(Received for publication, January 14, 1997)
From the Arthritis and Rheumatism Branch, NIAMS, National Institutes of Health, Bethesda, Maryland 20892
Activation of cells mediated by the high affinity
receptor for IgE leads to rapid phosphorylation of tyrosines (and later other residues) on the receptor's and
subunits, and there is
circumstantial evidence that the tyrosines modified are in the
so-called immunoreceptor tyrosine-based activation motifs (ITAMs). We
identified and quantitated the residues phosphorylated on the subunits
of the native receptor by comparing the properties of peptides derived
from the receptors radiolabeled in vivo or in
vitro with those of synthetic peptides. Our results with
receptors labeled in vivo confirm that only the tyrosines
in the ITAMs of
and
became phosphorylated, and preferentially,
those in the canonical YXX(L/I) sequences. The extent of
phosphorylation of the canonical tyrosines was of the same order of
magnitude, but the amino-terminal canonical tyrosine in the ITAM of the
subunit was consistently phosphorylated to a lesser degree. The
non-canonical ITAM tyrosine in the
subunit was considerably less
phosphorylated. Phosphorylation of serine (on
) and threonine (on
) also occurred mainly in the ITAMs, but selectively at some
positions whose characteristics seem to be conserved among other
receptors containing ITAMs. The studies with receptor complexes
isolated and radiolabeled in vitro gave similar results for
phosphorylation of tyrosines, suggesting that the latter, much simpler
system is a useful model for more detailed studies.
The receptor with high affinity for IgE, FcRI, belongs to a
family of "multichain immunorecognition receptors"
(MIRR)1 (1). Members of this family share
important functional and structural features. Upon interaction of the
bound immunoglobulin (Fc receptors) or the immunoglobulin-like part of
the receptor (B- and T-cell antigen receptors) with a multivalent
antigen, each of them initiates cellular responses through a process
dependent upon aggregation of the receptors (2, 3). Following such aggregation, one of the earliest events is phosphorylation of protein
tyrosines, even though all these receptors lack an intrinsic kinase
activity. Instead, they initiate such phosphorylation through their
association with one or more non-receptor Src family tyrosine kinase(s)
(4, 5). The cytoplasmic extensions of one or more of the subunits of
each of these receptors contain a highly conserved sequence (6), now
referred to as the immunoreceptor tyrosine-based activation motif
(ITAM) (7), which contains a tyrosine residue in two canonical
YXX(L/I) sequences. There is evidence that these tyrosines
are substrates for the Src family tyrosine kinases associated with the
receptors, and that their phosphorylation initiates signal propagation
by recruiting proteins containing SH2 domains; in particular, the
related tyrosine kinases Zap and Syk, phosphoinositide-related enzymes
such as phospholipase C and phosphoinositide-3 kinase, and adapter
proteins such as Vav, Crk, and Grb-2, which connect the receptors to
several metabolic pathways (8, 9).
The molecular details of the events triggered by FcRI and by related
receptors have been explored using genetically engineered chimeric
proteins and mutational analysis. The results from such studies have
varied somewhat, depending on the system studied and compared with what
is observed with the intact receptor. For example, Jouvin et
al. (10) found that upon aggregation, chimeric constructs of the
individual cytoplasmic domains of the
and
subunits of Fc
RI
elicited some cellular responses, and that mutation of the tyrosine
residues in the ITAMs inhibited the binding of Lyn kinase to
and of
Syk kinase to
. Notably, however, no phosphorylation of the
unmutated chimeras was observed, even though this is observed when
intact receptors are aggregated. In the B-cell antigen receptor, a
chimera containing the ITAM of the Ig
subunit was sufficient to
elicit cellular responses upon aggregation, and mutation of either of
the two ITAM tyrosines resulted in the loss of the response (11, 12).
However, when these mutations were expressed in the context of the
whole B-cell receptor, the loss of activity was much less dramatic
(12). In that study, phosphorylation of only one of the ITAM tyrosines
seemed to suffice for certain responses. In a related system, chimeras
of the CD3-
subunit of the T-cell receptor were found to interact
with different kinases, depending on which tyrosine residue was
phosphorylated in the ITAM (13, 14). In still another study,
phosphorylation of the CD3-
chimeras upon aggregation was not
detected, even when downstream proteins became phosphorylated (15).
It must be recalled that despite the power of protein engineering, the
evidence that is derived from those approaches is largely circumstantial particularly for mutants showing a loss of function. For
example, substituting tyrosine with an alanine or phenylalanine not
only substitutes a residue that can be phosphorylated for one that
cannot, but also for one that has very different hydropathicity and
hydrogen bonding properties; significant conformational changes in the
protein produced by such a substitution might contribute to or even
account for the functional alterations observed. Second, genetic
approaches can only indirectly give clues about the quantitative aspects of any modifications. We decided that to complement the published mutational studies, it was important to examine the phosphorylation of the different subunits in the native FcRI directly, a kind of analysis that has not been previously reported for
this or any of the other MIRR.
We recently compared the phosphorylation of the receptors and other substrates in vivo and after an in vitro assay. Those results supported a model in which receptors constitutively associated with kinase become transphosphorylated upon aggregation and are thereby able to recruit additional molecules of kinase (16, 17). A further rationale for undertaking the present study was to determine whether the receptor aggregates isolated under the special conditions we used would show a pattern of phosphorylation similar to what is observed in the much more complex situation in vivo.
RBL-2H3 cells were grown (18), sensitized with mouse monoclonal anti-2,4-dinitrophenyl (DNP) IgE (19), and stimulated with 0.5 µg/ml DNP-bovine serum albumin (25 mol of DNP/mol of protein) for 2 min as described previously (16). When appropriate, the cells were activated in buffer supplemented with 0.4 mM MgSO4 and 1 mM CaCl2.
Phosphorylation of ReceptorsFor studies in
vivo, cells were incubated with 1-3 mCi/ml
[32P]orthophosphate (acid-free, carrier-free, Amersham)
for 3-4 h in a medium that was otherwise free of phosphate. This
extended incubation in the absence of phosphate did not affect the
antigen-induced phosphorylation of receptor tyrosines, as assessed with
an anti-phosphotyrosine antibody by Western blotting of
immunoprecipitated receptors (data not shown). Cells were lysed as
described (20), except that 1 mM hapten
(DNP--N-aminocaproate) was added after lysis
to ensure by disaggregation, more complete immunoprecipitation of the
receptors aggregated during activation of the cells (16). The
immunoprecipitated receptors were isolated and washed as described
(20). For the in vitro labeling of the receptor, the
immunoprecipitates were isolated and incubated as described (16). The
immunoprecipitates from in vivo and in vitro
labeled receptors were submitted to SDS-polyacrylamide gel
electrophoresis and transferred to nitrocellulose membranes as reported
previously (16, 20).
The radioactive bands
corresponding to the and
subunits of the receptor, identified
by autoradiography, were excised from the nitrocellulose membrane and
submitted to proteolytic digestion as described (21). Alternatively,
the proteolysis was performed on specimens obtained by high pressure
liquid chromatography (HPLC) (below) after drying the fractions and
resuspending the material in the appropriate buffer. The buffers and
temperatures of incubation employed were as follows: (i) for
L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated
trypsin (Worthington), 50 mM
NH4HCO3, pH 8.0, 37 °C; (ii) for
Staphylococcus aureus V8 protease (ICN Pharmaceuticals Inc.,
Costa Mesa, CA), 50 mM NH4HCO3, pH
7.6, 37 °C; (iii) for proline-specific endopeptidase (ICN
Pharmaceuticals Inc.), 50-100 mM sodium phosphate, pH 7.2, 30 °C; (iv) for thermolysin (Calbiochem), 100 mM
NH4HCO3 and 1 mM CaCl2,
pH 8.0, 55 °C.
Peptides were synthesized on a model 431A peptide synthesizer (Applied Biosystems). Amino acid analyses of the purified synthetic peptides were performed with a model 420A amino acid analyzer (Applied Biosystems). Phosphoamino acid analysis was performed as described (22, 23).
Separation of PeptidesThe released peptides were dried in a Speed-Vac, resuspended in 0.2-0.5 ml of H2O, and analyzed by HPLC. The samples, in a volume ranging from 50 to 500 µl, were applied to a reverse phase column (Delta Pak 15µ C18 30 nm, 3.0 × 300 mm, Waters Corp., Milford, MA) after injection of the sample into a 1-ml loop. The column was developed with a discontinuous linear gradient from Buffer A (0.1% v/v trifluoroacetic acid in water) to 0.1% v/v trifluoroacetic acid in 60% v/v acetonitrile in water (Buffer B) at a flow rate of 0.7 ml/min. The gradient was (in percent buffer B): 0-0.06 min, (10%); 7.3 min, (15%); 20 min, (30%); 35 min, (40%); 40 min, (100%); 50 min, (100%); 60 min, (10%); 70 min, (10%). The Dynamax HPLC System (Rainin Instrument Co. Inc., Woburn, MA) was employed to process the column. Throughout these studies, the fraction size was 0.49 ml (0.7 min), except for the thermolysin digest, for which 0.21-ml (0.3 min) fractions were used to improve the separation of the peptides.
Analysis of PeptidesRadioactivity in the HPLC fractions was measured with scintillation liquid for direct quantitation, or by Cerenkov counting when the peaks required further processing. HPLC peaks identified by absorbance at 214 nm (synthetic peptides) or by radioactivity (from the phosphorylated receptor subunits) were dried in 1.5-ml polypropylene tubes in a Speed-Vac overnight, at the low or intermediate temperature setting. Higher temperatures resulted in poorer subsequent solubilization of some peptides.
In some instances we needed to determine whether specific cleavage products of a synthetic peptide contained non-radioactive phosphotyrosine or not. Lyophilized peptide was resuspended in H2O, adjusted to neutral pH, and its absorbance scanned between 220 and 320 nm. The solution was then adjusted to pH 11 with NaOH and rescanned. Phosphotyrosine-containing peptides show a peak of absorbance at 265 nm, which is unchanged at basic pH; peptides containing unphosphorylated tyrosine show a peak of absorbance at 274 nm at neutral pH, and at 290 nm at basic pH.
Identification and Characterization of the 32P-Labeled Peptides
The number of receptors we analyzed was too small to allow for conventional direct compositional analysis of the peptides generated by proteolysis and separated by HPLC. Instead, we identified the phosphorylated peptides by comparing their properties to those of synthetic peptides both with respect to their behavior in HPLC and by their sensitivity to various endoproteases. This approach, combined with analysis of which amino acid had become phosphorylated, allowed us to identify and quantitate the phosphorylated tyrosines and, in some instances, serines and threonines.
Choice of Synthetic Peptides
We selected proteases that would be likely to generate peptides that would be useful for distinguishing in particular between different tyrosines. We then prepared appropriate synthetic peptides based on the sequence of the receptors and on the particular proteases selected. Because our preliminary data suggested that the ITAMs were the main or exclusive sites of tyrosine phosphorylation, only peptides including all or part of those motifs were synthesized.
The subunit contains five tyrosines in the cytoplasmic portions of
the receptor: one in the loop between the second and third
transmembrane domains (Tyr125), and four in the
COOH-terminal extension (Tyr201, Tyr218,
Tyr224, and Tyr228) (24) (Fig.
1). Two of the latter, Tyr218 and
Tyr228, are in the canonical YXXL sequences of
the
chain's ITAM, and a third, Tyr224, falls between
them at a non-canonical site. We selected the endoprotease Glu-C from
S. aureus (V8) to hydrolyze this subunit. This enzyme
hydrolyzes peptide bonds carboxyl-terminal to glutamic and aspartic
acids, but in appropriate buffers cleaves exclusively next to glutamic
acid residues (25). Under the latter conditions, four peptides
containing the five cytoplasmic tyrosines of the
subunit are
expected: two containing the ITAM's tyrosines, and two others
containing transmembrane fragments. The non-canonical Tyr224 would be present in peptide
-2 together with the
canonical Tyr228. Appropriate phosphotyrosine peptides were
synthesized based on these considerations (Fig. 1 and Table
I).
|
In the chain, three of the four tyrosines are in or proximal to the
chain's cytoplasmic extension (Tyr25, Tyr47,
and Tyr58), and only the latter two are in the canonical
sequence. Trypsin is expected to yield three tyrosine-containing
peptides (Fig. 1).
Characterization of Synthetic Peptides
We synthesized the phosphotyrosine analogues of the expected
peptides shown in Fig. 1 (Table I). To assess which if any of the two
tyrosines in peptide -2 (Fig. 1) becomes phosphorylated, three
alternative peptides were prepared (
-2a,
-2b, and
-2c). For
each of the peptides used in this study, the major peak from a
preparative HPLC was isolated and re-purified by HPLC. The composition and properties of the purified synthetic peptides are summarized in
Table I. Sequence data were in good agreement with those expected.
Analysis of Tyrosines Phosphorylated in Subunit
The strips of nitrocellulose corresponding to the position of the
subunit were digested with V8 under conditions where cleavage carboxyl-terminal to glutamic acid occurs exclusively. Forty to 60% of
the radioactivity was released. Addition of up to 15% acetonitrile during treatment with V8 enhanced the release to 55-75%, but higher concentrations were inhibitory. Analysis of these peptides by HPLC,
from receptors labeled in vivo and in vitro,
consistently showed only two principal radioactive components with
peaks at fractions 37 and 64; a component at fraction 60-61 was less
consistently observed (Fig. 2, A,
bottom panel and B, top panel). Except
where noted, the same characterizations were performed with receptors labeled in vivo and in vitro.
Evidence for Phosphorylation of Tyr218
A peptide 11 residues in length containing the
NH2-terminal YXXL motif of the ITAM would be
expected to be generated by digestion of the subunit by V8 (Fig.
1). We identified the radioactive component in fraction 37 as the
phosphotyrosine analogue of this peptide (
-1 in Table I) on the
basis of the following observations. (a) The synthetic
phosphotyrosine analogue of
-1 eluted in the same fractions as this
radioactive component. (b) Fraction 37, isolated from
receptors labeled either in vivo or in vitro,
contained phosphotyrosine but no other phosphoamino acid.
(c) The radioactive material was sensitive to trypsin (Fig.
1 and Table I), and upon rechromatography of the tryptic digest, the
counts shifted to the same fractions (fractions 34 and 35) as those
containing the phosphotyrosine (assessed by UV spectral analysis) when
the synthetic peptide
-1 was similarly treated with trypsin. Because
of the low levels of phosphorylation in vivo, it was
impractical to perform the latter analysis on the
chains labeled
in vivo.
The small shift in the elution fraction is unexpected if tryptic
cleavage had occurred predominantly after Arg216 resulting
in the peptide LpYE (Fig. 1). Indeed, when a synthetic peptide of the
latter composition was prepared, it eluted at fraction 24. It appears
that cleavage occurred after Arg209 or Lys211
preferentially, possibly because of the cluster of acidic residues (Asp214, Asp215, Tyr(P)218, and
Glu219) surrounding Arg216 (Fig. 1). These
alternative cleavage sites will produce a peptide only one or three
amino acids shorter than the parental NH2-terminal peptide.
Similar results were obtained by treatment of a tryptic digest of with V8 (below). Only phosphotyrosine was identified in the peak at
fraction 35 when the latter treatment was performed on receptors
labeled in vivo and in vitro.
Evidence for Phosphorylation of Tyr228
A dodecapeptide containing the phosphorylated COOH-terminal
YXXL motif of the ITAM would also be expected after
digestion of the subunit by V8 (Fig. 1). The radioactive peak at
fraction 64 obtained from the same V8 digest was identified as the
phosphotyrosine analogue of this peptide as follows. (a) By
HPLC it eluted in the same fractions as the alternative synthetic
phosphopeptides
-2a and
-2b (Table I), both of which eluted in
the same fraction. (b) This fraction, isolated from
receptors phosphorylated in vivo or in vitro,
contained phosphotyrosine. Similarly, when this peak was isolated after
treatment of the tryptic digest with V8 (below) phosphotyrosine was
identified. (c) It was sensitive to prolylendopeptidase. This enzyme should cleave at Pro226 to yield the peptides
LHVYSP and IYSALE in which one or the other tyrosine would be
phosphorylated (Table II). When the alternative synthetic peptides
-2a and
-2b were subjected to cleavage with this endopeptidase, they each yielded two principal peptides (Table II,
column 3), one of which contained tyrosine and the other
phosphotyrosine by analysis of their UV spectra (see "Experimental
Procedures"). Reaction with prolylendopeptidase of fraction 64 from
receptors phosphorylated either in vivo or in
vitro, generated only one radioactive peptide, whose mobility
coincided with the phosphotyrosine-containing peptide generated from
-2a (fraction 36), and not from
-2b (Table II). Similar results
were obtained by digestion with prolylendopeptidase of the radioactive
material in fraction 64 of the combined trypsin plus V8 digest (Fig.
2A, bottom panel).
|
The digests of material phosphorylated in vivo sometimes
revealed an additional peak at fractions 65-66 (Fig.
2B, top panel). The relative intensity of this
peak was variable, and usually this component was only evident as a
widening of the major peak with a maximum at fraction 64. We think it
likely that this component arises by a partial alternative cleavage by
the V8 protease at Glu236 instead of Glu232. In
support of this suggestion, a synthetic peptide comprising residues
Leu221 to Glu236 (Fig. 1) eluted at fractions
65-66 and was only partially hydrolyzed by V8, under the same
conditions employed to digest from the nitrocellulose membrane.
Furthermore, analysis of the phosphoamino acids revealed phosphoserine
and phosphotyrosine as did the peak at fraction 64 (below).
Occasionally, it was also generated after treatment of the tryptic
digest with V8.
Relative Phosphorylation of Canonical Tyrosines in Subunit
We regularly observed that the amount of radioactivity
representing phosphorylated Tyr228 was considerably greater
than that representing Tyr218 (fraction 64 versus fraction 37 in the top panels of Fig. 2). To determine if this difference simply represented a difference in
recovery, we first digested with trypsin, which should release a
19-residue peptide containing the entire ITAM (Fig. 1). Tryptic digestion of the blotted
subunit led to release of 75-95% of the
counts, and by HPLC only a single peak was observed (middle panels in Fig. 2). Digestion of this component with the V8
protease was expected to yield the same COOH-terminal peptide as
generated by the treatment of
with V8 directly (Fig. 1). Typical
HPLC patterns from such a digestion are shown in the bottom
panels of Fig. 2. Again fraction 64 contained about 75% of the
radioactivity. Irregularly, the elution pattern from such a digestion
appeared very similar to the digestion in which V8 alone was used,
except that the peak at fraction 37 shifted to fractions 35-36 (Fig. 2, A and B, bottom panels). As already
noted, this likely results from an alternative tryptic cleavage. In any
case, it is clear that the difference in radioactivity in the two
fractions reflects a difference in the phosphorylation of the two
canonical tyrosines in the
ITAM rather than an artifact of
recovery.
Evidence for Phosphorylation of Non-canonical Tyr224
As already noted, and as illustrated in the bottom
panel of Fig. 2A and the top panel of Fig.
2B, a radioactive component was also observed in fractions
60 and 61, albeit less consistently. This is the same position in which
the bisphosphorylated synthetic peptide -2c elutes (Table I).
Phosphoamino acid analysis of this radioactive peak revealed only
phosphotyrosine after labeling in vivo and in
vitro. When the prolylendopeptidase digest of this peak was
subjected to HPLC, radioactive peaks were observed at fractions 36 and
45. Fraction 36 is where the phosphotyrosine-containing peptide derived
from the synthetic peptide
-2a elutes, whereas fraction 45 is where
the phosphotyrosine-containing peptide derived from the synthetic
peptide
-2b (Table II) is found. The amount of radioactivity in the
two peaks was similar. The radioactivity was too low to permit us to
perform the same analyses on receptors labeled in vivo.
These results indicate that the non-canonical as well as the canonical
tyrosine in the ITAM of the
subunit can become phosphorylated.
Similar results with prolylendopeptidase were obtained when this peak
was isolated from the V8 digest of the
subunit, or after V8
treatment of the tryptic digest (Fig. 2A, bottom
panel).
Analysis of Tyrosines Phosphorylated in Subunit
Tryptic cleavage of was performed on reduced samples of the
receptor, to promote the accessibility to the proteolytic enzyme. However, similar results were obtained with or without reduction. Trypsin released 85-95% of the radioactivity from the region of the
nitrocellulose membrane containing the
chains. Analysis of the
digest radiolabeled in vivo and in vitro by HPLC
consistently revealed only two discrete radioactive components, with
peaks at fractions 30 and 36 (Fig. 3, top and
bottom). For the receptors labeled in vitro the
peak at fraction 30 consistently contained a larger percent of the
radioactivity, whereas the opposite was true for the receptors labeled
in vivo. However, the difference was smaller than that
observed with the peptides derived from
. Except as noted, the same
characterizations were done for the receptors labeled in
vivo and in vitro.
Evidence for Phosphorylation of Tyr47
The following observations indicate that the canonical tyrosine at
position 47 became phosphorylated during both in vitro and
in vivo incubation with 32P. (a) By
HPLC the synthetic phosphorylated analogue of the tryptic fragment
-1 (Fig. 1) eluted in fraction 36: the same fraction in which the
principal radioactive components released by trypsin from the
chain
were found. (b) Phosphoamino acid analysis of the material
in fraction 36 from receptors labeled in vivo and in
vitro identified tyrosine as the main phosphorylated residue (see
also below). (c) Brief treatment with thermolysin of the receptor-derived material in fraction 36 generated three new
radioactive peaks by HPLC. This would be expected for cleavage
NH2-terminal to Val46 or Leu50, or
both, yielding VpYTGLNTR, SDAVpYTG, and VpYTG (Fig. 1). Treatment of
the synthetic
-1 peptide under these conditions produced five peptides, three of which contained phosphotyrosine, as assessed by UV
spectral analysis, in the same positions as the radioactive peptides
(data not shown). On the other hand, more complete digestion led to an
increase in the radioactive peak that eluted first at the expense of
the two others, as would be expected from a hydrophobic reverse phase
column. (d) Under the conditions we used, V8 cleaves only
after glutamic acid; therefore, as expected, the synthetic
-1 was
resistant to the action of V8. The material in fraction 36 was likewise
insensitive to the action of V8. (e) By digesting
first
with V8, one expects a 15-residue peptide containing the canonical
tyrosine 47 and the tripeptide containing the other canonical tyrosine
at position 58 (Fig. 1). The V8 digest of labeled
revealed a single
radioactive peak at fraction 35, likely because the highly polar TpYE
tripeptide, containing Tyr(P)58 (below) was only poorly
retained on the column. Subsequent digestion of the 15-mer with trypsin
should yield a peptide identical to
-1 (Fig. 1). When the
radioactive component in fraction 35 was treated with trypsin, this is
exactly what was observed. It was only practical to perform the
analysis described in (e) with the peptides derived from
receptors labeled in vitro.
Evidence for Phosphorylation of Tyr58
The evidence that the other canonical tyrosine, Tyr58,
becomes phosphorylated is based on the following observations.
(a) The synthetic peptide -2 eluted in the same fraction
as the other major component derived from the digest, i.e.
fraction 30 (Fig. 3). (b) By phosphoamino acid analysis,
tyrosine was the only phosphorylated residue identified in fraction 30 in the digest of the
chains derived from receptors labeled in
vivo and in vitro. (c) In addition, the
radioactive material in fraction 30 showed the expected sensitivity to
V8. Treatment with the latter led to a shift of the radioactivity from
fraction 30 to the void volume, as expected for the polar peptide TpYE
(Fig. 1) generated by hydrolysis at Glu56 and
Glu59.
Kinetics of Phosphorylation
The in vivo and in vitro phosphorylation of
the receptors was analyzed multiple times. The extent of in
vitro incorporation of 32P made it practical to
examine the kinetics at which the four canonical tyrosines in the ITAMs
of and
became phosphorylated. Such an experiment is shown in
Fig. 4 for the
(A) and
(B) subunits. The data are plotted to show the degree of labeling relative
to the levels achieved at 60 min. Several points are notable. The two
canonical tyrosines within the ITAM of an individual subunit achieve
their final levels of phosphorylation at more or less equivalent rates.
Similarly, the two types of subunits reach their final values at more
or less equivalent rates. Finally, in agreement with our previously
reported results, in both cases a plateau is achieved even though the
kinase remains active (as assessed by its activity toward an exogenous
substrate) (16). The low level of incorporation of 32P in
the labeling carried out in vivo made it impractical to
pursue a similar kinetic study of such labeling. All the results
reported here are for receptors that had been aggregated by antigen for 2 min, a time at which the phosphorylation in vivo and the
association of tyrosine kinase activity to the receptor, reach a
maximum under the conditions of activation employed (16).
Relative Phosphorylation of Canonical Tyrosines
The ratios of phosphorylation of the COOH-terminal and
NH2-terminal peptides and the individual tyrosines in the
ITAMs of the and
we observed are summarized in Table
III. Several aspects of these results are notable. With
respect to the ITAM in the cytoplasmic domain of
, it is apparent
that in vivo, the relative incorporation of 32P
into the membrane distal COOH-terminal peptide is larger than in
vitro (column 3). However, when the radioactivity is corrected for
incorporation of 32P into other amino acids (below), the
ratio for the in vivo incorporation into tyrosines falls to
approximately 3-4 (column 6). This ratio is close to that observed for
the incorporation observed in vitro, where phosphotyrosine
accounted for at least 95% of the incorporation of 32P
(below).
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With respect to the ITAM of , the ratio of phosphorylation of the
COOH-terminal peptide relative to the NH2-terminal peptide was roughly unity in vivo and in vitro (Table
III, column 4). In the case of the
chain, the fraction of the
counts that were incorporated in vivo that were
not due to tyrosine was sufficiently small that it did not
change significantly the ratio for the incorporation into tyrosines
shown in Table III, columns 7 and 8. However, the relative
incorporation into tyrosines on
in vivo was consistently 2-4 times larger than in vitro (Table III, columns 7 and 8, first versus second row).
We noted a roughly inverse relationship between the extent of labeling
observed in vivo and in vitro. This would be
expected if there were a limited number of tyrosines within an
aggregate that could be phosphorylated, if the level of phosphorylation in vivo at any one time was determined by a balance between
the activity of kinase(s) and of phosphatase(s) and if the
phosphorylation in vitro mimicked that in vivo
except for the absence of phosphatase. We reasoned that if the action
of phosphatases was also suppressed in vivo, then the level
of phosphorylation achieved should approximate that observed in
vitro. Fig. 5 shows the result of such an analysis using the phosphatase inhibitor phenylarsine oxide. In this experiment, we assessed phosphotyrosine by Western blotting to be able to compare
phosphorylation in vivo and in vitro. Although
the reactivity of the anti-phosphotyrosine antibody with phosphorylated
and phosphorylated
may not be identical (20), the correlation between phosphorylation and antibody binding should allow rough comparisons to be made between levels of phosphorylation on the individual subunits.
The phosphatase inhibitor resulted in an increased level of phosphorylation as shown previously (20, 26). The new finding is that this enhanced phosphorylation achieved in vivo approximates the maximum level achieved in vitro when the aggregated receptors are incubated under conditions that are permissive for further phosphorylation by the receptor-associated kinase.
In Vivo Phosphorylation of ITAM Serines and Threonines
The principal purpose of our studies was to analyze the
phosphorylation of tyrosines on the receptor that occurs as an early response to its aggregation. However, analysis of the and
subunits labeled in vivo also allowed us to identify and in
some cases to localize, other phosphorylated residues. Similar results were obtained in the presence or absence of extracellular
Ca2+ or Mg2+. Earlier work had indicated that
the
subunit became phosphorylated on one or more serines and the
subunit on one or more threonines (27-29). In the present study,
all of the radioactive peaks contained phosphotyrosine. It is possible
that serines and threonines outside the ITAM also become
phosphorylated, but at least in our studies, their steady state level
is likely to be less than 5% of the total phosphorylation because we
should have been able to observe larger amounts as discrete additional
peaks.
No phosphoserine was observed in the
NH2-terminal peptide derived from treatment of the subunit with V8 (
-1 in Fig. 1), but about 50% of the radioactivity
observed in the COOH-terminal peptide ((
-2), fraction 64 in Fig. 2),
was accounted for by phosphoserine. This peptide contains serines at
positions 225 and 229. As noted above, when this peptide from in
vivo labeled subunits was reacted with prolylendopeptidase, a
single radioactive peak was observed at fraction 36. This result is
consistent with the predominant if not exclusive phosphorylation of
Ser229 (in addition to Tyr228).
About 10% of the radioactivity in the
NH2-terminal peptide derived from tryptic digestion of the
subunit (
-1 in Fig. 1) from receptors labeled in vivo
was found in phosphothreonine with the remainder in phosphotyrosine,
whereas less than 1% was in phosphothreonine in the COOH-terminal
peptide. No incorporation of 32P into threonine was
observed in either peptide after labeling in vitro.
After treatment with thermolysin, phosphoamino acid analysis of the two
more hydrophobic peptides derived from the NH2-terminal peptide (fraction 36) revealed only phosphotyrosine, whereas the
remaining undigested peptide contained both phosphotyrosine and
phosphothreonine. These results favor Thr52 as the
principal and possibly exclusive threonine that becomes phosphorylated
in the
chain.
Both canonical tyrosines
present in the ITAM of were phosphorylated, although not to the
same degree. The severalfold greater phosphorylation of
Tyr228 than of Tyr214, was observed both
in vivo and in vitro (Fig. 2) and to a similar degree (Table III, column 6). In vivo, the level of
phosphorylation is the result of a dynamic process of phosphorylation
and dephosphorylation (20, 26, 30). It might be supposed that in
vivo the different levels of phosphorylation result from a
different susceptibility of the two tyrosines to a phosphatase.
However, this would not explain why a similar differential was observed
in vitro in the presence of the inhibitor vanadate, where
all tyrosine phosphatase activity was eliminated. Alternatively, the
differential might reflect a different accessibility of the two
tyrosines to the receptor-bound kinase(s). If so, then the difference
must be more or less absolute for some of the aggregated receptors
because the phosphorylation in vitro reached a plateau value
at similar rates for both tyrosines (Fig. 4). We have no additional
data to support other explanations. Recently, various downstream
signaling molecules were shown to interact with a diphosphorylated
ITAM peptide (31). Possibly, alternative phosphorylations of the
ITAM tyrosines leads to differential interactions with such distal
components.
The analyses reported here cannot determine what fraction (if any) of
the ITAMs were simultaneously phosphorylated on both tyrosines. It has been shown that both tyrosines must be phosphorylated simultaneously in order for an ITAM to interact with either Syk and
ZAP-70 to propagate an activation signal (9). Although those kinases
are thought to interact chiefly with the
subunit (below),
diphosphorylation may also favor interaction of SH2-containing molecules with
(31). The results on the
subunit resemble those
found with the Ig
chain of the B-cell antigen receptor, where only
the canonical tyrosines in the ITAM were phosphorylated, and to
different extents (12). The level of phosphorylation in Ig
were
minimally affected when one of the canonical tyrosines was mutated to
phenylalanine (12).
Our data show that the non-canonical Tyr224 (which is
conserved in the human, mouse, and rat ITAM) can also become
phosphorylated. This tyrosine is at position
4 from the COOH-terminal
canonical tyrosine. Among the members of the MIRR family, only murine
CD3
has a non-canonical tyrosine within the ITAM, at position
3 from the COOH-terminal canonical tyrosine. In neither instance is the
tyrosine surrounded by a sequence that is typically found in substrates
for Src family kinases (32). Phosphorylation of Tyr224 was
found only in the bisphosphorylated COOH-terminal peptide, suggesting that its phosphorylation occurs only after the canonical tyrosine has been phosphorylated. In those receptors in which the
COOH-terminal peptide eluting at fraction 64 contained phosphoserine, the bisphosphorylated peptides at fraction 60 contained only
phosphotyrosine. It is as if the double phosphorylations were mutually
exclusive. We are unaware of other reports of in vivo or
in vitro phosphorylation at non-canonical tyrosines in the
ITAMs.
An unexpected finding was that the principal serine(s) that became
phosphorylated were also in the ITAM. On HPLC we observed no
radioactive fractions that did not contain phosphotyrosine. Such
fractions should have been observed in the V8 or tryptic digest if any
of the numerous non-ITAM serines in the carboxyl- and/or amino-terminal
cytoplasmic domains of the had been phosphorylated. In
vivo, the phosphorylation of Ser229 accounts for about
50% of the radioactivity found in the COOH-terminal peptide of the
ITAM. It is potentially significant that serines at an equivalent
position (+1 from the COOH-terminal tyrosine) are found also in T-cell
receptor
and CD3
,
, and
, and in the viral protein BLV
gp30a (see Fig. 1 in Ref. 7). When phosphorylated, the serine would
have a negative charge, and it is noteworthy that the corresponding
residue in all the other ITAM-containing subunits of the multichain
immune recognition receptor family is a negatively charged glutamic or
aspartic acid (Fig. 1 in Ref. 7). Interestingly, in the receptor for
transforming growth factor
, mutation of a phosphorylatable
threonine to an aspartic or glutamic acid residue constitutively
activates the receptor (33). Again, our current studies do not reveal
whether any of the
subunits are phosphorylated on tyrosine and
serine simultaneously. It is possible that in the V8 digest, the
additional peak at fraction 66, frequently found as a widening of the
peak at fraction 64 was only observed on receptors labeled in
vivo (Fig. 2, B, top panel) because the
presence of phosphoserine at position 229 affects the hydrolysis by V8
at Glu232 (Fig. 1). Indeed, when we were able to isolate
this peak, its relative content of phosphoserine was larger than in
peak 64.
The phosphorylation of the
canonical tyrosines in the ITAM of was more equivalent
than in the case of
. In vivo, the relative phosphorylation of Tyr47 (NH2-terminal peptide)
was always larger than Tyr58 (COOH-terminal peptide), and
the opposite was true in vitro (Fig. 3), but in both cases
the ratio was reasonably close to 1. The extent of phosphorylation of
the
tyrosines relative to
Tyr218, was 2-3 times
larger in vivo than in vitro (Table III, columns 7 and 8, top versus bottom row). If one takes into account
that about 90% of the radioactivity in the NH2-terminal
peptide in vivo is phosphotyrosine, the average of the
ratios in vivo and in vitro of phosphorylation of
the two ITAM tyrosines in
is roughly 1 (Table III, column 4). This
is consistent with the results shown in Fig. 5.
Several reports suggest that it is the subunit that interacts with
Syk kinase. Thus, Syk as well as its isolated SH2 domains preferentially bind to the isolated
compared with the
subunit (10, 31, 34). It has also been shown that peptides containing the
ITAM bind and activate Syk kinase (35). Syk, and the related ZAP-70
tyrosine kinases, contain two consecutive SH2 domains whose integrity
is required for binding to ITAMs and activation (9). Furthermore, Syk
and the related Zap-70 used by other MIRR family members interact only
with bisphosphorylated ITAMs (9). Our observations, that the
phosphorylation of the two tyrosines in the ITAM of
were
approximately equivalent, is consistent with the mechanistic model
these data imply.
Our data indicate that the phosphorylation of threonine in also
occurs at sites proximal to a canonical tyrosine as was the case of
serine in
. However, whereas in
the phosphoserine generates the
negative charge that in virtually all other known ITAMs is conferred by
a highly conserved aspartic or glutamic acid residue (above), the
residues homologous to Thr48 are polar but not necessarily
charged, and to a similar extent the same is true for those homologous
to Thr52. Nevertheless, in all ITAMs either one of these
two positions is polar or charged. These residues are conserved in all
subunits analyzed so far, and its phosphorylation may regulate the
interaction of the
ITAMs with proteins containing SH2 domains.
Aggregated
receptors that were incubated under conditions permissive for tyrosine
kinase activity (and in the presence of phosphatase inhibitor) became
phosphorylated on their ITAM tyrosines (Figs. 2 and 3). The kinetics
were similar for the canonical tyrosines within each ITAM and for the
compared with the
subunits (Fig. 4). As noted previously for
the intact subunits, a plateau level was reached relatively rapidly
even though under the conditions used, phosphorylation of an exogenous
substrate continues unabated (16). Furthermore, under these conditions
only a fraction of the receptors are phosphorylated as judged by their
precipitability by anti-phosphotyrosine.2
Notably, the level of phosphorylation achieved in vitro
approximates that achieved in vivo when phosphatase activity
is inhibited under both conditions (Fig. 5). These new data are
consistent with the following formulation. 1) At a given point in time,
phosphorylation of aggregated Fc
RI results from a finite number of
receptor tyrosines being accessible to a finite number of
receptor-associated kinases. 2) In vivo, the steady state
level of phosphorylation results from the opposing actions of
phosphatases and the associated kinase. 3) In the presence of
phosphatase inhibitors, the level of phosphotyrosine achieved in
vitro does not substantially exceed the level observed in
vivo (Fig. 5). This observation suggests that under the conditions used for these studies, we recovered most of the kinase that was associated with Fc
RI in vivo. Together, these aspects
suggest that with respect to the initial phosphorylations of receptor tyrosines, the immunoprecipitates of the aggregated receptors isolated
under conditions that maintain their associated kinase(s) are a
reasonable analogue of the in vivo complex.
We thank George Poy for synthesizing the peptides, Patricia J. Spinella for the amino acid analyses, and Dr. Patrick Swann for assistance in setting up the HPLC system. We also thank Drs. J. Rivera and P. Swann for careful reading of the manuscript and suggestions.