(Received for publication, June 7, 1995)
From the
We recently reported that interleukin-3 (IL-3) stimulation of
the murine IL-3-responsive cell line, B6SUtA, results in
the rapid phosphorylation of the
subunit of the IL-3 receptor
(IL-3R), not only on tyrosine residues but on serine/threonine
(Ser/Thr) residues as well. Since this occurred even at 4 °C, it
suggested that a Ser/Thr-specific kinase might be closely associated
with the IL-3R. To test this possibility, IL-3R complexes were isolated
with anti-IL-3R (
IL-3R) antibodies, and in vitro phosphorylation studies were undertaken. These revealed the
presence of a 110-kDa protein that was heavily phosphorylated in
vitro on serine and threonine residues and that bound selectively
to
-ATP-Sepharose beads. Moreover, this protein, which was not the
110-kDa subunit of phosphatidylinositol 3-kinase, was tyrosine
phosphorylated in response to IL-3 and was specifically labeled in
vitro with azido-[
P]ATP. These data,
together with in vitro kinase inhibitor studies, suggest that
an as yet uncharacterized H7- and staurosporine-sensitive 110-kDa
Ser/Thr kinase may be constitutively associated with the IL-3R and
activated following IL-3 stimulation. A comparison of IL-3R and
erythropoietin receptor complexes suggests that this 110-kDa protein
may be preferentially associated with the IL-3R.
The identification of receptor-associated proteins is an
essential step in elucidating downstream signals initiated by the
binding of growth factors to their receptors. In the case of
interleukin-3 (IL-3), ()a cytokine that stimulates the
proliferation and differentiation of various hemopoietic cell lineages
and activates the functions of mature macrophages, eosinophils, and
mast cells(1) , its high affinity receptor consists of two
subunits, designated
and
(1) . While the 70-kDa
chain is specific for IL-3(2) , the 140-kDa
subunit
(
) is shared by IL-3, granulocyte-macrophage
colony-stimulating factor, and IL-5(3) . In the mouse, there is
a second
subunit called
, which shares 91%
amino acid identity with
and is specific for
IL-3(4) . Although none of these receptor subunits possess
intrinsic kinase activity, IL-3 binding rapidly induces the tyrosine
phosphorylation of a group of cellular proteins, including its own
subunits(5, 6, 7, 8, 9, 10, 11, 12, 13) .
Concomitant with tyrosine phosphorylation, IL-3 receptors (IL-3Rs) also
become phosphorylated on serine and threonine (Ser/Thr)
residues(8, 9) . Since many IL-3-induced
phosphorylations occur very rapidly, even at 4 °C, it has been
suggested that the protein kinases responsible for these
phosphorylations and many of their protein substrates may be associated
with the IL-3R prior to ligand binding(1, 14) .
In
early studies to identify components of the IL-3R, I-IL-3
cross-linking studies revealed the presence of 140- and 70-kDa
proteins(15, 16, 17, 18, 19, 20) that
were subsequently shown to correspond to the IL-3R
and
(plus a cleavage product of the
)
chains(2, 4, 8, 21) , respectively.
More recently, the search for IL-3R-associated proteins has revealed
that Jak2, a member of the Jak family of tyrosine kinases(22) ,
binds to the membrane proximal region of the
subunit and becomes
activated following IL-3 binding(23) . This tyrosine kinase is
most likely responsible, at least in part, for the rapid tyrosine
phosphorylations that occur following IL-3 stimulation. In this regard,
another protein, which has been shown to bind to the IL-3R following
IL-3 stimulation, is hematopoietic cell
phosphatase(24, 25) . Association of this
tyrosine-specific phosphatase with the IL-3R
subunit appears to
lead to dephosphorylation of the IL-3R and Jak2 and down-regulation of
the response of hemopoietic cells to IL-3(24, 25) .
The kinase(s) responsible for the rapid Ser/Thr phosphorylation of the
IL-3R, however, has not as yet been identified, but the fact that these
phosphorylations have been observed at 4 °C suggests that it might
be receptor associated as well.
In this study, we have investigated
whether a Ser/Thr kinase is associated with the IL-3R using both
anti-IL-3R (IL-3R) antibody and biotinylated IL-3/streptavidin
agarose-based approaches. Our results suggest that an H7- and
staurosporine-sensitive 110-kDa Ser/Thr kinase is constitutively
associated with the IL-3R, becomes tyrosine phosphorylated in vivo in response to IL-3, and may be more strongly associated with the
IL-3R than the EpR.
Figure 1:
Identification of IL-3R-associated
proteins via in vitro phosphorylation of IL-3R
immunoprecipitates. A, B6SUtA
cells, incubated
with IL-3 for 5 min at 37 °C, were lysed, and proteins were
immunoprecipitated with
IL3R (or aGST, as control) antibodies in
the presence or absence of antigen, i.e. a GST fusion protein
containing the intracellular domain of the IL-3R
subunit (FU). In vitro phosphorylations were performed using
[
-
P]ATP, and the labeled proteins were
identified using SDS-PAGE, with a 10% polyacrylamide gel, and
autoradiography. B, lysates from the same cells were
immunoprecipitated with
IL3R antibodies (lane1)
or incubated directly with protein A-Sepharose (lane2), and the eluted proteins were identified using
SDS-PAGE, with a 6% polyacrylamide gel, and silver
staining.
To compare the intensities of
the in vitro phosphorylated protein bands in our IL-3R
immunoprecipitates with their protein levels, these precipitates were
subjected to SDS-PAGE and silver staining (Fig. 1B). As
expected, the IL-3R
subunit was the most intensely staining band
(apart from the IgG heavy chain). Interestingly, though, the heaviest
P-labeled band, migrating with an apparent molecular mass
of 110 kDa, appeared to be only a minor component within the IL-3R
complex, as assessed by silver staining.
To determine if these
IL-3R-associated proteins were present within the IL-3R complex prior
to ligand binding, IL-3R immunoprecipitates from IL-3-stimulated
and -unstimulated cells were subjected to in vitro phosphorylation with [
P]ATP and analyzed
using SDS-PAGE and autoradiography. As can be seen in Fig. 2A (leftpanel), all of the
proteins detected following IL-3 stimulation were present before
stimulation. However, on occasion, an increase in the intensity of some
of the bands was observed following exposure to IL-3. This
pre-association is consistent with previous 4 °C
results(9, 14) .
Figure 2:
The IL-3R-associated proteins are
constitutively associated, and p110 binds specifically to
-phosphate-linked ATP-Sepharose and is phosphorylated on Ser/Thr
residues. A, B6SUtA
cells, incubated with or
without IL-3, were lysed, and the proteins were immunoprecipitated with
IL-3R antibodies and either subjected directly to in vitro phosphorylation with [
-
P]ATP and
SDS-PAGE (lanes1 and 2) or dissociated by
freezing and thawing, incubating with ATP-Sepharose, and in vitro phosphorylating with [
-
P]ATP. The
labeled proteins were then released by boiling in SDS-sample buffer (lanes3 and 4) or eluted with 1 mM ATP (lanes5 and 6) and subjected to
SDS-PAGE and autoradiography. B, phosphoamino acid analysis of
p110, phosphorylated while bound to
ATP-Sepharose.
Figure 3:
The IL-3R-associated p110 is specifically
labeled with azido-[P]ATP. B6SUtA
cells were treated with bIL-3, lysed, and incubated with
streptavidin agarose beads. The beads were washed extensively and
exposed to azido-[
P]ATP, in the presence or
absence of excess ATP, and boiled in SDS-sample buffer for SDS-PAGE and
autoradiography.
Figure 4:
The IL-3R-associated 110-kDa protein
kinase is inhibited by staurosporine and H7 and is not the 110-kDa
subunit of PI 3K. B6SUtA cells were treated with or without
IL-3 for 5 min at 37 °C, and IL-3R complexes were
immunoprecipitated with
IL-3R antibodies. The complexes were
dissociated by freeze-thawing and were incubated with ATP-Sepharose.
Bound proteins were phosphorylated in vitro in the presence
and absence of various kinase inhibitors. A, lanes1 and 2, no inhibitor; lane3,
20 mM compound 3; lane4, 1 µM staurosporine; lane5, 100 µM H7; lane6, 100 µg/ml genistein; lane7, 8 µM herbimycin A; lane8, 1 µM H89; lane9, 4
µM chelerythrine; lane10, 10 µM tyrphostin B42; lane11, 10 µM tyrphostin B46. B, the effect of different concentrations
of H7 and staurosporine on the in vitro phosphorylation of
p110. lanes1 and 2, no inhibitor; lane3, 50 µM H7; lane4, 25
µM H7; lane5, 10 µM H7; lane6, 0.01 µM staurosporine, lane7, 0.03 µM staurosporine; lane8, 0.1 µM staurosporine. C,
B6SUtA
cell lysates were immunoprecipitated with
IL-3R
antibodies or
p85 (of PI 3K) antibodies, and the precipitates were
phosphorylated in vitro with
[
-
P]ATP in the presence or absence of 1
µM Wortmannin and subjected to SDS-PAGE and
autoradiography.
Since it was recently shown that the 110-kDa subunit of PI 3K
phosphorylates not only phospholipids but proteins as
well(34, 35) , and since we and others have reported
that PI 3K associates with certain cytokine
receptors(31, 36, 37) , we investigated
whether the IL-3R-associated 110-kDa protein might be the p110 subunit
of this enzyme. To test this, B6SUtA cells, incubated with
or without IL-3, were lysed and immunoprecipitated with
IL-3R or
p85 (of PI 3K) antibodies, and the precipitates were subjected to in vitro phosphorylation in the presence and absence of
Wortmannin, a specific inhibitor of PI 3K(38) . As can be seen
in Fig. 4C, Wortmannin dramatically inhibited the
phosphorylation of the p110 subunit of PI 3K but not the p110 present
in the IL-3R complex, strongly suggesting that the heavily
phosphorylated 110-kDa protein in the IL-3R complex was not the 110-kDa
subunit of PI 3K. This gel also revealed that the p110 subunit of PI 3K
migrated slightly more slowly than the 110-kDa IL-3R-associated
protein, further proving their non-identity.
Figure 5:
The 110-kDa serine/threonine kinase is
tyrosine phosphorylated following IL-3 stimulation. A,
B6SUtA cells, treated with or without IL-3 for 5 min at 37
°C, were lysed, and proteins were immunoprecipitated with
IL-3R antibodies. SDS-PAGE and Western analysis was then carried
out with the
PY, 4G10 (leftpanel). The blot was
stripped and reprobed with
IL-3R antibodies to demonstrate equal
loading (rightpanel). B, B6SUtA
cells were treated with bIL-3, lysed without prior DSP
cross-linking, and incubated with streptavidin beads. The beads were
washed extensively, treated with azido-[
P]ATP in
the presence and absence of excess ATP, and then boiled in 50 µl of
1% SDS. The eluted proteins were diluted to 1 ml with 0.5% Nonidet P-40
lysis buffer and subjected to immunoprecipitation with
PY beads.
The
PY-immunoprecipitated proteins were eluted by boiling in
SDS-sample buffer for SDS-PAGE and
autoradiography.
To investigate whether IL-3
activated the IL-3R-associated p110 Ser/Thr kinase, B6SUtA cells were labeled with [
P]orthophosphate,
cooled to 4 °C, and incubated for 10 min at 4 °C with or
without IL-3. The cells were then lysed, and proteins were
immunoprecipitated with
IL-3R antibodies and subjected to SDS-PAGE
and autoradiography. As can be seen in Fig. 6A, several
IL-3R-associated proteins showed increased phosphorylation, even at 4
°C, in response to IL-3. Phosphoamino acid analysis of one of these
proteins (i.e. the IL-3R
subunit itself) revealed that
this increase was due to increases in both tyrosine and Ser/Thr
phosphorylation (Fig. 6B). This finding, together with
our other results to this point, suggested that p110 becomes activated
following IL-3 stimulation.
Figure 6:
The IL-3R subunit is phosphorylated
on Ser/Thr residues at 4 °C in response to IL-3. A,
B6SUtA
cells, labeled with
[
P]orthophosphate, were treated with or without
IL-3 for 10 min at 4 °C and lysed; the proteins were subjected to
immunoprecipitation with
IL-3R antibodies. The immunoprecipitated
proteins were then electrophoresed on SDS-gels and subjected to
autoradiography. B, the 140-kDa IL-3R
subunit band from
the gel was eluted and subjected to phosphoamino acid
analysis.
Figure 7:
The 110-kDa protein kinase is associated
more with the IL-3R than with the EpR. A, B6SUtA and BA-ER cells were stimulated with IL-3 or Ep and lysed;
proteins were immunoprecipitated with
IL-3R or
EpR
antibodies, subjected to in vitro phosphorylation, SDS-PAGE,
and autoradiography. B, BA-ER cells were stimulated with bIL-3
or biotinylated Ep, cross-linked with DSP, and lysed; the proteins were
exposed to streptavidin-agarose beads. Bound proteins were labeled with
[
P]ATP, boiled off with SDS, diluted, and
subjected to immunoprecipitation with
PY beads, SDS-PAGE, and
autoradiography.
In the present study, we have demonstrated that there is a
Ser/Thr kinase constitutively associated with the IL-3R (as evidenced
by the in vitro phosphorylation of p110 on serines and
threonines (Fig. 2, A and B)) and that this
kinase is most likely the 110-kDa IL-3R-associated protein. Preliminary
evidence for the latter comes from our in vitro phosphorylation studies with IL-3R immunoprecipitates, which
showed that this minor protein, as assessed by silver staining, was the
most heavily labeled. Since many protein kinases have been shown to be
heavily phosphorylated in vitro, this prompted us to
investigate this possibility further. Further evidence for this p110
being a kinase was obtained from
-ATP-Sepharose binding studies
(which demonstrated a marked enrichment of the 110-kDa protein from
disrupted IL-3R complexes) and specific labeling of p110 with
azido-[
P]ATP. Our results are consistent with an
earlier report by Schreurs et al.(44) who
demonstrated that a Ser/Thr kinase remains associated with the IL-3R
following bIL-3/streptavidin agarose enrichment of the latter. We also
attempted to demonstrate that this protein was a kinase by SDS-PAGE
renaturation assays(45) , but, while this procedure
successfully identified kinases present in total cell lysates, it did
not renature the kinase(s) present in our
IL-3R
immunoprecipitates. Characterization of this 110-kDa protein kinase
with various kinase inhibitors revealed that it was markedly inhibited
by the Ser/Thr kinase inhibitors, staurosporine and H7, but not by the
protein kinase C-specific inhibitors compound 3 or chelerythrine, nor
the protein kinase A-specific kinase inhibitor H89, nor the tyrosine
kinase inhibitors genistein, herbimycin A, and tyrphostins B42 and 46.
Interestingly, in this regard, when we added either H7 or staurosporine
to
IL-3R immunoprecipitates to inhibit the in vitro phosphorylation of p110, we inhibited the Ser/Thr phosphorylation
of all the other proteins in the complex, consistent with all the
phosphorylations being carried out by one Ser/Thr kinase. Based on its
molecular mass and its sensitivity to these kinase inhibitors, p110
does not appear to be either one of the known protein kinase C isoforms
or protein kinase A and may therefore be an as yet uncharacterized
Ser/Thr kinase. In this regard, IL-3 has been shown to modulate the
activity of several Ser/Thr kinases, including
Raf-1(46, 47) , protein kinase C(48) , and
interferon-inducible double-stranded RNA-dependent kinase(49) ,
but none of these kinases have been shown to associate with the IL-3R
and none possess a molecular mass close to 110 kDa.
To determine
whether the p110 reported here was the Ep and IL-3-induced major
90-100-kDa tyrosine-phosphorylated protein previously reported (10, 50, 51) , we co-electrophoresed PY
immunoprecipitates from Ep and IL-3-stimulated BA-ER cell lysates with
our in vitro
P-labeled
IL-3R
immunoprecipitates on two-dimensional O'Farrell gels and found
that the p110 detected by autoradiography was distinct from the
90-100-kDa protein detected by anti-PY Western blots (data not
shown).
In summary, we have demonstrated that a group of proteins specifically and constitutively associates with the IL-3R and that one of these proteins is an as yet unidentified 110-kDa serine/threonine kinase. Comparison of IL-3R and the EpR immune complexes suggests that this kinase has a higher affinity for the IL-3R than for the EpR, and this may provide a clue to the differences seen when these two receptors are triggered in Ba/F3 cells.