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and Tyrosine-Phosphorylated Stat31
* Department of Cell Biology, Cleveland Clinic Foundation and Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine, Case Western Reserve University, Cleveland, OH 44195;
Department of Adult Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115;
Division of Monoclonal Antibodies, Office of Therapeutics, Research and Review, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Bethesda, MD 20892
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Abstract |
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. IL-13 stimulated the phosphorylation of Stat3 on both Tyr705 and Ser727. In this study we show that IL-13 induces the association of PKC with Stat3, not with Stat1, and is required for Stat3 Ser727 phosphorylation. We found a novel IL-13-dependent cytosolic signaling complex of PKC and tyrosine-phosphorylated Stat3. A tyrosine kinase inhibitor blocked PKC association with Stat3 as well as Stat3 Ser727 phosphorylation. We therefore hypothesized that tyrosine phosphorylation was required for Stat3 interaction with PKC and subsequent PKC-dependent phosphorylation of Stat3 Ser727. We developed an efficient transfection protocol for human monocytes. Expression of Stat3 containing a mutation in Tyr705 inhibited the association of PKC with Stat3 and blocked Stat3 Ser727 phosphorylation, whereas transfection with wild-type Stat3 did not. Furthermore, by transfecting monocytes with Stat3 containing mutations in Tyr705 or Ser727 or with wild-type Stat3, we demonstrated that both Stat3 tyrosine and serine phosphorylations are required for optimal binding of Stat3 with DNA and maximal expression of 15-lipoxygenase, an important regulator of inflammation and apoptosis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Cytokines induce a variety of changes in cellular gene expression. The paradigm for cytokine activation of cells requires formation of a heterodimeric receptor with associated Jak tyrosine kinases. Upon engagement and receptor component interaction, the Jaks autophosphorylate and then phosphorylate the receptor chains (10, 11). Stat is then recruited to the receptors through binding of the Src homology 2 domain to the phosphotyrosine residue on the receptors. Recruited Stats can then be phosphorylated on specific C-terminal tyrosine residues by the activated Jak molecules (10, 11, 12, 13, 14, 15). Tyrosine-phosphorylated STATs then dimerize via Src homology 2 phosphotyrosine interactions and subsequently translocate to the nucleus, where they bind specific DNA promoter sequences leading to the transcriptional activation of responsive genes (9, 12, 15, 16, 17, 18). In addition to tyrosine phosphorylation, Stat1 and Stat3 have also been reported to be phosphorylated on another amino acid, Ser727. Recent reports suggest the role of different serine-threonine kinases, including MAPKs (ERK1/2, p38 MAPK, and JNKs) and protein kinase C (PKC), in serine phosphorylation of Stat1 and Stat3 (9, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30). Although the role of Ser727 phosphorylation is not clearly established, in certain situations it definitely enhances Stat3 transcriptional activity (31, 32).
Our recent studies have characterized the monocyte IL-13R and the immediate downstream signaling pathways (13). The heterodimeric receptor is comprised of IL-13R
1 and IL-4R. Three Jaks are associated with the receptor complex, but only two, (Jak2 and Tyk2), are phosphorylated upon receptor engagement (13). Several Stat family members are also activated, as determined by IL-13-induced tyrosine phosphorylation in human monocytes. These include Stat1, Stat3, Stat5A, Stat5B, and Stat6 (13).
Several recent observations prompted the studies in this report. First, we have shown that PKC is activated by IL-13 and is required for IL-13-induced 15-LO expression in human monocytes (33). PKC activation by a variety of stimuli has been shown to activate various transcription factors including Stats (28, 29, 34, 35). We have also recently shown that IL-13 induces Stat1 and Stat3 Ser727 phosphorylation in addition to tyrosine phosphorylation (9). Others have reported that in several cell lines, PKC activity appears to regulate Stat Ser727 phosphorylation (23, 28, 29). We therefore designed a series of experiments to explore the potential involvement of PKC in regulating Ser727 phosphorylation of Stat1 and Stat3 in IL-13-stimulated human monocytes. In this study, we demonstrate for the first time that in primary human monocytes, PKC binds tyrosine-phosphorylated Stat3 but not Stat1 in the cytosol before nuclear translocation and is required for Stat3 Ser727 phosphorylation. The dually phosphorylated Stat3 is then translocated to the nucleus where it mediates more efficient DNA binding activity and finally regulates the expression of 15-LO in IL-13-stimulated primary human monocytes.
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Materials and Methods |
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Recombinant human IL-13 was purchased from BioSource International. Abs specific for Stat1 phosphorylated on Ser727 (phospho-Ser727 Stat1) and for Stat3 phosphorylated on Ser727 (phospho-Ser727 Stat3) were generated in rabbits (36). Anti-phosphotyrosine Stat Abs were obtained from Upstate Biotechnology. Abs to PKC, Stat1, Stat3, and PY99 were from Santa Cruz Biotechnology. Pharmacological inhibitors such as rottlerin, Go 6976, H-7, and genistein were purchased from Biomol. The inhibitors were dissolved in DMSO and stored at –20°C as stock solutions. Untagged Stat3 wild-type (WT) plasmid, Stat3 serine mutant (Stat3S727A), and dominant negative Stat3 tyrosine mutant (Stat3Y705F) plasmids (cloned in pRC/CMV) were obtained as a gift from N. Reich (State University of New York, Stony Brook, NY). Flag-tagged Stat3 WT and dominant negative Stat3Y705F constructs (cloned in pcDNA3.1-Hygro(+); Invitrogen Life Technologies) were provided by R. Arceci (Johns Hopkins Kimmel Cancer Center, Baltimore, MD).
Isolation of human monocytes
Human peripheral blood monocytes (PBM) were isolated either by separation of mononuclear cells followed by adherence to bovine calf serum-coated flasks as earlier described (37) or by Ficoll-Hypaque sedimentation followed by countercurrent centrifugal elutriation (38, 39). PBM purified by these two methods were identical in their response to IL-13 and consistently >95% CD14+. These studies complied with all relevant federal guidelines and institutional policies regarding the use of human subjects.
Immunoprecipitation and immunoblotting
PBM were pretreated with the inhibitors (30 min) and then treated with IL-13 (500–1000 pM) for different time intervals as indicated. Total, cytosolic and nuclear extracts were prepared by previously published protocols (13, 40, 41). Lysates were resolved by 8% SDS-PAGE, transferred to a polyvinylidene difluoride membrane, blocked with 5% nonfat milk in PBS with 0.1% Tween 20 and subjected to immunoblotting with phospho-Ser727 Stat1 or phospho-Ser727 Stat3 Ab (diluted 1/1000 in 3% BSA in PBS with 0.1% Tween 20) overnight. The hybridization signal was detected using ECL (Pierce). 15-LO protein was detected on Western blots following a previously described protocol (37). For immunoprecipitation, the lysates were incubated with PKC or Stat3 Abs and precipitated with prewashed protein A-Sepharose beads (Sigma-Aldrich) at 4°C overnight. The precipitates were boiled for 5 min in SDS sample buffer and subjected to immunoblotting as described. In several experiments, immunoblots were stripped and reprobed to assess equal loading according to our previously published protocol (13).
Treatment of cells with PKC antisense oligodeoxyribonucleotide (ODN)
PBM were treated with PKC antisense ODN (5 or 10 µM) for 72 h with one re-feed at 24 h before the addition of IL-13 as previously described (33) with the exception that the cells were treated with only one PKC antisense ODN sequence. The PKC antisense ODN sequence was 5'-GAAGGCGATGCGCAGGAA-3'. A complementary PKC sense ODN sequence was used as a control. The antisense ODN sequence was selected based on prior literature (42).
In vitro phosphorylation of Stat3 by recombinant PKC
Stat3 proteins were immunoprecipitated from monocyte lysates using Stat3 Ab. The immune complexes were mixed with 2 pmol of recombinant PKC (Upstate Biotechnology) in 20 µl of final reaction volume containing 20 mM Tris-HCl (pH 7.5), 0.5 mM EDTA and EGTA, 400 µM MgCl2, 10 µM CaCl2, lipid activator (10 µM PMA and 0.28 mg/ml phosphatidylserine in a 0.3% Triton X-100 mixed micelle suspension), 1 µM ATP, 1x protease inhibitor mixture, and 1x phosphatase inhibitors. The mixture was incubated at 37°C for 30 min, and the reaction was terminated by adding 5x sample buffer. After boiling for 5 min, the samples were resolved by 8% SDS-PAGE and subjected to immunoblotting with phospho-Ser727 Stat3 Ab to investigate the status of Stat3 phosphorylation.
Transfection of primary human monocytes
Monocytes (5 x 106 in 2 ml of 10% bovine calf serum/DMEM) were kept in polypropylene tubes in a 37°C incubator with 10% CO2 for 2 h. The tubes were then centrifuged at 1200 rpm for 10 min. The supernatant was completely aspirated off and the cell pellet was resuspended in 100 µl of room temperature Human Dendritic Cell Nucleofector Solution (Amaxa). Highly purified plasmid DNA (1–2 µg) was mixed well with this 100-µl cell suspension and transferred into an Amaxa-certified cuvette. The cuvette was inserted in the cuvette holder of the Amaxa Nucleofector apparatus, and the nucleofection was performed using the program U-02. After nucleofection the cells were diluted in 1x Opti-MEM I solution (2 ml/well in six-well plates, preincubated in a 37°C incubator with 10% CO2 for 2 h) from the cuvettes using plastic pipettes, and the nucleofected cells were incubated for another 24 h followed by IL-13 addition for the desired time. The pmax GFP vector (2 µg) was used as a positive control.
Nuclear protein extraction and EMSA
To assess the role of Stat3 Ser727 phosphorylation in IL-13-induced Stat3 DNA binding activity, EMSA was performed using nuclear extracts and a specific Stat3 probe (Santa Cruz Biotechnology). Monocytes were nucleofected with Stat3 WT, Stat3 tyrosine mutant (Stat3Y705F), or Stat3 serine mutant (Stat3S727A) for 24 h followed by the addition of IL-13 for 30 min. Nuclear proteins were extracted, and the EMSA was performed following our previously published protocol (9, 13).
RNA extraction and real-time RT-PCR
Primary human monocytes were transfected with 2 µg of Stat3 WT, Stat3 tyrosine mutant (Stat3Y705F), or Stat3 serine mutant (Stat3S727A) (1 or 2 µg as indicated) for 24 h followed by IL-13 treatment for 16 h. Cells were collected and washed with PBS. Total cellular RNA was extracted using the RNeasy Mini kit from Qiagen, and real-time quantitative RT-PCR was performed according to our previously published protocols (9).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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is required for Stat3, but not for Stat1 Ser727 phosphorylation
We have recently shown that IL-13 induces Ser727 phosphorylation of Stat1 and Stat3 and that blocking this phosphorylation inhibits IL-13-induced 15-LO expression (9). We have also demonstrated that PKC expression and activity regulates this process. To explore whether PKC (a serine-threonine kinase) is involved in the serine phosphorylation of Stat1 and/or Stat3, we treated cells with the PKC inhibitor rottlerin followed by IL-13 stimulation. The status of serine phosphorylation of both Stat1 and Stat3 was determined by Western blot analysis using phospho-Ser727 Stat-specific Abs. The results indicate that, although pretreatment with rottlerin (a PKC-selective inhibitor) had no detectable effect on IL-13 induction of Stat1 Ser727 phosphorylation, it substantially inhibited IL-13-induced Stat3 Ser727 phosphorylation in a dose-dependent manner (Fig. 1, A and B). In contrast, pretreatment of cells with Go 6976, a conventional PKC-selective (PKC and PKC
) inhibitor, had no inhibitory effect on the level of Stat3 Ser727 phosphorylation (Fig. 1C). To evaluate whether rottlerin had any effect on IL-13-induced Stat1 and Stat3 tyrosine phosphorylation, the same cell lysates were analyzed by immunoblotting with phospho-Tyr701 Stat1 or phospho-Tyr705 Stat3 Abs. Pretreatment with rottlerin did not inhibit either IL-13-induced Stat1 Tyr701 or Stat3 Tyr705 phosphorylation (Fig. 1D), suggesting the specificity of the inhibition for Stat3 Ser727 phosphorylation.
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-specific antisense ODN on the phosphorylation of both Stat1 and Stat3 on Ser727. Our results demonstrate that although PKC antisense ODN had no effect on IL-13-mediated Stat1 Ser727 phosphorylation (Fig. 2B), it caused profound inhibition of IL-13-induced Stat3 Ser727 phosphorylation (
75% inhibition with 5 µM and almost complete (98–99%) inhibition with 10 µM antisense ODN after normalization to Stat3 protein expression levels) (Fig. 2A). Antisense ODN inhibition of PKC expression was also verified (Fig. 2, lower panels). These data corroborate the experiments using pharmacological inhibitors and indicate that PKC expression/activity, but not the activity of conventional PKC isoforms, is required for IL-13-stimulated phosphorylation of Stat3 Ser727.
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antisense ODN showed subtle changes in PKC expression levels using the 5 vs 10 µM doses, yet these subtle changes using different doses of PKC antisense ODN appeared to change IL-13-induced Stat3 Ser727 phosphorylation dramatically. There could be several possible explanations for this effect. In our experiments we observed an average decrease in PKC expression of 70% (43), which may alter kinase/phosphatase balance, subcellular localization, or PKC activation kinetics. Any of these factors could be responsible for the lack of exact correlation between PKC protein expression level and Stat3 phosphorylation level.
PKC directly phosphorylates Stat3 on Ser727
To examine whether PKC can directly mediate Stat3 Ser727 phosphorylation, we performed in vitro kinase assays using recombinant, active PKC and Stat3 protein immunoprecipitated from unactivated monocytes. As shown in Fig. 3, when the data are normalized for loading (as assessed by Stat3 immunoblotting), the addition of recombinant active PKC caused strong phosphorylation of Stat3 on Ser727 (a 6.3-fold increase as compared with control). The phosphorylation of Stat3 by recombinant, active PKC was substantially inhibited (67% after normalizing for loading differences) by pretreatment with rottlerin but not by Go 6976. These data indicate that recombinant PKC can directly phosphorylate Stat3 on Ser727.
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The Stat3 nuclear import was detectable around 5–10 min, reached a maximal level around 1 h, and then diminished (Fig. 4A, bottom). IL-13-induced Stat3 Ser727 phosphorylation was detected in both the nuclear as well as cytosolic fractions (Fig. 4B). In the cytosolic fraction, Stat3 Ser727 phosphorylation reached a maximum level at 15 min, whereas in the nuclear extracts, Ser727-phosphorylated Stat3 was first detectable from 10 min onward up to 4 h, reaching a maximum level around 1 h. In contrast, Stat3 Tyr705 phosphorylation occurred with faster kinetics. It was detected in the cytosol within 2 min, reached the maximum level around 10–15 min and then declined (Fig. 4C, top). The Tyr705-phosphorylated Stat3 was detected in the nuclear fractions from 10 min onward reached the maximum level at 15 min. The signal was maintained until 1 h and then gradually diminished (Fig. 4C, bottom). These data indicate that IL-13 induces Stat3 phosphorylation on Tyr705 followed by phosphorylation on Ser727.
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activity does not affect Stat3 nuclear translocation
In our previous studies, we reported the inhibition of DNA binding of Stat3 after blocking PKC (33). We, therefore, investigated whether inhibiting PKC activity blocks Stat3 nuclear translocation. To test this hypothesis, we first checked the effect of different kinase inhibitors on IL-13-driven nuclear translocation of Stat3. Genistein (a general tyrosine kinase inhibitor) inhibited the IL-13-mediated Stat3 nuclear translocation, whereas H-7 (a general serine-threonine kinase inhibitor) had no effect (data not shown). As expected, this result suggests that tyrosine phosphorylation is absolutely required for nuclear import of Stat3 molecules, whereas serine-threonine phosphorylation of Stat3 is not necessary for its transport from the cytosol to nucleus. To examine the specific effect of PKC on Stat3 (and also Stat1) nuclear translocation, we performed Western blot analysis of nuclear extracts from rottlerin-treated cells. IL-13 treatment strongly facilitated the nuclear import of both Stat1 and Stat3, whereas rottlerin did not inhibit their translocation (Fig. 4D).
PKC is associated with Stat3 in an IL-13-dependent manner
To investigate the potential interaction between Stat3 and PKC, cell lysates prepared from either untreated or IL-13-treated cells were immunoprecipitated with PKC-specific Ab. The immunoprecipitates were then subjected to immunoblotting using Stat3 Ab. As shown in Fig. 5A, Stat3 coimmunoprecipitated with PKC only in IL-13-treated cells. This association was strongly increased by IL-13 at 15 min and then decreased slowly over time (Fig. 5A, top). The association of Stat3 and PKC upon IL-13 stimulation was also found in the reciprocal experiment in which cell lysates were immunoprecipitated with Stat3 Ab and immunoblotted with PKC Ab (Fig. 5B). Similar experiments failed to detect any association of either Stat3 with another novel PKC, PKC
(Fig. 5C), or PKC with Stat1 (Fig. 5D). These results indicate that IL-13 induces PKC and Stat3 association and suggest that PKC may be directly involved in Stat3 Ser727 phosphorylation.
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is selectively induced by IL-13 stimulation, we used IL-6 as a negative control. As shown in Fig. 5E, IL-6 failed to induce the association of Stat3 with PKC, whereas Stat3 coimmunoprecipitated with PKC in IL-13-treated monocytes. We also found that the PKC inhibitor rottlerin did not inhibit IL-6-induced Stat3 Ser727 phosphorylation, suggesting that PKC activity is not involved in IL-6 induction of Stat3 Ser727 phosphorylation (data not shown). To further investigate the functional role of PKC in Stat3 Ser727 phosphorylation in monocytes, we performed a time-course study of Ser727 phospho-Stat3 to PKC association upon IL-13 stimulation. In untreated cells, the association of these two molecules was barely detectable. Upon treatment with IL-13, the association was strongly increased around 10–20 min and then diminished (Fig. 5F, upper). The same blot was then stripped and reprobed with phosphotyrosine Ab PY99 to investigate the time-dependent association of phosphotyrosine Stat3 with PKC (Fig. 5F, middle). To confirm equal amounts of PKC in the precipitates, the blot was further probed with PKC (Fig. 5F, bottom). These results indicate that upon IL-13 stimulation, both phospho-Ser727 Stat3 and phosphotyrosine Stat3 are associated with PKC in a time-dependent manner.
PKC-Stat3 complex formation occurs exclusively in the cytosol
To investigate the location of PKC-Stat3 complex, we evaluated nuclear and cytosolic fractions. Using our method of fractionation we found that proliferating cell nuclear Ag or cyclin appeared in the nuclear fraction but not in the cytosolic fraction, whereas -tubulin appeared in the cytosolic fraction but not in the nuclear fraction (data not shown). PKC was almost exclusively distributed in the cytosol in both untreated and IL-13-treated cells using our method of fractionation (Fig. 6A, bottom). PKC-Stat3 association was barely detected in cytosolic and nuclear fractions in untreated monocytes (Fig. 6A, top), whereas IL-13 induced complex formation that occurred almost exclusively in the cytosolic fraction (Fig. 6A, top). The Stat3 that was associated with PKC in the cytosol was tyrosine phosphorylated as verified by probing the PKC immunoprecipitates with the phospho-Tyr705 Stat3 Ab (Fig. 6B). By performing a similar experiment to that in Fig. 6B we further showed that PKC-associated Stat3 was also phosphorylated on serine at this time point (Fig. 6C).
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-Stat3 association
As shown in Fig. 4, IL-13-induced Stat3 Tyr705 phosphorylation can precede Stat3 Ser727 phosphorylation. We hypothesized that tyrosine phosphorylation might be required for Stat3 interaction with PKC and subsequent PKC-dependent Ser727 phosphorylation of Stat3. To test this hypothesis, we performed additional experiments. These studies showed that the association of tyrosine-phosphorylated Stat3 with PKC was profoundly induced in the cytosolic fraction after 5 min of IL-13 stimulation (Fig. 6D, upper). In that time point, we barely detected any induction of association between serine-phosphorylated Stat3 and PKC after IL-13 stimulation (Fig. 6D, middle). To assess equal immunoprecipitation, we further stripped and reprobed the same blot with PKC (Fig. 6D, lower). These data support our previous finding that tyrosine phosphorylation precedes serine phosphorylation on Stat3. We conducted several other experiments to explore this hypothesis. If the hypothesis is valid, one would predict that Stat3 association with PKC would be blocked by tyrosine kinase inhibitors, thereby blocking Stat3 Ser727 phosphorylation. Genistein, a general tyrosine kinase inhibitor, inhibited the IL-13-induced association of PKC with Stat3 in the cytosolic fraction (Fig. 6E). Secondly, if the hypothesis is valid, a serine-threonine kinase inhibitor would not influence PKC-Stat3 complex formation. We observed that the IL-13-induced association of PKC-Stat3 in the cytosol was not affected by H-7, a general serine-threonine kinase inhibitor (Fig. 6E), and furthermore that rottlerin had no effect on complex formation (Fig. 6A, top) at the same dose, which inhibited PKC activity (43). Thirdly, because genistein inhibited the association of PKC with Stat3, we were interested as to whether this affected the Ser727 phosphorylation of Stat3. Our results showed that genistein caused a dose-dependent inhibition of Stat3 Ser727 phosphorylation (Fig. 6F).
Tyrosine phosphorylation is required for Stat3 to interact with PKC and subsequent PKC-mediated Stat3 Ser727 phosphorylation
To more rigorously test our hypothesis that tyrosine phosphorylation might be required for Stat3 to form a molecular complex with PKC and allow subsequent PKC-dependent Ser727 phosphorylation of Stat3, we transfected human PBM with Stat3 containing mutations in Tyr705 (the site of tyrosine phosphorylation that is required for Stat3 dimer formation and translocation) and Ser727 (the site of serine phosphorylation that is required for maximal transcriptional efficiency for Stat3). In primary monocytes, transient transfection with the Stat3Y705F mutant (untagged) inhibited the association of PKC with total Stat3 (both endogenous and overexpressed) in a dose-dependent manner, whereas transfection with WT Stat3 had no inhibitory effect compared with the IL-13-treated control (Fig. 7A). In contrast the Stat3S727A mutant docks to the complex (with PKC) similarly to WT Stat3 (Fig. 7B). Transfection with Flag-tagged Stat3 WT and Stat3Y705F mutant showed the direct effect of these constructs on the formation of the molecular complex between PKC and Stat3. In monocytes transfected with Flag-tagged Stat3 WT, PKC formed a complex with Stat3 upon IL-13 stimulation. Transfection with the Stat3Y705F mutant interfered with the IL-13-induced complex formation (Fig. 7C). We also investigated whether tyrosine phosphorylation of Stat3 affected the PKC-mediated Stat3 Ser727 phosphorylation. Transfection with Stat3Y705F (untagged) completely inhibited the IL-13-induced Stat3 Ser727 phosphorylation level, whereas transfection with WT Stat3 (untagged) showed little, if any effect on the serine phosphorylation level of Stat3 compared with the IL-13-treated control (Fig. 8A, upper). In another experiment we showed that the Stat3S727A mutant had no effect on the IL-13-mediated tyrosine phosphorylation of Stat3 (Fig. 8A, middle). Flag-tagged Stat3 WT and Stat3Y705F mutant showed similar effects on IL-13-induced Stat3 Ser727 phosphorylation as they exerted on complex formation. IL-13 stimulated the Stat3 Ser727 phosphorylation level of Flag-tagged Stat3 WT, whereas the Stat3Y705F mutant (Flag-tagged) was not phosphorylated on Ser727 (Fig. 8B).
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To examine whether Stat3 Ser727 phosphorylation is required for optimal binding of Stat3 with DNA, we transfected monocytes with Stat3 containing mutations in Tyr705 and Ser727 along with WT Stat3. An EMSA was performed with nuclear extracts of posttransfected IL-13-treated monocytes and a 32P-labeled Stat3-specific probe. The results presented in Fig. 9A, indicate that transfection with the Stat3 tyrosine mutant completely prevented IL-13-induced Stat3 DNA binding activity and transfection with the Stat3 serine mutant markedly reduced Stat3 DNA binding. The PKC selective inhibitor rottlerin, which also inhibited PKC-mediated Stat3 Ser727 phosphorylation, profoundly inhibited the IL-13-induced Stat3 DNA binding activity, suggesting that the activation of PKC is required for optimal Stat3 DNA binding activity. To confirm this observation we performed an EMSA experiment using cells with reduced expression of PKC (PKC antisense transfected cells) (Fig. 9C). Our results showed that the IL-13-induced Stat3 DNA binding activity is indeed regulated by PKC expression/activity. In contrast transfection with WT Stat3 had no inhibitory effect on IL-13-induced Stat3 DNA binding activity. To further test that the effect of the Stat3 serine mutant on Stat3 DNA binding is not because of the blocking of IL-13-driven nuclear translocation of Stat3, we performed an immunoblotting experiment using the same nuclear extracts used for the EMSA experiment. Our result showed that Stat3 tyrosine phosphorylation is absolutely necessary for Stat3 nuclear translocation, whereas Stat3 serine phosphorylation has no effect on IL-13-stimulated nuclear import of Stat3 (Fig. 9B) yet both sites are critical for optimal DNA binding.
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To directly examine the functional role of Stat3 Tyr705 and Ser727 phosphorylation in IL-13-induced 15-LO expression, we transfected monocytes with Stat3 containing mutations in Tyr705 and Ser727 along with WT Stat3. As indicated in Fig. 10A, IL-13 induced the 15-LO mRNA expression by 6742-fold in primary monocytes, whereas transfection of monocytes with Stat3Y705F and Stat3S727A (2 µg each) mutants induced 15-LO mRNA expression around 27.7- and 333-fold, respectively, in presence of IL-13. In contrast, IL-13-stimulated 15-LO mRNA expression was not inhibited when monocytes were transfected with Stat3 WT plasmid. Stat3 phosphomutants (2 µg each) also inhibited IL-13-induced 15-LO protein expression by 55% for Stat3S727A and 68% for Stat3Y705F when transiently transfected in monocytes compared with the levels in untransfected (IL-13-induced) cells (Fig. 10B). These findings are the first direct evidence that phosphorylation of both Stat3 Tyr705 and Ser727 events regulate the 15-LO expression in IL-13-treated human monocytes.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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in the serine phosphorylation of Stat proteins in IL-13-stimulated primary human monocytes. In lymphoblastic cell lines, PKC has been reported to interact with Stat1 and regulate its phosphorylation on Ser727 in response to IFN- (29). In contrast, results from our studies demonstrate that in PBM, PKC associates with Stat3, but not Stat1, in an IL-13-inducible manner and mediates its phosphorylation on Ser727. Although it has previously been reported that PKC associates with and phosphorylates Stat3 on the Ser727 residue and that this complex is found in the cytosol (23). In that report, Jain et al. (23) studied the IL-6-induced response of cell lines (A431 epidermoid carcinoma cells, HepG2 human hepatoma, and 32D a myeloid progenitor cell line) and two of these are transformed cell lines. The use of cell lines and particularly transformed cell lines often reveals that they use different signal transduction pathways than primary cells and further, cells of different lineage use unique signaling pathways. This finding is particularly true for monocytic cell lines that are rapidly proliferating unlike their primary counterparts. Our work is entirely performed with primary PBM and therefore is a highly relevant cell type to mechanisms involved in inflammation and innate immunity. Our studies are thus the first to report a detailed signal transduction cascade downstream of IL-13 in monocytes and this manuscript is the first to use efficient transfection to perform mechanistic studies related to Stat3 phosphorylation.
Importantly the IL-6-induced Stat3 Ser727 phosphorylation, observed by Jain et al. (23), caused negative regulation of DNA binding rather than enhanced DNA binding as we have reported. The Jain studies on this issue were in COS cells with overexpressed Stat3 and PKC. Our study used endogenous levels of these components in a relevant primary cell type. Our prior data have shown a critical role for Stat3 Ser727 phosphorylation in regulating IL-13-induced 15-LO expression and are consistent with our findings in this study that PKC-dependent Stat3 Ser727 phosphorylation enhances DNA binding. Clearly IL-6 regulation of gene expression is very different from that mediated by IL-13. Furthermore our study goes beyond that of Jain et al. (23) in that we have shown that Ser727 phosphorylation by PKC requires Stat3 tyrosine phosphorylation both for docking to PKC and for Ser727 phosphorylation. Further we have performed novel transfection studies previously unfeasible in primary monocytes, to uncover the mechanisms for cytosolic retention of Stat3, allowing Ser727 to become phosphorylated by a cytosolic enzyme. The data showing the requirement for tyrosine phoshorylation to allow Stat3 to dock to PKC and become phosphorylated are entirely new.
In resting cells Stats, including Stat3, reside predominantly in the cytosol (44, 45, 46, 47, 48). After cytokine stimulation, Stats quickly become phosphorylated on tyrosine, dimerized, and translocate to the nucleus where they facilitate target gene expression. Nuclear Stat3 is then exported back to the cytosol after subsequent period of time for next round of signaling (48). Our results indicate that the formation of the molecular complex between PKC and Stat3 is an early event in IL-13 signal transduction pathway when a significant level of Stat3 resides in the cytosol and PKC is almost exclusively located in the cytosol. Although data presented in this study argue for predominantly cytoplasmic localization of Stat3 in untreated cells, a recent report suggests otherwise in different other primary cells and cell lines (49). Moreover, our time course experiment demonstrates that the maximal level of Stat3 tyrosine phosphorylation in the cytosol coincides with the maximal level of complex formation between these two molecules in the cytosol. Taken together, our data suggest that the tyrosine phosphorylation of Stat3, but not the activation of PKC or the serine phosphorylation of Stat3 is necessary to form this signaling complex.
Our in vitro kinase assays using recombinant active PKC and immunoprecipitated Stat3 protein from monocytes provide evidence that PKC can directly phosphorylate Stat3 on Ser727. Although those studies do not rule out the contribution by other kinases that are resistant to Go 6976 yet inhibited by rottlerin, our studies using PKC-specific antisense ODN treatment confirm that PKC is a major kinase regulating IL-13-induced Stat3 Ser727 phosphorylation in monocytes. We speculate that PKC phosphorylates Ser727 on Stat3 and then dissociates from the complex before Stat3 is translocated to the nucleus. Hence this cytosolic complex formation is likely to be important to allow serine phosphorylation on Stat3 rather than its immediate translocation upon tyrosine phosphorylation. After nuclear translocation, phosphorylated Stat3 (phosphorylated on both Tyr705 and Ser727 residues) binds DNA and regulates transcription (32).
Interestingly, treatment with rottlerin markedly reduced Stat3 DNA binding activities (33), even though it had no effect on IL-13-induced Stat3 tyrosine phosphorylation or nuclear import. Using our transfection protocol we investigated whether the expression of either Tyr705 or Ser727 mutants of Stat3 interfered with DNA binding to Stat3 consensus sites. Our recent results and those of others (9, 50) thus indicate that Ser727 phosphorylation of Stat3 contributes to or at least correlates with enhanced DNA binding activities in primary human monocytes. The precise mechanism by which Ser727 phosphorylation affects Stat3 DNA binding deserves further investigation.
Our previous results indicated that IL-13 induction of Stat1 and Stat3 Ser727 phosphorylation is dependent on p38 MAPK (9). Further studies with pharmacological inhibitors revealed that IL-13-induced PKC and p38 MAPK are activated in parallel with neither enzyme affecting the activation of the other (33). Our recent work suggests that IL-13 induces the formation of a complex containing p38 MAPK and PKC with Stat3 (data not shown). We are currently exploring the relationship among these three components in this complex. We predict that p38 MAPK facilitates the interaction between PKC and tyrosine-phosphorylated Stat3. Using an online program to search for predicted phosphorylation sites (Phosphobase v.2.0) (51), Ser727 on Stat3 is predicted to be a PKC site. Because we observed Stat3 Ser727 phosphorylation by PKC in vitro we predict that p38 MAPK may phosphorylate PKC to allow the docking of Stat3 into the complex, and then this might allow PKC phosphorylation of Stat3. This hypothesis is currently being tested. Hence, although the p38 activity inhibitor does not inhibit the phosphorylation of sites on PKC that are associated with activation (33), it may phosphorylate additional sites to allow protein-protein interactions (such as PKC-Stat3). Thus, inhibition of p38 MAPK could indirectly affect Stat3 Ser727 phosphorylation.
Finally by transfecting the Stat3 phosphomutants (both Stat3Y705F and Stat3S727A) in monocytes, we provided direct evidence for the first time that both tyrosine and serine phosphorylations of Stat3 are necessary for maximal expression of IL-13-induced 15-LO in primary human monocytes. Our studies therefore showed the novel role of PKC in regulating 15-LO gene expression in primary monocytes in response to IL-13 stimulation by forming a novel complex with tyrosine-phosphorylated Stat3 and by mediating Stat3 serine phosphorylation.
Although many studies have focused on the role of Stat6 in 15-LO expression (52, 53, 54, 55), we have investigated the functional role of Stat3 in IL-13-induced 15-LO gene expression and found that Stat3 is an important regulator of 15-LO gene expression. Using Stat3-specific decoy ODNs we provided direct evidence that activation of Stat3 by IL-13 is required for 15-LO gene expression (9). Earlier we also conducted experiments to determine that Stat3 was tyrosine phosphorylated in monocytes as a result of IL-13 stimulation (13). Using PKC-specific antisense ODNs we discovered that PKC regulates IL-13-induced 15-LO gene expression (33), and PKC expression/activity is required for optimal binding of Stat3 with DNA (Fig. 9C). In this experiment, we correlated all of these observations by our finding that a novel, IL-13-dependent, cytosolic signaling complex between PKC and tyrosine-phosphorylated Stat3 regulates 15-LO expression in human PBM. We continued to explore the mechanisms involved in this signaling process and found that Stat3 tyrosine phosphorylation is required for the specific interaction of Stat3 with PKC and PKC-mediated serine phosphorylation of Stat3 on Ser727 (Figs. 7 and 8). We further showed that PKC-mediated Stat3 serine phosphorylation is required for maximal Stat3 DNA binding (Fig. 9A) and for optimal expression of 15-LO (Fig. 10). Altogether these findings clearly show the importance of this novel PKC-Stat3 complex for inducing maximal 15-LO gene expression in primary human monocytes.
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Acknowledgments |
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Disclosures |
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Footnotes |
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1 This work is supported by Grant HL51068 from the National Institutes of Health (to M.K.C.) and by the General Clinical Research Center Grant M01 RR-018390. 
2 Address correspondence and reprint requests to Dr. Martha K. Cathcart, Department of Cell Biology, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195. E-mail address: cathcam{at}ccf.org
3 Abbreviations used in this paper: 15-LO, 15-lipoxygenase; PBM, peripheral blood monocyte; ODN, oligodeoxyribonucleotide; PKC, protein kinase C; WT, wild type.
Received for publication December 28, 2005. Accepted for publication June 28, 2006.
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receptor components. Science 263: 92-95. associates with and phosphorylates Stat3 in an interleukin-6-dependent manner. J. Biol. Chem. 274: 24392-24400. 
uses a different signaling pathway. Proc. Natl. Acad. Sci. USA 96: 13956-13961. induces serine phosphorylation of Stat-1 in Ewing’s sarcoma cells and mediates apoptosis via induction of IRF-1 and activation of caspase-7. Oncogene 19: 3372-3383. [Medline] is required for interleukin-6-induced STAT3 Ser-727 phosphorylation and transactivation. J. Biol. Chem. 276: 27709-27715. (PKC-) is activated by type I interferons and mediates phosphorylation of Stat1 on serine 727. J. Biol. Chem. 277: 14408-14416. -dependent phosphorylation of STAT1 on serine 727 and activation of gene expression. J. Biol. Chem. 276: 33361-33368. regulates thrombin-induced ICAM-1 gene expression in endothelial cells via activation of p38 mitogen-activated protein kinase. Mol. Cell Biol. 21: 5554-5565. 
Rn). Each cycle represents a 2-fold difference in mRNA amounts. After normalization with GAPDH amplification, the fold induction of 15-LO mRNA expression after transfection with Stat3 phosphomutants and WT Stat3 (in presence of IL-13) was compared with the IL-13-stimulated untransfected control (bottom). Data were collected from two independent experiments and shown as the mean ± data range. B, Western blot analysis shows 50 µg of protein from each sample was resolved by SDS-PAGE for immunoblotting with 15-LO Ab. The same blot was then stripped and reprobed with PKC