(Received for publication, November 19, 1996)
From the Department of Inflammation/Autoimmune Diseases, Hoffmann-La Roche Inc., Nutley, New Jersey 07110-1199
The role of the cytoplasmic regions of
interleukin-12 receptors (IL-12R) 1 and
2 in stimulating
proliferation was examined. The transmembrane and cytoplasmic regions
of IL-12R
1 or IL-12R
2 were fused to the extracellular domain of
the epidermal growth factor (EGF) receptor, yielding chimeric receptors
E12R1 and E12R2, respectively. These chimeras were stably transfected
into BaF3 cells, a factor-dependent murine pro-B cell line.
Only E12R2 or E12R1+E12R2 transfectants were capable of
EGF-dependent proliferation. EGF-dependent
phosphorylation of E12R2, JAK2, Tyk2, and STAT3 was observed. JAK2 was
phosphorylated in E12R1-, E12R2-, and E12R1+E12R2-expressing cells.
However, direct associations were detectable only between E12R2 and
JAK2. Tyk2 phosphorylation was observed only in cells expressing E12R1
or E12R1+E12R2. In parallel with this activation pattern, direct
interactions only between Tyk2 and E12R1 were demonstrable.
Phosphorylation of STAT3 was observed in cells expressing E12R1, E12R2,
and E12R1+E12R2. The expression levels of STAT4 protein in BaF3 cells
are undetectable by the methods employed here; therefore, STAT4
phosphorylation was not observed. Taken together, the data indicate
that differential interactions take place between the cytoplasmic
regions of the two IL-12R subunits and JAK2/Tyk2 and that the
cytoplasmic region of IL-12R
2 alone is capable of delivering a
proliferative signal.
Interleukin-12 (IL-12)1 is a 75-kDa
heterodimeric cytokine composed of two disulfide-bonded subunits, p35
and p40 (1, 2). IL-12 is primarily produced by macrophages and
dendritic cells. It has pleiotropic effects on T and natural killer
cells, including the induction of interferon- secretion, the
stimulation of cell proliferation, and the promotion of a
Th1-type response (3).
IL-12 manifests its biological functions through interaction with
cell-surface IL-12 receptors (IL-12R). Three classes of IL-12-binding
sites were identified on the surface of PHA-activated human T
lymphoblasts and the human T cell line Kit225/K6 (4, 5): high affinity
(Kd = 5-20 pM), intermediate affinity (Kd = 50-200 pM), and low affinity
(Kd = 2-6 nM). The cloning of a
cDNA encoding a human IL-12R subunit that belongs to the gp130
subgroup of the cytokine receptor superfamily (IL-12R1; see below)
has been reported (6). However, when expressed in COS cells, IL-12R
1
binds IL-12 with only low affinity (Kd = 2-5
nM). We have recently cloned the cDNA for a second
IL-12R subunit (7). This newly identified IL-12R subunit is also
strongly related to gp130. These IL-12R subunits have therefore been
classified as IL-12R
1 and IL-12R
2 (7). Similar to IL-12R
1,
IL-12R
2 binds IL-12 with only low affinity when transfected into COS
cells. However, coexpression of
1 and
2 subunits in COS cells
gives rise to both high and low affinity IL-12-binding sites and a
receptor complex capable of signaling (7).
The signal transduction pathways utilized by IL-12 have not been fully characterized. Previous studies indicate that IL-12R can signal through the JAK/STAT signal transduction pathway (8). It has been reported that IL-12 induces tyrosine phosphorylation of JAK2 and Tyk2 in PHA-activated T lymphocytes (8). IL-12 also induces tyrosine phosphorylation and activation of STAT3 and STAT4 in Th1 cells and PHA-activated T lymphocytes (9, 10). Other reports suggest that IL-12 signaling may involve more than one pathway since IL-12 was shown to induce tyrosine phosphorylation of Lck in natural killer cells (11) and of the 44-kDa mitogen-activated protein kinase in T cells (12). Using EGFR/IL-12R chimeras, we now report an initial analysis of the interactions between the cytoplasmic regions of the two known IL-12R subunits and molecules belonging to the JAK/STAT signaling pathways.
Recombinant human EGF; polyclonal rabbit antisera against JAK1, JAK2, and JAK3; and mouse monoclonal antibodies against phosphotyrosine (clone 4G10) and against human EGFR (clone LA22) were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Polyclonal rabbit IgG preparations directed against human and mouse Tyk2, STAT1, STAT2, STAT3, and STAT4 were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Mouse mAb 13A9 specific for human EGFR (13) and the EGFR cDNA were the generous gifts of Brian Fendly (Genentech, South San Francisco, CA). Recombinant IL-12 was supplied by Dr. A. Stern (Hoffmann-La Roche).
Cell LinesThe BaF3 cell line, an IL-3-dependent murine pro-B cell line, has been described (14). Transfection of BaF3 cells was performed by electroporation (15) with linearized plasmids (80 µg of the plasmid encoding the EGFR/IL-12R chimera and 4 µg of the plasmid encoding a puromycin resistance marker (16)). Transfected cells were selected by culture in medium containing either 3 µg/ml puromycin or 10 ng/ml EGF. Transfections of the COS-7 cell line were performed as described previously (17).
Construction of Plasmids Encoding EGFR/IL-12R ChimerasA
polymerase chain reaction-based overlap extension technique (18) and
Pfu polymerase (Stratagene) were used for this approach. A
cDNA encoding the 645-amino acid-long extracellular domain of EGFR
was fused in frame to cDNA fragments encoding the transmembrane and
cytoplasmic domains of either human IL-12R1 (122 amino acids; see
Ref. 6) or human IL-12R
2 (216 amino acids; see Ref. 7) to yield the
chimeric receptors E12R1 (767 amino acids) and E12R2 (861 amino acids),
respectively. The chimeric DNAs were subcloned into the expression
vector pEF-BOS (19), and the sequences were confirmed by DNA
sequencing.
A conventional [3H]thymidine incorporation assay was performed as described (20). Cells were incubated for 48 h with various concentrations of EGF before an 8-h tritiated thymidine pulse was performed. All samples were assayed in triplicate.
Detection of EGFR/IL-12R Chimeras Expressed on BaF3 Cells by Flow CytometryA modification of a previously described method (4) was employed. Briefly, 5 × 105 cells were washed twice with fluorescence-activated cell sorting buffer (phosphate-buffered saline, 3% fetal calf serum, and 0.01% NaN3) and incubated on ice for 1 h with 5 µg/ml mouse mAb 13A9. After a wash step, the cells were incubated with a 200 µg/ml concentration of an R-phycoerythrin-conjugated goat anti-mouse IgG (Cappel, Durham, NC) for 30 min on ice before the flow cytometric analysis was carried out using a FACSort instrument (Becton Dickinson Advanced Cellular Biology, San Jose, CA).
Immunoprecipitation and ImmunoblottingCells
(108 BaF3 transfectants or PHA-activated lymphoblasts/100
ml) were washed in acidified RPMI 1640 medium (pH 6.4) and incubated
overnight in RPMI 1640 medium containing 1% fetal calf serum as
described (8). The cells were then resuspended in 10 ml of RPMI 1640 medium and 1% fetal calf serum and stimulated with 100 ng/ml EGF or
104 units/ml IL-12 for 10 min at 37 °C. Subsequently,
the BaF3 transfectants were washed with cold phosphate-buffered saline
containing 2 mM NaVO4 and then lysed in 1 ml of
lysis buffer (1% Triton X-100 (or 1% Brij 96 for the analysis of the
association between JAK kinases and IL-12R), 50 mM NaCl, 50 mM Tris (pH 8), 4 mM EDTA, 10 mM
Na4P2O7, 2 mM
Na3VO4, 100 mM NaF, 0.02%
NaN3, 0.1% bovine serum albumin, 1 mM
phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 0.5 µg/ml
leupeptin, and 1 µg/ml pepstatin A). For experiments shown in Fig. 5,
an additional chemical cross-linking step was introduced. Cells were
resuspended at 5 × 107 cells/ml in cold
phosphate-buffered saline (pH 8.3) and 1 mM MgCl2, cross-linked with 1 mM
dithiobis(succinimidyl propionate) (Pierce) for 20 min at 4 °C, and
then washed in phosphate-buffered saline containing 20 mM
Tris-HCl (pH 7.5) and 5 mM EDTA. The cells were then lysed
as described above. Clarified lysates were incubated with the specific
antibodies and protein G-coupled Sepharose beads (Pharmacia Biotech
Inc.). The immunoprecipitates were fractionated on 6 or 8%
SDS-polyacrylamide gels and transferred to nitrocellulose membranes.
Probing of membranes with anti-phosphotyrosine mAb 4G10 was performed using a modification of a previously described procedure (10), using horseradish peroxidase-conjugated goat anti-mouse IgG as the secondary antibody. Immunoblotting with other antibodies was performed in a similar fashion (21). Enhanced chemiluminescence (ECL, Amersham Corp.) was used as the detection system for all the immunoblots.
To study
the functional role of the cytoplasmic domains of IL-12R subunits 1
and
2, we constructed two chimeric receptors in which the
extracellular domain of EGFR was fused to the transmembrane and
cytoplasmic domains of either IL-12R
1 or IL-12R
2. EGF is expected
to dimerize the chimeric receptors, thereby triggering an intracellular
IL-12-specific signal. The chimeric receptors were termed E12R1 and
E12R2 and can be detected using anti-EGFR antibodies. E12R1 is composed
of 767 amino acids (predicted molecular mass of ~84 kDa), and E12R2
is composed of 861 amino acids (predicted molecular mass of ~95 kDa).
As shown in Fig. 1, the receptors, when expressed in COS
cells, had apparent molecular masses of ~125 kDa (E12R1) and ~140
kDa (E12R2). The differences between predicted and observed molecular
masses are most likely due to glycosylation. In COS cells, the
expression efficiency tended to be higher for E12R1 than for E12R2
(data not shown). Therefore, to equalize expression levels, only
one-tenth the amount of E12R1 plasmid was used in the experiment shown
in Fig. 1. Fig. 1 also shows that COS cells express an endogenous EGFR
with a molecular mass of ~175 kDa (22). The EGFR/IL-12R chimeras were
subsequently transfected alone or in combination into the
IL-3-dependent pro-B cell line, BaF3. Stable transfectants
were selected either in EGF-supplemented medium or in medium containing
puromycin and IL-3. Flow cytometric analysis demonstrated that the
chimeric receptors could be detected on the surface of the BaF3
transfectants, again with different expression efficiencies as
previously observed in COS cells (Fig. 2). In cells
cotransfected with E12R1+E12R2, the anti-EGFR monoclonal antibody
cannot distinguish between the two different chimeric receptors. Flow
cytometric analysis did not detect any EGFR expression on the parental
BaF3 cells.
E12R2 Alone Is Sufficient to Transmit a Proliferative Signal in Transfected BaF3 Cells
Only transfectants expressing either
E12R1+E12R2 or E12R2 were able to maintain long-term growth in medium
containing EGF alone, while BaF3 transfectants expressing E12R1 by
itself were not capable of long-term proliferation under these
conditions. After the initial cell selection period, more quantitative
cell proliferation assays were performed. These results are shown in Fig. 3. Again, cells expressing either E12R1+E12R2 or
E12R2 alone undergo a dose-dependent proliferation in
response to EGF, with a very similar EC50. In contrast,
transfectants expressing E12R1 alone respond only very weakly and
transiently to EGF, while parental BaF3 cells do not respond to EGF at
all. In control experiments, all transfectants proliferated equally
well in the presence of WEHI-3-derived IL-3 (data not shown). The
inability of E12R1 to signal a proliferative response in EGF-containing
medium was not due to a lack of expression of the chimeric receptors at
the cell surface (Fig. 2). Overall, these findings support and extend
our earlier results that showed that the wild-type IL-12R2 subunit can indeed signal and sustain long-term IL-12-induced proliferation in
the absence of IL-12R
1 (7).
Tyrosine Phosphorylation of Tyk2 Occurs Only in Cells Transfected with E12R1
It was previously reported that IL-12 induces tyrosine
phosphorylation of JAK2 and Tyk2 (8). Thus, the phosphorylation patterns of JAK2 and Tyk2 were examined in the chimera-expressing BaF3
cells. As shown in Fig. 4A, EGF induced
tyrosine phosphorylation of JAK2 in all the transfectants (E12R1,
E12R2, and E12R1+E12R2). In contrast, Tyk2 was phosphorylated only in
cells expressing E12R1 or E12R1+E12R2 and not in cells expressing E12R2
alone (Fig. 4C). The amounts of JAK2 or Tyk2 were roughly
equivalent in the transfectants analyzed. These findings therefore
suggest that Tyk2 interacts directly with E12R1 and not with E12R2. It
is tempting to speculate that because E12R1 cells do not proliferate,
Tyk2 activation might not be necessary for a proliferative signal. However, additional data will be needed to confirm this hypothesis.
In control experiments, tyrosine phosphorylation of both Tyk2 and JAK2 was induced by IL-12 in human PHA-activated lymphoblasts as expected (Fig. 4, B and D). In agreement with a previous report (8), tyrosine phosphorylation of JAK1 or JAK3 was not induced either in IL-12-treated PHA-activated lymphoblasts or in EGF-treated E12R1 or E12R2 transfectants (data not shown).
Specific Interactions between E12R1 and Tyk2 and between E12R2 and JAK2 Are DetectableWe next set out to determine if the observed
phosphorylation patterns for JAK2 and Tyk2 were the results of direct
receptor subunit/kinase interactions, using coimmunoprecipitation
techniques and lysates from the BaF3 cells that had been transfected
with either E12R1 or E12R2 alone. To increase the likelihood of
detecting receptor-kinase complexes, cell lysates were cross-linked
with dithiobis(succinimidyl propionate) prior to the
immunoprecipitations. We first examined the proteins associated with
E12R1. E12R1 protein was immunoprecipitated with anti-EGFR mAb and
subjected to immunoblotting. Blots were probed with antibodies against
EGFR, phosphotyrosine, Tyk2, and JAK2. Fig.
5A (first and second
lanes) shows that equivalent amounts of E12R1 protein (~125 kDa)
were present in BaF3 transfectants whether or not the cells had been
stimulated with EGF. As expected, tyrosine phosphorylation of E12R1 was
not detected since the cytoplasmic domain of IL-12R1 contains no Tyr
residue (third and fourth lanes; see Ref. 6).
However, a protein corresponding in size to Tyk2 was strongly
tyrosine-phosphorylated when E12R1 transfectants were stimulated with
EGF (fourth lane). Bands of equal intensity in samples
prepared with or without EGF stimulation were detected using anti-Tyk2
antibodies (fifth and sixth lanes). JAK2 protein was not detected (seventh and eighth lanes), even
though EGF-dependent phosphorylation of JAK2 had been
observed previously (Fig. 4). Overall, the results suggest that Tyk2
was associated with E12R1 prior to EGF stimulation and became
tyrosine-phosphorylated upon stimulation with EGF. The failure to
detect any E12R1/JAK2 associations could be because (i) the amounts of
JAK2 are too small to detect or (ii) the interactions between E12R1 and
JAK2 are too weak or too short-lived to allow cross-linking and
coimmunoprecipitation. It is also conceivable that a third, as yet
unidentified signaling component acts as an intermediate between JAK2
and E12R1.
We next examined the association between JAK2/Tyk2 and E12R2 in transfectants expressing E12R2 (Fig. 5B). Consistent with the flow cytometric results (Fig. 2), expression of E12R2 was barely detectable when probing blots with the anti-EGFR antibody (Fig. 5B, first and second lanes). However, the anti-phosphotyrosine blot showed ligand-dependent phosphorylation of E12R2 (third and fourth lanes). We can rule out that the slowest migrating band in the fourth lane is phosphorylated Tyk2 because Tyk2 is not activated in E12R2 cells (Fig. 4C, third and fourth lanes). A protein corresponding in size to JAK2 was tyrosine-phosphorylated when cells were stimulated with EGF (Fig. 5B, fourth lane). The amounts of JAK2 associated with E12R2 appear to be equivalent in lysates from cells with or without EGF stimulation (fifth and sixth lanes). Reprobing the blot with anti-Tyk2 antibodies failed to detect Tyk2 (seventh and eighth lanes). Overall, the results suggest that JAK2 preassociates with E12R2 and becomes phosphorylated upon ligand stimulation. The failure to detect Tyk2 is in agreement with the observed absence of phosphorylation of Tyk2 in cells expressing E12R2 (Fig. 4C).
To compare these findings with the IL-12R complex naturally expressed
on PHA-activated lymphoblasts, we used the same techniques and the 2B10
antibody that reacts with IL-12R1 (23). Western blots were probed
with antibodies to phosphotyrosine, JAK2, and Tyk2. Fig. 5C
(first and second lanes) shows the
IL-12-dependent phosphorylation of two proteins with
molecular masses of of ~125 and 135 kDa. As expected, the 125-kDa
protein corresponds to JAK2 (third and fourth
lanes), while the 135-kDa protein is Tyk2 (fifth and
sixth lanes). The use of this receptor subunit-specific
antibody allows us to conclude that both JAK2 and Tyk2 are
preassociated with the IL-12R complex. However, since the IL-12R
complex is composed of both IL-12
1 and IL-12R
2 and was
cross-linked prior to the immunoprecipitations, this experiment does
not distinguish among the various subunit/kinase interactions.
Both STAT3 and STAT4 have been shown to be part of
the IL-12 signaling pathway. Interestingly, IL-12 is so far the only
cytokine that has been reported to induce tyrosine phosphorylation of
STAT4 (10, 24), suggesting a specific role of STAT4 in IL-12 signaling (9, 10). We therefore examined tyrosine phosphorylation of STAT1-STAT4
in the BaF3 transfectants. Cell lysates were immunoprecipitated with
antibodies against STAT1-STAT4, and the resulting blots were probed
with anti-phosphotyrosine antibodies. The phosphorylation patterns
detected for STAT3 are shown in Fig. 6. STAT3
phosphorylation is strictly dependent on ligand stimulation in BaF3
transfectants expressing E12R1, E12R2, or E12R1+E12R2. Tyrosine
phosphorylation of STAT1, STAT2, and STAT4 was not detected (data not
shown). Thus, the activation patterns observed for JAK2 and STAT3
appear indistinguishable for E12R1-, E12R2-, or E12R1+E12R2-expressing cells, despite the fact that only cells containing E12R2 are capable of
long-term proliferation in response to EGF. The crucial importance of
STAT4 in IL-12-induced signaling of proliferation was recently highlighted (25, 26). Expression of STAT4 protein and mRNA was thus
analyzed in more detail. Levels of expressed STAT4 protein appear to be
very low in BaF3 cells, as they were undetectable by our standard
immunoblotting procedures. In control experiments, the same antibody
easily detected levels of STAT4 protein in lysates from PHA-activated
lymphoblasts. In contrast to the Western blotting results, however,
reverse transcription-polymerase chain reaction analysis did detect
STAT4 mRNA in the parental BaF3 cell line (data not
shown). Therefore, the inability to detect an interaction between
phosphorylated STAT4 and the receptor subunits is in all likelihood due
to these very low levels of STAT4 protein present in BaF3 cells. The
interactions of these "catalytic" levels of STAT4 with the E12R2
chimera must still be sufficient to transmit a proliferative signal. It
should also be pointed out that the participation of non-JAK/STAT
components in the transmission of this signal cannot be excluded at
this point.
It is interesting to note that in E12R1-expressing cells, STAT3 is phosphorylated upon EGF stimulation, even though the cytoplasmic region of E12R1 does not contain any tyrosine residues. Similarly, an IL-4R mutant devoid of cytoplasmic tyrosines has been described that is able to activate receptor-associated JAK kinases and still transmit a proliferative signal (27). Therefore, the current thinking that such tyrosine residues serve as docking sites for STAT proteins that are in turn activated by receptor-associated kinases may have to be modified (28).
In conclusion, we have shown the following. (i) The cytoplasmic region
of only IL-12R2, but not of IL-12R
1, when fused to the
extracellular domain of EGFR, can signal sustained proliferation. This
situation is identical to that observed for the wild-type IL-12R
1
and IL-12R
2 subunits. (ii) Only the cytoplasmic region of IL-12R
2
is phosphorylated upon ligand binding. (iii) Ligand stimulation gives
rise to phosphorylation of Tyk2 in the presence of the IL-12R
1
cytoplasmic region only. In contrast, JAK2 is phosphorylated in the
presence of either IL-12R
1- or IL-12R
2-derived cytoplasmic tails.
(iv) Consistent with these phosphorylation patterns, we can detect
specific and differential interactions between E12R1 and Tyk2 and
between E12R2 and JAK2. Direct interactions between E12R1 and JAK2,
however, could not be detected. These results should lead the way to
further clarification of what components of the IL-12 receptor complex
are absolutely required for IL-12-mediated signaling and what other
signaling pathways may be involved.