From the Divisions of Cell Biology and Cellular Immunology, La Jolla Institute for Allergy and Immunology, San Diego, California 92121
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ABSTRACT |
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In cells expressing the oncogenic Bcr-Abl tyrosine kinase, the regulatory p85 subunit of phosphatidylinositol 3-kinase is phosphorylated on tyrosine residues. We report that this phosphorylation event is readily catalyzed by the Abl and Lck protein-tyrosine kinases in vitro, by Bcr-Abl or a catalytically activated Lck-Y505F in co-transfected COS cells, and by endogenous kinases in transfected Jurkat T cells upon triggering of their T cell antigen receptor. Using these systems, we have mapped a major phosphorylation site to Tyr-688 in the C-terminal SH2 domain of p85. Tyrosine phosphorylation of p85 in vitro or in vivo was not associated with detectable change in the enzymatic activity of the phosphatidylinositol 3-kinase heterodimer, but correlated with a strong reduction in the binding of some, but not all, phosphoproteins to the SH2 domains of p85. This provides an additional candidate to the list of SH2 domains regulated by tyrosine phosphorylation and may explain why association of phosphatidylinositol 3-kinase with some cellular ligands is transient or of lower stoichiometry than anticipated.
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INTRODUCTION |
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Phosphatidylinositol 3-kinases
(PI3Ks)1 are a family of
enzymes involved in a multiplicity of cellular functions, including cell proliferation and transformation (1-3), lymphocyte activation (4-7), G protein signaling (8), DNA repair (9), intracellular vesicle
trafficking (10, 11), and inhibition of programmed cell death (12, 13).
The currently best characterized type of PI3K is the heterodimeric
enzymes that consist of a 110-kDa catalytic subunit (p110 or
p110
; Refs. 14 and 15) and a 85-kDa regulatory subunit (p85
or
p85
; Refs. 16-18), and that are utilized for signaling by activated
growth factor, cytokine, and antigen receptors. In these heterodimeric
PI3Ks, the p85 subunit also functions as an adaptor protein that
mediates protein-protein interactions through its two Src homology 2 (SH2) domains, one SH3 domain, two proline-rich sequences, and a region
with similarity to the breakpoint cluster region gene (16-18). The two
SH2 domains of p85 are involved in recruitment of PI3K to activated
growth factor receptors (3, 16) or other proteins having the general motif phosphotyrosine Tyr(P)-X-X-methionine (19), or, in
some cases, Tyr(P)-X-X-leucine (20, 21). In T cells, the
physiologically relevant ligands for p85 include
tyrosine-phosphorylated CD28 (22), subunits of the T cell antigen
receptor (20, 21, 23), CD5 (24), CD7 (25), and the c-Cbl proto-oncogene
product (26). In addition to causing a subcellular relocation of PI3K,
these SH2 ligands cause an allosteric activation of the catalytic p110 subunit, which is bound to the region between the two SH2 domains of
p85 (15, 27-29).
Several additional modes of PI3K regulation have been demonstrated, and it is likely that they act in concert to regulate the production of 3-phosphorylated inositol phospholipids in response to a variety of stimuli. The catalytic p110 interacts with activated GTP-bound Ras proteins through a region adjacent to its p85-binding NH2 terminus (30, 31). Active Ras enhances PI3K activity in intact cells (30, 31), but some data indicate that Ras also acts downstream of PI3K (32). In T cells, the two p85 isoforms have been shown to undergo phosphorylation on both serine and threonine (33, 34). Tyrosine phosphorylation of the p85 subunit has been shown to occur in many different systems, such as in response to platelet-derived growth factor (3), insulin (35), B cell antigen receptor ligation (4), interleukin-2 (36), and in cells transformed by the Bcr-Abl fusion protein-tyrosine kinase (37-40). The sites of phosphorylation in p85 have been mapped to tyrosines 368, 508, and 607 in insulin-stimulated cells (35), but the physiological function of this phosphorylation has remained unknown. Tyrosine phosphorylation of p85 seems not to be required for the enzymatic activity of PI3K. Instead, tyrosine phosphorylation of p85 has been reported to correlate with the dissociation of PI3K from the activated insulin receptor kinase (41).
We have studied the tyrosine phosphorylation of p85 in hematopoietic cells, and report that phosphorylation occurs at least at Tyr-688 in the C-terminal SH2 domain. This event does not detectably affect the catalytic activity of PI3K per se, but causes a change in the binding properties of the SH2 domain. This change is likely to modify the function of PI3K in intact cells.
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MATERIALS AND METHODS |
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Antibodies and Reagents--
Antibodies against p85 of PI3K and
the anti-Tyr(P) mAb 4G10 were from Upstate Biotechnology Inc. (Lake
Placid, NY) and the anti-HA epitope mAb 12CA5 was from Boehringer
Mannheim (Indianapolis, IN). The hybridoma that produces the OKT3
anti-CD3 mAb was from American Type Cell Collection.
Phosphatidylinositol was from Upstate Biotechnology Inc. and
TPCK-treated trypsin was from Worthington Biochemicals (NJ).
Recombinant Abl protein-tyrosine kinase domain (Abl-K) was from New
England Biolabs (Beverly, MA) and purified Lck from Upstate
Biotechnology Inc. The protein-tyrosine kinase expression plasmids have
been used before (42, 43), and we recently described the cloning of the
HA-tagged p85 constructs (5). In addition to an N-terminal
hemagglutinin (HA) tag, the constructs contain the following amino
acids of bovine p85
: N-SH2 (329-439), C-SH2 (563-724), NC
(329-439 + 563-724), NiC (329-724), p85
iSH2 (1-439 + 563-724),
and wild-type p85 (1-724). GST fusion proteins containing the same
fragments of p85 were ligated into the glutathione
S-transferase (GST) fusion vector pGEX-4T-2 (Pharmacia, Sweden) by standard procedures. Expression was induced and the fusion
proteins were purified using glutathione-Sepharose beads (Pharmacia).
Cells--
Jurkat T leukemia cells, and their simian leukemia
virus 40 large T antigen-expressing variant, J-TAg (kind gift from Dr. M. Karin), were kept at logarithmic growth in RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum, L-glutamine,
and antibiotics. HL-60/Bcr-Abl cells were obtained by retroviral
transfection of HL-60 promyelocytic leukemia cells with
pSRMSVp185bcr-abl tkneo as described elsewhere (37). COS-1
cells were maintained in Dulbecco's modified Eagle's medium with 10%
fetal calf serum.
Transient Transfections-- 15 × 106 J-TAg cells, HL-60, or HL-60/Bcr-Abl were transfected with a total of 10-20 µg of DNA by electroporation at 960 microfarads and 240 V. Typically, cells were transfected with 15 µg of HA-tagged p85 construct. Empty vector was added to control samples to make a constant amount of DNA in each sample. Cells were harvested two days after electroporation. COS-1 cells were transfected by lipofection with 10 µg of DNA and grown for 48 h prior to the experiments as described (42, 44-47).
Immunoprecipitation-- These procedures were as reported before (5, 42, 46, 47). All steps were carried out at 0-4 °C. Cells were lysed in 20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA containing 1% Nonidet P-40, 1 mM Na3V04, 10 µg/ml aprotinin and leupeptin, 1 mM phenylmethylsulfonyl fluoride, and 100 µg/ml soybean trypsin inhibitor and clarified by centrifugation at 13,000 × g for 10 min. The clarified lysates were preabsorbed on agarose-conjugated goat anti-rabbit IgG or protein G-Sepharose. The lysates were then incubated with antibody for 2-4 h, followed by agarose-conjugated goat anti-rabbit IgG or protein G-Sepharose. Immune complexes were washed three times in lysis buffer, once in lysis buffer with 0.5 M NaCl, again in lysis buffer, and either suspended in SDS sample buffer or washed further for kinase assays.
SDS-PAGE and Western Blotting-- Proteins were separated by SDS-PAGE and transferred onto nitrocellulose filters. The antisera were used at 1:500-1:2000 dilution and the blots developed by the enhanced chemiluminescence technique (ECL kit, Amersham) according to the manufacturer's instructions.
In Vitro Phosphorylation of GST Fusion Proteins--
The GST
fusion proteins were dissolved in kinase buffer (10 mM
HEPES pH 7.5, 0.1% Triton X-100, 20 mM MgCl2,
1 mM dithiothreitol, and 0.1 mM
Na3VO4). After addition of 2 mM
cold ATP or 1 µM ATP and 10 µCi of
[-32P]ATP and 1 µl of purified Lck or 100 units of
Abl-K, the reaction was carried out for 30 min at 30 °C or overnight
at room temperature (for higher stoichiometry of phosphorylation). The
reaction was stopped by adding SDS sample buffer or by adding cold
lysis buffer. In the latter case, the mixture was incubated with
glutathione-Sepharose beads for 1 h, and the beads washed
extensively before they were added to precleared cellular lysates.
Binding of Cellular Proteins to GST Fusion Proteins-- These experiments were done as before (48). Cell lysates were prepared as above, cleared by centrifugation, and preadsorbed to glutathione-Sepharose beads. After removal of the beads, the lysates were incubated on ice with 5 µg of GST fusion protein and glutathione-Sepharose beads for 2 h, which were subsequently washed five times with lysis buffer. The bound proteins were eluted in SDS sample buffer, resolved by SDS-PAGE, and analyzed by immunoblotting as above.
PI3K Assay--
The assay for lipid kinase activity of
immunoprecipitated PI3K was as before (5, 6, 49). The assay mixture
contained 20 mM Tris/HCl, pH 7.5, 100 mM NaCl,
0.5 mM EGTA, 10 µg of phosphatidylinositol (stock
solution at 20 mg/ml in water), 20 mM MgCl2,
and 10 µCi of [-32P]ATP. Reaction products were
analyzed by ascending chromatography on Silica gel thin layer plates in
CHCl3, CH3OH, 25%
NH4OH/H2O (90:90:9:19) followed by
autoradiography.
Tryptic Peptide Mapping--
GST-p85-NiC protein phosphorylated
by Lck in the presence of [-32P]ATP was resolved on
10% SDS gels, transferred onto a nitrocellulose filter, exposed to
film, and the correct band excised. The filter piece was blocked and
digested with TPCK-treated trypsin as described in detail by Luo and
co-workers (50). The resulting phosphopeptides were separated by
electrophoresis on cellulose thin layer plates at pH 1.9 for 27 min
followed by ascending chromatography in
n-butanol/pyridine/acetic acid/water (75:50:15:60), and
exposed to film for 35 h.
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RESULTS |
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Tyrosine Phosphorylation of p85 in Bcr-Abl-expressing HL-60 Cells-- Bcr-Abl induces transformation of fibroblasts and hematopoietic cells (51-54) and confers resistance to apoptosis (55). Cells expressing the Bcr-Abl tyrosine kinase contain elevated amounts of proteins phosphorylated on tyrosine residues. At least in some Bcr-Abl expressing cells, such as HL-60 cells transfected with Bcr-Abl (37), a fraction of the p85 subunit is included among these substrates for enhanced tyrosine phosphorylation. When PI3K was immunoprecipitated from parental HL-60 cells with antibodies against the p85 subunit and immunoblotted with anti-Tyr(P) mAbs, no phosphorylation of p85 could be detected (Fig. 1, lane 1), even if the cells were pretreated with 100 µM pervanadate to increase intracellular Tyr(P) content (lanes 2 and 3). In contrast, when anti-p85 immunoprecipitates prepared from HL-60 cells stably transfected with Bcr-Abl (HL-60/Bcr-Abl cells) were analyzed in parallel, a sharp, but not very prominent, band at 85 kDa (in addition to several other phosphoproteins) was seen (Fig. 1, lane 4). Reprobing of the same filter with anti-p85 revealed that all immunoprecipitates contained equal amounts of p85, which co-migrated precisely with the Tyr(P)-containing 85-kDa band in lane 4. This result was obtained in several independent experiments.
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Tyrosine Phosphorylation of p85 Does Not Affect PI3K Activity-- To investigate whether this low level tyrosine phosphorylation of p85 in HL-60/Bcr-Abl cells has any effect on the catalytic activity of PI3K, we first immunoprecipitated PI3K from resting Jurkat T cells, and phosphorylated the immune complex in vitro with recombinant Abl-K, after which half of the sample was assayed for PI3K activity and the other half separated by SDS-PAGE for analysis by immunoblotting. Abl-K was found to strongly phosphorylate p85 (Fig. 2, left), but this phosphorylation had no effect on the catalytic activity of PI3K (Fig. 2, right, lanes 1 and 2). An anti-p85 blot of the same filter confirmed that the samples contained the same amount of PI3K. A comparison of the amount of p85 protein versus Tyr(P) content between Figs. 1 and 2 and several similar experiments, demonstrated that the stoichiometry of tyrosine phosphorylation was considerably higher in vitro than in vivo. Thus, the lower level of tyrosine phosphorylation of p85 in intact cells is even more unlikely to affect the catalytic activity of PI3K.
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Bcr-Abl Phosphorylates p85 in Its SH2 Domains--
To identify in
which part of the molecule p85 is tyrosine phosphorylated in
HL-60/Bcr-Abl cells, we transiently transfected these cells with a set
of HA-tagged truncated p85 constructs, which were subsequently
immunoprecipitated and analyzed by anti-Tyr(P) immunoblotting. In these
experiments (Fig. 3), wild-type p85 was phosphorylated on tyrosine, as was p85 lacking the inter-SH2 domain (p85iSH2) and the two constructs containing both SH2 domains with or
without the inter-SH2-region (NiC and NC, respectively). In contrast,
the individual SH2 domain proteins (N-SH2 and C-SH2) did not contain
detectable Tyr(P) even on very long exposures. All constructs were
expressed at comparable levels as judged by anti-HA tag immunoblotting
(Fig. 3, lower panel). Since the N-SH2 and C-SH2 proteins
together contain the same amino acids as the NC protein, we conclude
that the main phosphorylation occurs in one or both of these two
domains, but that both are required for phosphorylation in intact
HL-60/Bcr-Abl cells. This requirement correlates with the
co-immunoprecipitation of several cellular phosphoproteins with all
constructs having both SH2 domains (Fig. 3), but not the single SH2
domains. Thus, proper interaction of p85 with other proteins, perhaps
including Bcr-Abl itself, is important for efficient
phosphorylation.
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Activated Lck Phosphorylates p85 in the C-terminal SH2
Domain--
We have previously used a transient COS-1 cell
transfection system to study potential interactions between
protein-tyrosine kinases and the role of phosphorylation sites (42,
44-47). When full-length p85 or its truncated versions were expressed
in these cells together with an activated (56-58) Y505F-mutated Lck,
we observed that all constructs having two SH2 domains were efficiently phosphorylated in these cells (Fig. 4).
Also the C-SH2 protein reacted with anti-Tyr(P), although the
reactivity was weaker than that of the p85iSH2 (Fig. 4, lanes
2 and 3) or full-length p85 or NC (not shown). This
lower level of phosphorylation was not due to expression, which was
comparable (Fig. 4, lower panel). In contrast, the N-SH2
protein was completely unphosphorylated even on the very long exposure
shown in lanes 6-10 of Fig. 4. Co-expression of Bcr-Abl,
Bmx, or Jak2 was equally unable to cause tyrosine phosphorylation of
the N-SH2 protein, although these protein-tyrosine kinases caused some
phosphorylation of other p85 constructs (not shown). We conclude again
that phosphorylation of p85 is efficient when both SH2 domains are
present, but that the C-SH2 domain can become phosphorylated when
expressed alone at sufficiently high levels. Thus, the phosphorylation
of larger constructs is most likely to mainly occur in the C-terminal
SH2 domain.
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Receptor-induced Phosphorylation of p85-NC in T Cells--
Since
Lck-Y505F was very efficient at phosphorylating p85 proteins in
co-transfected COS-1 cells, we decided to examine whether endogenous
Lck or other protein-tyrosine kinases in T cells could also catalyze
this reaction. Triggering of the T cell antigen receptor on T cells
leads to a rapid phosphorylation of many cellular proteins on tyrosine
residues (59-63). When the HA-tagged NC construct was expressed in
J-TAg cells and immunoprecipitated following a brief stimulation of the
cells with an anti-CD3 mAb, we observed by anti-Tyr(P)
immunoblotting that the protein was phosphorylated to a low
stoichiometry prior to cell activation, but became increasingly phosphorylated with time (Fig. 5). Peak
tyrosine phosphorylation occurred at the 5-min time point. The NC
protein was equally immunoprecipitated in each sample (lower
panel), and it also co-precipitated a set of cellular
phosphoproteins at 36-38, 70, and 120 kDa. These proteins are similar
in size to those that co-immunoprecipitate with endogenous p85 (5, 49)
or that bind to GST fusion proteins of the two SH2 domains of p85 (see
below).
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Both Abl and Lck Phosphorylate the C-terminal SH2 Domain of p85 in Vitro-- As phosphorylation of the C-SH2 protein was relatively low in intact cells, perhaps due to less efficient recruitment or inappropriate subcellular location, we decided to test if Abl-K and Lck would display specificity for this region of p85 in vitro. GST fusion proteins were prepared of the N-SH2, C-SH2, NC, and NiC constructs, and these proteins (or control GST) were incubated with recombinant Abl-K or purified Lck in the presence of ATP. As shown in Fig. 6, all fusion proteins, except GST-N-SH2, were well and equally phosphorylated. The GST-C-SH2 was close in size to Abl-K making its phosphorylation difficult to appreciate in lane 5, but the size of Lck was larger and the phosphorylation of GST-C-SH2 is easily seen in lane 6. Control GST was very weakly phosphorylated by Abl-K or Lck, indicating that the phosphorylation of the p85 protein occurred in the p85-derived parts. Similar results were obtained in several independent experiments.
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Mapping of the Major Phosphorylation Site in the SH2 Domains of p85
to Tyr-688--
To determine how many tyrosine residues were
phosphorylated in the C-terminal SH2 domain of p85 in vitro,
we phosphorylated the GST-NiC protein by Abl-K in the presence of
[-32P]ATP and analyzed the reaction product by tryptic
peptide mapping. As shown in the bottom panel of Fig. 6, the
protein was phosphorylated at one major site (peptide a), a few minor
sites and a variable site (peptide b), which may be derived from the
GST part of the fusion protein as peptides with similar mobility can be
seen in other in vitro phosphorylated GST fusion proteins
(46). A similar map was obtained when GST-NiC was phosphorylated with
Lck (not shown). Since the maps contained only one major peptide, we
decided to map the site through site-directed mutagenesis of a few
plausible tyrosine residues. We generated the Y607F, Y685F, and Y688F
mutants of the GST-NiC protein, subjected them to the same in
vitro phosphorylation with Abl-K, and analyzed them by tryptic
peptide mapping. As can be seen in the bottom of Fig. 6, the Y607F and
Y685F mutants were still phosphorylated on peptide a, while this spot
was completely missing from the phosphorylated Y688F mutant. We
conclude that the major site for Abl- and Lck-mediated phosphorylation
of p85-NiC is Tyr-688 in the C-terminal SH2 domain. This suggests that
tyrosine phosphorylation of endogenous p85 and of the larger
transfected p85 constructs in HL-60/Bcr-Abl, COS-1, or J-TAg cells
occurs largely (but not necessarily exclusively) at Tyr-688 in the
C-terminal SH2 domain.
Tyrosine Phosphorylation Modifies the Binding Properties of the SH2
Domain--
The location of Tyr-688 in the C-terminal SH2 domain
prompted us to ask whether this phosphorylation may impact the
ligand-binding function of the SH2 domain. As a precedent for this
possibility, we recently reported that phosphorylation of the Lck SH2
domain at the equivalent residue in the EF loop, Tyr-192, causes a
dramatic decrease in its binding of ligands (47). To test this
possibility in the case of the p85 SH2 domains, we incubated the GST-NC
protein with or without ATP and Abl-K overnight at room temperature to obtain as high stoichiometry of phosphorylation as possible. Following this incubation, the GST fusion protein was adsorbed to
glutathione-Sepharose, washed extensively, and mixed with a precleared
lysate of pervanadate-treated Jurkat cells. After 2 h on ice, the
Sepharose beads were again washed extensively and bound proteins eluted
in SDS sample buffer, resolved on SDS gels, transferred onto
nitrocellulose filters, and analyzed by anti-Tyr(P) immunoblotting.
These experiments revealed that preincubation of the GST fusion protein
with Abl-K and ATP, but not ATP alone, caused several significant
changes to the set of cellular phosphoproteins subsequently bound to
the SH2 domains of p85 (Fig. 7). In
particular, a band at 21-kDa and another at 70 kDa decreased very
markedly, while other bands remained unchanged or even increased. An
anti-GST blot showed that the loading of GST fusion protein was equal
in all samples. The band at ~65 kDa contains the GST-NC itself, and
its phosphorylation can be seen in lanes 3 and 4.
This result was obtained in several independent experiments, two of
which are shown in Fig. 7. In other experiments we obtained similar
results with lysates from Jurkat cells treated with anti-CD3 instead
of pervanadate (not shown). In the other experiment shown, a similar
result was obtained with preincubation of GST-NC with Lck (lane
4) in addition to incubation with Abl-K (lane 3).
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DISCUSSION |
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Taken together, our findings indicate that PI3K can be phosphorylated at Tyr-688 in the C-terminal SH2 domain of the p85 subunit in Bcr-Abl expressing HL-60 cells, by active Lck in COS cells, by an unidentified receptor-activated protein-tyrosine kinase in T cells, and by both Abl and Lck in vitro. This phosphorylation does not measurably affect the lipid kinase activity of PI3K (at least at physiological stoichiometry), but was found to change the ligand binding properties of the SH2 domain(s). These results are in agreement with previous observations showing that expression of Bcr-Abl in NIH 3T3 cells induces tyrosine phosphorylation of PI3K without any significant increase in PI3K products in vivo (40).
The analysis of SH2 domain function in p85 is complicated by the tandem
arrangement of the two SH2 domains, resulting in their cooperative
binding to many ligands. Our results indicate that tyrosine
phosphorylation of the C-terminal SH2 domain reduced the affinity for
some ligands, while the binding of others was unchanged. Two
alternative explanations could be envisualized: either tyrosine
phosphorylation changed the ligand selection from the classical
Tyr(P)-X-X-methionine to something more or less different,
or only the C-terminal SH2 domain was inhibited, while the N-terminal
SH2 domain remained unchanged. In the latter case, only of those
ligands that bind exclusively to the C-terminal SH2 domain or that
require simultaneous binding to both domains will bind less strongly.
We have previously observed that expression of the HA-tagged p85
proteins in T cells resulted in the co-immunoprecipitation of
phospho-TCR only when both SH2 domains were present in the p85
protein. Such a requirement for two SH2 domains would explain why the
TCR
binds despite not having the optimal
Tyr(P)-X-X-methionine (19) motif. Apparently, two
Tyr(P)-X-X-leucine motifs in tandem in TCR
can bind the
two SH2 domains of p85 simultaneously and thereby increase the affinity
to physiologically relevant levels. The binding of PI3K to TCR
and
CD3 subunits (5, 20, 23), as well as to isolated phosphopeptides
derived from these proteins (21), has been reported. The findings
reported in the present paper may explain why p85 binding to these
receptor subunits is of low stoichiometry when assessed by
co-immunoprecipitation.
In the Bcr-Abl expressing cells, p85 was phosphorylated on tyrosine, but also co-immunoprecipitated with several Tyr(P)-containing proteins. While this may seem conflicting, it is clear that only a fraction of p85 is tyrosine phosphorylated, and it is impossible to judge if any of the co-immunoprecipitating protein bound to the phosphorylated minority of p85 molecules or (more likely) to the unphosphorylated majority. The requirement for both SH2 domains for efficient phosphorylation of p85 proteins in Bcr-Abl expressing cells, suggests that both are involved directly or indirectly in association with the protein-tyrosine kinase responsible for this phosphorylation. The simplest model predicts that the p85 SH2 domains bind directly to Bcr-Abl, but dissociate from it upon phosphorylation of the C-terminal SH2 domain. This would explain why the co-immunoprecipitation of Bcr-Abl and PI3K is of very low stoichiometry.
We recently reported that the Lck kinase is phosphorylated at Tyr-192 in the EF loop of its SH2 domain in activated T cells, and in COS-1 cells co-transfected with either Syk or Zap (45, 47). The phosphorylation of the SH2 domain, or the mutation of Tyr-192 to an acidic residue, caused a strong decline in the affinity of the domain for tyrosine-phosphorylated ligands (47). A similar change was reported by Stover and co-workers (65) for the c-Src SH2 domain upon its phosphorylation at Tyr-213 by the platelet-derived growth factor kinase. In this paper, we add a third example to the list of SH2 domains regulated by tyrosine phosphorylation, namely the C-terminal SH2 domain of PI3K p85. Interestingly, Tyr-688 resides in the same region of the SH2 domain as Tyr-192 in the Lck SH2 domain. Comparison of the amino acid sequences of SH2 domains from different proteins shows that many, but not all, contain tyrosine residues in the corresponding location in the EF loop (66). Notably, the N-terminal SH2 domain of p85 does not. Thus, it is tempting to speculate that the regulation of SH2 domains by tyrosine phosphorylation is a more general mechanism for the termination of SH2-ligand interactions, perhaps in part explaining their transient nature in intact cells.
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ACKNOWLEDGEMENT |
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We are grateful to Dr. Lewis C. Cantley for valuable discussions and advice.
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FOOTNOTES |
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* This work was supported by Finska Läkaresällskapet, the Finnish Cancer Organizations, National Institutes of Health Grant GM52735 and American Cancer Society Grant CB-82 (to D. R. G.), and National Institutes of Health Grants GM48960 and AI35603 (to T. M.). This is publication 185 from the La Jolla Institute for Allergy and Immunology.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Brazilian Research Council (CNPq) Fellow.
§ To whom correspondence should be addressed. Tel.: 619-558-3547; Fax: 619-558-3526; E-mail: tomas_mustelin{at}liai.org.
1 The abbreviations used are: PI3K, phosphatidylinositol 3-kinase; HA, hemagglutinin; Tyr(P), phosphotyrosine; SH2, Src homology 2 region; SH3, Src homology 3 region; mAb, monoclonal antibody; TPCK, L-1-tosylamido-2-phenylethyl chloromethyl ketone; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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