From the
Growth hormone (GH) treatment of cells promotes activation of
JAK2, a GH receptor (GHR)-associated tyrosine kinase. We now explore
JAK2 regions required for GHR-induced signaling. Wild-type (WT) JAK2
and JAK2 molecules with deletions of the amino terminus
(JAK2
Growth hormone (GH)
Recent evidence indicates that association of JAK2 with the GHR is
dependent on a perimembranous proline-rich region of the receptor
cytoplasmic domain(17, 18, 19) . Further,
removal of this region precludes GH-induced JAK2 and GHR tyrosine
phosphorylation, mitogen activated protein kinase activation, and gene
transcription(17, 18, 19, 20) . These
data underscore the necessity for the proper physical association
between the GHR and JAK2 to potentiate their GH-induced functional
coupling.
In this study, we explore by mutagenesis the regions of
JAK2 which are required for physical and functional interaction with
the GHR. JAK2 is a protein of 1129 amino acids(6) . Like the
other JAK family members (12-15) it is structurally characterized
by the presence of: (a) a consensus kinase domain in the
residue 849-1122 region; (b) a potential consensus motif for
tyrosine autophosphorylation (-EYYKVKE-) at residues 1006-1012; (c) a kinase-like (or ``pseudokinase'') domain of
unclear significance extending from residues 543 to 806; and (d) an amino-terminal half of the molecule with regions of
similarity among JAK family members, including a potential tyrosine
phosphorylation site (-IDGYYRL-) at residues 369-375. No clear
SH2 or SH3 domains are found.
We used a COS-7 cell-based transient
cotransfection system to test mutated JAK2 molecules in assays of
hGH-induced transcriptional activation, extracellular signal-regulated
kinase (ERK) SDS-PAGE mobility shift, JAK2 tyrosine phosphorylation,
and GHR-JAK2 coimmunoprecipitation. Our findings indicate that internal
deletion of the kinase-like domain does not prevent physical or
functional coupling of JAK2 with the rabbit (r) GHR. A deletion of the
JAK2 COOH terminus that disrupts the kinase domain prevents these
aspects of hGH signaling, but does not inhibit JAK2 association with
the rGHR. Amino-terminal truncation of one-fifth of the JAK2 molecule
abrogates both physical and functional coupling to the rGHR.
Further, expression and testing of rGHR/JAK2 chimeras indicates that
direct linkage of the COOH-terminal half of JAK2 (including the
kinase-like and kinase domain) or the COOH-terminal one-third of JAK2
(including only the kinase domain) to the portion of the GHR including
the external and transmembrane domains yields a receptor that can
confer a GH-dependent transcriptional activation. Inclusion of the
kinase-like domain only in such a chimera results in a receptor unable
to transduce this signal. These results complement the JAK2 deletional
analysis and support the conclusion that a role of the amino-terminal
region of JAK2 is to facilitate the native interaction with the GHR.
The p2FTL fos-luciferase reporter
plasmid (a gift of Dr. J. Kudlow, UAB, and Dr. W. Chen, Massachusetts
Institute of Technology.) has been described(21) . In brief,
from 5` to 3` it contains two copies of the c-fos 5`-regulated
enhancer element (-357 to -276), a fragment of the herpes
simplex virus thymidine kinase gene promoter (-200 to +70),
and the gene for firefly luciferase. Each copy of the c-fos enhancer element contains the sis-inducible element, the serum
response element, and an AP-1-binding site(22) .
pRc/CMV ERK2
The creation of the pRc/CMV WTrGHR and pRc/CMV rGHR
Creation of the chimera incorporating both the
kinase-like and kinase domains rGHR/JAK2
For
overexpression of mutant and WT mJAK2, 8
For assays of binding of mutant and WT mJAK2 to GST fusions,
fusion protein induction and affinity purification on
glutathione-Sepharose beads (Pharmacia) were as described
previously(19) . Bacterial extract containing roughly 20 µg
of the GST/hGHR
The
anti-JAK2 used for immunoprecipitation experiments has been
described.
The anti-hGHR serum used for
immunoprecipitation has been previously described(26) . For
coimmunoprecipitation experiments, hGH-stimulated (300 ng/ml for 5 min
at room temperature) COS-7 cells expressing the rGHR and either mutant
or WT mJAK2 were solubilized at approximately 1
Resolution of proteins under reduced conditions by SDS-PAGE,
Western transfer of proteins, and blocking of nitrocellulose membranes
(Schleicher and Schuell) with 2% bovine serum albumin were performed as
described previously(19, 26) . For immunoblotting,
anti-JAK2 peptide antiserum was used at 1:1000, and detection was
accomplished using biotinylated protein A (Amersham Corp.) and a
streptavidin alkaline phosphatase conjugate (Bethesda Reseach
Laboratories) along with the BCIP/NBT detection system (Promega,
Madison, WI), according to manufacturers' instructions. Anti-HA
12CA5 was used at 1:750 for immunoblotting and was detected with a goat
anti-mouse immunoglobulin G (Fab`)
As seen in Fig. 2, A and B, transient transfection of the rGHR, WT mJAK2, and p2FTL
into COS-7 cells resulted in an hGH-induced time- and
concentration-dependent accumulation of luciferase activity with
appreciable increases (greater than 2-fold) seen as early as 4 h into
hGH treatment. Maximal stimulation (3-4.6-fold) was routinely
achieved with a treatment of 18-24 h with 500 ng/ml hGH. Notably,
no hGH-induced augmentation of luciferase activity was detected if the
WT mJAK2 expression plasmid was omitted from the transfection (Fig. 2C). Therefore, we conclude that either the
expression level/cell of the endogenous COS-7 JAK2 or its particular
nature (monkey versus mouse) does not allow sufficient
coupling to the transfected rGHR to promote significant hGH-induced
activation of transcription. Consistent with our previous data
demonstrating a lack of endogenous hGH-binding sites in COS-7
cells(19) , no hGH-induced luciferase activation was detected if
the rGHR was omitted from the transfection mix (not shown).
The results of
comparisons of each JAK2 mutant with WT mJAK2 in this assay are shown
in Fig. 4, A-C. In each case, the hGH-induced appearance
of APT-precipitable WT mJAK2 is evident as a positive control.
Similarly, a robust signal is induced by hGH treatment of transfectants
expressing JAK2
In the experiments shown in Fig. 5, A-C, COS-7 cells transfected with the cDNAs for
the rGHR and mJAK2 (either WT or mutant) were treated with hGH,
solubilized in a detergent lysis buffer, cross-linked with a
thiol-cleavable cross-linker, and immunoprecipitated with anti-GHR (80%
of the extract) or anti-JAK2 (20% of the extract). The eluates were
resolved by SDS-PAGE under reducing conditions, thereby cleaving the
cross-linker, and immunoblotted with anti-JAK2 peptide serum. The
results reveal that WT mJAK2 (Fig. 5, A-C), JAK2
The rGHR/JAK2 chimeras were tested for
their ability to mediate the hGH-induced transcriptional activation of
the c-fos-luciferase reporter in COS-7 cells. Two
representative experiments are shown in Fig. 6C. Similar
to the results in Fig. 2, cotransfection of p2FTL, WT rGHR, and
WT mJAK2 enabled a 4.6-fold induction of luciferase activity after 22 h
of hGH treatment. Transfectants expressing rGHR/JAK2
Interactions between signaling molecules (activated
receptors, enzymes, docking molecules) are often dependent on the
presence in the interacting proteins of SH2 and/or SH3 domains and
their tyrosine phosphorylated or proline-rich binding
sites(36, 37, 38, 39) . Likely, the
hematopoietin receptors will also be shown to utilize such motifs in
their associations with some of the proteins involved in their
signaling complexes. However, the interaction between these receptors
and JAK family kinases appears to be independent of such known motifs
or modifications. JAKs have no obvious SH2 or SH3 domains and, though
studies of the GHR have indicated a ligand-induced enhancement of
receptor association with JAK2(1) , other hematopoietin
receptors appear to be constitutively associated with their JAK-family
kinase(2, 3, 5, 7, 9) . Indeed,
we have observed that the in vitro association of a GST fusion
protein containing the hGHR cytoplasmic domain with IM-9 cell-derived
JAK2 is not dependent on prior hGH-treatment of those cells or tyrosine
phopshorylation of either JAK2 or the hGHR(19) .
The
proline-rich perimembranous region of the GHR cytoplasmic domain has
been implicated by us and others as important in interaction with
JAK2(17, 18, 19) . Though rich in proline
residues, this so-called Box1 region does not constitute a canonical
SH3-binding site. Box1 has similar counterparts in various
hematopoietin receptors, which relate to association with
JAKs(5, 7, 8, 40, 41) . In this
study, we specifically map JAK2 regions involved in physical and
functional interaction of the GHR and JAK2. Using similar approaches,
we have also recently examined the JAK2 interaction with the
granulocyte macrophage-colony-stimulating factor receptor
Our findings of reconstitution experiments
in COS-7 cells support the contention that GH signaling requires proper
association of the GHR with JAK2 and enzymatic activation of JAK2 in
response to the GH-GHR interaction. Specific results that allow this
interpretation include the following. 1) Transient coexpression of WT
mJAK2 and the rGHR leads to anti-GHR coprecipitability of JAK2. 2)
Detectable hGH-induced biochemical events resulting from such
coexpression include JAK2 APT precipitability, retardation of migration
in SDS-PAGE (likely corresponding to phosphorylation) of an
epitope-tagged ERK2, and transactivation of a luciferase reporter with
c-fos enhancer elements. 3) None of the findings in 1 and 2
above occur to a significant degree if the rGHR is transiently
expressed without transfected JAK2, presumably relating to the
inadequate expression level or interacting capacity of the endogenous
COS-7 monkey JAK2.
Reconstitution experiments with amino- and
carboxyl-terminal deletion mutants of JAK2 further support these
notions. Carboxyl-terminal deletion (JAK2
Since JAK2
Interestingly, deletion of the kinase-like domain
(JAK2
The results obtained
with the chimeric GHR/JAK2 molecules (Fig. 6) are consistent with
the JAK2 deletional analysis. Reasoning that the amino terminus of JAK2
serves as an interacting domain, we eliminated it and directly fused
either the kinase-like domain, the kinase domain, or both domains to a
severely truncated rGHR. The rGHR component is almost identical to our
previously described rGHR
Each of our chimeras bound hGH at the cell surface. When assayed for
fos-luciferase transactivation capability, the chimeras that
incorporate the JAK2 kinase domain (rGHR/JAK2
We gratefully acknowledge Drs. J. Ihle, W. Wood, M.
Mulligan, W. Chen, J. Kudlow, J. Woodgett, and G. Yancoupoulos for
contribution of plasmids and Eli Lilly Co. for the hGH. We appreciate
helpful conversations with Drs. J. Kudlow, A. Paterson, T. Shin, and J.
J. O'Shea and critical review of the manuscript by J. Kudlow and
E. Chin.
Addendum-While this manuscript was under review, a study of
JAK2 regions involved in interaction with the GHR cytoplasmic domain
was reported. Those results also indicate importance for the
amino-terminal region of JAK2 in that interaction(43) .
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
), carboxyl terminus (JAK2
), or
kinase-like domain (JAK2
) were each transiently
coexpressed in COS-7 cells with the rabbit GHR. The following responses
were assayed: GH-induced transactivation of a luciferase reporter
governed by a c-fos enhancer element; GH-induced shift in the
molecular mass of a cotransfected epitope-tagged extracellular
signal-regulated kinase molecule; and GH-induced antiphosphotyrosine
immunoprecipitability of the transfected JAK2 form. In each assay,
WTJAK2 and JAK2
allowed GH-induced signaling, whereas
JAK2
and JAK2
did not. Anti-GHR serum
coimmunoprecipitated WTJAK2, JAK2
, and
JAK2
, but not JAK2
. Finally, a chimera in
which the JAK2 kinase domain replaced the GHR cytoplasmic domain
signaled GH-induced transactivation. We conclude: 1) kinase-like domain
deletion eliminates neither physical nor functional interaction between
JAK2 and the GHR; 2) kinase domain deletion eliminates functional but
not physical coupling of JAK2 to the GHR; 3) interaction with the GHR
appears dependent on the NH
-terminal one-fifth of JAK2; and
4) a GH-responsive signaling unit can include as little as the GHR
external and transmembrane domains and the JAK2 kinase domain.
(
)action is initiated
by specific interaction of the polypeptide hormone with a cell surface
glycoprotein, the GH receptor (GHR). A very early and apparently
obligate step in GHR signal transduction is the activation of the
GHR-associated cytoplasmic tyrosine kinase, JAK2(1) . In this
respect, the GHR is similar to other members of the hematopoietin
receptor family (such as receptors for prolactin, erythropoietin,
interleukin-3, granulocyte macrophage-colony-stimulating factor,
interleukin-6, leukemia inhibitory factor, oncostatin M, cillary
neurotrophic factor, and interferon
)(2, 3, 4, 5, 6, 7, 8, 9, 10, 11) .
These receptors also use JAK2 to facilitate ligand-induced signaling.
Still other members of this receptor family couple to the related
JAK-family kinases TYK2, JAK1, and
JAK3(12, 13, 14, 15, 16) .
Materials
Recombinant hGH was kindly provided by
Eli Lilly Co. Routine reagents were purchased from Sigma unless
otherwise noted. Restriction endonucleases were obtained from New
England Biolabs (Beverly, MA). Oligonucleotides were synthesized and
purified in the facility of Dr. J. Engler.
Cells and Cell Culture
COS-7 cells were maintained
as described previously(19) . CV-1 cells were maintained in
Dulbecco's modified Eagle's Medium (DMEM) (Biofluids,
Rockville, MD) supplemented with 10% newborn calf serum (Biofluids) and
50 µg/ml gentamicin sulfate, 100 units/ml penicillin, and 100
µg/ml streptomycin (all Biofluids).
Plasmid Construction
The murine JAK2 cDNA was
provided by Dr. J. Ihle (St. Jude Children's Research Hospital,
Memphis, TN) and was ligated into the pRc/CMV expression plasmid
(InVitrogen) as described previously(19) . Generation of the
mutant JAK2 cDNAs in pRc/CMV is described in detail
elsewhere.(
)In brief, the cDNA for JAK2
was created in pBluescript by replacing the region coding for
residues 1-294 (using an endogenous EcoRI site at
residue 294) with a PCR product encoding residues 240-294 with a
restriction site and an ATG start codon at the 5` end. Similarly, the
cDNA for JAK2
was created by replacing the region
encoding residues 932 (an NdeI site) to 1129 with a PCR
product encoding residues 932-999 with a termination codon on the
3` end. The cDNA for JAK2
was created by removal of the
residue 523-746 region at two in-frame BglII sites in
the cDNA and religation. All the cDNAs were subcloned into pRc/CMV, as
previously described(19) .
was constructed by ligating a PCR fragment of the entire
rat ERK2 cDNA (kindly provided by Dr. G. Yancoupoulos, Regeneron, Inc.)
coding region by a 5` NcoI site and a 3` ClaI site
into the pBluescript SK tag vector (a gift of Dr J. Woodgett, Ontario
Cancer Institute). The ERK2 coding region with the 5` tag sequence
(-YPYDVPDYASLGGP- of the influenza hemagglutinin) was removed from the
pBluescript SK and subcloned via NotI and ApaI sites
into the pRcCMV eukaryotic expression plasmid. The ERK2 and the 5` tag
coding region sequences were confirmed by dideoxy DNA sequencing.
plasmids has been described(19) . To construct the
rGHR/JAK2
and rGHR/JAK2
chimeras, we used the
pBluescript mJAK2 (referred to above) as a template for PCR of the
regions encoding residues 525-778 (called PK because it encodes
the kinase-like or pseudokinase domain) and residues 753-1129 (referred
to as K because it encodes the kinase domain). In each case restriction
sites for BamHI (5`) and HindIII (3`) were included
on the primers for PCR. After restriction digestion, the PK and K BamHI-HindIII fragments were ligated into the
previously described (19) pBluescript
rGHR
plasmid at the BglII and HindIII sites. This yielded in each case
cDNAs in pBluescript encoding the rGHR
devoid of
residues 278 to 292 (which harbor the Box1 element) directly fused
in-frame to regions of JAK2 containing either the kinase-like domain or
the kinase domain.
was
accomplished by removal via BglII and HindIII of the
region encoding residues 746-1129 from pBluescript mJAK2 and its
ligation into the BglII-HindIII digested pBluescript
rGHR/JAK2
. The rGHR/JAK2
cDNA thus encodes
the severely truncated and Box1-deleted rGHR fused to JAK2 residues
525-1129 (the COOH-terminal 53% of the JAK2 molecule). Correct PCR and
cloning were verified by dideoxy DNA sequencing of relevant regions,
and each cDNA was subcloned into pRc/CMV, as described(19) , for
eukaryotic expression. Notably, each of the three rGHR/JAK2 chimera
cDNAs encodes the JAK2 residue 758-776 region and would thus be
predicted to have reactivity with the anti-JAK2 peptide serum (see
below) raised against that sequence.
Protein Expression
COS-7 cells were transfected at
60-80% confluence in 10 ml of DMEM medium in 100 20-mm
tissue culture dishes (Becton-Dickinson) by the calcium phosphate
precipitation method, as described previously(19, 23) .
For individual experiments, the number of dishes transfected per
condition is indicated in the figure legend. For experiments comparing
the mutant and WT mJAK2, each dish was transfected with 30 µg of
wild type or mutant rGHR-containing pRC/CMV along either 9 µg of
the WT mJAK2, 36 µg of JAK2
, 30 µg of
JAK2
, or 9 µg of JAK2
cDNA in pRc/CMV.
In all cases, the total amount of DNA transfected was equalized by
addition of pRc/CMV. The rGHR/JAK2 chimeras in pRc/CMV were transfected
at 30 µg/dish. When used for cotransfection, p2FTL was added at 7
µg/dish and pRc/CMV ERK2
at 10 µg/dish.
10
CV-1
cells/100
20-mm dish were infected with vvT7 pol vaccinia virus (24, 25) (kindly provided by Dr. M. Mulligan, UAB) at
multiplicity of infections = 5 in 2 ml of DMEM without serum at
37 °C for 1 h. After infection, the cells were incubated in
serum-containing medium for 2 h and then transfected with the
above-mentioned JAK2 cDNAs in pRc/CMV using Lipofectin (Life
Technologies, Inc.) at a ratio of 3-7 µg of plasmid plus
20-40 µl of Lipofectin reagent/dish for 4 h. Twenty-four h
after transfection, the cells were harvested and solubilized as below.
c-fos-Luciferase Transactivation Assay
COS-7 cells
(8 10
/dish) were transfected as described above
along with p2FTL. At 18 h after transfection, the cells were split into
24-well plates (approximately 80,000 cells/well). Serum starvation
(substitution of 0.5% bovine serum albumin for fetal calf serum in the
medium) was begun at 24 h after transfection and hGH (at 500 ng/ml or
the dose indicated) or vehicle was added for the durations indicated,
such that all stimulations (done in triplicate) were terminated
simultaneously. Stimulations were ended by aspiration of the medium and
addition of luciferase lysis buffer (1% Triton X-100, 10% glycerol, 2
mM dithiothreitol, 2 mMtrans-1,2-diaminocyclohexane-N,N,N`,N`-tetraacetic
acid, 25 mM Tris-HCl, pH 7.8). Luciferase activity was assayed
by mixture of equal volumes of the resulting cell extract and
luciferase assay buffer (470 mM luciferin and 270 mM CoA) followed by counting for 10 s in a luminometer (Analytical
Luminescence Laboratory). Results are plotted as the hGH-induced
increase (mean ± 1 S.D.) relative to unstimulated (vehicle only)
controls. The control values at each point are considered 100%.
Cell Stimulation, Protein Extraction, and GST Fusion
Binding Assays
Serum starvation of COS-7 cells, usually at 24 h
after transfection, was accomplished by substitution of 0.5% bovine
serum albumin for fetal calf serum in the DMEM cuture medium for
another 16-20 h. The stimulation protocol has been
described(19) . Unless otherwise noted, hGH was used at a final
concentration of 500 ng/ml for 10 min at 37 °C. Cells were washed
once with ice-cold phosphate-buffered saline containing 0.4 mM
sodium orthovanadate (PBS-vanadate) and then harvested by scraping in
PBS-vanadate. After brief centrifugation, pelleted cells were then
solubilized for 15 min at 4 °C in either RIPA lysis buffer (1%
Nonidet P-40, 0.1% (w/v) SDS, 0.1% (w/v) sodium deoxycholate, 150
mM NaCl, 50 mM Tris-HCl (pH 7.4), 2 mM EDTA,
1 mM sodium orthovanadate, 50 mM NaF, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml aprotinin
(Boehringer Mannheim)) or WLB buffer (1% Triton X-100, 137 mM NaCl, 20 mM Tris-HCl (pH 8.0), 10% (v/v) glycerol, 2
mM EDTA, 1 mM sodium orthovanadate, 50 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml
aprotinin) as indicated. After centrifugation at 15,000 g for 15 min at 4 °C, the detergent extracts were either
immunoprecipitated or electrophoresed as below. CV-1 cells were
harvested similarly and solubilized in either RIPA or fusion lysis
buffer (1% Triton X-100, 150 mM NaCl, 10% glycerol, 50 mM Tris-HCl (pH 8.0), 100 mM NaF, 2 mM EDTA, 1
mM PMSF, 1 mM sodium orthovanadate, 10 µg/ml
aprotinin). After centrifugation, these extracts were processed as
below.
fusion (which includes the
entire hGHR cytoplasmic domain) bound to glutathione beads was
incubated with fusion lysis buffer extract from CV-1 cells expressing
either WT mJAK2 or JAK2
such that expression of the two
proteins was roughly normalized. After incubation for 90 min at 4
°C, the beads were then washed five times with fusion lysis buffer
and eluted in Laemmli sample buffer. 10% of the eluate was resolved by
SDS-PAGE on a 9.5% gel and immunoblotted with anti-GST. The remainder
was resolved on an 8% gel and immunoblotted with anti-JAK2 peptide
antiserum. Equal fractions of the extracts that were subjected to bead
binding were also electrophoresed and anti-JAK2 peptide antiserum
immunoblotted to indicate the presence of similar amounts of WT mJAK2
and JAK2
in the extracts.
Immunopreciptiation, Electrophoresis, and
Immunoblotting
The anti-JAK2 peptide antiserum (Upstate
Biotechnology, Inc., Lake Placid, NY), PY20 monoclonal
antiphosphotyrosine (APT) antibody (Transduction Laboratories,
Lexington, KY), and 12CA5 monoclonal anti-influenza hemagglutinin (HA)
peptide antibody (Berkeley Antibodies Co., Richmond, CA) were all
purchased commercially. Anti-JAK2 peptide antiserum is directed at
residues 758-776 of murine JAK2(6) . APT
immunoprecipitation of COS-7 cell RIPA detergent extracts was performed
with PY20 at 12 µg of antibody/immunoprecipitation as described
previously(19, 26) . Aliquots of CV-1 cell RIPA extracts
roughly normalized for similar expression levels of overexpressed
mutant and WT mJAK2 proteins were precipitated similarly with PY20 to
detect APT precipitability of these molecules, which were then
immunoblotted with the anti-JAK2 peptide antiserum as below.
In brief, this antiserum was raised in rabbits
against a gel-purified GST fusion protein incorporating residues
746-1129 of the murine JAK2. It was used at 18 µl/precipitation of
RIPA extracts (0.5-0.7 ml total volume) of either COS-7 or CV-1
cells. Protein A-Sepharose (Pharmacia) was used to adsorb immune
complexes and, after extensive washing with RIPA buffer, Laemmli sample
buffer eluates were resolved by SDS-PAGE and immunoblotted with
anti-JAK2 peptide antiserum.
10
/ml in WLB buffer in the presence of 1 mM 3,3`-dithiobis(sulfosuccinimidyl propionate) (Pierce), a
thiol-cleavable homobifunctional cross-linker, for 15 min on ice. After
quenching of cross-linking with 10 mM ammonium acetate for 5
min and centrifugation at 15,000
g for 15 min, 80% of
each detergent extract was immunoprecipitated with anti-hGHR or
nonimmune rabbit serum (40 µl/sample) in the presence of 0.1% SDS
and 0.1% sodium deoxycholate. For normalization purposes, 20% of each
extract was immunoprecipitated with anti-JAK2, as above. Eluates in
Laemmli sample buffer were resolved by SDS-PAGE under reducing
conditions and immunoblotted with anti-JAK2 peptide antiserum.
alkaline phosphatase
conjugate (Pierce), as described previously(19, 26) .
Competitive I-hGH Cross-linking Studies in rGHR/JAK2
Chimera-transfected COS-7 Cells
I-hGH
(NEN DuPont, specific activity 85-130 µCi/µg)
cross-linking experiments were performed on COS-7 cells transfected
with 30 µg of the cDNAs for either WTrGHR or each of the three
rGHR/JAK2 chimeras in the pRc/CMV expression plasmid according to
procedures previously described(19) . In brief, transfected
cells were incubated with
I-hGH (4 ng/ml or approximately
0.18 nM) either in the presence or absence of excess unlabeled
hGH (2 µg/ml) at 4 °C for 2 h. This was followed by incubation
with 0.5 mM fresh bis-(sulfosuccinimidyl)suberate
(BS
; Pierce), a noncleaveable water-soluble
homobifunctional cross-linker, for 15 min at room temperature.
Cross-linking was quenched for 10 min by addition of 10 mM ammonium acetate, and the cells were washed, harvested, and
solubilized directly in boiling Laemmli sample buffer. Proteins
radioiodinated by virtue of being specifically bound and cross-linked
by
I-hGH were resolved by SDS-PAGE on 9.5% gels and
detected by autoradiography.
Expression of Mutant JAK2 Molecules
To examine
regions of JAK2 that are required for GH-induced signaling, we used PCR
to generate cDNAs encoding the three deletion mutant murine (m) JAK2
molecules diagrammed with WT mJAK2 in Fig. 1A. The
details of the creation of these mutants are described elsewhere. JAK2
lacks residues 2-239 and is thus an
amino-terminal deletion. The carboxyl-terminal deletion mutant,
JAK2
, has a termination codon in place of residue 1000,
resulting in deletion of residues 1000-1129, disruption of the
tyrosine kinase domain, and removal of the consensus tyrosine
autophosphorylation motif (-EYYKVKE-) at residues 1006-1012.
JAK2
has in-frame internal deletion of residues
523-746 with removal of a large portion of the kinase-like (or
pseudokinase) domain but maintenance of the kinase and consensus
tyrosine autophosphorylation motifs.
Figure 1:
A, diagram of WT mJAK2 and three JAK2
mutants created to evaluate interaction with the GHR. The positions of
the kinase domain (residues 849-1122), the kinase-like (or
pseudokinase) domain (residues 543-806), and potential tyrosine
phosphorylation sites at the carboxyl-terminal (-EYYKVKE- at residues
1006-1012) and at the amino-terminal one-third (-IDGYYRL- at
residues 369-375) are indicated. JAK2 lacks
residues 2-239. JAK2
has a termination codon
replacing residue 1000 and is thus lacking residues 1000-1129.
JAK2
has an in-frame internal deletion of residues
523-746. B, expression of JAK2 mutants in COS-7 cells.
The pRc/CMV expression plasmid containing the cDNA for either WT mJAK2,
JAK2
, JAK2
, JAK2
, or the
vector alone was transfected into COS-7 cells, as under
``Experimental Procedures'' (one 100
15-mm
dish/lane). After harvesting and solubilization in RIPA lysis buffer,
extracts were immunoprecipitated with the anti-JAK2 serum.
Immunoprecipitated proteins were resolved by SDS-PAGE on an 8%
acrylamide gel, transferred to nitrocellulose, and immunoblotted with
anti-JAK2 peptide antiserum. The positions of migration of each of the
expressed JAK2 forms and of molecular mass standards are noted. Also
detected more faintly in each precipitate is the endogenous WT monkey
JAK2, which comigrates with the transfected WT mJAK2 at an molecular
mass of roughly 120 kDa.
Expression plasmids including
each cDNA were used to direct synthesis of the WT and mutant JAK2
molecules in COS-7 cells (Fig. 1B). Each protein was
detergent soluble and recognized by two different JAK2 antisera.
JAK2 and JAK2
species were detected at
approximately 100 kDa while JAK2
migrated at roughly 110
kDa, each consistent with its predicted molecular mass. Notably,
transfected WT mJAK2 comigrated with the endogenous monkey JAK2 (seen
in all samples) at approximately 120 kDa. Considering that only a small
fraction of the total cells examined are typically expressing the
transfected gene in such a transient assay, we conclude that the level
of the transfected JAK2 molecules greatly outweighs that of the
endogenous molecule in those cells that are transfected.
Signaling Properties of the Mutant JAK2
Molecules
Our previous work (19) indicated that
hGH-induced APT immunoprecipitability of JAK2 in COS-7 cells could be
detected if the rGHR was transiently cotransfected with WT mJAK2, but
not if the rGHR alone was expressed. This finding, along with the
robust relative expression of the mutant JAK2 molecules seen in Fig. 1B, led us to consider use of this system to
evaluate signaling and biochemical properties of these JAK2 mutants.
The hGH signaling events addressed have been previously shown to be
dependent on GHR-JAK2 association.
c-fos-Luciferase Transactivation
Others (27, 28, 29, 30, 31) have
demonstrated in various cells types the hGH-dependent acute and
transient increase in transcription of the c-fos gene. This
effect has been mapped to an upstream region (-361 to -264
base pairs) of the c-fos promoter(32) , a region that
includes the serum response element, the sis-inducible element, and an
AP-1 binding site(22) . To gauge the ability of our transiently
coexpressed rGHR and JAK2 molecules to mediate this gene activation, we
used the p2FTL luciferase reporter plasmid. p2FTL includes two tandem
copies of the murine c-fos enhancer fragment, the herpes
simplex virus thymidine kinase promoter, and the luciferase gene and
has been used in studies of epidermal growth factor receptor
signaling(21) .
Figure 2:
hGH-induced transactivation of a c-fos enhancer-driven luciferase reporter in transiently transfected
COS-7 cells. A, time course of transactivation mediated by WT
mJAK2. Cells (0.8 million cells/100 15-mm dish) were
transfected with pRc/CMV-WTrGHR, pRc/CMV-WT mJAK2, and p2FTL (as
described under ``Experimental Procedures''). At 18 h after
transfection, the cells were split into 24-well plates (approximately
80,000 cells/well). Under serum-starved conditions (as under
``Experimental Procedures''), hGH (500 ng/ml) or vehicle was
added for the durations indicated, such that all stimulations (done in
triplicate) were terminated simultaneously. Detergent extraction and
luciferase activity assay were as described under ``Experimental
Procedures.'' Results are plotted as the hGH-induced increase
(mean ± 1 S.D.) relative to unstimulated (vehicle only)
controls. The control values at each point are considered 100%. (There
was less than 5% deviation among the mean control values at each time
point.) B, hGH concentration dependence of transactivation
mediated by WT mJAK2. The experiment was the same as that in A except that the dose of hGH used was varied and all incubations
lasted 4.5 h at which point luciferase activity was measured. C, hGH-induced transactivation in COS-7 cells expressing JAK2
mutants. Cells transiently expressing the transfected WT rGHR and
either the transfected WT mJAK2 (as above in A and B), JAK2
, JAK2
,
JAK2
, or no transfected JAK2 (vector) along with p2FTL
were serum-starved and stimulated with hGH (500 ng/ml) for 22 h, as in A and B. The resulting hGH-induced luciferase
activity (the ratio of hGH-treated to untreated samples ± 1 S.D.
of triplicates) is shown. The luciferase activity (in relative light
units) of the unstimulated samples for each of the five transfections
shown were within 40% of each other (not shown), indicating roughly
comparable transfection efficiencies. The experiment shown is
representative of seven such experiments. Note that no significant
increase in luciferase activity was seen when the transfected rGHR was
expressed in the COS-7 cells with JAK2
or JAK2
or in the absence of transfected mJAK2 (vector
sample).
We
tested the ability of each of the JAK2 mutants to support this
hGH-induced gene activation when coexpressed with the rGHR (Fig. 2C). Using transfection conditions that resulted
in ample expression of each of the JAK2 molecules (not shown), neither
JAK2 nor JAK2
expression mediated
significant hGH-induced luciferase accumulation when compared to
expression of WT mJAK2. Expression of JAK2
, however,
resulted in consistently detectable hGH-induced augmentation of
luciferase activity, although levels were lower than for WT mJAK2 (1.8- versus 3.4-fold in the experiment shown).
Mobility Shift of Epitope-tagged ERK2
Another
GH-induced response that has been identified is the phosphorylation and
activation of ERKs, also known as mitogen-activated protein
kinases(17, 33, 34, 35) . When
phosphorylated, the ERKs characteristically exhibit retarded migration
on SDS-PAGE gels(33) . This change in mobility provides a
convenient way to monitor their phosphorylation state. We expressed an
epitope-tagged (influenza hemagglutinin) rat ERK2 molecule in COS-7
cells along with the rGHR and mJAK2. As seen in Fig. 3(top
panel), the appearance of an hGH-induced shift in ERK2 migration
was detected by anti-HA immunoblotting when the WT mJAK2 and rGHR were
coexpressed, but not when the mJAK2 was omitted from the transfection.
Testing of the JAK2 mutants in this assay (Fig. 3, bottom
panel) revealed a readily detectable ERK2 mobility shift in
response to hGH treatment when the WT mJAK2 (lane 2) or the
JAK2 mutant (lane 8) was expressed, but this
shift was not seen when either JAK2
or JAK2
were expressed (lanes 4 and 6). These results
do not prove that endogenous COS-7 cell ERK(s) is necessarily involved
in eliciting the hGH-induced transcriptional activation in the
transfected cells. However, the evidence does indicate that the JAK2
structural mutations have qualitatively similar effects on the ability
of hGH to activate signaling in both the transcriptional activation and
ERK2 mobility shift assays.
Figure 3:
hGH-induced shift in molecular mass of
epitope-tagged ERK2. Top panel, WT rGHR, along with either WT
mJAK2 or vector only and ERK2 or vector only (as
indicated), were coexpressed in COS-7 cells, as described under
``Experimental Procedures.'' After serum starvation, cells
were treated with hGH (500 ng/ml) (+) or vehicle only (-)
for 10 min at 37 °C. Cells were harvested and solubilized in WLB
lysis buffer. Detergent extracts (corresponding to one dish of
transfected cells/condition) were resolved by SDS-PAGE on a 10% gel,
transferred to nitrocellulose, and immunoblotted with the 12CA5 anti-HA
monoclonal antibody, as under ``Experimental Procedures.''
Note the hGH-induced appearance of an ERK2
band with
retarded migration only in the sample in which the WT mJAK2 was
expressed (lane 4). Bottom panel, the experiment was
the same as that in the top panel, except that either WT
mJAK2, JAK2
, JAK2
, or JAK2
(as indicated) was coexpressed with the rGHR and
ERK2
. Note the hGH-induced appearance of the shifted
(retarded) ERK2
only in the samples in which the WT mJAK2
or JAK2
was expressed (lanes 2 and 8,
respectively). The experiment shown is representative of four such
experiments.
JAK2 APT Precipitability
Since the initiating step
in multiple aspects of GH signaling appears to be JAK2 tyrosine kinase
activation with resultant tyrosine phosphorylation of JAK2 and other
cellular proteins, we examined the ability of the WT mJAK2 and the JAK2
mutants to undergo hGH-induced tyrosine phosphorylation. To enhance
sensitivity of detection, we relied on our previously characterized
ability to detect WT mJAK2 in APT immunoprecipitates of extracts of
hGH-treated COS-7 cell transfectants(19) . In each case,
cotransfection of cDNAs encoding the rGHR and WT mJAK2 or JAK2 mutant
was followed by treatment for 10 min at 37 °C with or without hGH,
detergent solubilization, and immunoprecipitation with the PY20 APT
monoclonal antibody (75% of the extract) or anti-JAK2 (25% of the
extract). Immunoprecipitated proteins were resolved by SDS-PAGE and
immunoblotted with anti-JAK2 peptide serum.
, although basal signal is also apparent
for this mutant (Fig. 4C). In distinction, despite
significant levels of expression, neither JAK2
nor
JAK2
became detectably APT precipitable in response to
hGH treatment (Fig. 4, A and B). We note that
while JAK2
has an interrupted kinase domain and lacks the
potential consensus tyrosine autophosphorylation site, this region is
intact in JAK2
. Indeed, as seen in Fig. 4D, JAK2
is immunoprecipitable with
the APT antibody when vastly overexpressed in CV-1 cells infected with
the vaccinia virus-encoded T
polymerase.
JAK2
, though amply expressed and anti-JAK2 precipitable,
is, as predicted, not detectable with the APT antibody in the same
assay. As also predicted, WT mJAK2 and JAK2
were APT
precipitable when similarly overexpressed (not shown). The inability of
JAK2
to become APT precipitable in response to hGH in
rGHR-cotransfected COS-7 cells, therefore, appears not to be due to a
defect in its potential to function as a tyrosine kinase and/or
substrate.
Figure 4:
APT precipitability of activated WT mJAK2
and JAK2 mutants. A, comparison of WT mJAK2 and JAK2 transiently expressed in COS-7 cells. The rGHR and either WT
mJAK2 or JAK2
(as indicated) were transiently coexpressed
in COS-7 cells, as under ``Experimental Procedures.'' After
serum starvation and treatment with (+) or without (-) hGH
(500 ng/ml) for 10 min at 37 °C, RIPA detergent extracts were
immunoprecipitated with the PY20 monoclonal APT antibody (the
equivalent of three transfected dishes/condition) (left panel)
or the anti-JAK2 antiserum (one dish/condition) (right panel),
resolved by SDS-PAGE, and immunoblotted with the anti-JAK2 peptide
serum. Note the failure of JAK2
to become detectably APT
precipitable in response to hGH treatment, despite ample expression.
The experiment shown is representative of three such experiments. B, comparison of WT mJAK2 and JAK2
transiently
expressed in COS-7 cells. The experiment was performed as in A, except that JAK2
was compared to WT mJAK2.
Note that JAK2
also fails to become detectably APT
precipitable in response to hGH treatment. The experiment shown is
representative of three such experiments. C, comparison of WT
mJAK2 and JAK2
transiently expressed in COS-7 cells. The
experiment was the same as in A and B, except that
JAK2
was compared to WT mJAK2. Note the easily detectable
augmentation of JAK2
APT precipitability in response to
hGH treatment. The experiment shown is representative of three such
experiments. D, JAK2
, but not
JAK2
, is APT precipitable when overexpressed.
JAK2
and JAK2
were each transiently
expressed in CV-1 cells that were also infected with the vvT7 pol
vaccinia virus, as described under ``Experimental
Procedures.'' RIPA extract of unstimulated cells was
immunoprecipitated with either PY20 (roughly 0.8 and 0.4 dish
equivalents/precipitation for JAK2
and
JAK2
, respectively) or anti-JAK2 (roughly 0.1 and 0.05
dish equivalents/precipitation for JAK2
and
JAK2
, respectively) and resolved and anti-JAK2 peptide
antiserum immunoblotted, as in A-C. Note that despite
evaluation of similar amounts of the two vastly overexpressed proteins,
only JAK2
exhibits APT precipitability. The band seen
above JAK2
in the APT precipitation and in the cell
extract sample of Fig. 5D is an artifact of the immunoblotting
detection system sometimes present when large amounts of CV-1 cell
extract are utilized and is independent of the particular JAK2 form
expressed (not shown).
Physical Association of Mutant JAK2 Molecules and the
GHR
To further explore the unresponsiveness of the JAK2 and JAK2
mutants, we examined their ability to
associate with the rGHR. Others have shown in murine preadipocytes and
in rat GHR Chinese hamster ovary cell transfectants that JAK2 becomes
coimmunoprecipitable with the GHR after GH treatment of the
cells(1, 17) . Likewise, we have shown that our anti-GHR
antibodies can specifically coimmunoprecipitate JAK2 from human IM-9
cells if a cleavable chemical cross-linker is used to stabilize the
interaction (19). Though present in the unstimulated state, the amount
of JAK2 that becomes anti-GHR coprecipitable from IM-9 cells is also
augmented by hGH treatment(19) .
(Fig. 5B), and JAK2
(Fig. 5C) were each coimmunoprecipitated with
similar efficiency with the anti-GHR antibody. To prove specificity, a
duplicate coexpression of rGHR and JAK2
was processed
identically, except that nonimmune serum was used instead of anti-GHR
for immunoprecipitation (Fig. 5C). Despite ample
directly precipitated JAK2
no JAK2
was
detected in the nonimmune precipitate. In contrast to the WT mJAK2 and
the other JAK2 mutants, JAK2
(Fig. 5A) was
not coimmunoprecipitable with anti-GHR, indicating a markedly
diminished interaction between this mutant and the rGHR. In the
experiment shown in Fig. 5D, this finding was confirmed
by the inability of JAK2
extracted from transfected
vaccinia virus infected-CV-1 cells to interact in vitro with
an immobilized GST fusion protein incorporating the full hGHR
cytoplasmic domain. WT mJAK2, similarly overexpressed, does interact
with this GST/hGHR
fusion, consistent with our
previous observations(19) . These findings indicate that the
amino-terminal 239 residues of JAK2, absent in JAK2
, are
required for physical and, therefore, functional interaction with the
GHR.
Figure 5:
Association of the rGHR with WT mJAK2 and
JAK2 mutants. A, comparison of WT mJAK2 and JAK2 transiently expressed in COS-7 cells. The rGHR and either WT
mJAK2 or JAK2
(as indicated) were transiently coexpressed
in COS-7 cells. After serum starvation and treatment with hGH (300
ng/ml) for 5 min at 25 °C, WLB detergent lysates were exposed to
the cross-linking agent 1 mM 3,3`-dithiobis(sulfosuccinimidyl
propionate (1 mM), as detailed under ``Experimental
Procedures.'' After quenching with 10 mM ammonium
acetate, extracts were immunoprecipitated with either anti-GHR (five
dishes/precipitation) or anti-JAK2 (one dish/precipitation) in the
presence of 0.1% SDS and 0.1% sodium deoxycholate. Eluted proteins were
resolved under reducing conditions by SDS-PAGE and immunoblotted with
the anti-JAK2 peptide serum. Note the lack of JAK2
coimmunoprecipitable with anti-GHR. The experiment shown is
representative of three such experiments. B, comparison of WT
mJAK2 and JAK2
transiently expressed in COS-7 cells. The
experiment was performed as in A, except that JAK2
was compared to WT mJAK2. Note the coimmunoprecipitability of
JAK2
with anti-GHR. The experiment shown is
representative of two such experiments. C, comparison of WT
mJAK2 and JAK2
transiently expressed in COS-7 cells. The
experiment was performed as in A and B, except that
JAK2
was compared to WT mJAK2. Note the
coimmunoprecipitability of JAK2
with anti-GHR. In
addition, when nonimmune serum was used instead of anti-GHR, no
detectable JAK2
was observed illustrating the specificity
of this coimmunoprecipitation procedure. The experiment shown is
representative of two such experiments. D, WT mJAK2, but not
JAK2
, interacts with a GST fusion protein containing the
hGHR cytoplasmic domain. WT mJAK2 and JAK2
were each
transiently expressed in CV-1 cells that were also infected with the
vvT7 pol vaccinia virus, as described under ``Experimental
Procedures'' and in Fig. 4D. Fusion lysis buffer extracts
were made of each population of transfected cells. This extract (0.9
dish equivalents for JAK2
and 0.1 dish equivalents for WT
mJAK2) was subjected to a binding assay with the glutathione bead-bound
fusion protein, GST/hGHR
(which contains the
entire hGHR cytoplasmic domain), as described under ``Experimental
Procedures'' and Ref. 19. Eluates of these precipitates were
separated by SDS-PAGE and immunoblotted with anti-JAK2 peptide serum
(90% of the eluate) or anti-GST antibodies (10% of the eluate), as
indicated. Unfractionated extract (one-tenth of the amount applied to
the beads for each sample) was also separated by SDS-PAGE and
immunoblotted with anti-JAK2 peptide serum for purposes of
normalization. Note that despite evaluation of similar amounts of each
JAK2 form and the presence of similar amounts of
GST/hGHR
in each sample, only WT mJAK2
interacted with the fusion protein. The experiment shown is
representative of two such experiments.
Expression of GHR/JAK2 Chimeras
In light of the
above findings that the JAK2 amino terminus contains region(s) required
for this interaction, we tested whether chimeric proteins incorporating
the GHR external and transmembrane domains and various JAK2 regions
other than the amino terminus could signal in response to hGH. The
chimeras created are diagrammed in Fig. 6A. Each
includes rGHR residues 1-277 and 293-296 (i.e. rGHR with deletion of the residue
278-292 region that includes Box1, the proline-rich motif in the
GHR that is essential for association with and activation of
JAK2(17, 18, 19) ). The chimera rGHR/JAK2
has this rGHR region fused to residues 525-778 of mJAK2, a
region inclusive of most of the kinase-like domain, but devoid of the
kinase domain. rGHR/JAK2
includes mJAK2 residues 753-1129,
the kinase domain, whereas rGHR/JAK2
contains mJAK2
residues 525-1129 (both the kinase-like and kinase domains).
Figure 6:
Expression of GHR/JAK2 chimeras in COS-7
cells. A, diagram of the WT rGHR, WT mJAK2, and three
rGHR/JAK2 chimeras created. The kinase-like (or pseudokinase) and
kinase domains of mJAK2 and the extracellular, transmembrane (horizontal line pattern), intracellular and proline-rich Box1 regions of the rGHR are indicated. The chimeras, rGHR/JAK2, rGHR/JAK2
, and rGHR/JAK2
, contain the extracellular and
transmembrane portions (but not the Box1 region) of the rGHR fused to
the indicated regions of mJAK2, as detailed under ``Experimental
Procedures'' and ``Results.'' B,
I-hGH cross-linking to rGHR and the rGHR/JAK2 chimeras
expressed on the surface of COS-7 cells. The rGHR and rGHR/JAK2
chimeras, as indicated, were expressed following transfection, as under
``Experimental Procedures.'' Cross-linking of radiolabeled
hGH in the absence (-) or presence (+) of excess unlabeled
hGH was accomplished using BS
. Total cellular proteins were
solubilized in boiling reduced Laemmli sample buffer, resolved by
SDS-PAGE, and subjected to autoradiography. C, hGH-induced
transactivation in COS-7 cells expressing rGHR/JAK2 chimeras. Cells
transiently expressing either the transfected WT rGHR and WT mJAK2 or
each of the three transfected rGHR/JAK2 chimeras (as indicated) along
with p2FTL were serum starved and stimulated with hGH (500 ng/ml) for
22 h, as in Fig. 2. The resulting hGH-induced luciferase activity (the
ratio of hGH-treated to untreated samples ± 1 S.D. of
triplicates) is shown. The results of the two experiments shown are
representative of eight similar experiments.
We
tested the integrity and hGH binding ability of each chimera by
performing radiolabeled hGH cross-linking studies (Fig. 6B). COS-7 cells transiently expressing each
chimera or the WT rGHR were incubated with I-hGH in the
presence or absence of excess unlabeled hGH and then exposed to
BS
, a noncleavable, membrane-impermeant chemical
cross-linker. SDS-PAGE and autoradiography revealed that each chimera
specifically bound
I-hGH at the cell surface and migrated
with its predicted molecular mass. Further, the indistinct nature of
the proteins likely reflects their glycosylation, presumably derived
from their rGHR component since JAK2 is not believed to be a
glycoprotein. Immunoprecipitation and immunoblotting (not shown)
revealed that the chimeras each reacted with the anti-JAK2 and/or
anti-JAK2 peptide sera in the pattern predicted, considering the JAK2
regions included in the particular chimera. Together, these data verify
the structural integrity of the chimeric molecules and their ability to
bind hGH at the cell surface.
, the
chimera that has only the kinase-like domain of JAK2, reproducibly
displayed no increase in luciferase activity in response to hGH.
However, inclusion in the chimeras of the JAK2 kinase domain alone
(rGHR/JAK2
) or in addition to the kinase-like domain
(rGHR/JAK2
) allowed significant hGH-induced
transactivation (1.8-3.0-fold). The signal transduced through
these chimeras was, however, consistently less than that seen with
cotransfection of the WT rGHR and WT mJAK2. Notably, coexpression of
the WT mJAK2 with the previously characterized truncated
rGHR
, which corresponds to the rGHR portion of
the chimeras, yielded no hGH-induced increase in luciferase activity
(not shown). This is consistent with its lack of the Box1 motif and its
inability to promote hGH-induced APT precipitability of
JAK2(19) . Thus, our data indicate that a minimal unit capable
of transmitting at least one type of hGH signal consists of the GHR
extracellular and transmembrane domains covalently fused to the kinase
domain of JAK2.
chain.
) does not
adversely affect physical association of JAK2 with the rGHR, but the
lack of hGH induction of the biochemical signals correlates with the
disruption of the kinase domain that results from this truncation. The
amino-terminal deletion mutant, JAK2
, was also unable to
couple hGH treatment to any of the activating events despite the
integrity of its kinase domain and its demonstrated ability to become
APT reactive when vastly overexpressed. In this case, the inability of
JAK2
to physically interact with the rGHR most likely
underlies its signaling defects.
lacks the
amino-terminal one-fifth of the JAK2 molecule, we infer that this
region contains elements necessary for interaction with the GHR.
Whether this region is solely sufficient to confer this ability to
interact with the GHR is not yet known. Likewise, the particular
area(s) within this amino-terminal region which promote this
interaction is yet to be determined. This same JAK2
is
also unable to associate with or promote tyrosine phosphorylation of
the GM-CSF receptor
chain in a transient expression
system.
This indicates a likely similarity in the mechanism
of interaction of JAK2 with each receptor. Given the topological
similarity of structure among JAK-family members, we might expect that
the amino termini of the other JAKs may also be involved in interaction
with their cognate receptors. Despite these likely similarities,
functional specificity of the receptor-JAK interaction is also evident.
We note that coexpression of JAK3 (provided by J. J. O'Shea,
NCI), which is 47% identical to JAK2(14, 15) , with the
rGHR was unable to mediate hGH-induced fos-luciferase transactivation
(not shown).
) did not abrogate the ability of the mutant JAK2 to
interact with the rGHR or to mediate hGH-induced fos-luciferase
transactivation, ERK2 mobilty shift, or APT precipitability of the
kinase itself. Although this implies that the kinase-like domain is not
essential for transduction of the hGH-induced activating signal, it is
conceivable that it subserves a modulatory function that would be
better discerned in other signaling assays. We note that JAK2
basal APT precipitability was reproducibly robust (Fig. 4C) and that our studies of the GM-CSF receptor
-JAK2 mutant interactions showed a GM-CSF-independent
GM-CSF receptor
tyrosine phopshorylation in CV-1
cells transiently coexpressing that receptor and JAK2
at
very high levels.
Although we are not certain, these
findings may indicate that the attenuated hGH-induced signal seen with
JAK2
relative to WT mJAK2 in the fos-luciferase
transactivation assay, for instance, could relate in part to a basal
disinhibition of kinase function in the mutant.
, having in addition
only residues 276-277 and 293-296 (none of which are
included in the Box1 region). We have shown that
rGHR
, though able to bind hGH normally, does not
mediate hGH-induced APT precipitability of cotransfected
JAK2(19) . Others have shown that the analogously truncated rat
GHR is also incapable of mediating GH-induced mitogen-activated protein
kinase activation, gene transcription, proliferation, JAK2 association,
substrate tyrosine phosphorylation, and insulin production in various
cell types(17, 18, 20, 33, 42) .
and
rGHR/JAK2
) transduced the hGH activating signal. The
rGHR/JAK2
chimera served as a negative control in that its
lack of elicitation of luciferase activity in response to hGH might be
predicted in the context of the findings with JAK2
.
Interestingly, inclusion of the kinase-like domain may influence the
signaling in that there was in general an increased ability of
rGHR/JAK2
to transduce hGH-induced transactivation when
compared to rGHR/JAK2
(not shown). We note, however, that
none of the chimeras signaled as well in this assay as did the WT rGHR
coexpressed with the WT mJAK2. We assume this reflects both the absence
of potentially critical regions of the GHR cytoplasmic domain and the
less than perfect assembly of unnatural chimeric molecules. Further
studies using the rGHR/JAK2 molecules may be helpful in discerning the
relative impact of the loss of these GHR region(s) on various measures
of signal transduction since the chimeras may serve as selective
activators of hGH-induced JAK2-mediated pathways.
bis-(sulfosuccinimidyl)suberate.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.