(Received for publication, December 13, 1996, and in revised form, February 20, 1997)
From the Division of Medical Oncology, Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado 80262
Granulocyte-macrophage colony-stimulating factor
(GM-CSF), interleukin (IL)-3, and IL-5 stimulate DNA synthesis and
proliferation and inhibit apoptosis in hematopoietic cells. Multiple
signal pathways are activated by binding of these ligands to their
receptors, which share a common subunit. Janus protein kinase 2 (Jak2) binds to the membrane proximal domain of the
chain and is
phosphorylated on receptor ligation. To explore the role of Jak2 in the
regulation of specific signal transduction pathways, we constructed
fusion proteins with a CD16 external domain, a CD7 transmembrane
region, and a Jak2 cytoplasmic domain. This cytoplasmic domain
consisted either of wild type Jak2 (CD16/Jak2-W) or Jak2 mutations with deletions of (a) the amino terminus (CD16/Jak2-N),
(b) kinase-like domain (CD16/Jak2-B), (c)
kinase domain (CD16/Jak2-C), or (d) amino-terminal and
kinase-like domains, leaving the kinase domain (CD16/Jak-K) intact. In
contrast to the CD16/Jak2-W fusion protein, which requires
cross-linking for activation, CD16/Jak2-N, CD16/Jak2-B, and CD16/Jak2-K
were constitutively phosphorylated, and they stimulated Shc
phosphorylation and increased binding of STAT to DNA in Ba/F3 cells.
Cell lines derived from IL-3-dependent Ba/F3 cells stably transfected with CD16/Jak2-W, CD16/Jak2-N, or CD16/Jak2-B mammalian expression vectors died at a rate similar to that of the parental cells
on IL-3 deprivation. In contrast, CD16/Jak2-K cell lines exhibited
increased expression of bcl-2 and pim-1
mRNA and maintained their viability when compared with control cell
lines. Thus, activation of tyrosine phosphorylation by creating a
CD16/Jak2-K fusion is sufficient to activate pathways that prevent cell
death.
Signal transduction and growth stimulation by GM-CSF,
IL-3,1 and IL-5 are mediated by a
heterodimeric receptor composed of and
subunits. The
subunit is specific for each cytokine (1), whereas the
subunit is
shared in common (2). Binding of the growth factors dimerizes the
receptor and induces signal transduction pathways that, in turn,
cooperate to induce cell growth. However, the mechanisms by which
dimerization of the
and
subunits transmit these signals remain
unclear. Various pathways, involving Jak-2, Pim-1, c-Myc, c-Fos, c-Jun,
and the Ras/Raf/MAP kinase pathway, have been implicated (3-5),
however, and their regulation and relative contributions to DNA
synthesis and proliferation and inhibition of apoptosis are areas of
active investigation. Deletion mutagenesis studies suggest that the
signal transduction pathways and, perhaps, functional outcomes are
specifically associated with the membrane-proximal (1-544) and distal
(544-763) regions of the
subunit (3-5). The proximal region has
been associated with binding of Jak2 and activation of Jak2 kinase (4-6), induction of c-myc transcription, and stimulation of
proliferation. The membrane distal region is associated with activation
of the Ras/Raf/MAP kinase pathway, induction of c-fos and
c-jun genes, and inhibition of apoptosis (4, 5).
Jak2 is a member of a family of protein kinases composed of Jak1, Jak2,
Jak3, and Tyk2 that are characterized by a kinase domain in the
carboxyl portion, a kinase-like domain (pseudokinase), and a large
amino-terminal domain characterized by seven highly conserved regions
(7-10). The amino-terminal domain appears to provide the site of
interaction between Jak2 (3, 6, 11, 12) and the subunit of the
hematopoietic growth factor receptors at a region proximal to the
membrane that contains a "box 1" sequence and 14 additional amino
acids (6). Deletion of this Jak2-binding region prevents transduction
of growth factor-induced signals (13-15). However, deletions of
specific portions of intracytoplasmic domains of the
subunit can
also block GM-CSF, IL-3, and IL-5 signaling and can inhibit the growth
factor-induced activation of Jak2, Pim-1, and c-Myc (16-19).
Transfection of cells with dominant negative forms of the Jak2 kinase
blocks both erythropoietin-induced mitogenesis and inhibition of
apoptosis, suggesting that Jak2 is necessary for both proliferation and
prevention of cell death (20, 21).
Although removal of the distal domain of the subunit does not block
IL-3- or GM-CSF-mediated cell growth, the cells undergo apoptosis at a
more rapid rate (22). Transfection of Ras into such cells restores
their normal growth pattern. If only a small portion of the
subunit
is removed (residues 626-763), then this cell growth defect can also
be restored by adding fetal calf serum, which presumably acts through
regulation of the Ras pathway (23). Mutation of tyrosine 750 in the
chain reduces the sensitivity of cells to GM-CSF and decreases the
tyrosine phosphorylation of Shc (24), suggesting that this is a docking
site for signal transduction molecules.
To investigate the role of Jak2 kinase in the regulation of signal
transduction pathways, in the absence of other signals generated by the
growth factor receptors, we previously fused Jak2 to the external
domain of CD16 (the low affinity FcIII IgG receptor) and the
transmembrane domain of CD7 (T cell differentiation gp40 protein)
(CD16/Jak2) (25, 26). This cDNA was encoded in a vaccinia virus
that was used to infect growth factor-dependent murine
Ba/F3 cells to generate high levels of fusion protein. Cross-linking
with anti-CD16 antibody stimulated the kinase activity of the fusion
protein but did not cause activation of endogenous Jak2 or
phosphorylation of the chain. Activation of the CD16/Jak2 fusion
protein stimulated increased phosphorylation of Shc and p145 (SHIP)
(27) and increased expression of c-fos, c-jun,
and jun-B mRNAs, caused only minor changes in MAP kinase
activity, and did not increase cellular levels of c-myc
mRNA. We concluded that activation of Jak2 is sufficient to
generate some, but not all, of the signals that arise upon binding of
growth factors to their receptors in factor-dependent
hematopoietic cells.
In the present study, we further examined the role of specific domains of the Jak2 protein in individual signal transduction pathways and, by creating deletion mutants of the Jak2 protein and cloning them in frame with CD16/CD7, determined whether activation of Jak2 is sufficient to sustain the growth of hematopoietic cells. Recombinant vaccinia viruses were prepared for rapid, high level expression of the fusion proteins, whereas mammalian expression vectors were used to establish stably transfected cell lines for analysis of biologic effects. Deletion of the amino-terminal two-thirds of the Jak2 protein, leaving only the protein kinase domain, did not change the pattern of signal transduction. However, in contrast to wild type fusion protein, which requires cross-linking for activity, the chimeric protein encoding only the kinase domain was constitutively fully active. In growth factor-deprived stably transfected cell lines, expression of the chimeric protein encoding only the kinase domain resulted in maintenance of cell viability, delayed cell death, and increased expression of pim-1 and bcl-2 mRNA.
The construction of the cDNA
for CD16/Jak2-W (wild type Jak2) and its cloning into a recombinant
vaccinia virus transfer vector have been described previously (26). The
cDNA for CD16/Jak2-N (N-terminal deletion) was created by replacing
the MluI-NcoI fragment (the region coding for
residues 1-717) with a polymerase chain reaction product encoding
residues 536-717 with a MluI site in the appropriate
reading frame at the 5 end. The cDNA for CD16/Jak2-B (pseudokinase
deletion) was created by removing the BglII-BglII fragment (the region coding for residues 523-746) and religation. The
cDNA for CD16/Jak2-C (kinase domain deletion) was made by replacing
the NdeI-NotI fragment (the region coding for
residues 932-1129) with a polymerase chain reaction product encoding
residues 932-999 with a terminal codon and the NotI site at
the 3
end. The cDNA for CD16/Jak2-K (both N-terminal homology
domain and kinase-like domain deletion) was created by replacing the
MluI-NotI fragment (the entire Jak2 coding
region) with a polymerase chain reaction product encoding residues
825-1129 with a MluI site in the appropriate reading frame
at the 5
end and a NotI site at the 3
end. Correct
polymerase chain reaction and cloning were confirmed by dideoxy-DNA
sequencing. For establishment of stably transfected cell lines, the
HindIII-MluI fragment (the region coding for
CD16/CD7) and the MluI-NotI fragment (the region
coding for wild type Jak2 or Jak2 deletion mutants) were ligated into a
HindIII-NotI site of the pcDNA3 expression
plasmid (Invitrogen, San Diego, CA).
Recombinant vaccinia viruses were constructed as described previously (26). Recombinant vaccinia virus transfer vector plasmids were introduced into BSC-40 cells by electroporation, followed by infection with wild type vaccinia virus. After a 24-h culture, viruses were harvested. Cells containing recombinant viruses were selected with guanine phosphoribosyltransferase and thymidine kinase selection. The presence of the recombinant vaccinia viruses was confirmed by polymerase chain reaction.
Infection with Recombinant Vaccinia Virus and Cross-linkingBa/F3 cells were infected for 1 h in
serum-free Dulbecco's modified Eagle's medium with recombinant
vaccinia virus at a multiplicity of infection of 100. After a 12-h
culture, infected cells were deprived of conditioned medium and
incubated for 4 h in RPMI 1640 medium containing 0.5% albumin.
Cells were incubated with 5 µg/ml of anti-CD16 antibody
F(ab)2 for 2.5 min, followed by incubation at 37 °C
with 25 µg/ml anti-mouse IgG F(ab)
2 for the indicated times.
The murine IL-3-dependent cell line, Ba/F3, was maintained in RPMI 1640 medium with 10% fetal calf serum, 10 mM 2-mercaptoethanol, 20 mM HEPES, pH 7.8, and 10% WEHI-3B conditioned medium as a source of IL-3. Ba/F3 cells were transfected by electroporation (960 microfarads; 350 V) with 30 µg of plasmid DNA. Selection with G418 (1 mg/ml) was initiated 48 h after electroporation, and G418-resistant clones were selected by limiting dilution. The expression of the cDNA products was examined by Western blot analysis using anti-Jak2 antibody and flow cytometric analysis using anti-CD16 antibody.
AntibodiesThe anti-Jak2 antibody used for
immunoprecipitation was raised against residues 747-1129 (6). The
anti-Jak2 antibody raised against residues 758-776 was purchased from
Upstate Biotechnology, Inc. (Lake Placid, NY), and the anti-Jak2
antibody (c-20) raised against residues 1110-1129 was purchased from
Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal anti-CD16
antibody 3G8 F(ab)2 was purchased from Medarex, Inc.
(Annandale, NJ) and used for cross-linking, immunoprecipitation, and
flow cytometric analysis. Anti-phosphotyrosine 4G10 was purchased from
Upstate Biotechnology, Inc. Anti-STAT1 antibody and affinity-purified
polyclonal antibody to Shc were obtained from Transduction Laboratories
(Lexington, KY). Anti-STAT3 antibody and anti-STAT5 antibody were
purchased from Santa Cruz Biotechnology. Immunoprecipitation and
immunoblotting were performed as described previously (26).
Nuclear extracts were prepared, and EMSA was carried out as described previously (26). For the supershift assay, 1 µg of specific antibody was added to the nuclear extract. Incubation was carried out at room temperature for 20 min prior to the addition of radiolabeled oligonucleotide.
Measurement of [3H]Thymidine IncorporationCells (5 × 104) were cultured in
RPMI 1640 medium and 10% fetal calf serum in the absence of
conditioned medium for 5 h. Cells were either cross-linked with 1 µg of anti-CD16 antibody F(ab)2 or 10 µg of anti-mouse
IgG F(ab)
2 or stimulated with 10% WEHI-3B conditioned
medium for 17 h. Subsequently, the cells were pulse-labeled with 1 µCi of [3H]thymidine for 4 h prior to harvest.
Northern blots were carried out as described
(26). After hybridization, identical filters were stripped and reprobed
with c-Fos, c-Jun, JunB, and -tubulin. The pim-1 cDNA
(1.0-kilobase XhoI/HindIII fragment) and the
bcl-2 cDNA were gifts, respectively, of Dr. T. Meeker
(University of Kentucky) and Dr. Michael Ruppert (University of Alabama
at Birmingham).
To explore the possibility that
specific signals are regulated by different domains of the Jak2
protein, we deleted specific segments of the protein and fused these
deletion mutants in frame with CD16/CD7. The segments deleted were as
follows (Fig. 1): the region of the protein that
interacts with the subunit of the receptor located at the amino
terminus of the Jak2 protein and encompassing amino acid residues
1-536 (CD16/Jak2-N); the majority of the kinase-like domain,
encompassing amino acid residues 522-747 (CD16/Jak2-B); a portion of
the kinase domain encompassing amino acid residues 999-1129
(CD16/Jak2-C); and the entire amino-terminal region and the kinase-like
domain encompassing residues 1-825 (CD16/Jak2-K) but leaving the
kinase domain intact.
To obtain rapid, high level expression of the fusion proteins, the
cDNAs were cloned into vaccinia virus, which was then used to
infect IL-3-dependent Ba/F3 murine hematopoietic cells. The expression of fusion proteins of the correct size was confirmed by
immunoprecipitation and Western blotting using a Jak2-specific antibody
(Fig. 2, A and B). On Western blot
analysis, the reactivity of antibodies specific for the carboxyl
terminus of the kinase-like domain or the Jak2 kinase domain confirmed
the expression of the kinase-like domain, but not the kinase domain, in
the CD16/Jak2-C fusion protein (Fig. 2, A and B).
Fluorescence-activated cell-sorting analysis using an antibody specific
for CD16 demonstrated that the fusion proteins were expressed at
equivalent levels on the cell surface (data not shown). Proteins of
lower molecular weight, which represent nonglycosylated, intracellular
forms of the fusion proteins, were also expressed (Fig. 2C,
major band below the arrow) as described
previously (28). The levels of intracellular fusion protein appear to
correlate with the multiplicity of viral infection. With lower protein
expression, similar levels are expressed on the cell surface, but
smaller amounts of protein are seen intracellularly (data not
shown).
The CD16/Jak2-K Fusion Protein Stimulates Markedly Higher Levels of Constitutive Protein Phosphorylation than Other Fusion Proteins
Jak2 is autophosphorylated on tyrosines upon ligation of the hematopoietic growth factors by their receptors. To investigate the effect on autophosphorylation caused by deleting specific domains of Jak2, we stripped the Western blot shown in Fig. 2, A and B, and reprobed the filter with anti-phosphotyrosine antibodies. The low molecular weight, intracellular, unglycosylated forms of the CD16/Jak2-N, -B, and K fusion proteins (Fig. 2C, arrows) were constitutively phosphorylated, but their phosphorylation state was unaffected by cross-linking of the fusion proteins. As previously shown, cross-linking of the CD16/Jak2-W fusion protein resulted in phosphorylation of the glycosylated protein (Fig. 2C, arrow), which was not phosphorylated in the absence of cross-linking. As expected, the CD16/Jak2-C fusion protein, which lacks the kinase domain was not phosphorylated whether cross-linked or not. Autophosphorylation of the glycosylated forms of the CD16/Jak2-N, -B, and -K fusion proteins was not markedly increased by cross-linking. Only the CD16/Jak2-B fusion protein, in which the kinase-like region is deleted, showed a slight increase in phosphorylation on cross-linking. Significantly, although the amounts of protein were equivalent (Fig. 2A), the level of constitutive tyrosine phosphorylation was greatly enhanced in the CD16/Jak2-K fusion protein compared with the other fusion proteins.
To investigate the levels of cellular protein phosphorylation, the identical amounts of cell extracts used to investigate CD16/Jak phosphorylation (Fig. 2, A-C) were subjected to anti-phosphotyrosine immunoblotting. As previously demonstrated, cross-linking of the CD16/Jak2-W fusion protein resulted in the phosphorylation of proteins with molecular masses of 145, 125, 97, 93, 67, and 54 kDa (Fig. 2D). Proteins of identical molecular weights have been shown to be phosphorylated after the addition of IL-3 and GM-CSF to factor-dependent cell lines (29-31). Expression of the CD16/Jak2-C fusion protein did not result in significant phosphorylation of these substrates, either constitutively or after cross-linking. In multiple experiments, the base-line phosphorylation in the non-cross-linked CD16/Jak2-W was variable, being similar to the low levels seen in CD16/Jak2-C containing cells.
In contrast, the expression of the CD16/Jak2-N, -B, or -K fusion proteins resulted in constitutive phosphorylation of proteins of similar molecular weights to those phosphorylated by activation of the CD16/Jak2-W fusion protein. However, because of the prominent phosphorylation of the CD16/Jak2 fusion proteins, it was not possible to identify all of these substrate proteins in the CD16/Jak2-N- and CD16/Jak2-N-B- containing cells. As demonstrated for the autophosphorylation of CD16/Jak2-B, cross-linking induced a slight increase in the level of substrate phosphorylation.
Although identical amounts of cell extracts were loaded on this SDS gel, the level of tyrosine phosphorylation of all substrates was greatly enhanced in the cells expressing the CD16/Jak2-K fusion protein. Because the levels of tyrosine phosphorylation were so elevated, it was difficult to identify the molecular weight of the specific phosphorylated protein extracts. Immunoblots carried out using lesser amounts of proteins demonstrated that the identical proteins to those seen in the CD16/Jak-W Ba/F3 cells (molecular mass 145, 125, 97, 93, 67, and 54 kDa) were phosphorylated (data not shown). Therefore, the infection of Ba/F3 cells with a vaccinia virus-expressing fusion protein containing only the kinase domain of Jak2 induced the constitutive phosphorylation of proteins with molecular weights identical to those of the proteins phosphorylated after the addition of GM-CSF.
Shc Is Constitutively Phosphorylated by CD16/Jak2 Deletion Fusion ProteinsAlthough the patterns of phosphorylation are
complicated, close examination of the anti-phosphotyrosine immunoblots
does not disclose major differences in the range of proteins
constitutively phosphorylated by the Jak2 deletion mutants. To
determine whether an identical protein is phosphorylated by each of the
mutants, Shc protein phosphorylation was examined. Shc is
phosphorylated after the binding of hematopoietic growth factors to
their receptors, suggesting that Shc is a natural target of the Jak2
cascade of protein kinases. Immunoprecipitation of Shc before and after
cross-linking indicated that cross-linking of CD16/Jak2-W, but not the
CD16/Jak2-C, fusion protein resulted in phosphorylation of Shc (Fig.
3). On the other hand, expression of the CD16/Jak2-N,
-B, and -K fusion proteins resulted in constitutive phosphorylation of
Shc, which was not increased by cross-linking. The phosphorylation of
Shc did not occur simply as a result of fusing a tyrosine kinase to CD16, since the CD16/Zap70, which was expressed at the same level as
the other fusion proteins (data not shown), did not highly phosphorylated Shc. The p145 protein, SHIP, which is associated with
Shc (27), was similarly phosphorylated, requiring cross-linking of the
CD16/Jak2 wild type but not the other fusion proteins (data not shown).
Reprobing of this Western immunoblot demonstrates that the CD16/Jak2
protein was coimmunoprecipitated with Shc (Fig. 4).
To examine whether these fusion proteins could phosphorylate specific
members of the STAT family, the activity of individual STAT family
members was examined. Individual members of the STAT family of proteins
are activated after the addition of specific growth factors to cells
(32-34), and phosphorylation is mediated by specific members of the
Jak family of proteins. These transcription factors require tyrosine
phosphorylation for dimerization and DNA binding. The addition of
GM-CSF to factor-dependent cell lines stimulates the
activity of STAT5 while having no effect on STAT1. To evaluate the
specificity of regulation of this family of proteins by the CD16/Jak2
chimera, cell extracts were subjected to electrophoretic mobility shift
assay (EMSA) using a interferon- activation site or
-activated
site (GAS oligomer) that is capable of binding multiple members of the
STAT family (35). As we reported previously (26), cross-linking of the
CD16/Jak2-W protein led to STAT protein binding to DNA (Fig.
5A). In contrast, cross-linking of either the
CD16/Jak2-C or the CD16/Zap70 had no effect on these assays (Fig.
5A). Expression of the CD16/Jak2-N, -B, and -K fusion
proteins resulted in a band shift, which was not affected by
cross-linking, although some slight variability was seen in this assay
(Fig. 5A). To determine which of the STAT family of proteins
was activated by these fusion proteins, antisera to STAT1, -3, and -5 were used to retard the EMSA complex. Using this technique, the
addition of conditioned medium (IL-3) to Ba/F3 cells stimulates the
activation of STAT5, whereas the addition of interferon-
activates
STAT1 (36) (Fig. 5B). Antisera to STAT5 but not antisera to
STAT1 and STAT3 supershifted the bands induced by cross-linking of the CD16/Jak2-W fusion protein (Fig. 5B), as well as the bands
constitutively induced by the expression of CD16/Jak2-N, -B, and -K
fusion proteins (data not shown). Therefore, each of the Jak2 fusion
proteins was able to activate STAT5 and not STAT1, thus mimicking the
biologic effects of binding of GM-CSF or IL-3 to its receptor.
Deletions of the amino-terminal and kinase-like domains of Jak2 led to
the constitutive activation of STAT DNA binding activity.
Expression of CD16/Jak2 Fusion Proteins in Stably Transfected Ba/F3 Cell Lines
In the experiments above, vaccinia virus vectors were
used to express the fusion proteins in Ba/F3 cells, permit high levels of expression of these proteins, and facilitate analysis of the phosphorylated substrates. Vaccinia virus infection affects Ba/F3 cell
growth; therefore, cells prepared using this technique could not be
used for analysis of the effects of the fusion proteins on cell growth.
Each of these fusion proteins was cloned into a mammalian expression
vector containing a CMV promoter, which was used to transfect Ba/F3
cells. Clones were selected and expanded to establish stably
transfected cell lines, and three or more independent isolates of each
fusion protein expressing clones were characterized. The presence of
the fusion proteins in these cells was confirmed by immunoprecipitation
with Jak2-specific antibodies, followed by Western blotting using
Jak2-specific antibodies (Fig. 6). Because background
proteins block the ability to easily detect the CD16/Jak2-N3 fusion,
fluorescence-activated cell-sorting analysis using anti-CD16 antibody
was used to confirm the equivalent surface expression of the fusion
proteins (data not shown). As with the recombinant virus-infected
cells, only the CD16/Jak2-W and CD16/Jak2-B fusion proteins exhibited
increased tyrosine phosphorylation upon cross-linking, and the
CD16/Jak2-K, -N, and -B mutants were constitutively phosphorylated. The
level of phosphorylation of the CD16/Jak2-K fusion protein was markedly
enhanced compared with that of other fusion proteins. CD16/Jak2-K
clones exhibited constitutive activation STAT and tyrosine
phosphorylation of Shc (data not shown). Phosphorylation of STAT by the
CD16/Jak2-N and -B clones was reduced when compared with the
CD16/Jak2-K clones. In stably transfected cell lines, as in recombinant
virus-infected cells, it is again evident that the non-kinase regions
of Jak2 play an important role in regulating Jak2 protein kinase
activity.
IL-3-deprived Ba/F3 Cells Stably Transfected with the CD16/Jak2-K Fusion Protein Incorporate [3H] Thymidine and Exhibit Delayed Cell Death
Ba/F3 cells depend on the presence of IL-3 to
suppress apoptosis and stimulate DNA synthesis and cell proliferation.
To determine whether Jak2 is capable of mediating all, or some, of the
biologic effects induced by binding of the hematopoietic growth
factors, the Ba/F3 cell lines expressing the fusion proteins were
deprived of conditioned media (IL-3). Parental cells deprived of
conditioned medium were unable to incorporate thymidine, but thymidine
uptake was stimulated by the addition of conditioned medium (Fig.
7A). In contrast, the CD16/Jak2-K cell line
(K-16) incorporated thymidine in the absence of conditioned medium,
resulting in a slight increase on cross-linking (Fig. 7A).
Similar results were obtained with all CD16/Jak2-K clones (K-1, K-4,
and K-18). Cell lines expressing the other fusion proteins behaved like
the parental Ba/F3 cells in that they were unable to incorporate
[3H]thymidine when deprived of conditioned medium. The
possibility that the cell line expressing the CD16/Jak2-K (K-16)
produced growth factors that stimulated thymidine uptake was excluded
by the absence of thymidine uptake in parental Ba/F3 cells incubated with conditioned medium obtained from the K-16 cells.
We next tested the effect of the fusion proteins on cell death as estimated by trypan blue exclusion. The CD16/Jak2-W, -B, and -N cell lines lost viability at the same rate as the IL-3-deprived parental cells (Fig. 7B). However, all four of the CD16/Jak2-K-transfected cell lines (K-1, K-4, K-16, and K-18) lost viability more slowly. Furthermore, the IL-3 deprived CD16/Jak2-K cell lines lost viability more rapidly than parental cells treated with conditioned medium.
Expression of CD16/Jak2-K Increases the Level of pim-1 and bcl-2 mRNATo obtain clues as to which genes might be involved in
the factor-independent survival of cells containing the CD16/Jak2-K fusion protein, we examined the level of expression of various mRNAs by Northern blot analysis of clone K-16 cells after growth factor deprivation, cross-linking, and growth in 10% FCS (Fig. 8A). When compared with growth
factor-deprived parental cells, deprived K-16 cells demonstrated slight
elevations in jun-B, but not c-fos or
c-jun mRNA. As in parental cells, cross-linking induced an increase in the level of expression of c-fos,
c-jun, and jun-B mRNA, although the level of
expression of jun-B mRNA was lower than that in parental
cells, whereas that of c-jun was slightly higher (Fig.
8A). As in parental cells, the addition of conditioned medium (IL-3) to clone K-16 cells stimulated increases in
c-jun, jun-B, and c-fos mRNA.
To approximate the conditions used to assess cell death of the CD16/Jak2-K cells, 10% FCS was added to the K-16 cells for 12 h (Fig. 8A, lanes 7-10). When compared with factor-deprived K-16 cells, the addition of 10% FCS appeared to result in a slight elevation in the levels of junB mRNA, whereas the levels of c-jun and c-fos mRNA were unchanged. Thus, the CD16/Jak2-K cells contain low levels of specific mRNAs associated with growth. Importantly, although the level of tyrosine phosphorylation of protein substrates does not appear to increase after cross-linking of this protein kinase fusion protein, substantial increases in the cellular levels of mRNAs were still seen.
Pim-1 expression is regulated by the addition of GM-CSF (37), and the
expression of this gene appears to be controlled by a domain of the chain close to the plasma membrane (5). Since Jak2 binds to a similar
region of the
chain, we evaluated the levels of pim-1
mRNA in CD16/Jak2 cells. The addition of conditioned medium (IL-3),
but not FCS, to deprived parental Ba/F3 cells markedly elevated the
level of pim-1 mRNA (Fig. 8B). CD16/Jak2-K
cells growing in FCS, in the absence of conditioned medium (IL-3),
maintained continuously elevated levels of pim-1 mRNA,
similar to those observed in the hormone-treated parental cells. Thus,
the CD16/Jak2-K fusion protein is capable of stimulating increases in
pim-1 mRNA levels.
The Bcl-2 family of proteins plays a role in the regulation of apoptosis (38), and overexpression of Bcl-2 prevents hematopoietic cells from undergoing apoptosis (39). Using Northern blots, we evaluated the levels of bcl-2 mRNA in two different clones of CD16/Jak2-K cells grown in 10% FCS (Fig. 8B). Parental cells incubated in either 0.5% albumin or 10% FCS expressed low levels of bcl-2 mRNA. The addition of conditioned medium to parental cells for 12 h markedly elevated the levels of bcl-2 mRNA. Strikingly, the levels of bcl-2 mRNA were also elevated in both cell lines, K-16 and K-18 (K-1 and K-4 data not shown), containing the CD16/Jak2-K fusion protein. Therefore, cells expressing the fusion protein that encodes the Jak2 kinase domain constitutively express Bcl-2 protein, which in turn inhibits cell death.
The addition of GM-CSF and IL-3 to factor-dependent
cells activates Jak2 protein kinase and stimulates specific signal
transduction pathways leading to cell growth. However, the exact role
of Jak2 activation in either the stimulation of specific
phosphorylation events or in the control of specific aspects of cell
growth, such as stimulation of DNA synthesis and proliferation or
suppression of apoptosis, remains ill-defined. To clarify the function
of Jak2 in these two processes, we have employed a CD16/Jak2 fusion protein that can be activated by cross-linking with CD16 antibodies. Under physiologic circumstances, Jak2 is activated when interacting with domains of hematopoietic growth factor receptors that are physically close to the plasma membrane. Similarly, in this model system, activation of Jak2 occurs physically adjacent to the plasma membrane. Previously, we demonstrated (26) that activation of this
fusion protein neither stimulates subunit phosphorylation nor
causes activation of endogenous Jak2, suggesting that this fusion
protein functions independently of the hormone receptor.
In the present studies, we used deletion mutations of Jak2 fused to CD16 to analyze the roles of the various domains of Jak2 in regulating phosphorylation of specific substrates. In these experiments, viral expression of CD16/Jak2 fusion gave large amounts of intracellular fusion protein, which is tyrosine-phosphorylated, with the level of intracellular protein being directly related to the multiplicity of infection. At lower multiplicity of infection (26), the intracellular levels of fusion proteins drop, while similar levels of protein are expressed on the plasma membrane.
In contrast to results obtained with the Jak2 wild type fusion protein,
deletion of the B or N region of the Jak2 protein allowed constitutive
phosphorylation of these substrates, including the fusion protein
itself in the absence of CD16 cross-linking. The proteins
phosphorylated by these deletion chimeras (for example, Shc) are known
substrates of Jak2 that are modified in response to the binding of
hematopoietic growth factors. These fusion proteins activate STAT5
specifically and not STAT1, demonstrating that these fusion proteins
are not phosphorylating cellular substrates randomly. One explanation
for the ability of these fusion proteins to modify Shc and STAT
proteins is that the fusion kinase is phosphorylating a transmembrane
hormone receptor protein that acts as a docking site for the Shc and
STAT. Recent data from other laboratories suggests that STAT5 directly
binds Jak2, which contains multiple phosphorylation
sites.2 The direct coprecipitation of Jak2
and Shc has been demonstrated in a cell line that responds to
erythropoietin (40). As well, if the subunit of the GM-CSF receptor
is deleted such that it does not contain any tyrosines, the addition of
GM-CSF can still activate STAT5 activity (41), suggesting that
additional proteins may be involved in STAT5 activation.
Deletion of neither the amino terminus nor the kinase-like region of Jak2 appears to change the general substrate specificity of the fusions, suggesting that these regions do not play a major role in defining the substrate specificity of these protein kinases within the CD16 fusion protein. The constitutive and enhanced phosphorylation induced by the CD16/Jak2 fusion suggests that the amino-terminal half and the kinase-like domain of the Jak2 protein might function as a regulatory region that inhibits Jak2 protein kinase activity. It is possible that Jak2 activity is normally inhibited by the amino-terminal half of the protein and that binding of Jak2 through the amino-terminal half of the protein to hematopoietic receptors relieves this inhibition. Alternatively, growth factor-induced phosphorylation of specific tyrosines within the amino-terminal half of the Jak2 protein might allow the docking of a phosphatase that inhibits the activity of the protein kinase. The PTP1C phosphatase binds to a tyrosine in the erythropoietin receptor tail and inhibits the activity of this receptor (42), with removal of this binding site greatly increasing the activity of the erythropoietin receptor (42). In addition, the two-hybrid yeast system has been used to show that a sequence in the kinase-like domain from the atrial natriuretic peptide receptor binds a phosphatase (43), suggesting that deletion of the kinase-like domain in Jak might remove a phosphatase binding site. The observation that a point mutation amino-terminal region of a Drosophila Jak-like protein leads to hyperactivation of this protein (44) again suggests that this portion of the molecule is important in regulating protein kinase activity. Our results support the idea that the amino terminus of Jak2 plays an important role in regulating Jak2 kinase activity.
We took advantage of the observation that CD16/Jak2 kinase domain fusion is constitutively highly active to examine the effects of this fusion protein on the growth of Ba/F3 cells. Cells expressing the CD16/Jak2-K fusion, but not the B or N deletion, demonstrated a delay in cell death after withdrawal from IL-3. This result was demonstrated in four independent clones, suggesting that blocking of cell death was not obtained because of clonal variation. These cells, but not those containing the B or N deletion, evidenced elevated levels of pim-1 and bcl-2 mRNAs that normally were found only in growth factor-treated cells.
The importance of pim-1 is suggested by the observation that bone marrow-derived mast cells purified from mice in which the pim-1 gene has been "knocked out" grow less well in IL-3 than control cells (45), suggesting an important role for pim-1 in regulating IL-3-induced growth. That Jak2 can regulate levels of Pim-1 has been suggested previously on the basis of the observation that erythropoietin receptors that cannot stimulate Jak2 kinase activation also cannot elevate the levels of pim-1 mRNA (46). The importance of pim-1 in hormone-mediated growth is demonstrated by the observation that dominant negative STAT5 inhibits the expression of Pim-1 and partially blocks the IL-3-dependent growth in Ba/F3 cells (34). Thus, the constitutive activation of STAT5 induced by the CD16/Jak2-K may mediate the induction of Pim-1.
Our results demonstrate that constitutive Jak2 activation alone (accomplished with our fusion proteins) is sufficient to activate the transcription of the bcl-2 and pim-1 mRNAs but is not sufficient to allow for continued cell growth. These findings are consistent with the previous observation that a dominant-negative Jak-2 mutation can block cell growth (20, 47) and hormone-induced rescue of cells from apoptosis (21). Other researchers have suggested that three pathways are necessary to stimulate factor-independent cell growth (48, 49). In these experiments, overexpression of Bcl-2 was not sufficient to induce factor-independent growth, but it was sufficient to inhibit cell death (48, 49). The addition of either c-Myc or p56lck to the cells carrying the Bcl-2 protein made these cells growth factor-independent and suggested that three stimuli were necessary for cell growth: increased Bcl-2, increased c-Myc, and the presence of an activator of the MAP kinase pathway, either Ras or Lck (48, 49). The mechanism by which both cell growth and cell death were delayed in our cells may be partly related to the elevation of bcl-2 mRNA. It is therefore likely that expressing additional genes, e.g. c-myc or p56lck, in the Ba/F3 cells containing CD16/Jak2-K may provide signals necessary for growth. Using this fusion protein, we demonstrate that the activation of Jak2 was not sufficient alone to stimulate continued cell growth.
We thank the Dr. Jeffrey Kudlow laboratory (University of Alabama at Birmingham) for assisting us with the vaccinia virus system and the laboratory of Dr. Brian Seed (Massachusetts General Hospital) for supplying us with the CD16/CD7 fusion vector. We appreciate the review of this manuscript by Drs. Fiona Hunter and Bill Weaver. We thank Y. Zhao for technical assistance.