(Received for publication, April 26, 1995; and in revised form, June 29, 1995)
From the
Three forms of rat JAK2 (type 2 Janus tyrosine kinase) were
produced via the baculovirus expression vector system. Recombinant
baculoviruses encoded either the full-length rat jak2 cloned
from the Nb2-SP cell line (rJAK2), a carboxyl-terminal deletion mutant
lacking the putative catalytic domain (rJAK2(C795)), or an
amino-terminal deletion mutant containing the putative catalytic domain
((N
661)rJAK2). The proteins produced in infected Sf21 cells were
assayed for phosphotyrosine content and autophosphorylating activity.
Tyrosine phosphorylation of rJAK2 was not observed 1 day postinfection
when rJAK2 was initially produced but was apparent 2 or more days
postinfection when the rJAK2 level had significantly increased.
Tyrosine phosphorylation of rJAK2(C
795) was not observed; further,
coproduction of rJAK2(C
795) with rJAK2 blocked tyrosine
phosphorylation of rJAK2, consistent with previously published results
(Zhuang, H., Patel, S. V., He, T-C., Sonsteby, S. K., Niu, Z., and
Wojchowski, D. M.(1994) J. Biol. Chem. 269,
21411-21414). Mutant (N
661)rJAK2 exhibited a robust tyrosine
phosphorylation signal. A second 62-kDa tyrosine phosphoprotein
co-immunoprecipitated with (N
661)rJAK2 but not with rJAK2 or
rJAK2(C
795). Both rJAK2 and (N
661)rJAK2 incorporated
phosphate under in vitro kinase assay conditions, but
rJAK2(C
795) did not. A JAK2 oligomer with interacting catalytic
sites and/or inhibitory sites would provide a simple model to describe
these results.
The JAK family of protein-tyrosine kinases (JAK1, JAK2, JAK3,
and TYK2; EC 2.7.1.112) appears to play a crucial role in the signal
transduction cascade initiated by activation of numerous cytokine
receptors(2) . Cytokines that signal through a common
chain, such as interleukin-2, interleukin-4, and interleukin-7, trigger
the tyrosine phosphorylation of JAK3 and may also induce the activation
of other Janus kinases(3, 4, 5) . Interferons
and
require both JAK1 and TYK2 in their signaling
cascade(6) , and interferon
signaling requires both JAK1
and JAK2(7) . A number of cytokine receptors are associated
with JAK2, and activation of these receptors by their cognate ligands
results in the rapid tyrosine phosphorylation of JAK2. Such receptor
systems include those for erythropoietin(8) ,
interleukin-3(9) , interleukin-6(10) , leukemia
inhibitory factor, ciliary neurotrophic factor, oncostatin
M(11) , granulocyte-macrophage colony-stimulating
factor(12) , growth hormone(13) , and
prolactin(14, 15) . It is noteworthy that deletion of
the membrane-proximal domains of most of these cytokine receptors
abrogates not only the transduction of a cytokine-initiated
proliferation but also the cytokine-triggered activation of
JAK2(8, 10, 12, 16, 17) .
The specific mechanism by which cytokine receptor activation converts
these kinases from a latent to an active state remains unclear, but in
systems involving the prolactin receptor(14, 15) , the
leukemia inhibitory factor receptor
chain, and the interleukin-6
signal transducer gp130(11) , JAK2 is associated with receptor
before ligand stimulation of the receptor. Thus, it does not appear
that receptor association per se is sufficient to activate the
kinase. The detailed enzymologic behavior of the
``activated'' Janus kinases has yet to be characterized, and
site-directed mutagenesis studies assessing the functional importance
of some of the structural domains of murine JAK2 (1) and of
human TYK2 (18) have only recently appeared in the literature.
As an initial step toward understanding the mechanism of enzymatic activation of the JAK protein-tyrosine kinases, we overexpressed the rat jak2 cDNA clone in Sf21 cells via the baculovirus expression vector system. We also overexpressed two deletion mutants of JAK2 that were predicted to be active and inactive forms based on the retention and deletion, respectively, of a highly conserved tyrosine kinase motif. The characterization of the resultant protein products is described.
A recombinant baculovirus was created with a transfer vector
(pBacPAK9:rJAK2) that contained approximately 150 base pairs between
the polyhedron promoter and the initiation codon of rat Jak2.
The mutants (N661)rJAK2 and rJAK2(C
795) were derived from
restriction endonuclease digests and religation of the transfer vector
pBacPAK9:rJAK2. A schematic diagram showing the JAK homology domains
retained in each of the three forms, rJAK2, (N
661)rJAK2, and
rJAK2(C
795), is presented in Fig. 1.
Figure 1:
Schematic comparison of recombinant
rJAK2 forms. rJAK2 is the protein that contains all seven JAK homology (JH) domains as defined by Harpur et
al.(34) ; the rat JAK2 amino acid residues contained
within each domain are JH7 (16-138), JH6 (144-284), JH5
(288-309), JH4 (322-440), JH3 (450-538), JH2
(542-824), and JH1 (836-1125). rJAK2 (C795) terminates
after Arg
and thus lacks 10% of JH2 and all of JH1.
(N
661)rJAK2 begins at Met
and thus includes 60% of
JH2 and all of JH1. The antibody recognition site is contained within
the JH2 segment retained in all three constructs. A map of the relevant
restriction sites of pBacPAK9:rJAK2 is aligned over the rJAK2 protein;
the dark arrow represents the polyhedron promoter, and the boxed area represents the jak2 coding
region.
A time course of protein production in Sf21 cells infected with the baculovirus-expressing rJAK2 is shown in Fig. 2. The cells were harvested at various times p.i., solubilized in a buffer containing 1% Triton X-100, pre-cleared with protein A-Sepharose CL-4B, then immunoprecipitated with antiserum directed against a synthetic peptide DSQRKLQFYEDKHQLPAPK(C) corresponding to amino acid residues 758-776 of murine and rat JAK2. At the saturating levels of virus used for this experiment, production of rJAK2 was observed as early as 27 h p.i. (Fig. 2, panelA), while tyrosine phosphorylation of rJAK2 was not detected at this time point (Fig. 2, panelB). Tyrosine phosphorylation of rJAK2 was first observed in this experiment at 51 h p.i. (Fig. 2, panelB), at a stage when rJAK2 production had increased (Fig. 2, panelA). The level of rJAK2 production and tyrosine phosphorylation of rJAK2 appeared to plateau at 71 h p.i. and was stable through 123 h p.i. The molecular mass deduced from the rat jak2 cDNA sequence is 130 kDa, but the apparent molecular mass of the native rat JAK2 is 120 kDa(23, 24) , which corresponds to the apparent molecular mass of the recombinant rat JAK2 produced via baculovirus expression vector system.
Figure 2:
Time
course of rJAK2 production and tyrosine phosphorylation. Plates
containing 2 10
Sf21 were infected with saturating
(M.O.I. = 50) levels of recombinant baculovirus expressing rJAK2
and then harvested 27, 51, 71, 97, or 123 h post-infection as
indicated. Control plates were left non-infected or were infected with
recombinant baculovirus expressing human prolactin receptor (hPRLR) and harvested 27, 51, 71, 97, or 123 h post-infection.
Cellular contents were solubilized and immunoprecipitated with
anti-JAK2 as described under ``Materials and Methods.''
Sample aliquots equivalent to approximately 6.7
10
cells were subjected to SDS-PAGE and transferred to Immobilon-P
membranes and immunodecorated with anti-JAK2 (panelA) or with anti-phosphotyrosine (panelB).
The JH1 domain contains a motif that is
recognized as a protein-tyrosine kinase consensus domain(25) .
Recombinant baculovirus producing rJAK2(C795), a mutant of rat
JAK2 containing amino acid residues 1 through 795 and thus lacking the
JH1 domain and most of the JH2 domain, was created with the initial
intention of testing whether active rJAK2 could transphosphorylate an
inactive rJAK2(C
795). A time course of protein production in Sf21
cells infected with baculovirus producing rJAK2(C
795) is presented
in Fig. 3. The molecular mass of rJAK2(C
795) deduced from
its cDNA is 91 kDa. As seen in panelA, a 90-kDa
protein was produced and recognized by anti-JAK2 antisera (Fig. 3, panelA) but not by the
antiphosphotyrosine monoclonal antibody (Fig. 3, panelB). As in Fig. 2, Sf21 cells infected with
recombinant baculovirus producing rJAK2 contained a 120-kDa protein
that was recognized by both polyclonal anti-JAK2 antisera and by the
monoclonal antiphosphotyrosine antibody. When Sf21 cells were
co-infected with equal M.O.I. levels of both baculoviruses, the
production of both rJAK2 and rJAK2(C
795) was detected by anti-JAK2
immunodecoration (Fig. 3, panelA), but
neither form was recognized by antiphosphotyrosine antibody (Fig. 3, panelB). One interpretation of these
data is that the inactive rJAK2(C
795) and rJAK2 form an inactive
oligomer, an interpretation originally proposed by Zhuang et
al.(1) , to account for similar observations obtained in a
COS cell expression system. Another concept that derives from such an
interpretation is that the amino-terminal portion of the enzyme may
contain an inhibitory or regulatory domain.
Figure 3:
Time course of rJAK2 and rJAK2(C795)
coproduction and tyrosine phosphorylation. 13 plates containing 1
10
Sf21 were infected either individually or
simultaneously with recombinant baculoviruses (M.O.I. = 20)
expressing rJAK2 and rJAK2(C
795). A control plate was left
non-infected. Samples were harvested 22, 47, 65, or 77 h post-infection
as indicated. Cellular contents were solubilized and immunoprecipitated
with anti-JAK2 as described under ``Materials and Methods.''
Sample aliquots equivalent to approximately 3.3
10
cells were subjected to SDS-PAGE and transferred to Immobilon-P
membranes and immunodecorated with anti-JAK2 (panelA) or with anti-phosphotyrosine (panelB). The blackarrows point to the
120-kDa rJAK2; the whitearrow points to the 90-kDa
rJAK2(C
795).
We sought to test this
concept, as well as to determine if sufficient information was
contained in the JH1/JH2 domains to confer catalytic activity, by
constructing the recombinant baculovirus that produced
(N661)rJAK2. (N
661)rJAK2, which contained amino acid residues
661 through 1132 and thus retained all of JH1 but only 60% of JH2, was
predicted to be a 472-amino acid protein with a molecular mass of 55
kDa. This protein comigrated with rabbit immunoglobulin G heavy chain,
and therefore immunodecoration of (N
661)rJAK2 with anti-JAK2
anti-sera following anti-JAK2 immunoprecipitation was obscured. A
demonstration of the time course of (N
661)rJAK2 production was
achieved by anti-JAK2 immunodecoration of detergent-solubilized
proteins that had not been immunoprecipitated before electrophoresis (Fig. 4, panelA). Under these conditions, an
endogenous cross-reactive 97-kDa protein was also observed. More
importantly, the (N
661)rJAK2 protein was detected as a 56-kDa
protein (or as a doublet of protein bands) between 27 and 49 h
post-infection (Fig. 4, panelA) and became
tyrosine phosphorylated by 49 h p.i. (Fig. 4, panelB). As the infection progressed, a distinct 62-kDa
protein in the anti-JAK2 immunoprecipitation complex was detected by
anti-phosphotyrosine immunodecoration at 72 and 93 h p.i. (Fig. 4, panelB). A Coomassie-stained band
was observed at this mobility in immunoblots obtained from cells
infected with baculoviruses producing rJAK2, rJAK2(C
795), or
(N
661)rJAK2 (data not shown), but tyrosine phosphorylation of the
62-kDa protein was only observed in immunoblots from cells infected
with baculovirus producing (N
661)rJAK2. It is not yet clear
whether the common Coomassie-detectable band, which was not detectable
by anti-JAK2 (data not shown), was a non-phosphorylated version of the
tyrosine-phosphorylated 62-kDa protein or whether it coincidentally
comigrated with it.
Figure 4:
Time course of (N661)rJAK2 production
and tyrosine phosphorylation. Four plates of 4
10
Sf21 cells were infected at a 4-fold M.O.I. with recombinant
baculovirus expressing (N
661)rJAK2. Cells were harvested at 27,
49, 72, and 93 h p.i. Cellular contents were solubilized as described
under ``Materials and Methods.'' PanelA,
following sample pre-clearance with protein A-Sepharose CL-4B but prior
to immunoprecipitation, aliquots equivalent to 1.5
10
cells were subjected to SDS-PAGE. The resolved proteins were
transferred to Immobilon-P membrane and then immunodecorated with
anti-JAK2. The blackarrow points to the
(N
661)rJAK2. PanelB, cellular contents were
immunoprecipitated with anti-JAK2, aliquots equivalent to 2
10
cells subjected to SDS-PAGE, transferred to Immobilon-P
membrane, and then immunodecorated with anti-phosphotyrosine. The blackarrow points to (N
661)rJAK2, and the whitearrow points to an unidentified 62-kDa
protein.
To demonstrate the relative intensities of the
anti-phosphotyrosine signal of rJAK2 and of (N661)rJAK2, as well
as to test whether co-expression of these two forms would influence
each other's phosphotyrosine state, co-infection studies were
carried out (Fig. 5). Sf21 cells were infected with varying
proportions of baculoviruses producing rJAK2 and (N
661)rJAK2, then
harvested and detergent-solubilized 88 h post-infection. The presence
of rJAK2 was determined via Western blot analysis of proteins that had
not been immunoprecipitated before electrophoresis (Fig. 5, panelA). As the proportion of baculovirus producing
rJAK2 increased, so did the intensity of the 120-kDa rJAK2 protein
band. The decrease in intensity of the 56-kDa (N
661)rJAK2 protein
band did not correlate as well with the decrease in the proportion of
(N
661)rJAK2-producing baculovirus. (
)It was still
apparent that the amount of rJAK2 produced at an M.O.I. of 18 or 20
equaled or exceeded the amount of (N
661)rJAK2 produced at an
M.O.I. of 2, and overall, the amounts of anti-JAK2 cross-reactive
material were comparable between the two proteins. This was not the
case, however, when the anti-phosphotyrosine signals were compared (Fig. 5, panelB). A 120-kDa phosphotyrosine
signal was first observed at an rJAK2 M.O.I. of 15, slightly
intensified at an rJAK2 M.O.I. of 18, and was clearly visible at an
rJAK2 M.O.I. of 20. In contrast, a very intense phosphotyrosine signal
was apparent in all samples in which (N
661)rJAK2 was produced, and
this intensity only slightly decreased when the (N
661)rJAK2 M.O.I.
was reduced to 2. The very intense signal was a combination of two
phosphotyrosine bands, as demonstrated by the data in Fig. 4. It
should be noted that other minor bands (migrating at approximately 135,
105, and 45 kDa) were detectable as phosphotyrosine-containing proteins
in the anti-JAK2 immunoprecipitate from (N
661)rJAK2-producing
cells.
Figure 5:
Co-production and tyrosine phosphorylation
of (N661)rJAK2 and rJAK2. Seven plates of 2
10
Sf21 cells were infected simultaneously with recombinant
baculoviruses expressing (N
661)rJAK2 and rJAK2 at a total M.O.I.
of 20. The proportion of the two viruses are listed above each
gel lane; the lane at the extremeleft was derived from Sf21 cells left uninfected. Cells were harvested
at 88 h p.i. Cellular contents were solubilized as described under
``Materials and Methods.'' PanelA,
following sample pre-clearance with protein A-Sepharose CL-4B but prior
to immunoprecipitation, aliquots equivalent to 1
10
cells were subjected to SDS-PAGE. The resolved proteins were
transferred to Immobilon-P membrane and then immunodecorated with
anti-JAK2. PanelB, cellular contents were
immunoprecipitated with anti-JAK2, aliquots equivalent to 1
10
cells subjected to SDS-PAGE, transferred to Immobilon-P
membrane, and then immunodecorated with anti-phosphotyrosine. In both panels, the blackarrow points to
(N
661)rJAK2 and the whitearrow points to the
rJAK2.
The variable intensity of a phosphotyrosine signal that
correlated to the retention or loss of a putative catalytic domain
suggested that the rJAK2 and (N661)rJAK2 proteins were active
enzymes but that the rJAK2(C
795) was not. In an attempt to verify
that these proteins were indeed active enzymes and not merely
substrates for phosphorylation, in vitro kinase assays were
conducted with proteins purified by immunoprecipitation (Fig. 6). This assay demonstrated incorporation of radioactive
phosphorus into the 120-kDa protein when rJAK2 was incubated with
[
-
P]ATP and a conspicuously greater
incorporation of radioactive phosphorus into the 56-kDa protein when
(N
661)rJAK2 was incubated with
[
-
P]ATP. However, no autophosphorylation of
the 90-kDa protein was observed upon incubation of the rJAK2(C
795)
with [
-
P]ATP, as was anticipated. This
demonstration of autophosphorylation verified that the rJAK2 and
(N
661)rJAK2 were active enzyme forms and that the rJAK2(C
795)
was an inactive form.
Figure 6:
Autophosphorylation of immunoprecipitated
rJAK2 forms. Four plates of 4 10
Sf21 cells were
infected at a 20-fold M.O.I. with recombinant baculoviruses expressing
one of the following: human prolactin receptor (hPRLR),
(N
661)rJAK2, rJAK2(C
795), or rJAK2. Infected cells were
harvested 112 h p.i. and then assayed for in vitro kinase
activity as described under ``Materials and Methods.''
Immunoprecipitated equivalents of 2
10
Sf21 cells
were loaded in each gel lane; the dried gel was exposed to
film for 9 h. The blackarrow points to the
full-length (120 kDa) rJAK2 in the gel autoradiograph, and the whitearrow points to the (56 kDa) (N
661)rJAK2
mutant.
The tyrosine phosphorylation of JAK2 overexpressed via baculovirus expression vector system has been previously reported(12) , and it appeared that the tyrosine phosphorylation occurs constitutively in such an overexpression system. By observing the time course of rJAK2 protein production (Fig. 2) and by observing JAK2 production at a fixed post-infection harvest time under varied multiplicities of infection (Fig. 5), one can amend this interpretation. It appears that once the JAK2 production exceeds a critical level, tyrosine autophosphorylation occurs spontaneously.
A simple, plausible
explanation of this observation is that monomers of JAK2 begin to form
catalytically competent oligomers (possibly simple dimers) once the
JAK2 concentration approaches a value that shifts the
JAK2(oligomer)/JAK2(monomer) equilibrium to a detectable amount of
oligomer. This explanation is currently plausible because it is
consistent with several pieces of information. First, the observation
that inactive JAK2 mutants are capable of inhibiting
autophosphorylation of wild-type JAK2 (initially observed by Zhuang, et al.(1) and demonstrated here in Fig. 3)
could be explained by the formation of an inactive hetero-oligomer.
Second, it has also been shown that JAK2-related receptors such as
growth hormone receptor (26, 27) dimerize subsequent
to ligand binding. Activation of tyrosine kinase activity is mediated
by dimerization of JAK2-related receptors such as prolactin receptor (28) and gp130(29) , which are associated with JAK2
before ligand binding(11, 14, 15) , leading
to the presumption that bringing the JAK2 proteins into close physical
proximity is crucial for the ``activation'' of the JAK2.
Finally, there is evidence that correlates a significant increase in jak2 mRNA (19) in the Nb2-SP pre-T lymphoma cell line
(as compared to the parental Nb2-11C cell line) to an increase of
tyrosine-phosphorylated JAK2 protein, ()a phenomenon that
may be relevant to the loss of absolute prolactin dependence in the
Nb2-SP cell line(30) .
The concept that the oligomeric form
of JAK2 is the catalytically active form must be verified by additional
independent experiments before it is acceptable as a part of the
mechanism of enzymatic activation. It should be noted that there is no
evidence yet to support transphosphorylation or inter-JAK2
phosphorylation; the full-length rJAK2 does not phosphorylate the
inactive rJAK2(C795), nor does the ``hyperactive''
(N
661)rJAK2 appear to phosphorylate rJAK2. These data are
admittedly inconclusive since we have not shown that rJAK2(C
795)
contains suitable phosphorylation sites nor have we demonstrated that
(N
661)rJAK2 and rJAK2 can form hetero-oligomers. Without a
``transphosphorylation mechanism,'' many scenarios remain
that would link oligomerization to activation. These include
cooperativity between multiple catalytic sites (e.g. the
proton-translocating ATP synthase(31) ), catalytic sites formed
at subunit interfaces (e.g. lipoamide
dehydrogenase(32) ), or relief of interactions between
inhibitory domains and catalytic domains (e.g. CaM kinase
II(33) ). The last scenario may also help to explain the
apparent hyperactivity of the (N
661)rJAK2 mutant.
There are
three pieces of evidence that suggest that the (N661)rJAK2 mutant
is both hyperphosphorylated and hyperactive. These data are as follows:
1) the exaggerated anti-phosphotyrosine signal intensity of this form
relative to that of the full-length protein (Fig. 5), 2) the
significant increase in radiolabel incorporation observed in in
vitro kinase assay autoradiography (Fig. 6), and 3) the
appearance of a novel 62-kDa protein that is recognized by
anti-phosphotyrosine, but not by anti-JAK2, on Western blots ( Fig. 4and Fig. 5). Although the variance in the
5`-untranslated region (which does not vary between rJAK2 and
rJAK2(C
795)) may have slightly increased expression of
(N
661)rJAK2, the apparent equal intensity of the anti-JAK2 signal
in samples that have obvious differences in anti-phosphotyrosine signal
intensities (Fig. 5) leads to the conclusion that
(N
661)rJAK2 is hyperphosphorylated relative to rJAK2. One
plausible explanation for hyperactivity is that an inhibitory or
regulatory domain is contained within the amino-terminal portion of the
enzyme. This model is consistent with data from Zhuang, et al.(1) that show that kinase-deficient JAK2 mutants inhibit
autophosphorylation of the wild-type JAK2 in the COS cell expression
system, as we also show in the baculoviral expression vector system (Fig. 3). The concept of such an inhibitory or regulatory domain
requires additional experimental support, since alternative
interpretations for the ``trans-inhibitory'' properties of an
inactive JAK2 still exist.
If such inhibitory or regulatory domains
exist in JAK2, one might expect other Janus kinases to possess them as
a consequence of the high degree of sequence conservation in these
proteins. Expression of TYK2 mutants TK (lacking JH1) and
KL
(lacking JH2) in cells derived from the 2fTGH cell line resulted in the
generation of inactive kinases (18) , which is consistent with
the presence of an inhibitory domain in the amino-terminal domains JH3
through JH7. A useful test of the inhibitory/regulatory hypothesis will
be to determine if expression of only the TK (JH1), KL (JH2), or
combined KL/TK (JH2/JH1) domains is sufficient to produce an active
kinase.
The nature and relevance of the 62-kDa band observed in Fig. 4pose intriguing questions. Although identification of this
protein is in progress, ()the identification process is not
yet complete. Thus, there remains some ambiguity as to whether this
protein is virally encoded, is produced in response to viral infection,
or whether it is a constitutive cellular product. It is also unknown
whether the appearance of an intense phosphotyrosine signal associated
with the 62-kDa protein in (N
661)rJAK2 (but not rJAK2)
immunoprecipitation complexes is due to an alteration in substrate
specificity or whether the apparent increase in catalytic turnover of
the (N
661)rJAK2 mutant increased the phosphotyrosyl forms of the
62-kDa protein beyond the capacity of endogenous phosphatases.
The dual hypotheses that the oligomeric form of JAK2 is the catalytically active form and that there is an inhibitory or regulatory domain in the amino terminus of the enzyme are currently under investigation in our laboratory. These hypotheses do, however, allow for the speculative prediction that dysfunctional regulation of JAK2 expression may be responsible for factor-independent and receptor-independent proliferation and hence may predict circumstances for JAK2-mediated oncogenesis.