From the
The binding of granulocyte-macrophage colony-stimulating factor
(GM-CSF) to its receptor stimulates JAK2 protein kinase activation,
protein phosphorylation, and JAK2 association with the
GM-CSF(
Although
the GM-CSF receptor does not encode a protein kinase, binding of GM-CSF
to its receptor stimulates the rapid tyrosine phosphorylation of
specific protein substrates and the GM-CSFR
To determine the regions of the JAK2 protein kinase that regulate
interaction with and phosphorylation of the GM-CSFR
The murine JAK2 cDNA was a gift from Dr. J. Ihle (St. Jude
Children's Hospital Memphis, TN). The JAK2 amino-terminal
deletion (ATD) was generated in Bluescript SK
GST fusion proteins expressing
four fragments of JAK2 (GST-J1, aa 1-294; GST-J2, aa
295-522; GST-J3, aa 523-746; GST-J4, aa 747-1127) were
generated by PCR and inserted into the BamHI/EcoRI
site of the pGEX2T vector. The sequence of the PCR products was
confirmed by sequencing.
Immunoprecipitations were carried out by lysing the cells in TNE
buffer containing inhibitors (see above). The cell lysate was then
microcentrifuged for 15 min to remove debris. Antibodies were added
into the supernatant and incubated at 4 °C for 1 h. The JAK2 and
GM-CSF antibodies produced in our laboratories were added at 1:40
dilution. 30 µl of protein A-Sepharose (Pharmacia Biotech Inc.)
slurry was used to adsorb immune complexes, and the beads were washed
five times in lysis buffer prior to elution in Laemmli sample buffer.
The eluted proteins were electrophoresed in 8% SDS-PAGE and, for
Western blots, electrophoretically transferred to a polyvinylidene
difluoride membrane filter (Millipore, Bedford, MA). The blotted filter
was incubated in TBS (20 mM Tris, pH 7.6, 137 mM
NaCl) containing 3% bovine serum albumin (Fraction V, Sigma) for 1 h.
The filter was incubated with either an anti-phosphotyrosine monoclonal
antibody (PY20), polyclonal anti-GM-CSFR
Bacteria expressing the four GST-JAK2
fusion proteins were lysed and the extract run on a 10% SDS-PAGE gel
and transferred to nitrocellulose. The filter was blocked in TBST (TBS
plus 1% Triton X-100) containing 2% nonfat dry milk. The filter was
then incubated for 2 h in the same buffer containing 1 µg/ml
biotintylated GST fusion protein, followed by extensive washing in TBST
followed by streptavidin-conjugated alkaline phosphatase (Boehringer
Mannheim) in TBST at a dilution of 1:5000 for 1 h. The blot was further
developed with the nitro blue tetrazolium and
5-bromo-4-chloro-3-indolyl phosphate detection system
(Promega)
(32) .
Previous studies have demonstrated the importance of the
membrane-proximal domain of many growth factor receptors for JAK2
binding. In the Epo receptor the region between box 1 and box 2 was
necessary for Epo-dependent growth, activation of JAK2 kinase activity,
and JAK2 binding
(19) . The results presented on the GM-CSF
receptor demonstrate that while a portion of the region between box 1
and 2 is necessary for JAK2 binding the sequence of amino acids between
box 1 and 2 does not alone bind this protein kinase. It has been
demonstrated that tryptophan (amino acid 494 in the GM-CSFR
To examine the potential regulatory role of GM-CSF in mediating
GM-CSFR
We thank Dr. J. Ihle and Dr. A. Miyajima for providing
the cDNA clones that were necessary for this work. We appreciate the
help of Dr. B. Weaver and Patsy Spitzer in editing and preparing this
manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
chain of the GM-CSF receptor. To better understand how different
domains of the JAK2 function to regulate association and
phosphorylation of the
receptor, the minimal portion
of the
receptor necessary for JAK2 binding has been
determined. Using glutathione S-transferase (GST) fusion
proteins expressing different portions of the membrane-proximal domain
of the
chain, we demonstrate that JAK2 binds to amino
acids 458-495, but showed little binding to fusion proteins
containing amino acids 483-559, 483-530, or 458-484.
The GST-
458-495 bound equally well to the wild
type (WT) JAK2, a carboxyl-terminal deletion of JAK2 removing the
protein kinase domain (amino acids 1000-1129), and a deletion of
the kinase-like domain (amino acids 523-746). However, an
amino-terminal JAK2 deletion (amino acids 2-239) markedly reduced
binding to this GST-
. Far Western blotting
demonstrated that a GST fusion protein containing amino acids
1-294 of JAK2, but not fusion proteins containing amino acids
295-522, 523-746, or 747-1127, bound GST-
458-559. When the JAK2 WT and deletions were transiently
expressed along with the
and
subunits of the
GM-CSF receptor and the cells were treated with GM-CSF, the following
results were obtained: 1) WT JAK2 phosphorylated the
subunit in a GM-CSF- dependent manner, 2) the kinase-like domain
deletion phosphorylated the
subunit, and 3) both the
kinase domain deletion and the amino-terminal deletion failed to
stimulate phosphorylation of the
subunit. Therefore,
phosphorylation of the
subunit requires the binding
of JAK2 through its amino terminus.
)
regulates the growth and
differentiation of hematopoietic cells by binding to a high affinity
receptor composed of an
chain and a
chain. The
chain
(60-80 kDa) alone is sufficient for the low affinity binding of
GM-CSF (1, 2), as it interacts with the
chain (140 kDa) to
constitute the high affinity receptor
(3, 4) . The
chain contains a short intracytoplasmic region of approximately 54
amino acids, which has been shown to be necessary for
hormone-stimulated growth
(5, 6, 7) . The
intracytoplasmic portion of the
chain (approximately 400 amino
acids) is much larger than that of the
chain. In the presence of
the
chain, a minimum membrane-proximal region of the
chain
containing 100 amino acids has been shown to be sufficient to mediate
GM-CSF-stimulated cell growth
(8, 9) . This near
membrane-proximal region of the
chain contains two stretches of
amino acids, denoted box 1 and box 2
(10) , which are highly
conserved in the large family of hemopoietin receptors, which includes
interleukin-6, interleukin-2
, erythropoietin (Epo), granulocyte
colony-stimulating factor, growth hormone, and prolactin.
chain
(9, 11, 12, 13) . This
GM-CSF-induced phosphorylation has been shown to be mediated by the
JAK2 protein tyrosine kinase
(9, 14) . The JAK family of
protein kinases consists of JAK1, JAK2, JAK3, and
Tyk2
(14, 15, 16, 17, 18) . These
protein kinases have a molecular mass of approximately 130 kDa and
contain a carboxyl-terminal kinase domain, a kinase-like (pseudokinase)
domain without SH2 or SH3 domains, and a potential carboxyl-terminal
phosphorylation site, VDGYFRL. JAK2 is phosphorylated, and its protein
kinase activity is stimulated in response to GM-CSF
(9) ,
Epo
(19, 20) , growth hormone
(21, 22) ,
interleukin-6
(24) , prolactin
(25) , and interferon
(26) . Mutagenesis experiments have demonstrated that JAK2
binds to the near membrane-proximal region of these receptors, which
contains both the box 1 and box 2 sequence of amino acids. Deletion of
this receptor region blocks JAK2 activation and mitogenesis by these
hormones
(9, 19, 22, 23, 24, 30, 31) .
chain, we
have mapped the domain of the membrane-proximal region of the
chain, which binds the JAK2 protein. Using this short sequence,
expressed as a GST fusion protein and amino, carboxyl, and pseudokinase
deletion mutants of the JAK2 protein kinase, we have determined the
region of JAK2 necessary for interaction with the
chain. By
expressing these mutants in vivo, we demonstrate that specific
portions of JAK2 protein kinase regulate hormone-inducible
chain
phosphorylation.
Plasmid Construction
cDNAs encoding GST fusion
proteins with the intracytoplasmic portion of the GM-CSFR subunit were generated by ligating the PCR fragments of GM-CSFR
subunit (a gift of Dr. A. Miyajima, DNAX Research
Institute, Palo Alto, CA) into the BamHI/EcoRI site
of the pGEX2T vector (Pharmacia Biotech Inc., Alameda, CA). The PCR
products correspond to amino acid numbers of the GM-CSFR
:458-495 (Box 1 plus), 483- 559 (Box 2),
458-484 (Box 1), 458-559 (Box 1 and 2), and 483-530
(Box bb). The GST fusion proteins were expressed in Escherichia
coli, and the proteins were purified as described
(6) .
vector
(Stratagene, La Jolla, CA) by replacing the DNA fragment of JAK2 from
the ATG start to the first EcoRI site (amino acid 294) with a
PCR fragment with a NotI site and a ATG start codon inserted
at the 5` end, and spanning amino acids 240-294. The pseudokinase
domain (PSKD) deletion was generated by cutting the JAK2 cDNA at the
two internal BglII sites, removing the fragment that encodes
amino acids (aa) 523-746, and religating the cDNA. The
carboxyl-terminal deletion (CTD) was generated by cleaving the cDNA
with NdeI and ApaI and removing aa 932-1129 of the
carboxyl terminus. This fragment was replaced with a PCR fragment
spanning aa 932-999, to which NdeI and ApaI
site, as well as a stop codon, had been added. Each cDNA was removed
from Bluescript SK and inserted into the NotI/ApaI
site of the PRC/CMV vector (Invitrogen, San Diego, CA). cDNA sequencing
confirmed the presence of the mutations and the fidelity of the
remaining regions subjected to PCR.
Overexpression of JAK2 Deletions in CV-1
Cells
CV-1 cells were maintained in DMEM containing 10% newborn
calf serum. 8 10
CV-1 cells in 10-cm dishes were
infected with vvT7 pol vaccinia virus (multiplicity of infection
= 5) (a gift of Dr. Mark Mulligan, UAB, Birmingham, AL) in 2 ml
of DMEM without serum at 37 °C for 1 h. After infection, the cells
were incubated in serum-containing media for 2 h and transfected with
the various JAK2 cDNAs using Lipofectin (Life Technologies, Inc.;
3-7 µg of plasmid plus 20-40 µl of Lipofectin
reagent/10-mm dish) for 4 h. Twenty-four hours after transfection the
cells were lysed, and the lysate was used as a source of expressed JAK2
proteins.
GST Fusion Protein Binding Assay
Equal amounts of
GST- fusion proteins immobilized on
glutathione-Sepharose (Sigma) were washed once in TNE buffer (50
mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40,
and 2 mM EDTA) containing 1 mM sodium vanadate, 10
µg/ml aprotinin, 10 µg/ml leupeptin, and 5 mM sodium
fluoride and once in TNE buffer without inhibitors.
Bis(sulfosuccinimidyl) suberate cross-linker (Pierce) was added to the
final concentration of 2.5 mM. The cross-linking reaction was
allowed to proceed on ice for 15 min and quenched with 10 mM
ammonium acetate for an additional 10 min on ice. CV-1 cell lysates
(see above) were used as a source of mutated or WT JAK2 proteins.
Approximately 10 µg of the Jak2 proteins (as judged by Western
blots) were incubated at 4 °C for 30 min with the cross-linked
GST-
fusion proteins immobilized on
glutathione-Sepharose. Resins were extensively washed in lysis buffer,
and associated proteins were then eluted in Laemmli sample buffer.
Eluted proteins were separated on SDS-polyacrylamide (10%) gels and
immunoblotted with various antisera.
Immunoprecipitation and Western Blotting
An
antibody to the JAK2 protein was raised by injecting an SDS
gel-purified fusion protein of glutathione S-transferase and
aa 747-1129 of JAK2 every 3 weeks into a New Zealand White rabbit. The
methods for immunogen injection have been described
(6) . This
antibody was used for all immunoprecipitations. The GM-CSFR antibody was raised in a similar manner to an external domain of
the receptor, as described previously
(6) . The PY20 mouse
monoclonal antibody was purchased from Transduction Laboratories
(Lexington, KY). An additional JAK2 antibody directed at
residues(758-776) was purchased from Upstate Biotechnology, Inc.
(UBI, Lake Placid, NY) and was used for Western blotting.
antibody, or
UBI anti-JAK2 antibody at concentrations of 1 µg/ml (1:100
dilution) and 1 µl/ml, respectively. Bound antibodies were
visualized using either peroxidase-conjugated rabbit anti-mouse
immunoglobulin and the enhanced chemiluminescence (ECL) system
(Amersham Corp.) or by biotinylated protein A (Amersham) and a
streptavidin alkaline phosphatase conjugate (BRL) along with the nitro
blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate detection
system (Promega, Madison, WI).
Far Western Blotting
The GST GM-CSFR 458-559 (Boxes 1 and 2) was biotinylated
(32) at a
protein concentration of 1-3 mg/ml at room temperature for 2 h
with biotinamidocaproate N-hydroxysuccinimide ester (Sigma) in
100 mM sodium borate, pH 8.8. The biotinylated protein was
purified by gel filtration.
The JAK2 Binding Region in GM-CSF
The membrane-proximal intracytoplasmic region
of hematopoietin receptors, including GM-CSFR Receptor
, has
been shown to be necessary for JAK2 protein binding
(9) . To map
in detail the minimal amino acid sequence necessary for physical
association with JAK2, we generated GST fusion proteins containing
short stretches of the membrane-proximal region of GM-CSF receptor
(Fig. 1A). The fusion proteins were
expressed in E. coli and then purified on
glutathione-Sepharose beads. These beads were then cross-linked with a
non-cleavable protein cross-linker to increase protein binding
efficiency (data not shown). Because simple transfection of CV-1 cells
with JAK2 cDNA did not yield a large amount of protein, these cells
were first infected with a vaccinia virus that produces the T7
polymerase
(27, 28) and then transfected with the JAK2
cDNA containing the T7 promoter. This system allows for massive
overexpression of the JAK2 protein. CV-1 extracts were then incubated
with the GST fusion protein on beads.
Figure 1:
The binding of WT JAK2 to GM-CSFR
fusion constructs. A, structure of the GST
fusion constructs. The amino acids that are conserved among
hematopoietin, receptors denoted as box 1 and box 2, are shown as a
closed area, and the hatched area contains the stretches of
amino acids between these two boxes. The stippled area
indicates the glutathione S-transferase protein. B,
extracts of CV-1 cells expressing WT JAK2 were incubated with
equivalent amounts of GST fusion proteins bound to
glutathione-Sepharose beads. The binding reaction was carried out as
described under ``Materials and Methods.'' The beads were
then boiled in Laemmli's buffer and transferred, and the blot was
probed with an anti-JAK2 Ab. The JAK2 is shown by an arrow.
The molecular sizes are shown on the y axis in
kilodaltons.
Because hematopoietin
receptors contain two conserved stretches of amino acids in the
membrane-proximal region, identified as box 1 and box 2, it was of
particular interest to examine the role of each of these sequences in
JAK2 binding. The various fusions tested are diagrammed in
Fig. 1A. The results (Fig. 1B)
demonstrate that JAK2 protein binding requires aa 458-495
(construct box 1 plus), which includes box1 and 14 amino acids between
box 1 and 2 of the GM-CSFR . Increasing the length of
the fusion protein including aa 458 to 559 (construct box 1 and 2) also
allowed this binding, but deletion of 11 amino acids (458-484,
construct box 1) including box 1 plus 3 amino acids markedly reduced
the binding of JAK2, suggesting that amino acids 484-495 play a
crucial role in this interaction. However, neither the region between
box 1 and 2 (aa 483-530, construct bb) nor the region between the
boxes combined with box 2 (aa 483-559, construct box 2) mediate
significant JAK2 binding. Therefore, JAK2 binding requires the
membrane-proximal region including box 1 and the next 14 amino acids.
), which is conserved in all hematopoietin receptors,
is important for both interleukin-6 and Epo-dependent growth and signal
transduction. This conserved tryptophan is found in the box 1 plus
fusion protein but not in the box 1 protein, suggesting that it might
also play an important role in the binding of JAK2 to the GM-CSF
receptor. Although it is also found in box bb, this tryptophan is not
sufficient alone to mediate binding.
The NH
A
similar approach was used to examine which region(s) of the JAK2
protein are important for interaction with the GST GM-CSF -terminal Domain of Jak2 Contains
the GM-CSF
Receptor Binding Region
receptor fusion (aa 458-495). Either the wild type or the
three JAK2 mutants (Fig. 2A) lacking either the
amino-terminal 239 residues (ATD), the carboxyl-terminal 130 residues
of the protein kinase domain (CTD) removing the phosphotransferase
motif, or a significant portion of the kinase-like domain (223 amino
acids out of 283 predicted) (PSKD) were expressed in CV-1 cells
(Fig. 2B). These four proteins were then individually
incubated with the box1 plus fusion protein. The PSKD, CTD, and WT JAK2
each bound to the box 1 plus fusion as well as the WT JAK2
(Fig. 2C), whereas no binding was detected when these
constructs were incubated with GST protein (data not shown). Notably,
deletion of the amino terminus of JAK2 (ATD) markedly reduced the
binding with the GST GM-CSF
receptor fusion,
suggesting that the JAK2 GM-CSF
receptor interaction
domain is in the amino terminus of the molecule
(Fig. 2C).
Figure 2:
The binding of wild type and JAK2 mutants
to the box 1 plus GST fusion protein. A, structure of the wild
type and JAK2 mutants. The kinase-like domain of the WT JAK2 is
closed, while the protein kinase domain is denoted with
stripes. The ATD is from amino acid 2 to 239. The CTD is from
1000 to 1129, and the PSKD is from 523 to 746. B, the WT and
deletion constructs were transfected into vaccinia-infected CV-1 cells.
Extracts of the cells were run on an SDS-PAGE gel, transferred, and
probed with anti-JAK2. The molecular sizes in kilodaltons (kDa) are
shown on the y axis. Arrows denote the four proteins.
C, extracts shown in B were mixed with box 1 plus
fusion protein beads, and binding reactions were conducted as described
under ``Materials and Methods.'' The beads were then boiled
in Laemmli's buffer, run on an SDS-PAGE, transferred, and Western
blotted with an anti-JAK2 antibody.
To demonstrate the physical association
between the amino terminus of JAK2 and the GM-CSF box
1 plus box 2 amino acids, we generated GST fusion proteins, each
containing approximately one-fourth of the JAK2 protein (GST-J1, aa
1-294; GST-J2, aa 295-522; GST-J3, aa 523-746; and
GST-J4, aa 747-1129). Western blotting with an anti-GST antibody
(Fig. 3A) demonstrated that these GST fusion proteins
have molecular masses ranging from approximately 50 to 68 kDa. To map
the region of JAK2 that associates with the box 1 plus box 2 region of
receptor, a far Western blot was carried out. The
GST-JAK2 fusion proteins in bacterial lysate were run on an SDS-PAGE
gel and transferred to nitrocellulose. This blot was then probed with a
biotin-labeled GST GM-CSF
(aa 458-559;
construct box 1 and 2) and the protein interaction identified by
developing the filter with streptavidin-conjugated alkaline
phosphatase. Only the GST-J1 fusion protein bound the probe,
demonstrating that amino acids 1-294 of JAK2 contain GM-CSF
receptor protein binding domain.
Figure 3:
Interaction of the amino terminus of JAK2
with GM-CSFR 458 to 559 (construct box 1 and 2).
A, bacterial extracts expressing GST-JAK2 fusion proteins
(lane1, GST-J1 aa 1-294; lane2, GST-J2 aa 295-522; lane3,
GST-J3, aa 523-746; and lane4, GST-J4 aa
747-1129) were run on an SDS-PAGE gel. This gel was then transferred
and probed with an anti-GST antibody 1:1000 dilution (Santa Cruz
Biotechnology, Santa Cruz, CA). B, a Western blot identical to
that described in panelA was probed with biotin
labeled GM-CSFR
458 to 559 (construct box 1 and 2)
and the far Western blot developed with streptavidin-conjugated
alkaline phosphatase. The arrow indicates the binding of the
labeled probe to GST-J1. Some nonspecific color reaction is seen
between lanes3 and
4.
Binding of JAK2 to the GM-CSFR
We sought to determine whether the Jak2
mutants had functional consequences on GM-CSFR Is
Required for GM-CSFR
Tyrosine
Phosphorylation
phosphorylation in vivo. After infection with the
vaccinia virus vvT7 pol
(27, 28) , we transiently
expressed the WT JAK2 and the JAK2 mutants along with GM-CSF
and
subunits in CV-1 cells (Fig. 4A). Because the
receptor cDNA expression plasmid contains the T7
polymerase initiation site, the receptor is also overexpressed
(Fig. 4C). In the absence of JAK2, no tyrosine
phosphorylation of the GM-CSFR
is seen
(Fig. 4D). Overexpression of the WT JAK2 or PSKD JAK2
mutant stimulated tyrosine phosphorylation of the GM-CSFR
in the absence of added GM-CSF, suggesting that overexpression of
JAK2 is sufficient to activate its protein kinase activity
(Fig. 4D). The absence of a significant portion of the
PSKD did not inhibit phosphorylation.
Figure 4:
Regulation of GM-CSFR
phosphorylation. A, CV-1 cells were infected with vaccinia
virus and then transfected with the WT JAK2 or the kinase-like domain
(PK, PSKD), carboxyl-terminal (CD), or
amino-terminal (AD) deletion JAK2 mutants as well as the
and
GM-CSF receptors. After 24 h, the JAK2 was
immunoprecipitated. The immunoprecipitate was run on an SDS-PAGE gel
and Western blotted with JAK2 antisera. The amino-terminal deletion
mutant is shown by an arrow. Molecular sizes are shown in
kilodaltons on the y axis. B, the Western blot in
A was stripped and reprobed with anti-Tyr(P) antibody. The
amino-terminal deletion is shown by an arrow. C,
one-third of the extracts described in A were
immunoprecipitated with an antibody to the
GM-CSF receptor. The
immunoprecipitate was run on an SDS-PAGE gel and Western blotted with
the same antibody. D, the Western blot in C was
stripped and probed with an anti-Tyr(P) antibody. The
receptor is
shown with an arrow.
CTD, lacking the COOH-terminal
130 amino acids that include the phosphotransferase motif
(PXXWYXPE), was not detectably phosphorylated in
these cells (Fig. 4B). This kinase-deficient Jak2 was
not able to promote tyrosine phophorylation of the GM-CSFR (Fig. 4D) despite its ability to interact with the
box 1 plus fusion protein (Fig. 2C) in vitro.
In contrast, when overexpressed the ATD can be phosphorylated on
tyrosine to a similar extent as the WT JAK2 (Fig. 4B,
arrow), but it failed to phophorylate GM-CSFR
(Fig. 4D), indicating that the phosphorylation of
the GM-CSFR
requires the amino terminus of JAK2.
phosphorylation, an identical experiment was
performed without vaccinia virus vvT7 pol infection. Twenty-four hours
after transfection, cells were serum-starved for 8 h and stimulated
with human GM-CSF (10,000 units/ml) for 10 min. A Western blot of the
immunoprecipitated GM-CSFR
with anti-phosphotyrosine
Ab demonstrates that transfection of either the ATD or the CTD of JAK2
did not mediate phosphorylation of the receptor, even in the presence
of GM-CSF (Fig. 5A). GM-CSF induced tyrosine
phosphorylation of the GM-CSFR
when WT JAK2 was
coexpressed. Coexpression of the GM-CSFR
with the
PKSD JAK2, in contrast, resulted in tyrosine phosphorylation of the
GM-CSFR
independent of GM-CSF treatment. Reprobing of
these Western blots with the GM-CSFR
antisera demonstrates
equivalent expression of the
receptor (Fig. 5B).
Since the expression level of JAK2 is a critical regulator of its
phosphorylation, it is not possible from these experiments to determine
whether the pseudokinase domain plays a specific role in regulating the
phosphorylation of JAK2. Recently it has been shown that a protein
kinase-like domain of atrial natriuretic peptide is capable of binding
a protein phosphatase
(29) . It is possible that a phosphatase
binds to the JAK2 kinase-like domain and is inhibited by GM-CSF
addition.
Figure 5:
GM-CSF-dependent phosphorylation of the
GM-CSF receptor. A, CV-1 cells were
transfected with the
and
GM-CSF receptors, and JAK2 WT and
the mutants. After 24 h, the medium was removed and the cells were
starved for 8 h in DMEM plus 0.5% bovine serum albumin. The cells were
treated with 10,000 units/ml GM-CSF for 10 min and then lysed and
immunoprecipitated with the
GM-CSF receptor antibody.
The immunoprecipitate was run on an SDS-PAGE gel, transferred, and
probed with anti-Tyr(P) antibody. B, the blots shown in A were stripped and reprobed with the
GM-CSF
receptor antibody.
In this communication, we demonstrate that the
amino-terminal 239 amino acids of JAK2 are necessary for binding to a
36-amino acid domain in the membrane-proximal region of the GM-CSFR
. This JAK2 binding is necessary for the
phosphorylation of the GM-CSFR
receptor in
vivo.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.