From the Division of Hematology, University of
Washington, Seattle, Washington 98195 and § The Puget
Sound Blood Center and Program, Seattle, Washington 98104
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The Janus family of tyrosine kinases (JAKs) plays
a critical role in signal transduction by members of the cytokine
receptor superfamily. In response to ligand-receptor interaction, these nonreceptor tyrosine kinases are rapidly phosphorylated and
activated, triggering tyrosine phosphorylation and activation of
downstream signaling intermediates. Upon binding to its receptor, the
product of the proto-oncogene c-mpl, thrombopoietin (TPO)
activates both JAK2 and TYK2 in multiple cell lines as well as
megakaryocytes and platelets. To study whether one or both of these
kinases are essential for TPO signal transduction, we engineered a
parental human sarcoma cell line (2C4) as well as sarcoma cell lines
that are deficient in JAK2 expression ( Tyrosine phosphorylation and activation of the Janus tyrosine
kinases (JAK)1 is an
essential element of signal transduction by all members of the
hematopoietic cytokine receptor family (reviewed in Refs. 1 and 2).
There are four members of the Janus kinase family (JAK1, JAK2, JAK3,
and TYK2), cytoplasmic proteins that associate with the intracellular
portion of cytokine receptors via conserved box 1 and box 2 motifs in
the membrane-proximal portion of the receptor. The box 1 motif is
defined by two characteristically spaced prolines (PXXP)
located within 30 residues of the transmembrane domain (3). Box 2 is
usually located 35-60 residues downstream of the transmembrane domain,
and contains 12-18 residues, rich in glutamic acid and serine (3). It
is likely that the specificity of JAK-receptor interactions is mediated
through these regions, although no definitive JAK binding motifs have
been recognized. Ligand binding leads to receptor multimerization and
permits trans-phosphorylation and activation of the associated kinases.
The utilization of a specific JAK or pair of JAKs is characteristic of
each receptor (reviewed in Refs. 1, 4, and 5).
Thrombopoietin (TPO), the primary regulator of platelet production (6),
is structurally and functionally related to a large family of hormones,
cytokines, and interleukins. Upon binding to its receptor, Mpl, TPO is
believed to induce receptor homodimerization. This hypothesis is
supported by the absence of interaction with the known common signaling
subunits (gp130, IL-2-R- To address these questions, we utilized human sarcoma cells that were
originally selected on the basis of their failure to transduce an
interferon-specific signal. These cells were found to lack normal
expression of either JAK2 ( Reagents--
Monoclonal phosphotyrosine antibody (4G10) and
polyclonal antiserum against JAK2 and Shc were purchased from Upstate
Biotechnology Inc. (Lake Placid, NY), and polyclonal antiserum against
TYK2 and STAT3 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal antiserum against Mpl was generously provided by
Zymogenetics (Seattle, WA).
Cell Lines--
Human sarcoma cell lines were generously
provided by George Stark (Cleveland Clinic, OH) and were each
maintained in Dulbecco's modified Eagle's medium plus 10%
heat-inactivated calf serum, antibiotics (penicillin, streptomycin,
fungizone), and L-glutamine. 2C4 (parental) and Preparation of Lysates--
When 15-cm tissue culture dishes
contained a nearly confluent cellular monolayer, the cells were washed
twice with phosphate-buffered saline and then incubated 12-16 h in
Dulbecco's modified Eagle's medium plus 0.5% bovine serum albumin,
antibiotics, and L-glutamine. Matched plates were either
unstimulated or exposed for 10 min to 15 ng/ml murine TPO (produced as
conditioned media by TPO-secreting baby hamster kidney cells). Cells
were scraped off the dishes with rubber spatulas and resuspended at
4 °C in 1 ml of lysis buffer (pH 7.4, 50 mM HEPES, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA, 100 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 1 mM NaVO4, 1 µg/ml leupeptin, and 1 µg/ml
aprotinin). After 10 min (4 °C, occasional vortexing), the
particulate material was removed by centrifugation (12,000 × g, 10 min, 4 °C), and the cleared lysates were stored at
Immunoprecipitation and Western Blot Analysis--
Protein
concentrations of lysates were determined by modified Lowry assay (D/C
Protein Assay, Bio-Rad); 1 mg of total protein was used for each
immunoprecipitation. The appropriate antibody was added (2.0 µg or
2.0 µl if concentration not available), the volume was adjusted to 1 ml using fresh lysis buffer, and the reactions were gently mixed at
4 °C for 2 h. Twenty-five µl of protein A-agarose beads
(Santa Cruz Biotechnology) were added for an additional 1-h incubation
at 4 °C. The beads were then collected by centrifugation, washed
three times in cold lysis buffer, and then boiled in sample loading
buffer, containing 2% SDS and 1% Electrophoretic Mobility Shift Assay--
The human
Sis Inducible Element (hSIE;
GATCCATTTCCCGTAAATCGATC), a STAT3-specific double-stranded DNA
sequence, was used as a probe for active STAT3 (20). The
Prolactin Response Element (PRE;
GATCAGATTTCTAGGAATTCAAATCGATC) was used as a probe for STAT5-specific binding activity (20). The probes were radiolabeled as described previously (18) and were separated from unincorporated
[32P]ATP using Centri-Sep columns (Princeton Separations,
Princeton, New Jersey). Detergent-free lysates were prepared from
unstimulated or TPO-stimulated cells as described previously (18). In a
final volume of 10 µl, 10 µg of protein were combined with 4 µg
of poly(dI·dC), binding buffer (final concentrations: 15 mM HEPES, pH 7.9; 125 mM NaCl; 8% glycerol; 1 mM dithiothreitol; 0.15 mM EDTA), and the
radiolabeled hSIE probe (0.6 pmol). After a 20-min incubation (22 °C), the total volume was subjected to electrophoresis on a
nondenaturing gel (4% acrylamide; 5% glycerol; 0.5× Tris-borate-EDTA buffer), using 0.5× Tris-borate-EDTA as the running buffer. When the
unbound probe had run into the buffer, the gel was dried and exposed to
x-ray film overnight to detect retarded bands.
JAK2 Is Required for TPO-dependent Tyrosine
Phosphorylation of Mpl--
Parental cells (2C4/mMpl) or those
deficient in either JAK2 ( Phosphorylation of JAK2 Does Not Require TYK2 Expression--
We
next wished to examine which of these cell lines sustained
TPO-dependent tyrosine phosphorylation of JAK2 and TYK2.
First, JAK2 was immunoprecipitated from lysates derived from each cell line either before or after TPO stimulation. These samples were analyzed by Western blot and probed to detect phosphotyrosine incorporation (Fig. 2A). We
found that the parental cell lines (2C4-Mpl) demonstrated JAK2
phosphorylation after TPO stimulation, and those cell lines lacking
JAK2 expression ( TYK2 Phosphorylation Requires JAK2 Expression--
Similarly,
immunoprecipitations were carried out with a TYK2-specific antibody to
determine which cells could support TPO-induced phosphorylation of
TYK2. Western blots were again probed with a phosphotyrosine-specific
antibody (Fig. 2B). Despite a low level of constitutive TYK2
phosphorylation, a marked increase in tyrosine phosphorylation was
observed in the TPO-stimulated parental cells (2C4-Mpl). As expected,
the TYK2-deficient cell lines (U1A-Mpl) had neither detectable TYK2
phosphorylation nor protein. Notably, the JAK2-deficient cell lines
( TPO-dependent Tyrosine Phosphorylation of Shc Requires
JAK2--
We next studied which JAK(s) were required for
phosphorylation of several downstream signaling proteins. The adapter
protein Shc that binds to a phosphotyrosine residue in the carboxyl
terminus of Mpl (21, 22) is believed to be directly phosphorylated by
JAKs and may play a critical role in TPO-dependent
development (21, 23). Immunoprecipitation and Western blot analysis
demonstrated that the phosphorylation of all three Shc isoforms
occurred in the absence of TYK2 but was not detectable in lysates
from JAK2-deficient cells (Fig.
3A).
Phosphorylation and Activation of STAT3--
In many cell lines
and human platelets, both STAT3 and STAT5 are directly tyrosine
phosphorylated and activated by Janus kinases (11, 12, 15), but in
murine megakaryocytes, STAT5 activation is minimal (18). Using lysates
from these cell lines, STAT3 was immunoprecipitated and tested for
TPO-dependent tyrosine phosphorylation by Western blot. As
for Shc and Mpl, STAT3 phosphorylation was dependent on functional JAK2
protein but not TYK2 (Fig. 3B). To further demonstrate that
Mpl was capable of mediating increased STAT3 DNA binding activity,
electrophoretic mobility shift assays were performed (Fig.
4). Using the STAT3-specific probe hSIE, TPO-dependent STAT3 binding was seen in the parental and
TYK2 nullizygous cells, but not in the JAK2-deficient cells (Fig.
4A). Although STAT5 was readily detected by immunoblotting
(data not shown), there was no activation of STAT5 binding activity in
any of these cells (Fig. 4B), a pattern of signaling
reminiscent of that observed in primary megakaryocytes (18).
Expression of Functional JAK2 in It is generally accepted that the activation of Janus kinases in
response to ligand binding is essential for signal transduction within
the hematopoietic cytokine receptor family. For the interferon- One previous study described a mutant form of Mpl that supports
TPO-dependent proliferation without phosphorylation of any JAKs, albeit requiring a much higher concentration of TPO (25). However, other reports suggest that mutations leading to the loss of
JAK activation result in nonfunctional receptors (14, 22, 26-28). It
is possible that additional tyrosine kinase families may be activated
by TPO stimulation (29). At present, however, the role of tyrosine
kinases other than JAKs has not been defined.
The sarcoma cell lines used in these studies are cytokine-independent
and highly primed for autonomous proliferation. Thus, it was not
possible to distinguish a proliferative response to TPO in these cell
lines (data not shown). Furthermore, as transformed fibroblasts, there
was no evident differentiation when grown in TPO for up to 1 week.
Specifically, the cells remained adherent with no alteration in
morphology or size (data not shown). Nevertheless, the reported data
clearly establish that JAK2 is essential for activation of all of the
tested secondary molecules implicated in TPO-induced signaling.
In both hematopoietic cell lines and human platelets, JAK2 and TYK2 are
tyrosine phosphorylated in response to TPO stimulation. To understand
how TPO and Mpl might activate two distinct JAK kinases, we constructed
three possible models of the active signaling complex. First, the
homodimeric receptor may associate with only one JAK, and the other
family member may be phosphorylated as a downstream or incidental event
(Fig. 6A). Second, both JAK2 and TYK2 may be able to bind to the box 1/box 2 motifs of Mpl and
function interchangeably during TPO signaling (Fig. 6B).
Third, there may be another, as yet unidentified, receptor subunit that specifically binds the second kinase molecule (Fig. 6C).
From each of these models of the active signaling complex, one can predict the effect if one of the two kinases were missing. In model B (center), the two kinases would be
redundant, and loss of one or the other might cause little change in
signal transduction. In the context of a heterodimeric receptor
(model C, right), both JAKs would be necessary to form an
active signaling complex, and loss of either one would disrupt
signaling. Finally, in model A (left), loss of
the JAK that directly binds the receptor would disrupt tyrosine
phosphorylation, whereas loss of the downstream kinase might have
little or no effect.
2A) or TYK2 expression (U1A) to express the wild-type Mpl receptor. The ability of TPO to induce tyrosine phosphorylation of Mpl and multiple intracellular substrates in each cell line was then examined. Our results demonstrate that JAK2-deficient cells (
2A-Mpl) are unable to initiate TPO-mediated signaling. In contrast, cells that are TYK2-deficient (U1A-Mpl) are
able to induce tyrosine phosphorylation of Mpl, JAK2, STAT3, and Shc as
efficiently as parental cells (2C4-Mpl). These data indicate that JAK2
is an essential component of Mpl signaling and that, in the
absence of JAK2, TYK2 is incapable of initiating TPO-induced
tyrosine phosphorylation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, and the common
-chain of the GM-CSF,
IL-3, and IL-5 receptors) as well as reports that Mpl homodimerization
is sufficient for signaling (7-11). In general, homodimeric receptors
utilize a single JAK (e.g. erythropoietin and prolactin
receptors utilize JAK2), whereas heterodimeric (or heterotrimeric)
receptors require two distinct JAKs (e.g. interferon-
uses JAK1 and TYK2; interferon-
uses JAK1 and JAK2) (reviewed in
Ref. 12). Thus, it was surprising when several reports demonstrated
that TPO induces phosphorylation of two distinct Janus family members,
JAK2 and TYK2. This has been observed both in cell lines engineered to
express the Mpl receptor (13, 14) as well as human platelets (15),
which likely represent a more physiologically relevant environment. Others have shown that, of the four Janus kinases, only JAK2 is tyrosine-phosphorylated in response to TPO (16, 17). In our previous
studies, we reported that there was a difference in JAK phosphorylation
between cell lines and purified, primary megakaryocytes. Whereas cell
lines, which proliferate in response to TPO, demonstrated abundant JAK2
and TYK2 phosphorylation after TPO stimulation, mature megakaryocytes
contained primarily JAK2 phosphorylation; TYK2 tyrosine phosphorylation
was barely detectable (18). This raised several important questions
about the earliest events during TPO signaling. Are JAK2 and TYK2 both
essential for Mpl signaling? What would be the consequence if JAK2 or
TYK2 were absent during TPO/Mpl signaling?
2A) or TYK2 (U1A) and interfere with
interferon-
or interferon-
signaling, respectively (12). These
cells have previously been used to demonstrate that although IL-6
stimulation induces tyrosine phosphorylation of three distinct Janus
kinases (JAK1, JAK2, and TYK2), JAK1 is most important for activation
of downstream signaling molecules (19). Based on this strategy, we
engineered each of these cell lines and the corresponding parental
cells to express the full-length murine Mpl receptor and then assessed
the ability of TPO to initiate signal transduction. Our results
demonstrate that JAK2, but not TYK2, is required for tyrosine
phosphorylation of Mpl, STAT3, and Shc. This suggests that TYK2
phosphorylation, although readily detected in various cell lines, is
not an essential step in TPO signaling. Dependence on a single Janus
kinase is consistent with our understanding of homodimeric receptors.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2A (JAK2
deficient) lines were grown in the presence of hygromycin, 250 micrograms/ml (Sigma). U1A (TYK2-deficient) cells were selected in
neomycin, 1 mg/ml (Sigma). 2C4 and
2A were transfected with a murine
Mpl expression vector (pCDNA3-mMpl) by the method of calcium
phosphate precipitation. Neomycin-resistant clones were selected by
addition of 1 mg/ml G418 and then tested for Mpl expression by Western
blot (Fig. 1) and fluorescence-activated cell sorter analysis (data not
shown). U1A cells were simultaneously transfected with pCDNA3-Mpl
and pCMV ouabain, a ouabain resistance plasmid (PharMingen, San Diego,
CA), and clones were selected by addition of 1 µM ouabain
(Sigma) to the culture media. Expression of Mpl was confirmed as above.
70 °C until use.
-mercaptoethanol. Samples were
loaded onto 7.5% acrylamide Laemmli mini-gels with prestained
molecular weight markers (Life Technologies, Gaithersburg, MD). Protein
was electrophoretically transferred to nitrocellulose which was then
blocked in Tris-buffered saline with 0.05% Tween 20 (TBST) and 3%
bovine serum albumin. Primary antibodies were used according to
manufacturer recommendations at room temperature for 2 h. The
membrane was washed three times with TBST (5 min per wash) then
incubated with goat anti-mouse or goat anti-rabbit IgG coupled to
horseradish peroxidase (1:5000, Bio-Rad, Hercules, CA) for 30 min. After three washes in TBST at 5 min each, chemiluminescent
reagents (Santa Cruz Biotechnology) were added for 1 min and the
membranes were exposed to x-ray film for 10-90 s. The nitrocellulose
was stripped prior to reprobing by incubating at 50 °C for 30 min in
62.5 mM Tris (pH 6.8), 100 µM
-mercaptoethanol, and 2% SDS.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2A/mMpl) or TYK2 (U1A/mMpl) were either
unstimulated or exposed to TPO as described under "Experimental
Procedures." Anti-Mpl antiserum was then used to immunoprecipitate
the receptor from whole cellular lysates; at least two independent
clones of each cell type were used. Immunoblotting with a
phosphotyrosine-specific antibody (4G10) was done to detect TPO-induced
Mpl phosphorylation (Fig. 1). We found
that the cells with all JAKs present as well as those lacking TYK2
expression were able to phosphorylate Mpl, whereas JAK2-deficient cells
were unable to phosphorylate Mpl.
View larger version (105K):
[in a new window]
Fig. 1.
Mpl phosphorylation in JAK2- and
TYK2-deficient cells. Cell lysates were prepared either before
( ) or after (+) stimulation with 15 ng/ml TPO for 10 min. Two
independently derived clones of cells expressing Mpl were tested for
each of the parental (2C4/Mpl), JAK2-deficient
(
2A/Mpl), and TYK2-deficient
(U1A/Mpl) lines. In each lane,
anti-Mpl antiserum was used to immunoprecipitate the receptor from 1 mg
of total cell lysate. The resulting Western blot was probed with an
anti-phosphotyrosine monoclonal antibody (4G10) to detect tyrosine
phosphorylation of Mpl. Below, the blot was stripped and
reprobed to demonstrate the presence of Mpl in all lanes. Essentially
identical results were obtained from three independent
experiments.
2A-Mpl) had neither phosphorylation nor detectable
protein (Fig. 2A, lower strip). However, cell lines
deficient in TYK2 expression (U1A-Mpl) retained JAK2 phosphorylation,
suggesting that JAK2 activation occurred upstream or independent of
TYK2.
View larger version (39K):
[in a new window]
Fig. 2.
Tyrosine phosphorylation of JAK2 and
TYK2. Cell lysates (1 mg total protein) from two clones of
parental (2C4/Mpl), JAK2-deficient
( 2A/Mpl), and TYK2-deficient
(U1A/Mpl) cell lines were immunoprecipitated with
anti-JAK2 (A) or anti-TYK2 (B) antibodies. The
resulting Western blots were probed to detect
phosphotyrosine-containing proteins. Each blot was then stripped and
reprobed with the same antibody used during immunoprecipitation
(panels below).
2A-Mpl) displayed only low level basal phosphorylation of TYK2,
which was not increased by TPO stimulation. This result demonstrates
that TPO-induced TYK2 phosphorylation requires JAK2 activity.
View larger version (33K):
[in a new window]
Fig. 3.
Tyrosine phosphorylation of Shc and
STAT3. Specific antibodies against Shc (A) and STAT3
(B) were used to immunoprecipitate these signaling proteins
from cell lysates derived from parental
(2C4/Mpl), JAK2-deficient
( 2A/Mpl), and TYK2-deficient
(U1A/Mpl) cells. The Western blots were probed to
detect phosphotyrosine incorporation after TPO stimulation. The
nitrocellulose membranes were then stripped and reprobed to demonstrate
equal immunoprecipitation in all lanes (lower panels). In
this particular experiment (B), only one U1A cell line is
shown for STAT3, but similar results were obtained from three distinct
U1A clones in other experiments.
View larger version (53K):
[in a new window]
Fig. 4.
Electrophoretic mobility shift assay.
Detergent-free lysates were generated either before ( ) or after (+)
TPO stimulation for 10 min. Ten µg of protein was incubated with
either the STAT3-specific hSIE probe (A) or the
STAT5-specific PRE probe (B), each radiolabeled with
32P. Extracts from Ba/F3-Mpl cells were used as positive
controls for STAT3 and STAT5 activation. The samples were resolved on
4% nondenaturing acrylamide gels, and the dried blots were exposed to
x-ray film for 12 h. The arrows indicate the position
of active STAT3/hSIE or STAT5/PRE complexes.
2A/Mpl Cells Restores TPO
Signaling--
To prove that JAK2 was essential for TPO-signaling, we
expressed wild-type JAK2 in
2A/Mpl cells (JAK2-deficient) by
transfecting them with the expression vector pCDNA3.1(Zeo)-JAK2
(JAK2 cDNA kindly provided by Stuart Frank, Birmingham,
AL and James Ihle, Memphis, TN). Cells resistant to Zeocin (500 µg/ml, Invitrogen) were screened for JAK2 expression by Western blot
and then tested for TPO-induced phosphorylation of Mpl. When Mpl was
immunoprecipitated from
2A-Mpl and
2A-Mpl/JAK2 cells,
TPO-dependent phosphorylation of the receptor was restored
in the cells expressing functional JAK2 (Fig.
5).
View larger version (57K):
[in a new window]
Fig. 5.
Phosphorylation of Mpl is restored by JAK2
expression. Mpl was immunoprecipitated from 2A/Mpl
(JAK2-deficient cells) and
2A/Mpl/JAK2 cells (engineered to express
native murine JAK2). Tyrosine phosphorylation of Mpl was analyzed by
Western blot either before (
) or after (+) TPO stimulation
(upper panel). The blot was then stripped and reprobed to
demonstrate equal amounts of Mpl in all lanes (middle
panel). On a separate blot, whole cell lysates (30 µg/lane) were
probed to detect JAK2 protein (bottom panel). The upper
band, seen in all lanes, is a nonspecific band.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
interferon-
receptors, two distinct JAKs are required for signal
transduction (4, 5, 12). Studies with the IL-6 receptor indicate that,
although three different JAKs are tyrosine phosphorylated after ligand
binding, only JAK1 is essential for downstream events (19). Similarly,
it was demonstrated that, although epidermal growth factor stimulation
activates JAK1, the resulting activation of STAT proteins and induction
of c-fos depends only on the intrinsic epidermal growth
factor receptor kinase activity (24). Thus, the functional role played
by tyrosine kinases cannot be predicted simply by studying the state of
phosphorylation or activation. Also, the receptor cytoplasmic domain
exhibits specificity regarding which JAKs are recruited to the cell
membrane and then activated through receptor subunit aggregation.
View larger version (18K):
[in a new window]
Fig. 6.
Models for the active TPO/Mpl signaling
complex. Diagrams are used to depict three hypothetical molecular
complexes after TPO-induced receptor aggregation. The white
and black ovals (labeled "JAK") indicate two
distinct Janus kinase family members. The gray bands in the
receptor membrane-proximal cytoplasmic domain represent box 1 and box
2. In model C, the receptor subunit with a
different shape and length represents a potential non-Mpl signaling
subunit.
Our results strongly support the model in Fig. 6A and suggest that JAK2 is essential for TPO-stimulated tyrosine phosphorylation of the receptor (Mpl), both Janus kinases (JAK2 and TYK2), and the downstream effector molecules (STAT3 and Shc). TYK2 is neither sufficient nor necessary for any of these events. Despite the fact that these studies were done in a transformed sarcoma cell system, the early signaling apparatus appears to be intact, and this same system has been used previously to determine the requirements for signal transduction by the interferon, IL-6, and epidermal growth factor receptors (12, 19, 24). Furthermore, our results are strengthened by the fact that signal transduction can be restored in the JAK2-deficient cell line by expression of functional JAK2.
These data reinforce the recent information obtained from the
development of JAK2-deficient mice (30). JAK2 knock-out animals were
not viable because of failure of embryonic erythrocyte development. However, study of the fetal liver hematopoietic progenitors
demonstrated that JAK2 /
cells were unresponsive to several
cytokines, including erythropoietin, thrombopoietin, interleukin-3, and
granulocyte-monocyte colony stimulating factor. However, responses to
granulocyte colony stimulating factor, interleukin-6, and
interferon-
were unaffected because the corresponding receptors
utilize other JAK family members. Thrombopoietin and Mpl, therefore,
belong to the subset of cytokine/receptor pairs that absolutely require
JAK2 for signaling activity.
In contrast, there have been no reports of TYK2 knock-out animals, and
data from cell lines as well as human platelets suggested that TYK2 is
tyrosine phosphorylated in response to TPO. In this report, we
establish that TYK2 is a secondary event in thrombopoietin signaling,
downstream of JAK2 activation, and not required for the phosphorylation
of other signaling molecules. It is possible that TYK2 is
phosphorylated either as a substrate of JAK2 or through receptor
cross-talk (i.e. Mpl activation might activate another associated receptor that itself specifically binds TYK2). However, we
have previously demonstrated that TYK2 is not phosphorylated to a
significant degree in mature murine megakaryocytes (18). These combined
pieces of evidence strongly suggest that the active signaling complex
for TPO and Mpl contains a homodimeric receptor in which each receptor
subunit binds an activated JAK2 molecule.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank George Stark (Cleveland, OH) for the
parental 2C4, 2A, and UIA cell lines; Don Foster (Seattle, WA) for
mMpl cDNA and anti-Mpl antiserum; Stuart Frank (Birmingham, AL) and
James Ihle (Memphis, TN) for the murine JAK2 cDNA; Chong Kim (Seattle, WA) for assistance with flow cytometry; and Michele Mehaffey and Jennifer Luthi (Seattle, WA) for technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant K08 HL03498-02 and by an American Society of Hematology Fellow Scholar Award.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Puget Sound Blood Center and Program, Research Division, 921 Terry Ave., Seattle, WA 98104. Tel.: 206-292-6510; Fax: 206-343-1776; E-mail: drachman{at}u.washington.edu.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: JAK, Janus tyrosine kinase; TPO, thrombopoietin; IL, interleukin; hSIE, human Sis-inducible element; PRE, prolactin response element.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|