1Department of Physiology and Functional Genomics, University of Florida College of Medicine, Gainesville, Florida 32610; and 2Department of Medicine, Division of Endocrinology and Metabolism, University of Alabama at Birmingham, Birmingham, Alabma 35294
Submitted 11 February 2004 ; accepted in final form 1 June 2004
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ABSTRACT |
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janus kinase 2
Previous studies (26, 28) demonstrated that mice lacking a functional JAK2 allele die during early embryonic development. These knockout mice are deficient in mandatory cytokine signaling as well as severely anemic, demonstrating a complete lack of erythropoiesis. The lethal effects associated with JAK2 knockout mice indicate the important physiological role JAK2 has in the development of animals in general and definitive erythropoiesis in particular. In addition to being activated by about 20 different cytokine receptors, JAK2 can also be activated by numerous tyrosine kinase growth factor and seven transmembrane-spanning receptors (2, 13, 17, 21, 22, 29, 30, 34).
Using gene-profiling technology, we sought to identify and characterize JAK2-dependent genes that are differentially expressed as a result of the presence, or absence, of JAK2. Specifically, Affymetrix microarray gene chips were used in these experiments. The chips contain probe sequences for 12,000 fully sequenced human genes. Analysis of two replicates demonstrated that 621 genes were shown to have a greater than twofold change in expression as a function of basally expressed JAK2, with 31 of these genes showing a sevenfold change or greater. Surprisingly, this differential expression pattern did not require the addition of exogenous ligand to activate a cell surface receptor but merely a basal level of JAK2 kinase function within the cell, as measured by a combination of Northern blot analysis, RT-PCR, and luciferase reporter assays. Subsequent ligand treatment of cells caused a further increase in JAK2-mediated gene transcription, but this increase was roughly of the same magnitude as that seen when basal JAK2 kinase function was reconstituted in these cells. These JAK2-dependent genes represent a wide range of ontological functions including transcription factors, signaling molecules, and cell surface receptors. Additionally, of the 621 genes identified in this study, 56 have already been shown to be cytokine responsive, thereby suggesting that these genes are true targets of JAK2 action. As such, this work demonstrates for the first time that, in addition to being a key mediator of ligand-activated gene transcription, JAK2 is also a critical mediator of basal level gene expression. Additionally, the large numbers of genes implicated in this study to be dependent on JAK2 for their transcriptional regulation indicate the critical and encompassing role that JAK2 has in transcriptional processes within the cell.
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METHODS |
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Western blot analysis.
Western blot analysis was performed exactly as detailed previously (35). Briefly, equal amounts of 2A/Zeo and
2A/JAK2 whole cell protein lysates were prepared from serum-starved cells and subsequently Western blotted with polyclonal anti-JAK2 antibody (Upstate Biotechnology) in 5% milk-TBST to determine JAK2 expression levels. Membranes were subsequently stripped and reprobed with polyclonal anti-STAT1 antibody (Santa Cruz Biotechnology) to confirm equal protein loading of all samples. For JAK2 tyrosine phosphorylation levels, cells were serum starved for 20 h, and protein whole cell lysates were prepared the next morning. The lysates were subsequently immunoprecipitated with monoclonal anti-phosphotyrosine antibody (clone PY20; BD Transduction Laboratories) and Western blotted with polyclonal anti-JAK2 antibody (Upstate Biotechnology).
Preparation of total and poly A+ mRNA. Cells were serum starved for 20 h, and total RNA was then isolated using the acid guanidine thiocyanate/phenol/chloroform method of extraction (4). For each of the two conditions, three confluent 100-mm culture dishes of cells were lysed, and extracted RNA was then pooled together to avoid artifacting that was unique to any one individual plate.
Poly A+ mRNA was isolated from both the 2A/Zeo and
2A/JAK2 cells using the Amersham Pharmacia Quick Prep mRNA purification kit. Three plates for each condition were again pooled to reduce the possibility of any artifact. Total and poly A+ mRNA were then used for Affymetrix analysis and Northern blot analysis as described below.
Probe preparation and Affymetrix chip hybridization. cRNA probes were prepared for hybridization to microarrays following the manufacturer's instructions (Affymetrix GeneChip Expresssion Analysis Manual). Briefly, double-stranded DNA was prepared from 10 µg of total RNA isolated from both cell lines using the Superscript double-stranded cDNA synthesis kit (Invitrogen). Newly synthesized double-stranded DNA was subsequently cleaned with phase lock gels-phenol/chloroform extraction. Five microliters of double-stranded DNA was then biotin-labeled following the Enzo Bioarray high-yield RNA transcript labeling kit protocol (Affymetrix). Biotinylated cRNA was subsequently cleaned using a Qiagen RNeasy column and quantitated. Twenty micrograms of unadjusted cRNA was then fragmented and hybridized to Affymetrix Test3 chips to verify the quality of each preparation. Samples having similar metrix values were then hybridized to U95A gene chips at the University of Florida MicroArray Core Laboratory.
Microarray data analysis. The data was analyzed using the Affymetrix Software Package, Microarray Suite version 4.0. Probe intensities for both cellular conditions were compared and reported in both tabular and graphical formats. The data was deposited in the Gene Expression Omnibus repository under accession no. GSM16418.
Northern blot analysis. Northern blot analysis was performed as previously described (36, 37). Briefly, 25 µg of total or 4 µg of poly A+ mRNA was separated on a 1% agarose-6% formaldehyde-containing gel. RNA samples were transferred onto nylon membranes, which were then hybridized to 32P-labeled cDNA probes. The cDNAs encoding for Pak1 (39), 4-1BBL (41), USA-CyP (16), and EphB6 (25) have been described.
Quantitative RT-PCR.
The two-step quantitative RT-PCR method was also used to confirm the differential expression results generated by the microarray experiments. Specifically, total RNA was extracted from either the 2A or the RASM-derived cell lines and subsequently reverse transcribed using the SuperScript II RNase H Reverse Transcriptase Kit (Invitrogen). Primers were designed for each gene using the Primer3 program (http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi/). PCR reactions were prepared using the SYBR Green PCR Core kit (Applied Biosystems) and performed on the GeneAmp 5700 Sequence Detector machine (Applied Biosystems). The 18s primers were used as a standard internal reference, and analyses were accomplished by calculating the 2
Ct values for each gene (14, 20).
Luciferase assay.
Cells were transfected with a luciferase reporter construct containing four tandem repeats of the -activation sequence element, upstream of a minimal tyrosine kinase promoter, in 10 µl of Lipofectin (Invitrogen). The cells were subsequently serum starved for 20 h and then treated as indicated. Luciferase activity was measured from detergent extracts in the presence of ATP and luciferin using the Reporter Lysis Buffer System (Promega) and a luminometer (Monolight model 3010). Luciferase values were recorded as relative light units per microgram of protein.
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RESULTS |
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Collectively, the results in Fig. 1 demonstrate that, although the 2A/Zeo cell line completely lacks JAK2 protein expression, the
2A/JAK2 cell line has readily detectable levels of this protein. Furthermore, the expressed JAK2 protein shows normal basal level tyrosine phosphorylation.
Microarray analysis demonstrates that JAK2 mediates the expression of many diverse genes.
We next wanted to determine whether the basal level expression of JAK2 in a cell changed the gene expression profile of that cell compared with the non-JAK2-expressing controls. To do this, total RNA was harvested from both cell lines and then prepared for Affymetrix microarray analysis as described in METHODS. The Affymetrix U95A GeneChip was used as the differential expression platform. This chip contains the probe sequences representing 12,000 fully sequenced human genes. The expression signals generated from the hybridization of probes from both cell lines were then compared and analyzed. Figure 2 shows a graphical illustration of the mRNA expression levels from this experiment (experiment 1). Each dot on the plot represents one of the 12,000 different genes on the chip. Genes falling outside the two parallel lines had a greater than twofold change in gene expression as a result of the presence of JAK2. Genes falling above the two parallel lines had increased gene expression, whereas those falling below the two parallel lines had decreased gene expression. The data indicated that basal level expression of JAK2 in a cell altered the expression of 1,251 genes by at least twofold.
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The results gathered in Fig. 2 were further analyzed using Venn diagram analysis. This analysis allows for the identification of genes that were present in both experiments. The results demonstrated that 621 genes were differentially expressed in both experiments. All of these genes had at least a twofold change in gene expression as a function of expressed JAK2. These 621 genes were further analyzed to distinguish upregulated genes from downregulated genes (Fig. 3A). A total of 474 genes were found to be upregulated in both experiments, whereas 147 genes were found to be downregulated. The fold changes for these genes ranged from 2- to 78-fold. Not surprisingly, the majority of genes found to be present in only one of the two experiments had induction numbers falling close to the twofold cutoff. In this case, they were picked up in one experiment with a value that was at twofold or higher, but not in the other experiment because the value was just under the twofold cutoff threshold.
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For our initial analysis, we started with the list of 621 genes and shortened it to include only those genes that were differentially expressed by at least sevenfold. Again, Venn diagram analysis was performed to identify those genes that had at least a sevenfold change in gene expression in both experiments (Fig. 3B). The results show that 76 genes in experiment 1 had at least a sevenfold change in expression, whereas 53 genes in experiment 2 were found to have at least a sevenfold change. Of these, 31 were common to both groups. Table 1 lists these 31 genes. For the genes found to be present in only one of the two experiments, the majority had induction numbers falling close to the sevenfold cutoff. As such, they were detected in one experiment with a value that was sevenfold or greater but not detected in the second experiment because the value was just below the sevenfold cutoff threshold. Overall, however, there was a strong concordance between the genes on both lists. Interestingly, this list of genes was found to encompass diverse categories of cellular function, including transcription factors, cell cycle control genes, cell surface receptors, and intermediate signaling molecules. As such, the data indicated that JAK2 was strongly regulating an important but diverse set of genes.
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To further validate the differential expression data generated by the microarray experiments, quantitative RT-PCR was also employed. Six separate genes were analyzed via quantitative RT-PCR. Graphs illustrating the derived fold changes between the 2A/Zeo and
2A/JAK2 cell lines are shown (Fig. 5). For the EphB6 gene, quantitative RT-PCR found the level of differential expression to be
12-fold greater in the JAK2-expressing cells (Fig. 5A). This was in close agreement with both the Affymetrix prediction and the Northern blot analysis shown in Fig. 4C. For the protein tyrosine kinase gene termed FBK III16, Affymetrix predicted that the JAK2-expressing cells would have 12-fold less mRNA compared with the non-JAK2-expressing controls. Quantitative RT-PCR found the difference to be
17-fold less (Fig. 5B). For the 13h9 gene, Affymetrix predicted a 78-fold decrease in mRNA levels in the JAK2-expressing cells. Quantitative RT-PCR actually found the level to be
10-fold less in these cells (Fig. 5C). For the trypsinogen IV-B gene, Affymetrix predicted a 15-fold increase in mRNA levels in the JAK2-expressing cells. Quantitative RT-PCR found the level to be
17-fold higher (Fig. 5D). For the microsomal glutathione S-transferase III gene, the microarray studies predicted a 34-fold increase in the mRNA levels in the JAK2-expressing cells. Quantitative RT-PCR found the level to be
10-fold higher (Fig. 5E). Finally, for the granulocyte macrophage colony-stimulating factor gene, Affymetrix predicted a 17-fold increase in mRNA levels in the JAK2-expressing cells compared with the non-JAK2-expressing controls. Quantitative RT-PCR actually found the level to be
107-fold higher in the JAK2-expressing cells (Fig. 5F).
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Suppression of endogenous JAK2 kinase activity via overexpression of a JAK2-dominant negative allele similarly inhibits JAK2-dependent gene expression.
One interpretation of the data in Figs. 4 and 5 is that basal level JAK2 tyrosine kinase activity within a cell, independent of exogenous ligand addition, can alter subsequent cellular gene expression. However, other interpretations might be that the results are due to artifact inherent to the 2A-derived clones or that the effect might be unique only to
2A-derived cells. To eliminate these alternate possibilities, we utilized RASM cells that stably express a JAK2-dominant negative cDNA (RASM DN). Expression of the dominant negative protein blocks function of wild-type JAK2 normally found in these cells. The full characterization of these cells has been reported (36). In short, JAK2-dependent signaling in the dominant negative-expressing cells is reduced by
90% compared with the control cells. The control cells are RASM cells that express only endogenous JAK2. Thus these cells allow for a determination of JAK2-dependent gene expression via a mechanism that is independent of the JAK2 null mutation.
Here, both sets of cells were serum starved for 48 h, and then total RNA was harvested. Quantitative RT-PCR was subsequently performed on several of the genes shown in Figs. 4 and 5. The results were consistent with both the Affymetrix-derived data in Fig. 2 and the validations in Figs. 4 and 5. Specifically, USA-CyP and 4-1BBL gene expression was 10-fold higher in the RASM WT cells compared with the RASM DN cells (Fig. 6, A and B, respectively). 13h9 gene expression was approximately eightfold less in the RASM WT cells compared with the RASM DN cells (Fig. 6C). Finally, trypsinogen IV-B gene expression was approximately sevenfold higher in the RASM WT cells compared with the RASM DN cells (Fig. 6D).
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JAK2 is a critical mediator of both basal level and ligand-induced gene transcription.
The data in Fig. 6 suggest that JAK2 is acting as a key mediator of gene expression, and this is occurring independent of exogenous ligand addition. This is a novel concept in that JAK2 has classically been viewed as a mediator of ligand-induced gene expression. We therefore hypothesized that JAK2 can act as a critical mediator of both basal level and ligand-induced gene transcription. To test this, we chose to measure the ability of angiotensin II to mediate mRNA gene expression as a function of both expressed JAK2 protein and exogenous ligand addition. Numerous independent laboratories, including our own, have shown that angiotensin II is a potent activator of JAK2, both in vitro and in vivo (1, 7, 12, 2224, 33, 36, 38). We recently created 2A-derived cell lines that stably express either the angiotensin II receptor alone (AT1) or the angiotensin II receptor along with JAK2 (AT1 + JAK2) via the stable integration of cDNA expression plasmids (33). In short, the AT1 cell line expresses the angiotensin II type AT1 on a background that is devoid of JAK2. However, the AT1 + JAK2 cell line expresses the AT1 receptor with similar affinity and abundance as the AT1 cell line but also expresses wild-type JAK2 protein. Thus these cells allow for a determination of the role of JAK2 in gene expression under both the basal and ligand-activated states.
To characterize both the basal and ligand-induced tyrosine phosphorylation levels of JAK2, both sets of cells were either left untreated or treated for 5 min with 100 nM angiotensin II. Equal amounts of whole cell lysate from each condition were then immunoprecipitated with anti-phosphotyrosine antibody and subsequently Western blotted with anti-JAK2 antibody (Fig. 7A). Because the AT1 cells lack JAK2, angiotensin II treatment failed to increase the tyrosine phosphorylation levels of this protein (lane 2 vs. 1). However, in the AT1 + JAK2 cells, JAK2 was found to be tyrosine phosphorylated before angiotensin II treatment (lane 3), and ligand treatment further increased its tyrosine phosphorylation levels (lane 4). Thus the data in Fig. 7A suggest that these cells appear to be suitable vehicles for studying gene expression that is both JAK2- and ligand-dependent.
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In Fig. 7B, although ligand treatment of the JAK2-expressing cells did increase EphB6 mRNA levels over untreated cells (lane 4 vs. lane 3), perhaps what is most striking is the large increase in EphB6 mRNA levels that is seen when JAK2 is simply expressed in the cell and not treated with ligand (lane 3 vs. lane 1). This suggests that JAK2 might perhaps be influencing gene expression independent of exogenous ligand addition. To determine whether this effect could be conferred onto a heterologous JAK2-responsive promoter, we transfected these same AT1 and AT1 + JAK2 cells with a luciferase reporter construct containing four tandem repeats of the JAK2-responsive, -activation sequence element upstream of a minimal tyrosine kinase promoter. The cells were subsequently serum starved for 20 h, treated with 100 nM angiotensin II for the indicated times, and then measured for luciferase activity (Fig. 7C). In the cells lacking JAK2, there was some basal level luciferase activity, and this increased modestly with the addition of ligand (lane 2 vs. lane 1). However, in the JAK2-expressing cells, there was a significant increase in luciferase activity, and this was independent of ligand addition (lane 3 vs. lane 1). Finally, receptor activation via the addition of exogenous ligand further increased luciferase activity compared with the unstimulated controls (lane 4 vs. lane 3). Clearly, however, of the four conditions, the largest relative fold increase in luciferase activity was seen in lane 3, where JAK2 expression significantly increased luciferase activity independent of exogenous ligand addition.
To demonstrate that this observation is not an artifact unique to the 2A/AT1 receptor-expressing cell lines, we transfected the same luciferase reporter construct into
2A cells that stably expressed either the GHR alone or the GHR + JAK2. The creation and characterization of these cells has been independently described (15). In short, both cell lines express the GHR at similar affinity and abundance, but only the second cell line expresses JAK2. In the absence of growth hormone, JAK2 displays low-level, basal tyrosine phosphorylation. On treatment with growth hormone, however, there is a marked increase in JAK2 tyrosine phosphorylation levels.
Here, both sets of cells were transfected with the luciferase reporter construct and then treated with ligand for the indicated times (Fig. 7D). In the 2A cells expressing only the GHR, ligand treatment failed to elicit any marked change in luciferase activity (lane 2 vs. lane 1). However, in the JAK2-expressing cells, there was a significant increase in luciferase activity, and this was once again independent of exogenous ligand addition; the level of luciferase activity in the cells expressing JAK2 was
2.5-fold higher than that in the equivalent cells lacking JAK2 (lane 3 vs. lane 1). Finally, receptor activation via the addition of exogenous ligand further increased luciferase activity compared with the unstimulated controls. In this case, addition of growth hormone increased luciferase activity approximately threefold above the untreated cells (lane 4 vs. lane 3). Thus the data demonstrate that the magnitude by which JAK2 increases ligand-dependent gene transcription (
3-fold) is nearly equivalent to the magnitude by which JAK2 increases ligand-independent gene transcription (
2.5-fold). As such, these data help strengthen the argument that JAK2 may act as a mediator of both ligand-independent and ligand-dependent gene transcription.
In summary, the data in Fig. 7 demonstrate that basal-level expression of JAK2 can greatly influence gene expression and that this is independent of exogenous ligand treatment. However, once ligand is added and JAK2 is activated to a higher catalytic state, JAK2 can further influence gene expression. Collectively, the data suggest that JAK2 may therefore act as a key mediator of gene expression in both the basal and ligand-activated states.
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DISCUSSION |
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This work is significant for several reasons. First, in the realm of cellular transcription, genes can be expressed at either basal levels or under activated conditions, such as when a ligand binds its receptor to increase the expression of a specific downstream target gene. JAK2 has long been regarded as a key mediator of this ligand-activated state of transcription and has never thought to have been important in basal transcriptional regulation. This paper shows for the first time that, when JAK2 is expressed in a cell at basal-level conditions, it appears to play a central role in cellular transcriptional regulation that is independent of exogenous ligand addition.
Second, a classification of these differentially regulated genes was done in an attempt to discover prominent classes of JAK2 signaling targets. Uncovering functional classes of genes could potentially lead to predictions about genomic targets of JAK2. Interestingly, however, no prominent class of genes appeared evident. The classification revealed a large assortment of genes encoding many diverse proteins such as transcription factors, intermediate signaling molecules, and cell surface receptors. The data suggest that JAK2 shows no single prominent function at the basal level but rather maintains a global influence within the cell.
Third, the JAK2 knockout mouse dies during development, therefore indicating that this tyrosine kinase is required for survival (26, 28). These same studies showed that JAK2 is required for proper signaling through a variety of cytokine receptors. Subsequent studies have also demonstrated that JAK2 is a critical mediator of tyrosine kinase growth factor and G protein-coupled receptor signaling (1, 7, 23, 24, 27, 29, 38, 40). However, the downstream target genes of JAK2 tyrosine kinase remain largely unknown. Here, we identified 621 genes that have at least a twofold change in gene expression as a function of expressed JAK2. As such, additional downstream target genes of JAK2 may now be known.
As mentioned previously, the major focus of this study did not include the genes falling within the differential signal expression range of two- to sevenfold. This does not suggest that these genes are not biologically important. To the contrary, genes having a twofold change in gene expression have previously been shown to have important biological consequences (5, 31). However, given the vast number of genes that were identified in this study, we narrowed our focus and chose to study genes having larger fold changes.
Interestingly, JAK2 has been regarded as an activator of ligand-dependent gene transcription. However, this study revealed that nearly one-quarter of all JAK2-dependent genes were downregulated. One possible explanation for this is that JAK2 is having an indirect effect on these gene promoters via the activation of transcriptional repressor genes. Once expressed, the repressors would subsequently bind other promoters and in turn reduce gene transcription. Alternatively, recent studies have shown that the JAK-STAT pathway itself is capable of directly inhibiting expression of specific gene promoters. Specifically, recent work (11) has shown that the -globin gene promoter is inhibited by the JAK-STAT signaling pathway in general, and STAT3
in particular. As such, further experiments are required to determine which of these scenarios might be happening in the
2A-derived cells.
As indicated above, a major finding of this work is that JAK2 may function as a critical mediator of ligand-independent gene transcription. An important question, however, is whether JAK2 is already in an activated state before exogenous ligand addition. For several reasons, we believe the answer is no. First, the level of JAK2 that is expressed in the 2A-derived cells used in these studies is at a level that is similar to cells that endogenously express JAK2, such as Jurkat cells. As such, this would tend to minimize JAK2 autophosphorylation in the absence of exogenously added ligand. Second, the cells were washed extensively with phosphate-buffered saline and serum starved before use. This made the cells quiescent and in turn minimized the tyrosine kinase activity of proteins such as JAK2 before any ligand treatment. Third, the addition of exogenous ligand subsequently activated JAK2, suggesting that JAK2 was not fully activated before ligand addition. Fourth, the phenomena of JAK2 mediating ligand-independent gene transcription was observed in multiple independent cell lines (
2A/AT1,
2A/GHR, and RASM), therefore suggesting that the effect is not due to clonal artifact. Fifth, in the case of the RASM-derived cells, when endogenous JAK2 tyrosine kinase activity was reduced via the expression of the dominant negative JAK2 allele, there was a subsequent alteration in gene expression that correlated with the microarray predictions. This demonstrates that when endogenously expressed JAK2 (i.e., nontransfected) tyrosine kinase function is reduced from its basal state, there is a corresponding change at the level of JAK2-dependent gene transcription. And sixth, a recent paper by Chatti et al. (3) demonstrated that, kinetically, the tyrosine kinase function of JAK2 exists in at least two independent states; namely, a basal state and a ligand-activated state. Specifically, they generated an activation loop mutant of JAK2 by changing the conserved tyrosines at positions 1,007 and 1,008 to phenylalanine. Although this JAK2 mutant was unable to propagate cytokine-dependent signaling, it was nonetheless able to bind ATP and autophosphorylate, albeit less efficiently than wild-type protein. As such, they concluded that JAK2 can exist in at least two kinetically distinct states of activity: a high-activity catalytic state and a low-efficiency basal catalytic state. However, what remained uncertain was whether this low-efficiency basal state had any biological consequence. Our data here suggest that the basal state of JAK2, characterized biochemically as being capable of binding ATP and tyrosine autophosphorylating, may in fact be an important mediator of cellular gene transcription.
In conclusion, this study showed that expression of JAK2 can alter the transcriptional regulation of 621 genes in 2A-derived cells. At least 56 of these genes have been classified as being cytokine responsive. These numbers are indicative of the critical role that JAK2 tyrosine kinase has within a cell and suggest that JAK2 plays a key role in basal, as well as ligand-activated, cellular gene transcription. As such, these studies suggest that JAK2 can significantly regulate gene expression outside of the classical, ligand-activated signaling paradigm.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
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2. Buggy JJ. Binding of alpha-melanocyte-stimulating hormone to its G-protein-coupled receptor on B-lymphocytes activates the Jak/STAT pathway. Biochem J 331: 211216, 1998.[ISI][Medline]
3. Chatti K, Farrar WL, and Duhé RJ. Tyrosine phosphorylation of the Janus kinase 2 activation loop is essential for a high-activity catalytic state but dispensable for a basal catalytic state. Biochem J 43: 42724283, 2004.[CrossRef]
4. Chomczynski P and Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156159, 1987.[CrossRef][ISI][Medline]
5. Cook SA, Matsui T, Li L, and Rosenzweig A. Transcriptional effects of chronic Akt activation in the heart. J Biol Chem 277: 2252822533, 2002.
6. Darnell JE Jr, Kerr IM, and Stark GR. Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 264: 14151421, 1994.[ISI][Medline]
7. Doan TN, Ali MS, and Bernstein KE. Tyrosine kinase activation by the angiotensin II receptor in the absence of calcium signaling. J Biol Chem 276: 2095420958, 2001.
8. Duhe RJ and Farrar WL. Structural and mechanistic aspects of Janus kinases: how the two-faced god wields a double-edged sword. J Interferon Cytokine Res 18: 115, 1998.[ISI][Medline]
9. Fan XD, Stark GR, and Bloom BR. Molecular cloning of a gene selectively induced by gamma interferon from human macrophage cell line U937. Mol Cell Biol 9: 19221928, 1989.[ISI][Medline]
10. Feng J, Witthuhn BA, Matsuda T, Kohlhuber F, Kerr IM, and Ihle JN. Activation of JAK2 catalytic activity requires phosphorylation of Y1007 in the kinase activation loop. Mol Cell Biol 17: 24972501, 1997.[Abstract]
11. Foley H, Ofori-Acquah SF, Yoshimura A, Critz S, Baliga S, and Pace BS. Stat3 beta inhibits gamma-globin gene expression in erythroid cells. J Biol Chem 277: 1621116219, 2002.
12. Frank GD, Saito S, Motley ED, Sasaki T, Ohba M, Kuroki T, Inagami T, and Eguchi S. Requirement of Ca2+ and PKC-delta for Janus kinase 2 activation by angiotensin II: involvement of PYK2. Mol Endocrinol 16: 367377, 2002.
13. Gadina M, Hilton D, Johnston JA, Morinobu A, Lighvani A, Zhou YJ, Visconti R, and O'Shea JJ. Signaling by type I and II cytokine receptors: ten years after. Curr Opin Immunol 13: 363373, 2001.[CrossRef][ISI][Medline]
14. Giulietti A, Overbergh L, Valckx D, Decallonne B, Bouillon R, and Mathieu C. An overview of real-time quantitative PCR: applications to quantify cytokine gene expression. Methods 25: 386401, 2001.[CrossRef][ISI][Medline]
15. He K, Wang X, Jiang J, Guan R, Bernstein KE, Sayeski PP, and Frank SJ. Janus kinase 2 determinants for growth hormone receptor association, surface assembly, and signaling. Mol Endocrinol 17: 22112227, 2003.
16. Horowitz DS, Lee EJ, Mabon SA, and Misteli T. A cyclophilin functions in pre-mRNA splicing. EMBO J 21: 470480, 2002.
17. Ju H, Venema VJ, Liang H, Harris MB, Zou R, and Venema RC. Bradykinin activates the Janus-activated kinase/signal transducers and activators of transcription (JAK/STAT) pathway in vascular endothelial cells: localization of JAK/STAT signalling proteins in plasmalemmal caveolae. Biochem J 351: 257264, 2000.[CrossRef][ISI][Medline]
18. Kohlhuber F, Rogers NC, Watling D, Feng J, Guschin D, Briscoe J, Witthuhn BA, Kotenko SV, Pestka S, Stark GR, Ihle JN, and Kerr IM. A JAK1/JAK2 chimera can sustain alpha and gamma interferon responses. Mol Cell Biol 17: 695706, 1997.[Abstract]
19. Liu B, Liao J, Rao X, Kushner SA, Chung CD, Chang DD, and Shuai K. Inhibition of Stat1-mediated gene activation by PIAS1. Proc Natl Acad Sci USA 95: 1062610631, 1998.
20. Livak KJ and Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2Ct method. Methods 25: 402408, 2001.[CrossRef][ISI][Medline]
21. Lukashova V, Chen Z, Duhe RJ, Rola-Pleszczynski M, and Stankova J. Janus kinase 2 activation by the platelet-activating factor receptor (PAFR): roles of Tyk2 and PAFR C terminus. J Immunol 171: 37943800, 2003.
22. Marrero MB, Schieffer B, Paxton WG, Heerdt L, Berk BC, Delafontaine P, and Bernstein KE. Direct stimulation of Jak/STAT pathway by the angiotensin II AT1 receptor. Nature 375: 247250, 1995.[CrossRef][ISI][Medline]
23. Mascareno E, Dhar M, and Siddiqui MA. Signal transduction and activator of transcription (STAT) protein-dependent activation of angiotensinogen promoter: a cellular signal for hypertrophy in cardiac muscle. Proc Natl Acad Sci USA 95: 55905594, 1998.
24. Mascareno E and Siddiqui MA. The role of Jak/STAT signaling in heart tissue renin-angiotensin system. Mol Cell Biochem 212: 171175, 2000.[CrossRef][ISI][Medline]
25. Matsuoka H, Iwata N, Ito M, Shimoyama M, Nagata A, Chihara K, Takai S, and Matsui T. Expression of a kinase-defective Eph-like receptor in the normal human brain. Biochem Biophys Res Commun 235: 487492, 1997.[CrossRef][ISI][Medline]
26. Neubauer H, Cumano A, Muller M, Wu H, Huffstadt U, and Pfeffer K. JAK2 deficiency defines an essential developmental checkpoint in definitive hematopoiesis. Cell 93: 397409, 1998.[ISI][Medline]
27. Pan J, Fukuda K, Kodama H, Makino S, Takahashi T, Sano M, Hori S, and Ogawa S. Role of angiotensin II in activation of the JAK/STAT pathway induced by acute pressure overload in the rat heart. Circ Res 81: 611617, 1997.
28. Parganas E, Wang D, Stravopodis D, Topham DJ, Marine JC, Teglund S, Vanin EF, Bodner S, Colamonici OR, Van Deursen JM, Grosveld G, and Ihle JN. JAK2 is essential for signaling through a variety of cytokine receptors. Cell 93: 385395, 1998.[ISI][Medline]
29. Park OK, Schaefer TS, and Nathans D. In vitro activation of Stat3 by epidermal growth factor receptor kinase. Proc Natl Acad Sci USA 93: 1370413708, 1996.
30. Peeler TC, Conrad KM, and Baker KM. Endothelin stimulates sis-inducing factor-like DNA binding activity in CHO-K1 cells expressing ETA receptors. Biochem Biophys Res Commun 221: 6266, 1996.[CrossRef][ISI][Medline]
31. Rome S, Clement K, Rabasa-Lhoret R, Loizon E, Poitou C, Barsh GS, Riou JP, and Laville H. Microarray profiling of human skeletal muscle reveals that insulin regulates approximately 800 genes during a hyperinsulinemic clamp. J Biol Chem 278: 1806318068, 2003.
32. Rozovskaia T, Ravid-Amir O, Tillib S, Getz G, Feinstein E, Agrawal H, Nagler A, Rappaport EF, Issaeva I, Matsuo Y, Kees UR, Lapidot T, Lo Coco F, Foa R, Mazo A, Nakamura T, Croce CM, Cimino G, Domany E, and Canaani E. Expression profiles of acute lymphoblastic and myeloblastic leukemias with ALL-1 rearrangements. Proc Natl Acad Sci USA 100: 78537858, 2003.
33. Sandberg EM, Ma X, VonDerLinden D, Godeny MD, and Sayeski PP. JAK2 tyrosine kinase mediates angiotensin II-dependent inactivation of ERK2 via induction of mitogen-activated protein kinase phosphatase 1. J Biol Chem 279: 19561957, 2004.
34. Sasaguri T, Teruya H, Ishida A, Abumiya T, and Ogata J. Linkage between 1 adrenergic receptor and the Jak/STAT signaling pathway in vascular smooth muscle cells. Biochem Biophys Res Commun 268: 2530, 2000.[CrossRef][ISI][Medline]
35. Sayeski PP, Ali MA, Harp JB, Marrero MB, and Bernstein KE. Phosphorylation of p130Cas by angiotensin II is dependent on c-Src, intracellular Ca2+, and protein kinase C. Circ Res 82: 12791288, 1998.
36. Sayeski PP, Ali MS, Safavi A, Lyles M, Kim SO, Frank SJ, and Bernstein KE. A catalytically active JAK2 is required for the angiotensin II-dependent activation of Fyn. J Biol Chem 274: 3313133142, 1999.
37. Sayeski PP and Kudlow JE. Glucose metabolism to glucosamine is necessary for glucose stimulation of transforming growth factor-alpha gene transcription. J Biol Chem 271: 1523715243, 1996.
38. Seki Y, Kai H, Shibata R, Nagata T, Yasukawa H, Yoshimura A, and Imaizumi T. Role of the JAK/STAT pathway in rat carotid artery remodeling after vascular injury. Circ Res 87: 1218, 2000.
39. Sells MA, Knaus UG, Bagrodia S, Ambrose DM, Bokoch GM, and Chernoff J. Human p21-activated kinase (Pak1) regulates actin organization in mammalian cells. Curr Biol 7: 202210, 1997.[ISI][Medline]
40. Von DerLinden D, Ma X, Sandberg EM, Gernert K, Bernstein KE, and Sayeski PP. Mutation of glutamic acid residue 1046 abolishes JAK2 tyrosine kinase activity. Mol Cell Biochem 241: 8794, 2002.[CrossRef][ISI][Medline]
41. Wen T, Bukczynski J, and Watts TH. 41BB ligand-mediated costimulation of human T cells induces CD4 and CD8 T cell expansion, cytokine production, and the development of cytolytic effector function. J Immunol 168: 48974906, 2002.
42. Zhuang H, Patel SV, He TC, Sonsteby SK, Niu Z, and Wojchowski DM. Dominant negative effects of a carboxy-truncated JAK2 mutant on Epo-induced proliferation and JAK2 activation. J Biol Chem 269: 2141121414, 1994.