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Division of Basic Sciences, Department of Pediatrics, National Jewish Medical and Research Center, Denver, CO 80206
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) and CD79b (Igß),
respectively. Aggregation of BCR by Ag results in tyrosyl
phosphorylation and/or activation of many proteins, including CD79a and
CD79b (1, 2, 3) and protein tyrosine kinases (PTKs) of the Src, Syk, and
Tec families (4, 5, 6, 7, 8, 9, 10). Activation of PTKs leads to phosphorylation of
other molecules, including Vav, PLC
, and Shc (11, 12, 13, 14, 15, 16, 17). These
effectors mediate downstream signaling leading to cell proliferation,
differentiation, and/or apoptosis.
The CD79a-CD79b heterodimer is an important part of the receptor
complex. These B cell-specific glycoproteins are required for transport
of mIg to the cell surface (18), and they also transduce signals
initiated by BCR aggregation (1). This latter function can be
ascribed to the ITAM, a motif found within the cytoplasmic domain not
only of CD79a and CD79b, but also of the 
-chain of TCR; , 
,
and 
of CD3 components; ß and subunits of FcRI; and
FcRIIA (19, 20). Although conservation of this motif in immune
system receptors was recognized in 1989 (19), the signaling potential
of ITAMs, specifically those of TCR- and FcRI chains, was only
demonstrated by a series of studies in 1992 (21, 22, 23). Mutational
analyses further delineated the signaling function of the motif itself
and revealed that the two motif tyrosines and the third residue
carboxyl to each tyrosine (Y+3) are critical for ITAM-mediated
signaling leading to certain biologic responses (24, 25, 26).
To study the signaling function of CD79a and CD79b, several groups
created chimeric molecules consisting of the ITAM-containing
cytoplasmic tail of CD79a or CD79b and extracellular and transmembrane
domains of other surface molecules, such as CD8 (27, 28), FcRII
(29), mutated mIg (30, 31, 32), and platelet-derived growth factor
receptors (33, 34). Using this approach, the ability of CD79a and CD79b
to signal independently was demonstrated.
Subsequent studies have begun to define the function of the tyrosyl residues within CD79a and CD79b. Sanchez et al. (31) showed that an mIgM/CD79b chimera bearing a mutation in the C-terminal ITAM tyrosine (Y206) could no longer mediate either Ca2+ mobilization or protein tyrosyl phosphorylation. This finding was extended by Williams et al. (32), who showed that a similar chimera mutated at the N-terminal CD79b-ITAM tyrosine (Y195) or at both Y195 and Y206 failed to elicit phosphorylation of cytoplasmic proteins. Together, these data suggest that CD79b requires both ITAM tyrosines to function. A similar study of CD79a-ITAM tyrosines in CD8-CD79a chimeras by Flaswinkel and Reth (35) also suggested the requirement for both CD79a-ITAM tyrosines in protein tyrosine phosphorylation and Ca2+ mobilization. However, using the J558L myeloma model, this group developed evidence that in the intact BCR, CD79a tyrosines may function differently in signaling. These results appear to differ from those of the study of CD8 chimeras, and it was assumed in this study that the wild-type CD79b present in the reconstituted BCR did not contribute to the signaling observed. Thus, the relative function of ITAM tyrosyl residues is unclear.
ITAMs transduce signals largely by virtue of their tyrosyl
phosphorylation and subsequent interaction with SH2 domain-containing
proteins. Screening of phosphopeptide libraries has revealed that SH2
domains of Src family kinases Src, Fyn, Lck, and Fgr preferentially
bind the sequence Y-E-E-I (36). This resembles the N-terminal sequence
Y-E-G-L, and particularly the C-terminal Y-E-D-I, found in both CD79a-
and CD79b-ITAMs. Indeed, binding of phosphorylated CD79a- and
CD79b-ITAMs to Src family kinases has been well documented (37, 38, 39, 40). In
addition, the CD79a-ITAM sequence L-Y182-E-G-L is predicted to be a
preferred substrate of Src family kinases Lyn and Blk (41). Taken
together, these findings predict that a Src family kinase may
phosphorylate the N-terminal CD79a ITAM tyrosyl residue initially,
creating a binding site for SH2 domains of Src family kinases (and
other effectors). Binding of Src family kinases to phosphorylated ITAMs
via their SH2 domains may be an important kinase activation/signal
amplification step, as phosphorylated ITAMs can activate Src family
kinases in vitro (40). Src family kinases with mutated SH2 domains that
can no longer bind to phosphotyrosyl residue also do not function
normally in signal transduction (42). Since ITAM tyrosines are
typically found in different amino acid contexts, they could
potentially be substrates for different kinases and recruit different
SH2 domain-containing proteins to the phosphorylated receptor complex.
The potential biologic importance of differential ITAM tyrosyl
phosphorylation and effector recruitment was illustrated by the finding
that TCR antagonists and agonists induce different patterns of TCR-
phosphorylation (43, 44).
To further examine the function of ITAM tyrosines in CD79a and CD79b,
we constructed chimeric molecules containing the extracellular and
transmembrane domains of MHC class II I-Ak and the
cytoplasmic domain of either CD79a or CD79b. In these chimeras, CD79a
is linked to I-Ak (designated I-AIg), while CD79b is
linked to I-Akß (designated I-AIgß). Similar constructs
bearing mutations at either one or both ITAM tyrosyl residues were
generated (designated I-AIgF182,
I-AIgF193, and I-AIgF182/193 for a
mutation(s) in CD79a, and I-AIgßF195,
I-AIgßF206, and I-AIgßF195/206 for a
mutation(s) in CD79b). Analysis of signaling by these chimeras
indicates that ITAM tyrosines may not be absolutely required for
receptor-mediated tyrosine phosphorylation of Src family kinases, but
both are required for Syk phosphorylation. In vitro, phosphorylation of
tyrosyl residues in CD79a and CD79b was not symmetrical, with >80% of
phosphorylation occurring on the N-terminal tyrosyl residue. In vivo,
similar asymmetric phosphorylation of CD79a was observed. Most
strikingly, the presence of the N-terminal tyrosine of CD79a-ITAM was
sufficient to allow CD79a to mediate phosphorylation of a subset of
downstream substrates and Ca2+ mobilization. However, both
ITAM tyrosines were required for most CD79b function.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and I-AIgß chimeric molecules
To prepare constructs encoding I-AIg chimeric molecules, cDNA
encoding the CD79a(Ig) cytoplasmic domain flanked by the 5'
StuI site and the 3' EcoRI site was generated by
PCR using construct containing the cytoplasmic domain of Ig in a
pGEX vector (Pharmacia, Piscataway, NJ) as template (37). The PCR
products were cloned into pCRII using the TA cloning kit (Invitrogen,
San Diego, CA) and sequenced. The correct insert was subsequently used
to replace the cytoplasmic domain of I-Ak, creating a
chimeric cDNA encoding fusion of I-Ak and Ig. This
construct was then shuttled into the EcoRI and
BamHI sites of a modified mammalian expression vector
pneoSR (DNAX, Palo Alto, CA) containing the polylinker region of
pBluescript SK (W. A. Jensen, unpublished observations). To make
I-AIgß chimeric molecules, the CD79b(Igß) cytoplasmic domain
flanked by the 5' BamHI site and the 3' EcoRI
site was generated by PCR using a pGEX-Igß cytoplasmic domain
construct as template (37). The products were cloned into pCRII
(Invitrogen) and sequenced. The correct insert was subsequently cloned
into vector containing I-Akß cDNA mutated previously to
introduce a BamHI at the end of its juxtamembrane region,
replacing the I-Akß cytoplasmic domain. The I-AIgß
chimeric construct was then shuttled into mammalian expression vector,
pCEP4 (Invitrogen), via HindIII and XhoI sites.
The truncated I-Ak and I-Akß molecules
were made by cloning the EcoRV/StuI fragment of
I-Ak into modified pneoSR vector and the
HindIII/BamHI fragment of I-Akß
into pCEP4 vector, respectively. Chimeras containing either one or two
tyrosine to phenylalanine mutations in the ITAM of Ig and Igß were
made similarly; however, mutations were introduced by PCR using
oligonucleotides containing the desired mutations and was confirmed by
sequencing.
Generation of cell lines expressing I-AIg and I-AIgß chimeric
molecules
Chimeric cDNA constructs were transfected into
phosphorylcholine-specific IgM-, IgG2-, and
I-Ad-expressing B lymphoma, M12.4g3r (V. Parikh and P.
Tucker, University of Texas, Austin, TX) by electroporation. Briefly,
cells were washed and resuspended in IMDM at 2 x
108/ml. Cell suspension equivalent to 1 x
107 cells was added to an electroporation cuvette (Bio-Rad,
Richmond, CA), incubated with 2 µg of each I-AIg chimeric construct
and truncated I-Ak cDNA at 4°C for 5 min, and
electroporated at 320 V and 960 µFd. After chilling the
electroporated cells on ice for 5 min, they were cultured for 36 to
48 h before addition of G418 (Life Technologies, Grand Island, NY;
2 mg/ml) and hygromycin B (Calbiochem, La Jolla, CA; 1000 U/ml) to
select for transfectants. Colonies resistant to both G418 and
hygromycin were then screened for surface expression of
I-Ak by FACS analysis and sorted to enrich for the
I-Ak-expressing population and, if necessary, to isolate
cells expressing similar levels of chimeric receptor. Since
I-Ak molecules are usually expressed as and ß
heterodimers on the cell surface, I-Ak chimeras were
always cotransfected with truncated I-Akß and vice versa.
Surface staining of cells was performed with cells at 2.5 x
106/ml in PBS and 0.2% azide with a 1/200–500 dilution of
Ab (1 mg/ml) and incubated at 4°C for 30 min. Stained cells were
washed three times in PBS and 0.2% azide before further staining with
secondary reagent.
Analysis of Ca2+ mobilization
Cells were loaded with indo-1/AM at 10 µM/5 x
106 cells/ml in balanced salt solution at 37°C for
1 h. After extensive washing, cells were resuspended in IMDM
containing 3% FCS at 1 x 106/ml, and analysis was
initiated. After establishing the basal intracellular free calcium
concentration, cells were stimulated by addition of premixed 10 µg of
biotinylated 39J (mouse mAb specific for I-Ak) and 20
µg of avidin (Sigma Chemical Co., St. Louis, MO) at 37°C, and
monitoring of the intracellular free calcium concentration was resumed
after about 30 s. Ca2+ mobilization was detected by
flow cytometry using Ortho 50H (Johnson and Johnson, Westbrook, MA)
with data analysis by Mtime software (Phoenix Flow Systems, San Diego,
CA).
Immunoprecipitation and immunoblotting
Cells at 1 x 108/ml IMDM were mock
stimulated with PBS or stimulated with either 1 µg
F(ab')2 of rabbit anti-mouse Ig heavy and light chains
or 4 µg of 39J, a mouse mAb against I-Ak, per 1
x 106 cells at 37°C for 1.5 min. Cells were lysed in
lysis buffer containing 1% Nonidet P-40, 150 mM NaCl, 10 mM Tris-HCl
(pH 8.0), 2 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 0.4 mM
EDTA, 10 mM NaF, 1 mM PMSF, and 1 µg/ml each of aprotinin, leupeptin,
and 1-antitrypsin. Lysates were centrifuged for 5 min at 12,000
x g at 4°C to pellet nuclei and other particulates.
Cleared lysates were boiled with 2x reducing sample buffer for 5 min
and analyzed by 10% SDS-PAGE, or were subjected to immunoprecipitation
by incubation with specific Abs for 1 h at 4°C. Immune complexes
were isolated by adsorption to protein A- or protein G-Sepharose beads
(Pharmacia) for 30 min at 4°C. Beads were washed four times with
0.5% Nonidet P-40 lysis buffer and boiled in 2x reducing sample
buffer for 5 min. These samples were subsequently fractionated by 10%
SDS-PAGE followed by electrophoretic transfer to polyvinylidene
difluoride (PVDF) membranes. The immunoblots were blocked with 5% BSA
in Tris-buffered saline (TBS; 10 mM Tris (pH 8.0) and 150 mM NaCl) for
1 h at room temperature, then incubated with
anti-phosphotyrosine Ab Py54 (Oncogene Science) at a 1/500 dilution
for 1 h. After incubation, the membranes were washed four times,
alternating TBS and TBS plus 0.05% Triton X-100, and further incubated
with horseradish peroxidase-conjugated rat anti-mouse IgG1
(Zymed, San Francisco, CA) at a 1/500 dilution in 5% nonfat milk for
30 min at room temperature. The blots were then developed using an
enhanced chemiluminescence (ECL) kit (Amersham, Arlington Heights, IL)
according to the manufacturer’s instructions. To quantitate the amount
of proteins in the immunoprecipitates, blots were stripped of Abs by
incubation in stripping buffer at 56°C for 30 min as indicated in the
ECL kit and washed extensively with TBS or TBS containing 0.05% Triton
X-100, alternatively. These membranes were then processed as before and
probed with specific Abs as indicated.
In vitro mapping of CD79a or CD79b sites of tyrosine phosphorylation
CD79a(Ig) or CD79b(Igß) ITAM-GST (wild-type or various Y to
F mutated) constructions were generated, and fusion proteins were
produced as previously described (38). Following extensive washing with
1% Nonidet P-40 lysis buffer, 25 µl of fusion protein-saturated
glutathione Sepharose (Pharmacia) adsorbates were washed twice with in
vitro kinase buffer (10 mM HEPES (pH 7.0), 10 mM
MgCl2, 2 mM sodium orthovanadate, and 1 mM PMSF),
resuspended in 100 µl of kinase buffer containing
baculovirus-produced Fyn or Lyn PTK (40) and 10 µCi of
[-32P]ATP (New England Nuclear-DuPont, Boston, MA),
and incubated overnight at room temperature with constant mixing.
Phosphorylation reactions were terminated by washing followed by
addition of reducing Lammlei sample buffer. Proteins were fractionated
by 10% SDS-PAGE, electrophoretically transferred to a PVDF membrane
(New England Nuclear-DuPont), and detected by autoradiography. Sites of
phosphorylation were determined as previously described (45). Briefly,
32P-labeled proteins prepared as described above, were
excised from the PVDF membrane, soaked in 0.5% PVP-360 (Sigma) in 100
mM acetic acid for 30 min at 37°C, and washed several times with
H2O followed by 0.05 M NH4HCO3.
Washed membrane slices were incubated overnight at 37°C with 20 µg
of thermolysin (Boehringer Mannheim, Indianapolis, IN), added in two
10-µg aliquots 6 h apart, in 500 µl of 0.05 M
NH4HCO3. The digestion was terminated with the
addition of 500 µl of H2O. Cleaved peptides were dried
completely in a Speed-Vac (Savant, Farmingdale, NY) and oxidized with
performic acid. Total yield was estimated by Cerenkov counting. Samples
were resuspended in a small volume of pH 1.9 buffer (2.5% formic acid
(88%) and 7.8% glacial acetic acid in deionized H2O), and
2500 counts of each was loaded on individual thin layer cellulose
plates (Merck-EM Science, Gibstown, N.J.). The first dimension
(electrophoretic separation) was run at 1.8 kV for 20 min in pH 3.5
buffer (0.05% glacial acetic acid and 0.5% pyridine in deionized
H2O). The second dimension (chromatographic separation) was
run for 16 h in isobutyric acid chromatography buffer (62.5%
isobutyric acid, 1.9% n-butanol, 4.8% pyridine, and 2.9%
glacial acetic acid in deionized H2O) for CD79a and phospho
chromatography buffer (37.5% n-butanol, 25.0% pyridine,
and 7.5% glacial acetic acid in deionized H2O) for CD79b.
Separated 32P-labeled peptides were detected by
autoradiography. Maps generated from fusion proteins containing various
tyrosine to phenylalanine mutations were compared with those generated
from wild-type ITAMs. Based on these comparisons,
32P-labeled spots were assigned as being derived from
specific tyrosines.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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wt and I-AIgßwt chimeric
receptors are competent to mediate signal transduction
Preliminary to analysis of the function of ITAM tyrosyl residues,
we compared the ability of wild-type CD79a and CD79b to transduce
signals when expressed as single chain receptors. Chimeric molecules
composed of extracellular and transmembrane regions of MHC class II
molecule I-A and I-Aß chains directly fused to the cytoplasmic
domains of CD79a and CD79b, respectively, were generated (Fig. 1
). Since MHC class II molecules are
expressed as heterodimers at the cell surface, cells were transfected
with truncated I-Ak and I-Akß,
lacking their cytoplasmic tails (designated I-Atr),
chimeric molecule I-AIg paired with tail-less I-Akß
(designated I-AIgwt), or chimeric molecule I-AIgß
paired with tail-less I-Ak (designated
I-AIgßwt). After drug selection, cell lines expressing
similar levels of chimeric receptors were isolated by FACS. Polyclonal
lines were used for experimentation rather than clones to avoid
potential artifacts resulting from clonal variation. Shown in Figure 2 is a FACS analysis illustrating
expression of the transfected molecules. Both wild-type CD79a and CD79b
containing chimeric receptors were competent to mediate signal
transduction as judged by Ca2+ mobilization (Fig. 3). Since aggregation of either
I-Atr or endogenous I-Ad failed to elicit this
response, cytoplasmic domains of CD79a and CD79b, but not extracellular
or transmembrane domains of MHC class II molecules, are responsible for
the signaling property of the chimeras. These data are in accordance
with previous studies that have demonstrated the ability of CD79a and
CD79b cytoplasmic domains to mediate signal transduction (27, 28, 29, 30, 31).
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mAb, 39J, and lysed in Nonidet P-40 lysis
buffer. Protein tyrosyl phosphorylation of molecules in whole cell
lysates or specific immunoprecipitated effectors was revealed by
anti-phosphotyrosine immunoblotting. As shown, ligation of the
chimeric I-AIgwt or I-AIgßwt resulted in
an increase in tyrosine phosphorylation of cellular proteins (Fig. 4). These receptors were also capable of
mediating tyrosine phosphorylation of Src family kinases Lyn, Fyn, and
Blk and Syk kinase (Fig. 5; data not
shown). In parallel experiments it was shown that both chimeras also
mediated phosphorylation of Shc and Vav (data not shown). Since
I-Atr molecules were unable to mediate detectable protein
tyrosyl phosphorylation, including the aforementioned signaling
effectors, the observed signaling functions must reside within the
cytoplasmic domains of CD79a and CD79b.
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). Cross-linking of the
chimeras led to their phosphorylation in a manner similar to that of
mIg-associated CD79a-CD79b heterodimers upon BCR ligation.
Surprisingly, mIg ligation led to some phosphorylation of chimeric
receptors, but the inverse was not seen. This may be because the
chimeric receptors are expressed at a higher copy number than
endogenous BCR; thus, more of this substrate is available for
BCR-activated kinases. Regardless, based on the parameters examined
here, CD79a and CD79b appear equivalent in their ability to transduce
signals. CD79a-ITAM tyrosines have distinct signaling functions
To determine the signaling function of each ITAM tyrosine in
CD79a, chimeric molecules with phenylalanine substitution at the
N-terminal ITAM tyrosine (F182), C-terminal ITAM tyrosine (F193), or
both ITAM tyrosines (F182/193) were generated and expressed along with
tail-less I-Aß in M12.4g3r (Fig. 1). Lines expressing chimeric
I-AIgF182, I-AIgF193, or
I-AIgF182/193 molecules at their surface were isolated
(Fig. 2), and the signal transducing capability of these chimeras was
assessed. As shown in Figure 3, while mutation of Y182 led to a
complete disruption of signaling leading to Ca2+
mobilization, F193 mutation still permitted a partial response. The
observed defect in signaling is not intrinsic to the cells, since
aggregation of endogenous BCR resulted in a robust Ca2+
mobilization. The ability of these chimeras to mediate tyrosyl
phosphorylation of cellular proteins was also determined (Fig. 6). Similar to the Ca2+
response, mutating Y182 prevented the signaling required for detectable
tyrosyl phosphorylation of virtually all cellular proteins. In
contrast, mutation of Y193 eliminated phosphorylation of some
substrates but not others (e.g., Vav; data not shown). Notably, an
unidentified 62-kDa substrate was more strongly phosphorylated in the
mutant, while a prominent substrate seen at 68 kDa with
IA-Igwt was not phosphorylated. Thus, the second
tyrosine in the ITAM of CD79a is not absolutely required for
receptor-mediated protein kinase activation. For optimal
Ca2+ mobilization, both tyrosines are essential.
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detectable tyrosyl phosphorylation of Lyn appeared independent of ITAM
tyrosines, since ligation of chimeras bearing either single or no ITAM
tyrosines still resulted in kinase phosphorylation. Fyn and Blk behaved
equivalently (data not shown). This suggests a mode of Src family
kinase activation that is dependent on receptor ligation but
independent of ITAM phosphorylation. Apparently, however,
phosphorylation of substrates (other than ITAMs) requires ITAM
tyrosines. Perhaps ITAM tyrosines must be phosphorylated to further
activate Src family kinases or to recruit substrates that can
then be phosphorylated. In contrast, mutating just one ITAM tyrosine to
phenylalanine disrupted the ability of the chimeras to
mediate Syk tyrosine phosphorylation (Fig. 7). This finding is
consistent with previous studies indicating that both SH2 domains are
required for Syk family kinases to bind doubly phosphorylated ITAMs and
to function in signaling (46, 47, 48).
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). While I-AIgF182 was barely
phosphorylated, aggregation of the I-AIgF193
chimera resulted in robust phosphorylation. As expected, no tyrosyl
phosphorylation of I-AIgF182/193 chimera could be
detected. These data are consistent with the relative tyrosyl
phosphorylation mediated by the chimeras and seen in whole cell lysates
(Fig. 6). These data suggest that in vivo, CD79a is
primarily phosphorylated at the N-terminal ITAM tyrosyl
residue, and this phosphotyrosine is likely to mediate the further
activation of the kinases that phosphorylate other cellular substrates
or mediate recruitment of substrates to the already active kinase.
Since both tyrosines are reportedly necessary for the ITAM-mediated
biologic responses that have been studied, it seems likely that both
tyrosyl residues are phosphorylated in physiologic situations.
Possibly, phosphorylation of Y193 may require preliminary
phosphorylation of Y182, and for this reason, it is not seen in this
model. To begin to address this possibility, we analyzed the tyrosyl
specificity of ITAM phosphorylation in vitro (Fig. 8). CD79a cytoplasmic tails expressed as
GST fusion proteins were phosphorylated with [32P]ATP
using baculovirus-expressed Lyn, Fyn, or Syk, and then analyzed by
phosphopeptide mapping. While Syk failed to phosphorylate the fusion
proteins (data not shown), both Lyn and Fyn phosphorylated the CD79a
cytoplasmic portion of the fusion proteins well, with >80% of
phosphorylation occurring at Y182 (Fig. 8). These data further support
the idea that Y182 is the primary site of CD79a phosphorylation by Src
family kinases in vivo, and thus most phosphorylated ITAMs can only
recruit effectors that can bind effectively via a single SH2
domain.
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To assess the function of CD79b-ITAM tyrosyl residues, cell lines
expressing I-AIgß chimeras bearing various tyrosine mutation(s),
namely I-AIgßF195, I-AIgßF206, and
I-AIgßF195/206, were created (Figs. 1 and 2), and
aggregation-induced phosphorylation and function of individual CD79b
tyrosyl residues were examined. Analysis of Ca2+
mobilization mediated by these receptors revealed that single mutation
of either tyrosine in CD79b has more severe functional consequences
than in CD79a, as no response could be observed upon aggregation of
either I-AIgßF195 or I-AIgßF206 receptors
(Fig. 3). In addition, neither single tyrosine mutant (nor the double
tyrosine mutant) appeared competent to mediate tyrosyl phosphorylation
of most proteins, including Shc and Vav (Fig. 9; data not shown).
I-AIgßF195 mediated no signal, and very weak, but
significant, phosphorylation of 62kDa and substrates of approximately
110 kDa was seen in I-AIgßF206.
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). Although mutated I-AIgß chimeras
failed to elicit detectable Ca2+ mobilization and most
protein tyrosyl phosphorylation, Src family kinases were nevertheless
tyrosyl phosphorylated upon receptor aggregation. Similar to that
mediated by I-AIg, I-AIgß-mediated Syk tyrosyl phosphorylation
required both ITAM tyrosines. Thus, for both I-AIg and I-AIgß
chimeras, distinct tyrosine requirements for Src and Syk family kinase
phosphorylation were observed. This implies that CD79a and CD79b ITAMs
may mediate activation of the respective kinases via common
mechanisms.
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I-AIgßwt chimera migrates more slowly than the mutants,
whose mobility is the same as that of endogenous Igß. The structural
basis of this differential mobility is unknown. Regardless, the results
indicate that phosphorylation of I-AIgß chimeras is largely dependent
on the presence of both tyrosines, since no phosphorylation of
I-AIgßF195 was seen, and phosphorylation of
I-AIgßF206 was only barely detectable. This result again
distinguishes I-AIgß from the I-AIg chimeras, in that the
C-terminal ITAM tyrosine mutation in the latter did not prevent robust
chimera tyrosyl phosphorylation, indicating that phosphorylation of
CD79a-and CD79-ITAMs is differently regulated. Y182 of CD79a may be
more efficiently phosphorylated and functional, simply because, as
predicted by Schmitz et al. (41), it is a better substrate for Lyn
(and Blk).
Interestingly, when CD79b cytoplasmic tail-containing GST fusion
proteins were phosphorylated in vitro by baculovirus-produced Fyn,
>80% of phosphorylation occurred on the N-terminal ITAM tyrosine, as
in CD79a fusion proteins (Fig. 8). Thus, the CD79b-ITAM can serve as a
substrate for Src family kinases, and the N-terminal tyrosine is
preferred.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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- or ß-chain of MHC class II
molecules, we analyzed various molecular signaling functions of CD79a
and CD79b with regard to the occurrence and functional consequences of
ITAM tyrosyl phosphorylation. The results indicate that in both CD79a
and CD79b, ITAM tyrosines are asymmetrically phosphorylated during BCR
signal transduction. The N-terminal tyrosine appears to be the
preferred site of phosphorylation of both chains. Consistent with its
preferred phosphorylation, Y182 in the CD79a-ITAM appears to be
particularly important in terms of function. Phenylalanine substitution
at this position effectively abolished all detectable tyrosyl
phosphorylation of the receptor and other substrates, except Src family
kinases, and prevented Ca2+ mobilization mediated by
the chimeric receptor. Mutating Y193 had a distinct effect on
phosphorylation and function. Phosphorylation of certain substrates,
including p62 and the receptor itself, was seen, and the
Ca2+ mobilization response was only partially disrupted.
Similar differences between the respective tyrosine mutants were
observed with CD79b; however, the effect of mutating the C-terminal
tyrosine was more severe, further quantitatively reducing p62 and
receptor phosphorylation and preventing Ca2+ mobilization.
For both CD79a and CD79b, neither tyrosyl residue was required for Src
family kinase phosphorylation, but both were required for Syk
phosphorylation. These findings lead to several conclusions regarding
molecular mechanisms in BCR signaling. One of the important conclusions of this work relates to the role of ITAM phosphorylation in kinase activation. Detectable (but generally not maximal) phosphorylation of Src family kinases appeared to not depend absolutely on either conserved ITAM tyrosine, supporting the possibility that these kinases are partially activated by aggregation of receptors to which they are tethered by virtue of kinase-unique region interactions with nonphosphorylated ITAMs (39, 49). The results described here also indicate that phosphorylation of ITAMs, at least Y182, may drive further activation of Src family kinases that now phosphorylate downstream targets. Alternatively, or more likely additionally, phosphorylated ITAMs may focus substrates to the already active kinase, which then phosphorylates them. Phosphorylation of both ITAM tyrosyl residues is required for Syk phosphorylation, presumably reflecting a previously demonstrated requirement that both SH2 domains must be functional for Syk to act in BCR signaling (50). It is noteworthy that binding to biphosphorylated ITAMs reportedly activates Syk (47, 48).
As supported by the findings reported here and from other studies, initial receptor phosphorylation is most likely mediated by Src family kinases. Analyses of Lck-deficient variants of Jurkat cell line (51, 52) and studies of COS cells reconstituted with receptor components and kinases (33) both indicate the necessity of Src family kinase activity in ITAM phosphorylation. Consistent with this, phosphorylation of Src family kinases was independent of CD79a and CD79b ITAM tyrosyl residues in the model presented here as well as in another model where CD79b was expressed in T cells (53). Thus, by virtue of their association with the resting receptors (37, 39, 49), Src family kinases probably subserve the role of endogenous kinase activity in growth factor receptors, functioning to quickly phosphorylate receptor tyrosines upon BCR aggregation, resulting in recruitment of other effectors (37, 54).
Y182 of CD79a appears to be the initial and preferred site of Ag
receptor phosphorylation by Src family kinases. In vitro, Src family
Lyn and Fyn predominantly phosphorylate this residue in CD79a, and Y195
does so in CD79 (35) (this study). Examination of phosphorylation of
I-AIg chimeras (this study) and that of CD79a-CD79b heterodimers
expressed as a part of BCR complex in J558L cells (35) also suggests
the same substrate preference in vivo. However, the possibility that
the presence of Y183 is somehow necessary for phosphorylation of Y193
in vivo cannot be excluded. This possibility is excluded in vitro,
since both tyrosil residues are present in the substrate. Other
features of nonphosphorylated CD79a ITAM support the idea that Y182 may
be the preferred substrate of Src family kinases. First, CD79a appears
to have higher affinity for Src family kinases than CD79b (37).
Secondly, analysis of kinase substrate specificity using phage display
has revealed that Src family kinases Blk and Lyn prefer substrate
peptides with sequence I/L-Y-D/E-x-L (41). This consensus is found in
Y182 of CD79a, in the context of L-Y182-E-G-L, but not in CD79b. Thus,
Src family kinases are selectively tethered to CD79a, where their
optimal substrate sequence is located. Nonetheless, until
phosphorylation of CD79a and CD79b is mapped in wild-type receptors,
the relative phosphorylation of various ITAM tyrosil residues in
physiologic settings cannot be firmly established.
Phosphorylation of Y182 in CD79a appears to play a critical role in signal propagation. As reported here, phosphorylation of Y182 alone can lead to further kinase activation and/or effector focusing necessary for phosphorylation of certain downstream targets, such as p62, p110, and Shc, but not others, such as Vav. As discussed earlier, phosphorylation of both tyrosines in a single ITAM seems necessary for Syk activation and full receptor function. Activation of Syk may be mediated solely as a consequence of tandem SH2 binding to a biphosphorylated ITAM, by Src family kinase-mediated phosphorylation of Syk, or by both (48, 55).
One of the surprising findings reported here is that activation of
Ca2+ mobilization can be demonstrated under conditions in
which Syk activation should not and apparently does not occur. Syk
kinases are thought to phosphorylate and activate PLC, as they
engage the SH2 domain of the lipase (56, 57). Consistent with this
possibility, PLC2 phosphorylation was not observed in the absence of
detectable Syk activation (data not shown). However, cross-linking of
I-AIgF193 resulted in detectable Ca2+
mobilization despite the absence of detectable Syk phosphorylation.
Perhaps a Syk- and PLC-independent Ca2+ mobilization
pathway can be activated by I-AIgF193 chimeras. However,
partial Syk activation as a result of interaction with two
trans-occurring phosphotyrosyl residues in aggregated
I-AIgF193 receptors cannot be excluded.
Finally, the results described here support the possibility that BCR phosphorylation is an ordered process involving initial phosphorylation of CD79a-Y182 followed, perhaps, by CD79b-Y195 and then the C-terminal tyrosines. The extent to which the sequence is completed may depend, for example, on the duration of receptor occupancy, more specifically on the Ag off-rate. The higher the off-rate, the more limited the progression through the phosphorylation program and, as a consequence, the more limited the spectra of effectors activated. Such a mechanism may provide a molecular basis by which cells make distinct biologic responses to high and low affinity Ags. Further studies using heterodimeric chimeras and ligands with varying affinities will be required to address this hypothesis.
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Footnotes |
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2 Abbreviations used in this paper: BCR, B cell Ag receptor; mIg, membrane Ig; PTK, protein tyrosine kinase; PLC, phospholipase C; ITAM, immunoreceptor tyrosine-based activation motif; IMDM, Iscove’s modified Dulbecco’s medium; PVDF, polyvinylidene difluoride; TBS, Tris-buffered saline; GST, glutathione-S-transferase.
Received for publication October 14, 1997. Accepted for publication December 9, 1997.
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, 187.1
(at 5 x 105 cells in 200 µl of PBS and azide with a
1/200 dilution of the Ab), followed by streptavidin-FITC (1/200
dilution). Endogenous receptor expression was determined by
anti-Ig
F) were phosphorylated in vitro with
baculovirus-produced Fyn, fractionated by SDS-PAGE, electrophoretically
transferred to PVDF membrane, and cleaved with thermolysin, and sites
of phosphorylation were analyzed by two-dimensional TLC peptide
mapping. A, Autoradiogram of in vitro phosphorylated GST
fusion proteins fractionated by SDS-PAGE. B, Representative
TLC peptide maps each loaded with 2500 counts of
32P-labeled and thermolysin-cleaved WT CD79a ITAM or Y
