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1 Is Essential for TCR-Induced Tyrosine Phosphorylation of Phospholipase C1
Laboratory of Immunobiology, Division of Monoclonal Antibodies, OTRR, Center for Biologics Evaluation and Research, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1 (PLC1) via a
tyrosine phosphorylation-dependent mechanism. PLC1 contains a pair
of Src homology 2 (SH2) domains whose function is that of promoting
protein interactions by binding phosphorylated tyrosine and adjacent
amino acids. The role of the PLC1 SH2 domains in PLC1
phosphorylation was explored by mutational analysis of an
epitope-tagged protein transiently expressed in Jurkat T cells.
Mutation of the amino-terminal SH2 domain (SH2(N) domain) resulted in
defective tyrosine phosphorylation of PLC1 in response to TCR/CD3
perturbation. In addition, the PLC1 SH2(N) domain mutant failed to
associate with Grb2 and a 36- to 38-kDa phosphoprotein (p36–38), which
has previously been recognized to interact with PLC1, Grb2, and
other molecules involved in TCR signal transduction. Conversely,
mutation of the carboxyl-terminal SH2 domain (SH2(C) domain) did not
affect TCR-induced tyrosine phosphorylation of PLC1. Furthermore,
binding of p36–38 to PLC1 was not abrogated by mutations of the
SH2(C) domain. In contrast to TCR/CD3 ligation, treatment of cells with
pervanadate induced tyrosine phosphorylation of either PLC1 SH2(N)
or SH2(C) domain mutants to a level comparable with that of the
wild-type protein, indicating that pervanadate treatment induces an
alternate mechanism of PLC1 phosphorylation. These data indicate
that the SH2(N) domain is required for TCR-induced PLC1
phosphorylation, presumably by participating in the formation of a
complex that promotes the association of PLC1 with a tyrosine
kinase. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1
(PLC1),4 a member of the
phosphoinositide-specific phospholipase C family, catalyzes the
hydrolysis of phosphatidylinositol 4,5-bisphosphate to the second
messengers, inositol 1,4,5-trisphosphate and 1,2-diacylglycerol (1, 2).
Inositol 1,4,5-trisphosphate initiates intracellular Ca2+
mobilization and 1,2-diacylglycerol activates protein kinase C (2).
Together, these messengers coordinate the expression of cellular
responses in several different biologic systems, including during T
lymphocyte activation (2). Tyrosine phosphorylation of PLC1 is
required for its activation (3, 4, 5, 6). Furthermore,
tyrosine-phosphorylated PLC1 displays increased enzymatic activity
in vitro (7) and could utilize profilin-bound phosphatidylinositol
4,5-bisphosphate as substrate (8).
PLC1 contains a pair of Src homology 2 (SH2) domains (9, 10). SH2
domains are structurally and functionally conserved protein modules
that promote protein interactions by binding phosphorylated tyrosine
and adjacent amino acids (11, 12, 13). Formal proof for the role of the
PLC1 SH2 domains in PLC1 activation has not been obtained.
Indirect experimental evidence, however, suggests a mechanism of
PLC1 activation by tyrosine kinase growth factor receptors that
involves recruitment of PLC1, a cytoplasmic protein, to the membrane
via binding of its SH2 domains to the autophosphorylated intracellular
tails of the receptor. This evidence includes the binding of PLC1 or
its SH2 domains to specific sites of phosphorylation of the
platelet-derived growth factor receptor (14, 15), the epidermal growth
factor receptor (16, 17, 18), or the fibroblast growth factor receptor
(19). Furthermore, PLC1 fails to bind to either the platelet-derived
growth factor receptor or the epidermal growth factor receptor with
mutations in the tyrosine residues critical for SH2 domain
interaction (15, 20).
Ag engagement or Ab-mediated perturbation of the TCR/CD3 complex
induces tyrosine phosphorylation of PLC1 in T lymphocytes (21, 22).
Induction of tyrosine kinase activity precedes PLC1 activation (23)
and tyrosine kinase inhibitors block TCR/CD3-induced inositol
phospholipid hydrolysis and Ca2+ mobilization (24). While
no component of the TCR/CD3 complex itself possesses enzymatic
activity, perturbation of the TCR/CD3 complex induces activation of
tyrosine kinases of the Src family, Fyn and Lck, and a T cell-specific
kinase, ZAP-70 (22). PLC1 has been found in complexes with the CD3
chains (25), kinases of the Src family (26, 27), and ZAP-70 (28). The
association of PLC1 with these proteins may be either direct or
mediated by adapter proteins, which lack enzymatic activity but mediate
the coupling of signaling proteins. PLC1 has been shown to interact
with a 62-kDa phosphorylated adapter, which was also found in
association with the Ras GTPase-activating protein (29). PLC1 can
also interact with p76 Slp (30) and with a yet unidentified
tyrosine-phosphorylated protein of 36 to 38 kDa (p36–38) (31). A
complex of PLC1, p36–38, and Grb2, another adapter, have also been
observed in activated T cells (28, 31, 32, 33, 34), suggesting a potential role
for Grb2 in PLC1 regulation, in addition to its well documented
function in the activation of Ras (35). Despite evidence of PLC1
interaction with numerous signaling proteins, an understanding of the
mechanism by which PLC1 is phosphorylated and activated after TCR
engagement is still lacking.
The interaction of the SH2 domains of PLC1 with
tyrosine-phosphorylated proteins is likely to play an important role in
assembling the initial complex that leads to PLC1 phosphorylation
and activation. To characterize the structural requirements for the
activation of PLC1 in T lymphocytes, we have performed a mutational
analysis of the SH2 domains in an epitope-tagged PLC1 protein
transiently expressed in Jurkat cells. As a first step, we studied the
role of the SH2 domains in PLC1 tyrosine phosphorylation induced by
TCR/CD3 perturbation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The anti-CD3 mAb (Ab), OKT3, was a gift from Ortho Biotech (Raritan, NJ). F(ab')2 fragments of OKT3 were prepared using immobilized pepsin (Pierce, Rockford, IL) followed by protein A column chromatography (Pierce). The anti-hemagglutinin (HA) mAb, 12CA5, was from Boehringer Manneheim (Indianapolis, IN). The anti-phosphotyrosine Ab, 4G10, was from Upstate Biotechnology (Lake Placid, NY). Goat affinity-purified Ab to mouse IgG F(ab')2 was from Cappel (West Chester, PA).
Plasmids
Bovine PLC1 cDNA was obtained from Dr. John Knopf (Genetics
Institute, Cambridge, MA). The cDNA was excised from the pTM-2 vector
and cloned into the XbaI site of pBluescript II-SK (pBluSK,
Stratagene, La Jolla, CA) to obtain pBluSK-PLC1, as well as into the
XbaI site of the expression vector, pCIneo (Promega,
Madison, WI), to obtain pCIneo-PLC1. A sequence encoding three
repeats of the HA epitope, Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala, was
added at the 3' end by PCR to obtain pCIneo-PLC1-HA. Mutations of
arginine in position 586 to lysine (R586K) within the SH2(N) domain or
of arginine in position 696 and/or 694 to lysine within the SH2(C)
domain (R694/6K and R694K, respectively) were introduced by PCR using
pBluSK-PLC1 as template, to obtain
pBluSK-PLC1[SH2(N)R586K],
pBluSK-PLC1[SH2(C)R694K],
pBluSK-PLC1[SH2(C)R694/6K], and
pBluSK-PLC1[SH2(N/C)R586/94/6K]. A
SacII/EcoRV fragment containing the mutations was
cloned into pCIneo-PLC1-HA, to obtain the corresponding mutated
pCIneo-PLC1-HA constructs. Fidelity of the PCR-amplified fragments
was verified by DNA sequencing.
Fusion proteins
The prokaryotic vector encoding the glutathione
S-transferase (GST) fusion protein, GST-Grb2, was a gift
from Dr. P. G. Pelicci (EOI, Milan, Italy) (36). GST-SH2(N) of
PLC1 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
GST-SH2(N)R586K and GST-SH2(C)R694/6K of
PLC1 were prepared by PCR using
pBluSK-PLC1[SH2(N)R586K] and
pBluSK-PLC1[SH2(C)R694/6K], respectively, as templates
and cloned into the pGEX-4T3 (Pharmacia Biotech, Piscataway, N). The
fusion proteins were obtained from lysed bacteria and purified on
glutathione-Sepharose beads (Pharmacia Biotech) according to the
manufacturer’s instructions.
Transient transfection and cell activation protocols
Jurkat T cells were maintained in RPMI 1640 medium with 7.5% FBS. Transfections were performed by electroporating (960 µF capacitance and 250 V) 107 cells (grown to log phase) in 200 µl of medium containing 20 µg of plasmid DNA (37). Transfected cells were cultured at 0.5 x 106/ml for 48 h. Before experimental use, transfected cells were treated with 0.1 mg/ml DNase (Sigma, St. Louis, MO) followed by removal of nonviable cells on Ficoll gradients.
In selected experiments, Jurkat cells were transfected with the
indicated PLC1-HA constructs together with 2 µg of pGreen
Lantern-1 (Life Technologies, Grand Island, NY), a plasmid DNA that
contains a mutated form of the gene coding for the green fluorescent
protein from Aequorea victoria (38). Transfected cells were
cultured for 24 h, treated with DNase, and then enriched for the
expression of the gene of interest by fluorescence activated cell
sorting of green fluorescent protein-expressing cells on a
FACStarPlus cell sorter equipped with an argon laser at
4880 Å (Becton Dickinson, Mountain View, CA). Sorted cells were
cultured overnight before experimental use.
For stimulation, 107 cells were coated with 10 µg OKT3 F(ab')2 for 15 min on ice, washed, and activated with 25 µg of affinity-purified goat Ab to mouse IgG F(ab')2 for the indicated times at 37°C. Alternatively, cells were activated with pervanadate (0.1 mM sodium orthovanadate and 0.3 mM hydrogen peroxide).
Precipitation and Western blot analysis
For protein analysis, cells were lysed in 60 mM Tris-HCl, pH 7.8, containing 150 mM NaCl, 5 mM EDTA, 10% glycerol, 2 mM Na3VO4, 25 mM NaF, 10 µg/ml leupeptin (Sigma), 10 µg/ml aprotinin (Sigma), 1 mM AEBSF (Sigma), 1 mM N-p-tosyl-L-lysine chloromethyl ketone (Sigma), 1 mM N-p-tosyl-L-phenylalanine chloromethyl ketone (Sigma), and 1% Triton X-100 (Calbiochem, La Jolla, CA). Cleared lysates were precipitated with either specific Ab prebound on protein A trisacryl beads (Pierce), or GST fusion proteins (100–200 pmol) bound to glutathione-Sepharose beads (Pharmacia Biotech). Washed precipitates were eluted with reducing Laemmli sample buffer, resolved by SDS-PAGE, and transferred to nitrocellulose membranes (Hybond-C super, Amersham, Arlington Heights, IL). Proteins were detected by primary Ab with or without a secondary Ab, followed by 125I-protein A (ICN, Costa Mesa, CA). When indicated, blots were reprobed after stripping at 60°C for 30 min in 50 mM sodium phosphate (pH 6.5), 10 M urea, and 100 mM 2-ME. Immunoblots were scanned on a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) and densitometry performed by ImageQuant software (Molecular Dynamics) using volume integration with background subtraction (local perimeter average). Photographic representations of the blots are PhosphorImager-generated computer images with no manipulation, except for the adjustment of the exposure range.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 GST-SH2 domain fusion proteins to p36–38 and
other phosphoproteins is abrogated by mutation of critical arginine
residues
We began our analysis of the function of the SH2 domains of
PLC1 by analyzing the proteins that bound to each isolated SH2
domain and assessing the effect of mutations of critical residues
within the binding pocket of each domain. This was accomplished by
generating GST fusion proteins containing the individual SH2 domains
and by creating mutants in which the arginine residue in the conserved
Phe-Leu-Val-Arg motif (13) of either domain (position 586 of the SH2(N)
domain and 694 of the SH2(C) domain) was mutated to lysine. In v-Src
SH2 domain, this conserved residue was shown to form an ion pair
through hydrogen bonding with the phosphate of the phosphorylated
tyrosine (39). A peculiarity of the PLC1 SH2(C) domain is the
presence of a second arginine in position 696. This residue, not
conserved in the SH2(N) domain, can interact with both the phosphate
and the aromatic ring of the phosphotyrosine (40). Because of this
structural feature, Arg694 and Arg696 may
potentially compensate for each other in binding phosphorylated
proteins. Therefore, a double mutant of Arg694 and
Arg696 was engineered for the GST-SH2(C) domain
construct.
The immobilized GST fusion proteins were used to precipitate
tyrosine-phosphorylated proteins from control (unstimulated) or
activated Jurkat cells. No tyrosine-phosphorylated proteins from
control cells bound any of the fusion proteins (Fig. 1
). From activated cells, the only
phosphoprotein that could be detected binding the GST-SH2(N) domain
fusion protein was p36–38, while the GST-SH2(C) pattern of GST-SH2
domain-binding proteins observed in Jurkat cells was consistent with
that previously reported for a murine T cell hybridoma transfected with
activated Lck (41). The GST-SH2(N)R586K or
GST-SH2(C)R694/6K fusion proteins failed to bind
phosphorylated proteins from activated Jurkat cells, confirming that
the amino acid substitutions abrogated the phosphotyrosine-binding
ability of either PLC1 SH2 domain.
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1
To explore the role of the SH2 domains in TCR-induced PLC1
activation, mutations of Arg586 of the SH2(N) domain as
well as of Arg694 or Arg694/Arg696
of the SH2(C) domain to lysine were introduced into an HA-tagged
PLC1 construct to create mutants that were transiently expressed in
Jurkat cells. We next established the levels of TCR-induced tyrosine
phosphorylation of WT and mutant PLC1. The phosphorylation kinetics
of transiently expressed PLC1-HA in response to TCR/CD3 ligation
closely matched that of endogenous PLC1 (data not shown), with a
maximum observed after 2 min followed by a decline, indicating that the
transfected and endogenous proteins behave similarly with respect to
immediate phosphorylation events. Hence, a 2-min time point was
selected for subsequent experiments.
Compared with the WT protein, the SH2(N) mutant
(PLC1[SH2(N)R586K]-HA) demonstrated substantially
reduced levels of tyrosine phosphorylation (Fig. 2). The level of phosphorylation of the
SH2(N) domain mutant was 17 ± 12% SD of that of the WT protein
(p = 0.0012 by two-tailed t test
comparison of PLC1[SH2(N)R586K]-HA with WT PLC1-HA,
adjusted for multiple comparisons), as determined by ImageQuant
densitometry of PhosphorImager scans of anti-phosphotyrosine
Western blots from five independent experiments, adjusted by the
corresponding levels of PLC1-HA of stripped blots reprobed with
anti-HA, and internally normalized as percentage of
TCR-induced phosphorylation of WT PLC1-HA. In contrast,
mutations of the SH2(C) domain had virtually no effect on
TCR/CD3-induced tyrosine phosphorylation of PLC1-HA (Fig. 2). No
difference in phosphorylation levels was observed between WT PLC1-HA
and either the single Arg694 or the double
Arg694/Arg696 SH2(C) domain mutants
(PLC1[SH2(C)R694K]-HA and
PLC1[SH2(C)R694/6K]-HA, respectively), effectively
excluding the possibility that the carboxyl-terminal Arg696
of the SH2(C) domain could compensate for the lack of effect observed
with the single Arg694 mutant. A PLC1 construct with
mutations of both the SH2(N) and SH2(C) domains
(PLC1[SH2(N/C)R586/694/6K]-HA) showed a reduced level
of phosphorylation in response to TCR/CD3 engagement and behaved
indistinguishably from the SH2(N) mutant,
PLC1[SH2(N)R586K].
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1[SH2(N)R586K]-HA mutant was not due to a delayed
response, since the modest level of phosphorylation paralleled that of
WT PLC1-HA over time, with a maximum still observed after 2 min of
activation (Fig. 3). The time course of
TCR-induced phosphorylation of PLC1[SH2(C)R694/6K]-HA
also did not differ from that of the WT protein (data not shown),
indicating that mutation of the SH2(C) domain was not associated with
changes in the kinetics of PLC1 phosphorylation.
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1 induced by treatment with
pervanadate does not require the function of the SH2 domains
Pervanadate is a pharmacologic agent commonly used to bypass
receptor/ligand interaction, which induces PLC1 phosphorylation and
activation in T lymphocytes (42). It was therefore of interest to
determine whether PLC1 phosphorylation induced by pervanadate
treatment showed structural requirements similar to those shown for
TCR/CD3 ligation.
The level of tyrosine phosphorylation of WT PLC1-HA induced by
pervanadate treatment was approximately twice (203 ± 82% SD,
n = 4) that induced by TCR/CD3 ligation (Fig. 4). Contrary to the defective
phosphorylation levels observed with TCR/CD3 perturbation, pervanadate
treatment induced the same level of phosphorylation of the SH2(N)
domain mutant (184 ± 46% SD, n = 4) as that of
the WT protein. Similar to what was seen with TCR stimulation, mutation
of the conserved Arg694 or the double mutation of
Arg694 and Arg696 of the SH2(C) domain resulted
in no significant difference in tyrosine phosphorylation of PLC1
induced by pervanadate treatment. Interestingly, pervanadate treatment
induced phosphorylation of the double SH2(N) and SH2(C) domains mutant
(PLC1[SH2(N/C)R586/694/6K]-HA) to the same level of
that of the WT protein (data not shown), further confirming that the
function of both SH2 domains is dispensable for phosphorylation induced
by treatment with pervanadate.
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1
To further understand the sequence of events and the interactions
governing the phosphorylation of PLC1 in response to TCR engagement,
we focused on the proteins that associate with PLC1 in the course of
activation. Since either GST-SH2 domain of PLC1 recognizes p36–38,
we investigated the association of this phosphoprotein with the
transiently transfected WT protein or PLC1 mutants. p36–38 was
prominently coprecipitated with WT PLC1-HA from activated cells but
was not detected in precipitates of
PLC1[SH2(N)R586K]-HA (Fig. 5A). Interestingly, normal
levels of p36–38 were present with the PLC1 SH2(C) mutants,
PLC1[SH2(C)R694/6K]-HA (Fig. 5A) or
PLC1[SH2(C)R694K]-HA (data not shown), indicating that
the function of the SH2(C) domain is not important for binding
p36–38.
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1[SH2(N)R586K]-HA
precipitates of pervanadate-treated cells, but normal levels were
coprecipitated with PLC1[SH2(C)R694/6K]-HA, confirming
that the association of p36–38 with PLC1 is exclusively dependent
upon the presence of a functional SH2(N) domain.
An important question is whether the expression of the PLC1 SH2(N)
domain mutant in Jurkat cells impairs the ability of these cells to
activate tyrosine kinases in response to CD3 ligation. If this were the
case, the phosphorylation of p36–38 would be primarily compromised,
while its defective binding to the PLC1-HA SH2(N) domain mutant
would be the indirect result of such an impairment. To rule out this
possibility, the degree of phosphorylation of p36–38 in cells
expressing PLC1[SH2(N)R586K]-HA was analyzed. Because
only a fraction of transiently transfected Jurkat cells expresses the
gene of interest (typically 15 to 20%, data not shown), a vector
expressing a modified green fluorescent protein from A.
victoria was cotransfected with PLC1-HA to allow for the
selection of the transfected population by fluorescence-activated cell
sorting (38). PLC1-HA was exclusively expressed in the positively
selected cell population and was completely excluded from the
negatively selected cells (data not shown). p36–38 was precipitated
from resting or CD3-activated, positively sorted cells by taking
advantage of its ability to bind GST-Grb2 (28) and probed with
anti-phosphotyrosine. The degree of phosphorylation of p36–38 did
not differ between cells expressing WT or
PLC1[SH2(N)R586K]-HA (Fig. 5
B), ruling out
a negative effect of the ectopic expression of the PLC1 SH2(N)
domain mutant on CD3-induced tyrosine kinase activation. These data
indicate that the primary defect of the PLC1 SH2(N) domain mutant is
its failure to interact with phosphorylated p36–38.
The PLC1 SH2(N) mutant fails to bind Grb2
Because our data suggest that the SH2(N) domain of PLC1 is
required for the formation of an early activation complex that includes
p36–38, we next explored the association of the SH2(N) domain PLC1
mutant with the adapter Grb2. Although the role of Grb2 in PLC1
activation is not known, coprecipitation with PLC1 has been
previously demonstrated (28, 31). Furthermore, the interaction of
PLC1 with Grb2 requires the association of both proteins with
p36–38 (28), suggesting that p36–38 acts by linking the two proteins
together. A GST-Grb2 fusion protein was used to precipitate PLC1
from lysates of Jurkat cells transiently transfected with WT PLC1-HA
or the SH2(N) mutant, PLC1[SH2(N)R586K]-HA. An
activation-dependent increase in GST-Grb2-bound WT PLC1-HA was
observed in cells activated via TCR/CD3 ligation or pervanadate
treatment (Fig. 6). No increase in the
association of GST-Grb2 with the SH2(N) mutant protein occurred when
cells were treated with either OKT3 or pervanadate. These results
demonstrate that the SH2(N) domain of PLC1 is required for the
association with Grb2.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 requires a functional PLC1 SH2(N) domain. In contrast,
pervanadate treatment induced the same levels of phosphorylation of the
SH2(N) domain mutant and the WT protein, suggesting that this
pharmacologic agent may bypass the physiologic requirements for
TCR-induced PLC1 phosphorylation.
The structural arrangement of SH2 domains in tandem, as in the case of
PLC1, suggests the potential for a coordinated interaction of the
two domains with target proteins. For instance, the tandem SH2 domains
of ZAP-70 kinase cooperate in simultaneously engaging the same
phosphorylated protein (43). The PLC1 SH2 domains, however, function
independently of each other, in that the SH2(N) domain is required and
sufficient for PLC1 tyrosine phosphorylation in response to TCR/CD3
engagement, while the SH2(C) domain is not critical for this early
event.
The SH2(N) domain may play a role in forming a protein complex critical
for PLC1 activation. The identity of the proteins involved with
PLC1 in this activation complex have not been established, although
the phosphoproteins recognized by the isolated SH2 domains of PLC1
represent logical candidates. Since the SH2(C) domain function is not
required for phosphorylation, it is unlikely that the phosphoproteins
selectively recognized by this domain are involved in TCR-induced
PLC1 phosphorylation. A role for p36–38, however, is suggested by
the correlation observed between the SH2(N) domain-dependent
interaction of PLC1 with this protein and the tyrosine
phosphorylation of PLC1 in response to TCR/CD3 ligation.
It is interesting that, in the context of the whole PLC1 molecule,
only the SH2(N) domain binds to p36–38, while both the isolated
GST-SH2(N) and GST-SH2(C) domains bind this phosphoprotein. PLC1 SH2
domains belong to the same category of domains that select the
consensus sequence, pTyr-hydrophobic-X-hydrophobic (where
X in position +2 represents any amino acid) (44, 45),
implying that either domain could potentially bind identical sites. The
SH2(N) domain, however, prefers an acidic residue in position +2, while
the SH2(C) domain prefers a hydrophobic residue in the same position
(44). This preference could account for the selectivity for p36–38
binding of the SH2(N) domain observed in vivo. The differences from the
results obtained in vitro with the GST fusion proteins may be explained
by the possibility that the artificially high stoichiometric ratio
between the recombinant SH2 domains and the target phosphoproteins used
in experiments that employ fusion proteins could facilitate binding of
all potential targets, including those with comparable lower affinity.
This is likely to reflect the targets’ relative abundance rather than
the true specificity of the interaction. Alternatively, the function of
the SH2(C) domain within the whole protein may be regulated, either by
the engagement of neighboring domains and/or post-translational
modification.
Our data support a model whereby TCR-induced phosphorylation of PLC1
proceeds via the interaction of the SH2(N) domain of PLC1 with the
phosphorylated p36–38, which acts as a scaffold protein responsible
for complexing PLC1 with Grb2 and other proteins, possibly its
tyrosine kinases. Previous evidence showed that PLC1-associated
p36–38 can be depleted by anti-Grb2 precipitation (31), consistent
with p36–38 binding simultaneously to PLC1 and Grb2. Moreover,
p36–38 binds the GST-SH2 domains of either Grb2 or PLC1 (28). In
contrast with this model, Motto et al. (46) were unable to block
TCR-induced PLC1 phosphorylation when p36–38 was artificially
dephosphorylated in vivo by means of a transmembrane chimeric
protein containing the Grb2-SH2 domain and the catalytic domain of CD45
phosphatase, although TCR-induced phosphoinositide hydrolysis was
inhibited. A role for p36–38 in TCR-induced PLC1 phosphorylation,
however, cannot be excluded on the basis of the above evidence. Since
the interaction of p36–38 with the chimeric phosphatase depends on it
being phosphorylated first and then recruited by the chimeric SH2
domain, a complex of PLC1 with phosphorylated p36–38 may form for a
time sufficient to promote PLC1 phosphorylation even under these
experimental conditions. Although it can still be argued that
phosphoproteins other than p36–38 may participate in TCR-induced
PLC1 phosphorylation through the interaction with its SH2(N) domain,
the high degree of selectivity of the GST-SH2(N) domain for p36–38
conflicts with this hypothesis.
The role that the SH2(N) domain can play in PLC1 activation is
further highlighted by the recent finding that a cell-permeable,
tyrosine-phosphorylated peptide with a sequence almost identical to
that selected by PLC1 SH2(N) domain inhibited basic fibroblast
growth factor-induced phosphoinositide hydrolysis in cultured
cerebellar neurons (47). The same peptide, however, failed to block
phosphoinositide hydrolysis induced by treatment with platelet-derived
growth factor (47), suggesting that the SH2(N) domain may be necessary
for coupling to some, but not all, receptors.
While the SH2(C) domain is dispensable for TCR-induced PLC1
phosphorylation, a role for this domain in events other than tyrosine
phosphorylation of PLC1 cannot be excluded. Such a role could
include the recruitment of additional proteins critical for the
enzymatic activation or the targeting of phosphorylated PLC1 to a
specific subcellular localization. Alternatively, the SH2(C) domain may
function to couple PLC1 to receptors other than the TCR. These
different possibilities are currently under investigation.
Neither PLC1 SH2 domain mutant appears to possess a
dominant-negative function, as determined by the phosphoinositide
hydrolysis levels of cells transiently transfected and sorted for the
expression of PLC1-HA (data not shown). This finding suggests that
overexpression of an SH2 domain mutant in Jurkat cells cannot
effectively compete with the endogenous PLC1 for its ability to
interact with the substrate or other regulatory proteins. This may be
due to the fact that the endogenous levels of PLC1 are sufficiently
high compared with the fraction of mutant enzyme expressed ectopically
and/or because the relative abundance of endogenous SH2 domain-binding
proteins exceeds the amount scavengeable by transient transfection of
mutant PLC1-HA.
The observation that treatment with pervanadate led to tyrosine
phosphorylation of the SH2(N) mutant at levels that were identical with
those of the WT protein suggests that the mutations introduced did not
interfere with the ability of the protein to act as a substrate for
tyrosine kinases. It is theoretically possible that pervanadate
treatment induces phosphorylation of PLC1 regardless of SH2
domain-mediated association with other proteins and on sites not
normally phosphorylated in response to TCR/CD3 engagement. Since
pervanadate treatment activates PLC1 and induces inositol phosphate
production and Ca2+ mobilization in T lymphocytes (42), it
is likely that the critical regulatory sites are phosphorylated.
Conversely, it is unlikely that treatment with pervanadate overcomes
the binding defect of the PLC1 SH2(N) domain mutant by inducing
phosphorylation of target proteins on nonphysiologic sites, since the
GST-SH2 domain mutants completely failed to bind phosphoproteins from
pervanadate-treated Jurkat cells (data not shown). Furthermore, PLC1
SH2(N) domain mutant failed to bind p36–38 even when both proteins
were phosphorylated in response to pervanadate treatment. This
observation also confirms that binding of p36–38 is not a direct
consequence of PLC1 phosphorylation.
The biochemical mechanism of action of pervanadate includes the
inhibition of protein tyrosine phosphatases together with the
activation of protein tyrosine kinases, such as Lck (48, 49).
Pervanadate treatment, in fact, is likely to activate a wide variety of
protein tyrosine kinases, some whose potential role may be that of
coupling PLC1 to receptors other than the TCR. This hypothesis
implies that PLC1 phosphorylation in T cells may be induced by
distinct mechanisms that differ in their requirement for the SH2(N)
domain function. That PLC1 tyrosine phosphorylation in T cells may
be mediated by different mechanisms is also suggested by the recent
observation that CD2 ligation induced PLC1 tyrosine phosphorylation
at levels comparable with those produced by TCR/CD3 activation,
although only minimal levels of phosphorylated p36–38 were induced by
CD2 perturbation (50). Further delineation of the mechanism of
pervanadate-induced phosphorylation of PLC1 and the role of the SH2
domains in CD2-induced PLC1 phosphorylation will provide important
insights in the mechanism of regulation of PLC1 in T cells. Our
demonstration of a requirement for the SH2(N) domain in TCR-induced
PLC1 phosphorylation and its role in binding to a potential
activation complex is a first step toward a definition of the
structural requirements for PLC1 activation in T lymphocytes. These
findings will form a basis for future elucidation of whether different
receptors trigger PLC1 activation by unique or distinct modes of
action.
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Acknowledgments |
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Footnotes |
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2 Present address: Departamento de Bioquimica y Biologia Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, 50009 Zaragoza, Spain.
3 Address correspondence and reprint requests to Dr. Ezio Bonvini, HFM-564, Center for Biologics Evaluation and Research, National Institutes of Health Campus, Building 29B, Room 3NN10, 8800 Rockville Pike, Bethesda, MD 20892.
4 Abbreviations used in this paper: PLC1, phospholipase C1; SH, Src homology; HA, influenza hemagglutinin; GST, glutathione S-transferase; WT, wild-type.
Received for publication July 3, 1997. Accepted for publication October 8, 1997.
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1 and phosphatidylinositol 3 kinase are the downstream mediators of the PDGF receptor’s mitogenic signal. Cell 73:321.[Medline]
and a 64-kilodalton protein, respectively. Mol. Cell. Biol. 13:133.1 activation in T cells. Mol. Cell. Biol. 16:2823.[Abstract]
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