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IMMUNOBIOLOGY
From the Lymphocyte Cell Biology Section, Arthritis and
Rheumatism Branch, National Institute of Arthritis and Musculoskeletal
and Skin Diseases; Oral and Pharyngeal Cancer Branch, National
Institute of Dental and Craniofacial Research; and Howard Hughes
Medical Institute-NIH Research Scholars Program, National Institutes of
Health, Bethesda, MD.
Interleukin-12 (IL-12) is a key immunoregulatory cytokine that
promotes Th1 differentiation and cell-mediated immune responses. The
transcription factor STAT4 (signal transducer and activator of
transcription 4) is an important element in mediating IL-12 signals, as
evidenced by the fact that STAT4 Interleukin (IL)-12 is a critical immunoregulatory
cytokine that plays a central role in cell-mediated immune responses,
enhancing proliferation and cytotoxic activity of natural killer (NK)
and T cells, inducing the production of proinflammatory cytokines, and
promoting Th1 cell differentiation.1 Like other cytokine receptors, the IL-12 receptor (IL-12R) lacks kinase activity but is
associated with 3 cytoplasmic Janus tyrosine kinases (JAKs), JAK2 and
TYK2.2,3 On ligand binding, the JAKs are activated and
phosphorylate tyrosine residues on IL-12R STATs are a family of latent cytosolic transcription factors that, on
binding to receptors, are themselves phosphorylated on a conserved
C-terminal tyrosine residue.11 This is critical for dimer
formation and DNA binding.12,13
In addition to tyrosine phosphorylation, it has been shown that most of
the STATs are also phosphorylated on serine residues in response to
cytokine stimulation. Serine phosphorylation appears to be important
for maximal transactivating potential,14-17 though its
role in STAT DNA binding is still somewhat
controversial.18,19 For STAT1 and STAT3, a conserved site
of serine phosphorylation has been mapped within the C-terminal
transcriptional activation domain.19 Although it is clear
that this site is phosphorylated in response to cytokine stimulation,
the identity of the kinase responsible for this event remains
elusive.17,20-22 This site resides in a consensus sequence
for proline-directed serine/threonine kinases-mediated phosphorylation.
Mitogen-activated family of protein kinases (MAPKs) comprises several
subfamilies of proline-directed serine/threonine kinases, including
extracellular signal-regulated kinases 1/2 (ERKs), c-Jun N-terminal kinases (JNKs), and p38.23-26 The MAPK
subfamilies are regulated by different extracellular stimuli. For
example, while ERKs are activated rapidly by growth factors, JNKs and
p38 are typically activated by environmental stress and proinflammatory cytokines.27 However, it has been recently demonstrated
that JNKs and p38 can also be activated by T-cell receptor-mediated signaling28 and by several cytokines, interleukins, and
colony-stimulating factors that regulate hemopoietic cell growth and
differentiation.29-36
Like STAT1 and STAT3, we have demonstrated that STAT4 is also
phosphorylated on both tyrosine and serine residues in response to
IL-12 stimulation.5,16 It too has a putative MAPK
phosphorylation site in its transcriptional activation
domain.37,38 However, the serine residue phosphorylated on
STAT4 and the kinase responsible for this phosphorylation had not been
identified. In this study, we show that, in response to IL-12
stimulation, STAT4 is phosphorylated on tyrosine 693 and serine 721 and
mutations of each of these residues strongly impair its transcriptional
activity. Although it had been argued that IL-12 can induce ERK
activity in human T cells,39 suggesting that ERKs are
responsible for STAT4 serine phosphorylation, here we show that IL-12
does not activate ERKs or JNKs in NK and T cells. On the contrary, we
demonstrate that stimulation of p38 Cytokines, antibodies, and reagents
Cell culture
Transfections The pEFBOS-IL-12R 1, pEFBOS-IL-12R2 (from Dr U. Gubler),
and pCDNA3-STAT4 (from Dr J. N. Ihle) were used to transfect 293T cells, a system that provides high efficiency of transfectability and
high levels of expression. Site-directed mutagenesis of STAT4 was
performed with the Transformer Site-Directed Mutagenesis Kit (Clontech,
Palo Alto, CA), according to manufacturer's instructions. Oligonucleotides were designed to change tyrosine to phenylalanine at
codon 693 (5'-GACAAGGGTTTCGTCCCTTCTGTTTTTATCCC-3'), serine to alanine
at codon 721 (5'-CAGACCTTCTCCCCATGGCTCCAAGTGCA-3'), and serine to
alanine at codon 723 (5'-CC ATGTCTCCAGCTGCATATGCTGTGC-3').
The 293T cells were transfected using a calcium phosphate transfection
kit (5 Prime 3 Prime, Boulder, CO). Thirty-six hours later,
cells were rested for at least 16 hours, then harvested, stimulated,
and lysed in a buffer containing 20 mmol/L HEPES pH 7.5, 2.5 mmol/L
MgCl2, 10 mmol/L EDTA, 1 mmol/L dithiothreitol, 40 mmol/L
Reporter gene assay NIH3T3 and Jurkat T cells were transfected by LipofectAMINE (Life Technologies, Gaithersburg, MD) and by SuperFect (Qiagen, Chatsworth, CA), respectively, following the manufacturer's protocol. The modest levels of expression of the transfected vectors in the cells (compared with 293T cells) were advantageous in permitting detection of ligand-dependent activation of the reporter. Except where indicated, cells were transfected with 0.2 µg p3 × GAS-luc (from Dr R. Pine), 0.2 µg pCMV--galactosidase (Clontech), 0.2 µg pEFBOS-IL-12R1, 0.6 µg
pEFBOS-IL-12R2, 0.2 µg pCDNA3-STAT4 wild-type or mutated. Cells
were cotransfected, as indicated, with various MAPK family members,
including pCDNA3-MEKEE, pCDNA3-MEKAA, pCDNA3-HA-ERK2, pCEV-MEKK1,
pCDNA3-HA-JNK1, pCEFL-GST-MKK6, pCEFL-GST-MKK6KR, pCEFL-HA-p38 ,
pCEFL-HA-p38 , pCEFL-MEK5DD, and pCEFL-HA-ERK5.40-44 As
required, the total amount of DNA was adjusted with vector with no
insert. The cells were rested overnight and, then, stimulated, where
indicated, with 10 ng/mL IL-12 for 8 hours. Where required, the cells
were pretreated with 20 µmol/L SB202190 or DMSO for 1 hour before the
stimulation with IL-12 for 6 hours. Cell were lysed and luciferase and
-galactosidase activities were determined using the Dual-Light Kit
(Tropix, Bedford, MA), following the manufacturer's
instructions. Luciferase activity in each sample was normalized by the
corresponding -galactosidase activity.
Cloning and bacterial expression of the glutathione S-transferase -STAT4 fusion protein The C-terminal portion of STAT4 (amino acids 538-749) was cloned in the vector pGEX-4T3 (Amersham Pharmacia Biotech, Buckinghamshire, England) in frame with the glutathione S-transferase (GST) gene. The vector was transformed in the BL 21 Lys strain of Escherichia coli. The transformed bacteria were grown until the optical density was 0.5, at which time isopropyl--thiogalactopyranoside (1 mmol/L final concentration) was
added for 3 hours. The GST-STAT4 fusion protein was purified using the
Bulk GST Purification Module (Amersham Pharmacia Biotech Inc),
following the manufacturer's instructions.
Immunoprecipitation, immunoblotting, and kinase assays Immunoprecipitation and immunoblotting were performed as previously described.2 For kinase assays, cleared lysates were immunoprecipitated at 4°C for 3 hours with the indicated Ab. The kinase reactions were performed as described.43
Phosphorylation of tyrosine 693 and serine 721 is required for IL-12-induced STAT4 activation Serine 721 of STAT4 resides in a putative MAPK phosphorylation site and is analogous to serine 727 of STAT1 and STAT3.14,19 To assess whether IL-12 stimulation was able to induce STAT4 serine 721 phosphorylation in vivo, we took advantage of a phosphospecific antibody that recognizes STAT3 phosphorylated on serine 727 and cross-reacts with STAT4 phosphorylated on serine 721. This was not unexpected, as the core sequence surrounding these serine residues is very conserved. To this end, purified lymphocytes were activated for 48 hours to induce IL-12R expression and restimulated, after serum starvation, with IL-12 for various periods. As shown in Figure 1A, no basal phosphorylation was observed in unstimulated cells (lane 1), whereas STAT4 was phosphorylated on serine 721 beginning at 5 minutes (lane 2) but did not peak until 45 minutes of IL-12 stimulation (lane 3). The specificity of this antibody for serine 721 was confirmed using a STAT4 S721A mutant (see below, Figure 6C). Of note, as we previously reported,16 the serine phosphorylated form of STAT4 had reduced electrophoretic mobility and phosphorylation on serine 721, correlated with the appearance of the shifted form of STAT4 (Figure 1A, compare upper and lower panels).
To ascertain whether serine 721 was a relevant site of phosphorylation, we next utilized site-directed mutagenesis and expression in a cell line, 293T cells, that lacks endogenous STAT4. We generated 2 STAT4 mutants in which the likely sites of phosphorylation, tyrosine 693 and serine 721, were mutated. We then transfected cells with constructs expressing the 2 chains of the human IL-12R, together with wild-type STAT4 or the STAT4 mutants. As shown in Figure 1B, wild-type STAT4 was tyrosine-phosphorylated after IL-12 stimulation (lane 4) but when tyrosine 693 of STAT4 was replaced by phenylalanine, no tyrosine phosphorylation was observed (lane 7). In addition, when serine 721 was replaced by alanine, tyrosine phosphorylation was unchanged, but serine phosphorylation of STAT4, assessed by its mobility shift, was significantly diminished (lane 10). As a control, we utilized another STAT4 mutant (S723A) in which the mutated serine does not reside in a proline-directed serine/threonine kinase consensus sequence. This mutant showed an identical phosphorylation and alteration of electrophoretic mobility compared with wild-type STAT4 (not shown). To further establish the importance of tyrosine 693 and serine 721, we transfected a T-cell line, Jurkat T, with a STAT reporter construct (3xGAS-luc), IL-12R, and wild-type or mutated STAT4 constructs. As shown in Figure 1C, strong IL-12-induced transactivation of the reporter gene was detected when wild-type STAT4 was expressed but not in its absence. Interestingly, expression of the S721A mutant permitted reduced IL-12-inducibility of the reporter construct, whereas the S723A mutant had no effect on the functional activity of STAT4. As expected from the biochemical analysis, the Y693F mutant was completely unable to transactivate the reporter. Thus, we conclude from these experiments that tyrosine 693 and serine 721 are important sites of phosphorylation in STAT4. Moreover, these results demonstrate that tyrosine and serine phosphorylation are required for maximal STAT4-mediated transcriptional activity in response to IL-12, and that serine 721 is likely to be the target of a proline-directed serine/threonine kinase. IL-12 activates p38 but not ERKs or JNKs As STAT4 serine 721 resides within a putative MAPK phosphorylation site, we next investigated the possible contributions of ERKs, JNKs, and p38 in IL-12 signaling and STAT4 serine phosphorylation. It has been suggested that IL-12 induces ERK activity in T cells39; thus, we thought it was important to ascertain whether the ERKs were responsible for STAT4 serine 721 phosphorylation. Early events in the activation of the ERK pathway are the phosphorylation of the adapter molecule Shc, which, in turn, recruits Grb2 and the exchange factor SOS, thereby stimulating Ras activity.45 We first asked if IL-12 could induce tyrosine phosphorylation of Shc in human T cells. As shown in Figure 2A, stimulation with IL-12 up to 1 hour resulted in no significant tyrosine phosphorylation of Shc (lane 2 and data not shown), whereas IL-2 was able to induce the phosphorylation of all the known isoforms of Shc (p46, p52, and p66) (lane 3).46 Consistent with the lack of Shc phosphorylation, Grb2 was not recruited into a complex with Shc after IL-12 stimulation of T cells (Figure 2B, lane 3), whereas strong Shc-Grb2 association was observed after treatment with IL-2 (lane 2). The same results were obtained using the NK3.3 cell line (not shown). In addition, we also directly assayed ERK activity on IL-12 stimulation of human T cells. As shown in Figure 2C, only IL-2, used as positive control,47 was able to induce MBP phosphorylation (upper panel, lane 2), whereas IL-12 clearly failed to stimulate ERK activity (lane 3). Moreover, ERK2 immunoprecipitates were tyrosine phosphorylated in response to IL-2 but not IL-12 stimulation (Figure 2C, middle panel). Finally, consistent with the preceding results, a specific MEK inhibitor, PD98059,48,49 did not abrogate IL-12-induced STAT4 serine phosphorylation as measured by retardation in its electrophoretic gel migration (Figure 2D). As a control, PD98059 was able to inhibit IL-2-induced ERK phosphorylation, as assayed using anti-phosphoERK antibody (not shown).
We next tested the activity of another subfamily of MAPKs, the JNKs, in response to IL-12. As shown in Figure 2E, IL-12 did not induce JNK activity in NK3.3 cells (lane 4). Both anisomycin (lane 2) and a more physiologic stimulus, IL-2 (lane 3), used as positive controls, stimulated the activity of these kinases, even if, as expected,50 IL-2 did to a much lesser extent than anisomycin. As IL-12 had no effect either on ERK or JNK activity, we next assayed p38 activation on IL-12 stimulation of NK3.3 cells. As shown in Figure 2F, p38 was activated by 15 minutes of IL-12 stimulation (lane 2) at levels comparable with that induced by anisomycin, used as a positive control (lane 7). The activation was detected for up to 45 minutes (lane 4), returning at basal levels in 1 hour (lane 5). To further confirm the ability of IL-12 to activate p38 as opposed to
ERKs and JNKs and to verify the utility of the cell line, we next
evaluated the ability of IL-12 to activate transiently transfected,
HA-tagged ERK2, JNK1, and p38
Thus, our results indicated that ERKs and JNKs were not activated in response to IL-12, however, p38 was. Thus, this latter member of the MAPK family is likely to participate in the IL-12 signaling pathway and could be responsible for STAT4 serine 721 phosphorylation. MKK6/p38 and its upstream activator, MKK6,51 IL-12 signaling
was markedly enhanced, as STAT4-dependent transactivation of the
3xGAS-luc reporter was increased 20-fold compared with cells
transfected with STAT4 alone. Interestingly, overexpression of MKK6
alone enhanced IL-12-mediated STAT4 transcriptional activity, without
altering the basal level of luciferase activity. To confirm the
specificity of this result, we next tested the other MAPK stimulators,
namely, MEK1/2, MEKK1, and MEK5, which activate ERKs, JNKs, and the
recently cloned MAPK ERK5, respectively. As shown in Figure 4A, a
constitutively active form of MEK, MEKEE,40 failed to
enhance STAT4 transactivation. Moreover, coexpression in NIH3T3 of a
truncated JNK kinase, MEKK1, a potent activator of
JNKs,52,53 and 2 different JNK isoforms, JNK1 or JNK2
(Figure 4A and data not shown), did not affect STAT4 transcriptional
activity. In addition, coexpression of a constitutively active form of
MEK5, MEK5DD, a strong stimulator of ERK5, had no effect on
IL-12-induced STAT4 transcriptional activity. As a control, these
kinases were tested for the ability to transactivate an Elk-1 (for
MEKEE and MEKK1)- or a MEF2C (for MEK5DD)-dependent reporter
construct43 and did so at the same concentration that
failed to transactivate STAT4 (not shown). These data therefore argue
that activation of the MKK6/p38 pathway is an important step in
IL-12 signal trasduction, leading to STAT4-regulated gene
expression.
Four mammalian p38 isoforms have been described: p38 To further confirm that the MKK6/p38 pathway is responsible for
enhancing STAT4 activity, we assessed the effect of a specific p38
inhibitor, SB202190. As shown in Figure
5A, IL-12-induced, STAT4-mediated
transactivation was completely abrogated on pretreatment of the cells
with SB202190. As shown in Figure 5B, the expression of a dominant
negative MKK6 construct, MKK6KR, was also able to significantly reduce
IL-12-induced STAT4 transcriptional activity. In contrast, a dominant
negative MEK construct, MEKAA, had no effect on STAT4 activity induced
by IL-12, confirming the specific importance of MKK6 for STAT4 function
on IL-12 stimulation.
Together, these results argue that MKK6/p38 are important elements that
affect IL-12 activation of STAT4. That is, overexpression of p38 Phosphorylation of STAT4 by p38 To confirm that p38 has the capacity to phosphorylate STAT4, we
next tested whether STAT4 was an in vitro substrate of p38. We cloned
the C-terminal portion of STAT4 in a vector for the expression of GST
fusion protein in bacteria. The GST-STAT4 fusion protein was used as a
substrate for an in vitro kinase assay on lysates from NK3.3 cells
stimulated with IL-12. As shown in Figure 6A, IL-12-activated p38 was
able to phosphorylate the STAT4-containing fusion protein
(lane 3).
Given this result, we next tested whether serine 721 of STAT4 was a
target for p38 To confirm that p38 has the capacity to phosphorylate STAT4 on serine
721, we transfected cells with wild-type STAT4, alone or with MKK6 and
p38 Nonetheless, taken together, our data argue strongly that STAT4 appears
an in vivo substrate for p38
In this report, we have shown that STAT4 is phosphorylated on
tyrosine 693 and serine 721 in response to IL-12 stimulation. Moreover,
we have demonstrated that phosphorylation of serine 721, a consensus
site for MAPK-mediated phosphorylation, is required for full
IL-12-induced STAT4 transcriptional activity. In addition, we have
shown that p38, but not ERKs or JNKs, is activated in response to
IL-12. Finally, we also provide evidence that the MKK6/p38 It has been previously reported that IL-12 can induce tyrosine
phosphorylation and activation of a 44 kd kinase in human T cells.39 In that report, Pignata et al39
concluded that this protein was one of the ERK isoforms based on (1)
the detected molecular weight, (2) the use of an anti-ERK antibody, and
(3) the observation that this kinase recognizes MBP as a substrate. In
contrast, here we show that IL-12 does not induce ERK tyrosine phosphorylation and activation. These seemingly disparate results could
be in agreement. Since the Pignata study was published, many new
members of the MAPK family, including p38, have been described. Most of
them share characteristics very similar to ERKs: molecular weight in
the 40 to 50 kd range, activation by tyrosine/threonine
phosphorylation, and very high sequence homology. In addition, MBP has
been recognized as a substrate for many MAPK family members, including
p38 If IL-12 does not activate ERKs but does activate p38, the question arises, how? Unfortunately, the molecular mechanisms that might serve to couple IL-12R to MKK6 and p38 are still very unclear. Although many molecules have been identified that link cell surface receptors to ERKs45 and, to a lesser extent, to JNKs,62 the mechanism of activation of p38 is still poorly understood. It has been suggested that p38 activity might be regulated in T cells by kinases of the src family, through tyrosine phosphorylation of the Vav exchange factor and activation of the Rac small GTP-binding protein.63 In this regard, one such src family member, Lck, is induced on IL-12 stimulation of NK cells.64 Indeed, the possibility of Lck bridging the IL-12R to Vav/Rac and, in turn, p38 activation, as well additional mechanisms, will warrant further investigation. We have shown that STAT4 serine phosphorylation peaks at 45 minutes after IL-12 stimulation (Cho et al16 and Figure 1A); on the other hand, p38 kinase activity begins at 15 minutes (Figure 2F). This discrepancy in kinetics is interesting and suggests, perhaps, that STAT4 and p38 both need to be translocated to the same intracellular compartment. It is unlikely that STAT4 serine phosphorylation is mediated by a de novo synthesized kinase induced by the p38 pathway, as pretreatment with cycloheximide does not affect STAT4 mobility shift (data not shown). Rather, it is appealing to speculate that STAT4 is phosphorylated by p38 in the nucleus in which both are described translocating after stimulation.11,25 The physiologic relevance of our data, showing that the MKK6/p38 Importantly, p38 may not be the sole MAPK family member that phosphorylates serine 721 in STAT4. It is entirely possible that the perfect consensus surrounding serine 721 in STAT4 may be recognized by other MAPKs. Nonetheless, we have proved that, among the different MAPKs, p38 is the only one activated by IL-12 and able to participate in IL-12-induced STAT4 transcriptional activity. It is therefore reasonable to speculate that other stimuli, such as IL-2, might activate other MAPKs, which in turn may induce STAT4 serine 721 phosphorylation and augment its transcriptional activity. In this scenario, STAT4 could represent a target for various signals, its final transcriptional response resulting from the integration of different biochemical pathways. Despite a considerable effort of this and other laboratories in unveiling IL-12 signaling pathways, very little is known about the nature of molecules, other than JAKs and STATs, mediating its biologic functions. The finding that a specific MAPK, p38, participates to the signal transduction pathways elicited by IL-12 is therefore very important and is expected to shed new light on the complexity of the mechanisms by which IL-12 exerts its biologic responses. In conclusion, our data suggests that MKK6 and p38 play an important
role in regulating STAT4 serine phosphorylation in response to IL-12.
Furthermore, our findings indicate that the optimal expression of the
genes induced by IL-12, including IFN- Note added in proof. While our manuscript was under review, multiple studies, linking STAT serine phosphorylation to the activation of various MAPKs have been published. Significantly, they report apparently divergent results, probably because of the differences in the STAT proteins investigated and in the systems used.68-73 Thus, in agreement with our data, it has been reported that p38, activated in response to IFNs, is indispensable for STAT1 serine 727 phosphorylation and transcriptional activity.71 In contrast, other findings indicate that JNKs, but not p38, mediate STAT3 serine 727 phosphorylation in response to various stress treatments and that this event results in the inhibition of STAT3 activity.72 The most plausible hypothesis at the moment is that the effect of serine phosphorylation of STAT proteins depends on the cell type and on the class of serine kinases activated in response to different extracellular stimuli.
We thank U. Gubler, J. N. Ihle, J. Kornbluth, R. Pine, C. Reynolds for kindly providing reagents; M. J. Marinissen, A. Mazzoni, S. Pece, R. Sanchez-Prieto, M. Santoro for useful discussions and critical experimental advice.
Submitted October 12, 1999; accepted May 2, 2000.
R.V. and M.G. contributed equally to this work.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Roberta Visconti, NIH-NIAMS, Building 10, Room 9N252, 10 Center Dr, MSC-1820, Bethesda, MD, 20892-1820; e-mail: viscontr{at}exchange.nih.gov.
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© 2000 by The American Society of Hematology.
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H. Ungefroren, W. Lenschow, W.-B. Chen, F. Faendrich, and H. Kalthoff Regulation of Biglycan Gene Expression by Transforming Growth Factor-beta Requires MKK6-p38 Mitogen-activated Protein Kinase Signaling Downstream of Smad Signaling J. Biol. Chem., March 21, 2003; 278(13): 11041 - 11049. [Abstract] [Full Text] [PDF]
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J. Valenzuela, C. Schmidt, and M. Mescher The Roles of IL-12 in Providing a Third Signal for Clonal Expansion of Naive CD8 T Cells J. Immunol., December 15, 2002; 169(12): 6842 - 6849. [Abstract] [Full Text] [PDF]
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Q. Han, J. Leng, D. Bian, C. Mahanivong, K. A. Carpenter, Z. K. Pan, J. Han, and S. Huang Rac1-MKK3-p38-MAPKAPK2 Pathway Promotes Urokinase Plasminogen Activator mRNA Stability in Invasive Breast Cancer Cells J. Biol. Chem., December 6, 2002; 277(50): 48379 - 48385. [Abstract] [Full Text] [PDF]
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R. Nishikomori, T. Usui, C.-Y. Wu, A. Morinobu, J. J. O'Shea, and W. Strober Activated STAT4 Has an Essential Role in Th1 Differentiation and Proliferation That Is Independent of Its Role in the Maintenance of IL-12R{beta}2 Chain Expression and Signaling J. Immunol., October 15, 2002; 169(8): 4388 - 4398. [Abstract] [Full Text] [PDF]
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M. Pesu, S. Aittomaki, K. Takaluoma, A. Lagerstedt, and O. Silvennoinen p38 Mitogen-activated Protein Kinase Regulates Interleukin-4-induced Gene Expression by Stimulating STAT6-mediated Transcription J. Biol. Chem., October 4, 2002; 277(41): 38254 - 38261. [Abstract] [Full Text] [PDF]
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J. Kwang Yoo, J. Ho Cho, S. Woo Lee, and Y. Chul Sung IL-12 Provides Proliferation and Survival Signals to Murine CD4+ T Cells Through Phosphatidylinositol 3-Kinase/Akt Signaling Pathway J. Immunol., October 1, 2002; 169(7): 3637 - 3643. [Abstract] [Full Text] [PDF]
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S. J. White, G. H. Underhill, M. H. Kaplan, and G. S. Kansas Cutting Edge: Differential Requirements for Stat4 in Expression of Glycosyltransferases Responsible for Selectin Ligand Formation in Th1 Cells J. Immunol., July 15, 2001; 167(2): 628 - 631. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
/
) or were
restimulated with IL-12 for the times indicated. Lysates were
immunoprecipitated (IP) with anti-STAT4 Ab, resolved by
SDS/polyacrylamide gel electrophoresis (SDS/PAGE), transferred to
membrane and blotted (IB) with antiphosphoserine 727 STAT3 Ab that also
detects serine phosphorylated STAT4 (upper panel). The blot was
stripped and reprobed with anti-STAT4, demonstrating equal levels of
proteins (lower panel). (B), Tyrosine 693 and serine 721 are important
sites of phosphorylation in STAT4. 293T cells were transfected with 0.5 µg pEFBOS-IL-12R
; +IL-12, 
.
, the first 2 having the highest level of homology with each other. Although these different isoforms share both some upstream
stimulators (such as MKK6) and downstream targets (ie, ATF2), recent
reports have clearly indicated that each isoform can recognize a
specific spectrum of substrates.
