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Originally published In Press as doi:10.1074/jbc.M003170200 on June 30, 2000

J. Biol. Chem., Vol. 275, Issue 36, 27634-27640, September 8, 2000
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The Rac1/p38 Mitogen-activated Protein Kinase Pathway Is Required for Interferon alpha -dependent Transcriptional Activation but Not Serine Phosphorylation of Stat Proteins*

Shahab UddinDagger , Fatima LekmineDagger , Niti SharmaDagger , Beata Majchrzak§, Ingrid MayerDagger , Peter R. Young, Gary M. Bokoch||, Eleanor N. Fish§, and Leonidas C. PlataniasDagger **

From the Dagger  Section of Hematology-Oncology, University of Illinois and West Side Veterans Administration Hospital, Chicago, Illinois 60607, the § Division of Cell & Molecular Biology, Toronto Research Institute, University Health Network and Department of Immunology, University of Toronto, Toronto, Ontario M5S 3E2, Canada, the  Department of Cardiovascular Diseases, DuPont Pharmaceuticals, Wilmington, Delaware 19880, and the || Departments of Immunology and Cell Biology, Scripps Research Institute, La Jolla, California 92037

Received for publication, April 13, 2000, and in revised form, June 22, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The p38 mitogen-activated protein (MAP) kinase is activated during engagement of the type I interferon (IFN) receptor and mediates signals essential for IFNalpha -dependent transcriptional activation via interferon-stimulated response elements without affecting formation of the ISGF3 complex. In the present study, we provide evidence that the small GTPase Rac1 is activated in a type I IFN-dependent manner and that its function is required for downstream engagement of the p38 MAP kinase pathway. We also demonstrate that p38 is required for IFNalpha -dependent gene transcription via GAS elements and regulates activation of the promoter of the PML gene that mediates growth inhibitory responses. In studies to determine whether the regulatory effects of p38 are mediated by serine phosphorylation of Stat1 or Stat3, we found that the p38 kinase inhibitors SB203580 or SB202190 or overexpression of a dominant negative p38 mutant do not inhibit phosphorylation of Stat1 or Stat3 on Ser-727 in several IFNalpha -sensitive cell lines. Altogether these data demonstrate that the Rac1/p38 MAP kinase signaling cascade plays a critical role in type I IFN signaling, functioning in cooperation with the Stat-pathway, to regulate transcriptional regulation of IFNalpha -sensitive genes and generation of growth inhibitory responses.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

It is well established that the type I interferon receptor activates the Jak1-Stat pathway (1-3) and other signaling cascades (3, 4) to mediate the pleiotropic biological effects of type I interferons. These pathways are regulated by the activated Tyk-2 and Jak-1 kinases, which are constitutively associated with the two subunits of the type I interferon receptor and are activated during IFNalpha or IFNbeta stimulation (reviewed in Ref. 1-3). The activated Jak kinases regulate downstream engagement of multiple Stat proteins that form homo- or heterodimers that translocate to the nucleus to regulate gene transcription via binding to specific elements in the promoters of interferon-stimulated genes (ISGs) (1-3).

The first characterized IFNalpha -activated Stat signaling pathway was that involving ISGF3, which is dependent on the formation of heterodimers between Stat1 and Stat2 and their association with a member of the IFN regulatory factor family, p48 (1, 2). The resulting ISGF3 complex translocates to the nucleus and binds to interferon-stimulated response elements (ISREs) in the promoters of ISGs (1-3). In addition, several other Stat proteins are involved in IFNalpha signaling. Stat1 and Stat3 homo- and heterodimers, as well as homodimers of Stat4, Stat5a, and Stat5b, bind palindromic sequences found in the promoters of IFN-stimulated genes to activate transcription (2, 3, 5). The CrkL adapter protein is also tyrosine-phosphorylated in a type I interferon-dependent manner (6) and forms heterodimers with Stat5 that translocate to the nucleus and bind to GAS elements in the promoters of certain IFN-stimulated genes (7).

In addition to tyrosine phosphorylation, which is required for translocation to the nucleus and DNA binding, serine phosphorylation is essential for full transcriptional activation of Stat proteins (8-10). Specifically, Ser-727 is located in the C terminus of Stat-1 in a consensus motif for a mitogen-activated protein kinase, and its phosphorylation is essential for transcriptional activation in response to IFNgamma and the generation of IFNgamma responses (8-10). Thus, in addition to tyrosine kinases, serine kinases play important roles in the regulation of IFN-dependent gene transcription and ultimately the generation of the biological effects of interferons.

Despite significant advances in identifying the functional roles of tyrosine kinases activated by the type I interferon receptor, much less is known regarding the serine kinases activated by interferons and their function in type I IFN-signaling cascades. Previous studies have established that the serine kinase phosphatidylinositol (PI) 3-kinase is activated downstream of the insulin receptor substrate-1 and insulin receptor substrate-2 multisite docking proteins (11-14). This pathway is selectively activated by type I, but not type II, interferons (12) and does not play a role in the activation of the ISGF3 complex and transcriptional regulation of genes that contain ISREs in their promoters (13). In addition, the p44 map kinase (Erk2) is activated during engagement of the type I interferon receptor (15), but there is no evidence to date that it regulates serine phosphorylation of Stat proteins.

Recently, the p38 MAP kinase, which is activated in response to stress (16-20) and mediates signals important for various biological responses (21-26), was shown to be activated by type I IFNs and to play an essential role in IFNalpha -dependent transcriptional regulation via ISREs (27, 28). Furthermore, it was demonstrated that an IFNalpha -regulated signaling cascade is activated downstream of p38, involving the MAPKAPK-2 and MAPKAPK-3 serine kinases (27). In the present study, we demonstrate that the small G-protein Rac1 is activated in an IFNalpha -dependent manner, suggesting that it is as an upstream regulator of the IFNalpha -dependent p38 pathway. We also demonstrate that, in addition to regulation of transcription via ISREs, p38 is required for IFNalpha -gene transcription via GAS elements and regulates the promoter of the IFN-inducible PML gene that mediates growth inhibitory responses. However, neither the serine kinase activity of p38 itself nor the downstream MAPKAPK-2 and MAPKAPK-3 kinases regulate phosphorylation of Stat1 or Stat3 on Ser-727, demonstrating that the regulatory effects of p38 occur via a mechanism distinct from serine phosphorylation of Stats.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cells and Reagents-- The Daudi (lymphoblastoid), KG-1 (acute myeloid leukemia) and Molt-4 (acute T-cell leukemia) cell lines were grown in RPMI 1640 supplemented with 10% fetal bovine serum and antibiotics. The U2OS human osteosarcoma cell line was grown in McCoy medium supplemented with 10% fetal bovine serum. Human recombinant IFNalpha 2 was provided by Hoffmann LaRoche. Human recombinant consensus IFNalpha was provided by Amgen Inc. Human recombinant IFNbeta was provided by Biogen Inc. (Cambridge, MA). The p38 MAP kinase inhibitors SB203580 and SB202190 were purchased from Calbiochem Inc.

Antibodies against the phosphorylated form of Stat1 on serine 727 (anti-Stat1Ser-727) and the phosphorylated form of Stat3 (anti-Stat3Ser-727) were obtained from Upstate Biotechnology. Monoclonal antibodies against Stat1 and Rac1 were obtained from Transduction Laboratories (Lexington, KY). A polyclonal antibody against the phosphorylated form of p38 was obtained from New England Biolabs. Polyclonal antibodies against p38 and Stat3 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). An anti-Stat1 antiserum was generously provided by Dr. Andrew Larner (Cleveland Clinic Research Foundation, Cleveland, OH) and was used for immunoprecipitations.

Cell Lysis, Immunoprecipitation, and Immunoblotting-- Cells were stimulated with 1 × 104 units/ml of the indicated interferons for the indicated times and lysed in phosphorylation lysis buffer as described previously (11-13). Immunoprecipitations and immunoblotting using an enhanced chemiluminescence (ECL) method were performed as described previously (11-13).

Luciferase Reporter Assays-- Cells were transfected with a beta -galactosidase expression vector and a luciferase reporter gene containing eight GAS elements linked to a minimal prolactin promoter (8×GAS) using the superfect transfection reagent as per the manufacturer's recommended procedure (Qiagen). The 8×GAS construct (29) was from Dr. Christofer Glass (University of California, San Diego, CA). The PromPML 1.44-Luc construct was kindly provided by Dr. Hugues de Thé (Center International de la Recherche Scientifique, Hôpital St. Louis, Paris, France) (30). Forty-eight hours after transfection, triplicate cultures were either left untreated or treated with 5 × 103 units/ml IFNalpha . In the experiments in which the effects of overexpression of a mutant p38 were determined, the cells were transfected with a mutated dominant-negative p38 DNA, subcloned in the pCMV5 vector (pCMV-p38AGF) (Ref. 31, kindly provided by Dr. Roger Davis (Howard Hughes Medical Institute, University of Massachusetts, Worcester, MA)) or pCMVHis vector (PCMV) (used as a control). The cells were washed twice with cold phosphate-buffered saline, and, after cell lysis, luciferase activity was measured using the protocol of the manufacturer (Promega). The measured luciferase activities were normalized for beta -galactosidase activity for each sample.

Genomic DNA Affinity Chromatography (GDAC)-- Genomic DNA affinity chromatography using cell extracts from untreated or IFNalpha -treated cells, in the presence or absence of the p38 inhibitor SB203580, was performed essentially as described previously (32).

Mobility Shift Assays-- 10 µg of nuclear extracts from untreated or IFNalpha -treated cells, were analyzed using electrophoretic mobility shift assays, as described previously (32, 33). A double-stranded oligodeoxynucleotide (ATTTCCCGTAAATCCC), representing a Sis-inducing element of the c-fos promoter, was synthesized and used in gel shift assays.

Rac1 Activation Assays-- The activation of Rac1 by IFNalpha was determined using a recently described methodology (34) with minor modifications. Briefly, the pGEX-4T3 construct encoding for the GTPase binding domain of human Pak1 (34) was expressed in Escherichia coli as a GST fusion protein (GST-PBD). The cells were treated with the indicated IFNs and lysed in phosphorylation lysis buffer. In some experiments, the cells were starved for 2 h prior to interferon treatment. Cell lysates were incubated with 5 µg of GST-PBD, and bound proteins were separated by SDS-PAGE and immunoblotted with a monoclonal antibody against Rac1 (New England Biolabs), to detect GTP-bound Rac1.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We initially sought to obtain information on the mechanisms of activation of the p38 MAP kinase during engagement of the type I IFN receptor. We first determined whether tyrosine kinase activity is required for activation of the p38 MAP kinase by type I IFNs. Cells were incubated in the presence or absence of the tyrosine kinase inhibitor genistein and the phosphorylation/activation of p38 in response to treatment of cells with IFNalpha was determined. As shown in Fig. 1A, genistein abrogated the IFNalpha -induced activation of p38, while there was no change in the amount of the p38 protein in cells either left untreated or treated with the inhibitor (Fig. 1B). On the other hand, pretreatment of cells with the PI 3-kinase inhibitor, LY294002, had no effect on the activation of the p38, indicating that engagement of p38 occurs independently of the activation of the PI 3-kinase by the type I IFN receptor (Fig. 1, A and B). Thus, engagement of the p38 MAP kinase by the type I IFN receptor requires the function of upstream tyrosine kinases, most likely the receptor-associated Tyk-2 and/or Jak-1 kinases. The IFN-regulated PI 3-kinase pathway (11-13) plays no role in p38 activation by type I IFNs.


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  		Fig. 1.   The type I IFN-dependent activation of the p38 MAP kinase pathway is tyrosine kinase-dependent. A, Motl-4 cells were preincubated for 60 min in the absence or presence of the tyrosine kinase inhibitor genistein (100 µg/ml) or the PI 3-kinase inhibitor LY294002 (50 µM). The cells were lysed, and equal amounts of total cell lysates were analyzed by SDS-PAGE and immunoblotted with an antibody against the phosphorylated/activated form of p38. B, the blot shown in A was stripped and re-probed with an anti-p38 antibody to control for loading.

Rac1 is a small G-protein, which initiates a cascade that regulates activation of the p38 pathway in response to stress (35-37). We sought to determine whether this small G-protein is engaged in IFN signaling to regulate phosphorylation/activation of p38. We used a recently described methodology (34), in which the extent of Rac1 activation is determined by its ability to associate with the GTPase binding domain of the Pak1 kinase, which is its downstream effector. Cells were treated with IFNalpha or IFNbeta , and lysates were bound to a GST fusion protein encoding for the GTPase binding domain of Pak1. The activated form of Rac1 was subsequently detected by immunoblotting with an anti-Rac1 antibody. IFNalpha or IFNbeta treatment of cells induced activation of Rac1 (Fig. 2, A and B), strongly suggesting that this small GTPase is engaged in a type I IFN signaling pathway to regulate downstream activation of Pak1 and p38 (35-37). Time-course studies demonstrated that the type I IFN-dependent activation of Rac1 was rapid, occurring within 1 min of type I IFN treatment, and persisted for at least 60 min (Fig. 2C). In addition, as in the case of p38 phosphorylation, the type I IFN-dependent activation of Rac1 was blocked by pretreatment of cells with genistein (Fig. 2D), indicating that the function of an upstream tyrosine kinase (s) is required for its activation.


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  		Fig. 2.   Type I IFNs induce activation of the small G-protein Rac1. A, KG-1 cells were incubated in the presence or absence of IFNalpha for 30 min at 37 °C. The cells were lysed, and lysates were bound to a GST fusion protein for the binding domain of Pak1 (GST-PBD). Bound proteins were analyzed by SDS-PAGE and immunoblotted with an anti-Rac1 antibody. B, serum-starved KG-1 cells were treated with IFNbeta for 30 min. Cell lysates were bound to GST-PBD, and bound proteins were analyzed by SDS-PAGE and immunoblotted with an anti-Rac1 antibody. C, serum-starved KG-1 cells were treated with IFNbeta for the indicated times at 37 °C. Cells were lysed, and lysates were bound to a GST fusion protein for the binding domain of Pak1 (GST-PBD) or GST alone as indicated. Bound proteins were analyzed by SDS-PAGE and immunoblotted with an anti-Rac1 antibody. D, serum-starved KG-1 cells were incubated for 30 min in the presence or absence of genistein as indicated. The cells were subsequently treated with IFNbeta for 30 min in the continuous presence or absence of genistein. Cells were lysed, and lysates were bound to a GST fusion protein for the binding domain of Pak1 (GST-PBD). Bound proteins were analyzed by SDS-PAGE and immunoblotted with an anti-Rac1 antibody.

We subsequently determined if the function of Rac1 is required for downstream activation of p38 by type I IFNs. We determined whether overexpression of a dominant-negative form of Rac1 abrogates activation of p38 by IFNalpha . Cells were transfected with either control empty vector (pCDNA3) or the pCDNA3-Rac1(T17N) construct, and after treatment with IFNalpha , the phosphorylation/activation of p38 was determined. As shown in Fig. 3, the dominant negative form of Rac1 inhibited the IFN-dependent activation of p38, establishing that its function is required for IFN-dependent p38 activation. Thus, Rac1 is activated during treatment of cells with IFNalpha to regulate downstream engagement of the p38 pathway by the type I IFN receptor.


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  		Fig. 3.   Inhibition of IFNalpha -dependent activation of p38 by a dominant-negative form of Rac1. A, Daudi cells were transiently transfected with either control vector alone (pCDNA3) or a dominant negative form of Rac1 (pCDNA3-Rac1T17N). 48 h after transfection, the cells were incubated for 30 min at 37 °C in the presence or absence of IFNalpha . Total cell lysates were analyzed by SDS-PAGE and immunoblotted with an antibody against the phosphorylated/activated form of p38. B, the blot shown in A was stripped and re-probed with an anti-p38 antibody to control for equal loading.

To further understand the functional role of the p38 pathway in IFN signaling, we determined whether inhibition of the activity of p38 blocks IFNalpha -induced transcriptional activation via GAS elements in luciferase reporter assays. We first examined the effect of SB203580, a p38-specific kinase inhibitor, on such IFNalpha -dependent transcriptional regulation. IFN-sensitive U2OS cells were transfected with a plasmid containing an 8×GAS luciferase construct and treated with IFNalpha in the presence or absence of the SB203580 inhibitor. As expected, IFNalpha treatment induced high levels of luciferase activity (Fig. 4A), while treatment of cells with SB203580 significantly decreased such an induction (Fig. 4A). We then measured IFNalpha -dependent luciferase activity in U2OS cells transiently transfected with a p38 kinase mutant that cannot undergo phosphorylation/activation (p38AGF), as the tyrosine and threonine phosphorylation sites have been substituted (31). As shown in Fig. 4B, overexpression of p38AGF blocked the IFNalpha -induced increase in luciferase activity, establishing that a functional p38 kinase is essential for transcriptional regulation via GAS elements.


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  		Fig. 4.   The p38 pathway is required for IFNalpha -dependent gene transcription via GAS elements. A, U2OS cells (2 × 105/plate) were transfected with an 8XGAS luciferase construct. 48 h after transfection, the cells were incubated with or without IFNalpha for 6 h in the absence or presence of 10 µM SB203580 at 37 °C as indicated. The cells were then harvested and assayed for luciferase activity. The data are expressed as -fold increase in luciferase activity in response to IFNalpha treatment in the absence or presence of SB203580. The -fold increase in each experiment was calculated by dividing the relative luciferase units (RLU) in IFNalpha -treated samples with the RLU in untreated samples. Mean values ± S.E. of two independent experiments are shown. B, U2OS cells (2 × 105/plate) were co-transfected with an 8XGAS luciferase construct and either control vector (pCMV) or the dominant negative p38 mutant (p38-AGF) construct as indicated. 48 h after transfection, the cells were incubated without or with IFNalpha for 6 h. The cells were then harvested and assayed for luciferase activity. The data are expressed as -fold increase in luciferase activity over background levels, in response to IFNalpha treatment in the pCMV- or p38 AGF-transfected cells. The -fold increase in each experiment was calculated by dividing the RLU in IFNalpha -treated samples with the RLU in IFNalpha -untreated samples. Mean values ± S.E. of three independent experiments are shown. C, U2OS cells (2 × 105/plate) were co-transfected with the PromPML 1.44-Luc construct and either control vector (pCMV) or the dominant negative p38 mutant (p38-AGF) construct as indicated. 48 h after transfection, the cells were incubated without or with IFNalpha for 6 h. The cells were then harvested and assayed for luciferase activity. The data are expressed as -fold increase in luciferase activity over background levels, in response to IFNalpha treatment in the pCMV- or p38 AGF-transfected cells. The -fold increase in each experiment was calculated by dividing the RLU in IFNalpha -treated samples with the RLU in IFNalpha -untreated samples. Mean values ± S.E. of three independent experiments are shown.

We next determined whether the dominant-negative p38 mutant has the ability to block transcriptional activation of the promoter of the PML gene, which contains both ISRE and GAS elements (30). This was of particular interest, as PML is an IFNalpha -inducible gene known to mediate growth inhibitory responses (30). Fig. 4C shows that, consistent with previous studies (30), IFNalpha treatment induced strong luciferase activity in assays using the PromPML1.44-Luc construct and that such IFNalpha -dependent promoter activity was strongly inhibited by overexpression of the dominant-negative p38 mutant.

We have previously shown that inhibition of the p38 pathway blocks ISRE-dependent gene transcription without affecting tyrosine phosphorylation and DNA binding of Stat1 and Stat2, which along with p48, form the active ISGF3 complex (27). We determined whether inhibition of the p38 pathway blocks tyrosine phosphorylation of Stat-3 and/or Stat5 that participate in the formation of complexes that regulate gene transcription via GAS elements, as well as the formation of DNA-binding complexes that contain these Stat proteins. We first determined whether treatment of cells with the SB203580 inhibitor blocks IFNalpha -dependent tyrosine phosphorylation of Stat3. Cells were incubated in the presence or absence of SB203580 and IFNalpha , cell lysates were immunoprecipitated with an antibody against Stat3, and immunoprecipitated proteins were analyzed by SDS-PAGE and immunoblotted with anti-phosphotyrosine. As shown in Fig. 5A, treatment of cells with SB203580 did not affect the IFNalpha -dependent tyrosine phosphorylation of Stat3. In a similar manner, when the effect of SB203580 on the IFNalpha -dependent tyrosine phosphorylation of Stat5 was studied, we found that SB203580 does not abrogate such phosphorylation (Fig. 5B).


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  		Fig. 5.   Inhibition of the type I IFN-dependent activation of the p38 MAP kinase does not abrogate tyrosine phosphorylation or DNA binding of Stat3 or Stat5 or formation of SIF complexes. A, KG-1 cells were pretreated with SB203580 (10 µM) for 30 min and subsequently incubated with or without IFNalpha for 10 min, in the continuous presence or absence of SB203580. Cell lysates were immunoprecipitated (IP) with an anti-Stat3 antibody as indicated. Immunoprecipitated proteins were analyzed by SDS-PAGE and immunoblotted with anti-phosphotyrosine (anti-PTyr). B, Molt-4 (lanes 1-4) or Daudi (lanes 5-8) cells were pretreated with SB203580 (10 µM) for 30 min and subsequently incubated with or without IFNalpha for 10 min, in the continuous presence or absence of SB203580. Cell lysates were immunoprecipitated with an anti-Stat5 antibody, and immunoprecipitated proteins were analyzed by SDS-PAGE and immunoblotted with anti-phosphotyrosine. C, Daudi cells were pretreated with SB203580 (10 µM) for 60 min and subsequently incubated with or without IFNalpha for 15 min, in the continuous presence or absence of SB203580. Nuclear extracts were prepared and analyzed for DNA-binding STAT complexes using GDAC. Eluates from genomic DNA were resolved by SDS-PAGE (7%) and, after immunoblotting, probed with an antibody against Stat3. D, Daudi cells were pretreated with SB203580 (10 µM) for 60 min and subsequently incubated with or without IFNalpha for 15 min, in the continuous presence or absence of SB203580. Nuclear extracts were prepared and analyzed for DNA-binding STAT complexes using GDAC. Eluates from genomic DNA were resolved by SDS-PAGE (7%) and, after immunoblotting, probed with an antibody against Stat5. E, U2OS cells were transfected with either pCMV control vector or the PCMV-p38AGF construct. 48 h after transfection the cells were treated with IFNalpha for 15 min, as indicated. Nuclear extracts were reacted with 40,000 cpm of a 32P-end-labeled Sis-inducing element, and complexes were resolved by native gel electrophoresis and visualized by autoradiography. F, U2OS cells were transfected with either pCMV control vector or the PCMV-p38AGF construct. 48 h after transfection, the cells were treated with IFNalpha for 15 min as indicated and equal amounts of cell lysates (100 µg/lane) were analyzed by SDS-PAGE and immunoblotted with an antibody against p38.

We subsequently determined whether inhibition of p38 activation has any effects on IFNalpha -dependent DNA binding of Stat3 and/or Stat5. In studies in which the IFNalpha -dependent DNA binding of Stat3 or Stat5 was evaluated using GDAC, we found that pretreatment of cells with the SB203580 inhibitor had no effect on Stat3 or Stat5 DNA binding (Fig. 5, C and D). In a similar manner, when U2OS cells were transfected with the pCMV-p38AGF construct and the formation of SIF complexes in response to IFNalpha treatment was determined, we found that there was no inhibition of SIF complex formation by overexpression of the dominant negative p38 (Fig. 5E), despite the fact that very high levels of mutant p38 expression are achievable in these cells (Fig. 5F), consistent with our previous report (27).

A question of considerable interest is whether the p38 MAP kinase, or a serine kinase activated downstream of it, mediate phosphorylation of Stat1 on serine 727, which has been previously shown to be essential for its full transcriptional activation (8). We examined whether Stat1 is serine-phosphorylated in response to IFNalpha treatment in several IFNalpha -sensitive cell lines, and whether treatment of cells with the specific p38 inhibitors SB203580 and SB202190 abrogates such phosphorylation. IFNalpha treatment induced strong phosphorylation of Stat1 on serine 727 in Daudi cells, which could be identified directly in total cell lysates (Fig. 6, A and B) or in anti-Stat1 immunoprecipitates (Fig. 6, C and D). SB203580, used at doses that specifically inhibit p38 activation (27), had no effect on such phosphorylation (Fig. 6, A-D). When similar studies were performed in Molt-4 (Fig. 6, E and F), U2OS (data not shown), and KG-1 cells (data not shown), similar results were obtained. We also examined whether another specific pharmacologic inhibitor of p38, SB202190, inhibits IFNalpha -dependent phosphorylation of Stat1 on Ser-727. Consistent with the results from the studies with SB203580, treatment of cells with SB202190 did not inhibit Stat1 serine phosphorylation (Fig. 7, A and B). On the other hand, treatment of cells with SB202190 blocked the serine phosphorylation of Stat1 induced by osmotic stress (NaCl) (Fig. 7, C and D), consistent with previous reports (38). Thus, although the function of the p38 pathway is required for serine phosphorylation of Stat1 in response to stress, p38 is not required for the IFNalpha -dependent serine phosphorylation of Stat1.


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  		Fig. 6.   Inhibition of p38 activation does not abrogate IFNalpha -dependent serine phosphorylation of Stat1. A, Daudi cells were preincubated for 30 min in the presence ot absence of SB203580 (10 µM) and were subsequently treated for 30 min with IFNalpha as indicated, in the continuous presence or absence of SB203580. The cells were subsequently lysed, and equal amounts of total lysates (100 µg/lane) were analyzed by SDS-PAGE and immunoblotted with an antibody that recognizes the serine phosphorylated form of Stat-1 at serine 727. B, the blot shown in A was stripped and re-probed with an antibody against Stat1 to control for loading. C, Daudi cells were preincubated for 30 min in the presence ot absence of SB203580 (10 µM) and were subsequently treated for 30 min with IFNalpha as indicated, in the continuous presence ot absence of SB203580. The cells were subsequently lysed, and cell lysates were immunoprecipitated with an anti-Stat1 antibody. Immunoprecipitated proteins were analyzed by SDS-PAGE and immunoblotted with an antibody that recognizes the serine phosphorylated form of Stat-1 at serine 727. D, the blot shown in C was stripped and re-probed with an antibody against Stat-1 to control for loading. E, Molt-4 cells were preincubated for 30 min in the presence ot absence of SB203580 (10 µM) as indicated and were subsequently treated with IFNalpha for 15 min, in the continuous presence or absence of SB203580. The cells were subsequently lysed, and equal amounts of total lysates (100 µg/lane) were analyzed by SDS-PAGE and immunoblotted with an antibody that recognizes the serine-phosphorylated form of Stat1 at serine 727. F, the blot shown in E was stripped and re-probed with an antibody against Stat1 to control for loading.


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  		Fig. 7.   SB202190 inhibits serine phosphorylation of Stat1 in response to osmotic stress, but not in response to IFNalpha treatment. A, Daudi cells were preincubated for 30 min in the presence ot absence of SB202190 as indicated and were subsequently treated for 15 min with IFNalpha as indicated, in the continuous presence ot absence of the SB202190 inhibitor. The cells were subsequently lysed, and equal amounts of total lysates (100 µg/lane) were analyzed by SDS-PAGE and immunoblotted with an antibody that recognizes the serine phosphorylated form of Stat-1 at serine 727. B, the blot shown in A was stripped and re-probed with an antibody against Stat-1 to control for loading. C, Daudi cells were preincubated for 30 min in the presence ot absence of SB202190 and were subsequently treated for 15 min with NaCl (0.5 M) as indicated, in the continuous presence ot absence of SB202190. The cells were subsequently lysed, and equal amounts of total lysates (100 µg/lane) were analyzed by SDS-PAGE and immunoblotted with an antibody that recognizes the serine phosphorylated form of Stat-1 at serine 727. D, the blot shown in C was stripped and re-probed with an antibody against Stat-1 to control for loading.

Stat3 is also activated by IFNalpha (1-3, 39) and binds to GAS elements. The protein also contains a serine phosphorylation site (Ser-727) (40) that is required for its full transcriptional activation (8). Although there has been no direct evidence to date that IFNalpha induces serine phosphorylation of Stat3, several other hormones, cytokines, and growth factors have been shown to induce phosphorylation of Stat3 on serine 727 (41-46). When Molt-4 cells were treated with IFNalpha and total cell lysates were analyzed by SDS-PAGE and immunoblotted with an anti-Stat3Ser-727 antibody, we found that Ser-727-phosphorylated Stat3 was inducible by IFNalpha treatment. However, pretreatment of cells with SB203580 had no effect on such IFNalpha -induced serine phosphorylation (Fig. 8, A and B). Similar results were obtained when KG1 cells were studied (Fig. 8, C and D). Consistent with these data, when cells were transfected with the dominant negative p38 mutant, there was no inhibition of the IFNalpha phosphorylation of the protein, confirming that the p38 kinase does not play a role in the regulation of IFNalpha -inducible Stat3 serine phosphorylation (Fig. 8, E and F).


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  		Fig. 8.   IFNalpha induces serine phosphorylation of Stat3 on serine 727 via a p38-independent pathway. A, Molt-4 cells were preincubated for 60 min in the presence or absence of SB203580 as indicated and were subsequently treated with IFNalpha for the indicated times, in the continuous presence ot absence of SB203580. The cells were subsequently lysed and equal amounts of total lysates (100 µg/lane) were analyzed by SDS-PAGE and immunoblotted with an antibody that recognizes the serine phosphorylated form of Stat3 at serine 727. B, the blot shown in A was stripped and re-probed with an antibody against Stat3 to control for loading. C, KG-1 cells were preincubated for 30 min in the presence ot absence of SB203580 as indicated and were subsequently treated with IFNalpha for the indicated times, in the continuous presence ot absence of SB203580. The cells were subsequently lysed, and equal amounts of total lysates (100 µg/lane) were analyzed by SDS-PAGE and immunoblotted with an antibody that recognizes the serine-phosphorylated form of Stat3 at serine 727. D, the blot shown in C was stripped and re-probed with an antibody against Stat3 to control for loading. E, U2OS cells were either transfected with control empty vector (pCMV) or the pCMV-p38AGF construct as indicated. The cells were subsequently incubated in the presence or absence of IFNalpha for 20 min at 37 °C as indicated. After cell lysis, equal amounts of total lysates (100 µg/lane) were analyzed by SDS-PAGE and immunoblotted with an antibody that recognizes the phosphorylated form of Stat3 on serine 727. F, the blot shown in E was stripped and re-probed with an anti-Stat3 antibody to control for loading.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The discovery of the Jak-Stat pathway has uncovered a critical mechanism by which gene regulation occurs in response to interferons and other cytokines. The mechanisms by which Stat proteins are activated have been elucidated to a large extent. An outstanding issue is whether other signaling cascades activated by the interferons cooperate with the Jak-Stat pathway to regulate gene transcription.

Recent studies have demonstrated that the p38 pathway is activated by type I interferons and plays a critical role in IFNalpha signaling (27, 28). In the present study, we identify the small GTPase Rac1 as a component of this signaling cascade, apparently upstream of IFNalpha -dependent MKK and p38 activation. Rac1, along with Cdc42, is an upstream regulator of the Pak1 serine kinase (47, 48), which regulates activation of MKK3/6 and p38 (35-37). Our data provide the first evidence for involvement of Rac1 in type I IFN signaling, and raise the possibility that the proto-oncogene for Vav may be the upstream regulator of the p38 pathway in response to interferons. Previous studies have demonstrated that Vav is a guanine exchange factor for Rac1 (37, 49, 51) and that its phosphorylation on tyrosine residues, ultimately regulates activation of the p38 MAP kinase (37). We have previously shown that the Vav proto-oncogene is tyrosine-phosphorylated by type I IFNs and is engaged in type I IFN signaling (52). Such IFN-dependent tyrosine phosphorylation of Vav appears to be regulated by type I IFN receptor-associated Tyk-2 kinase, which associates with Vav (53, 54). Other studies have also implicated Vav in the generation of the growth inhibitory effects of IFNalpha (55). It is likely that the tyrosine phosphorylation of Vav by interferons is required for Rac1 activation and downstream engagement of the p38 pathway, and this remains to be determined in future studies.

Our findings clearly establish that the p38 pathway plays a critical role in the transcriptional regulation of interferon-stimulated genes, containing GAS elements in their promoters. Thus, the function of p38 is necessary for transcriptional regulation of essentially all IFNalpha -dependent genes, as we and others have shown that p38 is also required for IFN-dependent gene transcription via ISREs (27, 28). However, the precise mechanisms by which such regulatory effects occur remain to be clarified. One potential mechanism may involve serine phosphorylation of Stat proteins, which has been previously shown to be required for the transcriptional activation of ISGs (8-10). Such a model is particularly attractive, as in other systems it has been established that the function of p38 is required for serine phosphorylation of Stat1 (38, 43, 56). Specifically, it has been shown that p38 mediates serine phosphorylation of Stat1 at Ser-727 in response to UV irradiation (56) or osmotic stress (38). Furthermore, it has been demonstrated that SB203580 completely inhibits the Stat1 and Stat3 serine phosphorylation induced by interleukin-12 and interleukin-2 and the functional synergy of these cytokines, without affecting tyrosine phosphorylation of Stat proteins (43).

We were interested to determine whether the p38 pathway regulates serine phosphorylation of Stat1 on Ser-727 in the IFN system. We performed extensive studies in several cell lines in which IFNalpha -dependent activation of p38 occurs (27). Using two different specific inhibitors for p38 (SB203580 and SB202190), we failed to observe any regulatory effects on phosphorylation of Stat1 on Ser-727. Similarly, experiments in which a dominant-negative p38 mutant was overexpressed and the IFNalpha -dependent phosphorylation of Stat1 on Ser-727 was examined established that p38 is not required for such phosphorylation.

In subsequent studies we determined whether Stat3 is phosphorylated on Ser 727 in response to IFNalpha treatment and whether the p38 MAP kinase plays a regulatory role on such phosphorylation. Our data directly establish, for the first time, that Stat-3 is phosphorylated on Ser-727, apparently to facilitate gene transcription via GAS elements. However, such an IFNalpha -dependent serine phosphorylation of Stat-3 was not abrogated by inhibition of the p38 pathway with pharmacological inhibitors or by expression of a dominant-negative mutant. Thus, although SB203580 and p38AGF clearly inhibit IFNalpha -induced transcription via ISRE (27) and GAS elements (current study), such effects are unrelated to serine phosphorylation of Stat1 or Stat3.

A recent study by Goh et al. (28) has suggested that the p38 MAP kinase may regulate serine phosphorylation of Stat1 in response to IFNalpha and IFNgamma . Their conclusions were based on studies with HeLaS3 cells, demonstrating partial inhibition of serine phosphorylation of Stat1 when cells were treated with SB203580 (28). We have been unable to demonstrate any effects of SB203580 or SB202190 on the serine phosphorylation of Stat1 or Stat3 in several cell lines tested. Although we cannot account for the differences between our study and the report by Goh et al., it is possible that in HeLa cells a different pathway is used by the type I IFN receptor, other than the one used in other IFNalpha -sensitive cell lines. Alternatively, the serine kinase that phosphorylates Stat1 in HeLa cells may be an unknown MAP kinase that is sensitive to SB203580, but does not correspond to p38. Nevertheless, our data clearly establish that serine phosphorylation of Stats is not the primary mechanism by which p38 regulates IFNalpha -dependent gene transcription.

Kovarik et al. (56) failed to demonstrate activation of p38 by IFNgamma in several different cell types, as well as an effect of SB203580 on the IFNgamma -induced serine phosphorylation of Stat1. Consistent with this, we have been also unable to demonstrate p38 phosphorylation/activation in response to IFNgamma .2 Thus, the p38 pathway is selectively activated by type I (alpha , beta ), but not type II (gamma ) IFNs, and regulates transcriptional activation independently of serine phosphorylation of Stat proteins. The mechanisms by which such transcriptional regulation occurs remain to be determined. It is possible that yet undetermined downstream effectors of p38 cooperate with the Stat-pathway in the nucleus, to regulate transcription of interferon-responsive genes, without modifying Stat activation. Such a model appears to also apply to cytokines that activate NF-kappa B, as it has been clearly established that inhibition of p38 blocks NF-kappa B-dependent transcriptional regulation without modifying NF-kappa B activation (50).

Recently, it was demonstrated that the activation of the p38 pathway is required for phosphorylation of histone H3 on serine 10 and HMG-14 on serine 6, which occur concomitantly with gene induction in response to a wide variety of stimuli (57). The downstream effector of p38 that regulates such activation was identified as the MSK-1 kinase (57). Thus, MSK-1 may be activated downstream of p38 in response to IFNalpha treatment to regulate histone phosphorylation. Such a model could explain the dichotomy between the lack of an effect of p38 inhibition on Stat serine phosphorylation and the inhibitory effects that it exhibits on transcriptional regulation. Independent of the precise mechanisms involved, our data indicate an important functional role of the p38 MAP kinase pathway in the type I IFN system. It is likely that this pathway plays a role in the induction of the growth inhibitory effects of interferons in vivo, as suggested by its regulatory effects on the transcriptional activation of the promoter of the PML gene in vitro, and this remains to be established in future studies.

    ACKNOWLEDGEMENTS

We thank Joanna Woodson for expert technical assistance. We also thank Dr. Roger Davis for providing the pCMV-p38AGF construct, Dr. Hugues de Thé for providing the PromPML 1.44-Luc construct, and Dr. Christopher Glass for the 8×GAS luciferase construct.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants CA73381 and CA77816 (to L. C. P.), by a grant from the Department of Veterans Affairs (to L. C. P.), and by Medical Research Council of Canada Grant MT15094 (to E. N. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

** To whom correspondence should be addressed: Section of Hematology-Oncology, University of Illinois, MBRB, MC-734, Rm. 3150, 900 S. Ashland Ave., Chicago, IL 60607. Tel.: 312-355-0155; Fax: 312-413-7963; E-mail: lplatani@uic.edu.

Published, JBC Papers in Press, June 30, 2000, DOI 10.1074/jbc.M003170200

2 S. Uddin and L. C. Platanias, unpublished data.

    ABBREVIATIONS

The abbreviations used are: Jak, Janus kinase; IFN, interferon; Stat, signal transducer and activator of transcription; Pak1, p21 activated kinase; GAS, interferon gamma -activated site; PI 3-kinase, phosphatidylinositol 3-kinase; MAP, mitogen-activated protein kinase; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; GDAC, genomic DNA affinity chromatography; RLU, relative luciferase unit(s); SIF, sis-inducible factor; ISG, interferon-stimulated gene; PML, promyelocytic leukemia; ISRE, interferon-stimulated response element; PBD, p21 binding domain.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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