JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M506178200 on October 28, 2005

J. Biol. Chem., Vol. 280, Issue 52, 42707-42714, December 30, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/52/42707    most recent
M506178200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhao, K.-W.
Right arrow Articles by Chen, G.-Q.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhao, K.-W.
Right arrow Articles by Chen, G.-Q.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Interferon-{alpha}-induced Expression of Phospholipid Scramblase 1 through STAT1 Requires the Sequential Activation of Protein Kinase C{delta} and JNK*

Ke-Wen Zhao{ddagger}§1, Dong Li{ddagger}1, Qian Zhao§, Ying Huang§, Robert H. Silverman¶, Peter J. Sims||, and Guo-Qiang Chen{ddagger}§2

From the {ddagger}Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis, Chinese Ministry of Education, Rui-Jin Hospital, Shanghai Jiaotong University School of Medicine (formerly Shanghai Second Medical University), Shanghai 200025, China, §Institute of Health Science, Shanghai Jiaotong University School of Medicine/Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200025, China, the Department of Cancer Biology, Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195, and the ||Department of Molecular and Experimental Medicine, The Scripps Research Institute, La, Jolla, California 92037

Received for publication, June 7, 2005 , and in revised form, October 18, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Phospholipid scramblase 1 (PLSCR1), a calcium-binding protein that either inserts into the plasma membrane or binds to genomic DNA in the nucleus, has been shown to contribute to the cell proliferation, differentiation, and apoptosis as well as antiviral activity of interferon (IFN). The expression of PLSCR1 protein is also known to be markedly increased in response to IFN and to some differentiation inducing agents such as all-trans retinoic acid, but the precise mechanisms of this response remain to be investigated. In this study, we show that the protein kinase C{delta} (PKC{delta})-specific inhibitor rottlerin and the dominant negative mutant of PKC{delta} significantly antagonized IFN-induced PLSCR1 expression. The influence of PKC{delta} on IFN-mediated induction of PLSCR1 was dependent upon the phosphorylation of STAT1 at Ser-727. Furthermore, PKC{delta}-mediated activation of STAT1 required the activation of JNK, as the inhibition of JNK activity by its specific inhibitor or transfection of its dominant negative mutant suppressed both serine phosphorylation of STAT1 and PLSCR1 expression but not the activation of PKC{delta}. In conclusion, our results suggest that the induction of PLSCR1 transcription through STAT1 depends upon sequential activation of PKC{delta} and JNK.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human phospholipid scramblase 1 (PLSCR1)3 is a multiply palmitoylated endofacial plasma membrane protein with a proline-rich cytoplasmic domain containing several Src homology 3 and WW domain binding motifs (1, 2). PLSCR1 protein is also phosphorylated by several tyrosine kinases such as c-Abl and c-Src, which participate in multiple growth factor receptor signaling pathways (3–5). As an example, tyrosine phosphorylation of PLSCR1 by c-Src occurs in response to epidermal growth factor, resulting in the association of phosphorylated PLSCR1 with Shc and the activated epidermal growth factor receptor complex (6). PLSCR1 was also reported to be a substrate of protein kinase C{delta} (PKC{delta}), although this could not be confirmed in our recent experiments with recombinant PKC{delta} and apoptosis induction by Fas ligation in Jurkat cells (7). When it fails to be palmitoylated, PLSCR1 can be imported into the nucleus where it binds to genomic DNA, suggesting a potential role for this protein in gene transcription (8, 9).

These biochemical studies strongly indicate that PLSCR1 exerts wide biological effects. Whereas the role of PLSCR1 in remodeling plasma membrane phospholipids and in cell surface exposure of phosphatidylserine, which was originally identified (10, 11), remains a topic of great controversy, there is increasing evidence to suggest a direct role of this protein in regulating cell proliferation and terminal differentiation. As an example, proliferation and terminal differentiation of myeloid precursor cells in response to selective growth factors are impaired in PLSCR1–/– mice (12). Furthermore, the suppression of PLSCR1 expression by small interfering RNA and antisense RNA inhibits the all-trans-retinoic acid (ATRA) and/or phorbol 12-myristate 13-acetate (PMA)-induced leukemic cell differentiation (7, 13).

However, the mechanisms of regulation of PLSCR1 expression remain largely unknown thus far. Recently, we reported that PKC{delta} mediates ATRA and PMA-induced PLSCR1 expression (7). PLSCR1 has also been shown to be among the most potently activated of the interferon (IFNs)-stimulated genes, suggesting that it possibly participates in cellular response to this important cytokine (14, 15). As recently reported, gene deletion of PLSCR1 or suppression of PLSCR1 expression by small interfering RNA in wild type cells was found to markedly attenuate the antiviral activity of IFNs, which appeared to be related to diminished expression of a subset of IFN-stimulated genes with known antiviral activities in cells failing to up-regulate PLSCR1 (16). However, the mechanism by which IFN induces PLSCR1 expression also remains unresolved. Because IFNs was also shown to activate PKC{delta} (17), in the present study we investigated whether and how activated PKC{delta} contributes to the transcriptional activation of PLSCR1 by IFN.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and Cell Lines—IFN{alpha}2a was purchased from Roche Applied Science. PMA and all specific inhibitors, including Gö6976, SB203580, U0126, and SP600125, were from Calbiochem, except for rottlerin (from Biomol, Plymouth, PA). Rabbit polyclonal antibodies for p38, ERK, c-Jun, JNK, and their phosphoylated forms as well as phospho-PKC{delta} (Ser-643), phospho-STAT1 (Ser-727 and Tyr-701), and horseradish peroxidase-linked secondary antibodies were purchased from Cell Signaling (Beverly, MA), except for anti-phospho STAT1 (Ser-727) used in the experiments shown in Fig. 7 (Upstate%20Biotechnology">Upstate Biotechnology, Inc., Waltham, MA). Rabbit polyclonal anti-PKC{delta} antibody and mouse monoclonal anti-STAT1 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA), but anti-STAT1 antibody to detect transfection of mutant STAT1-S727A was purchased from BD Biosciences. Mouse monoclonal anti-beta-actin antibody (Ab-5) was from NeoMarkers (Fremont, CA). Human monocytic leukemic cell line U937 (from Cell Bank of Shanghai Institutes for Biological Sciences, Shanghai, China) was cultured in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (Hyclone, Logan, UT). The human fibrosarcoma cell line HT1080 (also called 2fTGH) and its STAT1–/– cell line U3A (18) (kindly provided by Dr. George Stark, The Lerner Research Institute, Cleveland, OH) were each cultured in Dulbecco's modified Eagle's medium (Sigma-Aldrich) supplemented with 10% heat-inactivated fetal bovine serum. All cells were maintained in 5% CO2-95% air at 37 °C. During experiments, cell viability was kept at least 90% by trypan blue exclusion assay.


Figure 1
View larger version (61K):
[in this window]
[in a new window]
  		FIGURE 1.
The PKC{delta} inhibitor rottlerin suppresses IFN induction of PLSCR1. A, U937 cells were treated with IFN{alpha}2a (3000 IU/ml) for various times as indicated. PLSCR1 mRNA and protein were detected, respectively, by real-time quantitative RT-PCR (top) and Western blot (bottom) with beta-actin as internal controls. Increased -fold (means ± S.D.) for PLSCR1 mRNA in three repeated experiments with each triplicates and for PLSCR1 protein in three experiments are expressed. B and C, U937 cells were treated with 3000 IU/ml IFN{alpha}2a for various times as indicated (B) or with 3000 IU/ml IFN{alpha}2a in the presence or absence of rottlerin (10 µM) for 4 h (C), and phosphorylation of PKC{delta} on Ser-643 was detected by Western blot. Total PKC{delta} was used as an equal loading control. The -fold change (means ± S.D. from three independent tests) for phosphorylated PKC{delta}/total PKC{delta} against untreated cells is shown. *,p = 0.013, compared with column 1;&, p = 0.025, compared with column 3;#, p = 0.071, compared with column 1. D, PKC{delta} activity, in the same samples as shown in C, was measured in an in vitro kinase assay using histone H1 as substrates. U937 cells treated with 20 nM PMA for 1 h as a positive control. The -fold change against control cells is shown as means ± S.D. from three independent tests. *, p = 0.040, compared with column 2;&, p = 0.038, compared with column 4;#, p = 0.048, compared with column 2. E, U937 cells were treated with IFN{alpha}2a (3000 IU/ml) for various times as shown. Then cell lysates were immunoprecipitated with anti-PKC{delta} antibody. The immunoprecipitates were analyzed by Western blots respectively against the indicated antibodies. Input came from untreated U937 cells. F, after preincubation for 1 h with or without rottlerin (10 µM) (left) or Gö6976 (5 µM) (right), U937 cells were treated with 3000 IU/ml IFN{alpha}2a for 18 h. Then, real-time RT-PCR (upper panel) and Western blot (middle panel) were used to detect mRNA and protein levels of PLSCR1 as described in A. The -fold change (means ± S.D.) for PLSCR1 mRNA in three independent samples with the same treatment is expressed. The experiments were repeated three times with the same results. Upper left panel:*, p = 0.005, compared with column 1;&, p = 0.007, compared with column 3;#, p = 0.126, compared with column 1. Upper right panel:*, p = 0.005, compared with column 1; $, p = 0.101, compared with column 3; [caret], p = 0.002, compared with column 1. Then -fold change of PLSCR1 protein against untreated cells is shown in the lower panel as means ± S.D. from three independent experiments. Lower left panel:*, p = 0.013, compared with column 1;&, p = 0.009, compared with column 3;#, p = 0.070, compared with column 1. Lower right panel:*, p = 0.007, compared with column 1;$, p = 0.373, compared with column 3; [caret], p = 0.036, compared with column 1.

 
Plasmids and Transfection —Plasmid pcDNA3-PKC-DN{delta} carrying the dominant negative (DN) fragment of PKC{delta} and its empty vector pcDNA3 were generous gifts from Dr. Jae-Won Soh (Inha University, Incheon, Korea) (19). pcDNA3-DN-JNK1 was kindly provided by Dr. Young-Mi Ham (Seoul National University, Seoul, Korea) (20). pRc/CMV-STAT1 and pRc/CMV-STAT1-S727A were generously provided by Dr. James Darnell (Rockefeller University, New York) (21). These plasmids were transfected into H1080 and U3A cells using Polyfect transfection reagent (Qiagen, Valencia, CA) according to the manufacturer's instructions. Expression of transfected cDNAs was confirmed by Western blotting.


Figure 2
View larger version (19K):
[in this window]
[in a new window]
  		FIGURE 2.
Dominant negative PKC{delta} (DN{delta}) blocks IFN induction of PLSCR1. A, HT1080 cells were transiently transfected with either HPPP-luc or pGL3 control vector together with pRL-SV40 vector. Twenty-four hours later, IFN{alpha}2a with or without rottlerin was added for an additional 18 h. Then, the cells were harvested for measurement of luciferase activities. The bar graph shows the ratio of luciferase to Renilla activities (ordinate). Error bars denote mean ± S.D. of triplicates in an independent experiment. Each experiment was repeated at least three times with similar results. *, p = 0.024, compared with column 2; #, p = 0.021, compared with column 4; &, p = 0.239, compared with column 2. B, HT1080 cells were transiently transfected with either pcDNA3 empty vector or DN{delta} cDNA plasmid. Twenty-four hours after transfection, IFN{alpha}2a was added for an additional 18 h. PLSCR1 mRNA and protein were detected by real-time RT-PCR (top) and Western blot (bottom), respectively. The -fold change (means ± S.D.) for PLSCR1 mRNA in three independent samples with the same treatment and for PLSCR1 protein in three experiments is expressed. *, p < 0.05, compared with column 1; #, p < 0.05, compared with column 3; &, p > 0.05, compared with column 1. C, HT1080 cells were transiently transfected with either HPPP-luc luciferase plasmids or pGL3 control vector together with pRL-SV40 vector and DN{delta} mutant plasmids. After 24 h of transfection, cells were treated with IFN{alpha}2a for an additional 18 h. Cells were harvested for measurement of luciferase activities. The bar graph shows the ratio of relative luciferase activities. Error bars denote mean ± S.D. of triplicate assays in independent experiments, which were repeated at least three times and for which similar results were obtained. *, p = 0.004, compared with column 2;#, p = 0.008, compared with column 4;&, p = 0.068, compared with column 2. All experiments were repeated three times with the same results.

 
Real-time Quantitative RT-PCR for PLSCR1 mRNA—Total RNA was isolated by Trizol kit (Invitrogen) and treated with DNase (Promega, Madison, WI). Complementary DNA was synthesized by using the cDNA synthesis kit according to manufacturer's instructions (Applied Biosystems, Foster City, CA), and fluorescence real-time RT-PCR was performed as we described previously (7) with specific primers for PLSCR1 (sense strand, 5'-CTGACTTCTGAGAAGGTTGC-3'; antisense strand, 5'-GAATGCTGTCGGTGGATACTG-3') and for beta-actin (sense strand, 5'-CATCCTCACCCTGAAGTACCC-3'; antisense strand, 5'-AGCCTGGATAGCAACGTACATG-3'). The -fold change is shown as means ± S.D. in three independent samples with the same treatment.


Figure 3
View larger version (21K):
[in this window]
[in a new window]
  		FIGURE 3.
STAT1 is required for IFN-induced PLSCR1 transcription. STAT1-negative U3A cells (A) or STAT1-positive HT1080 cells (B) were treated with IFN{alpha}2a at 3000 IU/ml for different times as indicated. PLSCR1 mRNA and proteins (PLSCR1, STAT1 Ser-727, total STAT1, and beta-actin) were detected by real-time RT-PCR (top) and Western blot (bottom). The {Delta} symbol represents the non-specific band.

 


Figure 4
View larger version (35K):
[in this window]
[in a new window]
  		FIGURE 4.
IFN induction of PLSCR1 is dependent upon Ser-727 of STAT1. U3A cells were transiently transfected with pRc/CMV-STAT1 (left) and pRc/CMV-STAT1-S727A (right) or empty vector. Twenty-four hours later, cells were treated with 3000 IU/ml IFN{alpha}2a, and the phosphorylated form of STAT1 at Tyr-701 and Ser-727 and total STAT1, PLSCR1, and beta-actin protein were detected by Western blot. The {Delta} symbol points to the nonspecific band. Altered folds of PLSCR1 protein compared with untreated cells are shown as means ± S.D. in three independent experiments. *, p = 0.031, compared with column 1;#, p = 0.047, compared with column 3.

 
Luciferase Reporter Assays —pRL-SV40 vector and human PLSCR1 promotor-primed (HPPP)-luciferase plasmid, which was a pGL3-basic-luciferase reporter vector (Promega) cloned with a 4.18-kb DNA fragment consisting of the 5' flanking region (–1to –4120) and the first 60 bp of the first exon of the human PLSCR1 gene (GenBankTM AF153715) as described previously (14), were transfected into HT1080 cells with or without dominant negative JNK1 (DN-JNK1) or dominant negative PKC{delta} (DN-{delta}) using the Polyfect reagent (Qiagen) following the manufacturer's protocol. Twenty-four hours after transfection, triplicate cultures were treated with IFN{alpha}2a in the presence or absence of rottlerin (10 µM) or Gö6976 (5 µM) for 18 h. Luciferase activity was measured using a Dual-Luciferase reporter assay system (Promega) following the manufacturer's protocol. The measured luciferase activity was normalized against pRL-SV40 Renilla luciferase activity for each sample, and luciferase activity was expressed as -fold over empty pGL3 vector luciferase activity normalized by pRL-SV40 Renilla activity.


Figure 5
View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 5.
PKC{delta} inhibition prevents IFN{alpha}2a-induced Ser-727 phosphorylation of STAT1. HT1080 cells were preincubated with or without rottlerin for 1 h (left) or transfected with PKC-DN{delta} for 24 h (right). Cells were subsequently incubated with 3000 IU/ml IFN{alpha}2a for 2 h, and phosphoserine 727 STAT1 (Ser-STAT1) and total STAT1 were detected with beta-actin as a control. The {Delta} symbol denotes the nonspecific band. The altered -fold of Ser-STAT1/STAT1 against untreated cells is shown as means ± S.D. from three independent experiments. *, p < 0.05, compared with column 1;#, p < 0.05, compared with column 3; &, p > 0.05, compared with column 1.

 
PKC{delta} Activity Assays—U937 cells were lysed in phosphorylation lysis buffer (1% Triton X-100, 150 mM NaCl, 200 µM sodium orthovanadate, 10 mM sodium pyrophosphate, 100 mM sodium fluoride, 1 mM EDTA, 50 mM Hepes, 1.5 mM magnesium chloride, 10% glycerol, and protease inhibitor mixture). Cell lysates were immunoprecipitated with anti-PKC{delta} antibody, and immunoprecipitates were washed three times with phosphorylation lysis buffer and two times with kinase buffer (25 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 0.5 mM EDTA, 1 mM dithiothreitol, 20 µg of phosphatidylserine, and 20 µM ATP) and resuspended in 30 µl of kinase buffer containing 5 µg of histone H1 as an exogenous substrate, to which 1–2 µCi of [{gamma}-32P]ATP was added. The reaction was incubated for 15–30 min at room temperature and terminated by the addition of 2x SDS sample buffer. Proteins were analyzed by SDS-PAGE, and phosphorylated histone H1 was detected by autoradiography (22).


Figure 6
View larger version (69K):
[in this window]
[in a new window]
  		FIGURE 6.
Kinetics of the phosphorylation of ERK, p38, JNK, and compared with the induction of PLSCR1 in response to IFN. U937 cells were incubated with IFN{alpha}2a at 3000 IU/ml for different times as indicated. Total cell lysates were analyzed by Western blot with specific antibodies as shown. Levels of total ERK, p38, JNK, and c-Jun are shown beneath the phosphorylated forms of these proteins.

 
Western Blot—Cells were harvested and lysed with ice-cold phosphorylation lysis buffer as described above and an equal volume of 2xSDS sample buffer. Cell lysates were loaded on 10% SDS-polyacrylamide gel and electrophoretically transferred to nitrocellulose membrane (Amersham Biosciences). After being blocked with 5% nonfat milk in Tris-buffered saline, the membranes were incubated with monoclonal anti-human PLSCR1 4D2 antibody (14) and other indicated antibodies followed by horseradish peroxidase-linked secondary antibodies. Detection was performed by chemiluminescence phototope-horseradish peroxidase kit according to the manufacturer's instructions (Cell Signaling). Blots were stripped and reprobed with mouse monoclonal anti-beta-actin antibody to ascertain equal loading of protein. As necessary, the signal intensities of proteins tested were normalized against the indicated internal control using a densitometer (SmartView, version 5.0, software from Furi, Shanghai, China), and the -fold change was expressed compared with untreated cells.

Statistical Analysis —Student's t test was used to compare the difference between two different groups. A value of p < 0.05 was considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
As reported previously (14, 15), IFN{alpha}2a highly induced PLSCR1 mRNA and protein in leukemic U937 cells as determined by real-time quantitative RT-PCR and Western blotting, respectively (Fig. 1A). Prior to PLSCR1 expression, IFN{alpha}2a treatment rapidly and time-dependently resulted in an increase in PKC{delta} phosphorylation in intact cells. As shown in Fig. 1B, increased phosphorylated PKC{delta} began to appear at 5 min after IFN treatment and then became more significant. To determine the possible involvement of PKC{delta} in IFN induction of PLSCR1, leukemic U937 cells were treated with IFN-{alpha}2a in the absence and presence of the PKC{delta} inhibitor, rottlerin. Pretreatment with rottlerin for 1 h effectively inhibited IFN activation of PKC{delta} as evidenced by PKC{delta} phosphorylation (Fig. 1C) and the ability of immunoprecipitated PKC{delta} to phosphorylate histone H1 (Fig. 1D). It was noteworthy that in addition to PKC{delta} several other kinases, including p38, ERK, and Cdc2/Cdk1, could not be detected in such immunoprecipitates (Fig. 1E), indicating the specificity for in vitro kinase assays of PKC{delta}. More intriguingly, pretreatment with rottlerin prevented IFN induction of PLSCR1 mRNA and protein (Fig. 1F, left panel). In contrast, Gö6976, an inhibitor of conventional PKCs, was ineffective to IFN-induced PLSCR1 expression (Fig. 1F, right panel).


Figure 7
View larger version (46K):
[in this window]
[in a new window]
  		FIGURE 7.
Effects of specific inhibitors of ERK, p38, and JNK on STAT1 Ser-727 phosphorylation and PLSCR1 protein induction by 3000 IU/ml IFN{alpha}2a. U937 cells were incubated for 18 h with IFN{alpha}2a with or without pretreatment with the JNK inhibitor SP600125 at 25 µM (A), the ERK inhibitor U0126 at 5 µM (B), and the p38 inhibitor SB203580 at 1 µM (C). Protein levels were detected at the indicated times by Western blots probed with specific antibodies.

 
As reported previously, rottlerin directly uncouples mitochondrial respiration from oxidative phosphorylation and thus reduces cellular ATP levels that could block any number of ATP-dependent processes (23). On the other hand, rottlerin has also been shown to inhibit other kinases (24, 25). Therefore, the data gathered from using rottlerin should be evaluated cautiously. Considering this, we also tested the effect of a dominant negative mutant of PKC{delta} (DN{delta}), which selectively inhibited action of PKC{delta} (19). For the convenience of transfection, the human fibrosarcoma cell line HT1080 was used. First, a plasmid containing the PLSCR1 promoter linked to luciferase cDNA (HPPP-luc) or the empty vector was transfected into HT1080 cells. As seen in Fig. 2A, IFN treatment resulted in an increase in the promoter activity, which was significantly suppressed by rottlerin but not by Gö6976 (data not shown). In agreement with the effects of rottlerin, transient transfection of DN{delta} inhibited IFN induction of PLSCR1 mRNA/protein (Fig. 2B) and the PLSCR1 promoter-reporter construct (Fig. 2C). All of these findings indicate that IFN activation of PKC{delta} precedes to and contributes to the subsequent induction of PLSCR1. Additionally, rottlerin and DN{delta} also slightly abrogated the basal level of PLSCR protein and its promoter activity, suggesting that basal activity of PKC{delta} possibly contributes to the constitutive expression of PLSCR1.

Because PKC{delta} is known to phosphorylate STAT1 on Ser-727 in response to IFN, STAT1, Ser-727 phosphorylation, and PLSCR1 induction were monitored in HT1080 in comparison with its derivative, the STAT1-null U3A cell line (Fig. 3A) (18). In response to IFN, STAT1 Ser-727 phosphorylation increased at 30 min of treatment (lower, Fig. 3B), which appeared after activation of PKC{delta} (Fig. 1B), whereas the induction of PLSCR1 mRNA and protein was not seen until after 3 and 6 h of treatment, respectively (Fig. 3B). Similar results were obtained in U937 cells (data not shown). Consistent with our previous report (14), however, PLSCR1 was not induced by IFN in the STAT1-deficient U3A cells (Fig. 3A).

We further determined the role of STAT1 Ser-727 phosphorylation in IFN-induced PLSCR1 expression. For this purpose, wild type STAT1 and mutant STAT1-S727A were transfected into U3A cells. Western blots showed the effectiveness of transfection of wild type STAT1 and mutant STAT1-S727A, the former but not the latter being phosphorylated on Ser-727 (Fig. 4). The results showed that ectopic expression of STAT1 but not STAT1-S727A increased PLSCR1 expression in U3A cells (Fig. 4).

In the next phase of analysis, we extended our investigation to the potential relationship between PKC{delta} and STAT1 in the induction of PLSCR1. Toward this end, HT1080 cells were treated with IFN{alpha}2a in combination with either rottlerin or DN{delta} transfection (Fig. 5). The results showed that both rottlerin and DN{delta} transfection effectively inhibited IFN{alpha}2a-stimulated phosphorylation on STAT1 at Ser-727, suggesting that PKC{delta} contributes, directly or indirectly, to IFN{alpha}2a-induced STAT1 phosphorylation.

Previous studies (26, 27) showed that IFN treatment leads to activation of several serine-threonine kinases including mitogen-activated protein kinases (MAPKs) besides PKC{delta}, although the mechanism(s) is incompletely understood. Indeed, several members of the MAPK family including ERK1/2, p38 and JNK, as well as the JNK substrate transcription factor c-Jun, were rapidly but transiently phosphorylated upon IFN{alpha}2a treatment (Fig. 6). Thus, we sought to determine whether any of these MAPKs contribute to PLSCR1 expression in response to IFN{alpha}2a treatment. U937 cells were pretreated for 1 h with MAPK inhibitors U0126 (28), SB203580 (29), and SP600125 (30). The results showed that SP600125, which inhibited phosphorylation of c-Jun by JNK, blocked IFN{alpha}2a stimulation of STAT1 Ser-727 phosphorylation and PLSCR1 expression (Fig. 7A), but U0126 and SB203580 failed to do so. These two inhibitors diminished phosphorylation of ERK1/2 and p38, respectively, after IFN{alpha}2a treatment (Fig. 7, B and C). These results indicate that JNK, but not ERK1/2 or p38, contributes to STAT1 Ser-727 phosphorylation and PLSCR1 expression induced by IFN{alpha}2a. Consistent with this fact, DN-JNK, a dominant negative mutant JNK, also reduced phosphorylation of STAT1 Ser-727 induced by IFN{alpha}2a and blocked IFN-{alpha}2a-induced expression of both the PLSCR1 promoter construct and endogenous PLSCR1 in HT1080 cells (Fig. 8).


Figure 8
View larger version (28K):
[in this window]
[in a new window]
  		FIGURE 8.
Dominant negative JNK inhibits IFN{alpha}2a-induced phosphorylation of Ser-727 STAT1 and PLSCR1 transcription. A, HT1080 cells were transfected with the dominant negative mutant of JNK (DN-JNK) or empty vector. Twenty-four hours later, IFN{alpha}2a was added for an additional 18 h. Total cell lysates were used in Western blots for phosphoserine 727 STAT1, total STAT1, PLSCR1, and beta-actin. The {Delta} symbol shows the non-specific band. The altered -fold of Ser-STAT1/STAT1 or PLSCR1/beta-actin against untreated cells is shown as means ± S.D. from three independent experiments. *, p < 0.05, compared with column 1; #, p < 0.05, compared with column 3; &, p > 0.05, compared with column 1. B, HT1080 cells were transfected with either HPPP-luc luciferase plasmids or pGL3 control vector together with pRL-SV40 vector and DN-JNK plasmids. After 24 h, IFN{alpha}2a (3000 IU/ml) was added for an additional 18 h. The cells were harvested for the measurement of luciferase activities (see "Materials and Methods"). The values are expressed as mean ± S.D. of relative luciferase activities of triplicates from an independent experiment. The experiments were repeated at least three times, and similar results were obtained each time. *, p = 0.014, compared with column 2;#, p = 0.032, compared with column 4;&, p = 0.025, compared with column 2.

 
Finally, we sought to determine whether the effects of PKC{delta} and JNK on STAT1 Ser-727 phosphorylation are independent or in the same signaling pathway. Here we found that inhibition of PKC{delta} by rottlerin reduced IFN{alpha}2a-induced activation of JNK in U937 cells, as determined by reduced phosphorylation of c-Jun. However, inhibition of JNK with SP600125 had no effect on the IFN stimulation of PKC{delta} activation (Fig. 9). In agreement with the effects of these inhibitors, transfection of DN{delta} could inhibit the phosphorylation of JNK and c-Jun, whereas DN-JNK failed to affect the activation of PKC{delta} in HT1080 cells (Fig. 10). These results indicate that PKC{delta} is upstream of and required for JNK activation and that both kinases are involved in IFN signaling to the PLSCR1 gene.


Figure 9
View larger version (55K):
[in this window]
[in a new window]
  		FIGURE 9.
JNK activity is not required for IFN activation of PKC{delta}. After preincubation for 1 h in the presence or absence of rottlerin or SP600125, U937 cells were treated with IFN{alpha}2a for 4 h. A, phospho-c-Jun or phospho-PKC{delta} was measured in a Western blot. The altered -fold of phospho-c-Jun/c-Jun or phospho-PKC{delta}/PKC{delta} against untreated cells is shown as means ± S.D. from three independent experiments. Left panel: *, p = 0.045, compared with column 1; #, p = 0.039, compared with column 3; &, p = 0.062, compared with column 1; right panel:*, p = 0.045, compared with column 1;$, p = 0.459, compared with column 3; [caret], p = 0.141, compared with column 1. B, in vitro PKC{delta} kinase assay was performed as described under "Materials and Methods" with histone H1 as a substrate. U937 cell extract with PMA treatment for 1 h was used as the positive control.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Recently we reported that the activation of PKC{delta} by phosphorylation mediates ATRA and PMA-induced PLSCR1 expression in leukemic cells (7). In this work, we continued to show that the inhibition of PKC{delta} activation by the chemical inhibitor rottlerin and transfection of the dominant negative mutant of PKC{delta} significantly inhibited IFN{alpha}2a-induced expression of PLSCR1 mRNA and protein, whereas such inhibition was not observed for Gö6976, an inhibitor of the conventional PKC. These data, combined with our previous report demonstrating that transfection of the catalytic fragment of PKC{delta} directly up-regulates PLSCR1 expression (7), strongly suggest that PKC{delta} is a common mediator for PLSCR1 expression induced by IFN{alpha}2a, ATRA, or PMA. More recently, PLSCR1 expression has been shown to mediate the antiviral activity of IFNs (16). Our previous studies also showed that induced expression of PLSCR1 contributes to ATRA/PMA-induced leukemic cell differentiation (7). Furthermore, IFN{alpha}2a and ATRA act synergistically in the induction of cell differentiation and growth inhibition (22). Therefore, it is of interest that IFN{alpha}2a and ATRA both up-regulate PLSCR1 expression through PKC{delta}.

It is well known that IFN-{alpha}/beta binding to the type I IFN receptor initiates a tyrosine phosphorylation cascade involving JAK kinases (JAK1 and TYK2), resulting in the heterodimerization of tyrosine-phosphorylated STAT1 and 2. These phosphorylated STAT proteins are transported to the nucleus and, in a complex with IRF9, bind to IFN-stimulated response elements and activate the transcription of many different genes (31–33). However, an additional phosphorylation event on STAT1 Ser-727 is required for a full IFN-stimulated transcriptional response (34). Previously, IFN-{alpha}2a was reported to activate expression of the human PLSCR1 gene at the transcriptional level through a single IFN-stimulated response element located in the first exon (14). Accordingly, IFN-{alpha}2a failed to induce PLSCR1 in STAT1-negative U3A cells. Here we showed that complementation with wild type STAT1, but not STAT1 mutant S727A, almost completely restored IFN-{alpha}2a-induced PLSCR1 expression. These findings support an essential role for Ser-727 of STAT1 in IFN-{alpha}2a-induced PLSCR1 expression. Prior studies demonstrating that PKC{delta} can phosphorylate STAT1 on Ser-727 indicated that a role for PKC{delta} in PLSCR1 expression is likely (35). It has been proposed that PKC{delta} is activated during engagement of the Type I IFN receptor and that such an activation of PKC{delta} appears critical for phosphorylation of STAT1 on Ser-727 (17). DeVries et al. (36) also showed that PKC{delta} activates STAT1 through phosphorylation of STAT1 on Ser-727 in etoposide-induced apoptosis. We have shown here that the PKC{delta} inhibitor rottlerin or a dominant negative mutant of PKC{delta} significantly antagonized IFN{alpha}2a-induced STAT1 phosphorylation on Ser-727. Therefore, our findings demonstrate a role for PKC{delta} in the Ser-727 phosphorylation of STAT1 required for IFN induction of PLSCR1.


Figure 10
View larger version (43K):
[in this window]
[in a new window]
  		FIGURE 10.
Inhibition of PKC{delta} could abrogate JNK activation induced by IFN. HT1080 cells were transfected with a dominant negative mutant of PKC{delta} (DN{delta})(left) or dominant negative mutant of JNK (DN-JNK)(right). Twenty-four hours later, IFN{alpha}2a was added for an additional 4 h. Total cell lysates were used in Western blots for phosphorylation of PKC{delta}, JNK, and c-Jun and total PKC{delta}, JNK, and c-Jun. The altered -fold of phospho-JNK/JNK, phospho-c-Jun/c-Jun, or phospho-PKC{delta}/PKC{delta} against untreated cells is shown as means ± S.D. from three independent experiments. *, p < 0.05, compared with column 1; #, p < 0.05, compared with column 3; &, p > 0.05, compared with column 1 (for lower right panel:&, p = 0.049).

 
How PKC{delta} activation leads to Ser-727 phosphorylation of STAT1 is still unresolved. IFN{alpha} treatment results in phosphorylation of many different signaling proteins, including insulin receptor substrate (IRS)-phosphatidylinositol 3-kinase (37, 38), Crk (39), and MAPKs (40, 41). A recent report demonstrated that genotoxic agents such as arabino-furanosylcytosine-induced JNK activation is attenuated by treatment with rottlerin, expression of a kinase-inactive PKC{delta} mutant, and down-regulation of PKC{delta} by small interfering RNA, proposing that PKC{delta} is required, in part, for induction of JNK (42). We have demonstrated here that a JNK inhibitor, but not inhibitors of ERK1/2 and p38, blocked both STAT1 serine727 phosphorylation and PLSCR1 expression following IFN treatment. More importantly, transfection of a dominant negative mutant of JNK also reduced IFN-initiated Ser-727 phosphorylation of STAT1 and PLSCR1 expression. Our results indicated that JNK contributes to both STAT1 Ser-727 phosphorylation and an IFN transcriptional response as monitored by expression of PLSCR1. However, inhibition of JNK did not suppress IFN{alpha}2a-induced phosphorylation and activation of PKC{delta}. Therefore, JNK activation is downstream of PKC{delta}, whereas both of these kinases are required for maximal STAT1 Ser-727 phosphorylation. In summary, PKC{delta} inhibition suppressed IFN-initiated JNK activation, STAT1 Ser-727 phosphorylation, and expression of PLSCR1. Transcriptional induction of PLSCR1 through STAT1 that is induced by IFN{alpha}2a is dependent upon sequential activation of both PKC{delta} and JNK kinase.


   FOOTNOTES
 
* This work was supported by Grant NO2002CB512806 from the National Key Program (973) for Basic Research of China, Grant 2003DF000038 from the International Collaborative Items of Ministry of Science and Technology of China, Grants 90408009 and 30470736 National Natural Science Foundation of China, Grants 03XD14016, 04QMX1467, and 04DZ14901 from the Science and Technology Committee of Shanghai, and Grant 5R01CA089132 from NCI, National Institutes of Health (to R. H. S. and P. J. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 These authors were equal contributors to this work. Back

2 To whom correspondence should be addressed: Dept. of Pathophysiology, Shanghai Jiaotong University School of Medicine, No. 280, Chong-Qing South Rd., Shanghai 200025, China. Tel. and Fax: 86-21-64154900; E-mail: chengq{at}shsmu.edu.cn or gqchen{at}sibs.ac.cn.

3 The abbreviations used are: PLSCR1, phospholipid scramblase 1; ATRA, all-trans-retinoic acid; DN, dominant negative; MAPK, mitogen-activated protein kinase; PKC{delta}, protein kinase C{delta}; PMA, phorbol 12-myristate 13-acetate; STAT1, signal transducer and activator of transcription-1; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; IFN, interferon; RT, reverse transcription. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Jae-Won Soh, Young-Mi Ham, and James Darnell for generously providing the plasmids. We are also grateful to Dr. George Stark, who kindly provided us with HT1080 and U3A cell lines.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Sims, P. J., and Wiedmer, T. (2001) Thromb. Haemostasis 86, 266–275[Medline] [Order article via Infotrieve]
  2. Zhao, J., Zhou, Q., Wiedmer, T., and Sims, P. J. (1998) Biochemistry 37, 6361–6366[CrossRef][Medline] [Order article via Infotrieve]
  3. Sun, J., Zhao, J., Schwartz, M. A., Wang, J. Y., Wiedmer, T., and Sims, P. J. (2001) J. Biol. Chem. 276, 28984–28990[Abstract/Free Full Text]
  4. Nanjundan, M., Sun, J., Zhao, J., Zhou, Q., Sims, P. J., and Wiedmer, T. (2003) J. Biol. Chem. 278, 37413–37418[Abstract/Free Full Text]
  5. Frasch, S. C., Henson, P. M., Kailey, J. M., Richter, D. A., Janes, M. S., Fadok, V. A., and Bratton, D. L. (2000) J. Biol. Chem. 275, 23065–23073[Abstract/Free Full Text]
  6. Sun, J., Nanjundan, M., Pike, L. J., Wiedmer, T., and Sims, P. J. (2002) Biochemistry 41, 6338–6345[CrossRef][Medline] [Order article via Infotrieve]
  7. Zhao, K. W., Li, X., Zhao, Q., Huang, Y., Li, D., Peng, Z. G., Shen, W. Z., Zhao, J., Zhou, Q., Chen, Z., Sims, P. J., Wiedmer, T., and Chen, G. Q. (2004) Blood 104, 3731–3738[Abstract/Free Full Text]
  8. Chen, M. H., Ben-Efraim, I., Mitrousis, G., Walker-Kopp, N., Sims, P. J., and Cingolani, G. (2005) J. Biol. Chem. 280, 10599–10606[Abstract/Free Full Text]
  9. Ben-Efraim, I., Zhou, Q., Wiedmer, T., Gerace, L., and Sims, P. J. (2004) Biochemistry 43, 3518–3526[CrossRef][Medline] [Order article via Infotrieve]
  10. Basse, F., Stout, J. G., Sims, P. J., and Wiedmer, T. (1996) J. Biol. Chem. 271, 17205–17210[Abstract/Free Full Text]
  11. Zhou, Q., Zhao, J., Stout, J. G., Luhm, R. A., Wiedmer, T., and Sims, P. J. (1997) J. Biol. Chem. 272, 18240–18244[Abstract/Free Full Text]
  12. Zhou, Q., Zhao, J., Wiedmer, T., and Sims, P. J. (2002) Blood 99, 4030–4038[Abstract/Free Full Text]
  13. Nakamaki, T., Okabe-Kado, J., Yamamoto-Yamaguchi, Y., Hino, K., Tomoyasu, S., Honma, Y., and Kasukabe, T. (2002) Exp. Hematol. 30, 421–429[CrossRef][Medline] [Order article via Infotrieve]
  14. Zhou, Q., Zhao, J., Al-Zoghaibi, F., Zhou, A., Wiedmer, T., Silverman, R. H., and Sims, P. J. (2000) Blood 95, 2593–2599[Abstract/Free Full Text]
  15. Der, S. D., Zhou, A., Williams, B. R., and Silverman, R. H. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 15623–15628[Abstract/Free Full Text]
  16. Dong, B., Zhou, Q., Zhao, J., Zhou, A., Harty, R. N., Bose, S., Banerjee, A., Slee, R., Guenther, J., Williams, B. R., Wiedmer, T., Sims, P. J., and Silverman, R. H. (2004) J. Virol. 78, 8983–8993[Abstract/Free Full Text]
  17. Uddin, S., Sassano, A., Deb, D. K., Verma, A., Majchrzak, B., Rahman, A., Malik, A. B., Fish, E. N., and Platanias, L. C. (2002) J. Biol. Chem. 277, 14408–14416[Abstract/Free Full Text]
  18. McKendry, R., John, J., Flavell, D., Muller, M., Kerr, I. M., and Stark, G. R. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 11455–11459[Abstract/Free Full Text]
  19. Soh, J. W., and Weinstein, I. B. (2003) J. Biol. Chem. 278, 34709–34716[Abstract/Free Full Text]
  20. Ham, Y. M., Choi, J. S., Chun, K. H., Joo, S. H., and Lee, S. K. (2003) J. Biol. Chem. 278, 50330–50337[Abstract/Free Full Text]
  21. Zhang, J. J., Zhao, Y., Chait, B. T., Lathem, W. W., Ritzi, M., Knippers, R., and Darnell, J. E., Jr. (1998) EMBO J. 17, 6963–6971[CrossRef][Medline] [Order article via Infotrieve]
  22. Kambhampati, S., Li, Y., Verma, A., Sassano, A., Majchrzak, B., Deb, D. K., Parmar, S., Giafis, N., Kalvakolanu, D. V., Rahman, A., Uddin, S., Minucci, S., Tallman, M. S., Fish, E. N., and Platanias, L. C. (2003) J. Biol. Chem. 278, 32544–32551[Abstract/Free Full Text]
  23. Soltoff, S. P. (2001) J. Biol. Chem. 276, 37986–37992[Abstract/Free Full Text]
  24. Leitges, M., Elis, W., Gimborn, K., and Huber, M. (2001) Lab. Investig. 81, 1087–1095[Medline] [Order article via Infotrieve]
  25. Davies, S.P., Reddy, H., Caivano, M., and Cihen, P. (2000) Biochem. J. 351, 95–105[CrossRef][Medline] [Order article via Infotrieve]
  26. Platanias, L. C. (2003) Pharmacol. Ther. 98, 129–142[CrossRef][Medline] [Order article via Infotrieve]
  27. Kalvakolanu, D. V. (2003) Pharmacol. Ther. 100, 1–29[CrossRef][Medline] [Order article via Infotrieve]
  28. DeSilva, D. R., Jones, E. A., Favata, M. F., Jaffee, B. D., Magolda, R. L., Trzaskos, J. M., and Scherle, P. A. (1998) J. Immunol. 160, 4175–4181[Abstract/Free Full Text]
  29. Borsch-Haubold, A. G., Pasquet, S., and Watson, S. P. (1998) J. Biol. Chem. 273, 28766–28772[Abstract/Free Full Text]
  30. Bennett, B. L., Sasaki, D. T., Murray, B. W., O'Leary, E. C., Sakata, S. T., Xu, W., Leisten, J. C., Motiwala, A., Pierce, S., Satoh, Y., Bhagwat, S. S., Manning, A. M., and Anderson, D. W. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 13681–13686[Abstract/Free Full Text]
  31. Darnell, J. E., Jr. (1997) Science 277, 1630–1635[Abstract/Free Full Text]
  32. Platanias, L. C., and Fish, E. N. (1999) Exp. Hematol. 27, 1583–1592[CrossRef][Medline] [Order article via Infotrieve]
  33. Stark, G. R., Kerr, I. M., Williams, B. R., Silverman, R. H., and Schreiber, R. D. (1998) Annu. Rev. Biochem. 67, 227–264[CrossRef][Medline] [Order article via Infotrieve]
  34. Wen, Z., Zhong, Z., and Darnell, J. E., Jr. (1995) Cell 82, 241–250[CrossRef][Medline] [Order article via Infotrieve]
  35. Kaur, S., Parmar, S., Smith, J., Katsoulidis, E., Li, Y., Sassano, A., Majchrzak, B., Uddin, S., Tallman, M. S., Fish, E. N., and Platanias, L. C. (2005) Exp. Hematol. 33, 550–557[CrossRef][Medline] [Order article via Infotrieve]
  36. DeVries, T. A., Kalkofen, R. L., Matassa, A. A., and Reyland, M. E. (2004) J. Biol. Chem. 279, 45603–45612[Abstract/Free Full Text]
  37. Uddin, S., Fish, E. N., Sher, D. A., Gardziola, C., White, M. F., and Platanias, L. C. (1997) J. Immunol. 158, 2390–2397[Abstract]
  38. Uddin, S., Fish, E. N., Sher, D., Gardziola, C., Colamonici, O. R., Kellum, M., Pitha, P. M., White, M. F., and Platanias, L. C. (1997) Blood 90, 2574–2582[Abstract/Free Full Text]
  39. Fish, E. N., Uddin, S., Korkmaz, M., Majchrzak, B., Druker, B. J., and Platanias, L. C. (1999) J. Biol. Chem. 274, 571–573[Abstract/Free Full Text]
  40. Uddin, S., Majchrzak, B., Woodson, J., Arunkumar, P., Alsayed, Y., Pine, R., Young, P. R., Fish, E. N., and Platanias, L. C. (1999) J. Biol. Chem. 274, 30127–30131[Abstract/Free Full Text]
  41. Uddin, S., Lekmine, F., Sharma, N., Majchrzak, B., Mayer, I., Young, P. R., Bokoch, G. M., Fish, E. N., and Platanias, L. C. (2000) J. Biol. Chem. 275, 27634–27640[Abstract/Free Full Text]
  42. Yoshida, K., Miki, Y., and Kufe, D. (2002) J. Biol. Chem. 277, 48372–48378[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Proc. Natl. Acad. Sci. USAHome page
S. Kaur, A. Sassano, B. Dolniak, S. Joshi, B. Majchrzak-Kita, D. P. Baker, N. Hay, E. N. Fish, and L. C. Platanias
Role of the Akt pathway in mRNA translation of interferon-stimulated genes
PNAS, March 25, 2008; 105(12): 4808 - 4813.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Biol. CellHome page
T. Panaretakis, L. Hjortsberg, K. P. Tamm, A.-C. Bjorklund, B. Joseph, and D. Grander
Interferon {alpha} Induces Nucleus-independent Apoptosis by Activating Extracellular Signal-regulated Kinase 1/2 and c-Jun NH2-Terminal Kinase Downstream of Phosphatidylinositol 3-Kinase and Mammalian Target of Rapamycin
Mol. Biol. Cell, January 1, 2008; 19(1): 41 - 50.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. Kaur, L. Lal, A. Sassano, B. Majchrzak-Kita, M. Srikanth, D. P. Baker, E. Petroulakis, N. Hay, N. Sonenberg, E. N. Fish, et al.
Regulatory Effects of Mammalian Target of Rapamycin-activated Pathways in Type I and II Interferon Signaling
J. Biol. Chem., January 19, 2007; 282(3): 1757 - 1768.
[Abstract] [Full Text] [PDF]

Home page
 Microbiol. Mol. Biol. Rev.Home page
M. A. Bogoyevitch and B. Kobe
Uses for JNK: the Many and Varied Substrates of the c-Jun N-Terminal Kinases
Microbiol. Mol. Biol. Rev., December 1, 2006; 70(4): 1061 - 1095.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
K. E. Thomas, C. L. Galligan, R. D. Newman, E. N. Fish, and S. N. Vogel
Contribution of Interferon-beta to the Murine Macrophage Response to the Toll-like Receptor 4 Agonist, Lipopolysaccharide
J. Biol. Chem., October 13, 2006; 281(41): 31119 - 31130.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available