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The Journal of Immunology, 2001, 166: 6771-6775.
Copyright © 2001 by The American Association of Immunologists

A Retroviral-Derived Immunosuppressive Peptide Activates Mitogen-Activated Protein Kinases1

Akio Takahashi, Noorbibi K. Day2, Voravich Luangwedchakarn, Robert A. Good and Soichi Haraguchi

Department of Pediatrics, University of South Florida, All Children’s Hospital, St. Petersburg, FL 33701


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The highly conserved region within the retroviral transmembrane envelope proteins has been implicated in a number of retrovirus-associated mechanisms of immunosuppression. CKS-17, a synthetic peptide representing the prototypic sequence of the immunosuppressive domain, has been found to suppress numerous immune functions, disregulate cytokines, and elevate intracellular cAMP. In this report we show that using a human monocytic cell line THP-1, CKS-17 activates mitogen-activated protein (MAP) kinases extracellular signal-regulated kinase 1 and 2 (ERK1/2). Kinetic studies show that CKS-17 induces an acute increase of ERK1/2 activity followed by a rapid decrease and then a second sustained increase of ERK1/2. CKS-17 also activates MAP kinase/ERK kinase (MEK) with a similar induction pattern. Mutant THP-1 cells isolated in our laboratory, in which CKS-17 exclusively fails to activate cAMP, did not show the transient decrease of CKS-17-induced ERK1/2 phosphorylation. Pretreatment of THP-1 cells or mutant THP-1 cells with cAMP analog or forskolin followed by treatment with CKS-17 showed no activation of MEK or ERK1/2. These results indicate that CKS-17 activates the MEK/ERK cascade and that there is a cross-talk between CKS-17-mediated MEK/ERK cascade and cAMP in that the MEK/ERK cascade is negatively regulated by cAMP. These data present a novel molecular mechanism(s) by this highly conserved retroviral immunosuppressive component.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The transmembrane glycoproteins of numerous animal and human retroviruses share conserved structural features (1, 2, 3). One highly conserved region of the transmembrane proteins contains a leucine zipper-like domain comprising an {alpha} helical secondary structure that may play an important role in the processes of virus fusion, infectivity, and entry (4, 5, 6, 7). Of interest, this leucine zipper-like domain overlaps a unique region that has strong immunosuppressive potential (5, 7, 8). CKS-17, a synthetic peptide, represents the prototypic amino acid sequence of this immunosuppressive domain (9) and has been found to exhibit potent immunosuppressive activities in numerous immune reactions both in vitro and in vivo (reviewed in Ref. 8).

We have also reported that CKS-17 induces intracellular cAMP using a human monocytic cell line THP-1 and PBMCs (10). cAMP, an important intracellular second messenger, controls various immune functions, particularly of suppression of the Th1 type of immune responses (11, 12, 13, 14). Currently we are trying to elucidate another signal transduction pathway, i.e., mitogen-activated protein (MAP)3 kinase, induced by CKS-17. MAP kinase, also known as extracellular signal-regulated kinase (ERK) 1 and ERK2, cascades have been widely studied in view of their role as signal transduction pathways through receptor activation by ligand binding (15). Recently, it has been reported that activation of MAP kinase pathway plays a role in enhancing viral infection and replication (16) or in suppressing Th1-related cytokine production (17).

In this study, we show that CKS-17 activates MAP kinases, ERK1 and ERK2. CKS-17 also activates MEK. Kinetic studies demonstrate that CKS-17 induces an acute activation followed by a rapid, transient inactivation and a second activation of ERK1/2 or MEK. This provocative finding and the considerations described above prompted us to further assess the cross-talk between CKS-17-induced MAP kinase and cAMP. We show that pretreatment of THP-1 cells with cAMP-elevating agents suppresses significantly CKS-17-induced activation of ERK1/2 and MEK, indicating that cAMP negatively regulates MAP kinase activation by CKS-17 upstream of MEK.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and Abs

PD98059 (18, 19) (Alexis Biochemicals, Pittsburgh, PA), U0126 (20), forskolin, N6,2'-O-dybutyryladenosine-3',5'-cyclic monophosphate, and sodium salt monohydrate (dibutyryl-cAMP) (Biomol, Plymouth Meeting, PA) were used to treat cells. Mouse anti-phospho-ERK1/2 (p-ERK1/2) mAb (E10), rabbit anti-ERK1/2 Ab, rabbit anti-phospho-MEK1/2 (p-MEK1/2) Ab, rabbit anti-MEK1/2 Ab, rabbit anti-phospho-Elk1 Ab, inactive ERK2 full length recombinant protein, and inactive Elk-1 fusion protein were purchased from New England Biolabs (Beverly, MA). Goat anti-mouse IgG Ab conjugated to HRP and goat anti-rabbit IgG Ab conjugated to HRP were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Preparation of synthetic peptide

A dimer of CKS-17, termed MN10021, [(LQNRRGLDLLFLKEGGLC)2] was prepared by inclusion of the naturally occurring cysteine at the carboxyl terminus and dimerization via a cysteine-disulfide linkage. The monomer of CKS-17, termed MN10022 (MN22), [LQNRRGLDLLFLKEGGLC] and the reverse peptide dimer, termed MN20050 (MN50), [(LGGEKLFLLDLGRRNQLC)2] were prepared similarly. These peptides are the gifts of George Cianciolo (Duke University Medical Center, Durham, NC).

Cell line

THP-1, a human acute monocytic leukemia cell line (21), was obtained from American Type Culture Collection (Manassas, VA). THP-1 cells were routinely maintained in RPMI 1640 supplemented with 10% FCS (HyClone, Logan, UT), 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, and 5 x 10-5 M 2-ME at 37°C in a 5% CO2 incubator.

Establishment of mutant THP-1 cell lines

To select mutant cells that are unable to induce cAMP by treatment with CKS-17, THP-1 cells were mutagenized with 400 µg/ml of ethyl methanesulfonate and cloned under limiting dilution conditions. Mutant cells that did not induce intracellular cAMP by treatment with CKS-17 were screened by cAMP assays and further cloned under limiting dilution conditions. Mutant cell lines, THP-1:mC1.1 (C1) and THP-1:mG7.1 (G7), were selected for this study.

Treatment of cells

Subconfluent cells were cultured in RPMI 1640 without FCS for 24 h before treatment. Serum-starved cells were washed once with PBS and resuspended in serum-free and protein-free hybridoma medium (Sigma, St. Louis, MO), then treated with reagents as indicated in figure legends at 37°C using 1.5-ml microcentrifuge tubes.

Western blotting

Stimulated cells were lysed with ice-cold lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% (octylphenoxy)polyethoxyethanol CA-630, 0.25% sodium deoxycholate, 1 mM EGTA, 1 mM NaF, 1 mM sodium orthovanadate, 1 mM PMSF, and protease inhibitor mixture (Sigma). Protein determination was performed by using a protein assay kit (Bio-Rad, Hercules, CA). Equal amounts of protein were separated by SDS-PAGE and transferred to Hybond ECL (Amersham Pharmacia Biotech, Piscataway, NJ) using Trans-Blot transfer cell (Bio-Rad). Membranes were blocked with 5% nonfat milk/PBS with 0.1% Tween 20 for ERK1/2 and MEK, 1% bovine serum albumin/PBS for p-ERK1/2 and p-Elk1, and then incubated with an appropriate secondary Ab. After several washes with PBS containing 0.1% Tween 20, the membranes were incubated at room temperature for 1 h with goat anti-rabbit or anti-mouse IgG Ab conjugated to HRP, and then washed again. The protein on the membranes was detected by an ECL kit (Amersham Pharmacia Biotech). The detected bands were scanned and the density was determined using the multiscan-R (Interactive Technologies International, St. Petersburg, FL).

Immunoprecipitation

The cells were lysed with ice-cold lysis buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM {beta}-glycerolphosphate, 1 mM NaF, 1 mM sodium orthovanadate, 1 mM PMSF, 1 µg/ml leupeptin, and protease inhibitor mixture. The lysates were centrifuged, and the resulting supernatants were adjusted to equal amounts of protein. The supernatants were incubated for 1 h on ice with anti-p-MEK1/2, and then incubated with 20 µl of protein A-Sepharose 4B beads (Zymed Laboratories, South San Francisco, CA) overnight at 4°C with gentle rocking. To immunoprecipitate active ERK, the supernatants were incubated with anti-p-ERK1/2-immobilized agarose beads (New England Biolabs) overnight at 4°C with gentle rocking. The beads were washed twice with lysis buffer and subjected to in vitro kinase assay or Western blotting.

Immunocomplex in vitro kinase assays

The kinase activities of ERK1/2 and MEK were determined by nonradioactive protein kinase assay system (New England Biolabs). The precipitated immunocomplexes were washed with kinase buffer containing 20 mM Tris-HCl (pH 7.5), 5 mM {beta}-glycerolphosphate, 2 mM DTT, 0.1 mM sodium orthovanadate, and 10 mM MgCl2. The anti-p-ERK1/2 immunoprecipitates were resuspended in 50 µl of kinase buffer with 200 µM ATP and 2 µg inactive Elk-1 fusion protein. Similarly, immunoprecipitates prepared with anti-p-MEK1/2 were resuspended in 50 µl of kinase buffer with 200 µM ATP and 2 µg inactive ERK2 recombinant protein. All mixtures were incubated for 30 min at 30°C. The reactions were then terminated by addition of SDS sample buffer and analyzed by Western blotting probed with anti-p-Elk1 Ab for ERK1/2 activity or anti-p-ERK1/2 Ab for MEK activity.

Quantitation of cAMP levels

Intracellular cAMP levels were determined as described previously (10). Briefly, cells (1 x 106) were treated with CKS-17 at 37°C and centrifuged. Five hundred microliters of ice-cold 65% ethanol were added to the pellet, which was vortexed and centrifuged at 2000 x g for 15 min at 4°C. The supernatant was transferred to a new tube, dried in a SpeedVac concentrator (Savant, Holbrook, NY), and stored at -20°C. Just before use, the dried extracts were dissolved in 1 ml assay buffer, and cAMP levels were measured by using a cAMP enzyme immunoassay system (Amersham Pharmacia Biotech).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CKS-17 phosphorylates MAP kinases ERK1 and ERK2

The influence of CKS-17 on MAP kinase (ERK1 and ERK2) activation using THP-1 cells is shown in Fig. 1Go. THP-1 cells were cultured in serum-starved medium for 24 h and then stimulated with 30 µM of CKS-17 (dimer), MN22 (monomer), or MN50 (reverse dimer). Kinetic studies showed that CKS-17 induced an acute increase of p-ERK levels followed by a rapid decrease and a second activation that sustains elevated levels of p-ERKs determined by Western blotting and scanning densitometry (Fig. 1Go, A and B). This increase of p-ERK levels was observed within 30 s of treatment and at least 24 h after treatment (data not shown). The control monomer peptide MN22 and reverse peptide dimer MN50 did not induce the increase of p-ERK levels. Fig. 1Go, C and D, shows the influence of various concentrations (3, 10, and 30 µM) of CKS-17 on MAP kinase phosphorylation. As shown, p-ERK levels were remarkably induced by CKS-17 in a dose-dependent manner both at 2 and 60 min after treatment. MAP kinase phosphorylation by CKS-17 was inhibited by PD98059 and U0126, specific inhibitors of MAP kinase/ERK kinase (MEK) (Fig. 1Go, C and D).



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  		FIGURE 1. CKS-17 activates MAP kinase (ERK1 and ERK2) using human monocytic cell line THP-1. Serum-starved THP-1 cells were treated as indicated. A, Time course experiment. THP-1 cells were treated with 30 µM CKS-17 or 30 µM control monomer peptide MN22 or reverse peptide dimer MN50 for 2, 10, 60, and 120 min. Whole cell extracts (10 µg of protein) were prepared, and the phosphorylation of MAP kinase was determined by Western blotting using a monoclonal Ab specifically raised against threonine-202 and tyrosine-204 phosphorylated human ERK1 (p-ERK1) corresponding identically to phosphorylated ERK2 (p-ERK2) (upper lane). The membrane was reprobed by a polyclonal Ab to whole ERK1/2 molecules to confirm that equal amounts of ERKs were present in each lane (lower lane). B, The phosphorylation of MAP kinase in the time course experiments was quantified by scanning densitometry. Data represent a fold increase of relative p-ERK1/2 levels compared with nonstimulated medium control. Experiments were performed at least three times each. Error bars show SD of the mean. C, Dose response experiment. Serum-starved THP-1 cells were treated with indicated concentrations of CKS-17 for 2 or 60 min. Specific MEK inhibitors, PD98059 (50 µM) and U0126 (10 µM), were used for pretreating cells for 1 h before CKS-17 treatment. Total cell lysates (10 µg of protein) were subjected to Western blotting probed by anti-p-ERK1/2 Ab (upper lane). The membranes were reprobed by anti-ERK1/2 Ab (lower lane). The experiment was repeated three times and one of the three typical results is shown. D, The phosphorylation of MAP kinase in the dose response experiments was quantified by scanning densitometry. Data represent a fold increase of relative p-ERK1/2 levels compared with nonstimulated medium control.

 
CKS-17 activates kinase activity of MAP kinase and MEK

We next examined ERK and MEK kinase activity using an in vitro kinase assay. Immunoprecipitated active ERKs and MEK were incubated with an inactive Elk-1 fusion protein, a substrate of ERKs (22), and an inactive recombinant ERK2 protein, a substrate of MEK, respectively. Phosphorylation of Elk-1 or ERK2 was determined by Western blotting using a phospho-Elk-1 (serine 383)-specific Ab or p-ERK1/2 Ab, respectively. As shown in Fig. 2Go, A and B, the in vitro kinase assay shows that CKS-17 activates MAP kinase and MEK in a similar pattern to the induction of p-ERK levels indicated in Fig. 1Go, A and B. In addition, both PD98059 and U0126 inhibit MAP kinase phosphorylation by CKS-17 (see Fig. 1Go, C and D). These results suggest that CKS-17-induced activation of MAP kinase is mediated by MEK activation.



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  		FIGURE 2. MAP kinase and MEK activation were monitored using an in vitro kinase assay. A, Serum-starved THP-1 cells were treated with 30 µM of CKS-17 for 2, 10, 60, and 120 min. Immunoprecipitated ERK1/2 was incubated with an inactive Elk-1 fusion protein as a substrate, and phosphorylated Elk-1 was detected by Western blotting using anti-p-Elk1 Ab (upper lane). Similarly, immunoprecipitated MEK was incubated with an inactive ERK2 recombinant protein as a substrate, and phosphorylated ERK2 was detected by Western blotting with anti-p-ERK1/2 Ab (lower lane). B, The in vitro kinase assay was quantified by scanning densitometry. Data represent a fold increase compared with nonstimulated median control. Experiments were performed three times. Error bars show SD of the mean.

 
Mutant THP-1 cell lines in which CKS-17 fails to increase cAMP levels do not exhibit initial decrease of p-ERK1/2 levels induced by CKS-17

Because previous experiments in our laboratory have shown that CKS-17 induces increased intracellular levels of cAMP in THP-1 cells (10), we next investigated the influence of cAMP on MAP kinase activation by CKS-17. First, we established mutant THP-1 cell lines C1 or G7, which were unable to produce cAMP by CKS-17 (see Materials and Methods). Fig. 3GoA shows that while CKS-17 significantly induces cAMP in THP-1 cells, CKS-17 completely fails to elevate intracellular cAMP levels in mutant cells, C1 or G7. These mutant cell lines are able to produce cAMP by forskolin, a direct activator of adenylate cyclase (data not shown). Next, both C1 or G7 cells were treated separately with CKS-17 for various times, and p-ERK1/2 levels were determined by Western blotting. As shown in Fig. 3GoB, the initial decline of p-ERK1/2 levels by treatment of CKS-17 that was observed using THP-1 cells (see Fig. 1Go, A and B, and Fig. 3GoB) was abolished using C1 and G7 cells.



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  		FIGURE 3. A, Failure of CKS-17 to induce intracellular cAMP levels in mutant THP-1 cells. THP-1, C1, and G7 were treated with 30 µM CKS-17 for 2, 10, 30, 60, and 120 min. Cell extracts were prepared and monitored for cAMP. Error bars show SD of the mean. B, MAP kinase phosphorylation by CKS-17 in mutant THP-1 cells. Serum-starved THP-1, C1, and G7 cells were treated with 30 µM CKS-17 for 2, 10, 30, 60, and 120 min. Total cell extracts (10 µg of protein) were prepared, and the phosphorylation of MAP kinase was determined by Western blotting. The phosphorylation of MAP kinase was quantified by scanning densitometry and is shown as a fold increase. The data represent mean of at least three separate experiments. Error bars show SD of the mean.

 
cAMP inhibits MAP kinase and MEK activation induced by CKS-17

Based on these results suggesting that cAMP may be involved in the initial decline of MAP kinase activation, we next determined whether elevated intracellular cAMP suppresses CKS-17-induced MAP kinase phosphorylation using C1 or G7 cells. These cells were pretreated with forskolin or dibutyryl-cAMP (db-cAMP), a membrane-permeable cAMP analog, and then incubated with CKS-17 for 2, 10, 60, and 120 min. Fig. 4Go, A and B, shows that both forskolin and db-cAMP dramatically reduced ERK1/2 phosphorylation induced by CKS-17 using C1 or G7 cells. Thus, our experiments using mutant THP-1 cells had suggested that cAMP was responsible for the initial decline of MAP kinase activation by CKS-17. Experiments were then designed to further investigate this hypothesis. Fig. 5Go, A and B, shows that the elevated intracellular cAMP also suppressed kinase activity of MAP kinase or MEK using THP-1 cells.



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  		FIGURE 4. Inhibitory effect of cAMP on CKS-17-stimulated MAP kinase activity. A, Forskolin and db-cAMP inhibit CKS-17-induced phosphorylation of MAP kinase. Serum-starved mutant C1 and G7 cells were preincubated with 50 µM forskolin or 4 mM db-cAMP for 20 min and then stimulated with 30 µM CKS-17 for 2, 10, 60, and 120 min. After stimulation, total cell lysates (10 µg of protein) were prepared, and levels of phospho-ERK1/2 protein were determined by Western blotting using anti-p-ERK1/2 monoclonal Ab (top lane, C1; third lane, G7). These same membranes were reprobed by anti-ERK1/2 Ab as a control (second lane, C1; bottom lane, G7). B, The phosphorylation of MAP kinase was quantified by scanning densitometry and is shown as a fold increase (left panel, C1; right panel, G7). The data represent mean of at least three separate experiments. Error bars show SD of the mean.

 


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  		FIGURE 5. Elevation of cAMP levels inhibits activation of MAP kinase and MEK by CKS-17. A, THP-1 cells were preincubated with 50 µM forskolin for 20 min and then stimulated with 30 µM CKS-17 for 2, 10, 60, and 120 min. Immunoprecipitation and in vitro kinase assay of ERK1/2 (upper lane) or MEK (lower lane) were performed as indicated in Fig. 2Go. The experiment was repeated three times and one of the three typical results is shown. B, The in vitro kinase assay was quantified by scanning densitometry. Data represent a fold increase compared with nonstimulated median control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report, we have added another novel modality for the action of a highly conserved retroviral peptide on the immune system. The most important issue of this study is that CKS-17, known for its immunosuppressive characteristics, for its regulation of cytokines, and for its induction of cAMP, activates MAP kinase, a crucial signaling molecule that is activated by a variety of extracellular stimuli through its receptor (15). In these experiments we used THP-1 cells in which CKS-17 induces a dramatic increase of intracellular cAMP levels and mutant THP-1 cells that are unable to increase cAMP levels when treated with CKS-17. Using THP-1 cells we show that CKS-17 induces an initial phosphorylation and kinase activity of ERK1/2 or MEK followed by a rapid decline of activation. With THP-1 mutant cells this rapid decline of activation is abolished, suggesting that cAMP is responsible for the inhibition of activation of MEK and ERK1/2 by CKS-17 observed in THP-1 cells. Pretreatment of both THP-1 and mutant cells with cAMP analog or forskolin inhibited MEK and ERK1/2 activation induced by CKS-17.

It is noteworthy that the activation of MAP kinase signaling pathway enhances HIV-1 replication and infectivity (16), suggesting that activation of MAP kinase is beneficial to viral infection. Thus with our new evidence, it is possible that viruses or the highly conserved domain of retroviral transmembrane proteins may interact with a putative molecule, activate the MAP kinase cascade, and contribute to the immunopathogenesis associated with enhancement of retroviral infections, viral infectivity, and replication. Further studies, which are beyond the scope of these experiments, are necessary to determine this hypothesis.

From an immunological point of view it is interesting to note that using a murine system activation of the Ras/MAP kinase pathway has been shown to up-regulate Th2 cell differentiation and down-regulate Th1 cell differentiation (23). Furthermore, enhanced activation of ERK results in inhibition of macrophage IL-12 production (17). Previously we and others have reported that CKS-17 primarily down-regulates Th1-type immune response, including inhibition of IL-12 production (reviewed in Ref. 8).

As indicated earlier, in our current studies we show that the activation of MAP kinase by CKS-17 is inhibited by pretreatment of a cAMP analog or forskolin. Using mutant THP-1 cells isolated in our laboratory, which unlike the original THP-1 cells do not induce cAMP upon activation by CKS-17, we have demonstrated clearly that the rapid and significant transient down-regulation of MAP kinase phosphorylation is abolished in these cells, thus supporting our hypothesis that elevated cAMP levels are responsible for the brief decline in the process of MEK and ERK1/2 activation by CKS-17. Our finding is also in agreement with the observations from other laboratories using many types of cells, where an increase in intracellular cAMP levels is associated with down-regulation of the Ras/Raf-1/MEK/ERK pathway (24, 25, 26, 27, 28, 29, 30). Also, our provocative observations showing that increased levels of cAMP down-regulate MAP kinase challenge us to understand the cross-talk between CKS-17-induced MAP kinase and cAMP.


   Acknowledgments
 
We thank Michelle James-Yarish and Remi Hitchcock for technical assistance, and Letitia Ferguson and Chad Edmisten for preparation of the manuscript.


   Footnotes
 
1 This work was supported by Pediatric Cancer Foundation to the Children’s Research Institute, American Cancer Society Florida Division Inc. Grant F96USF-3, American Cancer Society Institutional Research Grant 202, Ito Foundation, Mochida Memorial Foundation for Medical and Pharmaceutical Research, and Eleanor Naylor Dana Charitable Trust. Back

2 Address correspondence and reprint requests to Dr. Noorbibi K. Day, Department of Pediatrics, University of South Florida, All Children’s Hospital, 801 6th Street South, St. Petersburg, FL 33701. E-mail address: dayn{at}allkids.org Back

3 Abbreviations used in this paper: MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; MEK, MAP kinase/ERK kinase; p-ERK1/2, anti-phospho-ERK1/2; p-MEK1/2, anti-phospho-MEK1/2; C1, THP-1:mC1.1; G7, THP-1:mG7.1; db-cAMP, dibutyryl-cAMP. Back

Received for publication June 6, 2000. Accepted for publication March 26, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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