Constitutive Activation of JAK2 Confers Murine Interleukin-3-independent Survival and Proliferation of BA/F3 Cells*

Chang-Bai LiuDagger , Tohru ItohDagger §, Ken-ichi AraiDagger , and Sumiko WatanabeDagger parallel

From the Dagger  Department of Molecular and Developmental Biology, Institute of Medical Science, University of Tokyo and  CREST, Japan Science and Technology Corp. (JST), 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan

    ABSTRACT
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Abstract
Introduction
References

The Janus tyrosine kinase 2 (JAK2) plays an essential role of cytokine receptor signaling, including that of the human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor. We reported earlier that the activation of JAK2 is essential for all the examined signals induced by human GM-CSF through the box1 region of beta c, such as promotion of cell survival and proliferation. To elucidate the role of JAK2 in cell survival and proliferation, we generated an artificial activation system by constructing a chimeric molecule (beta /JAK2) consisting of beta c extracellular and transmembrane regions fused with JAK2, and we analyzed various signaling events in interleukin-3-dependent mouse pro-B cell, BA/F3. The beta /JAK2 was constitutively phosphorylated in the absence of human GM-CSF and murine interleukin-3, and this led to proliferation and cell survival. Western blot analysis showed that STAT5, Shc, and SHP-2 were not phosphorylated in the cells, and the consistent activation of beta -casein and c-fos promoters was not enhanced. In contrast, c-myc transcription was constitutively activated. We propose that the activation of beta /JAK2 suffices for survival and proliferation and that the activation of STAT5 and mitogen-activated protein kinase cascade is not required for these activities in BA/F3 cells.

    INTRODUCTION
Top
Abstract
Introduction
References

The Janus tyrosine kinases (JAK) family has four known members, JAK1, 2, 3 and Tyk2, that play an essential role in the signal transduction of cytokine receptors and interferon receptors (1). The importance of their roles was first recognized in studies using mutant cell lines defective in interferon signaling (2, 3). Subsequent studies showed that JAKs were activated in response to a wide variety of cytokines including granulocyte-macrophage colony-stimulating factor (GM-CSF)1 and interleukin-3 (IL-3) (2, 4-6). Targeted disruption of JAKs in mice revealed the essential roles of JAKs in cytokine signals in vivo. JAK2 knockout mice are embryonic lethal because of the absence of definitive erythropoiesis (7, 8). The myeloid progenitors from the fetal liver of JAK2-deficient mice failed to respond to erythropoietin, IL-3 and GM-CSF; hence, the essential role of JAK2 in these cytokine signals became evident.

The GM-CSF receptor (GM-CSFR) consists of an alpha  subunit and a common beta  (beta c) subunit, shared by the receptors for GM-CSF, IL-3 and IL-5 (9). We reported that although the addition of GM-CSF induces oligomerization of alpha  and beta  subunits, the beta  subunit is present as a homodimer even before the addition of GM-CSF (10). It is also reported that GM-CSF induced dimerization of beta c (11). beta c does not contain intrinsic kinase activity; rather, GM-CSF activates JAK2 through the box1 region (4), and dominant negative JAK2 blocked all the examined activities of human (h) GM-CSF in BA/F3 cells (5). In studies done using a series of beta c mutants, multiple signaling pathways were induced by GM-CSFR, and all required the box1 region of beta c (12-14). One pathway is the activation of the Ras/Raf/mitogen-activated protein kinase (MAPK) cascade followed by c-fos/c-jun transcriptional activation. This pathway requires a membrane distal region of beta c containing tyrosine residues in addition to the box1 region. Phosphorylation of tyrosine residues of beta c by JAK2 in response to GM-CSF stimulation and the following recruitment of SH2-containing effectors such as Shc, SHP-2 through association of SH2 domain, and phosphorylated tyrosine residues of beta c has been proposed (14-16). In contrast, cell survival and low level proliferation can be induced through a beta c mutant having no tyrosine residue or only the membrane proximal region containing box1 and box2 regions. These activities are abrogated by deletion of the box1 region or co-expression of dominant negative JAK2, suggesting that cell survival and low level proliferation require JAK2 activation but no cytoplasmic region of beta c. Nevertheless, only limited knowledge is available to explain the promotion of proliferation and survival induced by GM-CSF, because the precise mechanisms through which JAK2 transduces signals to downstream pathways remain to be elucidated. One approach to resolve such issues is to differentiate the activation of JAK2 from receptor phosphorylation-dependent signals. We generated a chimeric protein consisting of the beta c extracellular and transmembrane domains and JAK2 designated as beta /JAK2 and analyzed signaling in BA/F3 cells. Our evidence shows that beta /JAK2 is phosphorylated constitutively in BA/F3 cells, and the cells survive and proliferate without activation of STAT5 or the MAPK cascade.

    EXPERIMENTAL PROCEDURES

Reagents and Antibodies-- RPMI 1640 was purchased from Nikken BioMedical Laboratories Co. Ltd. Fetal calf serum was purchased from Biocell Laboratories Co. Ltd. Recombinant mIL-3 produced in silkworm, Bombyx mori was purified as described (17). Recombinant hGM-CSF and G418 were kindly provided by Schering-Plough. The anti-hGM-CSF receptor beta  chain (S-16, N-20), anti-JAK2 (HR-758, C-20), anti-STAT5a (L-20), STAT5b (C-17), and anti-SHP-2 (C-18) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Antiphosphotyrosine (4G10), anti-Shc, and anti-JAK2 antibodies were purchased from Upstate Biotechnology, Inc. The antiphospho-Akt (Ser-473) and anti-Akt antibodies were from New England BioLabs (Beverly, MA). Phosphatidylinositol and phosphatidylserine were purchased from Sigma. CIS cDNA and Bcl-xL cDNA used as probes in the Northern blot analysis were gifts from Drs. A. Yoshimura (Kurume University, Japan) and Y. Tsujimoto (Osaka University), respectively.

Plasmids Construction-- The JAK2 cDNA (pBSK-JAK2) was kindly provided by Dr. J. Ihle (St. Jude Children's Research Hospital). The beta c cDNA was originally cloned into pME18S vector containing SRalpha , as a promoter (18). The construction of pME-beta /JAK2, a chimera gene encoding beta c extracellular and transmembrane regions fused with the N terminus of the full-length JAK2 was done as follows. Two fragments containing the coding region of JAK2, corresponding to amino acid positions 4 to 455 and 456 to 1129, were isolated from pBSK-JAK2 by digesting in HaeI/XhoI and XhoI/NheI, respectively. The coding region of beta c extracellular and transmembrane domains (corresponding to amino acid positions 1 to 455) was prepared by isolating the XhoI/FspI fragment from pME18S-beta c. The XhoI/NheI fragment of JAK2 was inserted into the expression vector pME18S using XhoI and SpeI sites, then the XhoI/FspI fragment of beta c and the HaeI/XhoI fragment of JAK2 were inserted at the XhoI site.

Cell Culture and Transfections-- COS7 cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 50 units/ml penicillin, and 50 µg/ml streptomycin. Transient transfection of COS7 cells with plasmid was done using LipofectAMINETM (Life Technologies, Inc.) according to the manufacturer's instruction. A mIL-3-dependent pro-B cell line, BA/F3, was maintained in RPMI 1640 medium containing 5% fetal calf serum, 0.25 ng/ml mIL-3, 50 units/ml penicillin, and 50 µg/ml streptomycin. To obtain stable transfectants, BA/F3 cells or BAFGMRalpha , which stably express the wild-type hGM-CSFR alpha  subunit, were cotransfected with pME-beta /JAK2 and the pKU-2Neo vector (containing a neomycin-resistant gene) by electroporation, as described (18). After a 2-week selection using 1 mg/ml G418, drug-resistant clones were screened for surface expression of beta /JAK2 by FACS analysis (Becton Dickinson), and size of beta /JAK2 was confirmed by Western blotting using an anti-JAK2 antibody.

DNA Fragmentation Assay by Agarose Gel-- Cells cultured with or without mIL-3 and/or hGM-CSF for 12 h were lysed with TTE buffer (0.5% Triton X-100, 5 mM Tris-HCl, pH7.4, 20 mM EDTA). Genomic DNA was extracted as described (19) and was then suspended in 10 mM Tris, 1 mM EDTA, pH 8.0, containing 20 µg of DNase-free RNase. Equal amounts of nucleic acids from each sample were separated through a 1.8% agarose gel then the fragmented DNA was visualized after ethidium bromide staining.

Proliferation Assay-- Analysis of incorporation of [3H]thymidine was done as described (5). Briefly, the cells were seeded into a 96-well plate (1 × 104 cells/well) with various concentrations of mIL-3 or hGM-CSF as indicated, then the cells were cultured for 24 h, labeled with [3H]thymidine (1 µCi/well) for 4 h, then harvested onto a glass fiber filter. Incorporation of [3H]thymidine was measured using a filter counter (model 1450 MicroBetaTM, Wallac, Turku, Finland).

Immunoprecipitation and Western Blot Analysis-- Immunoprecipitation and Western blotting were done as described (5). Briefly, cells (1 × 107 cells/sample) were lysed in 500 µl of lysis buffer (0.5% Nonidet P-40, 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride). Immunoprecipitation was carried out with the indicated antibody and protein A-Sepharose beads (Amersham Pharmacia Biotech). For Western blotting of total cell lysates, 4 × 105 cell were lysed with 20 µl of lysis buffer, mixed with a buffer containing 50 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 1% 2-mercaptoethanol, and 5 µg of bromphenol blue/ml, and boiled. Samples were separated through 10% SDS-polyacrylamide electrophoresis gels, and transferred to a Immobilon polyvinylidene difluoride membrane (Millipore), and Western blottings were performed using appropriate antibodies. The immuno reactive bands were visualized by the enhanced chemiluminescence kit (ECL, Amersham Pharmacia Biotech) according to the manufacturer's instruction.

Analysis of Phosphoinositide 3-Kinase (PI3-K) and Akt Activities-- PI3-K activity associated with antiphosphotyrosine antibody immunoprecipitates were assayed as follows. Cells (1.5 × 107/sample) were lysed and immunoprecipitated with antiphosphotyrosine antibody (4G10) as described above except that the lysis buffer contained 1% Nonidet P-40, 50 mM sodium fluoride, 2 µg/ml leupeptin, and 1 µg/ml pepstatin A. Immunoprecipitates were washed four times with the lysis buffer, twice with 0.5 M LiCl in 50 mM Tris-HCl, pH 7.5, and twice with 100 mM NaCl in 50 mM Tris-HCl, pH 7.5. Lipid kinase assays were performed on the beads in a 50-µl reaction mixture containing 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EGTA, 200 µM adenosine, 0.1 mg/ml phosphatidylinositol, and 0.1 mg/ml phosphatidylserine. The kinase reaction was initiated by the addition of 10 µCi of [gamma -32P]ATP and MgCl2 to final concentrations of 10 µM and 10 mM, respectively. After incubation at 25 °C for 15 min, the reactions were terminated by the addition of 125 µl of 1N HCl, and the reaction mixtures were extracted with CHCl3:CH3OH (2:1, v/v), spotted onto oxalate-treated thin layer chromatography plates (Silica Gel 60, Merck), and developed using a solvent system of CHCl3, CH3OH, H2O, 25% NH4OH (90:65:8:12, v/v/v/v). Phosphatidylinositol 3-phosphate was visualized by autoradiography, and 32P incorporation was quantified using a FUJI image analyzer (model BAS-2000). Activity of Akt was analyzed by Western blotting of total cell lysate using antiphosphorylated-Akt antibody.

Luciferase Assay-- Cells (4.5 × 106 cells/sample) were transfected with 30 µg of beta -casein luciferase (20) or 3 µg of c-fos luciferase (18) plasmids by electroporation as described (18). The cells were separated to three groups and incubated with depletion medium for 6 h then stimulated with mIL-3 (1 ng/ml), hGM-CSF (10 ng/ml), or left unstimulated for another 6 h. The sample indicated as random culture was cultured in mIL-3 (0.25 ng/ml)-containing media for 12 h immediately after transfection. The cells were lysed with 20 µl of 250 mM Tris-HCl, pH 7.4, and protein concentration was determined using the BCA protein assay kit (Pierce). Luciferase activity was measured using a luminometer (model LB9501; Berthold Lumat Co. Ltd., Japan) and a luciferase assay substrate (Promega, Madison, WI).

Northern Blot Analysis-- Northern blots were performed with mRNA prepared using the Fast Track 2.0 kit (Invitrogen, CA). Briefly, 1 µg of mRNA was separated on a 1% agarose gel containing 6% formaldehyde and transferred onto a nylon membrane (Hybond-N, Amersham Pharmacia Biotech) by capillary blotting. The blots were hybridized with cDNA probes (c-fos, c-jun, c-myc, bcl-xL, CIS, and glyceraldehyde-3-phosphate dehydrogenase genes) labeled by the Ready-To-GoTM kit (Amersham Pharmacia Biotech) using [alpha -32P]dCTP. The blotted membrane was visualized using a FUJI image analyzer (model BAS-2000).

    RESULTS

Construction and Expression of the beta /JAK2-- To analyze the direct signals from JAK2, we constructed a JAK2 chimera, beta /JAK2, in which the extracellular and transmembrane domains of beta c were fused to the N terminus of full-length JAK2. The total length of the beta /JAK2 is 1580 amino acids (Fig. 1A). We first examined the expression and size of beta /JAK2 in COS7 cells. COS7 cells were harvested after 24 h of culture after transfection of beta /JAK2 or wild-type JAK2 as a control, then subjected to immunoprecipitation using anti-beta c N terminus (S16) or anti-JAK2 antibodies followed by Western blotting using an anti-JAK2 antibody (Upstate Biotechnology, Inc). A 170-180-kDa band that corresponds to the expected size of beta /JAK2 appeared (Fig. 1B). We next prepared BA/F3 cells stably expressing beta /JAK2 (BAFbeta /JAK2) and beta /JAK2 together with the hGM-CSFR alpha  subunit (BAFalpha ,beta /JAK2). The expression of beta /JAK2 in BA/F3 cells was confirmed by flow cytometry assay (Fig. 1C) and Western blotting (Fig. 2).


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Fig. 1.   Construction and expression of beta /JAK2 chimera. A, a schematic diagram of beta /JAK2 chimera. The extracellular and transmembrane regions of beta c are fused to the N terminus of full-length JAK2. a.a., amino acid. B, transient expression of beta /JAK2 in COS7 cells. The beta /JAK2 or wild-type JAK2 as a control was expressed in COS7 cells, and expressed proteins were examined by immunoprecipitation followed by Western blotting using an anti-beta c and JAK2 antibodies. Sizes of beta /JAK2 (170-180 kDa) and JAK2 (120 kDa) correspond to the predicted ones. C, surface expression of beta /JAK2 in BA/F3 cells stably expressing beta /JAK2 (BAFbeta /JAK2) or beta /JAK2 and GM-CSFR alpha  subunit (BAFalpha ,beta /JAK2) was analyzed by flow cytometry using an anti-beta c antibody. The shaded trace indicates control staining using mouse IgG, and the open trace indicates anti-beta c antibody staining.


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Fig. 2.   Phosphorylation of the beta /JAK2 in BA/F3 cells. The BAFGMR (BA/F3 cells expressing wild-type hGM-CSFR), BAFbeta /JAK2, and BAFalpha ,beta /JAK2 cells were depleted of mIL-3 for 6 h, stimulated with mIL-3 or hGM-CSF for 10 min, and subjected to immunoprecipitation using an anti-JAK2 antibody followed by Western blotting. beta /JAK2 and endogenous wild-type JAK2 are indicated by arrows. anti-PTyr, antibody against phosphorylated Tyr.

Phosphorylation of beta /JAK2 in BA/F3-- To examine the activity of beta /JAK2, we first examined tyrosine phosphorylation of beta /JAK2 in BA/F3 cells by immunoprecipitation followed by Western blotting. We used BA/F3 cells expressing wild-type hGM-CSFR (BAFGMR) or parental BA/F3 cells as a control in the following experiments. Fig. 2 shows Western blotting patterns obtained with either an antiphosphotyrosine antibody (4G10) or anti-JAK2 antibodies of anti-JAK2 immunoprecipitants of BAFGMR, BAFbeta /JAK2, or BAFalpha ,beta /JAK2 cells. The anti-JAK2 antibody immunoprecipitated beta /JAK2 as well as the endogenous JAK2, and beta /JAK2 was phosphorylated even in the absence of mIL-3 or hGM-CSF in both BAFbeta /JAK2 cells and BAFalpha ,beta /JAK2 cells. In contrast, endogenous JAK2 was phosphorylated only after mIL-3 stimulation. hGM-CSF stimulation induced phosphorylation of endogenous JAK2 in BAFGMR cells but not in BAFbeta /JAK2 or BAFalpha ,beta /JAK2 cells. Therefore, beta /JAK2 is constitutively phosphorylated in BA/F3 cells regardless of the presence of hGM-CSFR alpha  subunit or hGM-CSF stimulation. In addition, transphosphorylation to endogenous JAK2 from beta /JAK2 did not occur.

BA/F3 Cells Expressing beta /JAK2 Survived and Proliferated in the Absence of mIL-3-- Because there was a constitutive phosphorylation of beta /JAK2, we next examined survival and proliferation of the BAFbeta /JAK2 and BAFalpha ,beta /JAK2 cells. Fig. 3A shows DNA fragmentation patterns of BAFGMR, BAFbeta /JAK2 and BAFalpha ,beta /JAK2 cultured in the absence or presence of mIL-3 or hGM-CSF. As expected, the DNA from BAFGMR cells cultured in growth factor-free media for 12 h was fragmented. When BAFGMR cells were cultured either in mIL-3 or hGM-CSF, no fragmentation was observed. In the case of BAFbeta /JAK2 and BAFalpha ,beta /JAK2 cells, only a residual ladder DNA was observed.


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Fig. 3.   Cell proliferation and survival of BA/F3 cell expressing beta /JAK2. Cell survival and proliferation were analyzed by DNA fragmentation (A), [3H]thymidine incorporation (B), or trypan blue dye exclusion (C) assays. The BAFGMR, BAFbeta /JAK2, and BAFalpha ,beta /JAK2 cells were incubated for 12 h (A), 24 h (B), or the indicated days in the presence of mIL-3 (closed circle) or hGM-CSF (open circle) or the absence of factor (open squares) (C).

We next analyzed the proliferation of BAFbeta /JAK2 and BAFalpha ,beta /JAK2 cells by [3H]thymidine incorporation analysis. As shown in Fig. 3B, in BAFGMR, incorporation of [3H]thymidine was not evident in the absence of factor, and incorporation was stimulated in the presence of mIL-3 or hGM-CSF in a dose-dependent manner. Both BAFbeta /JAK2 and BAFalpha ,beta /JAK2 cells had a significant level of [3H]thymidine incorporation in the absence of mIL-3 or hGM-CSF. When cells were cultured in mIL-3-containing media, increased [3H]thymidine incorporation occurred in a mIL-3 dose-dependent manner. In contrast, hGM-CSF did not stimulate [3H]thymidine incorporation in BAFbeta /JAK2 or in BAFalpha ,beta /JAK2 cells. These results indicate that the expression of beta /JAK2 supports DNA replication in BA/F3 cell in addition to cell survival. It should be noted that the level of proliferation was less than that observed with mIL-3 stimulation.

We then examined long term proliferation of these cells by examining cell number and viability by making use of trypan blue dye exclusion (Fig. 3C). As expected, the BAFGMR cells all died within one day after mIL-3 depletion (left upper panel). In contrast, more than 70% of BAFbeta /JAK2 cells and 90% of BAFalpha ,beta /JAK2 cells survived without mIL-3 for at least 4 days. The cell number of BAFbeta /JAK2 and BAFalpha ,beta /JAK2 cells constantly increased, but the growth rate was slower than that of the cells cultured in mIL-3. Furthermore, we were able to maintain both BAFbeta /JAK2 and BAFalpha ,beta /JAK2 cells in medium containing no mIL-3 for more than four months (data not shown).

Neither PI3-K nor Akt Was Activated in BAFbeta /JAK2 or BAFalpha ,beta /JAK2 Cells-- A role for PI3-K followed by Akt activation in cell survival was reported in various cells. Because we found that both PI3-K and Akt are activated by mIL-3 or hGM-CSF stimulation in BAFGMR cells,2 we examined whether these kinases are activated in BAFbeta /JAK2 and BAFalpha ,beta /JAK2 cells. PI3-K activity co-immunoprecipitated with antiphosphotyrosine antibody was determined by in vitro kinase assay. As shown in Fig. 4A, residual kinase activity was observed after depletion of mIL-3 for 5 h, and activity was induced by mIL-3 stimulation in all the three cells. Random culture samples showed almost the same level of activity as that of the depleted sample. The addition of hGM-CSF into BAFalpha ,beta /JAK2 cells did not affect the PI3-K activity. Akt is assumed to be downstream of PI3-K, and we next analyzed Akt activity by Western blotting using antiphospho-Akt antibody (Fig. 4B). Consistent with the results of PI3-K, almost no activity was observed from depleted samples of any of the cells. Activity was induced by the addition of mIL-3, and random-cultured samples showed very weak activity in comparison to the depleted cells. Taken together, these results indicate that no constitutive activation of the PI3-K pathway in BAFbeta /JAK2 and BAFalpha ,beta /JAK2 cells exists in the absence of factors. We also examined the effect of wortmannin, a PI3-K-specific inhibitor. The addition of wortmannin up to 200 nM, which completely suppressed the GM-CSF-induced activation of PI3-K and Akt in BAFGMR cells, did not show any inhibitory effect on factor-independent proliferation of BAFbeta /JAK2 and BAFalpha ,beta /JAK2 cells (data not shown), further eliminating the possible involvement of this pathway within these cells.


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Fig. 4.   Analysis of PI3-K and Akt. PI3-K activity was analyzed by immunoprecipitation followed by in vitro kinase assay (A), and Akt activity was analyzed by Western blotting of total cell lysate using antiphospho-Akt antibody (B). A, the cells were depleted of mIL-3 and stimulated either by mIL-3 or hGM-CSF for 1 min. The sample indicated as random culture is a sample cultured continuously in mIL-3 media. Immunoprecipitation using antiphosphotyrosine antibody (4G10) and then lipid kinase assay were done as described under "Experimental Procedures." Lipid products were separated by TLC. PIP indicates phosphatidylinositol phosphate. B, the upper panel shows the blotting pattern of antiphospho-Akt, and the lower panel shows the blotting pattern using anti-Akt antibody of the same membrane.

STAT5, Shc, or SHP-2 Was Not Phosphorylated in BAFbeta /JAK2 and BAFalpha ,beta /JAK2 Cells-- To examine whether cellular proteins are phosphorylated in BAFbeta /JAK2 and BAFalpha ,beta /JAK2 cells, we did Western blot analysis of total cell lysates using antiphosphotyrosine antibody (Fig. 5A). Cells were depleted of mIL-3 for 6 h and restimulated by mIL-3 or hGM-CSF for 10 min. In all the cells, the addition of mIL-3 induced tyrosine phosphorylation of various cellular proteins. Random-cultured cells in the presence of mIL-3 showed significant tyrosine phosphorylation in every cell lines. In contrast, although BAFbeta /JAK2 and BAFalpha ,beta /JAK2 cells grew in the absence of factors, no significant tyrosine-phosphorylated band was observed.


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Fig. 5.   Tyrosine phosphorylation of cellular proteins in BAFbeta /JAK2, BAFalpha ,beta /JAK2, and parental BA/F3 cells. BAFbeta /JAK2, BAFalpha ,beta /JAK2, and BA/F3 cells were depleted of mIL-3 and stimulated either by mIL-3 or hGM-CSF. The sample indicated as a random culture is a sample cultured continuously in mIL-3 media. A, total cell lysates were subjected to Western blotting using antiphosphotyrosine antibody (4G10). B, immunoprecipitation using the indicated antibodies followed by Western blotting were done as described under "Experimental Procedures." In the bottom panel, the arrow indicates SHP-2.

We previously found that STAT5, Shc, and SHP-2 are tyrosine-phosphorylated in response to hGM-CSF in BA/F3 cells expressing hGM-CSFR, and this phosphorylation requires the tyrosine residues of beta c (14, 15). We analyzed the tyrosine phosphorylation of these molecules in both BAFbeta /JAK2 and BAFalpha ,beta /JAK2 cells by immunoprecipitation followed by Western blotting. When BA/F3 cells were depleted of mIL-3 for 5 h, no residual phosphorylation of STAT5 was observed, and the STAT5 was tyrosine-phosphorylated in response to mIL-3, in accord with our previous reports (Fig. 5B). In BAFbeta /JAK2 and BAFalpha ,beta /JAK2 cells, no phosphorylation of STAT5 was observed with samples of factor-depleted cells. The addition of hGM-CSF induced little tyrosine phosphorylation of STAT5 in BAFalpha ,beta /JAK2 cells. Because tyrosine phosphorylation of STAT5 is transient, it is possible that phosphorylation of STAT5 is not observed with unsynchronized cells. We examined the state of tyrosine phosphorylation of STAT5 from random-cultured BA/F3 cells. STAT5 is weakly but significantly tyrosine-phosphorylated within the sample of random-cultured cells, thus indicating that beta /JAK2 cannot phosphorylate STAT5 in the absence of mIL-3 in BAFbeta /JAK2 and BAFalpha ,beta /JAK2 cells. Although both BAFbeta /JAK2 and BAFalpha ,beta /JAK2 cells grew without mIL-3, tyrosine phosphorylation of STAT5 was never observed.

Similarly, we analyzed the tyrosine phosphorylation of Shc and SHP-2 by immunoprecipitation followed by Western blotting under the same conditions used for STAT5. Both Shc and SHP-2 were phosphorylated by mIL-3 stimulation in all the cells, and weak phosphorylation was observed in the random-cultured BA/F3 cells. In contrast, in mIL-3-depleted BAFbeta /JAK2 and BAFalpha ,beta /JAK2 cells, neither Shc nor SHP-2 was phosphorylated. A positive role for the activation of Shc and SHP-2 in the MAPK pathway was proposed (15). The results suggested that neither STAT5 nor MAPK pathway is required for survival and proliferation of BA/F3 cells in this system.

To determine whether STAT5 and MAPK pathways are functionally defective in BAFbeta /JAK2 and BAFalpha ,beta /JAK2 cells, we further analyzed the activities of beta -casein and c-fos promoters, assumed to be targets of STAT5 and the MAPK cascade, respectively (21). A transient transfection analysis was made using beta -casein or c-fos promoters fused to the luciferase-coding region. Luciferase activity of random-cultured cells, factor-depleted cells, and factor restimulated cells was examined. As shown in Fig. 6A, the beta -casein luciferase activity was induced about 5-fold in all the cells after depletion and readdition of mIL-3. The beta -casein luciferase activity of random-cultured BA/F3 cells showed higher luciferase activity than was observed in mIL-3 restimulated cells. In contrast, hGM-CSF stimulation did not induce the activity of beta -casein luciferase in BAFbeta /JAK2 and BAFalpha ,beta /JAK2 cells. These results are consistent with those observed in experiments examining tyrosine phosphorylation of STAT5. We suggest that constitutively activated beta /JAK2 does not activate STAT5 in BAFbeta /JAK2 and BAFalpha ,beta /JAK2 cells. Similarly, c-fos luciferase activity in unstimulated BAFbeta /JAK2 and BAFalpha ,beta /JAK2 cells was significantly lower than that observed in cells subjected to mIL-3 stimulation (Fig. 6B). In contrast, the c-fos luciferase activity of random-cultured BA/F3 cells was higher than that in cells stimulated with mIL-3.


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Fig. 6.   Induction of gene expression in BAFbeta /JAK2, BAFalpha ,beta /JAK2, and parental BA/F3 cells. A and B, examination of promoter activities of beta -casein and c-fos using transient transfection analysis. The beta -casein promoter luciferase (A) or c-fos promoter luciferase (B) plasmids were transiently transfected to BA/F3, BAFbeta /JAK2, and BAFalpha ,beta /JAK2 cells, and luciferase activities of indicated conditions were examined. Values are expressed as relative to the values obtained by mIL-3-stimulated samples. C, Northern blot analysis of c-fos, c-jun, CIS, bcl-xL, and c-myc. The cells were depleted for 6 h and stimulated for 30 min (c-fos, c-jun, CIS) or 3 h (bcl-xL, c-myc) by mIL-3 or hGM-CSF. random culture indicates samples cultured continuously in mIL-3. G3PDH, glyceraldehyde-3-phosphate dehydrogenase.

c-myc but Not c-fos, c-jun, CIS, and bcl-xL Was Constitutively Activated in BAFbeta /JAK2 and BAFalpha ,beta /JAK2 Cells-- We previously found that immediate early genes, c-fos, c-jun, bcl-xL, and c-myc are activated either by mIL-3 or hGM-CSF stimulation in BA/F3 cells. For c-fos and c-jun gene activation, the membrane distal region of beta c is required in addition to the membrane proximal region. In contrast, c-myc activation requires only the membrane proximal region, and this requirement is the same as that required for cell proliferation. To analyze whether these genes are activated or not in BAFbeta /JAK2 and BAFalpha ,beta /JAK2 cells, we performed Northern blot analysis. Factor-depleted cells were restimulated for 30 min for c-fos, c-jun, and CIS analysis and for 3 h for c-myc and bcl-xL analysis. As shown in Fig. 6C, by 6 h of mIL-3 depletion, levels of c-fos, c-jun, bcl-xL, and CIS fell to background levels, and mIL-3-induced these genes in all the three cells. In contrast, even after mIL-3 depletion, the level of c-myc expression remained significantly higher in BAFbeta /JAK2 and BAFalpha ,beta /JAK2 cells than in the parental BA/F3 cells. These results indicate that c-myc transcription is selectively activated by the constitutively active beta /JAK2 molecule.

    DISCUSSION

In the present study, we found that activation of JAK2 using a chimeric beta /JAK2 supports long term survival and proliferation of BA/F3 cells in the absence of mIL-3. Chimeric JAK2 is tyrosine-phosphorylated constitutively in BA/F3 cells regardless of the presence of hGM-CSFR alpha  subunit or hGM-CSF. Similar to the model proposed for the receptor-type tyrosine kinase (22), JAKs are assumed to be activated by homodimer formation followed by transphosphorylation by JAKs themselves (2, 3). Thus, constitutive tyrosine phosphorylation of beta /JAK2 in the present work may occur through constitutive dimer formation of the beta /JAK2. We reported that hGM-CSF receptor beta  subunit forms a dimer even in the absence of hGM-CSF in BA/F3 cells and that the cytoplasmic region of beta c is not required for this dimer formation (10). Although attempts to show the presence of a dimer form of beta /JAK2 using a chemical cross-linker were not successful (data not shown), we speculate that the chimeric beta /JAK2 forms a dimer through transmembrane and extracellular regions of beta c.

Analyses using mutant receptors as well as mutant JAKs showed an essential role of JAK2 in the IL-3 and GM-CSF signaling pathway (5). The JAK2 knock-out mice revealed the essential role of JAK2 for IL-3 signals in vivo (7, 8). Our initial idea to isolate the activity of JAK2 was based on the finding that there are several activities of hGM-CSF that require JAK2 activation but not the C-terminal region of beta c. During mutation analysis of beta c, we found distinct signaling pathways of hGM-CSFR (13). The signaling pathway that requires the receptor C terminus region was initiated by phosphorylation of beta c tyrosine residues, and then a cascade of signaling molecules such as the MAPK pathway by protein-protein interactions followed. STAT activation also requires tyrosine residues, because it is assumed that STAT binds to phosphorylated tyrosine through its SH2 domain. On the other hand, there are signaling pathways that require only JAK2 activation through the box1 region but not the receptor tyrosine residues, leading to cell survival, cell proliferation, and c-myc activation. The only event that is constitutively activated in BAFbeta /JAK2 and BAFalpha ,beta /JAK2 cells was c-myc transcription. Taken together, an important role for c-myc in cell survival and proliferation is strongly suggested. In the present work, the activation of JAK2 occurred without involvement of the cytoplasmic region of beta c. beta /JAK2 transduces signals for cell survival and weak proliferation. We previously revealed that a mutant beta c lacking all the tyrosines that cannot activate STAT5 or the MAPK cascade is still capable of maintaining cell survival and weak but significant proliferation. In addition, we recently found that PI3-K and Akt activation induced by IL-3 or GM-CSF does not play an essential role in cell survival in BA/F3 cells,2 which is consistent with the current observation that no induction of PI3-K and Akt occurs in BAFbeta /JAK2 and BAFalpha ,beta /JAK2. Taken together, it is strongly suggested that activation of the MAPK and PI3-K cascades as well as STAT5 by cytokines is not essential for survival and proliferation of BA/F3 cells.

We constructed and analyzed another type of chimeric JAK2 using Gyrase B as an artificial dimerizer (23, 24). With this fusion protein of Gyrase B and JAK2 (GyrB/JAK2), which is assumed to be inducibly dimerized by binding to the chemical compound coumermycin, we obtained different results from beta /JAK2. The addition of coumermycin results in activation of this chimeric JAK2 molecule and induces phosphorylation of STAT5 but does not support long term survival, proliferation, and MAPK cascade activation of BA/F3 cells. It is of interest that phosphorylation of STAT5 and proliferation are antiparallel events in GyrB/JAK2 and beta /JAK2. This finding supports the notion that STAT5 may not be involved in signaling for proliferation or survival of BA/F3 cells. The different activities of these chimeras may be explained by differences in subcellular localization of these molecules, because GyrB/JAK2 may exclusively locate in cytoplasm, and beta /JAK2 is the transmembrane protein. There are other examples of JAK2 fusions with transmembrane molecules such as the EGF receptor and the erythropoietin receptor (25, 26). In both cases, the chimeras can mediate proliferative signaling without activating the MAPK cascade. Finally, naturally occurring TEL-JAK2 fusion proteins, as a result of chromosomal translocations, have been found via patients with acute lymphoblastic leukemia and chronic myelogenous leukemia and are assumed to cause malignancy within hematopoietic cells (27, 28). TEL-JAK2 exhibits constitutive tyrosine kinase activity, and its expression confers mIL-3-independent proliferation to BA/F3 cells, yet the molecular pathways that mediate the signals from this molecule remain to be clarified. Further studies using our beta /JAK2 chimera may help gain an insight into the mechanism of JAK2 signaling in hematopoiesis as well as in leukemogenesis.

    ACKNOWLEDGEMENTS

Authors grateful to Yutaka Aoki and Yukitaka Izawa for excellent technical assistance and Marty Dahl and Mariko Ohara for critical reading of the manuscript.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ A recipient of a research fellowship from the Japan Society for the Promotion of Science for Young Scientists.

parallel To whom correspondence should be addressed. Tel.: 81-3-5449-5660; Fax: 81-3-5449-5424; E-mail: sumiko{at}ims.u-tokyo.ac.jp.

2 R. Liu, T. Itoh, and S. Watanabe, unpublished results.

    ABBREVIATIONS

The abbreviations used are: GM-CSF, granulocyte-macrophage colony-stimulating factor; GM-CSFR, GM-CSF receptor; IL-3, interleukin-3; MAPK, mitogen-activated protein kinase; hGM-CSF, human GM-CSF; mIL-3, murine IL-3; PI3-K, phosphoinositide 3-kinase; CIS, cytokine-inducible SH2-containing protein.

    REFERENCES
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Abstract
Introduction
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