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J Biol Chem, Vol. 274, Issue 42, 30127-30131, October 15, 1999


Activation of the p38 Mitogen-activated Protein Kinase by Type I Interferons*

Shahab UddinDagger , Beata Majchrzak§, Joanna WoodsonDagger , Pony ArunkumarDagger , Yazan AlsayedDagger , Richard Pine, Peter R. Youngparallel **, Eleanor N. Fish§, and Leonidas C. PlataniasDagger Dagger Dagger

From the Dagger  Section of Hematology-Oncology, The University of Illinois at Chicago and West Side Veterans Affairs Hospital, Chicago, Illinois 60607, the § Department of Medical Genetics and Microbiology, University of Toronto, Toronto, Ontario M5S 3E2, Canada, the  Public Health Research Institute, New York, New York 10016, and the parallel  Department of Molecular Biology, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

The p38 mitogen-activated protein (Map) kinase plays a critical role in the generation of signals in response to stress stimuli, but its role in interferon (IFN) signaling and its potential regulatory role in the activation of Jak-signal transducer and activator of transcription (Stat) pathway are not known. In the present study, we provide evidence that the p38 Map kinase is rapidly phosphorylated and activated during treatment of cells with Type I interferons (IFNalpha and IFNbeta ). Furthermore, the Type I IFN-dependent activation of p38 regulates induction of the catalytic domains of MapKap kinase-2 and MapKap kinase-3, strongly suggesting the existence of an IFNalpha signaling cascade activated downstream of the p38 kinase. The engagement of this pathway in interferon signaling plays a critical role in interferon-dependent transcriptional regulation, as evidenced by the fact that inhibition of p38 activation results in abrogation of interferon-dependent gene transcription via interferon-stimulated response elements. Interestingly, inhibition of the kinase activity of the p38 blocks IFNalpha -induced gene transcription without inhibiting DNA binding or tyrosine phosphorylation of Stat proteins, suggesting that the p38 pathway acts in cooperation with the Stat pathway. Thus, the p38 kinase signaling cascade is activated by the Type I interferon receptor and plays a critical role in interferon signaling and interferon-dependent transcriptional regulation.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Interferons are pleiotropic cytokines that exhibit multiple biological effects on cells and tissues, including growth inhibitory and antiviral effects. It is well established that Jak-activated pathways play a critical role in the generation of interferon alpha  (IFNalpha )1 signals (1-3). Two tyrosine kinases of the Janus family, Tyk-2 and Jak-1, are constitutively associated with the IFNaR1 and IFNaR2 chains of the Type I IFN receptor, respectively (reviewed in Refs. 1-3). Upon binding of Type I interferons to the Type I IFNR, Tyk-2 and Jak-1 are tyrosine-phosphorylated and activated to regulate tyrosine phosphorylation of several downstream signaling elements, including Stat proteins (reviewed in Refs. 1-3), insulin receptor kinase proteins (4, 5), the CrkL adaptor protein (6, 7), and the vav proto-oncogene product (8, 9). In addition, the p42/44 Map kinases (10) and the PI 3'-kinase serine kinase (11) have been reported to be activated and participate in the generation of interferon signals.

The family of p38 Map kinases are serine-threonine protein kinases, which are activated in response to hyperosmolarity, heat shock, and other cellular stress responses, as well as in response to treatment of cells with proinflammatory cytokines, thrombin, or hematopoietic growth factors (12-16). The p38 Map kinase pathway plays a critical role in various signaling systems and has been shown to mediate signals for the generation of important biological responses, such as phosphorylation of transcription factors that regulate transcriptional regulation (17, 18), induction of cytokine production (17, 18), platelet aggregation (16), and induction of apoptosis in neuronal cells and fibroblasts (19-23).

Despite the important role that the p38 family of kinases plays in the generation of biological responses in various systems, its role in the generation of interferon signals is unknown. Furthermore, there has been no link established to date between the p38 Map kinase and the Type I IFN-activated Jak-Stat pathway. In the present report, we provide the first evidence that p38 is phosphorylated and that its catalytic domain is activated in response to treatment of target cells with Type I interferons. In addition, we demonstrate that IFN-dependent gene transcription via IFN-stimulated response elements (ISREs) is inhibited by blocking the activation of p38. Viewed together, these findings suggest a critical role for p38 in the generation of Type I interferon signals and the induction of interferon responses.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Cells and Reagents-- The Daudi (lymphoblastoid), Molt-4 (acute T-cell lymphoblastic leukemia), and KG-1 (acute myeloid leukemia) cell lines were grown in RPMI 1640 medium (Life Technologies, Inc.) supplemented with fetal bovine serum (Life Technologies, Inc.) and antibiotics. Human recombinant IFNalpha 2 was provided by Hoffmann LaRoche. Human recombinant IFNalpha -consensus was provided by Amgen Inc. Human recombinant IFNbeta was provided by Biogen Inc. (Cambridge, MA). A polyclonal antibody against p38 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against the MapKap-kinase-2 or MapKap-kinase-3 kinases were obtained from Upstate Biotechnology. An anti-Stat-1 antiserum was provided by Dr. Andrew Larner (Cleveland Clinic Research Foundation, Cleveland, OH) and was used for immunoprecipitations. A monoclonal antibody against Stat-1 was obtained from Transduction Laboratories (Lexington, KY) and was used for immunoblotting. An antibody that recognizes specifically the tyrosine-phosphorylated form of Stat-1 at tyrosine 701 was obtained from Upstate Biotechnology and was used for immunoblotting. A polyclonal antibody against the phosphorylated/activated form of p38 was obtained from New England Biolabs and was used for immunoblotting. A polyclonal antibody that recognizes the phosphorylated/activated form of ATF-2 was obtained from New England Biolabs. The SB203580 inhibitor was obtained from Calbiochem Inc.

Cell Lysis and Immunoblotting-- Cells were stimulated with 1 × 104 units/ml of the indicated interferons for the indicated times, and the cells were lysed as described previously (4, 5). Immunoprecipitations and immunoblotting using an enhanced chemiluminescence method were performed as described previously (4, 5).

p38 map Kinase Assay-- Cells were incubated in the presence or absence of the indicated interferons for the indicated times at 37 °C. The cells were subsequently lysed in phosphorylation lysis buffer (11). Cell lysates were immunoprecipitated with an antibody against p38 using protein G-Sepharose (Amersham Pharmacia Biotech). The immunocomplexes were subsequently washed three times with phosphorylation lysis buffer containing 0.1% Triton X-100 and two times with kinase buffer (25 mM Hepes, 25 mM MgCl2, 25 mM beta -glycerophosphate, 2 mM dithiothreitol, 0.1 mM Na3VO4, 20 µM ATP) and resuspended in 30 µl of kinase buffer containing 5 µg of glutathione S-transferase-ATF-2 fusion protein, and 30 µCi of [gamma -32P]ATP was added. The reaction was incubated for 30 min at room temperature and was terminated by the addition of SDS-sample buffer. Proteins were analyzed by SDS-PAGE, and the phosphorylated form of ATF-2 was detected by immunoblotting with an anti-phospho-ATF-2 antibody.

MapKap Kinase-2 and MapKap Kinase-3 Kinase Assays-- Cells were serum-starved by overnight incubation in RPMI medium-1% fetal calf serum. They were subsequently incubated in RPMI medium without serum for 2 h and then treated with the indicated IFNs for the indicated times, in the presence or absence of 10 µM SB203580, which was added 30 min prior to IFN treatment. The cells were then lysed in phosphorylation lysis buffer, lysates were immunoprecipitated with antibodies against MapKap kinase-2 or MapKap kinase-3, immunoprecipitated proteins were washed three times in phosphorylation lysis buffer and two times in kinase buffer (25 mM Hepes, pH 7.4, 25 mM MgCl2, 25 mM beta -glycerophosphate 100 µM sodium orthovanadate, 2 mM dithiothreitol, 20 µM ATP), and the immune complex kinase assays were initiated by the addition of 30 µl of kinase buffer containing 5 µg of Hsp-25 protein (Stress Gen Laboratories) as a substrate and 25 µCi of [gamma -32P]ATP. The reaction was incubated for 30 min at room temperature and was terminated by the addition of SDS-sample buffer. Proteins were subsequently analyzed by SDS-PAGE, and the phosphorylated form of Hsp-25 was detected by autoradiography.

Luciferase Reporter Assays-- Cells were transfected with a beta -galactosidase expression vector and an ISRE-luciferase plasmid using the Superfect transfection reagent as per the manufacturer's recommended procedure (Qiagen). The ISRE-luciferase construct included the wild type ISG15 ISRE (TCGGGAAAGGGAAACCG AAACTGAAGCC) cloned via cohesive ends into the BamHI site of the pZtkLuc vector. Forty-eight hours after transfection, triplicate cultures were either left untreated or treated with 5000 units/ml of IFNalpha , in the presence or absence of 10 µM SB203580, that was added to the cultures 30 min prior to IFNalpha treatment. 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) (24) (kindly provided by Dr. R. Davis, Howard Hughes Medical Institute, University of Massachusetts, Worcester, MA) or the 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 from untreated or IFNalpha -treated cells, in the presence or absence of the p38 inhibitor SB203580, was performed essentially as described previously (25).

Mobility Shift Assays-- 10 µg of nuclear extracts from untreated or IFNalpha -treated cells, in the presence or absence of the p38 inhibitor SB203580, were analyzed using electrophoretic mobility shift assays, as described previously (26). The composition of the ISGF3 complex was confirmed by supershifting with antibodies against Stat-1 and Stat-2.

    RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

We sought to determine whether the p38 Map kinase is tyrosine-phosphorylated/activated during IFNalpha treatment of IFNalpha -sensitive cell lines. Molt-4 or Daudi cells were treated in the presence or absence of IFNalpha or IFNbeta , and after cell lysis, total lysates were analyzed by SDS-PAGE and immunoblotted with an antibody against the phosphorylated form of p38 (New England Biolabs). In both Molt-4 and Daudi cells, we noticed that the phosphorylated form of p38 was induced after IFNalpha or IFNbeta treatment (Fig. 1), suggesting that this member of the MAP family of kinases is a substrate for upstream MKK kinases and possibly is activated by Type I IFNs to transduce downstream signals. Similarly, Type I IFN-dependent tyrosine phosphorylation of p38 was seen in the IFN-sensitive KG-1 myeloid cell line.2 The kinetics of this Type I IFN-dependent phosphorylation of p38 were such that peak phosphorylation of the kinase occurred at 30 min, with the signal declining by 60 min, suggesting that the activation/phosphorylation of p38 by interferons is rapid and transient (Fig. 2).


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  		Fig. 1.   Type I interferons induce phosphorylation of the p38 Map kinase. A, Molt-4 cells were incubated in the presence (+) or absence (-) of IFNalpha or IFNbeta for 30 min at 37 °C as indicated. Total cell lysates, corresponding to 1 × 106 cells, were analyzed by SDS-PAGE and immunoblotted with an antibody against the phosphorylated form of p38 (left panel). The blot was subsequently stripped and reprobed with an antibody against p38 (right panel). B, Daudi cells were incubated in the presence or absence of IFNalpha or IFNbeta for 30 min at 37 °C as indicated. Total cell lysates, corresponding to 1 × 106 cells, were analyzed by SDS-PAGE and immunoblotted with an antibody against the phosphorylated form of p38 (left panel). The blot was then stripped and reprobed with an antibody against p38 (right panel).


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  		Fig. 2.   Kinetics of the Type I IFN-dependent phosphorylation of p38. Molt-4 cells were treated with IFNbeta for the indicated times. Total cell lysates, corresponding to 1 × 106 cells, were analyzed by SDS-PAGE and immunoblotted with an antibody against the phosphorylated form of p38 (left panel). The blot was then stripped and reprobed with an antibody against p38 (right panel).

To determine whether the kinase activity of p38 is induced by Type I IFN treatment, KG-1 or Daudi cells were treated with IFNbeta , cell lysates were immunoprecipitated with an anti-p38 antibody, and in vitro kinase assays were performed using a glutathione fusion protein encoding for ATF-2 as an exogenous substrate. IFN treatment resulted in activation of the kinase and phosphorylation of ATF-2 in both cell lines, indicating that the phosphorylation of p38 results in activation of its catalytic domain (Fig. 3).


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  		Fig. 3.   Type I interferon-dependent induction of the kinase activity of p38. A, KG-1 cells were incubated in the presence (+) or absence (-) of IFNbeta for 30 min at 37 °C as indicated. Cell lysates were immunoprecipitated with an antibody against p38, and immunoprecipitates were subjected to an in vitro kinase assay using glutathione S-transferase-ATF-2 as a substrate. Proteins were analyzed by SDS-PAGE, and phosphorylated proteins were detected by immunoblotting with an anti-phospho-ATF-2 antibody (left panel). The blot from the kinase assay was stripped and probed with an antibody against p38 to control for loading (right panel). B, Daudi cells were incubated in the presence or absence of IFNbeta for 30 min at 37 °C as indicated. Cell lysates were immunoprecipitated with an antibody against p38, and immunoprecipitates were subjected to an in vitro kinase assay using glutathione S-transferase-ATF-2 as a substrate. Proteins were analyzed by SDS-PAGE, and phosphorylated proteins were detected by immunoblotting with an anti-phospho-ATF-2 antibody (left panel). The blot from the kinase assay was stripped and probed with an antibody against p38 to control for loading (right panel).

Previous studies have identified MapKap kinase-2 and MapKap kinase-3 as the in vivo substrates for the kinase activity of p38 in response to stress and other stimuli (15, 27-30). To determine whether these kinases are also activated downstream of the p38 kinase during engagement of the Type I IFN receptor, lysates from IFNbeta -treated or untreated cells were immunoprecipitated with specific antibodies against MapKap kinase-2 or MapKap kinase-3 and in vitro kinase assays were performed on the immunoprecipitates. The results in Fig. 4 demonstrate that both downstream effectors of the p38 MAP kinase pathway are activated by IFN treatment and that such activation is blocked by treatment of cells with the specific p38 inhibitor SB203580, suggesting that the regulatory effects of the p38 pathway in IFN signaling are mediated, at least in part, by these kinases.


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  		Fig. 4.   Activation of MapKap kinase-2 and MapKap kinase-3 by IFNbeta . A, Molt-4 cells were incubated in the presence (+) or absence (-) of IFNbeta for 60 min at 37 °C, in the presence or absence of SB203580 as indicated. Cell lysates were immunoprecipitated with an antibody against MapKap kinase-2, and in vitro kinase assays were carried out on the immunoprecipitates using Hsp25 as an exogenous substrate. Proteins were analyzed by SDS-PAGE, and the phosphorylated form of Hsp25 was detected by autoradiography. B, Daudi cells were incubated in the presence or absence of IFNbeta for 60 min at 37 °C and in the presence or absence of SB203580 as indicated. Cell lysates were immunoprecipitated with an antibody against MapKap kinase-3 and an in vitro kinase assay was carried on the immunoprecipitates using Hsp25 as an exogenous substrate. Proteins were analyzed by SDS-PAGE, and phosphorylated proteins were detected by autoradiography.

We subsequently sought to identify the functional consequences of this Type I IFN-dependent activation of the p38 pathway. In the IFN system, it is well established that gene transcription is regulated by the Stat pathway. A major signaling cascade involves association of activated Stat-2 and Stat-1 with p48 to form the mature ISGF3 complex, which then translocates to the nucleus to regulate gene transcription via binding to ISREs (1-3). We examined whether inhibition of the activity of p38 kinase blocks IFN-induced gene transcription via ISREs in gene reporter assays. IFN-sensitive U2OS cells, in which the p38 kinase is also activated by Type I IFNs,2 were transfected with a plasmid containing an ISRE-luciferase construct and treated with IFNalpha in the presence or absence of the SB203580 inhibitor. As expected, IFNalpha treatment of cells resulted in a significant increase in luciferase activity (Fig. 5A). Treatment of cells with SB203580 clearly reduced such induction (Fig. 5A), suggesting that the p38 pathway mediates signals required for ISRE-regulated gene transcription during activation of the Type I IFN receptor. To further establish the role of p38 in the induction of IFNalpha gene transcription via ISREs, we measured IFNalpha -dependent induction of luciferase activity in cells overexpressing a p38 kinase that cannot undergo phosphorylation/activation (p38AGF), as the tyrosine and threonine phosphorylation sites have been mutated (24). As shown in Fig. 5B, overexpression of p38AGF blocked the IFNalpha -induced increase in luciferase activity, establishing that a functional p38 kinase is essential for transcriptional regulation via ISREs.


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  		Fig. 5.   p38 is required for IFNalpha gene transcription via ISREs. A, U2OS cells (2 × 105/plate) were transfected with an ISRE luciferase construct. 48 h after transfection the cells were incubated without or with 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 over background levels, 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 in IFNalpha -treated samples with the relative luciferase units in IFNalpha -untreated samples. Mean values ± S.E. of four independent experiments are shown. B, U2OS cells (2 × 105/plate) were co-transfected with an ISRE 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 relative luciferase units in IFNalpha -treated samples with the relative luciferase units in IFNalpha -untreated samples. Mean values ± S.E. of three independent experiments are shown.

It is well established that IFN regulation of gene transcription in the interferon system is dependent on phosphorylation of Stat proteins and the formation of DNA binding complexes by activated Stat proteins (1-3). IFN-induced complexes include Stat 1:2 heterodimers that participate in the formation of the active ISGF3 complexes that regulate gene transcription via ISREs (1-3). As SB203580 and overexpression of a dominant-negative p38 construct inhibited induction of IFNalpha -dependent gene transcription, we sought to determine whether the IFNalpha activation of p38 affects tyrosine phosphorylation and activation of the DNA binding activity of Stat proteins that form the ISGF3 complex.

We determined whether treatment of cells with SB203580 inhibits detection of the IFNalpha -induced tyrosine-phosphorylated/activated form of Stat-1. Daudi cells were treated with IFNalpha in the presence or absence of SB203580, and total cell lysates were analyzed by SDS-PAGE and immunoblotted with an antibody against the phosphorylated/activated form of Stat-1. As shown in Fig. 6A, SB203580 had no effect on the IFNalpha -dependent tyrosine phosphorylation of Stat-1 on tyrosine 701. 


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  		Fig. 6.   Lack of an effect of SB203580 or overexpression of a dominant-negative p38 mutant on the IFNalpha -depended induction of the tyrosine-phosphorylated form of Stat-1. A, Daudi cells were incubated in the presence (+) or absence (-) of IFNalpha for 30 min at 37 °C as indicated. 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 tyrosine-phosphorylated form of Stat-1 at tyrosine 701 (left panel). The blot was then stripped and reprobed with an antibody against Stat-1 to control for loading (right panel). B, U20S cells were transfected with either 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 tyrosine-phosphorylated form of Stat-1 at tyrosine 701 (left panel). The blot was then stripped and reprobed with an antibody against Stat-1 to control for loading (middle panel). Equal amounts of protein (100 µg/lane) were analyzed in parallel by SDS-PAGE and immunoblotted with an anti-p38 antibody (right panel).

We then sought to determine whether overexpression of the dominant-negative p38-AGF mutant also blocks the IFNalpha -dependent induction of the phosphorylated form of Stat-1. Overexpression of the p38AGF mutant in U2OS cells had no effect on the induction of the tyrosine-phosphorylated form of Stat-1 (Fig. 6B), consistent with the findings using the SB203580 inhibitor. We also determined the effect of the SB203580 inhibitor on the IFNalpha -induced tyrosine phosphorylation of Stat-1 and Stat-2 in Daudi cell lysates, in which Stat-1 was directly immunoprecipitated by an anti-Stat-1 antibody. Incubation of the cells with the inhibitor, at doses that selectively block p38 activation and inhibit IFNalpha -induced gene transcription, did not have a significant effect on tyrosine phosphorylation of Stat-1 and Stat-2 (Fig. 7, A-C). Some minimal inhibition of the phosphorylation of Stat-1 protein at 30 min of IFNalpha treatment seen in this experiment was not consistently seen (data not shown).


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  		Fig. 7.   Inhibition of the Type I IFN-dependent activation of the p38 map kinase does not abrogate tyrosine phosphorylation of Stat-1 or Stat-2 or their DNA binding activities. A, 32P-labeled Daudi cells were preincubated for 30 min in the presence (+) or absence (-) of 10 µM SB203580 as indicated and then treated with IFNalpha for the indicated times in the continuous presence of SB203580. Cell lysates were immuroprecipitated with an anti-Stat-1 antiserum, and immunoprecipitated proteins, were analyzed by SDS-PAGE and transferred to an Immobilon membrane. The membrane was treated for 1 h in 1 M KOH to select for tyrosyl phosphoproteins, and phosphorylated proteins were detected by autoradiography. B, antiphosphotyrosine immunoblot of the Immobilon membrane of the experiment shown in A. C, the blot shown in B was stripped and reprobed with an anti-Stat-1 antibody to control for loading. D, U2OS cells were incubated with or without IFNalpha for 15 min, in the presence or absence of 10 µM SB203580. Nuclear extracts were prepared and analyzed for DNA-binding STAT complexes by GDAC. Eluates from GDAC were resolved by SDS-PAGE and, after Western blotting, probed with antibodies to Stat-1 and Stat-2. IFNalpha -induced Stat proteins are indicated by arrows. E, U2OS cells were treated with IFNalpha in the presence or absence of SB203580 as indicated. Nuclear extracts were reacted with 40,000 cpm of a 32P-end-labeled ISRE, and complexes were resolved by native gel electrophoresis and visualized by autoradiography.

We next performed GDAC studies to determine whether nuclear translocation and DNA binding activity of ISGF3 are regulated by p38. Nuclear extracts from IFNalpha -treated cells, incubated in the presence or absence of SB203580, were analyzed using GDAC and the high salt eluate fractions were resolved by SDS-PAGE and transferred to nitrocellulose. Anti-Stat immunoblotting revealed that after IFNalpha treatment, nuclear extracts from U2OS cells contained inducible DNA binding factors that correspond to the Stat proteins, Stat-1 and Stat-2, the induction of which was not affected by treatment of cells with SB203580 (Fig. 7D). To further characterize the IFNalpha -inducible Stat-containing DNA binding activities, we performed gel mobility shift assays, using the ISRE recognition element. Cells were incubated in the presence or absence of SB203580, and the formation of ISGF3-ISRE complexes in response to IFNalpha was determined. As shown in Fig. 7E, the formation of DNA binding Stat-complexes was not blocked by inhibition of the kinase activity of p38. Thus, although the function of the p38 kinase is essential for Type I IFN-dependent gene transcription, its activation is not required for Stat-tyrosine phosphorylation and DNA binding.

In the present study, we provide the first evidence for the existence of a Type I IFN-dependent signaling pathway involving activation of the p38 kinase and downstream regulation of the MapKap-2 and Mapkap-3 kinases. This pathway is apparently regulated upstream by a member of the MKK family of kinases, as evidenced by the rapid Type I IFN-dependent phosphorylation of p38. Previous studies have established that MKK3 and MKK6 are selective activators of p38 (17), whereas MKK4 activates both p38 and JNK. It remains to be seen whether any of the known MKK family members, or a novel MKK, regulate the Type I IFN-dependent activation of this pathway.

Our findings also provide direct evidence that the p38 pathway acts in coordination with the Jak-Stat pathway to regulate IFN-dependent gene transcription. It is well known that tyrosine phosphorylation of Stats is required for their translocation to the nucleus and DNA binding. As inhibition of p38 activation blocks IFN gene transcription without affecting Stat DNA binding, our data establish that the p38 pathway does not affect Jak kinase activity and tyrosine phosphorylation of Stats.

It has been reported that maximal activation of transcription by Stat-1 in response to IFNgamma requires serine phosphorylation of Stat-1 in addition to tyrosine phosphorylation (31-33), but the serine kinase regulating Stat-1 phosphorylation is unknown. Although there is no direct evidence so far that serine phosphorylation of Stat-1 and/or Stat-2 also occurs in the Type I IFN system, it is possible that such phosphorylation occurs and is regulated by a serine kinase downstream of p38, therefore modifying the transcriptional activity of Stat1, Stat2, or both. Another explanation, however, is that the p38 pathway converges with the Stat pathway further downstream, possibly at the nucleus, and cooperates with it to regulate transcription of interferon sensitive genes. Such a model for a synergism between these two pathways is similar to the previously described effects of p38 on NF-kappa B dependent pathways, where pharmacological inhibition of p38 has been shown to block NF-kappa B-dependent gene transcription, without affecting NF-kappa B-dependent binding activity (34). Viewed together, these data strongly suggest that the p38 pathway regulates gene transcription without affecting the DNA binding activity of transcription factors. The results presented herein provide the first evidence for such effects on gene products regulated by the IFN-activated Jak-Stat pathway.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants CA73381 and CA77816 (to L. C. P.) and 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.

** Current address: Dept. of Molecular Biology, Division of Cardiovascular Diseases, DuPont Pharmaceuticals, Wilmington, DE 19880.

Dagger Dagger To whom correspondence should be addressed: Section of Hematology-Oncology, University of Illinois at Chicago, MBRB, MC-734, Rm. 3150, 900 S. Ashland Ave., Chicago, IL 60607-7173. Tel.: 312-355-0155; Fax: 312-413-7963; E-mail: Lplatani@uic.edu.

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

    ABBREVIATIONS

The abbreviations used are: IFN, interferon; Stat, signal transducer and activator of transcription; ISGF3, interferon-stimulated gene factor-3; ISRE, interferon-stimulated response element; Map, mitogen-activated protein; PAGE, polyacrylamide gel electrophoresis; GDAC, genomic DNA affinity chromatography.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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