©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
JAK2 Is Essential for Activation of c-fos and c-myc Promoters and Cell Proliferation through the Human Granulocyte-Macrophage Colony-stimulating Factor Receptor in BA/F3 Cells (*)

(Received for publication, August 21, 1995; and in revised form, February 23, 1996)

Sumiko Watanabe Tohru Itoh Ken-ichi Arai (§)

From the Department of Molecular and Developmental Biology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Interleukin-3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GM-CSF) is known to activate JAK2 in various cells, but the role of JAK2 in IL-3 or GM-CSF receptor signal transduction is largely unknown. We have now examined the role of JAK2 in GM-CSF-induced signaling events in BA/F3 cells. In BA/F3 cells expressing hGMR, activation of JAK2 by hGM-CSF requires the box1 region of hGMRbeta. Dominant negative JAK2 (DeltaJAK2), which lacked the kinase domain suppressed mIL-3- or hGM-CSF-induced c-fos promoter activation as well as c-myc promoter activation/cell proliferation, thereby suggesting that JAK2 is involved in the signaling of both pathways. Further analyses of the role of JAK2 in c-fos gene activation in BA/F3 cells expressing hGMR revealed that DeltaJAK2 inhibited hGM-CSF-induced phosphorylation of Shc and protein tyrosine phosphatase 1D. Within hGMRbeta, the several tyrosine residues which exist are related to activation of Shc or protein tyrosine phosphatase 1D, and are phosphorylated in response to hGM-CSF stimulation. In addition, we observed that DeltaJAK2 inhibited hGM-CSF-induced phosphorylation of hGMRbeta. Taken together, our results suggest that JAK2 activated by the box1 region of hGMR mediates hGM-CSF-induced c-fos promoter activation through phosphorylation of hGMR.


INTRODUCTION

Granulocyte-macrophage colony-stimulating factor (GM-CSF) (^1)is a cytokine that stimulates proliferation and differentiation of various hematopoietic cells(1) . IL-3 exhibits biological activities similar to those of GM-CSF. Receptors of IL-3 and GM-CSF consist of alpha and beta subunits, both of which are members of a cytokine receptor superfamily(2) . The alpha subunit is specific for each cytokine, and the beta subunit is shared by IL-3 and GM-CSF in addition to IL-5(3) . IL-3 and GM-CSF induce early response genes and cell proliferation in both hematopoietic cells and fibroblasts(4) . With IL-3 or GM-CSF stimulation, phosphorylation of tyrosine residues of several cytoplasmic proteins and the beta subunit itself occurs(5, 6, 7, 8) . Because the cytokine receptor family, including IL-3R or GMR, does not contain a kinase domain or kinase activity in the receptor itself, cellular tyrosine kinase may be involved. Phosphorylation or activation of tyrosine kinases such as src family tyrosine kinases and janus kinase (JAK) 2 by IL-3 or GM-CSF stimulation has been reported(9, 10, 11, 12, 13, 14) but with no direct evidence of the involvement of these tyrosine kinases(s) in IL-3 and GM-CSF activities, the exact roles of these kinases have remained unknown.

The JAK family kinase consists of JAK1, JAK2, JAK3, and Tyk2 in mammalian species(15) , but what role they have in hematopoiesis remained to be determined. Interestingly, the dominant mutation of Drosophila homolog, hop gene (hopscotch) resulted in hematopoietic defects(16) . Much attention has been directed to JAK family kinases because their functions in interferon signals were recognized(17) . Studies with interferon receptor signals revealed that JAK family kinases are involved in interferon-specific gene expression in cooperation with STAT proteins(18, 19, 20) . Subsequent studies on IL-6 and MGF signaling revealed that the JAK-STAT system plays a role in cytokine-specific gene expression(21, 22) . However, it is unclear whether or not JAK is involved in activities shared by many cytokines, for example induction of cell proliferation or activation of immediate response genes.

JAK2 is phosphorylated or activated by many cytokines including IL-3 and GM-CSF(9, 10, 23) , and association of JAK2 with the common beta subunit of IL-3R and GMR was noted(24) . We reported that two distinct signaling pathways function in hGMR signaling, one for activation of c-myc promoter/proliferation and the other for activation of c-fos/c-jun promoters(25, 26) . The membrane-proximal region of hGMRbeta containing box1 and box2 motifs conserved among members of the cytokine receptor family is essential for both signaling pathways, and in addition, the membrane-distal region is required for activation of c-fos/c-jun promoters. A role for tyrosine kinases in these two signals has been considered, as deduced from studies using kinase inhibitors(26) . Genistein and herbimycin almost completely suppressed activation of the c-myc promoter and cell proliferation, whereas herbimycin only partially suppressed activation of c-fos/c-jun promoters and genistein did not suppress the induction of c-jun mRNA; it even augmented activation of the c-fos promoter. These findings suggest an essential role for genistein/herbimycin sensitive kinase(s) in cell proliferation and in c-myc mRNA induction. However, whether or not tyrosine kinase is involved in activation of c-fos/c-jun genes remained to be clarified. Because the membrane-proximal region of hGMRbeta is required for phosphorylation of JAK2(10, 27) , we asked whether or not JAK2 is involved in activation of both signaling pathways. An approach using dominant negative JAK2 indicated a role for JAK2 in erythropoietin-induced proliferation and the partial requirement of JAK2 in IL-3-induced cell proliferation(28) , but the role of JAK2 in signaling pathway leading to activation of c-fos promoter is not known. In the present work, we attempted to determine whether or not JAK2 is involved in hGMR signals using BA/F3 cells. We found that JAK2, which is activated through the box1 region of hGMRbeta, plays essential roles in both signaling pathways.


MATERIALS AND METHODS

Chemicals, Media, and Cytokines

Fetal calf serum was from Biocell laboratories Co. Ltd. RPMI 1640 and Dulbecco's modified Eagle's medium were from Nikken BioMedical Laboratories Co. Ltd. Recombinant hGM-CSF was kindly provided by Schering-Plough. mIL-3 produced by silkworm, Bombyx mori, was purified as described elsewhere(29) . Genistein was from Wako Pure Chemical Industries, Ltd. G418 was a gift from Schering-Plough.

Plasmids and Genes

JAK2 cDNA (pBSK-JAK2) was kindly provided by Dr. J. Ihle (St. Jude Children's Research Hospital). Construction of the plasmid containing JAK2 under the control of the SRalpha promoter was as follows. The coding region of JAK2 was isolated from pBSK-JAK2 at NotI and SalI sites. The insert, which was blunt-ended using the Klenow fragment and attached to the NotI linker, was subcloned into pME18S at the NotI site. Dominant negative JAK2 (DeltaJAK2) was constructed as follows. DeltaJak2 that lacks the C terminus kinase domain was isolated at NotI and AvrII(2724) sites, and the NotI site was blunt-ended by the Klenow fragment before AvrII digestion. The fragment was inserted at blunt-ended EcoRI site and intact SpeI site of pME18S. hIL-2Rbeta and hIL-2R under the SRalpha promoter were kind gifts from Dr. K. Sugamura (Tohoku University, Japan). Dominant negative ras gene (MMTV promoter-N17 Ras; pMT64AA) was kindly provided by Dr. G. M. Cooper (Harvard Medical School). To construct a plasmid containing N17 Ras under the control of the SRalpha promoter, the coding region of N17 Ras was isolated from pMT64AA at BamHI sites. The insert was blunt-ended using the Klenow fragment and was subcloned into pME18S at blunt-ended XhoI sites. Construction of hGMRbeta mutants Deltabox1 (lacking amino acid positions 458-465) and Deltabox2 (lacking amino acid positions 518-530) are described elsewhere(50) .

Cell Lines and Culture Methods

A mIL-3-dependent proB cell line, BA/F3 was maintained in RPMI 1640 medium containing 10% fetal calf serum, 1 ng/ml mIL-3, 100 units/ml penicillin, and 100 µg/ml streptomycin. Various BA/F3 cell clones expressing hGMRalpha and hGMRbeta (BA/FGMR) or hGMRbeta mutants (BA/FDeltabox1, BA/FDeltabox2, BA/F589, BA/F 544) were grown in the same type of medium but supplemented with 500 µg/ml G418.

Immunoprecipitation, SDS-Polyacrylamide Gel Electrophoresis, and Western Blotting

BA/F3 cells (1 times 10^7 cells/sample) were harvested, washed with phosphate-buffered saline containing 1 mM sodium orthovanadate, and lysed for 30 min in 500 µl of ice-cold lysis buffer (0.5% Nonidet P-40 (for BA/F3), 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride). Nuclei and cell debris were removed by centrifugation for 5 min at 4 °C in a microfuge. Protein A-Sepharose (Pharmacia Biotech Inc.) or protein G-Sepharose (Pharmacia), 30 µl of 50% slurry equilibrated in phosphate-buffered saline, and antibodies were added to the supernatant followed by rotation for 2 h at 4 °C. The protein A or G bound immunoprecipitates were washed four times with lysis buffer and eluted in 2 times SDS loading buffer by boiling. Samples were separated on 7.5% SDS-polyacrylamide gels, transferred electrophoretically to Immobilon(TM) polyvinylidene difluoride membrane (Millipore, MA). Membranes were blocked with 4% bovine serum albumin (Fraction V) in TBST (20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.1% Tween 20) for 1 h, washed in TBST, incubated with the relevant primary antibody for 1 h, and then washed in TBST. Protein antibody complexes were detected and visualized with horseradish peroxidase-coupled secondary antibodies (anti-rabbit or anti-mouse, as appropriate, Amersham Corp.) using a chemiluminescence system (ECL(TM), Amersham Corp.). To reprobe immunoblots, the filters were incubated in 62.5 mM Tris-HCl, pH 6.7, 100 mM 2-mercaptoethanol, 2% SDS at 50 °C for 30 min. Antibodies used for immunoprecipitation were polyclonal rabbit antiserum against PTP 1D (sc-280, Santa Crutz, CA), JAK2 (HR-758, c-20, Santa Crutz, CA), Shc (Upstate Biotechnology, Inc.), monoclonal rat anti-hGMRbeta (5A5). Antibodies used for immunoblotting were polyclonal rabbit antiserum against PTP 1D (sc-280), JAK2 (Upstate Biotechnology, Inc.) and hGMRbeta (MBL, Nagoya, Japan) and monoclonal mouse anti-phosphotyrosine (Tyr(P)) antibody 4G10 (Upstate Biotechnology, Inc.).

Transfection and CAT/Luciferase Assay in BA/F3 Cells

BA/FGMR cells were transfected either with 5 µg of SRE-CAT reporter plasmid(30) , 15 µg of pmycPCAT or 3 µg of c-fos-luciferase, and 10 µg of control vector, JAK2, or DeltaJAK2 plasmids. c-fos-luciferase plasmid contains a 0.4-kilobase pair c-fos promoter upstream of the initiation site and luciferase coding region(4) . In some cases, receptor plasmids, 2 µg each of hGMRalpha and hGMRbeta, were co-transfected with these genes to BA/F3 cells(4) . Transfection was done by electroporation as described elsewhere(25) . Briefly, cells suspended in 0.2 ml of Opti MEM (3 times 10^6 cells) were transferred to a cuvette (0.4-cm electrode gap, Bio-Rad) and mixed with DNA. Cells were electroshocked using 960 microfarads at 200 V using a Gene Pulser electroporation apparatus (Bio-Rad). After 30 min of incubation at room temperature, cells were divided into three portions and transferred to 6-well plates in mIL-3-depleted medium. After 10 h of restimulation with 5 ng/ml of mIL-3, hGM-CSF, or hIL-2, cells were harvested and lysed by three cycles of freezing and thawing in liquid N(2). Each sample containing approximately 20 µg of total protein was subjected to luminescence assay or CAT assay. For luciferase assay, the substrate was automatically injected into the sample in the luminometer (model LE9501; Berthold Lumat Co. Ltd.), and luminescence of 30 s was counted and expressed as arbitrary units. CAT assay was done by diffusion analysis(31) . Protein concentration was estimated using the BCA protein assay reagent (Pierce) according to the manufacturer's instructions.

Replication Assay Using Plasmid Containing Polyoma (Py) Replication Origin

Activity to induce DNA replication by mIL-3 or hGM-CSF in BA/F3 cells was analyzed by replication of the transfected plasmid containing the Py replication origin, using an assay involving DpnI analysis, as described elsewhere(32) . Briefly, plasmids (1 µg of indicated template, 10 µg of RSV-LTag, and 10 µg of control vector, JAK2, or DeltaJAK2) were introduced into semiconfluent BA/F3 cells by the DEAE dextran method. After factor depletion for 5 h, cells were stimulated with 5 ng/ml of either mIL-3 or hGM-CSF. After incubation for an additional 24 h, cells were harvested, and low molecular weight DNA was isolated by the Hirt extraction method(33) . Ten µl of extracted DNA solution was digested with HindIII, which linearizes template plasmid, and DpnI. DpnI digests only the methylated or hemimethylated recognition site of DNA, and newly synthesized DNA is resistant to DpnI digestion. Southern blotting of digested DNA were done using with HindIII-digested pPyOICAT DNA as a probe. Blots were washed and exposed to an imaging plate for 15 min and visualized using a FUJI Image Analyzer (model BAS-2000).

Incorporation of [^3H]Thymidine

Plasmids of hGMRalpha (5 µg), hGMRbeta (5 µg), and either vector or DeltaJAK2 (15 µg) were transfected to BA/F3 cells (10^6/sample) by electroporation. Cells were cultured for 8 h and seeded into 96-well plates (10^4 viable cells/well) with various concentrations of hGM-CSF. After 48 h of culture, [^3H]thymidine (1 µCi/well) was added, and cells were harvested after 4 h of incubation. [^3H]Thymidine incorporation was measured using a liquid scintillation spectrophotometer.


RESULTS

Phosphorylation of JAK2 by hGM-CSF Requires box1 Motif but Not box2 Motif of hGMRbeta in BA/FGMR Cells

It had been reported that the membrane-proximal region of hGMRbeta is required for phosphorylation of JAK2(10, 27) . This region contains both box1 and box2 motifs, which are conserved among several cytokine receptors, including gp130 and erythropoietin receptor(2, 34) . We first examined the requirement of these motifs in activating JAK2 kinase using hGMRbeta mutants that lacked either box1 or box2 motif (betaDeltabox1 and betaDeltabox2), as schematically shown in Fig. 1. BA/F3 cells expressing wild type hGMRalpha and the wild type hGMRbeta, betaDeltabox1, or betaDeltabox2 (BA/FGMR, BA/FDeltabox1, BA/FDeltabox2, respectively) were depleted of mIL-3 for 5 h and restimulated with 5 ng/ml of either mIL-3 or hGM-CSF. After 5 min of incubation, immunoprecipitation of JAK2 was performed followed by Western blotting using monoclonal anti-Tyr(P) antibody 4G10. JAK2 was phosphorylated in response to either mIL-3 or hGM-CSF stimulation. Deletion of 8 amino acids at the box1 region resulted in a complete loss of hGM-CSF-induced JAK2 phosphorylation (Fig. 2A). On the other hand, deletion of the box2 region had no effect on JAK2 phosphorylation. As indicated in the bottom panel of Fig. 2A, the box1 motif is essential, whereas the box2 motif is not required for activation of the c-fos promoter by hGM-CSF.


Figure 1: Schematic drawing of various hGMRbeta mutants.




Figure 2: IL-3/GM-CSF induced phosphorylation of JAK2 in BA/FGMR cells. A, BA/F3 cells expressing wild type hGMRalpha and wild type hGMRbeta, betaDeltabox1, or betaDeltabox2 were depleted mIL-3 for 5 h and restimulated with either mIL-3 (5 ng/ml) or hGM-CSF (5 ng/ml) for 5 min. Cells were harvested and immunoprecipitated with anti-JAK2 antibody. The immunoprecipitant was electrophoresed and subjected to Western blot analysis with anti-Tyr(P) (4G10) or anti-JAK2 antibodies. Bands were visualized using a chemiluminescence system (ECL(TM)). For c-fos-luciferase assay, 2 µg of c-fos-luciferase plasmid was transfected, and luciferase activities induced by either mIL-3 or hGM-CSF were analyzed as described under ``Materials and Methods.'' The values given at the bottom of the figure are the averages of three samples. B, effect of tyrosine kinase inhibitor genistein on the hGM-CSF-induced phosphorylation of JAK2. Factor depleted BA/FGMR cells were treated with the indicated concentrations of genistein for 30 min and then stimulated with 5 ng/ml either mIL-3 (data not shown) or hGM-CSF for 5 min. JAK2 protein were immunoprecipitated and analyzed by Western blotting using anti-Tyr(P) or anti-JAK2 antibodies.



Tyrosine Kinase Inhibitor Genistein Did Not Inhibit hGM-CSF-induced Phosphorylation of JAK2

In previous work, we noted that genistein completely suppressed cell proliferation or activation of c-myc gene in response to IL-3/GM-CSF. In contrast, activation of the c-fos gene was not suppressed; rather it was augmented by genistein(26) . We next examined the effect of genistein on the phosphorylation of JAK2. After depletion of mIL-3 for 5 h, BA/FGMR cells were treated with various concentrations of genistein for 30 min before GM-CSF stimulation. The cells were then stimulated with hGM-CSF (5 ng/ml) and harvested after 5 min of incubation. As shown in Fig. 2B, hGM-CSF induced phosphorylation of JAK2 even in the presence of 10 µg/ml of genistein, a concentration that completely suppressed hGM-CSF-induced proliferation(26) . At 20 µg/ml genistein, the level of phosphorylation decreased but the level of protein was also reduced, suggesting that genistein inhibited protein synthesis rather than tyrosine phosphorylation. Essentially the same results were obtained in response to mIL-3 stimulation (data not shown). Lack of any appreciable inhibition of JAK2 phosphorylation by genistein is consistent with the notion that JAK2 is involved in hGM-CSF-induced activation of the c-fos promoter, which is not inhibited by genistein.

Dominant Negative JAK2 (DeltaJAK2) Suppressed c-myc Gene Activation and DNA Replication Induced by mIL-3/hGM-CSF

To examine the involvement of JAK2 in mIL-3 and hGM-CSF signals, we constructed mutant JAK2 lacking kinase activity. Kinase domain at the C terminus (nucleotide position 2714) was deleted from JAK2 (DeltaJAK2) based on an earlier report(28) . Similar to the construct described by Wojchowsky and co-workers(28) , our DeltaJAK2 construct inhibits autophosphorylation of wild type JAK2, in a dominant negative manner in the COS7 cells. We also examined the effects of DeltaJAK2 on activation and autophosphorylation of JAK1 or JAK3 in COS7 cells and found that they were not inhibited by the co-expression of DeltaJAK2 (data not shown). We next examined the effect of DeltaJAK2 on hGM-CSF-induced JAK2 activation in BA/F cells by transient transfection assay. Plasmids carrying hGMRalpha and beta subunits were co-transfected with or without DeltaJAK2. After stimulation of BA/F3 cells by hGM-CSF for 5 min, immunoprecipitation of JAK2 followed by Western blotting was performed. As shown in Fig. 3, JAK2 was phosphorylated through transiently expressed hGMR in BA/F3 cells, and this phosphorylation was suppressed by the co-expression of DeltaJAK2. Fig. 3(lower panel) shows the pattern of anti-JAK2 antibody blotting, and bands indicated by the upper arrow have a molecular weight corresponding to the wild type. The bands indicated by the lower arrow have a molecular weight that corresponding to the DeltaJAK2. We next examined the effect of DeltaJAK2 on mIL-3 or hGM-CSF activities in BA/FGMR cells. We previously reported that DNA replication and c-myc activation are mediated through the membrane-proximal region of hGMRbeta. A kinase-negative JAK2 mutant used by Wojchowsky and co-workers (28) partially inhibited the mIL-3-induced proliferation of DAER cells. We first examined the effect of DeltaJAK2 on c-myc gene activation using c-myc reporter plasmid (pmyPCAT)(25) , which contains the 2.6-kilobase pair fragment of the c-myc promoter fused to the CAT coding region. In this construct, the mycI site (E2F recognition site) was seen to be a major site responding to mIL-3 or hGM-CSF signals(25) . Fig. 4A shows that co-expression of DeltaJAK2 completely suppressed mIL-3- or the hGM-CSF-induced c-myc-CAT activity. We previously described T antigen-dependent replication of Py origin in BA/FGMR cells as a model system for initiation of DNA replication(32) . As shown in Fig. 4B, DeltaJAK2 suppressed Py origin-dependent DNA replication induced by mIL-3 or hGM-CSF in BA/F3GMR. We also examined effect of DeltaJAK2 on thymidine incorporation promoted by hGM-CSF via transiently expressed hGMR in BA/F3 cells. We found that co-expression of DeltaJAK2 suppressed hGM-CSF-induced [^3H]thymidine incorporation almost completely even in the presence of excess amounts of hGM-CSF (Fig. 4C).


Figure 3: Effect of dominant negative JAK2 (DeltaJAK2) on hGM-CSF-induced JAK2 phosphorylation in BA/F3 cells transiently expressing hGMR. hGMRalpha and beta subunit plasmids were transfected to BA/F3 cells with either vector control or DeltaJAK2 plasmids. Cells were cultured overnight with mIL-3 containing medium and depleted for 5 h. Immunoprecipitation was done with anti-JAK2 antibody after stimulation by hGM-CSF for 5 min. The upper panel shows blotting pattern with anti-Tyr(P) (4G10), and the lower panel shows blotting pattern with anti-JAK2 (Upstate Biotechnology, Inc.). The blot was visualized using a chemiluminescent system as described under ``Materials and Methods.''




Figure 4: Characterization of DeltaJAK2 in BA/FGMR cells. A, effects of DeltaJAK2 on hGM-CSF-induced c-myc activation in BA/FGMR cells were analyzed by transient assay of c-myc promoter CAT. pmycPCAT plasmid was transfected with either control vector or DeltaJAK2, and CAT activities induced by mIL-3 (hatched bars) or hGM-CSF (shaded bars) were analyzed by diffusion assay as described under ``Materials and Methods.'' B, mIL-3- or hGM-CSF-induced Py origin-dependent DNA replication in the presence or the absence of DeltaJAK2 in BA/FGMR cells were analyzed as described under ``Materials and Methods.'' The arrows indicate DpnI-resistant replicated plasmid and DpnI-sensitive unreplicated transfected plasmids. C, [^3H]thymidine incorporation into BA/F3 cell transiently expressing hGMR was analyzed in the presence or the absence of DeltaJAK2 as described under ``Materials and Methods.'' The values are the average of duplicate samples. The standard deviation is shown as error bars in the figure.



DeltaJAK2 Completely Suppressed Activation of the c-fos Gene Induced by mIL-3/hGM-CSF but Not by hIL-2

We next determined the effect of DeltaJAK2 on activation of the c-fos gene, which is mediated through the box1 region and the more membrane-distal region of hGMRbeta. c-fos-luciferase, hGMRalpha, and hGMRbeta plasmids were co-transfected with control vector, JAK2, or DeltaJAK2 into BA/F3 cells, and luciferase activities induced by mIL-3 or hGM-CSF were analyzed. As shown in Fig. 5A, hGM-CSF stimulated c-fos-luciferase activity in BA/F3 cells as noted previously(4) , and co-expression of DeltaJAK2 completely abolished the hGM-CSF-induced c-fos activation. Essentially the same results were obtained with mIL-3 stimulation (data not shown). IL-2 activated JAK1 and JAK3 but not JAK2 (35, 36) and, in response to IL-2, IL-2Rbeta and IL-2R transduced signals to activate the c-fos promoter even in the absence of IL-2Ralpha(37) . To determine whether or not the observed effect of DeltaJAK2 is specific to IL-3/GM-CSF signals, we examined the effect of DeltaJAK2 on hIL-2-induced c-fos-luciferase activity. hIL-2Rbeta and hIL-2R were transiently expressed in BA/F3 cells together with the c-fos-luciferase plasmid in the presence or the absence of DeltaJAK2. In contrast to a complete suppression of hGM-CSF-induced c-fos-luciferase activity, the same activity induced by hIL-2 was only partially inhibited by DeltaJAK2 (Fig. 5A). It should be noted, however, that wild type JAK2 co-transfected with hIL-2Rbeta and hIL-2R also partially suppressed the c-fos-luciferase activity induced by hIL-2 (data not shown). On the other hand, dominant negative Ras (N17-Ras) almost completely suppressed both hIL-2-induced and hGM-CSF-induced c-fos-luciferase activity. Taken together, these results suggest that suppression of c-fos-luciferase activity by DeltaJAK2 is specific to the mIL-3- or hGM-CSF-dependent pathway.


Figure 5: Effects of DeltaJAK2 on hGM-CSF-induced c-fos activation signaling pathway in BA/FGMR cells. A, effect of DeltaJAK2 on c-fos-luciferase activity induced by hGM-CSF or hIL-2. hGMRalpha and hGMRbeta or hIL-2Rbeta and hIL-2R plasmids were co-transfected with control vector, DeltaJAK2, or dominant negative Ras(N17) and c-fos-luciferase activities induced by either hGM-CSF (hatched bars) or IL-2 (shaded bars) were analyzed. B, plasmid containing three tandem SRE sites fused to the CAT coding region were transfected with or without DeltaJAK2, and mIL-3- (hatched bars) or hGM-CSF-induced (shaded bars) CAT activities were analyzed by diffusion assay as described in the legend to Fig. 4. C, effect of DeltaJAK2 on transiently expressed hGMR-dependent Shc and PTP 1D phosphorylation. Plasmids encoding hGMRalpha and hGMRbeta (10 µg each) were transfected to BA/F3 cells (2 times 10^7 cells/sample) with either 20 µg of control vector or DeltaJAK2and cultured overnight with medium containing 1 ng/ml of mIL-3. After 5 h of mIL-3 depletion, cells were restimulated with 5 ng/ml of hGM-CSF and harvested after 5 min of incubation. Immunoprecipitation with either anti-Shc or PTP 1D was done and followed by Western blotting.



The c-fos promoter contains SRE and SIE sites, and the latter is known to carry the GAS sequence(38) . Deletion analysis of c-fos promoter suggested that both SRE and SIE sites are essential for c-fos activation by IL-3/GM-CSF in BA/FGMR cells (data not shown). It should be noted that the SRE site also plays an important role for activation of egr1 by GM-CSF. (^2)To examine whether or not DeltaJAK2 exerts its effect through the SRE site, we next carried out similar experiments using SRE-CAT(30) . As shown in Fig. 5B, DeltaJAK2 inhibited CAT activity of SRE induced by mIL-3/hGM-CSF.

DeltaJAK2 Suppressed Shc and PTP 1D Activities Induced by mIL-3/hGM-CSF Stimulation

To analyze the role of Jak in activation of SRE or c-fos, we next examined the effect of DeltaJAK2 on signal transducing molecules known to be involved in activation of the c-fos promoter. SH2 containing tyrosine phosphatase PTP 1D (also called Syp, SH-PTP2, and PTP2C) (39) and adaptor molecule Shc (40) are well documented constituents of the Grb2-Sos cascade(41) , and they are phosphorylated upon mIL-3 or hGM-CSF stimulation(42) . To determine if JAK2 is involved in the phosphorylation of Shc or PTP 1D induced by hGM-CSF stimulation, hGMRalpha and hGMRbeta were transiently transfected into BA/F3 cells, and immunoprecipitation was done with either anti-Shc or PTP 1D antibodies. As shown in Fig. 5C, phosphorylation of Shc or PTP 1D through transiently expressed hGMR was evident but was abolished with the co-expression of DeltaJAK2. It appears that DeltaJAK2 interferes with signaling event(s) upstream of Shc or PTP 1D activation, thereby indicating that JAK2 plays an essential role in activation of both signaling molecules.

Phosphorylation of hGMRbeta Induced by hGM-CSF Is Abolished by DeltaJAK2

The hGMRbeta is tyrosine-phosphorylated in response to hGM-CSF stimulation, yet the nature of the tyrosine kinase involved is unknown. To determine whether or not JAK2 participates in hGMRbeta phosphorylation, we examined the effect of DeltaJAK2. hGMRalpha with control vector or with DeltaJAK2 were transiently transfected into BA/FGMbeta cells and cultured overnight. After depletion of mIL-3 for 5 h, cells were stimulated with hGM-CSF (5 ng/ml) for 5 min, and immunoprecipitation was done with anti-hGMRbeta antibody (Fig. 6). Transiently reconstituted hGMR was phosphorylated by hGM-CSF stimulation, and this phosphorylation was not observed when DeltaJAK2 was present. These results indicated that JAK2 is involved in ligand-induced phosphorylation of hGMRbeta.


Figure 6: Phosphorylation of hGMRbeta in the absence or the presence of DeltaJAK2. Plasmids encoding hGMRalpha (10 µg) were transfected to BA/FGMRbeta cells (2 times 10^7 cells/sample) with 20 µg of either control vector or DeltaJAK2 and cultured overnight. After 5 h of mIL-3 depletion, cells were restimulated with 5 ng/ml of hGM-CSF and harvested after 5 min of incubation. Immunoprecipitation with anti-hGMRbeta was done and followed by Western blotting.




DISCUSSION

JAK2 Is Apparently the Primary Kinase Regulating IL-3/GM-CSF Signals

We obtained evidence that in BA/FGMR cells, JAK2 is involved in activities controlled by hGM-CSF, including cell proliferation and activation of c-myc and c-fos promoters. We previously described two signaling pathways of hGMR, one for activation of c-fos/c-jun genes and the other for activation of c-myc gene/cell proliferation. We also reported that these two signaling pathways differ in sensitivity to tyrosine kinase inhibitor genistein, where c-fos gene activation is hardly inhibited by genistein(25) . Other investigators reported that gp130-dependent JAK2 activation is suppressed by genistein(43) . If this is also the case for IL-3/GM-CSF systems, JAK2 is unlikely to be involved in IL-3/GM-CSF-dependent activation of c-fos promoter. However, as noted in the present work, JAK2 activation in BA/F3 cells is insensitive to genistein, an observation that raises the possibility that JAK2 has a role in c-fos promoter activation. This view was more directly supported by experiments using dominant negative DeltaJAK2. DeltaJAK2 inhibited the activation of c-fos and c-myc promoters as well as cell proliferation. Complete inhibition of cell proliferation by genistein suggests that in addition to JAK2, other kinase(s) sensitive to genistein probably play(s) an essential role in the pathway downstream of JAK2. In fact, proliferation induced by mitogenic factors such as EGF and fetal calf serum were also inhibited by genistein(25) . These results suggest that tyrosine kinase, which is sensitive to genistein, has role in the common pathway responding to various mitogenic factors.

Activation of JAK2 Depends on the hGMRbeta box1 Region Essential for All Known GM-CSF Activities

box1 and box2 motifs in hGMRbeta are conserved among cytokine receptors(34) . We showed that the region including box1 is essential for hGM-CSF-dependent activation of JAK2 and the c-fos promoter, whereas box2 is not required for these activities. Similar analyses using the same constructs revealed that these regions affect DNA replication and egr1 promoter activation in a manner similar to that related to JAK2 activation.^2 The level of hGM-CSF-induced proliferation is significantly reduced by the removal of the box2 region of hGMRbeta, thereby implying an enhancing role for box2 for cell proliferation. A close correlation between the activation of JAK2, cell proliferation, and c-fos and c-myc promoters, all of which depend on the box1 region, is consistent with the notion that JAK2 is the primary kinase regulating GM-CSF signals. It should be noted that in COS7 cells, overexpression of JAK2 resulted in phosphorylation of hGMRbeta in a ligand-independent and box1-independent manner (data not shown). It is tempting to speculate that box1 facilitates establishment of an effective link between extracellular signals and intracellular events resulting in ligand-dependent activation of JAK2 in BA/F3 cells. Other receptors such as gp130 and erythropoietin receptor also require the box1 motif(44, 45, 46) .

JAK2 Induces Phosphorylation of hGMRbeta and Activates a Signaling Pathway Leading to Activation of c-fos Promoter

Our observation that the SRE sequence, which lacks the putative STAT recognition site, is suppressed by DeltaJAK2 is in keeping with the notion that JAK2 regulates the STAT-independent pathway involving c-fos promoter activation. Deletion analysis suggests that the SRE site is essential and the SIE site has an enhancing activity for IL-3/GM-CSF-induced c-fos promoter activation (data not shown). IL-3 or GM-CSF activated STAT5 and STAT6(47, 48, 49) , and dominant negative STAT5 partially suppressed endogenous c-fos gene activation in response to mIL-3 in BA/F3 cels. (^3)It is possible that the SIE site containing the GAS sequence is the target of STAT activation. Taken together, it is tempting to speculate that c-fos activation is regulated by STAT-dependent and -independent mechanisms via SIE and SRE sites, respectively, and that JAK2 plays an essential role in activation of both pathways.

Interestingly, DeltaJAK2 suppresses hGM-CSF-dependent phosphorylation of hGMRbeta in BA/F3 cells. To explain the primary role of JAK2 in hGM-CSF-dependent activation of c-fos promoter, we considered that JAK2 phosphorylates hGMRbeta itself. We further speculate that Shc or PTP1D interacts with phosphorylated tyrosine residue(s) of hGMRbeta and transduces signals downstream. Our finding that DeltaJAK2 inhibited activation of PTP1D and Shc supports this view.

In contrast to activation of the c-fos promoter, the hGMRbeta region containing phosphorylatable tyrosine residues is not required for activation of the c-myc promoter and cell proliferation. Dominant negative STAT5 did not affect c-myc activation by mIL-3 in BA/F3 cells.^3 This means that even if box1 and JAK2 are essential for activation of c-myc promoter/cell proliferation, the mechanism of JAK2 action differs from that related to activation of the c-fos promoter. Further work is under way to clarify the role of JAK2 in activation of cell proliferation and c-myc promoter.


FOOTNOTES

*
This work was supported in part by a grant-in-aid for scientific research on priority areas and cancer research from the Ministry of Education, Science, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 81-3-5449-5660; Fax: 81-3-5449-5424.

(^1)
The abbreviations used are: GM-CSF, granulocyte-macrophage colony-stimulating factor; IL, interleukin; CAT, chloramphenicol acetyltransferase; Py, polyoma; SRE, serum response element; SIE, c-sis-inducible element.

(^2)
S. Watanabe, K. Sakamoto, and K. Arai, manuscript in preparation.

(^3)
A. Miyajima, personal communication.


ACKNOWLEDGEMENTS

We are grateful to Drs. A. Miyajima and H. Mano for advice and to M. Ohara for comments on the manuscript.


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