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(Received for publication, May
10, 1995; and in revised form, July 20, 1995) From the
Signal transduction of cytokine receptors is mediated by the JAK
family of tyrosine kinases. Recently, the kinase partners for the
interleukin (IL)-2 receptor have been identified as JAK1 and JAK3. In
this study, we report the identification of splice variants that may
modulate JAK3 signaling. Three splice variants were isolated from
different mRNA sources: breast (B), spleen (S), and activated monocytes
(M). Sequence analysis revealed that the splice variants contain
identical NH
The JAKs ( Recently,
we and others have cloned a new member of the JAK family, JAK3, and
found that it is the IL-2 and IL-4 receptor-associated tyrosine
kinase(9, 10, 11) . This 120-kDa mouse JAK3
is highly homologous to the other JAK kinases, binds to the IL-2
receptor, and undergoes tyrosine phosphorylation upon IL-2 stimulation.
Thus, the JAK3 kinase is predicted to be a signaling molecule central
to immune function. We now report the cloning and characterization
of the human JAK3. We find that JAK3 exists as three splice variants
resulting in proteins with different carboxyl termini. One variant was
found to lack intrinsic tyrosine kinase activity, and may function to
modulate JAK3 downstream signaling. Although Jak3 transcripts are
mainly found in normal hematopoietic tissues, its expression is present
in epithelial cell lines and primary cancers. Thus, JAK3 may have a
role in epithelial cell biology in addition to its importance for
lymphoid function.
Primary human breast cancers and their matched
normal tissue counterparts were obtained through the Tissue Procurement
Facility of the UNC-SPORE in breast cancer. Tissues were homogenized
and disrupted in lysis buffer as described above. 50 µg of the
total cell lysate were electrophoresed and subjected to Western blot
analysis using the For IL-2 stimulation studies intended for
immunoprecipitation and immunoblot analysis, stimulation was done as
described previously (9, 10) for 10 min at 37 °C
and blot was stripped according to the ECL manufacturer's
instruction.
Figure 6:
Correlation of JAK3 isoforms and their in vitro kinase activty. A, structure of mouse Jak3
and chimeric Jak3 isoforms expression plasmids.
Figure 1:
Sequence of the
human Jak3 variants. A, complete nucleotide sequence and
predicted amino acid sequence of the human Jak3 variants. The deduced
amino acid sequence is shown below with the nucleotide
sequence. The ATG found in the most 5` end of the open reading frame
was assigned as an initiation site for translation. The nucleotide and
amino acid differences from the published L-Jak cDNA sequences are
indicated(11) . Splice variants diverge at amino acid position
1070. > represents the stop codon and two AATAAA polyadenylation
signals are underlined. B, comparison of the
COOH-terminal sequence motifs for subdomains X and XI among all known
JAK3 kinases with EGF receptor. B-HJAK3 represents human
B-form Jak3, M-HJAK3 represents human M-form Jak3, S-HJAK3 represents human S-form Jak3, and EGFR represents
epidermal growth factor receptor. Sequences used to make the
comparisons were derived from the following sources:
L-JAK(11) , mouse JAK3 (1MJAK3, (9) ), rat JAK3 (RJAK3, (19) ), second mouse JAK3 (2MJAK3, (20) ), and
EGFR(18) . Boxed residues represent the consensus
sequences in subdomains X and XI.
Figure 2:
Expression of human Jak3 transcript.
Northern blot analysis of Jak3 mRNA expression in: A, normal
human adult tissues and, B, human transformed cell lines. The
two Northern blots were obtained from Clontech Labs. Each lane contains
2 µg of poly(A)
In
transformed cell lines, however, Northern blot analysis (Fig. 2B) revealed that Jak3 is expressed more widely
in lymphoblastic leukemia MOLT-4, Burkitt's lymphoma Raji,
colorectal adenocarcinoma SW480, lung carcinoma A549, and melanoma G361
cell lines. This confirms our earlier expression data that Jak3 is
expressed in epithelial cell lines. In addition, the Jak3 transcripts
in epithelial cancer cell lines are smaller than those in lymphoid
tissues and cell lines (Fig. 2, A and B).
These data suggest that complex splicing of the Jak3 gene is involved
which may have significance in epithelial cell biology. All RNA samples
were normalized by either hybridization with a
To confirm that these different forms represent splice site
variants, we isolated a genomic clone of Jak3. This clone showed the
splice donor to be GCTGAG that encodes for amino acids 1068 and 1069
(alanine and glutamine) and that the B-form is a read-through
transcript. (
Figure 3:
RT-PCR analysis of Jak3 splice variant
expression in cell lines. The ethidium bromide-stained agarose gels
show Jak3 variants and Jak3 kinase domains amplified with a common
5`-Jak3 primer, but with 3`-primers specific for each splice variant:
3`-bJak3 primer specific for B-form Jak3 splice variant was used to
detect Jak3B transcripts; 3`-sJak3 primer specific for S-form Jak3
splice variant was used to detect Jak3S transcripts; and 3`-mJak3
primer specific for M-form Jak3 splice variant was used to detect Jak3M
transcripts. Whereas, the common 3`-Jak3 primer was used for Jak3
kinase domain to detect the expression of all three Jak3 isoforms. The
fragment sizes are 404 bp (B-form), 588 bp (S-form), 388 bp (M-form),
and 240 bp (common Jak3 kinase domain). A 201-bp
Figure 4:
Immunoblot analysis of human B-form JAK3. A, to test antibody specificity, total cell lysates from K562,
Jurkat, BT-474, and T-47D cell lines were triple loaded and resolved on
a 8% SDS-PAGE. They were electroblotted onto PVDF membrane and
immunoblotted with preimmune serum, UNC36, anti-peptide antibody to
B-form JAK3 (amino acids 1,077-1,094), or UNC36 in the presence
of the immunogenic peptide (10 µg/ml) to which the antiserum was
raised. JAK3B is seen as a single 125-kDa band. B, expression
of JAK3B in paired-samples of breast tumors. 50 µg of total cell
lysates from 5 primary human breast cancers (T) and their
match normal tissue (N) counterparts were used. Equal amounts
of BT-474 and T-47D total cell lysates were also run in parallel for
the purpose of serving as control samples. In three of five samples,
JAK3B is overexpressed in tumor tissues.
To confirm that UNC36 recognized a JAK
protein, the
Figure 5:
Immunoprecipitation and immunoblot
analysis of JAK3. A, human JAK3 protein was immunoprecipitated
from BT-474 total cell lysates with different sources of JAK3 antisera,
resolved by 8% SDS-PAGE, electroblotted, and probed with
anti-phosphotyrosine (G410) antibody. The results indicate that UNC36
recognizes a 125-kDa JAK3 protein which is tyrosine phosphorylated. B, JAK3B is linked to IL-2R. Total cell lysates from untreated
HUT-78 T-cells were immunoprecipitated with normal preimmune rabbit
serum or a monoclonal anti-IL-2R antibody, resolved on 8% SDS-PAGE,
electroblotted, and probed with UNC36 (specific for the B-form JAK3).
The result indicates that JAK3B is co-immunoprecipitated with the
IL-2R.
Using the various JAK3-specific
antibodies, we noticed that the JAK3S protein migrated more quickly
than the JAK3B isoform: JAK3S at 116 kDa and JAK3B at 125 kDa despite
the calculated molecular mass of the S-form (125 kDa) being slightly
larger than the B-form (121 kDa). These differences permitted easy
identification of the two JAK3 isoforms (Fig. 7, A-C, and see below). Although there is no clear
explanation for this discrepancy in the predicted and the actual
molecular weights, we suspect that differences in phosphorylation
status may account for the mobility shift.
Figure 7:
Kinase deficient B-form JAK3 is not
tyrosyl phosphorylated after IL-2 stimulation in HUT-78 cells. A, lysates from HUT-78 cells either unstimulated or stimulated
with IL-2 for 10 min were used for immunoprecipitation with the
indicated antisera. The immunoprecipitates were then resolved by 7.5%
SDS-PAGE, electroblotted, and probed with anti-phosphotyrosine (G410)
antibody. JAK3S is tyrosine phosphorylated after IL-2 stimulation,
whereas immunoprecipitable JAK3B is not phosphorylated on tyrosine. B, the blot was stripped and reprobed with anti-peptide
antibody to S-form JAK3 to examine the levels of S-form JAK3 protein. C, the blot was then reprobed with anti-peptide antibody to
B-form JAK3 (UNC 36). B and C demonstrate that JAK3S
coprecipitates with JAK3B.
We extended our
expression analysis to the protein level by Western blot and found
JAK3B to be expressed in SKBR-3, HeLa, SW480, A549, HUT-78, and MOLT-4
cell lines (data not shown). Because of its significant expression in
breast cancer cell lines, we asked whether JAK3B could be detected in
primary breast cancers. In five matched pairs of normal breast and
breast cancers, we found augmented JAK3B expression in three tumors
with absent expression in the normal breast epithelium (Fig. 4B). In two tumors, the level of JAK3B expression
was equivalent to that found in BT-474 known to be a high expressor of
JAK3B. Thus, the JAK3B isoform of JAK3 is expressed at significant
levels in both epithelial cell lines and in primary breast carcinomas.
An intriguing finding, however, is that when JAK3S was
immunoprecipitated using a COOH-terminal antibody, we found JAK3B in
the immune complex. Conversely, when JAK3B is imunopreciptiated, JAK3S
co-precipitates (Fig. 7, B and C). Since the
antibodies used are against the divergent COOH-terminal sequences and
do not appear to cross-react on Western blots, we surmise that JAK3B
and JAK3S reside in the same protein complex. By contrast, however,
we found that the immunoprecipitated JAK3B was noticeably tyrosine
phosphorylated in the breast cancer cell lines BT-474 as determined by
anti-phosphotyrosine Western blot analysis (Fig. 5A, lane
3). BT-474 cells do not express JAK3S as assessed by RT-PCR and
Western blot (Fig. 3, and data not shown), putatively lack IL-2
receptors, but express JAK3M by RT-PCR. This suggests that JAK3B may be
phosphorylated by another kinase, potentially JAK3M, which may be
activated by yet an unknown receptor. Cytokines function through receptors of the cytokine receptor
superfamily which include receptors for the interferons, IL-3, IL-6,
erythropoietin, growth hormone, prolactin, granulocyte
colony-stimulating factor, and granulocyte-macrophage
colony-stimulating factor. These ligand-receptor associations are
further related by their use of a novel subfamily of cytoplasmic
tyrosine kinases, called the Janus kinases (JAKs) to affect their
intracellular signaling. Structurally, the JAK kinases are unique
because of the presence of two kinase domains, an
NH During
this effort, we uncovered three splice variants that result in distinct
COOH termini all starting at amino acid 1070. The S-, M-, and B-forms
appear to be expressed in most cell lines as assessed by RT-PCR but at
differing ratios from tissue to tissue. The S-form is the Jak3 sequence
previously published as the signaling component of the IL-2 receptor in
lymphoid cells, and in our analysis appears to be expressed
predominantly in hematopoietic cell lines. The B- and M-forms, however,
have a wider expression profile, being detected in cell lines derived
from hematopoietic, and epithelial tissues (Fig. 3). Some cell
lines express only the B- and M-forms (e.g. BT-474), or only
the B-form Jak3 transcripts (e.g. SKBR-3). Predicted
protein sequences of the COOH-terminal kinase domains of the JAK3
splice isoforms reveal that they represent alterations at subdomain XI
of the JAK3 kinase. The B-form JAK3 is predicted to lack a recognizable
subdomain XI. A protein sequence data base search indicated that the
amino acid sequence at the COOH terminus of the B-form JAK3 shows no
homology with any other known proteins. We have identified the genetic
basis for the generation of the Jak3 splice variants by analyzing Jak3
genomic clones; whereas the S-form and M-form Jak3 are the result of
differential splicing using GCTGAG, encoding for amino acids 1068 and
1069 (alanine and glutamine), as the splice donor, the B-form Jak3 does
not use this splice donor and generates a read-through transcript. Although transcribed, it is possible that these splice variants
would not be translated. To prove that a functional protein product is
generated, we raised antibodies specific to the COOH terminus of the
most divergent of the splice isoforms, JAK3B, and found that in cell
lines known to express the Jak3B transcript, a 125-kDa protein could be
detected by Western blot analysis which can be competed by excess
antigenic peptides (Fig. 4A). Immunoprecipitations
using JAK3 antibodies raised against non-COOH-terminal residues
recognized the JAK3B-specific isoform (Fig. 5A),
suggesting that the 125-kDa immunoreactive band is indeed a JAK3
protein. In addition, JAK3B co-precipitates with the IL-2 receptor in
unstimulated cells much like the previously described associations
between JAK3S/L-JAK and the IL-2 receptor Previous studies have demonstrated the importance of the protein
tyrosine kinase COOH terminus in substrate selection and in the control
of kinase
activity(24, 25, 26, 27, 28) .
COOH-terminal sequences can act intramolecularly to regulate intrinsic
kinase activity. 3`-Truncations of c-src sequences through
retroviral transduction or sequestration of the src COOH-terminal peptides by polyoma middle T result in kinase
activation and induction of src's transforming
potential. In addition, the COOH-terminal tail domain of the EGF
receptor is the site of substrate recruitment. Phospholipase C- The B-form of JAK3 is of
particular interest because its sequence shows an absence of a
recognizable kinase subdomain XI consensus. Earlier structure-function
analyses of the EGF receptor showed that COOH-terminal deletions to
amino acid residue 944 completely abolished its kinase
activity(21, 22) . The EGF receptor subdomain XI has
been described to reside between amino acids 920 and 951 with its
subdomain XI consensus motif at residues 927-934. Since JAK3B
represents a more drastic change in this region, we suspected that
JAK3B would lack kinase activity. Our results confirm this hypothesis
in that neither immunoprecipitated JAK3B nor a recombinant JAK3B
expressed in COS-7 cells show any autokinase activity. Furthermore, the
JAK3B protein that co-precipitates with the ligand-stimulated IL-2
receptor is not significantly tyrosine phosphorylated as compared to
the JAK3S isoform (Fig. 7, A and C). These
data raise the possibility that JAK3B may function as a transdominant
negative in JAK3 signaling. The current model of IL-2 receptor and JAK
interactions shows the IL-2R Alternatively, the
three JAK3 isoforms may function to enrich the complexity of IL-2
signaling by recruiting different intracellular proteins and
substrates. The function of a kinase-deficient dimerization partner is
still unclear, but has precedence in the EGF receptor family of
receptor tyrosine kinases. c-erbB3 is homologous to
c-erbB4 but is unique among the kinases in that its wild-type
sequence in the catalytic domain predicts for an enzymatically
deficient tyrosine kinase. Confirming this prediction is the finding
that although baculovirus expressed p180 Despite the evidence for the absence of kinase activity in the JAK3B
isoform, several intriguing possibilities are raised by our results.
Immunoprecipitations of JAK3B from HUT-78 cell lysates (which contain
all JAK3 isoforms) show no tyrosine phosphorylation even after IL-2
treatment; however, in the breast cancer cell line, BT-474 (which
expresses only the B- and M-forms), the JAK3B appears constitutively
tyrosine phosphorylated. This suggests that other kinases or other
receptors may phosphorylate JAK3B. In addition, our co-precipitation
data shows that the S- and B-forms of JAK3 can potentially form a
ternary complex with the IL-2 receptor in HUT-78 cells (Fig. 5B and Fig. 7, B and C).
If verified, this finding changes the current model of IL-2 signaling
that describes only binary JAK1 and JAK3S interactions. In this
paper, we describe the cloning of human Jak3 cDNAs. Sequence comparison
between our human Jak3S sequence and the recently published human Jak3
cDNA (previously called L-Jak) revealed a number of nucleotide
discrepancies resulting in eight non-conservative amino acid changes.
Since both cDNA sequences were derived from multiple cDNA libraries and
from transformed (HUT-78, YT, Jurkat, and SKBR-3 cell lines) and
non-transformed cells (spleen, activated monocyte, and
phytohemagglutinin-activated T cells), these discrepancies may be due
to somatic mutations, polymorphisms, and mutational artifacts
engendered during the reverse transcriptase step, or a combination of
all three. Until the significance of these sequence variants are
elucidated, biological studies transfecting Jak3 cDNAs should be
interpreted with caution, and the source of the cDNA should be taken
into account.
The sequences of
human Jak3 isoforms have been deposited in GenBank, accession numbers
U31601[GenBank],
U31602[GenBank], and U31317 [GenBank]for hJak3B, hJak3M, and hJak3S isoforms,
respectively.
Volume 270,
Number 42,
Issue of October 20, 1995 pp. 25028-25036
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-terminal regions but diverge at the COOH
termini. Analyses of expression of the JAK3 splice isoforms by reverse
transcriptase-polymerase chain reaction on a panel of cell lines show
splice preferences in different cell lines: the S-form is more commonly
seen in hematopoietic lines, whereas the B- and M-forms are detected in
cells both of hematopoietic and epithelial origins. Antibodies raised
against peptides to the B-form splice variant confirmed that the
125-kDa JAK3B protein product is found abundantly in hematopoietic as
well as epithelial cells, including primary breast cancers. The lack of
subdomain XI in the tyrosine kinase core of the B-form JAK3 protein
suggests that it is a defective kinase. This is supported by the lack
of detected autokinase activity of the B-form JAK3. Intriguingly, both
the S and B splice isoforms of JAK3 appear to co-immunoprecipitate with
the IL-2 receptor from HUT-78 cell lysates. This and the presence of
multiple COOH-terminal splice variants co-expressed in the same cells
suggest that the JAK3 splice isoforms are functional in JAK3 signaling
and may enrich the complexity of the intracellular responses functional
in IL-2 or cytokine signaling.
)are cytoplasmic tyrosine kinases with a
unique structure consisting of two kinase domains lacking SH2 or SH3
motifs(1, 2, 3, 4, 5) .
Recently, members of this kinase family have been implicated in the
signaling of a number of cytokine receptor superfamily members. For
instance, JAK2 associates with the erythropoietin receptor(6) ,
JAK1 interacts with TYK2 to signal in the interferon-
pathway, and
JAK1 associates with JAK2 to signal in the interferon-
pathway(7, 8) . These scenarios suggest that following
ligand binding, receptor dimerization or oligomerization brings two JAK
molecules into close proximity, resulting in their activation by
tyrosine phosphorylation. Thus, JAK kinases may associate in a
homologous manner with another JAK of the same kind, or heterologously
with other family members. Once activated, the JAKs phosphorylate their
associated receptors and cellular substrates, including a novel class
of transcriptional activators called STAT proteins which transduce JAK
signals after translocation to the nucleus. There appear to be specific
STAT proteins that interact with the receptors that bind different JAK
kinases. Taken together, the combinatorial interactions between the
cytokine receptors and members of the JAK family appear to enhance the
complexity of intracellular responses to related ligands.
Isolation of Human Jak3 cDNA
Based on sequences
obtained from the 210-bp cDNA sequence of Jak3 (previously named TK5, (12) ), 5` kinase-specific primers were synthesized and used in
the 3`-RACE (Rapid Amplification of 3`cDNA Ends, Life Technologies,
Inc.) procedure as performed according to the manufacturer's
specifications. This resulted in the isolation of a 645-bp cDNA clone
of human Jak3 gene from SKBR-3 cells, p3R7. A gt11 SKBR-3 human
breast adenocarcinoma cDNA library (Clontech), a
gt10 human spleen
cDNA library (Clontech), and a
-ZAP activated human monocyte cDNA
library (kindly provided by Dr. H. Shelton Earp) were screened with the
p3R7 cDNA probe, labeled by random priming according to the published
procedure(13) . Approximately 1
10
recombinants from each library were screened and multiple
overlapping cDNA clones were either subcloned into the pBluescript II
vector (Stratagene) or excised with R408 helper phage (Stratagene). To
obtain the 5` end of the human Jak3 sequence, murine 5` Jak3 SacI cDNA fragment was used as a probe to isolate clones from
a -ZAP human Jurkat cDNA library (Stratagene) in addition to those
isolated by the 5`-RACE procedure. RT-PCR primers for filling in a gap
sequence within the NH
-terminal kinase were flanking
oligonucleotides 5`-CME primer (5`-GAACTGCATGGAGTCATTCC-3`) and 3`-HGN
primer (5`-CATTGCCATGGGGCAGGCCTTTG-3`). Sequence analysis was performed
using the dideoxy method with Sequenase Version 2.0 (U. S. Biochemical
Corp.). The sequences obtained were confirmed by sequencing both
strands of DNA with different primers and compressions were resolved by
using 7-deaza-dGTP. Sequences were analyzed using the GCG program from
the University of Wisconsin.Preparation of Peripheral Blood Cell Populations and Bone
Marrow Mononuclear Cells
Mononuclear cells were isolated from
peripheral blood of two healthy donors using Histopaque 1077 (Sigma).
Following Histopaque treatment, the red blood cell/granulocyte pellet
was washed several times with a red blood cell lysing solution (0.1555 M NH
Cl, 0.01 M KHCO
, pH 7.0,
and 0.1 mM NaEDTA) to yield purified granulocytes.
The bone marrow mononuclear cells from a healthy transplant donor were
prepared by Ficoll-Paque (Pharmacia) treatment. Monocytes were
separated from lymphocytes using two different methods: (i) adherence
to plastic for 40 min or (ii) flow cytometry using an EPICS V system
(Coulter) with appropriate light scatter gates for monocytes. T cells
(CD3/CD19
) and B cells
(CD3
/CD19
) were purified by flow
cytometry from peripheral blood mononuclear cells with appropriate
light scatter gates for lymphocytes as described previously (14) . FACS reanalysis of sorted cells revealed a suspension
purity of >98% for lymphocyte samples (data not shown). To further
ascertain the purity of the flow cytometric sort, PCR analysis was
performed on the sorted cells using primers which span the region of
alternative splicing in the extracellular domain of CD45 that can
distinguish between T and B lymphocyte ( (15) and (16) , CD452F = bp 140-162,
5`-GTATTTGTGACAGGGCAAAGCC-3`; CD452R = bp 1146-1169,
5`-ATGTTGGGTTCAAGGTTTTCTAA-3`). This analysis revealed no detectable
contamination of the two lymphocyte subpopulations.
Cell Lines
All cell lines used in the RT-PCR,
immunoblot analysis, and in vitro kinase assays were obtained
from the American Type Culture Collection and maintained according to
the ATCC recommended medium. The lineages of cell lines used are
summarized as follows: BT-20, BT-474, MCF-7, SKBR-3, T-47D, and 600PEI
are breast carcinoma cell lines; HeLa is a cervical carcinoma cell
line; SW480 is a colon carcinoma cell line; Jurkat, MOLT-4, and HUT-78
are T lymphocyte leukemia cell lines, K562 is a chronic myelogenous
leukemia cell line, and COS-7 is a SV-40 transformed monkey kidney cell
line.RNA Isolation and RT-PCR Amplification
Total
cellular RNA was isolated by the RNAzol method (Cinna/Biotecx
Laboratories, Inc.) according to the manufacturer's instructions.
Approximately 10 µg of total RNA was first incubated with DNase
enzyme at 37 °C for 1 h, extracted with phenol-chloroform, and
ethanol precipitated. The DNase-treated RNA was then used as a template
for cDNA synthesis. cDNA synthesis was performed as described (12, 14, 17) using Superscript reverse
transcriptase (Life Technologies, Inc.). For PCR amplification, the
sense oligonucleotide primer used was 5`-Jak3 primer:
5`-GAGAGCGAGGCACACGTCAA-3` corresponding to nucleotides 2967-2986
which is common among all splice forms of Jak3. The antisense
oligonucleotide used in the blood and bone marrow expression
experiments was 3`-NIF primer: 5`-CTGGCGAGAGAAGATGTTGTC-3`,
corresponding to nucleotides 3096 to 3116 (a region common among the
Jak3 splice variants). 3`-bJak3 primer: 5`-AGCTGGCTTGCCCGAGA-3`
specific for the B-form Jak3 splice variant, corresponding to
nucleotides 3355 to 3371, was used to detect Jak3B transcripts.
3`-sJak3 primer: 5`-CTAAGGTCACACAGCCAGTC-3` corresponding to S-form
Jak3 nucleotides 3506 to 3525 was used to detect S-form Jak3
expression. 3`-mJak3 primer: 5`-GCCACCAGCCCATGGAG-3` corresponding to
nucleotides 3338 to 3354 was used to assess M-form Jak3 expression. The
3`-Jak3 primer: 5`-TCCCATCATCCGCAGGAACTCG-3` corresponding to
nucleotides 3191 to 3212 was a second common primer employed to detect
the expression of all three Jak3 isoforms. The various oligonucleotide
primer pairs were sensitive in RT-PCR analyses but did not PCR amplify
using genomic DNA because of the presence of introns between the
5`-Jak3 and the different antisense primers. PCR was run for 33 cycles
(freshly isolated hematopoietic populations), 35 cycles (FACS sorted T
and B lymphocytes), and 36 cycles (different transformed cell lines).
An annealing temperature of 60 °C was used for the Jak3 PCR
amplification, but a higher annealing temperature of 68 °C was used
to analyze the expression pattern of Jak3 splice variants. Each PCR
reaction contained a reverse transcriptase negative control to rule out
any genomic amplification, and a no-template control. All PCR results
were repeated a minimum of three times. To normalize the RNA levels of
RT-PCR, -actin primers (12, 14, 17) were
used for 23 cycles at 95 °C for 1 min, 60 °C for 1 min, and 72
°C for 1 min. The PCR products were analyzed by electrophoresis on
a 1.0 or 1.5% agarose gel containing ethidium bromide.Northern Blot Analysis
Human multiple tissue
Northern blots (Clontech) were probed with the EcoRI-restricted DNA fragment from p3R7 labeled by random
priming. The p3R7 probe was identical to the cDNA and genomic libraries
probe which spanned the COOH-terminal kinase domain. Hybridization was
performed in 50% formamide, 5 SSPE, 0.5% SDS at 42 °C.
Filters were given final washes at 68 °C in 0.1 SSC and
0.5% SDS.Antibodies
Synthetic peptides corresponding to the
carboxyl-terminal amino acid sequence of human B-form JAK3 variant
(SVSQSVDWAGVSGKPAGA) were coupled to keyhole limpet hemocyanin by
glutaraldehyde and used for immunization into New Zealand female
rabbits. Antibodies (UNC36) were purified using the ImmunoPure(A/G) IgG
purification kit from Pierce according to the manufacturer's
instructions. Antibodies to mouse JAK3 and JAK families were from Dr.
James Ihle(9) , antibodies to human S-form JAK3 was purchased
from Santa Cruz Biotechnology, Inc. (CA), whereas anti-phosphotyrosine
(4G10) and monoclonal anti-human IL-2 receptor antibodies were
purchased from UBI (New York). The -murine JAK3 antibodies were
raised against amino acids 966-982 (kinase subdomain VII, (9) ), the -JAK family antiserum were raised against TYK2
amino acids 819-838 (9) , and the human S-form JAK3
antibody were raised against the COOH terminus of the JAK3S protein
(residues 1105-1124, (10) ). Anti-HA monoclonal antibody
(clone 12CA5) were purchased from Boehringer Mannheim, which recognizes
the HA peptide sequence (YPYDVPDYA).Immunoprecipitation and Western Blot
Analysis
Cells (approximately 10
cells) were lysed
in Nonidet P-40 buffer containing 50 mM Tris, pH 7.5, 150
mM NaCl, 0.5% Nonidet P-40, 50 mM sodium fluoride, 1
mM sodium orthovanadate, 1 mM dithiothreitol, 25
µg/ml leupeptin, 25 µg/ml aprotinin, and 1 mM
phenylmethylsulfonyl fluoride. The cell lysates were shaken in the cold
room for 10 min and clarified by centrifugation. The postnuclear
supernatants were collected and incubated at a 1:100 dilution with JAK
antibodies (UNC 36, -mouse JAK3 and -JAK families), a 1:1000
dilution with anti-phosphotyrosine antibodies (4G10, UBI), and a 1:250
dilution with monoclonal anti-human IL-2 receptor antibodies (UBI) at 4
°C for 4 h. The immunoprecipitates were then mixed with protein
A-Sepharose beads at 4 °C for 1 h, washed, resolved on SDS-PAGE,
and electrophoretically transferred to Immobilon-P PVDF membranes
(Millipore). The blots were then hybridized with the appropriate
antibodies. For Western blot, the proteins were prepared in a similar
way, except that 50 µg of total protein from the lysates were
resolved by 8% SDS-PAGE. For peptide competition, the membrane was
incubated with antibody against human B-form JAK3 peptides in the
presence of excess antigenic peptides (10 µg/ml) from which the
antiserum was raised. The immunoblot was subsequently incubated with
the appropriate horseradish peroxidase-coupled sheep anti-rabbit IgG or
anti-mouse IgG for detection with enhanced chemiluminescence reagents
(ECL; Amersham).-hJAK3B antibody (UNC 36) at a dilution of
1:3,000.Construction of Expression Plasmids
A pcDNA3
eukaryotic expression vector (Invitrogen) for mouse Jak3 and mouse
Jak3:hJak3 chimeric isoforms was first tagged in-frame with three
repeats of the HA epitope sequence via cloning of the HA PCR product
into KpnI and NotI sites. Full-length mouse Jak3 cDNA
sequence (9) was cloned into the NotI site to create
pc3HA-moJak3. A NotI-SacI partial restriction digest
of mouse Jak3 yielded a 3-kb fragment (encoding regions homologous to
human Jak3 from amino acids 1-1018), which was ligated to the
different hJak3 isoforms: SacI-XbaI fragments from
amino acid 1019 to 1124, amino acid 1019 to 1109 corresponding to the
hJak3B and hJak3S isoforms, respectively, and a SacI-ApaI fragment from amino acid 1019 to 1131
corresponding to the hJak3M isoform. The ligation products were then
cloned into the appropriate sites of pcDNA3 (NotI-XbaI for hJak3B and hJak3S, and NotI-ApaI for Jak3M), resulting in chimeric clones of
mouse Jak3 and human Jak3 isoforms designated pc3HA-Jak3B, pc3HA-Jak3S,
and pc3HA-Jak3M (Fig. 6A). All chimeras were sequenced
through the junctions to confirm that no introduced deletions or
mutations were introduced during the subcloning steps.
represents
HA-tagged epitope or different 3`-Jak3 isoforms, &cjs2112; represents
mouse Jak3 cDNA, and &cjs2098; represents the common 5` region of human
Jak3 cDNA sequence. B, COS-7 cells were transiently
transfected with the above plasmids. The cells were subsequently lysed
and the extracts were immunoprecipitated with -HA. The
immunoprecipitates were then used for in vitro kinase assays
and immunoblot analysis. In vitro kinase activity detected the
autophosphorylation of JAK3 isoforms and mouse JAK3 by autoradiography.
The level of protein expression was determined by probing with
-HA.
Transient Transfection of COS-7 Cells
3 µg of
plasmid DNA was introduced into COS-7 cells by transient transfection
with Lipofectamine reagent as described by the manufacturer (Life
Technologies, Inc.). After 60 h, cells were harvested and lysed in
Nonidet P-40 buffer. Protein concentrations were determined by the
Bradford (35) (Bio-Rad) method. Approximately 300 µg of
total cellular protein was isolated for immunoprecipitation with
anti-HA monoclonal antibodies (Boehringer Mannheim) at a dilution of
1:500. Immunoprecipitates were isolated using protein A-Sepharose, and
immunoblots were probed as described by the manufacturer. In vitro kinase assays were performed as described by Witthuhn et
al.(6) .
Cloning and Characterization of Jak3 cDNA
In
order to isolate a full-length human Jak3 cDNA sequence, PCR
amplification and cDNA library screening approaches were pursued. Using
the 3`-RACE method, a cDNA clone called p3R7 extending from the JAK3
kinase domain to the poly(A) tail was isolated and
both nucleotide and predicted protein sequence reveal a high homology
with the published JAK kinase family sequences. Then a 645-bp cDNA
clone (p3R7) was used as a probe to screen two other libraries, one
from human activated monocytes and the other from human adult spleen.
Multiple overlapping cDNAs were isolated and found to have a common 5`
end sequence; however, the 3` end of these sequences diverged. Since
the putative 5`-most sequence by homology with the published mouse
sequences was still lacking, 5`-RACE was utilized to clone the 5` end
of the human Jak3. Unfortunately, the longest isolated clone was a 1-kb
cDNA which still lacked the authentic 5` end sequence. Since no
libraries had cDNA clones that extended beyond amino acid 639 in the
JAK consensus sequences, we then used the murine 5` Jak3 SacI
cDNA fragment (9) as a probe to screen a human Jurkat cell line
cDNA library (Stratagene). This yielded a cDNA of approximately 2 kb
which encodes the most 5`-coding sequence, and included a small 113-bp
insertion at nucleotide position 1536 containing a premature stop
codon. By RT-PCR and direct sequencing, we now know that this insertion
is an intron (data not shown). RT-PCR was also employed to isolate the
missing cDNA sequences within the NH
-terminal kinase-like
domain in order to obtain a full-length human Jak3 cDNA. The multiple
overlapping cDNA clones allowed for a composite Jak3 sequence,
generating a sequence corresponding to a single large open reading
frame, but with three divergent sequences at the COOH terminus begining
at amino acid position 1069. These three variants were isolated from
cDNA libraries from the breast cancer cell line SKBR-3 (B-form), normal
adult spleen (S-form), and activated monocytes (M-form), respectively.
During these studies, a sequence from human Jak3 (11) was
published which corresponded to the S-form of Jak3. A comparison of the
sequences indicate that the S-form Jak3 is very similar to that
published as L-Jak (11) but with some differences in the
5`-untranslated region from nucleotides 1 to 73, and in the
3`-untranslated region of the S-form Jak3 from nucleotides 3636 to the
3` end. In addition, there were a total of 15 nucleotide differences in
the coding region, which resulted in 8 amino acid changes: amino acids
34 (Gly versus Ala), 212 (Arg versus Ala), 222 (Arg versus Pro), 610 (Ile versus Met), 845 (Gly versus Ala), 846 (Asp versus His), 896 (Gln versus Pro), 897 (Ser versus Glu) as shown in Fig. 1A. These discrepancies are not sequencing
ambiguities since all clones were sequenced from both directions, and
some were confirmed by multiple cDNA clones.
Analysis of Putative Jak3 Protein
Structure
Comparison of the predicted amino acid sequence of the
JAK family members revealed that the human Jak3 gene is most closely
related to murine Jak2, showing 69% amino acid sequence identity
followed by Jak1, showing 62% amino acid sequence identity, and Tyk2,
showing 60% amino acid sequence identity. The three isoforms encode a
1094-, 1124-, and 1131-amino acid polypeptide with calculated molecular
weights of 121,434, 124,764, and 125,541 corresponding to B-form,
S-form, and M-form JAK3 proteins, respectively. The deduced primary
protein structures reveal features typical for the JAK kinase family: a
518-amino acid NH terminus followed by two kinase domains
with the COOH-terminal domain possessing catalytic function. In
addition, a pair of tyrosine residues at amino acid positions
980-981 as well as the FWYAPE motif in subdomain VIII at amino
acid positions 992-997, which so far have been only found in the
JAK kinase family, were also observed(12) . Subdomain VI,
HRDLAA at amino acid positions 947 to subdomain IX, DVW, are tyrosine
kinase specific motifs originally used in our design of the targeted
degenerate oligonucleotides for identification of the Jak3
sequence(12) . Analysis of the COOH-terminal kinase domain
showed that the M and S splice isoforms harbor the 11 major conserved
kinase subdomains (I-XI); however, the B-form JAK3 variant lacks a
recognizable subdomain XI characterized by the arginine at position
1085 for the S-form and position 1092 for the M-form that is invariant
among the tyrosine kinases (Fig. 1B). Highly conserved
individual amino acids within the catalytic domain are expected to play
important roles in kinase function since deletions within subdomain XI
of the EGF receptor abolish kinase activity(21, 22) .
Thus, the absence of an intact subdomain XI in the B-form JAK3 suggests
that this isoform lacks kinase activity.Expression of Human Jak3 mRNA
Tissue expression of
human Jak3 was determined by probing human multiple tissue Northern
blots using p3R7 random-primed radiolabeled cDNA. Fig. 2A shows that two major transcripts of sizes 7.6 and 5.8 kb were
detected in normal blood, spleen, and thymus, but a 4.8-kb transcript
was detected at low levels in the spleen. These results confirm that
Jak3 is normally expressed predominantly in hematopoietic tissues. By
RT-PCR, we sought to determine the expression of Jak3 in primary
hematopoietic cells fractionated by FACS. Fig. 2C indicates that Jak3 is expressed in the bone marrow and in all
hematopoietic populations derived from the peripheral blood including
monocytes, granulocytes, and T-lymphocytes
(CD3/CD19
), but at low levels in B
lymphocytes (CD3
/CD19
).
RNA. The cell lines represented
are: MOLT-4, lymphoblastic leukemia cell line; Raji, Burkitt's
lymphoma cell line; SW480, colorectal adenocarcinoma cell line; A549,
lung carcinoma cell line; and G361, melanoma cell line. C,
RT-PCR analysis of Jak3 expression in normal blood and bone marrow. The
ethidium bromide-stained agarose gel indicates the 143-bp Jak3 fragment
and a 201-bp
-actin band as a reference band for amount of
template in each reaction. PBMC, peripheral blood mononuclear
cells; CD3/CD19
and CD3
/CD19
lanes are from sorted lymphocytes (see ``Experimental
Procedures'').
-actin probe (data
not shown) or RT-PCR with -actin primers to ensure that
comparisons were made according to the same amount of input RNA.Identification of Jak3 Splice Variants
We noticed
that our original Jak3 cDNA clone had COOH-terminal sequences that
dramatically differed from the published human and mouse
sequences(9, 10) , suggesting rearrangements or splice
variants. Our subsequent screening of breast (B), spleen (S), and
activated monocytes (M) cDNA libraries identified a total of three
distinct cDNA clones with different 3` termini: S-form, M-form, and
B-form. The fact that all clones were identical until amino acid 1070
where the sequences diverged (Fig. 1) suggested that these
represented splice variants. As shown in Fig. 1B, the
S-form Jak3 (S-HJAK3) is identical to the published human Jak3 (L-JAK, (11) ) sequences. The M-form (M-HJAK3) is related to the S-form
only by the presence of the Trp and Arg residues common to subdomain XI
of the tyrosine kinases. However, as mentioned above, the B-form Jak3
(B-HJAK3) lacks subdomain XI of the COOH-terminal catalytic domain. )To determine the expression pattern of the
three splice variants, we employed RT-PCR using a common 5`-Jak3
primer, a common 3`-Jak3 primer, and three primers specific to the
splice variants. The results indicate that a 404-bp cDNA of B-form Jak3
is detected in near uniform levels in most cell lines tested (Fig. 3) but there is no detectable or a very low amount of this
cDNA in MCF-7 and T-47D. This conforms to our earlier published RT-PCR
findings (12) as well as our B-form antibody Western blot
analysis (Fig. 4). The detectable low level of B-form transcript
in T-47D by RT-PCR at 36 cycles associated with absent protein
expression as noted in Fig. 4A is not unexpected since
RT-PCR is semi-quantitative, very sensitive, and therefore can detect
transcript levels that may not be biologically relevant. A 588-bp PCR
fragment of S-form Jak3 is detected in all the hematopoietic cell
lines, but only in three of eight epithelial cancer cell lines. The
388-bp cDNA of M-form Jak3 is detected in a wider range of cell lines
but is absent in MCF-7 and SKBR-3, and seen at a low level in K562
cells. Some cell lines expressed different combinations of the splice
forms. BT-20 and HUT-78 cells express all splice forms equally, whereas
BT-474 cells express mainly B- and M-forms, and SKBR-3 cells express
only the B-form Jak3 (Fig. 3). The presence of multiple splice
variants at the COOH terminus co-expressed in the same cell suggests
complexity in the downstream signaling of JAK3.
-actin band was
amplified in parallel from the same template as a reference
band.
Characterization of the B-form Jak3 Protein
By
homology with the mouse JAK3, the S-form of the human JAK3 is predicted
to directly interact with the IL-2 and IL-4 receptors and is involved
in the IL-2/IL-4 signaling response. In order to determine whether the
other splice forms are translated and whether they are involved in IL-2
signaling as well, a polyclonal antiserum was raised against the B-form
COOH-terminal peptide at amino acid positions 1077 to 1094. The B-form
was chosen for study because it represented the most divergent sequence
from the JAK consensus. As shown in Fig. 4A, the
anti-B-form JAK3 antiserum readily recognized a 125-kDa protein in the
K562, BT-474, and Jurkat cell lines that also express Jak3 transcripts.
Conversely, T-47D, which has no detectable Jak3 mRNA by Northern blot
analysis (data not shown), has no detectable JAK3B protein. Antibody
binding by UNC36 (-hJAK3 B form antibody) could be competed by the
immunogenic peptide, attesting to the specificity of this antiserum (Fig. 4A).-mouse JAK3 and -JAK family antibodies (9) were used to immunoprecipitate BT-474 cell lysates and
immunoblotted with either anti-phosphotyrosine (4G10) or B-form JAK3
antibody (UNC 36). The results show that the anti-B-form antiserum,
UNC36, specifically recognizes a JAK3 protein immunoprecipitated by
these two anti-JAK antibodies. In addition, the 125-kDa JAK3 protein in
unstimulated HUT-78 cells immunoprecipitated by an anti-IL-2 receptor
monoclonal antibody was recognized by anti-B-form JAK3 antibodies
confirming that JAK3B is bound to the IL-2 receptor (Fig. 5B).
JAK3B Is a Kinase-deficient Form of the JAK Kinase That
Complexes with JAK3S
The structure of the JAK3B kinase suggests
that, unlike the S- and M-forms, this splice variant removes the kinase
subdomain XI (Fig. 1B) characterized by a consensus
motif CW(X)RP (the underlined residues being invariant; (18) ) and substitutes in sequences of unknown significance.
Deletions in the EGF receptor within this domain demarcated by residues
920-951 have been shown to eliminate kinase activity (22) suggesting that JAK3B, with more profound perturbations of
this subdomain, may also be kinase-defective. To confirm this
possibility, we immunoprecipitated JAK3B and determined that it
completely lacked in vitro autokinase activity (data not
shown). Moreover, using HUT-78 cell lysates, we immunoprecipitated
JAK3B and found no baseline or IL-2 inducible autophosphorylation
despite increased tyrosine phosphorylation of JAK3S after IL-2
induction. These results are consistent with the notion that JAK3B
lacks intrinsic kinase activity (Fig. 7, A and C). In addition, we transfected expression constructs of
HA-tagged JAK3 isoform cDNAs into COS-7 cells to directly determine the
kinase activity of each isoform (Fig. 6). Mouse-human JAK3
chimeras were constructed using the first 1014 amino acids encoded by
the mouse JAK3 cDNA and the terminal amino acids 1094, 1124, and 1131
corresponding to the B, S, and M human JAK3 isoforms. Chimeras were
used because of the significant differences in the 5`-sequences
reported for the human Jak3 cDNAs (see above), raising the possibility
that any of the cloned human Jak3 cDNAs may have altered enzymatic
function. Furthermore, immunoprecipitations directed against the HA
epitope were used to eliminate the possibility that antibody binding to
the COOH-terminal sequences would alter kinase activity. Lysates of
COS-7 cells transfected with the JAK3S, -M, and -B chimeras were
immunoprecipitated with anti-HA antibodies and subjected to in
vitro kinase assay and immunoblot analysis (Fig. 6). The
results show that JAK3S has the highest autokinase activity followed by
JAK3M. However, JAK3B is completely devoid of kinase activity as
measured by autophosphorylation. Taken together, our results show that
the B-form JAK3 is a kinase-deficient enzyme that interacts with the
IL-2R.
-terminal (JH2) domain which has unknown enzymatic
function followed by a COOH-terminal kinase domain (JH1) with catalytic
activity. In addition, the JAKs harbor no SH2 or SH3 domains and
contain a signature FWYAP(E) motif which is not found in other tyrosine
kinases. Recently, we and others have reported a new member of the JAK
family, murine JAK3, as the kinase involved in IL-2 and IL-4
signaling(9, 10) . To further examine the biology of
JAK3 in human cells, we sought to clone human Jak3 cDNAs.(23) . Thus, the
Jak3 3`-splice variants can be translated into functional proteins.
and Grb2 have been shown to interact via their SH2 domains with the
autophosphorylated COOH-terminal tail of activated EGF receptor (29, 30, 31) . Similarly, the COOH terminus
of nerve growth factor/Trk receptor tyrosine kinase appears to be
involved in receptor-substrate interaction. Deletion of the 15
COOH-terminal amino acids abrogated Trk receptor and phospholipase
C-
substrate phosphorylation activities (32) . Since the
level of expression in different cell types and the changes in an
important kinase structural subdomain are seen for JAK3 splice
variants, we suggest that expression of these isoforms may have
significant functional consequences.
-chain recruits JAK1 and the IL-2R
-chain binds JAK3(23, 33) , and that the
co-activation of JAK1 and JAK3 in the IL-2 receptor
complex is necessary to transduce IL-2 signals. The competition between
a kinase active (JAK3S) and a kinase-defective (JAK3B) JAK3 may
attenuate the IL-2 responses downstream of JAK3.
was able to
bind its ligand, neu differentiation factor, it showed no
autophosphorylation and kinase activities(34) . Despite its
deficient enzymatic function, p180
is involved in
heterodimerization with EGF receptor, p180
, or
p185
and is tyrosine phosphorylated in these complexes.
In cells co-expressing the EGF receptor and p180
, the
signaling protein phosphatidylinositol 3-kinase is recruited to the
heterodimeric complex only by the p180
component. Thus,
it is possible that JAK3B may function in an analogous fashion.
))
We are grateful to Gustavo Maroni for his support and
discussion of the work, and Harvey Mohrenweiser, H. Shelton Earp, and
Kevin Kelleher for critical reading of the manuscript. We thank Edward
Baptist for helpful assistance in the manuscript preparation.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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