(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-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.
The JAKs ()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.
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 -hJAK3B antibody (UNC 36) at a dilution of
1:3,000.
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. 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.
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) 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'').
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 -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.
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. ()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.
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 -actin band was
amplified in parallel from the same template as a reference
band.
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 -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).
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-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.
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 (23) . Thus, the
Jak3 3`-splice variants can be translated into functional proteins.
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-
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.
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 -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.
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 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.
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.