(Received for publication, July 19, 1994; and in revised form, January 19, 1995)
From the
The high affinity human interleukin-3 receptor is a
heterodimeric protein consisting of an and
subunit. The
subunit is responsible for
receptor signal transduction. We have shown that a membrane proximal
domain of the cytoplasmic tail of the human
subunit
(amino acids 451-517) is minimally required for human IL-3 to
signal DNA synthesis in quiescent transfected NIH 3T3 cells.
Glutathione S-transferase (GST) fusion proteins of this
451-517 region and another region 451-562 that includes an
acidic domain previously shown in other receptors to bind Src family
kinases were constructed. Purified Lyn and Lck kinase, but not Fes,
could phosphorylate tyrosines in both domains. Adsorption with lysates
from the human IL-3-dependent hematopoietic cell line (TF-1) or 3T3
cells and in vitro phosphorylation showed that both these
domains were intensely phosphorylated. Phosphoamino acid analysis,
however, revealed that the majority of phosphorylation was on serine
and threonine rather than tyrosine. Adsorption of these domains with
3T3 or TF-1 cell lysates, followed by immunoblotting, showed that
cytoplasmic tyrosine kinases Lyn, Fes, and JAK-2 could also stably
associate with both domains; however, Src family kinases are more
strongly recognized by both regions than JAK-2 kinase. In addition,
phosphatidylinositol 3-kinase from cell lysates was also found stably
associated with these domains, but GTPase activating protein, Vav,
Sos1, or Grb2 were not.
Human interleukin-3 (IL-3) ()and
granulocyte-macrophage colony stimulating factor (GM-CSF) are
hematopoietins that support the survival and differentiation of a large
number of myeloid progenitor cells(1) . Human interleukin-5
(IL-5) is a more restricted hematopoietin whose target is more mature
cells, especially eosinophils and some B-cells(2) . The
receptors for human IL-3, GM-CSF, and IL-5 comprise a family of
heterodimeric proteins that share a common structure, and their ligands
can cross-compete for receptor binding(3, 4) . The
high affinity receptor for each of these growth factors consists of a
unique
subunit coupled to a common
subunit
(
)(5) . The
subunits are
cytokine-specific and bind their respective ligand with low affinity.
The
subunit does not bind any ligand but allows for
the formation of a high affinity receptor and signal
transduction(5) . Transfection of the human receptor GM-CSF
and
subunits into murine hematopoietic cells
has reproducibly allowed human GM-CSF to support survival and
proliferation of these cells(5, 6) . Data on whether
the human GM-CSF receptor transfected into murine fibroblasts allows
these nonhematopoietic cells to proliferate in response to human GM-CSF
are conflicting(7, 8) . Thus, it is not yet clear
whether nonhematopoietic cells contain all the downstream effector
molecules required to allow the
subunit to signal
cell proliferation in such cells.
The human subunit is a 120-kDa glycoprotein with a large cytoplasmic
domain(6) . The cytoplasmic domain contains neither consensus
sequences for intrinsic tyrosine kinase activity nor SH2/SH3 domains;
however, IL-3, GM-CSF, and IL-5 all induce similar patterns of tyrosine
phosphorylation(9, 10, 11) . This tyrosine
phosphorylation appears to be required for signaling cell
proliferation(5, 11, 12, 13, 14, 15, 16, 17) .
The cytoplasmic tail of the
protein contains 431
amino acids(4) , and it has been suggested that the carboxyl
terminus of the protein can be truncated at amino acid 517
(
517), leaving a membrane proximal 67 amino acids,
without loss of signal transduction capability in hematopoietic cells;
however, the
517 signals a much reduced level of
tyrosine phosphorylation(18, 19) . Interestingly, the
cell proliferation signal transduced by this membrane proximal
mutant was blocked by the tyrosine kinase inhibitor
herbimycin A(16) . The
517 domain contains a
Pro-X-Pro amino acid motif (where X is Asn in
) that is shared with the p130 subunit of the IL-6
receptor and is important in signaling tyrosine phosphorylation and
cell proliferation(20) . Adjacent to amino acid 517 is a region
between amino acids 520 and 562 that is rich in acidic amino acids and
homologous to an acidic region in the p75 IL-2 receptor subunit that
appears to be required for binding
p56
(21) . It is thus possible that these
regions may contribute to binding cytoplasmic tyrosine kinases and
other signal transduction molecules. No data are available on the
interactions of these regions with downstream signaling proteins. Thus,
although deletion mutagenesis and sequence analysis have helped define
the organization of the
subunit and the presence of
motifs capable of associating with signaling proteins, no molecular
studies are available that directly investigate the physical
interaction of
subunit cytoplasmic domains with
signaling effector proteins from hematopoietic and nonhematopoietic
cell lines. In this study, we have investigated whether the intact
or membrane proximal mutants of this subunit can
signal quiescent murine fibroblasts to enter DNA synthesis. Further, we
have prepared glutathione S-transferase (GST) fusion proteins
containing these
membrane proximal domains to analyze
their interaction with purified effector proteins and effector proteins
in the cytoplasm of human hematopoietic (TF-1) and murine fibroblast
(NIH 3T3) cells.
Figure 1:
Structure of the IL-3
receptor subunit. A, the organization of the
IL-3 receptor
subunit is represented in schematic form: EC, extracellular domain; WSXWS,
Trp-Ser-variable-Trp-Ser box; TM, transmembrane domain; gp130 homology, region containing Pro-Asp-Pro motif; and Acid rich, region containing 11 acidic and 1 basic amino acid
IL-2 receptor homology region. The amino acids (a.a.) within
each domain are numbered. B, schematic representation of the
subunit mutants
455,
517,
562, and wild type (WT)
used to transfect NIH 3T3 cells. Amino acid 451 is the first amino acid
in the cytoplasmic domain. C, amino acid sequences from the
subunit that are expressed as fusion proteins. The starred residues identify a motif homologous in the IL-6
receptor gp130, the boxed motif is shared between the human
and murine IL-3 receptor
subunits, and the underlined amino acids are the acidic residues homologous to
the acidic rich region in the p75 subunit of the IL-2
receptor.
Figure 2:
In vitro phosphorylation of
cytoplasmic domains after adsorption with TF-1 cell
lysates. A, cytoplasmic domain fusion proteins or GST alone
were coupled to GST-Sepharose beads. The beads were loaded with
20-60 µg of GST or GST fusion protein without equalizing the
protein loadings. GST alone was incubated with native TF-1 cell lysate (lane 1), GST
517 with boiled cell lysate (lane 2) or native TF-1 lysate (lanes 3 and 4), and GST
562 with native TF-1 lysate (lanes 5 and 7) or boiled TF-1 lysate (lane
6) overnight at 4 °C. After washing, the absorbed beads were
resuspended in kinase buffer for in vitro kinase assay with
[
-
P]ATP. In some of the kinase assays the
protein kinase inhibitor FSBA was included (lanes 4 and 7). Samples were washed five times after in vitro kinase assay and solubilized in sample buffer for resolution on
reducing SDS polyacrylamide gels. The dried gels were autoradiographed
at -70 °C using Kodak XAR film to visualize phosphorylated
proteins. B, equal amounts of each protein (GST alone or GST
fusion protein) were coupled to beads, adsorbed with TF-1 cell lysate,
and subjected to in vitro kinase assay as described in A.
Figure 3:
Phosphorylation of
cytoplasmic domains by purified Src family kinases. Cytoplasmic domain
fusion proteins or GST alone (25-35 µg) were coupled to
GST-Sepharose beads and suspended in the appropriate reaction buffer
with 30 units of either purified p56
(A), p56
(B), or
p93
(C), and
[
-
P]ATP. The reaction mixture was incubated
for 30 min at 30 °C and then solubilized in reducing sample buffer.
Proteins were resolved by SDS-polyacrylamide gel electrophoresis.
Phosphoproteins were detected by autoradiography at -70 °C
using Kodak XAR film.
Figure 4:
Phosphoamino acid analysis of cytoplasmic domains phosphorylated by TF-1 cell lysates.
Cytoplasmic domains of the
subunit were subjected to in vitro kinase assays after adsorption with TF-1 cell lysates
and resolved by gel electrophoresis as described in the legend to Fig. 3. The separated proteins were transferred to
polyvinylidene difluoride membranes and detected by autoradiography.
Portions of the membrane containing phosphorylated cytoplasmic domain
fusion proteins were cut out and hydrolyzed using constant boiling HCl.
Phosphoamino acids were resolved by two-dimensional thin layer
electrophoresis and detected by autoradiography of the thin layer
plates.
Figure 5:
Stable complexes of Src and Janus family
kinases from TF-1 or 3T3 cell lysates with
cytoplasmic domains. Cytoplasmic domain fusion proteins or GST alone
(25-35 µg) coupled to beads were incubated overnight at 4
°C, with TF-1 cell lysates. After incubation, the beads were
extensively washed and solubilized in reducing sample buffer.
Solubilized proteins were resolved in 10% SDS-polyacrylamide gels.
Resolved proteins were transferred to nitrocellulose, and
immunoblotting was performed with rabbit polyclonal
anti-p56
antibody (A), rat monoclonal
anti-93
antibody (C), and rabbit
polyclonal anti-p130 JAK-2 antibodies (D). In B, the
lane labeled
is a
non-preimmunoprecipitated control while lane
is preimmunoprecipitated with
anti-Lyn antibody before gel electrophoresis and immunoblotting.
Immunoreactive bands were detected by
chemiluminescence.
Figure 6:
Stable complex of PI 3-kinase from TF-1 or
3T3 cell lysates with cytoplasmic domains. Adsorption
of fusion protein coupled beads with TF-1 or 3T3 cell lysates, and
immunoblot analysis was performed as in the legend to Fig. 5. A, immunoblotting was performed with antibody against the p85
subunit of PI 3-kinase. B, immunoblotting was performed with
rabbit polyclonal antibody against human GAP residues 171-448
that also recognizes mouse GAP. Immunoreactive bands were detected by
chemiluminescence.
Whether the human IL-3 receptor requires
lineagespecific downstream effector molecules or can signal in a
nonhematopoietic fibroblast background has been controversial. Watanabe et al.(7) have reported that the human GM-CSF
receptor reconstituted in NIH 3T3 cells induced tyrosine
phosphorylation, immediate early response gene induction, and DNA
synthesis in response to human GM-CSF. Eder and co-workers (8) also observed tyrosine phosphorylation and immediate early
response gene induction in response to human GM-CSF in NIH 3T3 cells
transfected with the human GM-CSF receptor complex, but were unable to
measure any increases in DNA synthesis. Thus, it has been unclear if
this hematopoietin receptor system could signal cell proliferation
ubiquitously in nonhematopoietic cells. Previous work from our
laboratory has shown that NIH 3T3 cells transiently transfected with
the human IL-3 receptor respond to human IL-3 by increases in tyrosine
phosphorylation and phosphatidylcholine-specific phospholipase C
activity, as well as translocation of protein kinase C(31) .
The experiments with transient transfection reported here extend these
findings and confirm that the human IL-3 receptor indeed can induce DNA
synthesis in NIH 3T3 cells in response to human IL-3. It is thus
possible that the inability of GM-CSF to signal cell proliferation in
the cell lines selected by Eder and co-workers (8) for stable
expression of the GM-CSF receptor may represent a loss of cellular
function acquired during the selection for stable receptor expression.
The transient transfection experiments that we have performed show that
both the
562 and
517 membrane
proximal domain truncations could also signal cell proliferation in
response to human IL-3 after transfection into NIH 3T3 cells. Thus,
these truncations function in a nonhematopoietic environment as they do
in murine hematopoietic BaF3 cells. In addition, our experiments also
showed that IL-3 could signal DNA synthesis through the wild type
or
517 even in the complete absence
of serum. Thus, the effector molecules activated by the IL-3 receptor
alone are sufficient to complete the signal pathway required to move
the cells from G
through to S phase.
Using GST fusion
proteins containing the membrane proximal regions of the cytoplasmic
tail of the subunit, we showed that both the
517 and
562 were intensely
phosphorylated in kinase assays after adsorption with hematopoietic
(TF-1) and fibroblast (NIH 3T3) cell lysates. The cytoplasmic domains
were also phosphorylated by the Lyn and Lck tyrosine kinases.
Interestingly,
562 was reproducibly a better substrate
for tyrosine phosphorylation by the purified Src family kinases. This
is consistent with our phosphoamino acid analysis of cytoplasmic domain
phosphorylation which revealed that tyrosines 451 and 453 were poorly
if at all phosphorylated by kinases after adsorption with hematopoietic
cell lysates and in vitro phosphorylation. Addition of the
518-562 region enhanced the phosphorylation of the two tyrosines
in the 451-517 region to 2% of the total phosphoamino acid
content. The fact that the
517 domain could be
phosphorylated by purified kinases is probably due to the far greater
amounts of enzyme activity units added to the in vitro kinase
assay compared to those present in the cell lysates. Thus, although the
major site for tyrosine phosphorylation on the
subunit is between Ser
and
Ser
(16) , the tyrosines at positions 451 and 453
may play a role in signaling after the binding of tyrosine kinases in
the full length
subunit.
The phosphoamino acid
analysis also revealed that both cytoplasmic domains can also bind
serine/threonine kinases. In fact, the major proportion of total
phosphorylation observed after adsorption with hematopoietic cell
lysates was serine/threonine phosphorylation. Such phosphorylation of
the subunit has not been well investigated in in
vivo studies, and it is not clear which kinase(s) contribute to
this phosphorylation. Similar results have been reported, however, for
the B-cell antigen receptor(24) . GST fusion proteins
containing the antigen receptor homology I (ARH1) motif bind both Src
family and serine/threonine kinases, and the majority of
phosphorylation of the ARH1 domains is on serine and threonine after
cell lysate adsorption and in vitro phosphorylation. In
IgM-associated Ig
and Ig
chains of the B-cell antigen
receptor there is also constitutive in vivo phosphorylation of
serine and threonine residues(24) . Further, the gp130 subunit
of the IL-6 receptor is constitutively phosphorylated on serine and
threonine residues, and activation of the IL-6 receptor increases the
amount of serine/threonine phosphorylation of this
protein(20) . Thus, both the IL-3 and IL-6 receptor signal
transducing subunits can associate with serine/threonine kinases,
although the role of serine/threonine phosphorylation in signaling
through the
subunit is unclear.
Further
investigation of the ability of these domains to associate with
cytoplasmic signaling molecules revealed that at least two Src family
kinases, p93 and p56
, can form stable
associations with
517 and
562 in
cytosolic preparations from TF-1 cells. The presence of Src family
kinases in a complex with this region is consistent with recent data
showing that the Src family kinases p53/p56
,
p62
, and p93
are activated in vivo during biological signaling through the IL-3/GM-CSF receptor
subunit (14, 15, 32) .
Further evidence that Src family kinases are critical to coupling
IL-3/GM-CSF receptor
subunits to biological responses
comes from the work of Linnekin et al.(33) examining
the effects of the Src family kinase HCK in GM-CSF signaling in HL-60
cells. HL-60 cells express high affinity receptors for GM-CSF, but
GM-CSF cannot signal cell proliferation in these cells unless the hck gene is induced with dimethyl sulfoxide or overexpressed
after transfection. Thus, our finding of Src family kinases complexed
with the membrane proximal domain accounts for the activation of these
kinases during biological signaling observed by other laboratories.
It has also been shown recently that the Janus family kinase JAK-2
associates with the IL-3 receptor subunit, and it has
been suggested that JAK-2 is central to cytokine receptor signaling (34, 35) . We could reproducibly detect JAK-2 binding
to both IL-3 receptor
cytoplasmic domains. The
association, however, appeared to be significantly weaker than the
association with Src family kinases. It is possible that a stronger
association with JAK-2 may occur at other sites in the IL-3 receptor
subunit that provide better structural recognition than the domains
examined here. This suggestion is completely consistent with the data
of Quelle and co-workers (36) who have shown that JAK-2 is
strongly activated by mutants of the IL-3 receptor
subunit truncated at amino acids 763 or 626 in transfected BaF3
cells; however, the
subunit truncated at amino acid
517 merely produced a weak activation of JAK-2. The weak association of
JAK-2 with the
517 region in vitro thus
correlates well with the weak activation of JAK-2 observed with the
517 mutant in vivo. Although Silvennoinen et al.(17) detected only JAK-2 associated with the
IL-3 receptor
subunit, our work and that of several
other groups would support the association of multiple tyrosine kinases
from both the Src and Janus family with the
subunit.
Recent studies of the initial events in GM-CSF receptor signal
transduction have shown that GM-CSF receptor occupancy additionally
results in the formation of a complex between
p53/p56/p62
and an 85-kDa protein
(immunologically related to the 85-kDa subunit of PI 3-kinase) with an
accompanying increase in PI 3-kinase activity. The tyrosine
kinase
PI 3-kinase complex can be immunoprecipitated with
antiphosphotyrosine antibodies(29) . Our experiments directly
showed that the minimal signaling domain
517 indeed
forms a stable complex with Src family kinases and PI 3-kinase. We
could find no immunological evidence for the presence of GAP, Sos1,
Vav, or Grb2 complexed with these domains. Thus, the interaction of the
minimal signaling domain with tyrosine kinases and PI 3-kinase appears
specific. The
517 domain does not contain any PI
3-kinase consensus binding sequence. It is, therefore, likely that PI
3-kinase does not bind directly to the peptide, but to specific domains
on the tyrosine kinases that have associated directly with the
cytoplasmic domains. Interactions of effector
molecules with such domains on Lyn, Fyn, and Blk have recently been
directly demonstrated in in vitro fusion protein binding
studies(37) . PI 3-kinase appears to bind specifically to
p56
or p60
, and this binding appears to
involve amino-terminal SH3 domains on these protein kinase
molecules(38) . Our data also correlate well with recent
studies in transfected BaF3 cells showing that PI 3-kinase is activated
after stimulation of GM-CSF receptors containing a
subunit truncated at amino acid 517, and that this kinase may
play a role in signaling cell proliferation(18) . In fact,
activation of PI 3-kinase and tyrosine phosphorylation are the only
measurable biochemical response to activation of this
517 mutant in transfected BaF3 cells(19) . Our
data would suggest that the activation of PI 3-kinase occurs in a
direct physical association with the
subunit domain. The
association of this region
517 with PI 3-kinase may in
fact be quite important to signaling cell proliferation because mutants
of the platelet-derived growth factor receptor(38) , CSF-1
receptor(39, 40) , and insulin receptor (41) that have reduced association with PI 3-kinase are
defective in their ability to induce mitogenesis.