteins were phosphorylated on tyrosine detected by APT
immunoblotting. The positions of protein markers are indicated. B, DNP-HSA (Ag
) was added at the
time indicated. The concentrations of DNP-HSA used were as follows: 2.5
ng/ml, dot-dashedline; 10 ng/ml, dottedline; 25 ng/ml, closedashedline; 50 ng/ml, dashedline; 100 ng/ml, solidline.
The reactivity of IgE-sensitized PT-18 cells to DNP-HSA was
also examined by monitoring changes in the concentration of
intracellular free calcium
[Ca]
. The
concentration-response curve for DNP-HSA is Gaussian in that addition
of low levels of ligand produce small increases in
[Ca
]
, moderate levels produce a
maximal increase in [Ca
]
that
is sustained, and higher levels produce a maximal
[Ca
]
rise that lacks the
sustained phase. The response of PT 18 cells to moderate (2.5 ng/ml) to
high (100 ng/ml) concentrations of DNP-HSA are shown in Fig. 1B.
Figure 2:
Time course of FcRI-
(A) and Fc
RI-
(B) tyrosine phosphorylation.
PT18 cells were stimulated with IgE/DNA, lysed at the indicated times,
and immunoprecipitated with antibodies specific for the indicated
proteins. Tyrosine phosphorylation was evaluated by APT immunoblotting (upperpanels), and relative abundance was estimated
by anti-
or anti-
immunoblotting (lowerpanels). Positions of Fc
RI-
, Fc
RI-
,
phosphorylated Fc
RI-
, and protein size markers are
indicated.
Figure 3: Time course of Lyn, Syk, and Fer enzyme activation. PT18 cells were stimulated with IgE/DNA, lysed at the indicated times and immunoprecipitated with antibodies specific for the indicated protein-tyrosine kinases. The protein kinase activities of the immunoprecipitated protein-tyrosine kinases were evaluated in immune complex autophosphorylation assays (upperpanels), or immune complex kinase assays using rabbit muscle enolase as an exogenous substrate (lowerpanels).
The catalytic activity of Syk was
also found to increase following FcRI engagement. Syk activity was
elevated immediately, but it did not reach its peak level of activity
until about 30 s poststimulation (Fig. 3B, upperpanel). The activity then slowly decayed, returning to
basal levels by 30 min.
The kinase activity of Fer was also examined to determine if Fer could be responsible for the protein band(s) at approximately 94 kDa detected by APT immunoblotting. The protein kinase activity of Fer was found to increase following addition of DNP-HSA. Fer activity was increased by 10 s, with a maximum level attained by 1 min. Fer activity was reduced to unstimulated levels by 10-30 min.
The activity profiles of Lyn, Syk, and Fer determined by autophosphorylation were confirmed by evaluating the ability of these enzymes to phosphorylate an exogenous substrate. In each case, the ability of each enzyme to catalyze the phosphorylation of enolase roughly paralleled changes in autophosphorylation demonstrated in the previous assays. Lyn was again activated immediately, but the magnitude of the difference between unstimulated and stimulated activities toward enolase is much smaller than observed in the autophosphorylation assays. The capacity of Syk to phosphorylate enolase correlated with its ability to catalyze autophosphorylation. Similarly, the kinase activity profile of Fer toward enolase matched that observed in the autophosphorylation assay.
The correlation between the enzymatic
activation and tyrosine phosphorylation states of these enzymes was
established by determining reactivity of each protein with APT
antibodies following FcRI engagement. The results of these
experiments are presented in Fig. 4. Syk was unreactive to APT
antibody when isolated from unstimulated cells, but its
immunoreactivity increased significantly following receptor engagement (Fig. 4A). APT immunoreactivity was high from 30 s to 3
min, and then it decreased to a low but detectable level by 30 min. The
abundance of Syk was not altered in the course of these experiments.
Thus, the reactivity of Syk with APT antibody paralleled its enzymatic
activation as determined by both autophosphorylation and
phosphorylation of exogenous substrate. The APT reactivity of Fer also
increased upon stimulation of the PT18 cells, reaching a maximum at 30
s to 1 min and decreasing to unstimulated level by 30 min. The
abundance of Fer was constant over this time course. As observed with
Syk, changes in the tyrosine phosphorylation state of Fer correlate
with changes in its enzymatic activity.
Figure 4: Time course of tyrosine phosphorylation of Syk (A) and Fer (B). PT18 cells were stimulated with IgE/DNA, lysed at the indicated times, and immunoprecipitated with antibodies specific for the indicated protein-tyrosine kinases. Tyrosine phosphorylation was evaluated by APT immunoblotting (upperpanels), and relative abundance was estimated by anti-protein-tyrosine kinase immunoblotting (lowerpanels). Positions of Syk, Fer, IgG heavy chain, and protein size markers are indicated.
Protein tyrosine phosphorylation is a well established initiator of signal transduction pathways triggered in response to receptor engagement in hemopoietic cells. The concept of an ordered activation of enzymes being necessary to propagate such signals has been recently established in both B- and T-cells(17, 24) . We have found that this observation extends to mast cells, albeit with a compressed time scale. Furthermore, we report evidence that in addition to Lyn and Syk, the nonreceptor protein-tyrosine kinase Fer participates in mast cell signaling and is part of the ordered activation scheme.
The results
of our immune complex kinase assays show that the enzymatic activation
of the Src family member Lyn after FcRI cross-linking is immediate
and transient. The activation of Src family enzymes in MIRR signal
transduction is a well established phenomenon. Lck and Fyn are
activated in response to T-cell receptor ligation(24) , the
enzymatic activities of Lyn and Blk are increased following stimulation
of B-cell lines through the B-cell receptor(17) , and Lyn has
been shown to be activated in stimulated mast cell and basophilic cell
lines(7, 15) . Lyn is the most abundant Src family
member in PT 18 cells and other mast cell and basophilic cell lines and
appears to send the initiating signal following Fc
RI engagement.
The location of Lyn at the cytoplasmic subsurface of the plasma
membrane and the observation that this protein-tyrosine kinase is
constitutively bound to the Fc
RI-
chain places this enzyme in
the appropriate subcellular location to respond rapidly to receptor
engagement.
The peak levels of Syk and Fer activation were found to
follow that of Lyn. The peak of Syk activity coincides with the time at
which FcRI-
tyrosine phosphorylation is approaching maximal
levels. This finding is consistent with a model where Lyn is somehow
activated by receptor cross-linking and phosphorylates the
and
subunits of the receptor. Tyrosine-phosphorylated
ITAMs
would provide a docking site for Syk via its tandem SH2
domains(21, 22) . Recent evidence indicates that
binding of Syk to phosphorylated ITAMs induces a conformational change
in the protein, leading to its enzymatic activation(30) .
The overall timing of the tyrosine-phosphorylation response and the
characteristics of Syk activation in mast cells are quite different
from those observed in B-cells(17) . B-cells present a less
complex pattern of tyrosine-phosphorylated proteins following
engagement of the B-cell antigen receptor, and the tyrosine
phosphorylation develops more slowly. Furthermore, this response is
more sustained in nature. In WEHI 231 cells for instance, tyrosine
phosphorylation of Ig and activation and tyrosine phosphorylation
of Syk are both maintained for more than 1 h. The Fc
RI-mediated
response in PT 18 mast cells is characterized by a more compressed time
frame, the total cell tyrosine phosphorylation response as well as the
activation of protein-tyrosine kinases returns to unstimulated levels
of activity by 30 min poststimulation. This time frame is consistent
with the physiological role of the mast cell to provide an immediate
hypersensitivity reaction and then turn off.
The protein-tyrosine
kinase Fer is also activated following engagement of the FcRI. The
catalytic activation and tyrosine phosphorylation of Fer follows that
of Syk. This is the first known example of Fer activation in response
to external stimuli. Additional studies are being performed to clarify
the role of Fer in hemopoitic cell signal transduction. Another
protein-tyrosine kinase reported to play a role in mast cell signal
transduction is Btk(31) . Although we found Btk to be an
abundant enzyme in PT18 cells, we have been unable to detect activation
or an increase in tyrosine phosphorylation of Btk following stimulation
of PT18 cells (data not shown). The Btk present in PT 18 cells appears
to have a high level of constitutive tyrosine phosphorylation. This
high level of tyrosine phosphorylation in the absence of stimulation
may mask changes in the activation state of Btk following receptor
cross-linking or limit our ability to detect such changes.
In
summary, we have shown that tyrosine phosphorylation in PT 18 mast
cells is rapid and transient. Three different classes of
protein-tyrosine kinases are activated in a time-dependent manner
following FcRI engagement. The Src family member Lyn is activated
early, followed by activation of Syk and Fer. These experiments suggest
that Lyn is the initiator of the protein-tyrosine kinase cascade that
results in mast cell degranulation.