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Vaccinia virus-mediated high level expression and single step purification of recombinant Jak2 protein
Protein Expression and PuriWcation 35 (2004) 181–189 www.elsevier.com/locate/yprep
Vaccinia virus-mediated high level expression and single step puriWcation of recombinant Jak2 protein夽 Xianyue Ma and Peter P. Sayeski¤ Department of Physiology and Functional Genomics, University of Florida College of Medicine, P.O. Box 100274, Gainesville, FL 32610, USA Received 7 May 2003, and in revised form 16 January 2004
Abstract Jak2 functions as a non-receptor tyrosine kinase and has been linked to pathologies such as cancer and cardiovascular disease. Because of this, many studies have tried to better understand its function. Unfortunately, very little information is known about its catalytic or biochemical properties as puriWcation of signiWcant amounts of functional Jak2 protein has been exceedingly diYcult. Here, Jak2 was expressed in BSC-40 cells using a vaccinia virus-mediated expression system. SigniWcant amounts (»10 g) of Jak2 protein were expressed from a single 100-mm cell culture dish. The protein was Wrst harvested using three diVerent methods of extraction to determine the relative eYciency of each lysis method with respect to Jak2 protein yield and catalytic activity. We found that lysis methods utilizing detergents increased the eYciency of protein extraction about 3-fold when compared to a method lacking detergent. However, with respect to catalytic activity, Jak2 isolated from cells using detergent-containing lysis buVers had signiWcantly less catalytic activity than when compared to the method that was detergent free. Expression was then scaled up and Jak2 protein was puriWed via a one step immunoaYnity puriWcation scheme using both the detergent-free and a modiWed detergentcontaining method of extraction that maintained catalytic activity. In vitro kinase assays demonstrated that the puriWed product was highly catalytic as measured by its ability to tyrosine phosphorylate Stat1. Collectively, the results show that (1) Jak2 can be expressed at very high levels in mammalian cells, (2) it can be puriWed to homogeneity via a single step puriWcation scheme, and (3) the puriWed product is biologically active. 2004 Elsevier Inc. All rights reserved.
Jak2 is a member of the Janus family of tyrosine kinases (Jaks) that also includes Jak1, Jak3, and Tyk2. Jak2 is »130 kDa in mass and contains seven conserved Jak homology (JH) domains. It is known to have important roles both in the physiology and pathophysiology of animals. For example, mice lacking the Jak2 allele die embryonically, thereby demonstrating that Jak2 is essential for life [1]. As such, no other tyrosine kinase in the mouse genome, including other Jak kinases, can function in its place. With respect to pathophysiology, studies have demonstrated a clear link between Jak2 and diseases such as cancer, heart failure, and neointimal formation following balloon angioplasty [2–4]. Jak2 functions as non-receptor tyrosine kinase and so far has been best characterized in cytokine-induced 夽
US Patent Application No. 60/443,042. Corresponding author. Fax: 1-352-846-0270. E-mail address: [email protected] (P.P.Sayeski). URL: http://www.med.ufl.edu/phys/sayeski.shtml.
¤
1046-5928/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2004.02.009
signal transduction pathways [5]. In this model, Jak2 is constitutively bound to the cytoplasmic tail of cytokine receptors. Ligand binding induces receptor aggregation which results in the tyrosine autophosphorylation of Jak2 molecules via a juxta-positioning of the associated Jak proteins. Activated Jak2 then phosphorylates tyrosine residues on the cytoplasmic tails of these receptors which serve as docking sites for the Signal Transducers and Activators of Transcription (STAT) proteins. Activated Jak2 subsequently phosphorylates the receptorbound STAT proteins. Phosphorylated STAT proteins then translocate from the receptor complex to the nucleus where they facilitate gene transcription [6]. Thus, Jak2 is a key intermediate linking ligand binding at the cell surface with gene transcription in the nucleus. Although a reasonable amount of information is known about the mechanisms by which Jak2 mediates gene transcription, little is known about the biochemical and kinetic properties of Jak2 protein, as expression and
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puriWcation of signiWcant quantities of functional Jak2 protein has been exceedingly diYcult [7,8]. Furthermore, the only commercially available sources of Jak2 protein are sold as antibody conjugates which limit the biochemical analysis that can be performed on Jak2. In the present study, an HA-tagged Jak2 protein was successfully expressed in BSC-40 cells using the vaccinia virus-mediated expression system. About 10 g of Jak2 protein was yielded on a small culture scale (one 100-mm cell culture dish). The protein was puriWed to homogeneity in a single immunoaYnity chromatography step using an anti-HA antibody. A Jak2 in vitro kinase assay system was developed to test the function of the puriWed protein. Stat1, a Jak2 substrate, was phosphorylated by both Jak2 immunocomplexes and the puriWed Jak2 protein product. Moreover, the puriWed Jak2 product phosphorylated a GST-Stat1 fusion protein substrate with a speciWc activity of 111,500 pmol/min/mg protein. As such, these results demonstrate that the vaccinia virus expression system followed by anti-HA aYnity chromatography provides an eYcient means of obtaining large amounts of pure and functional Jak2 protein.
strand primer (50-GTAATACGACTCACTATAGGG C-30) and a bottom strand primer (50-CGCGGATCCCT AAAGGGAAGCGGCCGC-30). The blunt-end PCR product was inserted into the TOPO cloning vector using the Zero Blunt TOPO PCR Cloning Kit (Invitrogen). The HA-tagged Jak2 cDNA was then excised from TOPO via NcoI and BamHI restriction digest and then cloned into the vaccinia recombination vector, pTM3, to yield pTM3-Jak2-WT-HA.
Materials and methods
Preparation of cell lysate containing Jak2-WT-HA
Chemicals and supplies
For protein expression, twelve 100-mm dishes of conXuent BSC-40 cells were infected with 0.5 moi of vJak2WT-HA and 1.0 moi of vTF7-3 recombinant viruses in serum-free media. One hour later, the media were aspirated and the cells were incubated overnight in media supplemented with 10% fetal bovine serum. The following morning, the cells were washed once with phosphatebuVered saline containing 1 mM Na3VO4 and then lysed using one of three diVerent methods so that both protein yield and functional activity could be assessed. For method one, each plate was lysed in 1 ml RIPA buVer (20 mM Tris–HCl, pH 7.5, 10% glycerol, 1% Triton X100, 1% deoxycholate, 0.1% SDS, and 2.5 mM EDTA) containing phosphatase and protease inhibitors (4 mM benzamidine, 1 mM AEBSF (4-(2-amonoethyl)-benzenesulfonyl Xuoride), 1 mM Na3VO4, and 10 g/ml aprotinin). For method two, each plate was lysed in 1 ml Triton lysis buVer (100 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, and 5 mM EDTA) containing the same inhibitors. In both cases, the samples were brieXy sonicated and incubated on ice for at least 30 min. The lysates were then cleared by centrifugation and stored in ¡80 °C for future use. For method three, lysates were prepared using a Dounce homogenizer. SpeciWcally, the cells were detached from the plates in cold harvest buVer (40 mM Tris–HCl, pH 7.5, 10 mM EDTA, and 150 mM NaCl) using a disposable cell scraper. The cells were then collected by centrifugation (1000g, 5 min) and washed once with harvest buVer. The cells from twelve 100-mm dishes were resuspended in 3 ml of BuVer A (10 mM
DMEM cell culture media and antibiotic supplements were purchased from Life Technologies/Invitrogen. Heat inactivated fetal bovine serum was from Hyclone. All other reagents were from either Fisher ScientiWc or Sigma Chemical. Construction of vaccinia recombination plasmid pTM3Jak2-HA The HA-tagged Jak2 cDNA was subcloned into the vaccinia recombination vector pTM3 in a multi-step process. The Jak2 expression vector, pCI-Neo-Jak2-WTHA [9], was modiWed via the QuikChange site-directed mutagenesis system (Stratagene) in order to convert the 50 SalI to NcoI and to destroy the internal EcoRI and NcoI sites. The top and bottom strand oligonucleotides used for this were 50-GGTACCTCTAGAGCCATGG GAATGGCCTGC-30 and 50-GCAGGCCATTCCCA TGGCTCTAGAGGTACC-30; 50-ACTGGAAACGGT GGTATTCAGTGGTCAAGA-30 and TCTTGACCAC TGAATACCACCGTTTCCAGT-30; 50-CAGGAGAG AATACCTTGGGTACCTCCTGAA-30 and 50-TTCA GGAGGTACCCAAGGTATTCTCTCCTG-30, respectively. Although the DNA sequence of the modiWed template was changed, the Jak2 amino acid sequence was not. Using this as a template, a 3.3 kb HA-tagged Jak2 cDNA fragment containing 50 NcoI and 30 BamHI ends was ampliWed via PFU DNA polymerase using a top
Generation of recombinant vaccinia virus vJak2-WT-HA To generate a recombinant vaccinia virus containing the HA-tagged Jak2 cDNA under the control of the T7 promoter, an 80% conXuent monolayer of BSC-40 cells (100 mm dish) was transfected for 5 h with 20 g of pTM3-Jak2-WT-HA in 20 l of Lipofectin (Life Technologies) following the manufacturer’s instruction. Serum-containing media were then added to the plate and the cells were infected with 0.5 multiplicities of infection (moi) of wild-type vaccinia virus. Virus was harvested two days later and recombinants (vJak2-WT-HA) were selected for gpt and against tk as described [10].
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Hepes, pH 7.9, 5 mM MgCl2, 0.1 mM. EDTA, 10 mM NaCl, 10 mM NaF, 1 mM dithiothreitol, and 1 mM AEBSF) and allowed to swell on ice for 5 min. The cells were then lysed in a Dounce homogenizer (pestle B, 20– 40 strokes). The lysates were brieXy sonicated, cleared by centrifugation (10,000g, 10 min), and stored in ¡80 °C for future use. COS-7 cell lysates were prepared with the Dounce homogenizer in a similar manner, but without any virus infection.
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was stopped by adding 0.3 ml of 4£ Triton lysis buVer and placing the plates on ice. Soluble lysates were prepared as described above. Four milliliters of cleared lysate was then mixed with 4 ml of 2£ binding buVer and incubated with 16 g anti-HA monoclonal antibody (Santa Cruz Biotechnology) and 120 l of a 50% slurry of protein A/G–agarose beads (Santa Cruz Biotechnology) for 2 h at 4 °C with constant shaking. The Jak2WT-HA protein was then pelleted, washed, eluted, and frozen as described above.
Immunoprecipitation of expressed Jak2-WT-HA protein In vitro kinase assay For this, 0.5 ml of cell lysate was mixed with 0.5 ml of 2£ binding buVer (50 mM Hepes, pH 7.6, 20% glycerol, 200 mM KCl, 25 mM MgCl2, 0.2 mM EDTA, 0.2 mM IGEPAL CA-630, 2 mM DTT, and 2 mM AEBSF) and incubated with 2 g anti-HA monoclonal antibody (Santa Cruz Biotechnology) and 20 l of a 50% slurry of protein A/G–agarose beads (Santa Cruz Biotechnology) for 2 h at 4 °C with shaking. The beads were washed three times with 1£ binding buVer and then in vitro kinase assays were performed as described below. For Stat1 immunoprecipitation, 0.5 ml COS-7 cell lysate was prepared by the Dounce homogenizer method and then immunoprecipitated with 2 g anti-Stat1 polyclonal antibody (Santa Cruz Biotechnology), and 20 l of a 50% slurry of protein A/G–agarose beads (Santa Cruz Biotechnology) for 2 h at 4 °C. The beads were washed with binding buVer and then kinase assays were performed as described below. PuriWcation of Jak2-WT-HA protein For large scale puriWcation of recombinant Jak2 protein, we utilized both the detergent-free (Dounce) and a modiWed Triton lysis method of extraction. For the Dounce method, 4 ml of prepared lysate was mixed with 4 ml of 2£ binding buVer and incubated with 37 g antiHA polyclonal antibody (Santa Cruz Biotechnology) and 160 l of a 50% slurry of protein A/G–agarose beads (Santa Cruz Biotechnology) for 2 h at 4 °C with constant shaking. The immune complexes were then pelleted (2800g, 5 min, 4 °C) and washed three times with binding buVer to remove non-speciWc proteins. The pellet was resuspended in 200 l HA elution buVer (binding buVer containing 1 mg/ml HA peptide [YPYDVPDYA]) and then incubated for 2 h at 4 °C on an orbital shaker. The sample was centrifuged (2800g, 5 min, 4 °C) and the supernatant containing eluted Jak2-WT-HA protein was collected, aliquoted, and frozen at ¡80 °C. For the Triton lysis method of extraction, each 100mm plate of cells was Wrst incubated in 0.9 ml kinase reaction buVer (50 mM Hepes, pH 7.6, 5 mM MgCl2, 5 mM MnCl2, 100 mM NaCl, 0.5 mM DTT, 1 mM Na3VO4, and 10 g/ml aprotinin) for 1 h at 23 °C to activate the Jak2 protein prior to cell lysis. The incubation
For the Jak2 immunoprecipitates, the beads were rinsed once with kinase reaction buVer and then incubated with the Stat1 immune complexes in the same buVer containing 2.5 mM ATP in a total volume of 63 l. The samples were incubated for 30 min at 23 °C on an orbital shaker. Reactions were stopped by the addition of 4£ SDS sample buVer. For puriWed Jak2 protein kinase reactions, »100 ng of eluted product was added directly to the kinase reaction buVer containing the Stat1 immune complexes and incubated as described. After adding SDS sample buVer, both sets of samples were boiled for 5 min, separated by SDS–PAGE, transferred onto nitrocellulose membranes, and Western blotted as described. Western blot analysis Western blots were visualized with the enhanced chemiluminescence system (NEN Life Science Products) following the manufacturer’s instructions. The polyclonal antibody used for Western blotting Jak2 was purchased from Upstate Biotechnology (HR-758). The anti-HA (clone F-7) and anti-phospho-Stat1 (pTyr 701) blotting antibodies were from Santa Cruz Biotechnology and Upstate Biotechnology, respectively. The anti-phosphotyrosine blotting antibodies were a mixture of PY99, PY20, and 4G10 monoclonal antibodies from Santa Cruz Biotechnology, BD Transduction Laboratories, and Upstate Biotechnology, respectively. Expression and puriWcation of the GST/Stat1 fusion protein The GST/Stat1 fusion protein was a kind gift from Dr. M. Showkat Ali (Emory University). The protein was expressed in DH5 cells and puriWed using Sepharose 4B–glutathione beads as previously described [11]. 32
P tyrosine kinase assay
The puriWed Jak2 product was resuspended in a 50 l reaction volume containing 50 mM Hepes, pH 7.6, 5 mM MgCl2, 5 mM MnCl2, 100 mM NaCl, 0.5 mM DTT,
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1.0 g GST/Stat1 fusion protein, 6 M ATP, and 1.0 Ci [-32P]ATP. The samples were incubated for 20 min at room temperature, a time within the linear reaction range. Reactions were terminated via the addition of 5 l of 0.5 M EDTA. Each sample was then spotted onto Whatman GF/C glass Wber Wlters and washed three times (10 min each) with 5% trichloroacetic acid (TCA) at 65 °C. The Wlters were air-dried overnight and then counted in a liquid scintillation counter. To account for 32 P incorporation into Jak2 itself, parallel reactions were performed without the GST/Stat1 substrate and this activity was subtracted away from the samples containing both enzyme and substrate.
Results Overview of Jak2 expression using the vaccinia virus expression system Previous attempts to express recombinant Jak2 protein have utilized approaches such as the baculovirusmediated expression system [7,8]. While these attempts were fruitful, they had some limitations regarding the purity and yield of the expressed Jak2 protein. Here, we elected to express Jak2 using the vaccinia virus expression system. This system has the advantage of expressing large amounts of protein that require post-translational modiWcation such as tyrosine phosphorylation [12]. The cartoon representing Fig. 1A illustrates the essential
elements that make up the recombinant Jak2 vaccinia virus termed, vJak2-WT-HA. The HA-tagged Jak2 cDNA is under the control of the T7 promoter. This promoter drives high level transcription of the Jak2-HA mRNA. The EMC leader sequence then allows for rapid and eYcient translation of these mRNA transcripts. Recombinant Jak2 was expressed using the vaccinia virus co-infection protocol [12,13]. This protocol is depicted as Fig. 1B. In this system, cells are infected with two independent vaccinia viruses. The Wrst virus, vTF73, has the cDNA encoding T7 RNA polymerase under the control of the constitutively active Promoter11. Once it is made inside the cell, the T7 RNA polymerase then binds and promotes high level expression from the T7 promoter which drives Jak2-HA expression. The translated Jak2 protein product is expressed with an HA epitope tag thus allowing for immunoaYnity puriWcation using anti-HA antibody. Expression of Jak2-HA protein using a recombinant Jak2 vaccinia virus BSC-40 cells were infected with increasing multiplicities of infection (moi) of vJak2-WT-HA to identify the amount of virus that resulted in the highest level of expressed Jak2 protein. Whole cell lysates were subsequently prepared and immediately subjected to immunoblot analysis with either anti-Jak2 or anti-HA antibodies. Immunoreactive bands at »130 kDa in mass (the size of Jak2) were clearly seen with both
Fig. 1. Cartoon summarizing the Jak2 expression system. (A) Diagram of the modiWed thymidine kinase (TK) locus in the recombinant vaccinia virus termed, vJak2-WT-HA. TK L, TK left arm; Prom T7, bacteriophage T7 promoter sequence; EMC, encephalomyocarditis virus derived element used for high level mRNA translation; Term T7, bacteriophage T7 transcription termination sequence; P7.5, vaccinia virus constitutively active promoter 7.5; Eco gpt, E. coli derived guanine phosphoribosyl transferase gene used for positive selection; TK R, TK right arm. (B) As T7 RNA polymerase is made from the vTF7-3 virus, it binds the T7 promoter on the vJak2-WT-HA virus and in turn drives high level expression of an HA-tagged Jak2 protein. The HA tag is then used for purifying the protein via anti-HA antibody.
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Fig. 2. Production of Jak2-HA protein using the vaccinia virus expression system. BSC-40 cells were infected with vaccinia viruses vJak2WT-HA and vTF7-3 at the indicated moi. Protein lysates were then separated by SDS–PAGE and subjected to immunoblot analysis using rabbit polyclonal antibodies directed against either Jak2 (upper panel) or the HA tag (lower panel). Shown is one of three representative experiments.
antibodies (Fig. 2). The data indicated that the amount of vJak2-WT-HA virus needed for maximal Jak2-HA expression was 0.5 moi. Collectively, these results demonstrate that the vJak2WT-HA recombinant vaccinia virus is able to express HA-epitope-tagged Jak2 protein in a readily detectable manner. Furthermore, the amount of vJak2-WT-HA virus needed to express maximal protein is 0.5 moi. Determination of Jak2 tyrosine kinase activity as a function of cell lysis buVer Numerous buVers have been described for lysing cultured cells [14–16]. Most diVer mainly by the amount of solubilizing detergent found in each. While detergents are ideal for solubilizing the plasma membrane of a cell, they can have the unintended consequence of suppressing the biological function of cellular proteins. Therefore, we wanted to determine which method of cell lysis was appropriate for producing suYcient yields of Jak2-HA protein that were also biologically active. To do this, BSC-40 cells were infected with 1.0 moi of vTF7-3 and 0.5 moi of vJak2-WT-HA. The following morning, the cells were lysed using one of three diVerent cellular lysis buVers (see Materials and methods for details). The RIPA buVer contained several detergents while the Triton lysis buVer contained only one. The Dounce lysis buVer had no detergents. An equal portion of each lysate was then Western blotted with anti-HA antibody to detect expressed Jak2HA protein. We found that lysis buVers containing detergents modestly increased the eYciency of Jak2-HA protein extraction »3-fold when compared to the method lacking detergent (data not shown). We then chose to measure the relative kinase activity for each of the three methods of lysis. Jak2 was immunoprecipitated from the respective lysates and then resuspended in a kinase reaction buVer either with, or without, ATP. Incubation with ATP will promote increased levels of Jak2 tyrosine phosphorylation if the Jak2 is catalytically active. Jak2-HA tyrosine phosphorylation levels were then measured via anti-phosphotyro-
Fig. 3. Determination of Jak2 tyrosine kinase activity as a function of cell lysis buVer. After viral infection, protein samples were prepared using either the Triton, Dounce or RIPA methods of cell lysis. (A) Jak2 immunoprecipitates were resuspended in kinase reaction buVer either containing (+) or lacking (¡) ATP. Jak2 was then allowed to autophosphorylate. The samples were separated by SDS–PAGE and Jak2 tyrosine phosphorylation levels were measured by immunoblotting the samples with anti-phosphotyrosine antibody (top) or with anti-Jak2 antibody to conWrm equal precipitation of Jak2 protein across all lanes (bottom). (B) Jak2 immunoprecipitates were resuspended in kinase reaction buVer containing ATP. Stat1 protein was then added as indicated. The samples were subsequently separated by SDS–PAGE and Jak2 and Stat1 tyrosine phosphorylation levels were measured by antiphosphotyrosine immunoblotting (top). The same membrane was then immunoblotted with either anti-Jak2 (middle) or anti-Stat1 (bottom) antibodies so that levels of these proteins could be determined. Shown is one of three representative experiments.
sine Western blotting (Fig. 3A, top). We found that ATP signiWcantly increased the tyrosine phosphorylation levels of Jak-HA protein derived from the Dounce lysis method, but only marginally increased the tyrosine phosphorylation levels of Jak2-HA derived from detergent solubilized methods (RIPA and Triton). To demonstrate that all
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lanes had an equal amount of precipitated Jak2-HA protein, the membrane was subsequently Western blotted with anti-Jak2 antibody (Fig. 3A, bottom). To determine whether the increased tyrosine phosphorylation levels of Jak2-HA correlated with its ability to increase the tyrosine phosphorylation levels of a known Jak2 substrate, we repeated the in vitro kinase assay, but this time included a Wxed amount of Stat1 protein, as Stat1 is a known substrate of Jak2 [17]. For this set of experiments, all Jak2-HA kinase reactions contained ATP. In addition, immunopuriWed Stat1 was added as indicated. The tyrosine phosphorylation levels of both Jak2 and Stat1 were then measured by Western blotting the samples with anti-phosphotyrosine antibody (Fig. 3B, top). Two important results were observed. First, the phosphotyrosine levels of Jak2-HA were once again signiWcantly greater for the Dounce extracted samples than they were for the detergent extracted ones, thus recapitulating the results shown in Fig. 3A. Second, the tyrosine phosphorylation levels of Stat1 were also signiWcantly higher when the Stat1 was incubated with Jak2HA derived from Dounce solubilized lysates as opposed to detergent solubilized lysates. As such, this suggests that the Jak2-HA protein derived from the Dounce solubilized cells has greater catalytic activity than Jak2-HA protein derived from detergent solubilized cells. Longer exposures of the membrane did indicate that the Jak2HA, derived from either the Triton or RIPA buVer treated cells, was able to tyrosine phosphorylate Stat1, but the eYciency of that phosphorylation was signiWcantly reduced when compared to the Dounce harvested cells (data not shown). To demonstrate equal levels of Jak2 protein across all lanes, the same membrane was stripped and Western blotted with anti-Jak2 antibody (Fig. 3B, middle). Finally, the same membrane was reprobed with anti-Stat1 antibody to conWrm equal addition of Stat1 protein to lanes 2, 4, and 6 (Fig. 3B, bottom). Collectively, the data in Fig. 3 demonstrate that the Dounce method of lysis yields about one-third the amount of Jak2 protein when compared to the RIPA and Triton lysis buVer methods. However, the catalytic activity of the Jak2-HA protein derived from the Dounce lysis method is approximately 100–1000-fold more biologically active than the detergent-derived Jak2-HA protein, as measured by its ability to autophosphorylate and tyrosine phosphorylate Stat1. One step puriWcation of soluble Jak2-HA protein Taking advantage of the HA tagging, we now wanted to generate soluble Jak2-HA protein on a larger scale. BSC-40 cells were infected and the cells were subsequently lysed via the Dounce method. The expressed Jak2-HA protein was then bound to anti-HA antibody as described under Materials and methods. After washing, the Jak2-HA protein was eluted by the addition of
competing HA peptide. The eluted protein sample was then applied to an SDS–PAGE gel and Coomassie stained (Fig. 4A). The results showed the presence of two major bands that were approximately130 and 55 kDa in mass. The 130 kDa band was Jak2 as it was immunoreactive against both anti-Jak2 and anti-HA antibodies while the 55 kDa protein was not (data not shown). Apparently, this method of puriWcation yielded Jak2 and an unidentiWed 55 kDa protein. We also puriWed Jak2-HA protein using the Triton buVer method of extraction. The data in Fig. 3 demonstrated that the Jak2 protein derived from this method of lysis had reduced catalytic activity when compared to the Dounce method. However, we found that when the cells were pre-incubated in kinase reaction buVer prior to Triton lysis, the catalytic activity of Jak2 could be markedly increased. This observation is shown in Fig. 4B. Here, prior to Triton lysis, the cells were either left untreated (¡) or incubated for 1 h in kinase reaction buVer (+). Lysates were then prepared and Jak2 was immunoprecipitated via the addition of anti-HA antibody. The Jak2 protein was then resuspended in buVer containing Stat1 and in vitro kinase reactions were performed similar to those shown in Fig. 3. When the samples were then Western blotted with anti-phosphotyrosine antibody, the results showed that kinase reaction buVer pre-treatment greatly increased the ability of Jak2 to both autophosphorylate and tyrosine phosphorylate Stat1, as determined by the increased phosphotyrosine signal of these two proteins (Fig. 4B, top). The same membrane was subsequently Western blotted with antiJak2 (Fig. 4B, middle) and anti-Stat1 (Fig. 4B, bottom) antibodies to conWrm the presence of these proteins in both lanes. Collectively, the results in Fig. 4B demonstrate that when cells are placed in kinase reaction buVer prior to Triton lysis, Jak2 can be activated to levels similar to those seen with the detergent-free lysis method. Twelve plates of infected cells were then subjected to the same kinase reaction pre-incubation and Jak2-HA protein was puriWed as described under Materials and methods. After aYnity binding and washing, the eluted Jak2-HA protein sample was applied to an SDS–PAGE gel and Coomassie stained (Fig. 4C). The results show that the Jak2-HA protein was puriWed to homogeneity due to the presence of a single band in the 130 kDa size range. When the protein was similarly silver stained, a single band was also observed (Fig. 4D). The eluted protein sample was subsequently quantitated using a Bradford protein assay. We estimated that twelve 100-mm dishes of infected cells yielded »4 g of pure Jak2-HA protein. We now wanted to quantitate the total amount of Jak2-HA protein that was expressed from a single 100mm dish of infected cells. For this, known amounts of the eluted Jak2-HA protein sample were separated by SDS–PAGE along with increasing amounts of original lysate from both the Triton and Dounce methods. The
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Fig. 4. AYnity puriWcation of Jak2-HA protein. (A) Jak2-HA protein was puriWed via the Dounce method as described. The original lysate and the puriWed protein product were then separated by SDS–PAGE and visualized by Coomassie blue staining. There was a 55 kDa protein of unknown identity that co-puriWed with Jak2. (B) Cells were either left untreated (¡) or incubated for 1 h in kinase reaction buVer (+) as described. Lysates were prepared using the Triton lysis method and immunoprecipitated Jak2 protein was then allowed to phosphorylate Stat1, in vitro. Stat1 and Jak2 tyrosine phosphorylation levels were then determined by Western blotting the samples with anti-phosphotyrosine antibody (top). The levels of Jak2 and Stat1 protein in each lane were then determined by blotting with either anti-Jak2 (middle) or anti-Stat1 (bottom) antibodies. (C) Jak2-HA protein was puriWed via the Triton method of extraction after Wrst receiving the kinase reaction pre-incubation. The original lysate, Xow through, and puriWed product were separated by SDS–PAGE and visualized by Coomassie blue staining. Also included on the gel were known amounts of bovine serum albumin (BSA) which served as an internal standard. (D) The total lysate and eluted protein product were also analyzed by silver staining.
samples were then Western blotted with anti-HA antibody to determine the relative amount of protein in each sample (Fig. 5). From this, we estimated that the Triton method of lysis yielded »10 g of soluble Jak2-HA protein per 100-mm dish while the Dounce method of lysis yielded »3.5 g per dish. Thus, for the overall expression, when twelve 100-mm plates of cells were lysed by the Triton method of extraction, »120 g Jak2-HA protein was produced. The 16 g anti-HA antibody column in turn yielded 4 g pure Jak2-HA protein. Determination of Jak2-HA speciWc activity We now wanted to determine the speciWc activity of the biologically active product. For this, we utilized a GST/ Stat1 fusion protein. Jak2 is known to phosphorylate
Stat1 at tyrosine residue 701 [18,19]. To demonstrate that the Jak2 expressed via the vaccinia virus expression system could phosphorylate the GST/Stat1 fusion protein at position 701, a Wxed amount of the fusion protein was incubated with increasing amounts of total lysate or puriWed Jak2-HA protein product. The samples were subsequently separated via SDS–PAGE and Western blotted with an anti-phospho-Stat1 (Tyr 701) antibody (Fig. 6). The results show that both the total lysate and the puriWed product generated via the Triton method of extraction utilizing the kinase reaction pre-incubation, phosphorylated the GST/Stat1 fusion protein at Tyr 701, in a dose-dependent manner. Similar reactions were then performed using the GST/ Stat1 fusion protein in the presence of [-32P]ATP so that speciWc activity could be determined. These results
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Fig. 5. Estimation of expressed Jak2 protein as a function of method of lysis. Known amounts of puriWed Jak2-HA elution product (20 and 40 ng) were separated by SDS–PAGE along with increasing amounts of cell lysate prepared from both the Triton and Dounce methods of lysis. The samples were subsequently Western blotted with anti-HA antibody to estimate the amount of Jak2-HA protein contained in the lysates. Shown is one of two representative results. Table 1 PuriWcation of recombinant Jak2 protein Step
Total Total activity SpeciWc activity Recovery protein (mg) (pmol/min) (pmol/min/mg) (%)
Lysate 3.867 Elution 0.004 product
28,344 446
7329 111,500
100 2
The in vitro kinase assays were performed using a GST-Stat1 fusion protein as the Jak2 speciWc substrate. The ATP concentration was 6 M.
are shown in Table 1. In summary, the puriWed Jak2-HA protein product generated by the Triton method of lysis was enzymatically active with a speciWc activity of 111,500 pmol/min/mg protein.
Discussion Jak2 is a critically important molecule in animals. We know that it is essential for life as animals lacking the
Jak2 allele die during early development [1]. Additionally, Jak2 has been linked to diseases such as cancer, heart failure and neo-intimal formation following vascular injury [2–4]. As a result of this, many studies have attempted to study the function of Jak2 at the DNA, mRNA, and protein levels. Of these three areas of research interest, it appears that the least amount of knowledge regarding Jak2 function exists at the protein level. The reasons for this are several fold. One such reason is that Jak2 is a large protein. By nature, larger proteins are harder to express and subsequently purify. Work by Farrar and co-workers [7,8] did express Jak2 in insect cells. However, while Jak2 expression was better in this system, problems still persisted. For example, Jak2 was expressed as a GST fusion and cleavage from GST was exceedingly diYcult [8]. Here, we attempted to address this problem by expressing Jak2 in a vaccinia virus-mediated expression system. The expression is phenomenal as a single dish of cells yielded about 10 g of protein. We facilitated the puriWcation of Jak2 by engineering a nine amino acid, HA epitope tag, on the C-terminal end of the protein. When cells were lysed using the Triton method of extraction, this one step isolation process puriWed Jak2 to homogeneity. More importantly, the puriWed product was biologically active as it could speciWcally tyrosine phosphorylate Stat1. Finally, given the fact that there are numerous commercially available anti-HA antibodies and hybridoma cell lines which make anti-HA antibody, the capacity of a potential anti-HA column can extend into milligram quantities. Another important observation from this work is the apparent eVect that detergents have on Jak2 kinase function. Our results clearly demonstrate that when Jak2 is isolated from cells that have been lysed with detergent, the catalytic activity of Jak2 is markedly reduced when compared to Jak2 that has been puriWed via a detergentfree lysis method. However, in the case of the Triton lysis method, this can be overcome by Wrst incubating the cells in kinase reaction buVer prior to lysis. This
Fig. 6. The Jak2-HA product phosphorylates Stat1 on tyrosine residue 701. Increasing amounts of either total lysate or puriWed Jak2-HA protein product prepared from the Triton method of extraction were incubated with 1.0 g GST/Stat1 fusion protein, in vitro. The samples were subsequently separated via SDS–PAGE and Western blotted with anti-phospho-Stat1 (Tyr 701) antibody to measure the levels of phosphorylation at position 701. Shown is one of three representative results.
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observation may provide an explanation as to why previous studies have had a diYcult time expressing a recombinant Jak2 protein with readily detectable levels of tyrosine phosphorylation. As such, our work suggests that future studies aimed at expressing and purifying functional Jak2 protein should either utilize a detergentfree puriWcation scheme or, when using detergent, Wrst perform the kinase incubation reaction prior to cell lysis. In summary, we have created a vaccinia virus-mediated expression system which allows for high level expression and single step puriWcation of recombinant Jak2 protein. More importantly, we have identiWed the conditions by which Jak2 protein should be prepared so that kinase activity can be maintained at maximum level. Collectively, these observations should help advance current studies that are aimed at understanding the biochemical and catalytic properties of Jak2. Acknowledgments We thank Drs. Pipsa Saharinen and Olli Silvennoinen for kindly providing the pCI-Neo-Jak2-WT-HA expression plasmid and Dr. M. Showkat Ali for the GST/Stat1 construct. The recombinant vaccinia virus clone vTF7-3 was graciously provided by Dr. Bernard Moss and was used in compliance with the Materials Transfer Agreement. We are grateful to Eric M. Sandberg and TiVany A. Wallace for critically reviewing the manuscript. This work was supported by a Biomedical Research Support Program for Medical Schools Award to the University of Florida College of Medicine by the Howard Hughes Medical Institute, an American Heart Association National Scientist Development Grant (#0130041N), and National Institutes of Health Awards K01DK60471 and R01-HL67277. References [1] E. Parganas, D. Wang, D. Stravopodis, D.J. Topham, J.C. Marine, S. Teglund, E.F. Vanin, S. Bodner, O.R. Colamonici, J.M. van Deursen, G. Grosveld, J.N. Ihle, Jak2 is essential for signaling through a variety of cytokine receptors, Cell 93 (1998) 385–395. [2] N. Meydan, T. Grunberger, H. Dadi, M. Shahar, E. Arpaia, Z. Lapidot, J.S. Leeder, M. Freedman, A. Cohen, A. Gazit, A. Levitzki, C.M. Roifman, Inhibition of acute lymphoblastic leukaemia by a Jak-2 inhibitor, Nature 379 (1996) 645–648. [3] J. Pan, K. Fukuda, H. Kodama, S. Makino, T. Takahashi, M. Sano, S. Hori, S. Ogawa, Role of angiotensin II in activation of the JAK/STAT pathway induced by acute pressure overload in the rat heart, Circ. Res. 81 (1997) 611–617.
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[4] Y. Seki, H. Kai, R. Shibata, T. Nagata, H. Yasukawa, A. Yoshimura, T. Imaizumi, Role of the JAK/STAT pathway in rat carotid artery remodeling after vascular injury, Circ. Res. 87 (2000) 12–18. [5] M. Gadina, D. Hilton, J.A. Johnston, A. Morinobu, A. Lighvani, Y.J. Zhou, R. Visconti, J.J. O’Shea, Signaling by type I and II cytokine receptors: ten years after, Curr. Opin. Immunol. 13 (2001) 363–373. [6] C. Schindler, J.E. Darnell Jr., Transcriptional responses to polypeptide ligands: the JAK–STAT pathway, Annu. Rev. Biochem. 64 (1995) 621–651. [7] R.J. Duhé, W.L. Farrar, Characterization of active and inactive forms of the JAK2 protein–tyrosine kinase produced via the baculovirus expression vector system, J. Biol. Chem. 270 (1995) 23084– 23089. [8] R.J. Duhé, E.A. Clark, W.L. Farrar, Characterization of the in vitro kinase activity of a partially puriWed soluble GST/JAK2 fusion protein, Mol. Cell. Biochem. 236 (2002) 23–35. [9] P. Saharinen, K. Takaluoma, O. Silvennoinen, Regulation of the Jak2 tyrosine kinase by its pseudokinase domain, Mol. Cell. Biol. 20 (2000) 3387–3395. [10] Earl et al., Generation of recombinant vaccinia viruses, in: Current Protocols in Molecular Biology, Wiley, New York, 1998, pp. 16.17.1–16.17.19. [11] M.S. Ali, P.P. Sayeski, L.B. Dirksen, D.J. Hayzer, M.B. Marrero, K.E. Bernstein, Dependence on the motif YIPP for the physical association of Jak2 kinase with the intracellular carboxyl tail of the angiotensin II AT1 receptor, J. Biol. Chem. 272 (1997) 23382– 23388. [12] B. Moss, Vaccinia virus: a tool for research and vaccine development, Science 252 (1991) 1662–1667. [13] T.R. Fuerst, B. Moss, Structure and stability of mRNA synthesized by vaccinia virus-encoded bacteriophage T7 RNA polymerase in mammalian cells. Importance of the 50 untranslated leader, J. Mol. Biol. 206 (1989) 333–348. [14] P.P. Sayeski, M.S. Ali, A. Safavi, M. Lyles, S.O. Kim, S.J. Frank, K.E. Bernstein, A catalytically active Jak2 is required for the angiotensin II-dependent activation of Fyn, J. Biol. Chem. 274 (1999) 33131–33142. [15] P.P. Sayeski, M.S. Ali, S.J. Frank, K.E. Bernstein, The angiotensin II-dependent nuclear translocation of Stat1 is mediated by the Jak2 protein motif 231YRFRR, J. Biol. Chem. 276 (2001) 10556– 10563. [16] D. VonDerLinden, X. Ma, E.M. Sandberg, K. Gernert, K.E. Bernstein, P.P. Sayeski, Mutation of glutamic acid residue 1046 abolishes Jak2 tyrosine kinase activity, Mol. Cell. Biochem. 241 (2002) 87–94. [17] X. Zhu, Z. Wen, L.Z. Xu, J.E. Darnell Jr., Stat1 serine phosphorylation occurs independently of tyrosine phosphorylation and requires an activated Jak2 kinase, Mol. Cell. Biol. 17 (1997) 6618– 6623. [18] K. Shuai, A. Ziemiecki, A.F. Wilks, A.G. Harpur, H.B. Sadowski, M.Z. Gilman, J.E. Darnell Jr., Polypeptide signalling to the nucleus through tyrosine phosphorylation of Jak and Stat proteins, Nature 399 (1993) 580–583. [19] A. Stephanou, T.M. Scarabelli, B.K. Brar, Y. Nakanishi, M. Matsumura, R.A. Knight, D.S. Latchman, Induction of apoptosis and Fas receptor/Fas ligand expression by ischemia/reperfusion in cardiac myocytes requires serine 727 of the STAT-1 transcription factor but not tyrosine 701, J. Biol. Chem. 276 (2001) 28340–28347.
Vaccinia virus-mediated high level expression and single step puriWcation of recombinant Jak2 protein夽 Xianyue Ma and Peter P. Sayeski¤ Department of Physiology and Functional Genomics, University of Florida College of Medicine, P.O. Box 100274, Gainesville, FL 32610, USA Received 7 May 2003, and in revised form 16 January 2004
Abstract Jak2 functions as a non-receptor tyrosine kinase and has been linked to pathologies such as cancer and cardiovascular disease. Because of this, many studies have tried to better understand its function. Unfortunately, very little information is known about its catalytic or biochemical properties as puriWcation of signiWcant amounts of functional Jak2 protein has been exceedingly diYcult. Here, Jak2 was expressed in BSC-40 cells using a vaccinia virus-mediated expression system. SigniWcant amounts (»10 g) of Jak2 protein were expressed from a single 100-mm cell culture dish. The protein was Wrst harvested using three diVerent methods of extraction to determine the relative eYciency of each lysis method with respect to Jak2 protein yield and catalytic activity. We found that lysis methods utilizing detergents increased the eYciency of protein extraction about 3-fold when compared to a method lacking detergent. However, with respect to catalytic activity, Jak2 isolated from cells using detergent-containing lysis buVers had signiWcantly less catalytic activity than when compared to the method that was detergent free. Expression was then scaled up and Jak2 protein was puriWed via a one step immunoaYnity puriWcation scheme using both the detergent-free and a modiWed detergentcontaining method of extraction that maintained catalytic activity. In vitro kinase assays demonstrated that the puriWed product was highly catalytic as measured by its ability to tyrosine phosphorylate Stat1. Collectively, the results show that (1) Jak2 can be expressed at very high levels in mammalian cells, (2) it can be puriWed to homogeneity via a single step puriWcation scheme, and (3) the puriWed product is biologically active. 2004 Elsevier Inc. All rights reserved.
Jak2 is a member of the Janus family of tyrosine kinases (Jaks) that also includes Jak1, Jak3, and Tyk2. Jak2 is »130 kDa in mass and contains seven conserved Jak homology (JH) domains. It is known to have important roles both in the physiology and pathophysiology of animals. For example, mice lacking the Jak2 allele die embryonically, thereby demonstrating that Jak2 is essential for life [1]. As such, no other tyrosine kinase in the mouse genome, including other Jak kinases, can function in its place. With respect to pathophysiology, studies have demonstrated a clear link between Jak2 and diseases such as cancer, heart failure, and neointimal formation following balloon angioplasty [2–4]. Jak2 functions as non-receptor tyrosine kinase and so far has been best characterized in cytokine-induced 夽
US Patent Application No. 60/443,042. Corresponding author. Fax: 1-352-846-0270. E-mail address: [email protected] (P.P.Sayeski). URL: http://www.med.ufl.edu/phys/sayeski.shtml.
¤
1046-5928/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2004.02.009
signal transduction pathways [5]. In this model, Jak2 is constitutively bound to the cytoplasmic tail of cytokine receptors. Ligand binding induces receptor aggregation which results in the tyrosine autophosphorylation of Jak2 molecules via a juxta-positioning of the associated Jak proteins. Activated Jak2 then phosphorylates tyrosine residues on the cytoplasmic tails of these receptors which serve as docking sites for the Signal Transducers and Activators of Transcription (STAT) proteins. Activated Jak2 subsequently phosphorylates the receptorbound STAT proteins. Phosphorylated STAT proteins then translocate from the receptor complex to the nucleus where they facilitate gene transcription [6]. Thus, Jak2 is a key intermediate linking ligand binding at the cell surface with gene transcription in the nucleus. Although a reasonable amount of information is known about the mechanisms by which Jak2 mediates gene transcription, little is known about the biochemical and kinetic properties of Jak2 protein, as expression and
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puriWcation of signiWcant quantities of functional Jak2 protein has been exceedingly diYcult [7,8]. Furthermore, the only commercially available sources of Jak2 protein are sold as antibody conjugates which limit the biochemical analysis that can be performed on Jak2. In the present study, an HA-tagged Jak2 protein was successfully expressed in BSC-40 cells using the vaccinia virus-mediated expression system. About 10 g of Jak2 protein was yielded on a small culture scale (one 100-mm cell culture dish). The protein was puriWed to homogeneity in a single immunoaYnity chromatography step using an anti-HA antibody. A Jak2 in vitro kinase assay system was developed to test the function of the puriWed protein. Stat1, a Jak2 substrate, was phosphorylated by both Jak2 immunocomplexes and the puriWed Jak2 protein product. Moreover, the puriWed Jak2 product phosphorylated a GST-Stat1 fusion protein substrate with a speciWc activity of 111,500 pmol/min/mg protein. As such, these results demonstrate that the vaccinia virus expression system followed by anti-HA aYnity chromatography provides an eYcient means of obtaining large amounts of pure and functional Jak2 protein.
strand primer (50-GTAATACGACTCACTATAGGG C-30) and a bottom strand primer (50-CGCGGATCCCT AAAGGGAAGCGGCCGC-30). The blunt-end PCR product was inserted into the TOPO cloning vector using the Zero Blunt TOPO PCR Cloning Kit (Invitrogen). The HA-tagged Jak2 cDNA was then excised from TOPO via NcoI and BamHI restriction digest and then cloned into the vaccinia recombination vector, pTM3, to yield pTM3-Jak2-WT-HA.
Materials and methods
Preparation of cell lysate containing Jak2-WT-HA
Chemicals and supplies
For protein expression, twelve 100-mm dishes of conXuent BSC-40 cells were infected with 0.5 moi of vJak2WT-HA and 1.0 moi of vTF7-3 recombinant viruses in serum-free media. One hour later, the media were aspirated and the cells were incubated overnight in media supplemented with 10% fetal bovine serum. The following morning, the cells were washed once with phosphatebuVered saline containing 1 mM Na3VO4 and then lysed using one of three diVerent methods so that both protein yield and functional activity could be assessed. For method one, each plate was lysed in 1 ml RIPA buVer (20 mM Tris–HCl, pH 7.5, 10% glycerol, 1% Triton X100, 1% deoxycholate, 0.1% SDS, and 2.5 mM EDTA) containing phosphatase and protease inhibitors (4 mM benzamidine, 1 mM AEBSF (4-(2-amonoethyl)-benzenesulfonyl Xuoride), 1 mM Na3VO4, and 10 g/ml aprotinin). For method two, each plate was lysed in 1 ml Triton lysis buVer (100 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, and 5 mM EDTA) containing the same inhibitors. In both cases, the samples were brieXy sonicated and incubated on ice for at least 30 min. The lysates were then cleared by centrifugation and stored in ¡80 °C for future use. For method three, lysates were prepared using a Dounce homogenizer. SpeciWcally, the cells were detached from the plates in cold harvest buVer (40 mM Tris–HCl, pH 7.5, 10 mM EDTA, and 150 mM NaCl) using a disposable cell scraper. The cells were then collected by centrifugation (1000g, 5 min) and washed once with harvest buVer. The cells from twelve 100-mm dishes were resuspended in 3 ml of BuVer A (10 mM
DMEM cell culture media and antibiotic supplements were purchased from Life Technologies/Invitrogen. Heat inactivated fetal bovine serum was from Hyclone. All other reagents were from either Fisher ScientiWc or Sigma Chemical. Construction of vaccinia recombination plasmid pTM3Jak2-HA The HA-tagged Jak2 cDNA was subcloned into the vaccinia recombination vector pTM3 in a multi-step process. The Jak2 expression vector, pCI-Neo-Jak2-WTHA [9], was modiWed via the QuikChange site-directed mutagenesis system (Stratagene) in order to convert the 50 SalI to NcoI and to destroy the internal EcoRI and NcoI sites. The top and bottom strand oligonucleotides used for this were 50-GGTACCTCTAGAGCCATGG GAATGGCCTGC-30 and 50-GCAGGCCATTCCCA TGGCTCTAGAGGTACC-30; 50-ACTGGAAACGGT GGTATTCAGTGGTCAAGA-30 and TCTTGACCAC TGAATACCACCGTTTCCAGT-30; 50-CAGGAGAG AATACCTTGGGTACCTCCTGAA-30 and 50-TTCA GGAGGTACCCAAGGTATTCTCTCCTG-30, respectively. Although the DNA sequence of the modiWed template was changed, the Jak2 amino acid sequence was not. Using this as a template, a 3.3 kb HA-tagged Jak2 cDNA fragment containing 50 NcoI and 30 BamHI ends was ampliWed via PFU DNA polymerase using a top
Generation of recombinant vaccinia virus vJak2-WT-HA To generate a recombinant vaccinia virus containing the HA-tagged Jak2 cDNA under the control of the T7 promoter, an 80% conXuent monolayer of BSC-40 cells (100 mm dish) was transfected for 5 h with 20 g of pTM3-Jak2-WT-HA in 20 l of Lipofectin (Life Technologies) following the manufacturer’s instruction. Serum-containing media were then added to the plate and the cells were infected with 0.5 multiplicities of infection (moi) of wild-type vaccinia virus. Virus was harvested two days later and recombinants (vJak2-WT-HA) were selected for gpt and against tk as described [10].
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Hepes, pH 7.9, 5 mM MgCl2, 0.1 mM. EDTA, 10 mM NaCl, 10 mM NaF, 1 mM dithiothreitol, and 1 mM AEBSF) and allowed to swell on ice for 5 min. The cells were then lysed in a Dounce homogenizer (pestle B, 20– 40 strokes). The lysates were brieXy sonicated, cleared by centrifugation (10,000g, 10 min), and stored in ¡80 °C for future use. COS-7 cell lysates were prepared with the Dounce homogenizer in a similar manner, but without any virus infection.
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was stopped by adding 0.3 ml of 4£ Triton lysis buVer and placing the plates on ice. Soluble lysates were prepared as described above. Four milliliters of cleared lysate was then mixed with 4 ml of 2£ binding buVer and incubated with 16 g anti-HA monoclonal antibody (Santa Cruz Biotechnology) and 120 l of a 50% slurry of protein A/G–agarose beads (Santa Cruz Biotechnology) for 2 h at 4 °C with constant shaking. The Jak2WT-HA protein was then pelleted, washed, eluted, and frozen as described above.
Immunoprecipitation of expressed Jak2-WT-HA protein In vitro kinase assay For this, 0.5 ml of cell lysate was mixed with 0.5 ml of 2£ binding buVer (50 mM Hepes, pH 7.6, 20% glycerol, 200 mM KCl, 25 mM MgCl2, 0.2 mM EDTA, 0.2 mM IGEPAL CA-630, 2 mM DTT, and 2 mM AEBSF) and incubated with 2 g anti-HA monoclonal antibody (Santa Cruz Biotechnology) and 20 l of a 50% slurry of protein A/G–agarose beads (Santa Cruz Biotechnology) for 2 h at 4 °C with shaking. The beads were washed three times with 1£ binding buVer and then in vitro kinase assays were performed as described below. For Stat1 immunoprecipitation, 0.5 ml COS-7 cell lysate was prepared by the Dounce homogenizer method and then immunoprecipitated with 2 g anti-Stat1 polyclonal antibody (Santa Cruz Biotechnology), and 20 l of a 50% slurry of protein A/G–agarose beads (Santa Cruz Biotechnology) for 2 h at 4 °C. The beads were washed with binding buVer and then kinase assays were performed as described below. PuriWcation of Jak2-WT-HA protein For large scale puriWcation of recombinant Jak2 protein, we utilized both the detergent-free (Dounce) and a modiWed Triton lysis method of extraction. For the Dounce method, 4 ml of prepared lysate was mixed with 4 ml of 2£ binding buVer and incubated with 37 g antiHA polyclonal antibody (Santa Cruz Biotechnology) and 160 l of a 50% slurry of protein A/G–agarose beads (Santa Cruz Biotechnology) for 2 h at 4 °C with constant shaking. The immune complexes were then pelleted (2800g, 5 min, 4 °C) and washed three times with binding buVer to remove non-speciWc proteins. The pellet was resuspended in 200 l HA elution buVer (binding buVer containing 1 mg/ml HA peptide [YPYDVPDYA]) and then incubated for 2 h at 4 °C on an orbital shaker. The sample was centrifuged (2800g, 5 min, 4 °C) and the supernatant containing eluted Jak2-WT-HA protein was collected, aliquoted, and frozen at ¡80 °C. For the Triton lysis method of extraction, each 100mm plate of cells was Wrst incubated in 0.9 ml kinase reaction buVer (50 mM Hepes, pH 7.6, 5 mM MgCl2, 5 mM MnCl2, 100 mM NaCl, 0.5 mM DTT, 1 mM Na3VO4, and 10 g/ml aprotinin) for 1 h at 23 °C to activate the Jak2 protein prior to cell lysis. The incubation
For the Jak2 immunoprecipitates, the beads were rinsed once with kinase reaction buVer and then incubated with the Stat1 immune complexes in the same buVer containing 2.5 mM ATP in a total volume of 63 l. The samples were incubated for 30 min at 23 °C on an orbital shaker. Reactions were stopped by the addition of 4£ SDS sample buVer. For puriWed Jak2 protein kinase reactions, »100 ng of eluted product was added directly to the kinase reaction buVer containing the Stat1 immune complexes and incubated as described. After adding SDS sample buVer, both sets of samples were boiled for 5 min, separated by SDS–PAGE, transferred onto nitrocellulose membranes, and Western blotted as described. Western blot analysis Western blots were visualized with the enhanced chemiluminescence system (NEN Life Science Products) following the manufacturer’s instructions. The polyclonal antibody used for Western blotting Jak2 was purchased from Upstate Biotechnology (HR-758). The anti-HA (clone F-7) and anti-phospho-Stat1 (pTyr 701) blotting antibodies were from Santa Cruz Biotechnology and Upstate Biotechnology, respectively. The anti-phosphotyrosine blotting antibodies were a mixture of PY99, PY20, and 4G10 monoclonal antibodies from Santa Cruz Biotechnology, BD Transduction Laboratories, and Upstate Biotechnology, respectively. Expression and puriWcation of the GST/Stat1 fusion protein The GST/Stat1 fusion protein was a kind gift from Dr. M. Showkat Ali (Emory University). The protein was expressed in DH5 cells and puriWed using Sepharose 4B–glutathione beads as previously described [11]. 32
P tyrosine kinase assay
The puriWed Jak2 product was resuspended in a 50 l reaction volume containing 50 mM Hepes, pH 7.6, 5 mM MgCl2, 5 mM MnCl2, 100 mM NaCl, 0.5 mM DTT,
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1.0 g GST/Stat1 fusion protein, 6 M ATP, and 1.0 Ci [-32P]ATP. The samples were incubated for 20 min at room temperature, a time within the linear reaction range. Reactions were terminated via the addition of 5 l of 0.5 M EDTA. Each sample was then spotted onto Whatman GF/C glass Wber Wlters and washed three times (10 min each) with 5% trichloroacetic acid (TCA) at 65 °C. The Wlters were air-dried overnight and then counted in a liquid scintillation counter. To account for 32 P incorporation into Jak2 itself, parallel reactions were performed without the GST/Stat1 substrate and this activity was subtracted away from the samples containing both enzyme and substrate.
Results Overview of Jak2 expression using the vaccinia virus expression system Previous attempts to express recombinant Jak2 protein have utilized approaches such as the baculovirusmediated expression system [7,8]. While these attempts were fruitful, they had some limitations regarding the purity and yield of the expressed Jak2 protein. Here, we elected to express Jak2 using the vaccinia virus expression system. This system has the advantage of expressing large amounts of protein that require post-translational modiWcation such as tyrosine phosphorylation [12]. The cartoon representing Fig. 1A illustrates the essential
elements that make up the recombinant Jak2 vaccinia virus termed, vJak2-WT-HA. The HA-tagged Jak2 cDNA is under the control of the T7 promoter. This promoter drives high level transcription of the Jak2-HA mRNA. The EMC leader sequence then allows for rapid and eYcient translation of these mRNA transcripts. Recombinant Jak2 was expressed using the vaccinia virus co-infection protocol [12,13]. This protocol is depicted as Fig. 1B. In this system, cells are infected with two independent vaccinia viruses. The Wrst virus, vTF73, has the cDNA encoding T7 RNA polymerase under the control of the constitutively active Promoter11. Once it is made inside the cell, the T7 RNA polymerase then binds and promotes high level expression from the T7 promoter which drives Jak2-HA expression. The translated Jak2 protein product is expressed with an HA epitope tag thus allowing for immunoaYnity puriWcation using anti-HA antibody. Expression of Jak2-HA protein using a recombinant Jak2 vaccinia virus BSC-40 cells were infected with increasing multiplicities of infection (moi) of vJak2-WT-HA to identify the amount of virus that resulted in the highest level of expressed Jak2 protein. Whole cell lysates were subsequently prepared and immediately subjected to immunoblot analysis with either anti-Jak2 or anti-HA antibodies. Immunoreactive bands at »130 kDa in mass (the size of Jak2) were clearly seen with both
Fig. 1. Cartoon summarizing the Jak2 expression system. (A) Diagram of the modiWed thymidine kinase (TK) locus in the recombinant vaccinia virus termed, vJak2-WT-HA. TK L, TK left arm; Prom T7, bacteriophage T7 promoter sequence; EMC, encephalomyocarditis virus derived element used for high level mRNA translation; Term T7, bacteriophage T7 transcription termination sequence; P7.5, vaccinia virus constitutively active promoter 7.5; Eco gpt, E. coli derived guanine phosphoribosyl transferase gene used for positive selection; TK R, TK right arm. (B) As T7 RNA polymerase is made from the vTF7-3 virus, it binds the T7 promoter on the vJak2-WT-HA virus and in turn drives high level expression of an HA-tagged Jak2 protein. The HA tag is then used for purifying the protein via anti-HA antibody.
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Fig. 2. Production of Jak2-HA protein using the vaccinia virus expression system. BSC-40 cells were infected with vaccinia viruses vJak2WT-HA and vTF7-3 at the indicated moi. Protein lysates were then separated by SDS–PAGE and subjected to immunoblot analysis using rabbit polyclonal antibodies directed against either Jak2 (upper panel) or the HA tag (lower panel). Shown is one of three representative experiments.
antibodies (Fig. 2). The data indicated that the amount of vJak2-WT-HA virus needed for maximal Jak2-HA expression was 0.5 moi. Collectively, these results demonstrate that the vJak2WT-HA recombinant vaccinia virus is able to express HA-epitope-tagged Jak2 protein in a readily detectable manner. Furthermore, the amount of vJak2-WT-HA virus needed to express maximal protein is 0.5 moi. Determination of Jak2 tyrosine kinase activity as a function of cell lysis buVer Numerous buVers have been described for lysing cultured cells [14–16]. Most diVer mainly by the amount of solubilizing detergent found in each. While detergents are ideal for solubilizing the plasma membrane of a cell, they can have the unintended consequence of suppressing the biological function of cellular proteins. Therefore, we wanted to determine which method of cell lysis was appropriate for producing suYcient yields of Jak2-HA protein that were also biologically active. To do this, BSC-40 cells were infected with 1.0 moi of vTF7-3 and 0.5 moi of vJak2-WT-HA. The following morning, the cells were lysed using one of three diVerent cellular lysis buVers (see Materials and methods for details). The RIPA buVer contained several detergents while the Triton lysis buVer contained only one. The Dounce lysis buVer had no detergents. An equal portion of each lysate was then Western blotted with anti-HA antibody to detect expressed Jak2HA protein. We found that lysis buVers containing detergents modestly increased the eYciency of Jak2-HA protein extraction »3-fold when compared to the method lacking detergent (data not shown). We then chose to measure the relative kinase activity for each of the three methods of lysis. Jak2 was immunoprecipitated from the respective lysates and then resuspended in a kinase reaction buVer either with, or without, ATP. Incubation with ATP will promote increased levels of Jak2 tyrosine phosphorylation if the Jak2 is catalytically active. Jak2-HA tyrosine phosphorylation levels were then measured via anti-phosphotyro-
Fig. 3. Determination of Jak2 tyrosine kinase activity as a function of cell lysis buVer. After viral infection, protein samples were prepared using either the Triton, Dounce or RIPA methods of cell lysis. (A) Jak2 immunoprecipitates were resuspended in kinase reaction buVer either containing (+) or lacking (¡) ATP. Jak2 was then allowed to autophosphorylate. The samples were separated by SDS–PAGE and Jak2 tyrosine phosphorylation levels were measured by immunoblotting the samples with anti-phosphotyrosine antibody (top) or with anti-Jak2 antibody to conWrm equal precipitation of Jak2 protein across all lanes (bottom). (B) Jak2 immunoprecipitates were resuspended in kinase reaction buVer containing ATP. Stat1 protein was then added as indicated. The samples were subsequently separated by SDS–PAGE and Jak2 and Stat1 tyrosine phosphorylation levels were measured by antiphosphotyrosine immunoblotting (top). The same membrane was then immunoblotted with either anti-Jak2 (middle) or anti-Stat1 (bottom) antibodies so that levels of these proteins could be determined. Shown is one of three representative experiments.
sine Western blotting (Fig. 3A, top). We found that ATP signiWcantly increased the tyrosine phosphorylation levels of Jak-HA protein derived from the Dounce lysis method, but only marginally increased the tyrosine phosphorylation levels of Jak2-HA derived from detergent solubilized methods (RIPA and Triton). To demonstrate that all
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lanes had an equal amount of precipitated Jak2-HA protein, the membrane was subsequently Western blotted with anti-Jak2 antibody (Fig. 3A, bottom). To determine whether the increased tyrosine phosphorylation levels of Jak2-HA correlated with its ability to increase the tyrosine phosphorylation levels of a known Jak2 substrate, we repeated the in vitro kinase assay, but this time included a Wxed amount of Stat1 protein, as Stat1 is a known substrate of Jak2 [17]. For this set of experiments, all Jak2-HA kinase reactions contained ATP. In addition, immunopuriWed Stat1 was added as indicated. The tyrosine phosphorylation levels of both Jak2 and Stat1 were then measured by Western blotting the samples with anti-phosphotyrosine antibody (Fig. 3B, top). Two important results were observed. First, the phosphotyrosine levels of Jak2-HA were once again signiWcantly greater for the Dounce extracted samples than they were for the detergent extracted ones, thus recapitulating the results shown in Fig. 3A. Second, the tyrosine phosphorylation levels of Stat1 were also signiWcantly higher when the Stat1 was incubated with Jak2HA derived from Dounce solubilized lysates as opposed to detergent solubilized lysates. As such, this suggests that the Jak2-HA protein derived from the Dounce solubilized cells has greater catalytic activity than Jak2-HA protein derived from detergent solubilized cells. Longer exposures of the membrane did indicate that the Jak2HA, derived from either the Triton or RIPA buVer treated cells, was able to tyrosine phosphorylate Stat1, but the eYciency of that phosphorylation was signiWcantly reduced when compared to the Dounce harvested cells (data not shown). To demonstrate equal levels of Jak2 protein across all lanes, the same membrane was stripped and Western blotted with anti-Jak2 antibody (Fig. 3B, middle). Finally, the same membrane was reprobed with anti-Stat1 antibody to conWrm equal addition of Stat1 protein to lanes 2, 4, and 6 (Fig. 3B, bottom). Collectively, the data in Fig. 3 demonstrate that the Dounce method of lysis yields about one-third the amount of Jak2 protein when compared to the RIPA and Triton lysis buVer methods. However, the catalytic activity of the Jak2-HA protein derived from the Dounce lysis method is approximately 100–1000-fold more biologically active than the detergent-derived Jak2-HA protein, as measured by its ability to autophosphorylate and tyrosine phosphorylate Stat1. One step puriWcation of soluble Jak2-HA protein Taking advantage of the HA tagging, we now wanted to generate soluble Jak2-HA protein on a larger scale. BSC-40 cells were infected and the cells were subsequently lysed via the Dounce method. The expressed Jak2-HA protein was then bound to anti-HA antibody as described under Materials and methods. After washing, the Jak2-HA protein was eluted by the addition of
competing HA peptide. The eluted protein sample was then applied to an SDS–PAGE gel and Coomassie stained (Fig. 4A). The results showed the presence of two major bands that were approximately130 and 55 kDa in mass. The 130 kDa band was Jak2 as it was immunoreactive against both anti-Jak2 and anti-HA antibodies while the 55 kDa protein was not (data not shown). Apparently, this method of puriWcation yielded Jak2 and an unidentiWed 55 kDa protein. We also puriWed Jak2-HA protein using the Triton buVer method of extraction. The data in Fig. 3 demonstrated that the Jak2 protein derived from this method of lysis had reduced catalytic activity when compared to the Dounce method. However, we found that when the cells were pre-incubated in kinase reaction buVer prior to Triton lysis, the catalytic activity of Jak2 could be markedly increased. This observation is shown in Fig. 4B. Here, prior to Triton lysis, the cells were either left untreated (¡) or incubated for 1 h in kinase reaction buVer (+). Lysates were then prepared and Jak2 was immunoprecipitated via the addition of anti-HA antibody. The Jak2 protein was then resuspended in buVer containing Stat1 and in vitro kinase reactions were performed similar to those shown in Fig. 3. When the samples were then Western blotted with anti-phosphotyrosine antibody, the results showed that kinase reaction buVer pre-treatment greatly increased the ability of Jak2 to both autophosphorylate and tyrosine phosphorylate Stat1, as determined by the increased phosphotyrosine signal of these two proteins (Fig. 4B, top). The same membrane was subsequently Western blotted with antiJak2 (Fig. 4B, middle) and anti-Stat1 (Fig. 4B, bottom) antibodies to conWrm the presence of these proteins in both lanes. Collectively, the results in Fig. 4B demonstrate that when cells are placed in kinase reaction buVer prior to Triton lysis, Jak2 can be activated to levels similar to those seen with the detergent-free lysis method. Twelve plates of infected cells were then subjected to the same kinase reaction pre-incubation and Jak2-HA protein was puriWed as described under Materials and methods. After aYnity binding and washing, the eluted Jak2-HA protein sample was applied to an SDS–PAGE gel and Coomassie stained (Fig. 4C). The results show that the Jak2-HA protein was puriWed to homogeneity due to the presence of a single band in the 130 kDa size range. When the protein was similarly silver stained, a single band was also observed (Fig. 4D). The eluted protein sample was subsequently quantitated using a Bradford protein assay. We estimated that twelve 100-mm dishes of infected cells yielded »4 g of pure Jak2-HA protein. We now wanted to quantitate the total amount of Jak2-HA protein that was expressed from a single 100mm dish of infected cells. For this, known amounts of the eluted Jak2-HA protein sample were separated by SDS–PAGE along with increasing amounts of original lysate from both the Triton and Dounce methods. The
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187
Fig. 4. AYnity puriWcation of Jak2-HA protein. (A) Jak2-HA protein was puriWed via the Dounce method as described. The original lysate and the puriWed protein product were then separated by SDS–PAGE and visualized by Coomassie blue staining. There was a 55 kDa protein of unknown identity that co-puriWed with Jak2. (B) Cells were either left untreated (¡) or incubated for 1 h in kinase reaction buVer (+) as described. Lysates were prepared using the Triton lysis method and immunoprecipitated Jak2 protein was then allowed to phosphorylate Stat1, in vitro. Stat1 and Jak2 tyrosine phosphorylation levels were then determined by Western blotting the samples with anti-phosphotyrosine antibody (top). The levels of Jak2 and Stat1 protein in each lane were then determined by blotting with either anti-Jak2 (middle) or anti-Stat1 (bottom) antibodies. (C) Jak2-HA protein was puriWed via the Triton method of extraction after Wrst receiving the kinase reaction pre-incubation. The original lysate, Xow through, and puriWed product were separated by SDS–PAGE and visualized by Coomassie blue staining. Also included on the gel were known amounts of bovine serum albumin (BSA) which served as an internal standard. (D) The total lysate and eluted protein product were also analyzed by silver staining.
samples were then Western blotted with anti-HA antibody to determine the relative amount of protein in each sample (Fig. 5). From this, we estimated that the Triton method of lysis yielded »10 g of soluble Jak2-HA protein per 100-mm dish while the Dounce method of lysis yielded »3.5 g per dish. Thus, for the overall expression, when twelve 100-mm plates of cells were lysed by the Triton method of extraction, »120 g Jak2-HA protein was produced. The 16 g anti-HA antibody column in turn yielded 4 g pure Jak2-HA protein. Determination of Jak2-HA speciWc activity We now wanted to determine the speciWc activity of the biologically active product. For this, we utilized a GST/ Stat1 fusion protein. Jak2 is known to phosphorylate
Stat1 at tyrosine residue 701 [18,19]. To demonstrate that the Jak2 expressed via the vaccinia virus expression system could phosphorylate the GST/Stat1 fusion protein at position 701, a Wxed amount of the fusion protein was incubated with increasing amounts of total lysate or puriWed Jak2-HA protein product. The samples were subsequently separated via SDS–PAGE and Western blotted with an anti-phospho-Stat1 (Tyr 701) antibody (Fig. 6). The results show that both the total lysate and the puriWed product generated via the Triton method of extraction utilizing the kinase reaction pre-incubation, phosphorylated the GST/Stat1 fusion protein at Tyr 701, in a dose-dependent manner. Similar reactions were then performed using the GST/ Stat1 fusion protein in the presence of [-32P]ATP so that speciWc activity could be determined. These results
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Fig. 5. Estimation of expressed Jak2 protein as a function of method of lysis. Known amounts of puriWed Jak2-HA elution product (20 and 40 ng) were separated by SDS–PAGE along with increasing amounts of cell lysate prepared from both the Triton and Dounce methods of lysis. The samples were subsequently Western blotted with anti-HA antibody to estimate the amount of Jak2-HA protein contained in the lysates. Shown is one of two representative results. Table 1 PuriWcation of recombinant Jak2 protein Step
Total Total activity SpeciWc activity Recovery protein (mg) (pmol/min) (pmol/min/mg) (%)
Lysate 3.867 Elution 0.004 product
28,344 446
7329 111,500
100 2
The in vitro kinase assays were performed using a GST-Stat1 fusion protein as the Jak2 speciWc substrate. The ATP concentration was 6 M.
are shown in Table 1. In summary, the puriWed Jak2-HA protein product generated by the Triton method of lysis was enzymatically active with a speciWc activity of 111,500 pmol/min/mg protein.
Discussion Jak2 is a critically important molecule in animals. We know that it is essential for life as animals lacking the
Jak2 allele die during early development [1]. Additionally, Jak2 has been linked to diseases such as cancer, heart failure and neo-intimal formation following vascular injury [2–4]. As a result of this, many studies have attempted to study the function of Jak2 at the DNA, mRNA, and protein levels. Of these three areas of research interest, it appears that the least amount of knowledge regarding Jak2 function exists at the protein level. The reasons for this are several fold. One such reason is that Jak2 is a large protein. By nature, larger proteins are harder to express and subsequently purify. Work by Farrar and co-workers [7,8] did express Jak2 in insect cells. However, while Jak2 expression was better in this system, problems still persisted. For example, Jak2 was expressed as a GST fusion and cleavage from GST was exceedingly diYcult [8]. Here, we attempted to address this problem by expressing Jak2 in a vaccinia virus-mediated expression system. The expression is phenomenal as a single dish of cells yielded about 10 g of protein. We facilitated the puriWcation of Jak2 by engineering a nine amino acid, HA epitope tag, on the C-terminal end of the protein. When cells were lysed using the Triton method of extraction, this one step isolation process puriWed Jak2 to homogeneity. More importantly, the puriWed product was biologically active as it could speciWcally tyrosine phosphorylate Stat1. Finally, given the fact that there are numerous commercially available anti-HA antibodies and hybridoma cell lines which make anti-HA antibody, the capacity of a potential anti-HA column can extend into milligram quantities. Another important observation from this work is the apparent eVect that detergents have on Jak2 kinase function. Our results clearly demonstrate that when Jak2 is isolated from cells that have been lysed with detergent, the catalytic activity of Jak2 is markedly reduced when compared to Jak2 that has been puriWed via a detergentfree lysis method. However, in the case of the Triton lysis method, this can be overcome by Wrst incubating the cells in kinase reaction buVer prior to lysis. This
Fig. 6. The Jak2-HA product phosphorylates Stat1 on tyrosine residue 701. Increasing amounts of either total lysate or puriWed Jak2-HA protein product prepared from the Triton method of extraction were incubated with 1.0 g GST/Stat1 fusion protein, in vitro. The samples were subsequently separated via SDS–PAGE and Western blotted with anti-phospho-Stat1 (Tyr 701) antibody to measure the levels of phosphorylation at position 701. Shown is one of three representative results.
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observation may provide an explanation as to why previous studies have had a diYcult time expressing a recombinant Jak2 protein with readily detectable levels of tyrosine phosphorylation. As such, our work suggests that future studies aimed at expressing and purifying functional Jak2 protein should either utilize a detergentfree puriWcation scheme or, when using detergent, Wrst perform the kinase incubation reaction prior to cell lysis. In summary, we have created a vaccinia virus-mediated expression system which allows for high level expression and single step puriWcation of recombinant Jak2 protein. More importantly, we have identiWed the conditions by which Jak2 protein should be prepared so that kinase activity can be maintained at maximum level. Collectively, these observations should help advance current studies that are aimed at understanding the biochemical and catalytic properties of Jak2. Acknowledgments We thank Drs. Pipsa Saharinen and Olli Silvennoinen for kindly providing the pCI-Neo-Jak2-WT-HA expression plasmid and Dr. M. Showkat Ali for the GST/Stat1 construct. The recombinant vaccinia virus clone vTF7-3 was graciously provided by Dr. Bernard Moss and was used in compliance with the Materials Transfer Agreement. We are grateful to Eric M. Sandberg and TiVany A. Wallace for critically reviewing the manuscript. This work was supported by a Biomedical Research Support Program for Medical Schools Award to the University of Florida College of Medicine by the Howard Hughes Medical Institute, an American Heart Association National Scientist Development Grant (#0130041N), and National Institutes of Health Awards K01DK60471 and R01-HL67277. References [1] E. Parganas, D. Wang, D. Stravopodis, D.J. Topham, J.C. Marine, S. Teglund, E.F. Vanin, S. Bodner, O.R. Colamonici, J.M. van Deursen, G. Grosveld, J.N. Ihle, Jak2 is essential for signaling through a variety of cytokine receptors, Cell 93 (1998) 385–395. [2] N. Meydan, T. Grunberger, H. Dadi, M. Shahar, E. Arpaia, Z. Lapidot, J.S. Leeder, M. Freedman, A. Cohen, A. Gazit, A. Levitzki, C.M. Roifman, Inhibition of acute lymphoblastic leukaemia by a Jak-2 inhibitor, Nature 379 (1996) 645–648. [3] J. Pan, K. Fukuda, H. Kodama, S. Makino, T. Takahashi, M. Sano, S. Hori, S. Ogawa, Role of angiotensin II in activation of the JAK/STAT pathway induced by acute pressure overload in the rat heart, Circ. Res. 81 (1997) 611–617.
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