(Received for publication, June 1, 1995; and in revised form, June 27, 1995)
From the
Tyrosine phosphorylation of cellular proteins is an early and an
essential step in T cell receptor-mediated lymphocyte activation.
Tyrosine phosphorylation of transmembrane receptor chains (such as
and CD3 chains) and membrane-associated proteins provides docking
sites for SH2 domains of adaptor proteins and signaling enzymes,
resulting in their recruitment in the vicinity of activated receptors.
pp36/38 is a prominent substrate of early tyrosine phosphorylation upon
stimulation through the T cell receptor. The tyrosine-phosphorylated
form of pp36/38 is membrane-associated and directly interacts with
phospholipase C-
1 and Grb2, providing one mechanism to recruit
downstream effectors to the cell membrane. Here, we demonstrate that in
Jurkat T cells, pp36/38 associates with the p85 subunit of
phosphatidylinositol 3-kinase (PI-3-K p85) in an activation-dependent
manner. Association of pp36/38 with PI-3-K p85 was confirmed by
transfection of a hemagglutinin-tagged p85
cDNA into Jurkat cells
followed by anti-hemagglutinin immunoprecipitation. In vitro binding experiments with glutathione S-transferase fusion
proteins of PI-3-K p85 demonstrated that the SH2 domains, but not the
SH3 domain, mediated binding to pp36/38. This binding was selectively
abrogated by phosphopeptides that bind to p85 SH2 domains with high
affinity. Filter binding assays demonstrated that association between
pp36/38 and PI-3-K p85 SH2 domains was due to direct binding. These
results strongly suggest the role of pp36/38 in recruiting PI-3-K to
the cell membrane and further support the idea that pp36/38 is a
multifunctional docking protein for SH2 domain-containing signaling
proteins in T cells.
Engagement of the T cell receptor (TCR) ()by the
major histocompatibility complex-bound antigenic peptides leads to T
cell activation, a prerequisite for effective immune responses. One of
the earliest and obligatory biochemical steps in T cell activation is
the tyrosyl phosphorylation of cellular proteins including the receptor
components themselves(1, 2) . Studies with receptor
tyrosine kinases, such as the epidermal growth factor receptor, have
elucidated the role of tyrosine phosphorylation in recruiting
downstream signaling proteins. Specific phosphotyrosyl peptide motifs
serve as docking sites for the SH2 (Src homology 2) domains of
signaling proteins, allowing them to form stable complexes with the
activated receptors (3, 4, 5) . However,
unlike growth factor receptors(4) , the TCR/CD3 components lack
intrinsic tyrosine kinase domains and signal through noncovalently
associated cytoplasmic tyrosine kinases(1, 2) . Two
Src family kinases, the TCR/CD3-associated p59
(Fyn) (6) and the CD4/8-associated p56
(Lck)(7, 8) , and a distinct cytoplasmic
tyrosine kinase, ZAP-70(9, 10) , have been
demonstrated to play critical and apparently nonoverlapping roles in T
cell activation. It is now widely accepted that these cytoplasmic
tyrosine kinases phosphorylate the cytoplasmic tails of CD3 and
chains and other membrane-associated proteins, which serve as docking
sites for SH2 domain-containing proteins(1, 2) . For
example, tyrosine phosphorylation of CD3 and
chains by the Src
family kinases allows binding to SH2 domains of ZAP-70 (10) and
adaptor protein Shc(11, 12) . The latter protein is
itself phosphorylated, providing a binding site for Grb2. This is
thought to translocate Grb2-bound guanine nucleotide exchanger m-Sos to the cell membrane, thus leading to Ras
activation(11) , although
-Shc-Grb2-Sos complexes have
been difficult to demonstrate(12) . Grb2 also associates with
other proteins through its SH3 domains, such as the p120
(13, 14, 15) and p76-SLP (16) proteins, which are also likely to be recruited to the
cell membrane in this fashion.
While tyrosine-phosphorylated
CD3/ chains provide an important set of motifs to recruit
signaling proteins, these are unlikely to directly interact with all
signaling proteins that are recruited to the TCR, given the preference
of various SH2 domains for specific peptide sequences at positions
+1 to +3 following phosphotyrosine(17) . Thus, it is
important to identify additional SH2 domain targets and to characterize
their specific preferences for different SH2 domains. Recent studies
have revealed a likely role for a polypeptide (pp36/38) in recruiting
downstream effectors to the cell membrane in activated T cells. pp36/38
represents a dominant substrate of TCR-associated tyrosine kinases in
primary T cells as well as in the Jurkat human T cell
line(18, 19, 20, 21) . Importantly,
pp36/38 was shown to coimmunoprecipitate with phospholipase C-
1
and Grb2, and SH2 domains of these proteins directly complexed with
pp36/38 in vitro(18, 19, 20, 21) . Since
tyrosine-phosphorylated pp36/38 was found to be
membrane-associated(20, 21) , these results strongly
suggested a possible role of this protein in recruiting critical
signaling proteins to the membrane.
The phosphatidylinositol 3-kinase (PI-3-K) enzyme, which phosphorylates the D-3 position of the inositol ring to generate a unique class of lipid second messengers, has been implicated in signaling through a number of growth factor, cytokine, and other receptors(22, 23) . In T lymphocytes, stimulation through the TCR leads to an increase in the PI-3-K activity and activation-dependent incorporation of this enzyme into phosphotyrosyl protein complexes(24, 25, 26, 27, 28, 29) . However, p110 or p85 subunits of PI-3-K are not tyrosine-phosphorylated upon TCR triggering(25, 27) , indicating that incorporation of PI-3-K into phosphotyrosyl complexes occurs through interactions with other proteins. The regulatory p85 subunit of PI-3-K (PI-3-K p85) possesses an SH3 domain and two SH2 domains (30) and has been implicated in mediating protein-protein interactions that regulate the activity and localization of PI-3-K(22, 23) . Here, we demonstrate that pp36/38 associates with the p85 subunit of PI-3-K. In vitro binding experiments demonstrate that this binding is direct and involves the PI-3-K p85 SH2 domains. These results provide further evidence for the role of pp36/38 as a multifunctional SH2 domain docking adaptor in TCR signaling.
Fusion proteins were affinity-purified on
glutathione-Sepharose beads (Pharmacia Biotech Inc.) using the Triton
X-100-soluble fraction of
isopropyl-1-thio--D-galactopyranoside-induced Escherichia coli (DH5
strain) as
described(13, 38, 39) . Proteins were
quantitated by the Bradford assay (Bio-Rad) against a bovine serum
albumin standard and analyzed on Coomassie gels to confirm quantitation
and to assess purity (usually >95%).
Figure 1:
In vivo association of pp36/38
with PI-3-K p85 in anti-CD3-stimulated Jurkat T cells. A,
immunoprecipitates from lysates of 5 10
unstimulated (-) or anti-CD3 (SPV-T3b)-stimulated (+)
Jurkat cells with the indicated antibodies (I.P.; shown on
top) or whole cell lysates (10
cells) were resolved by
SDS-9% PAGE, transferred to PVDF membranes, and subjected to
immunoblotting with the antibodies indicated on the right, followed by
protein A-horseradish peroxidase and ECL detection. Immunoprecipitated
species are indicated on left. PLC
1, phospholipase
C-
1; Ig, immunoglobulin heavy chain; NRS, normal
rabbit serum. The threelowerpanels represent a reprobing of different parts of the filter shown in
the toppanel. B, association of the PI-3-K
activity with phosphotyrosyl proteins. Immunoprecipitations, carried
out as described for A, were subjected to lipid kinase assay
as described under ``Materials and Methods.'' The reaction
products were subjected to TLC and visualized by autoradiography. PIP, phosphorylated inositides.
Figure 2:
In vivo association of pp36/38
with transfected HA-tagged PI-3-K p85. JMC-T cells were either
mock-transfected or transfected with pCG.85-HA plasmid DNA (p85-HA
transfectant) and analyzed after 72 h. Immunoprecipitates from 5
10
unstimulated(-) or anti-CD3
(SPV-T3b)-stimulated (+) cells were subjected to immunoblotting
with the indicated antibodies, followed by protein A-horseradish
peroxidase and ECL detection. I.P., immunoprecipitate; NRS, normal rabbit serum; HA, influenza hemagglutinin
epitope recognized by monoclonal antibody 12CA5. Immunoprecipitated
species are shown on the left. The p85 and Grb2 blots
represent serial reprobing of the filter used for the anti-pY
blot.
Figure 3:
Time
course of tyrosine phosphorylation of PI-3-K p85- and Grb2-associated
pp36/38 upon T cell activation. Whole cell lysates (10 cells; toppanel), anti-Grb2 immunoprecipitates
(5
10
cells; second and thirdpanels), or anti-PI-3-K p85 immunoprecipitates (fourth and fifthpanels) from
unstimulated(-) or anti-CD3 (2Ad2)-stimulated (+) Jurkat
cells were resolved by SDS-PAGE and subjected to immunoblotting with
the antibodies shown on the right, followed by protein A-horseradish
peroxidase and ECL detection. Time of anti-CD3 stimulation is shown in
seconds (s) or minutes (m). Anti-Grb2 and anti-p85
immunoblots represent reprobing of the respective filters. I.P., immunoprecipitate.
Figure 4:
Binding of pp36/38 to GST fusion proteins
of PI-3-K p85 and selective inhibition of binding by p85 SH2-specific
phosphotyrosyl peptides. A, SH2 domains (but not the SH3
domain) of PI-3-K p85 are capable of binding to pp36/38. Binding
reactions were carried out by incubating lysate from 2 10
anti-CD3 (SPV-T3b)-stimulated Jurkat cells (in a 2-ml volume)
with 10 µg of the indicated GST fusion proteins noncovalently
immobilized on glutathione-Sepharose beads (5 µl of packed beads)
for 1 h. Whole cell lysate (10
cells) or binding reactions
were subjected to anti-pY immunoblotting, followed by protein
A-horseradish peroxidase and ECL detection. Only the pp36/38 portion of
the blot is shown. B, binding of pp36/38 to GST-p85
SH2(N+C) is selectively abrogated by PI-3-K p85 SH2-specific
phosphotyrosyl peptides. Competing peptides were separately added to
bead-bound fusion proteins and cell lysate at the indicated
concentrations (shown in µM). After 15 min, beads and
lysate were mixed, and binding reactions and immunoblotting were
carried out as described for A. Peptides were EPQpYEEIPIYL
(pYEEI), EPQYEEIPIYL (YEEI), PSpYVNVQNL (pYVNV), PSpYVAVQNL (pYVAV),
HSDpYMNMTPR (pYMNM), and HSDYMNMTPR (YMNM). -, no peptide. pYMNM
is p85 SH2-specific; pYVNV is Grb2 SH2-specific; and pYEEI is Src
SH2-specific.
Figure 5:
Direct binding of PI-3-K p85 SH2(N+C)
fusion protein to membrane-immobilized pp36/38. Anti-pY
immunoprecipitates from 10 unstimulated(-) or
anti-CD3 (SPV-T3b)-stimulated (+) Jurkat T cells were resolved by
SDS-PAGE, transferred to PVDF membrane, and incubated with 2.5
µg/ml GST (leftpanel, lanes 1 and 2), GST-Grb2 (middlepanel, lanes 5 and 6), or GST-p85 SH2(N+C) (right panel, lanes 9 and 10) in lysis buffer containing 1 mg/ml
gelatin. After 1 h of incubation at 4 °C, filters were probed with
anti-GST antibody, followed by protein A-horseradish peroxidase and ECL
detection. Each blot was reprobed with anti-pY antibody (lanes 3 and 4, 7 and 8, and 11 and 12) and shows similar phosphotyrosyl protein signals. pp36/38
and p120
are
indicated.
Stimulation through the TCR leads to an increase in the
PI-3-K activity and activation-dependent incorporation of this enzyme
into phosphotyrosyl protein
complexes(24, 25, 26, 27, 28, 29) .
Since p110 or p85 subunits of PI-3-K are not tyrosine-phosphorylated
upon TCR triggering (25, 27) (data not shown), the
incorporation of this enzyme into phosphotyrosyl complexes must occur
through its interactions with other proteins. It has been shown that
SH3 domains of the Src family tyrosine kinases can interact in
vitro with PI-3-K p85 by recognizing its proline-rich peptide
motifs(26, 37, 43) . However, we observed a
significantly lower level of PI-3-K activity associated with
immunoprecipitates of Fyn tyrosine kinase compared with that observed
in anti-pY immunoprecipitates (13) (data not shown). An
alternate mechanism to recruit PI-3-K into signaling complexes is
through binding of the SH3 and SH2 domains of its p85 subunit to
signaling proteins. Recent studies have identified one such protein. We (13) and others (15) have demonstrated a primarily SH2
domain-mediated association of PI-3-K p85 with
p120, a rapidly tyrosine-phosphorylated protein
in T cells, and a substantial level of PI-3-K activity
coimmunoprecipitated with p120
. In the present
report, we have identified and characterized the interaction of PI-3-K
p85 with pp36/38, a major substrate of TCR-dependent tyrosine
phosphorylation that has been shown to also associate with Grb2 and
phospholipase C-
1 in T
cells(18, 19, 20, 21) .
Using both parental and transfected Jurkat T cells, we have demonstrated in vivo association between pp36/38 and PI-3-K p85. pp36/38 was the most prominent phosphotyrosyl protein associated with PI-3-K p85. In vitro binding experiments using GST fusion proteins demonstrated that pp36/38 (at least the fraction that is detectable with the anti-pY antibody) interacts specifically with the SH2 domains (but not the SH3 domain) of PI-3-K p85. Peptide competition experiments further demonstrated the requirement for phosphotyrosyl peptide motif recognition. Finally, filter binding assays clearly demonstrated that pp36/38 directly binds to PI-3-K p85. Consistent with this result, PI-3-K p85-associated pp36/38 was still observed (although reduced) after complete immunodepletion of Grb2 and its associated pp36/38 (data not shown).
It is likely that pp36/38, together with other
p85-associated proteins like
p120(13, 15) , plays an
important role in recruiting PI-3-K into TCR-induced signaling
complexes. We suggest that the interaction of PI-3-K p85 with
TCR-induced phosphotyrosyl proteins is likely to complement other
previously described mechanisms, such as Fyn SH3 binding to
proline-rich regions of PI-3-K
p85(26, 37, 43) , to recruit this enzyme into
TCR signaling. Binding of tyrosine-phosphorylated proteins or peptides
to PI-3-K p85 has been demonstrated to activate the associated 110-kDa
catalytic subunit of PI-3-K(44, 45) . Therefore, it is
quite possible that binding of pp36/38 to PI-3-K p85 may also activate
the PI-3-K enzyme.
The pp36/38-PI-3-K interaction was primarily observed in activated cells, reflecting the fact that at present we can detect pp36/38 only as a phosphotyrosyl protein. Consistent with a TCR activation-dependent association between pp36/38 and PI-3-K p85, in vitro binding and peptide competition experiments demonstrated that pp36/38-PI-3-K p85 interactions were exclusively SH2-mediated. However, we cannot rule out an activation-independent complex of unphosphorylated pp36/38 with PI-3-K. Such analyses await identification of the pp36/38 protein.
It is of significant interest
that pp36/38 interacts directly with the SH2 domains of PI-3-K p85
(this study) as well as those of phospholipase C-1 and Grb2 ( (18, 19, 20, 21) and this study).
Given the specificity of these different SH2 domains for distinct
phosphotyrosyl peptide motifs(17) , it is likely that various
SH2 domain-containing signaling proteins can concurrently interact with
pp36/38. Coimmunoprecipitation between Grb2 and PI-3-K p85 (Fig. 1A) and between Grb2 and phospholipase C-
1 (Fig. 1A) (20) is consistent with this
proposal. In additional experiments, we have observed that pp36/38
coimmunoprecipitates with Lck when Jurkat T cells are stimulated and
that the Lck SH2 (but not SH3) domain can bind to pp36/38 in vitro (data not shown). Thus, pp36/38 and other proteins with multiple
SH2-binding sites (e.g. p120
) (13) are likely to substitute for multiple SH2 docking sites
that are created on growth factor receptors (such as the epidermal
growth factor receptor) by autophosphorylation. In this regard, it is
noteworthy that pp36/38 in its tyrosine-phosphorylated form has been
demonstrated to be cell membrane-associated(20, 21) .