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Blood, Vol. 91 No. 3 (February 1), 1998:
pp. 844-851
Cloning and Characterization of the Human Homolog of Mouse Jak2
By
Ilan Dalal,
Enrico Arpaia,
Harjit Dadi,
Shaila Kulkarni,
Jerami Squire, and
Chaim M. Roifman
From the Division of Immunology/Allergy, Department of Pediatrics,
and the Department of Pathology, University of Toronto and the The
Hospital for Sick Children, Toronto, Ontario, Canada.
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ABSTRACT |
Members of the Jak family play a critical role in signal
transduction mediated by cytokine and hormone receptors. In this study,
we report the cloning and characterization of human Jak2. The predicted
amino acid sequence shows 91% homology to the described murine Jak2,
but with a significant difference in the extreme C-terminal sequence.
Using the human cDNA as a probe, we localized the gene for human Jak2
to chromosome 9p23-24. Human Jak2 mRNA is highly expressed in the
spleen, lymph nodes, and peripheral blood lymphocytes (PBLs). A
polyclonal antibody raised against the unique C-terminus of human Jak2
was used to characterize Jak2 protein. Levels of Jak2 protein
expression increased significantly in mitogen- and anti-IgM-stimulated
B cells and to a lesser degree in activated T cells. In addition, high
levels of Jak2 protein were detected in pre-B leukemia cells.
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INTRODUCTION |
CYTOKINES CONTROL proliferation and
differentiation by binding to specific receptors expressed on
hematopoietic cells.1,2 Because cytokine receptors do not
possess a kinase domain, great effort has been made to identify the
cytoplasmic protein tyrosine kinases (PTKs) responsible for the rapid
increase in PTK activity seen upon ligand-receptor
interaction.3 A commonly used technique has involved
polymerase chain reaction (PCR) with degenerate oligodeoxynucleotide primers directed to conserved tyrosine kinase catalytic domain motifs.
Many new PTKs have been discovered by this method, including members of
the Jak family.4,5 It has been shown recently that in
addition to src PTKs, cytokine receptors activate one or more Jak
kinases. Cytokines using receptors consisting of a single chain, such
as erythropoietin, prolactin, and growth hormone, primarily activate
Jak2,6-8 whereas the interferons activate two members of
the Jak family.9-11 The receptors for interleukin-2 (IL-2),
IL-4, IL-7, IL-9, and IL-15 are functionally coupled to Jak1 and
Jak3,12,13 where the common  -chain (c) selectively recruits Jak3. The cytokines ciliary neurotropic factor,
leukemia-inhibiting factor, oncostatin M, and IL-6 comprise a unique
cytokine subfamily on the basis of their predicted structural
similarity and shared  signal-transducing receptor component (gp
130). This family of receptors is capable of recruiting different
members of the Jak family (Jak1, Jak2, and Tyk2) in different cell
types.14,15
The Jak kinase family is unusual in possessing two distinct kinase
domains.5,16 The kinase domain proximal to the carboxyl terminal contains all of the recognized essential PTK motifs and is
therefore likely to be catalytically active.17 However, the function of the second kinase domain remains unknown, as it lacks several residues believed to be essential for catalytic
activity.16,17 Members of the Jak family lack src homology
2 (SH2) and src homology 3 (SH3) domains,18 but they show
other homology regions of yet unknown significance.5,16
Jak3 expression appears limited to the myeloid and lymphoid
lineages,19,20 and it appears to increase in T cells and B
cells after mitogenic stimulation.19-21 In contrast, Tyk2,
Jak1, and Jak2 are believed to be widely expressed.4,5,22 However, for Jak2, experiments were performed with murine reagents and
the assumption was made that similar expression patterns hold for
humans.
Recently, we have shown that pre-B leukemia cells express elevated
levels of Jak2 that appears to be constitutively
activated.23 Inhibition of Jak2 activity with a specific
inhibitor resulted in cell death, suggesting a critical role for this
enzyme in leukemia cell growth.23 Indeed, a mutation in the
Jak homolog was found to cause leukemia in
Drosophila.24,25 To study the role of human Jak2 in
signaling, we cloned the human Jak2 cDNA. The sequence was found to be
highly homologous to murine Jak2, but with an alternative 13 amino
acids at the extreme carboxy terminus. Antibodies raised against this
unique sequence were used to analyze the expression of human Jak2 in
various cells and tissues. In lymphoid cells, the level of Jak2 protein
expression was found to be low in peripheral resting B and T cells.
However, there was a significant increase in the level of Jak2 protein in B cells upon ligation of the B-cell antigen receptor (BCR) and after
mitogenic stimulation with SAC. In T cells, the increase in Jak2 levels
following ligation of the T-cell receptor or activation with
phytohemagglutinin (PHA) was less significant.
Currently, controversy exists as to the exact chromosomal localization
of the human Jak2 gene. Using murine cDNA, Jak2 was localized to
chromosome 9p24.26 However, the murine Jak2 gene is
genetically linked to Fas on chromosome 19,16 which
corresponds to human chromosome 10q23-q24.16 Using the
human Jak2 cDNA as a probe, we localized the human Jak2 gene to
chromosome 9p23-24, confirming that the murine and human Jak2 genes are
indeed located on different chromosomes.
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MATERIALS AND METHODS |
Library screening.
A human thymus cDNA  gt11 library (Clontech, Palo Alto,
CA) was screened according to standard
protocols27 using the full-length murine Jak2 cDNA as a
probe. Plaques were transferred to ICN (Plainview, NY)
Biotrans nylon filters and screened by hybridization at 65°C in 5×
SSC, 5× Denhardt solution, and 0.1% SDS. The final wash was
performed at 55°C in 2× SSC and 0.1% SDS. Filters were
autoradiographed overnight using Kodak (Rochester, NY) XAR-5 x-ray
film. Phage DNA was prepared from positive plaques. cDNA
inserts were excised, subcloned, and sequenced.
Reverse transcriptase-PCR cloning.
For reverse transcriptase (RT)-PCR cloning, total
RNAs were extracted from relevant cell types according to Chomczynski
and Sacchi.28 The RNAs were reverse-transcribed using
standard protocols.27 The resulting cDNAs were used as
substrates for PCRs using Elongase (GIBCO-BRL, Gaithersburg,
MD) as the thermoresistant amplifying enzyme. The PCR
products were subcloned into a pUC19 vector and sequenced with
Sequenase (Amersham, Arlington Heights, IL).
To isolate the 5 end of the cDNA, total RNA from peripheral blood
lymphocytes (PBLs) was reverse-transcribed with oligo dT. A 5
degenerate oligonucleotide primer to the mouse sequence beginning at
the start codon [ATG GG(ACGT) ATG GC(ACGT) TG(CT) CT(ACGT) A] was
used with an antisense nondegenerate primer to the human sequence
obtained from the library Jak2 clones (nucleotides 1416 to 1394; GAA
GTT CTT CTT TGT CCC ACT G). The reaction cycle conditions were as
follows: 94°C (30 seconds), 50°C (30 seconds), and 68°C (90 seconds) for five cycles, followed by 35 cycles of 94°C (30 seconds),
62°C (30 seconds), and 68°C (90 seconds).
To isolate the 3 end of the cDNA, total RNA from PBLs was
reverse-transcribed using oligo (dT)12GC as a primer. We
designed a nondegenerate primer to the human Jak2 sequence from
nucleotide 3055 to 3085 (ATA TTC TGG TAT GCT CGA CAA TCA CTG ACA) and
an antisense nondegenerate primer to the murine Jak2 untranslated sequence (TTC TGC CTA GCT AGC ATC ATG ATA AGG ATG). The reaction cycle
conditions were as follows: five cycles at 94°C (30 seconds), 40°C
(30 seconds), and 68°C (60 seconds), followed by 35 cycles at 94°C
(30 seconds), 50°C (30 seconds), and 68°C (60 seconds).
To isolate the complete cDNA, total RNAs from thymocytes, PBLs, and G2
cells (pre-B leukemia cell line) were reverse-transcribed with oligo dT
primer. A 5 primer beginning at the start codon of human Jak2 (ATG GGG
ATG GCT TGC CTT ACG ATG ACA GAA) was used with an antisense primer
starting at the stop codon of human Jak2 (TCA TCC AGC CAT GTT ATC CCT
TAC TTG ATC). The PCR cycling conditions were 30 cycles at 94°C (30 seconds), 60°C (30 seconds), and 68°C (5 minutes).
Chromosomal localization.
Positional mapping of the Jak2 gene was performed by fluorescence in
situ hybridization (FISH)29 to normal human lymphocyte chromosomes counterstained with propidium iodide and
46-diamidin-2-phenylindole-dihydrochloride (DAPI). A 1.5-kb Jak2 cDNA
probe and a corresponding genomic probe obtained by screening a
P1-derived artificial chromosome (PAC) library were used. Both were
labeled with biotin and detected with avidin-fluorescein isothiocyanate
(FITC).29 Images of 20 well-spread metaphase preparations
were captured by a thermoelectrically cooled charge-coupled camera
(Photometrics, Tucson, AZ). Separate images of DAPI-banded chromosomes
and FITC-targeted chromosomes were obtained.30
Hybridization signals were acquired and merged using image-analysis
software, and pseudocolored blue (DAPI) and yellow (FITC) and overlaid
electronically.31
Northern blot analysis.
Membranes with 2 µg polyadenylated mRNA per lane from various human
tissues were purchased from Clontech and probed with a 400-bp cDNA
corresponding to the 3 end of the human Jak2. The DNA probes were
labeled with 32P by random priming (Boehringer, Mannheim,
Germany) to a specific activity of 2 × 108
cpm/µg DNA. Membranes were hybridized in ExpressHyb hybridization solution (Clontech) at 68°C for 1 hour, and washing was performed in
0.1× SSC and 0.1% SDS at 50°C twice for 40 minutes.
Membranes were exposed to Kodak BMR-1 x-ray film at  70°C for 4 days. Subsequently, membranes were probed with -actin to check RNA
loading.
Antibody preparation.
The rabbit polyclonal anti-human Jak2 antibody was raised against a
synthetic peptide, VLRVDQVRDNMAG, which is the unique C-terminus of
human Jak2. This antibody was found to immunoprecipitate a 130-kD
protein, which was recognized by three different commercially available
anti-mouse Jak2 antibodies (made by PharMingen [San Diego, CA], UBI
[Lake Placid, NY], and Santa-Cruz Biotech [Santa-Cruz, CA]).
Because the anti-human Jak2 antibody we prepared was unable to
recognize denaturated Jak2 protein in Western blots, we used anti-mouse
Jak2 antibody (PharMingen) for immunoblotting after immunoprecipitation
with our antibody.
Cell preparation and stimulation.
Peripheral blood mononuclear cells were obtained from healthy
volunteers. Mononuclear cells were isolated by Ficoll-Hypaque gradient
centrifugation. Adherent cells were removed by adherence to plastic
dishes for 60 minutes at 37°C. Separation of T cells from B cells was
performed by Ficoll-Hypaque centrifugation of cells rosetting with
neuroaminidase-treated sheep erythrocytes.32 The purity of
the B- and T-cell population was assessed by flow cytometry. The B-cell
population was always 60% to 70% CD19+ with less than 5%
CD3+ cells. The T-cell population was always 95% to 99%
CD3+ with less than 2% CD19+ cells. T cells
were stimulated with 10 µg/mL PHA or 20 µg/mL anti-CD3. B cells
were stimulated with 2 mg/mL staphylococcus protein A cowan (SAC) or 20 µg/mL anti-IgM antibody for the indicated times at 37°C in RPMI
with 10% fetal calf serum.
Immunoprecipitation and immunoblotting.
Immunoprecipitation was performed as described before.33
Briefly, immunoprecipitates using the antibody to human Jak2 were prepared from lysates of 2 × 107 T or B cells in 1 mL
lysis buffer containing 20 mmol/L Tris (pH 7.5), 150 mmol/L NaCl, 1%
Triton X-100, and 1 mmol/L Na3VO4 buffer with
antisera to human Jak2, and subjected to 6% SDS-PAGE. Where different
cell types were used, the total amount of protein in the cell lysate
was measured by the Lowry method and 750 µg was used for each
immunoprecipitation. After transfer to supported nitrocellulose
membrane (Amersham), immunoblotting was performed with anti-murine Jak2
antibody from PharMingen and then detected by ECL (Amersham).
In vitro transcription and translation.
Full-length Jak3 and Jak2 cDNAs were inserted into the pcDNA3 vector
(Invitrogen, San Diego, CA) downstream from a T7
promoter. Approximately 1 µg luciferase control, Jak3, and Jak2 cDNAs
in pcDNA3 were added to a combined transcription and translation reticulocyte lysate system (Promega, Madison, WI) in the
presence of T7 polymerase. The protein products were labeled by
inclusion of (35S) methionine in the reaction. Aliquots (5 µL) of each product were assayed on 6% SDS-PAGE, and the remaining
45 µL lysate from each reaction was diluted in 1 mL 1% Triton X-100
lysis buffer (as before) and immunoprecipitation was performed with
antisera to human Jak2. The precipitates were washed with lysis buffer and separated on a 6% SDS-PAGE gel, followed by electrotransfer to
nitrocellulose membrane and visualization by autoradiography.
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RESULTS |
Cloning of human Jak2 cDNA.
The murine Jak2 cDNA was used to screen a human thymus cDNA library to
isolate the human Jak2 sequence. This screening yielded a series of
overlapping fragments combining to form a 1.8-kb open reading frame
(ORF) that was 89% homologous to the kinase domain of the translated
murine Jak2 cDNA (from nucleotide 1398 to nucleotide 3207). Noting the
extensive homology between our isolated clones and the murine Jak2
cDNA, we used RT-PCR with degenerate primers to murine Jak2 to complete
the cloning of the 5 and 3 ends of the human cDNA. Figure
1 summarizes the cloning strategy used. To
obtain the 5 end of human Jak2, we used a degenerate oligonucleotide primer to the murine start codon region and an antisense nondegenerate primer to human Jak2 in a PCR on human PBL first-strand cDNA. This
produced a fragment of approximately 1.4 kb extending from the ATG and
overlapping the 5 end of our original 1.8-kb human Jak2 ORF. To obtain
the 3 end of Jak2, RNA extracted from human PBLs and
reverse-transcribed with oligo(dT)12 GC was used as a substrate for PCR. We designed a nondegenerate primer on the 1.8-kb ORF
and an antisense nondegenerate primer to murine Jak2 untranslated sequence. This amplification generated a sequence of approximately 450 bp containing a stop codon and overlapping the 3 end of the 1.8-kb
human Jak2 ORF. The RT-PCR-generated fragments and the 1.8-kb ORF
combined to form a 3.4-kb ORF encoding a polypeptide of 1,134 amino
acids with a predicted molecular weight of 130 kD.
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| Fig 1.
Strategy used to clone human Jak2. A combination of
library screening using the mouse Jak2 cDNA as a probe and RT-PCR was used to obtain the full-length cDNA of human Jak2. The screening yielded a series of overlapping fragments combining to form a 1.8-kb
ORF (nucleotides 1398 to 3207). A RT-PCR with primers to the published
murine sequence and to the 1.8-kb ORF obtained from the library
screening was used to complete the cloning of the 5 (nucleotides 1 to
1416) and 3 (nucleotides 3055 to 3503) ends of the human Jak2. A stop
codon was found in position 3402, as indicated by the arrow.
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This complete sequence demonstrated 87% homology with murine Jak2
cDNA. Although the predicted amino acid sequence of the human cDNA
showed an overall homology of 91% with murine Jak2, a significant
difference was noted at the extreme carboxy terminus. An insertion of
one base in position 3361 causes a frameshift relative to the murine
sequence that creates an alternate last 13 amino acids and with the
addition of a further three amino acids. Using oligodeoxynucleotides to
the 5 and 3 ends of the human sequence, we cloned (by RT-PCR) a
full-length Jak2 cDNA from both human thymocytes and human G2 cells
(pre-B leukemia cell line) and found them to be identical to the
previously isolated human Jak2 sequence.
Figure 2 shows a comparison between the
predicted amino acid sequence of human Jak1, Jak3, and Tyk2 and the
human Jak2 sequence. The alignment shows that the human Jak2 exhibits
the seven domains of homology (JH1 to JH7) that have been shown to
exist among other members of the Jak family, confirming that this novel
human cDNA is indeed a member of the human Jak family. The homology
between the predicted amino acid sequence of our clone and that of
murine Jak2 indicates that our clone represents the human homolog of the murine Jak2 gene.
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| Fig 2.
Comparison of the predicted amino acid sequence of
members of the human Jak family kinases. Alignment was performed with
the PILEUP program (Genetics Computer Group, Madison, WI). Identical residues are shown on a black background, and related residues are
shaded. Gaps were introduced for optimal alignment and are indicated by
hyphens. Boundaries of the Jak homology (JH) domains are denoted by
arrows.
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Chromosomal localization.
A 1.5-kb DNA fragment corresponding to the 5 end of the human Jak2
cDNA was used as a probe to localize the chromosomal position of the
human Jak2 gene. This probe was chosen in a region of relatively low
homology to other members of the Jak family. This probe was used in
both FISH analysis and in obtaining a corresponding genomic PAC probe.
Images of 20 well-spread metaphase preparations were captured by a
thermoelectrically cooled charge-coupled camera, and separate images of
DAPI-banded chromosomes and FITC-targeted chromosomes were obtained.
Both probes mapped to chromosome 9p23-24 in greater than 95% of the
cells.
The human Jak2 gene was previously localized on chromosome 9p24 using
the murine Jak2 cDNA as a probe.26 However, the fact that
the murine Jak2 gene itself was localized to chromosome 19, which
corresponds to human 10q23-24, had cast doubt on the accuracy of the
human localization. Our data obtained with a human sequence probe
confirm localization of the human Jak2 gene to chromosome 9 (Fig
3).
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| Fig 3.
Chromosomal localization of the human Jak2 gene.
Positional mapping was performed by FISH to normal human lymphocyte
metaphase chromosomes. Both a 1.5-kb Jak2 cDNA probe and a
corresponding genomic probe obtained by screening a PAC library showed
positive signals in >95% of the cells on both chromatids of each
homolog of chromosome 9p23-24, as indicated by the arrows.
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Human Jak2 mRNA expression in various tissues.
To assess human Jak2 tissue distribution, we used a 400-bp fragment of
the human Jak2 cDNA to probe a series of membranes containing
polyA+ mRNA from different tissues. This Northern blot
analysis revealed the presence of three transcripts of approximately
7.0, 5.4, and 4.8 kb. These transcripts were found to be expressed
ubiquitously (Fig 4). Densitometric
scanning analysis corrected by reference to the -actin signal showed
that expression of the 7-kb mRNA is low (RNA to actin ratio, 0.01 to
0.05) in the thymus, prostate, testis, small intestine, colon,
appendix, lymph node, and bone marrow, whereas the level of this
transcript in the spleen and ovary is relatively high (RNA to actin
ratio, 0.20 and 0.12, respectively). Similarly, densitometric scanning
of the 5.4- and 4.8-kb transcripts showed the highest expression in the
testis, spleen, lymph node, and PBLs (RNA to actin ratio, 1.26, 0.63, 0.64, and 0.28, respectively). The thymus and bone marrow showed a RNA
to actin ratio of 0.25 and 0.16, respectively. The results suggest
there is a higher expression of Jak2 in organs such as the spleen,
PBLs, and lymph nodes that contain mature lymphocytes, whereas the
thymus and bone marrow, which contain predominately immature
lymphocytes, show lower levels of Jak2. Neither the structure nor the
significance of the three human Jak2 transcripts are currently known.
They may represent the products of alternative splicing or,
alternatively, the use of different polyadenylation sites.
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| Fig 4.
Northern blot analysis of human Jak2 expression.
Commercial membranes containing 2 µg Poly A+ mRNA per
lane of various human tissues (Clontech) were probed with a 400-bp cDNA
corresponding to the 3 end of human Jak2. Hybridization and washing of
the membranes were performed according to the manufacturer's
specifications (Clontech). -Actin was used for comparison of RNA
loading.
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Jak2 protein expression in hematopoietic cells.
To examine Jak2 protein expression, we generated a polyclonal
antibody against a peptide consisting of the 13 amino acids (VLRVDQVRDNMAG) located at the carboxy terminal of the Jak2
protein. This portion of human Jak2 does not exhibit homology with any other member of the human Jak family (Fig 2). The antibody was found to
immunoprecipitate a 130-kD protein, which was recognized by
commercially available anti-mouse Jak2 antisera from PharMingen (peptide 758), UBI (peptide 758), and Santa-Cruz Biotech (peptide 758).
Because the anti-human Jak2 antibody was unable to recognize the
denaturated Jak2 protein in Western blots, we used anti-mouse Jak2
(PharMingen) to detect the immunoprecipitated protein. To further
confirm the specificity of the anti-human Jak2 antibody, we tested its
ability to recognize an in vitro synthesized Jak2 protein. Jak3 and
Jak2 cDNAs were added to a combined transcription and translation
reticulocyte lysate system, and the products were labeled with
(35S) methionine. Aliquots (5 µL) of each product were
assayed on SDS-PAGE gel and visualized by autoradiography to examine
translation. The reactions yielded two major proteins of 120 and 130 kD
corresponding to Jak3 and Jak2 (lanes 1 and 2, Fig
5), respectively. Anti-human Jak2 antibody
was added to the remaining (45 µL) lysate from each reaction, and the
immunoprecipitate was resolved on SDS-PAGE followed by transfer to
nitrocellulose membrane and visualization by autoradiography. Only the
Jak2 protein was immunoprecipitated by the anti-human Jak2 antibody
(Fig 5, lane 4) with no cross-reactivity to Jak3 (Fig 5, lane 3). The
immunoprecipitated Jak2 could then be recognized by immunoblotting with
the anti-mouse Jak2 antibody (PharMingen) (Fig 5, lane 6).
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| Fig 5.
In vitro translation and detection of Jak2.
Approximately 1 µg Jak3 and Jak2 cDNAs were translated in a
combined transcription and translation reticulocyte lysate system
(Invitrogen) including (35S) methionine. Aliquots of each
product were resolved on SDS-PAGE gel, followed by transfer to
nitrocellulose and visualization by autoradiography. The reactions
yielded proteins of 120 and 130 kD, corresponding to Jak3 and Jak2,
respectively (lanes 1 and 2). The remaining lysate of each reaction was
precleared with Protein A-Sepharose CL-4B and subsequently
immunoprecipitated with anti-human Jak2 antibody, resolved by SDS-PAGE
gel, electrotransferred to nitrocellulose membrane, and visualized by
autoradiography. Only Jak2 was immunoprecipitated by the anti-human
Jak2 antibody with no cross-reactivity to Jak3 (lanes 3 and 4). Only
the immunoprecipitated Jak2 in lane 4 was detected by ECL (lane 6)
after the same membrane was probed with an anti-mouse Jak2 antibody
(PharMingen).
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Using the anti-human Jak2 antibody for immunoprecipitation, we
demonstrated low levels of Jak2 expression in resting B and T cells
separated from PBLs, and even lower levels in thymocytes. As
expected, Jak2 expression in pre-B ALL cell lines was
high,23 serving here as a positive control, while the
monocytic leukemia cell line U937 showed virtually undetectable levels
of Jak2, providing a negative control (Fig
6). We showed here that the anti-human Jak2
antibody that was produced against the unique C terminus of this
enzyme specifically recognizes human Jak2.
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| Fig 6.
Expression of human Jak2 protein. Immunoprecipitates were
prepared from lysates containing 750 µg protein from each type of cell with antisera to human Jak2 and subjected to 6% SDS-PAGE. After
transfer to nitrocellulose membrane, immunoblotting was performed with
anti-Jak2 antibody (PharMingen) and detection was made by ECL.
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Expression of Jak2 is increased in stimulated lymphocytes.
Northern blot analysis of hematopoietic and lymphoid organs suggested
relatively high expression of Jak2 in tissues that contain activated
lymphocytes. We have therefore examined whether human Jak2 is inducible
in stimulated PBLs. There was a significant increase in Jak2 expression
in B lymphocytes stimulated with anti-IgM (Fig
7A). The time course of anti-IgM-induced
B-cell activation showed (by densitometric scanning of the
autoradiographs from four separate experiments) that the maximum
expression of Jak2, an increase of fourfold, was reached within 1 hour
(Fig 7A) with no significant change over the course of 48 hours. These
results are in contrast to previous observations by Saouaf et
al,38 who showed a threefold to fourfold increase in the
level of Jak2 within 5 to 10 minutes of B-cell antigen receptor (BCR)
ligation in murine WEHI 231 cells but reported no expression of Jak2 in mature lymphoma B-cell lines. These differences may be explained by a
possible aberrant expression of Jak2 in malignant cell lines or
interspecies differences between mouse and human tissues. B cells
stimulated for 12 hours with SAC showed a sixfold to sevenfold increase
in the level of Jak2, with no significant change in this level over the
course of 48 hours (Fig 7B). Although there is an increased expression
of Jak2 protein, this does not appear to be tyrosine-phosphorylated in
either T or B cells (data not shown). The results suggest that the
increased expression of Jak2 protein in stimulated B cells might
reflect the use of this kinase by growth and differentiation factor
receptors needed for the terminal maturation of these cells. In
contrast, the time course of Jak2 expression in peripheral T cells
stimulated with anti-CD3 or PHA showed a modest twofold increase after
24 hours, with no further change apparent up to 48 hours (Fig 7C and
D).
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| Fig 7.
Induction of Jak2 expression in activated peripheral
blood B and T lymphocytes. Human B or T cells (2 × 107
cells per lane) were incubated for the indicated times with (A) 20 µg/mL anti-IgM antibody, (B) 2 mg/mL SAC, (C) 20 µg/mL anti-CD3 antibody, or (D) 10 µg/mL PHA, respectively. Cell lysates were prepared, followed by immunoprecipitation with anti-human Jak2 and
immunoblotting with anti-mouse Jak2 antibody (PharMingen).
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DISCUSSION |
We have cloned the human Jak2 cDNA by a combination of library
screening and RT-PCR. The human Jak2 cDNA demonstrates an overall homology of 87% with murine Jak2, and the predicted amino acid sequences are 91% identical. As previously observed for the three other known Jak kinases, human Jak2 contains kinase and kinase-like domains (JH1 and JH2, respectively) at its C terminus along with five
other conserved domains (JH3 to JH7). This close homology to the murine
sequence suggests that we have isolated the human Jak2 gene. Using the
human cDNA as a probe, we localized the human Jak2 to chromosome
9p23-24. Although previous data using the murine Jak2 as a probe had
localized the human Jak2 on chromosome 9p24,26 the accuracy
of this location had been questioned, as the mouse Jak2 gene is
genetically linked to Fas on chromosome 19, which corresponds to human
chromosome 10q23-24.1.16 Our data confirm that the human
Jak2 gene is indeed located on chromosome 9.
Northern blot analysis of murine Jak2 had previously demonstrated the
presence of two transcripts of 5.0 and 5.3 kb in a wide variety of
tissues, with the relative level of the two transcripts differing in
some tissues.22 Northern blot analysis of human tissues
reveals three polyA+ transcripts of 7.0, 5.4, and 4.8 kb in
a variety of tissues. At present, it is not possible to explain the
biologic significance of the three transcripts. In the absence of
further data, it is not clear whether the transcripts are the result of
alternative splicing or the use of different promoters or different
polyadenylation sites. Analysis of immune relevant tissues showed that
Jak2 appears to be primarily expressed in the spleen, lymph nodes, and
PBLs, but to a lesser degree in the thymus and bone marrow. This is in
sharp contrast to results obtained in mouse tissues, where expression
of murine Jak2 mRNA was found to be high in the thymus and much lower
in the spleen.22 Our findings suggest that expression of
human Jak2 is likely to be more important in mature rather than
immature lymphocytes and in lymphoid organs that contain activated
lymphocytes.
To analyze Jak2 protein expression in resting and activated
lymphocytes, we have generated a polyclonal antibody against the C
terminus of Jak2, an area that does not exhibit homology to the other
known members of the human Jak family. We examined Jak2 protein
expression in peripheral resting T and B cells before and after
stimulation with anti-CD3 or PHA and with anti-IgM or SAC,
respectively. Using this antibody, we found a striking difference in
the pattern of Jak2 expression in mature T and B cells. In both resting
T and B cells, Jak2 protein appears to be expressed at low levels.
However, whereas activated T cells show a small increase of Jak2
expression, activated B cells exhibit a dramatic increase of Jak2
expression. The increase of Jak2 is probably required to accommodate
signal transduction through growth and differentiation factor
receptors. Indeed, the cytokine receptors for IL-2, IL-4, IL-5, IL-6,
and IL-10 act at various stages in the growth and differentiation of
mature B cells and require Jak kinases for signal
transduction.12,14,16,34-36 Although most PTKs are not
inducible by mitogens, recently yet another Jak kinase, Jak3, was found
to be markedly upregulated following stimulation of B cells with SAC or
anti-CD40 antibodies,21 and also in PHA-stimulated T
cells.19,20
We confirm here that Jak2 is highly expressed in human pre-B leukemia
cells (Fig 6). We have previously shown that inhibition of Jak2
activity by a specific tyrosine kinase blocker selectively killed
leukemic cells, suggesting that Jak2 activity is essential for the
survival of these cells.23 Similar suggestions for the contribution of Jak kinases to transformation were obtained in other
systems such as T cells infected with HTLV-I37 and also in
hematopoietic malignancy (fly leukemia) in
Drosophila.24,25
We report here the cloning and characterization of human Jak2. Although
the predicted amino acid sequence of the human cDNA showed 91% overall
homology with the murine Jak2, a significant difference in the extreme
C-terminal sequence was noted. The gene was localized to chromosome
9p23-24. Northern blot analysis revealed that Jak2 mRNA was highly
expressed in the spleen and lymph nodes, which contain activated
lymphocytes. In complete agreement with these results, Jak2 protein
levels were markedly increased in mitogen-stimulated B lymphocytes and
to a lesser degree in T cells, highlighting the need for an enhanced
presence of Jak2 to accommodate the lymphokine receptor signal
transduction required for terminal differentiation of these
cells.
| |
FOOTNOTES |
Submitted May 1, 1997;
accepted September 25, 1997.
Supported by the Medical Research Council of Canada and the National
Cancer Institute of Canada.
Address reprint requests to Chaim M. Roifman, MD, Division
of Immunology/Allergy, The Hospital for Sick Children, 555 University Ave, Toronto, Ontario, M5G 1X8.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely
to indicate this fact.
| |
ACKNOWLEDGMENT |
We are indebted to Nigel Sharfe for helpful discussion and for
reviewing the manuscript, and to Bill Fox for the illustrations. The
GenBank accession no. for human Jak2 cDNA is AF001362.
| |
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Abstract
Introduction
Abstract