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(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
Human interleukin-3 (IL-3) ( The human
Figure 1:
Structure of the IL-3
receptor
Figure 2:
In vitro phosphorylation of
Figure 3:
Phosphorylation of
Figure 4:
Phosphoamino acid analysis of
Figure 5:
Stable complexes of Src and Janus family
kinases from TF-1 or 3T3 cell lysates with
Figure 6:
Stable complex of PI 3-kinase from TF-1 or
3T3 cell lysates with
Whether the human IL-3 receptor Using GST fusion
proteins containing the membrane proximal regions of the cytoplasmic
tail of the 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 Further
investigation of the ability of these domains to associate with
cytoplasmic signaling molecules revealed that at least two Src family
kinases, p93 It has also been shown recently that the Janus family kinase JAK-2
associates with the IL-3 receptor 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
Volume 270,
Number 12,
Issue of March 24, 1995 pp. 6886-6893
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Subunit That Signals DNA Synthesis in NIH 3T3 Cells Specifically
Binds a Complex of Src and Janus Family Tyrosine Kinases and
Phosphatidylinositol 3-Kinase (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
)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. 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.
DNA Construction
cDNAs for the and
subunits of the human IL-3 receptor were cloned from
TF-1 cells by reverse transcription and polymerase chain reaction
(PCR). DNA sequencing was performed to confirm the fidelity of the
amplified DNA products. They were then cloned into mammalian cell
expression vectors pREP10 and pcDNA1neo (Invitrogen), respectively, as
described previously by Weiss et al.(22) . Mutants of
the human , 562,
517, and 455 were prepared by the
polymerase chain reaction method also as described
previously(22) . Primers were synthesized by phosphoramidite
chemistry on an Applied Biosystems Automated DNA synthesizer. All PCR
products were sequenced to confirm the fidelity of amplification. The
mutant subunits were also cloned into the pcDNA1neo
expression vector. To construct fusion proteins, DNA fragments encoding
the IL-3 receptor subunit domains were amplified from
cDNA by using the following primer pairs in
polymerase chain reactions (PCR): 517, 5` ATC GAA TTC
CCC TCG CTG TCC CCG AA 3` and 5` ATC GGA TCC TAT ACG GGT ACA GGC TGC G
3`; 562, 5`ATC GAA TTC CCC TCA GGT GTC TGG GA 3` and
5` ATC CGA TCC TAT ACG GGT ACA GGC TGC G 3`. The primers encode the
unique restriction sites underlined (BamHI GGATCC and EcoRI GAATTC) which facilitate subsequent cloning and 2 extra
base pairs at the 5` end for stability and increased efficiency of
restriction digestion. First strand cDNA synthesis was performed by
previously described methods(22) . The PCR was continued for 30
cycles at: 95 °C for 1 min, 50 °C or 43 °C for 2 min (for
517 and 562, respectively), and 72
°C for 3 min in a buffer containing 2 mM MgCl
.
The fidelity of all PCR products was determined by cloning the PCR
products into the vector PCR 1000 (Invitrogen), as recommended by the
manufacturer, and sequencing the cloned fragment using an Applied
Biosystems automated DNA sequencer (Applied Biosystems). All PCR
sequences were found to be 100% faithfully amplified.Expression of GST-
The cloned PCR fragments were restricted with EcoRI and BamHI and ligated into the vector pGEX3X
(Pharmacia Biotech Inc.) to produce GST fusion proteins. The ligated
pGEX3X vector was used to transform Escherichia coli strain
DH5517 and GST
562 and Coupling to Glutathione-Sepharose
Beads for fusion protein expression. Transformants were grown,
induced for fusion protein expression, and lysed by methods recommended
by the vector manufacturer (Pharmacia). Clarified lysates were
incubated with a 50:50 (v/v) slurry of glutathione-Sepharose beads
(Pharmacia) in equilibration buffer (20 mM HEPES, 100 mM KCl, 0.5 mM EDTA, 1 mM dithiothreitol, pH 7.5)
and then washed extensively with phosphate-buffered saline containing
1% Triton X-100. Washed beads were stored in equilibration buffer.Transient Transfection of NIH 3T3 Cells
The human
IL-3 receptor subunit cloned into pREP10 or subunit mutants cloned into pcDNA1neo (5 µg of each) were
transiently transfected into subconfluent cultures of NIH 3T3 cells
using DEAE-dextran essentially as described previously by Kriegler (23) and Watanabe(7) . The method can yield
transfection efficiencies up to 80% (20) . The percentage of
IL-3 receptor positive cells was determined and used as an index of
transfection efficiency. This was done for each experiment, and
transfection efficiencies of 60-80% were obtained. We have also
found a constant level of IL-3 receptor expression for up to 72 h after
transfection.Measurement of DNA Synthesis
Twenty four h after
transfection cells were trypsinized, washed in serum-free
Dulbecco's modified Eagle's medium, and replated in
Dulbecco's modified Eagle's medium containing 0.5% calf
serum in 96-well plates (10
cells/well). In some
experiments, the cells were cultured in the complete absence of serum.
The cells were serum-deprived for 24 h and then stimulated with IL-3
(20 ng/ml) or 3% calf serum for 15 h.
[
H]Thymidine (1 µCi/well) was then added, and
the cells were incubated for an additional 4 h. Cells were then
harvested in a Packard Filtermate 196, and radioactivity was determined
by direct counting in a Packard Matrix 9600 Direct Beta Counter.Mammalian Cell Culture and Preparation of Cell
Lysates
TF-1 and NIH 3T3 cells were propagated as described
previously(7) . For preparation of lysates, 2 
10
cells were collected by centrifugation and resuspended in 1.0 ml
of lysis buffer (0.5% Nonidet P-40, 150 mM NaCl, 10 mM Tris-HCl, pH 7.3, 2.0 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 0.4 mM EDTA, 10 mM NaF, 2
µg/ml each of aprotinin and leupeptin, and 1 mM phenylmethylsulfonyl fluoride) and incubated on ice for 30 min.
Following centrifugation, the clarified cell supernatants were used in
further adsorption experiments.In Vitro Phosphorylation of IL-3 Receptor
Aliquots of the
clarified supernatants were incubated overnight at 4 °C with 50
µl of packed beads preadsorbed with 25-35 µg of fusion
protein or GST alone. The beads were then washed as described
previously and resuspended in kinase buffer (10 mM HEPES, pH
7.4, 10 mM MgCl Subunit Domains by Cell Lysate Adsorbates, 2 mM sodium
orthovanadate, and 1 mM phenylmethylsulfonyl fluoride)
containing 10 µCi of
[
-
P]ATP(22) . The kinase reaction
was incubated for 10 min at 30 °C. Samples were then washed with
lysis buffer, and proteins were solubilized in 2
Laemmli's sample buffer and boiled for 5 min. Proteins were
resolved by 7.5% SDS-polyacrylamide gel electrophoresis under reducing
conditions. After the gels were dried, phosphoproteins were detected by
autoradiography using Kodak XAR film at -70 °C.Factor Xa Cleavage of GST Fusion Proteins
Fusion
proteins coupled to beads were adsorbed with cell lysates and
phosphorylated in vitro as described above. The phosphorylated
fusion proteins were washed three times with lysis buffer, twice in
phosphate-buffered saline containing 1% Triton X-100, once in wash
buffer (50 mM Tris-Cl, 150 mM NaCl, pH 7.5), and
finally once in cleavage buffer (wash buffer containing 1 mM CaCl). Factor Xa (New England Biolabs) was used on a
2% (w/w of fusion protein) basis, and digestion was carried out at 25
°C on an end over end rotator. Aliquots were drawn at hourly
intervals, boiled in sample buffer, and resolved by SDS-polyacrylamide
gel electrophoresis under reducing conditions (12.5% polyacrylamide, 8 M urea). Very low molecular weight standards were used to
calibrate the protein molecular weights. Proteins were detected in
dried gels by autoradiography at -70 °C using Kodak XAR film. Phosphorylation of
Purified Src family kinases
were purchased from Upstate Biotechnology (Lake Placid, NY). For
phosphorylation by the purified Fes kinase, beads coupled to fusion
protein (25-35 µg) or GST were added to a buffer composed of
200 mM HEPES, pH 7.4, 100 mM MgCl Cytoplasmic Domains
with Purified Src Family Kinases, 100
mM MnCl, and, for Lyn and Lck, the buffer was 50
mM Tris, pH 7.0, 25 mM MgCl, 5 mM MnCl, and 0.05 mM NaVO. To each reaction 0.5 mM ATP
containing 2 µCi of [-P]ATP and 30
units of purified tyrosine kinase were added in a total volume of 50
µl. The reaction mixture was incubated for 30 min at 30 °C. At
the end of this incubation, the reaction mixture was solubilized in 2
Laemmli's sample buffer. The solubilized protein was
loaded on to 7.5% SDS-polyacrylamide gels for electrophoretic
separation under reducing conditions. Phosphoproteins were detected by
autoradiography at -70 °C after drying the gels.Association of Tyrosine Kinases and Signal Transduction
Proteins in Cell Lysates with IL-3 Receptor
Cytoplasmic domain fusion proteins or GST
(25-35 µg) coupled to beads were incubated with cell lysate
(2 Subunit
Cytoplasmic Domains 10 cells) overnight at 4 °C. After
incubation, the beads were extensively washed as described
previously(24) , and the washed beads were then boiled for 5
min in 2 Laemmli's buffer. Solubilized proteins were
resolved in 10% SDS-polyacrylamide gels under reducing conditions and
transferred to nitrocellulose membranes (Amersham). Immunoblotting was
performed using commercially characterized polyclonal and monoclonal
antibodies by procedures prescribed by the manufacturers. For all
primary polyclonal antibodies, the secondary antibody was sheep
anti-rabbit IgG. When a rat monoclonal antibody (anti-v-Fes) was used,
a mouse anti-rat antibody was used as a bridging antibody followed by
horseradish peroxidase-labeled goat anti-mouse IgG. Immunoreactive
bands were revealed by chemiluminescence (ECL System, Amersham Corp.).
All antibodies were purchased from Upstate Biotechnology except the
anti-v-Fes monoclonal antibody that was purchased from Oncogene
Sciences (Uniondale, NY).Phosphoamino Acid Analysis of Phosphorylated Cytoplasmic
Domains
The procedure of Cooper et al.(25) was
used for phosphoamino acid analysis. Fusion protein adsorbates from
cell lysates were phosphorylated by in vitro kinase assays,
and the proteins were resolved by SDS-polyacrylamide gel
electrophoresis. The separated proteins were transferred from the gel
to polyvinylidene difluoride membranes in CAPS buffer (150 mM CAPS, pH 11.0, and 10% methanol), and detection of phosphoproteins
was by autoradiography. The portions of the membranes containing the
phosphorylated cytoplasmic domains were cut out and digested using
constant boiling 6 N HCl (Pierce). Phosphoamino acids were
resolved by two-dimensional thin layer electrophoresis using a pH 1.9
buffer (2.5% formic acid, 7.8% acetic acid, 89.7% HO (v/v))
and a pH 3.5 buffer (15% acetic acid, 0.5% pyridine, 94.5%
HO (v/v/v)) in the first and second dimensions,
respectively. Detection of phosphoamino acids was by autoradiography of
the thin layer plate. Densitometric analysis of autoradiograms was
performed using a flat bed Epson ES 300C scanner and the NIH software
package Image 1.57.
Transient Transfection of NIH 3T3 Cells with Mutated
Human IL-3 Receptors
Watanabe and co-workers (7) have
reported that transient transfection of NIH 3T3 with both subunits of
the human GM-CSF receptor allows human GM-CSF to drive these cells into
DNA synthesis. Eder et al.(8) , however, were unable
to observe GM-CSF stimulation of DNA synthesis in NIH 3T3 cells after
stable transfection and selection of cell lines stably expressing the
receptor. We therefore chose to examine the response of NIH 3T3 cells
to human IL-3 after transfection of human IL-3 receptor constructs
using a DEAE-dextran transient transfection protocol similar to that
used successfully by Watanabe et al.(7) . Table 1shows that NIH 3T3 cells transiently transfected with the
intact IL-3 receptor and subunits and
subsequently serum-deprived (0.5% calf serum) will enter DNA synthesis
in response to stimulation with human IL-3. In addition, truncation of
the subunit at either amino acid 562 or 517 still
allowed the reconstructed human IL-3 vector to signal DNA synthesis in
quiescent cells. Truncation of the subunit membrane
proximal domain to amino acid 455, however, completely abrogated the
response to human IL-3. Thus, the membrane proximal 67 amino acids of
the subunit are a minimal domain for signaling DNA
synthesis. All transfectants showed a similar increase in
[H] thymidine incorporation in response to 3%
calf serum. Neither the full length 517 nor
562 mutations required the presence of 0.5% serum to
signal DNA synthesis in NIH 3T3 cells. These latter three constructions could signal DNA synthesis even if the transfected
cells were cultured in the complete absence (0%) of serum and the IL-3
was added in serum-free medium (data not shown).
Construction of
Having
established that these membrane proximal domains of the IL-3 receptor
can signal DNA synthesis, we wished to determine how they might
interact with downstream effector molecules. We therefore constructed
GST fusion proteins containing membrane proximal domains of the
Subunit Membrane
Proximal Domain Fusion Proteins and In Vitro Phosphorylation Assay of
Domains 517 and 562 subunit (Fig. 1). The domain
517 contains a motif PNPSKSH that is shared with
gp130, the signal-transducing subunit of the IL-6
receptor(20) . Within this motif, the sequence PNP appears to
be important for signaling in the IL-6 receptor. The
562 domain includes the Pro-X-Pro motif, but
also contains a region homologous to the p75 subunit of the IL-2
receptor that mediates binding of p56
to
this IL-2 receptor subunit(21) . We expressed these domains as
fusion proteins with GST and coupled them to glutathione-Sepharose
beads. These coupled constructs were then used as probes to identify
molecules in cell lysates that can bind to the subunit domains. Fusion protein (25-35 µg) coupled to
beads was incubated with aliquots of Nonidet P-40 lysates from
unstimulated TF-1 cells equal to 2 10 cells/ml. The
beads were extensively washed and subjected to an in vitro kinase assay with [-P]ATP. At the end
of the assay, beads were further washed to remove free ATP, and the
phosphorylated proteins were analyzed by polyacrylamide gel
electrophoresis and autoradiography. In Fig. 2A, lane 1, shows that GST protein alone (50 µg) coupled to
beads was not phosphorylated; however, GST fusion proteins containing
either the domain 517 (lane 3) or
562 (lane 5) were intensely phosphorylated in
the in vitro kinase assay. In addition to the domain
themselves, several kinases or kinase substrates with molecular masses
of 46-69 kDa or greater were also bound and phosphorylated. It
should be noted that in this experiment unequal amounts of fusion
protein were used in the in vitro kinase assay, and this
accounts for the difference in phosphorylation intensity between the
lanes (see Fig. 2B). Boiling the TF-1 lysate before
adsorption with either fusion protein (lanes 2 and 6)
reduced the in vitro phosphorylation to undetectable levels
demonstrating that the fusion proteins themselves have no intrinsic
protein kinase activity and their phosphorylation is due to protein
kinase activity. This was verified by addition of 100 µM FSBA, a protein kinase inhibitor to the in vitro kinase
assays for 517 (lane 4) and
562 (lane 7) adsorbates, respectively. The
inhibitor completely blocked phosphorylation of the adsorbed fusion
proteins. Thus, the phosphorylation is enzymatically mediated by
protein kinases. Fig. 2B shows that about equal
intensities of phosphorylation are achieved when equal amounts (35
µg) of either fusion protein are incubated with TF-1 cell lysate.
Preadsorption of the lysates with an excess of GST-Sepharose did not
result in any qualitative alteration in the precipitation of kinase
activity by the fusion proteins. Similar results were obtained when
phosphorylation experiments were performed in vitro with NIH
3T3 cell lysates (data not shown). Finally, to verify that the
subdomains themselves were being phosphorylated, we
cleaved both fusion proteins with Factor Xa after adsorption with TF-1
cell lysate and in vitro kinase assay. SDS-polyacrylamide gel
electrophoresis revealed that only those proteolytic fragments
representing cytoplasmic domain were significantly
phosphorylated (data not shown). These experiments indicate that the
517 and 562 domains can both
associate with protein kinases and are also themselves substrates for
phosphorylation by the associating kinases.
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.
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.
Phosphorylation of IL-3 Receptor
Since
residues 451 and 463 of the IL-3 receptor Cytoplasmic Domains by Purified Src Family Kinases cytoplasmic
domain are tyrosine residues, we determined whether these tyrosines are
available for phosphorylation by Src family tyrosine kinases. We chose
the Src family kinases, Lyn and Fes, reported to be associated with and
activated by the IL-3 receptor in hematopoietic cells, and Lck, a
related kinase present specifically in lymphocytic cells. Fig. 3shows that both Lyn and Lck are capable of phosphorylating
the tyrosines present in both 517 and
562, but Fes was only weakly active or inactive with
these fusion protein substrates. None of the kinases phosphorylated GST
alone. Thus, some but not all Src family kinases are capable of
phosphorylating these domains. Finally, it is important to note in Fig. 3that 562 was reproducibly a better
substrate for Src kinases than 517.
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.
Phosphoamino Acid Analysis of
Although it appears that purified Src family
kinases can phosphorylate these Cytoplasmic Domain after TF-1 Lysate Adsorption and in Vitro
Phosphorylation cytoplasmic domains,
previous investigations have shown that the major sites of tyrosine phosphorylation is not in this region(18) .
Therefore, we further investigated the nature of the residues
phosphorylated after adsorption of these cytoplasmic domains with TF-1
lysates and in vitro kinase assay. Phosphoamino acid analysis
was performed on acid hydrolysates of gel-purified phosphorylated
cytoplasmic domains using two-dimensional thin layer electrophoresis. Fig. 4compares the phosphoamino acid patterns obtained from
domains 517 and 562. In
562, about 77% of the phosphorylation is on threonine,
13% is on serine, and 2% on tyrosine. In 517, about
85% of the phosphorylation is on serine, 15% on threonine, and
phosphotyrosine was undetectable. Thus, serine/threonine kinase
activity accounts for a major proportion of the total phosphorylation
of these domains, although the region between 517 and 562 appeared to
enhance the phosphorylation of tyrosine residues 451 and 453. The
increased availability of 5 more threonines in the 520-562 region
may account for the shift to threonine phosphorylation in
562 compared to 517, or the
additional residues could alter peptide conformation and contribute to
the longer domain being a better substrate. The phosphorylation of
517 by the purified Src kinases but not by
TF-1-adsorbed tyrosine kinases is probably due to the greater amounts
of tyrosine kinase enzyme activity added to the in vitro assays compared to the low levels of tyrosine kinase actually
available in cell lysates.
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.
IL-3 Receptor Cytoplasmic
Although
the Domains
517 and 562 Associate with Src Family
and Janus Family Kinases in TF-1 and 3T3 Cell Lysates517 domain was not phosphorylated on tyrosine
after adsorption with TF-1 cell lysates, this domain could still be
capable of physically associating with cytoplasmic tyrosine kinases
important in signal transduction. To characterize complexes of
cytoplasmic tyrosine kinases with our subunit
cytoplasmic domains, immunoblotting of NIH 3T3 and TF-1 adsorbates was
performed. We chose antibodies against kinases that had been shown to
be activated by IL-3 or associated with the wild type subunit of
the IL-3 receptor. p56
is a hematopoietic cell-specific
kinase that had previously been shown to be activated by engagement of
the IL-3 receptor(15) . Immunoblotting of proteins associated
with 517 and 562 after incubation
with TF-1 lysates using rabbit anti-human Lyn polyclonal antibody
revealed the presence of Lyn immunoreactivity at the 56-kDa molecular
mass (Fig. 5A). No Lyn immunoreactivity was associated
with GST alone. If the TF-1 lysates were immunodepleted of Lyn protein
by pre-immunoprecipitation with anti-Lyn antibodies, prior to
incubation with cytoplasmic domain fusion proteins, then no Lyn
immunoreactivity could be detected associated with the cytoplasmic
domains (Fig. 5B). Immunoprecipitation of TF-1 lysates
with preimmune serum did not alter Lyn association with the fusion
protein (data not shown). Thus, we have confirmed that
p56 is associated with the subunit
minimal signaling domain. Other studies have shown that GM-CSF and IL-3
induce phosphorylation and activation of p93
, another
hematopoietic cell-specific non-receptor tyrosine kinase(16) .
We have thus performed similar experiments to determine whether
p93 associates with the subunit cytoplasmic domains
using a rat monoclonal antibody against v-Fes which recognizes all
mammalian c-Fes antigens. An immunoreactive band at the 93-kDa position
characteristic of c-Fes was physically associated with both cytoplasmic
domains of the subunit, but not with GST alone (Fig. 5C). Immunodepletion of the TF-1 lysates with
anti-Fes antibody prior to adsorption with fusion proteins also
abrogated the detection of Fes immunoreactivity confirming the identity
of the Fes band associated with the fusion proteins. Finally, it has
recently been suggested that the Janus family kinase JAK-2 associates
with the subunit and is crucial to hematopoietic receptor
signaling(17) . Therefore, we also probed for the association
of JAK-2 with these cytoplasmic domains using rabbit anti-mouse JAK2
polyclonal antibody that recognizes the human protein. Again,
25-35 µg of fusion protein was incubated with aliquots of
cell lysate equivalent to 2 10 TF-1 cells/50 ml,
and the adsorbed proteins were probed for the presence of p130 JAK-2
with JAK-2 polyclonal antibody. We could reproducibly detect JAK-2 at
the appropriate molecular weight associated with both the
517 region and the 562 region, but
not with GST alone in lysates of TF-1 or 3T3 cells (Fig. 5D). The immunological signal with JAK-2,
however, was always significantly weaker than with the Src family
kinase antibody probes. This is probably not due to a paucity of JAK-2
in our TF-1 lysates, because immunoblots of 5 µl of lysate from 2
10 TF-1 cells/ml yielded a strong signal with the
same antibody (Fig. 5D). Again, pre-immunodepletion of
the 3T3 or TF-1 lysates with anti JAK-2 antibody but not preimmune
serum abrogated the detection of JAK-2 activity after adsorption with
fusion protein.
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-p56antibody (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.
IL-3 Receptor
Engagement of the IL-3
receptor results in the tyrosine phosphorylation of a number of
proteins involved in signal transduction. Among these proteins are
p21 Cytoplasmic Domains Bind
Phosphatidylinositol 3-Kinase (PI 3-Kinase) but Not GTPase Activating
Protein (GAP), Vav, Sos1, or Grb2
-GAP(26, 27, 28) , PI
3-kinase(29) , and Vav and Shc(30) . In addition,
tyrosine-phosphorylated Shc has been shown to associate with the
docking protein Grb2 and the guanine nucleotide exchange protein
Sos1(29, 30) . We therefore determined whether either
the -subunit cytoplasmic domain 517 or
562 participates in the formation of such signaling
complexes. To examine this possibility, fusion protein adsorbates
obtained after incubation of cytoplasmic domains 517
and 562 with TF-1 lysates were analyzed by
polyacrylamide gel electrophoresis and immunoblotting. Rabbit
polyclonal antibodies against the p85 subunit of PI 3-kinase that
recognize the human protein detected an 85-kDa protein adsorbed to the
517 and 562 fusion proteins (Fig. 6A). No protein immunoreactivity was detected in
adsorbates from either beads coupled to GST alone or fusion protein
adsorbed with TF-1 cell lysate and probed with preimmune serum. In
contrast, adsorbates from 517 or 562
showed no immunoreactivity when a rabbit anti-GAP polyclonal antibody
that can recognize human and mouse antigens was used as a probe (Fig. 6B). Both the anti-PI 3-kinase antibody and the
anti-GAP antibody readily detected their respective antigens in total
cell lysate from TF-1 cells. We also probed both cytoplasmic domain
adsorbates for immunoreactivity associated with the signal transducing
proteins Vav, Grb2, or Sos1 but did not detect the presence of these
molecules in adsorbates from either cytoplasmic domain using
commercially available antibodies (data not shown).
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.
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. 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. 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. 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. 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. /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.
)
We thank Dr. Steven Ullrich for his assistance with
the phosphoamino acid analysis and Tammy Krogman for technical
assistance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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