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J. Biol. Chem., Vol. 275, Issue 41, 32214-32219, October 13, 2000
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From the Divisions of Hematology and Cardiology, University of
Washington School of Medicine, Seattle, Washington 98195
Received for publication, June 12, 2000, and in revised form, July 13, 2000
Thrombopoietin (TPO), the critical regulator of
platelet production, acts by binding to its cell surface
receptor, c-Mpl. Numerous studies have shown that TPO binding
leads to JAK2 kinase activation and Tyr phosphorylation of c-Mpl and
several intracellular signaling intermediates, events vital for the
biological activity of the hormone. In contrast, virtually nothing is
known of the role of Ser or Thr phosphorylation of c-Mpl. By using
phosphoamino acid analysis we found that Ser residues of c-Mpl were
constitutively phosphorylated in receptor-bearing cells, levels that
were increased following exposure of cells to TPO. To identify which
residues were modified, and to determine the functional consequences of their phosphorylation, we generated a series of Ser to Ala mutations of
a truncated c-Mpl receptor (T69) capable of supporting TPO-induced cell
growth. Of the eight Ser within T69 we found that at least four are
phosphorylated in TPO-stimulated cells. The mutation of each of these
residues alone had minimal effects on TPO-induced proliferation, but
substitution of all of the phosphoserine residues with Ala reduced the
capacity of the receptor to support cell growth by over 50%.
Additionally, the Ser at cytoplasmic position 18 is not detectably
phosphorylated. However, the mutation of Ser-18 to Ala nearly abrogates
TPO-induced proliferation and co-precipitation of JAK2 with Mpl. This
study provides the first systematic analysis of the role of Ser
residues in c-Mpl signaling.
Thrombopoietin is a hematopoietic cytokine regulating
megakaryopoiesis and platelet production (1). The receptor for
thrombopoietin, c-Mpl, is expressed on hematopoietic stem cells,
immature hematopoietic progenitor cells, megakaryocytes, and platelets.
The genetic elimination of either
TPO1 or c-Mpl in mice reduces
the levels of all of these cell types by 70-90%, establishing
that TPO-induced signaling plays a major physiological role in
hematopoiesis in general and megakaryopoiesis in particular (2, 3). The
intracellular signaling pathways utilized by c-Mpl have been
extensively studied, focusing primarily on tyrosine
phosphorylation events. After stimulation with TPO, the c-Mpl receptor
is believed to alter its conformation resulting in a homodimeric
receptor complex capable of supporting the transphosphorylation and
activation of JAK2 and TYK2 tyrosine kinases (4-6). Once activated,
JAK kinases phosphorylate a number of substrates, including c-Mpl
itself, providing docking sites for Src homology 2- and phosphotyrosine-binding motif-containing proteins, including the latent
transcription factors STAT3 and STAT5 and a number of adapter proteins
including Shc, Vav, and Cbl (7, 8). Other signaling intermediates have
also been reported to be activated by TPO in various cell lines and
primary cells including mitogen-activated protein kinase (MAPK),
protein kinase C, and phosphatidylinositol 3-kinase (9, 10).
Despite the many published reports on tyrosine phosphorylation of
cytokine receptors, it is apparent that other mechanisms for signal
transduction must exist. For instance, a mutant receptor of c-Mpl, in
which all of the tyrosine residues were eliminated by deletion or Phe
substitution, is capable of supporting cellular proliferation in
cytokine-responsive cell lines (4). Moreover, it has been reported that
elimination of the proximal nine residues of the Mpl receptor
eliminates JAK2 activation but is capable of supporting cell growth
(11). Candidate sites for supporting alternate signaling pathways are
phosphorylation of Ser and/or Thr residues.
The murine c-mpl gene encodes a polypeptide predicted to
contain a 25-residue secretory leader, a 457-amino acid extracellular domain, a 22-residue transmembrane domain, and a 121-amino acid cytoplasmic domain (12). The cytoplasmic domain of the Mpl receptor contains 5 Tyr, 8 Thr, and 13 Ser residues. However, it is clear from
both in vitro and in vivo studies that distal
truncation of up to one-half of the receptor cytoplasmic domain
maintains its capacity to support cell proliferation and
differentiation (4, 13). To facilitate our analysis of the Ser and Thr
phosphorylation of c-Mpl, we have utilized a truncated form of the
receptor bearing the membrane-proximal 69 residues of the cytoplasmic
domain (T69), and site-specific Ser and Thr to Ala mutants of this
receptor. T69 includes two Tyr residues that are not phosphorylated in
response to TPO stimulation in BaF3/mpl cells and contains four Thr and eight Ser residues. In this study, we demonstrated that at least four
of the Ser residues in T69 are phosphorylated and that the Thr residues
are not modified. Furthermore, multiple substitution of all Ser(P)
residues significantly reduced the capacity of the receptor to support
cellular proliferation. Moreover we found that the Ser at position 18 is critical for JAK2 binding to the receptor and cellular proliferation
although it is neither constitutively nor inducibly phosphorylated.
These results provide the first systematic structure-function analyses
of the Ser and Thr residues of the c-Mpl receptor.
Reagents--
Purified recombinant murine TPO (rmTPO) was the
generous gift of Dr. Akihiro Shimosaka (Kirin Pharmaceuticals, Tokyo,
Japan). Western blot chemiluminescence reagents were purchased from
PerkinElmer Life Sciences, and all other reagents were purchased from
Sigma unless otherwise indicated.
Cell Lines and Site-directed Mutagenesis--
The mammalian
expression vector, pcDNA3 (Invitrogen, Carlsbad, CA), containing
murine c-mpl, was used as a template to make multiple Ser
and Thr to Ala mutations using the Quickchange mutagenesis kit
(Stratagene, La Jolla, CA). The PCR products were sequenced to confirm
the result of mutagenesis with the BigDye Terminator cycle sequencing
kit (Applied Biosystems, Foster City, CA). The murine interleukin
(IL)-3-dependent cell line BaF3 was engineered to express
the full-length murine Mpl receptor (BaF3/Mpl), the T69 truncation
(BaF3/T69), and the site-directed T69 mutants by electroporation and
limiting dilution into 1 mg/ml G418-containing culture medium.
Individual clones of each cell type were selected, and multiple clones
for each receptor construct were analyzed by flow cytometry (Becton
Dickinson, San Diego, CA) using a rabbit anti-murine Mpl antibody (4).
Clones expressing similar levels of surface Mpl receptors were selected
for further analysis and were maintained in RPMI 1640 medium
(BioWhittaker, Walkersville, MD) with 10% heat-inactivated fetal calf
serum (HyClone, Logan, UT), murine IL-3, and G418.
Cell Proliferation Assays--
Cell proliferation was measured
by MTT assay as described previously (4). Briefly, the cells were
incubated with serial concentrations of rmTPO or an optimal level of
murine IL-3-conditioned medium for 48 h. Proliferation was
expressed as a percentage of maximal murine IL-3-induced growth.
Phosphoamino Acid Analysis and Phosphopeptide
Mapping--
Phosphorylation of c-Mpl was studied by incubating cell
lines expressing c-Mpl or its mutation in phosphate-free RPMI 1640 medium (Life Technologies, Inc.) with 0.5% bovine serum albumin for
14 h and labeling with 2.5 mCi of
[32P]orthophosphate (PerkinElmer Life Sciences) for
3 h. During the last 10 min of incubation, 200 µM
sodium orthovanadate was added to the culture media, and the cells were
treated with 25 ng/ml rmTPO or control medium. The receptor was then
immunoprecipitated with an anti-Mpl antibody (AMM2, a kind gift of Dr.
Takashi Kato (Kirin Pharmaceuticals, Takasaki, Japan)) and
size-fractionated by SDS-PAGE. The proteins were transferred to a
polyvinylidene fluoride membrane (Millipore, Bedford, MA) and exposed
to BioMax XR film (Eastman Kodak Co.). The radioactive bands
co-migrating with the 90-kDa c-Mpl or 80-kDa T69 were excised from the
membrane and boiled in 6 N HCl (Pierce) for 1 h at
110 °C to allow degradation to phosphoamino acids. Products of this
acid hydrolysis were analyzed by one-dimensional high voltage
electrophoresis using ninhydrin-stained phosphoamino acid standards
(14). For phosphopeptide mapping, BaF3/T69 cells were labeled with
[32P]orthophosphate, and T69 receptor proteins were
recovered by immunoprecipitation, separated by SDS-polyacrylamide gel
electrophoresis (SDS-PAGE), transferred to a nitrocellulose membrane
(Schleicher & Schuell), and excised from the membrane following
autoradiography. The radiolabeled proteins were incubated with sequence
grade trypsin (Promega, Madison, WI) at 37 °C for 18 h and
separated on cellulose coated 20 × 20-cm thin layer
chromatography (TLC) glass plates (Merck) in 10% acetic acid, 1%
pyridine (v/v) buffer at 1000 V for 2 h at 10 °C. The plate was
then used for TLC in the second dimension for 5 h in a buffer of
30% 1-butanol, 30% pyridine, 6% glacial acetic acid (v/v/v). The
radioactive spots were visualized and quantitated by phosphorimaging
(Molecular Dynamics, Sunnyvale, CA).
Immunoprecipitation and Western Blot Analysis--
BaF3/T69
cells and cells bearing the S18A mutation were incubated in serum- and
cytokine-free medium for 14 h, stimulated with or without 25 ng/ml
rmTPO for 10 min, and lysed as described previously (4). The protein
concentration of the lysate was measured using the Protein DC assay kit
(Bio-Rad). Specific proteins were immunoprecipitated with the indicated
antibodies and protein A/G-conjugated agarose beads (Santa Cruz
Biotechnology, Santa Cruz, CA). The immunoprecipitates were subjected
to SDS-PAGE and Western blot analysis as described previously using
anti-JAK2 and anti-phosphotyrosine (4G10) antibodies obtained from
Upstate Biotechnology (Lake Placid, NY) (4).
Phosphoamino Acid Analysis of the Mpl and T69 Receptors--
We
previously reported that two tyrosine residues in c-Mpl are
phosphorylated after stimulation with TPO (4). The cytoplasmic domain
of c-Mpl includes 13 Ser and 8 Thr residues, but little is known about
their phosphorylation status and functional role in TPO-induced
signaling. To explore whether some of these residues were
constitutively or inducibly phosphorylated, BaF3 cells were engineered
to express full-length c-Mpl receptor (BaF3/mpl) or a truncation mutant
containing the membrane-proximal 69 amino acids of the cytoplasmic
domain of c-Mpl (Fig. 1, T69)
and then metabolically labeled with [32P]orthophosphate.
The phosphorylation status of the receptor was analyzed before and
after stimulation with TPO by phosphoamino acid analysis (Fig.
2). In BaF3/mpl cells, Ser residues were
constitutively phosphorylated, and their radioactive intensities were
enhanced ~3-fold following stimulation with TPO (Fig. 2B).
Tyrosine phosphorylation was also markedly enhanced upon stimulation by
TPO, but Thr phosphorylation was barely detectable in BaF3/mpl cells
under any conditions. Like BaF3/mpl cells, constitutive and
TPO-inducible phosphorylation of Ser was also observed in T69 cells. As
anticipated from the results of our previous studies utilizing Western
blotting with a Tyr(P)-specific antibody (4), neither
constitutive nor inducible Tyr phosphorylation of the T69 receptor was
detected.
Tryptic Peptide Mapping of the T69 Mpl Receptor--
Since one or
more of the Ser residues of c-Mpl and T69 were found to be
phosphorylated, we performed additional studies to identify which Ser
residues were modified. As shown in Fig. 1, a predicted tryptic peptide
map suggests that the complete digestion of T69 with trypsin should
generate nine peptides, four of which bear one or more potential sites
of Ser phosphorylation. To begin to determine the sites of Ser(P), we
performed phosphopeptide mapping of BaF3/T69 cells (Fig.
3). We found that seven phosphopeptides were reproducibly generated by tryptic digestion of 32P
metabolically labeled T69. The intensity of all the radioactive spots
was enhanced after stimulation with TPO (Fig. 3B). Although it is possible that an extracellular domain peptide was labeled, it was
more likely that incomplete digestion of the intracellular domain of
Mpl was responsible for our finding more than the four predicted
labeled peptides. For example, although trypsin digests polypeptides
following lysine (Lys) and arginine (Arg) residues, it cuts poorly if
the basic residue is followed by a charged residue. Since one predicted
tryptic fragment (SSESTPLPL) has Ser in the first position (Fig. 1), if
phosphorylated it might prevent complete trypsin digestion at that site
and lead to an extra peptide. Subsequent analysis of a mutant Mpl
receptor bearing multiple Ser to Ala mutations (see below) confirmed
that all of the receptor phosphorylation events can be accounted for by
Ser phosphorylation within the cytoplasmic domain of Mpl.
To identify which of the 8 Ser residues within T69 are phosphorylated,
multiple Ser to Ala T69 receptor mutants were generated, and
phosphopeptide maps of each were determined (Fig.
4). As the phosphopeptide map of T69/S18A
was identical to T69, we established that Ser-18 was not constitutively
or inducibly phosphorylated (compare Figs. 4A and
3B). The remaining predicted tryptic phosphopeptides all
contained more than a single Ser residue, requiring the generation of
multiple Ser to Ala mutations. A phosphopeptide map of T69/S37A/S39A lost two radioactive spots compared with that of T69 (Fig.
4B), indicating that either one or both of Ser-37 and Ser-39
were phosphorylated in response to TPO. As this mutant retained full
proliferative activity (see below), we did not further analyze these
sites. Like T69/S37A/S39A, the T69/S46A/S53A double mutant also lost two 32P-radiolabeled spots in the phosphopeptide map (Fig.
4C). A single T69/S46A mutant also lost the same spots as
T69/S46A/S53A (Fig. 4D), indicating that Ser-46 is
phosphorylated. This conclusion could be extended; our finding an
identical phosphopeptide map of T69/S53A and T69 indicated that only
Ser-46 was phosphorylated within this peptide (compare Figs.
4E and 3B). There are three Ser residues and one
Thr residue in the last predicted tryptic peptide, SSESTPLL (Fig. 1).
Analysis of the T69/S61A/S62A/S64A/T65A mutant revealed the loss of two
radiolabeled peptides (Fig. 4F), consistent with at least
one of the three Ser sites being phosphorylated, with partial tryptic
digestion prior to Ser-61 accounting for the loss of two
phosphopeptides. Additional T69 mutants were analyzed to dissect the
phosphorylation status of this region. Interestingly, compared with
T69, T69/S61A/S62A (Fig. 4G) and T69/S64A/T65A (Fig. 4H) both lost the same spots as did the
T69/S61A/S62A/S64A/T65A mutants (Fig. 4F). It is possible
that like the p55 tumor necrosis factor (TNF) receptor, heat shock
factor-1, and rhodopsin proteins (15-17), phosphorylation of one Ser
residue might be responsible for phosphorylation of subsequent Ser
residues, helping to explain why the same spots were lost in different
mutations. Nevertheless, the results in Fig. 4, G and
H, indicate that Ser-61 (due to the partial digestion at
Lys-60/Ser-61) and Ser-64 (since Thr-65 is not phosphorylated), and
possibly Ser-62, are phosphorylated in TPO-stimulated BaF3 cells
bearing these mutant receptors. Finally, to be certain that all of the
sites of Ser phosphorylation were accounted for by Ser-37, Ser-39,
Ser-46, Ser-61, Ser-62 and Ser-64, a mutant Thr-69 receptor bearing Ser
to Ala changes at all these sites (and at Thr-65; referred as
T69/S37A/S39A/S46A/S61A/S62A/S64A/T65A) was tested and found to be
devoid of Ser(P) in 32P-labeled, TPO-stimulated BaF3 cells
(Fig. 5). In these experiments the same
number of 32P-labeled T69 cells were used as a positive
labeling control. This result confirmed that these residues contain the
sole sites for Ser phosphorylation of the proximal 69 residues of the
c-Mpl receptor.
Mutant T69 Receptor Cell Proliferation Assays--
To determine if
any of the Ser residues of T69 play a functional role in TPO-induced
BaF3 cells, the cell lines used in our Ser(P) analyses were evaluated
in cell proliferation assays. By using only clones that express the
same level of the mutant receptors as seen in BaF3/T69 cells, we found
that BaF3 cells bearing the S37A/S39A, S46A, S53A, S61A/S62A, and
S64A/T65A mutant T69 receptors displayed a nearly identical
dose-response proliferation curve as BaF3/T69 cells (Fig.
6, A and C-G). To
exclude the possibility that the function of these Ser residues is
redundant, we also tested T69/S37A/S39A/S46A/S61A/S62A/S64A/T65A cells;
we found >50% reduction of proliferation in response to TPO
stimulation (Fig. 6H). These results suggest that although
any single Ser(P) may not be crucial, the presence of some Ser(P)
within the first 69 cytoplasmic domain of Mpl contribute to the
capacity of the Mpl receptor to support the signals responsible for
cell proliferation.
Signaling Analysis of Hypoproliferative T69 Mutants--
Fig.
6B displays the proliferation response of BaF3/T69/S18A
cells; in essence, this single mutation nearly abrogated the capacity
of this receptor to support BaF3 cell growth. As Ser-18 resides within
box 1 of Mpl, the site of JAK2 binding, we examined the hypothesis that
loss of the JAK2 signal was responsible for extremely poor response of
BaF3/T69/S18A cells to TPO. JAK2 activation in BaF3/T69/S18A cells was
analyzed by Western blotting. In contrast to BaF3/T69 cells, where JAK2
kinase was inducibly tyrosine-phosphorylated within minutes of exposure
to TPO, its phosphorylation was markedly reduced in T69/S18A cells
(Fig. 7A) despite equal
protein loading (Fig. 7B). Although JAK2 binds to T69
constitutively, and their association is increased by TPO stimulation
(Fig. 7C), we could not detect JAK2 binding to the T69/S18A
receptor before or after stimulation with TPO (Fig. 7C).
These results indicate that Ser-18 contributes to JAK2 binding and is
thereby responsible for cellular proliferation in BaF3 cells. A similar
analysis was conducted using T69/S37A/S39A/S46A/S61A/S62A/S64A/T65A
cells, to test whether their impaired response to TPO is due to altered
JAK2 activation. As shown in Fig. 7, E and F, we
found that JAK2 activation was reduced in the mutant cells in
proportion to the level of TPO-induced proliferation.
The molecular basis for cytokine responsiveness and cellular
proliferation is an intensely studied topic. Several groups have worked
out many of the details of the signals that emanate from c-Mpl tyrosine
phosphorylation, but little is known of the Ser/Thr phosphorylation of
the receptor. In this study we demonstrate that both full-length c-Mpl
and T69 are constitutively phosphorylated on Ser, the levels of which
are enhanced following stimulation with TPO. The most important
findings in the studies presented here are as follows. 1) We have
mapped the Ser and Thr phosphorylation pattern of the region between
the transmembrane domain and Leu-69 of the murine Mpl receptor (Ser-37
and/or Ser-39, Ser-46, Ser-61, Ser-64, and possibly Ser-62 are
phosphorylated, and Ser-18 and Ser-53 are definitely not
phosphorylated), a region sufficient to support cellular proliferation.
2) Phosphorylation of Ser residues of Mpl in BaF3 cells contributes
substantially to the capacity of the Mpl receptor to support cellular
proliferation (Figs. 6H). 3) Ser-18 is critical for the
interaction of Mpl and JAK2 and thus is vital for TPO-induced signal transduction.
The erythropoietin (EPO) receptor is also known to be phosphorylated on
Ser and Tyr but not on Thr residues (18). Since TPO and EPO and their
receptors display sequence similarities and are almost certainly
related on an evolutionary basis, our finding of a conserved
phosphorylation pattern is interesting. In contrast, the
granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor Perhaps the most surprising result to come from our studies is that
alteration of Ser-18 nearly eliminated association of Mpl and JAK2 and
caused a profound reduction in the capacity of this receptor to support
cell proliferation. We do not feel this conclusion is due to trivial
technical problems, as the conclusion is based on the following: 1)
performing multiple proliferation assays employing three separate
clones of BaF3/T69/S18A cells; 2) these same cell clones grew well in
murine IL-3; 3) surface expression in each line was equivalent to that
of the truncated but otherwise wild type T69 receptor; and 4) the
Mr of the immunoprecipitated receptor matched
that of T69 (Fig. 7). Thus, there is ample evidence that technical
reasons do not account for our failure to observe a proliferative
response to TPO in these cells.
Murakami and colleagues (21) were the first to identify the "box 1"
PXXP or PXP motif conserved in the
intracytoplasmic, membrane-proximal region of all members of the
hematopoietic cytokine receptor family and recognized its importance in
cytokine-induced activation of cytoplasmic tyrosine kinases. However,
alignment of these sequences from multiple cytokine receptors that
employ JAK2 (EPO receptor, GM-CSF receptor, Mpl, and gp130) reveals
that the internal positions between the two Pro residues are poorly conserved, suggesting they play little role in JAK recruitment or
activation. Although cytokine receptors were initially thought to
recruit cytoplasmic JAK kinases to the receptor upon ligand-induced dimerization, more recent studies suggest that the Mpl receptor, like
most hematopoietic cytokine receptors, displays a basal level of
receptor-JAK interaction. The results shown in Fig. 7 indicate that
mutation of Ser to Ala at position 18 eliminates all or most of the
constitutive association of Mpl with JAK2, as well as that induced by
TPO binding. Thus, JAK2 is very minimally phosphorylated in response to
TPO stimulation of BaF3/T69/S18A cells. The loss of TPO-induced
proliferation in these cells is thus consistent with the majority of
studies of Mpl signaling indicating that JAK2 activation is critical
for TPO-induced cell signaling (4, 22). This result contradicts
that of Dorsch and colleagues (11) in which a membrane-proximal
deletion mutant of Mpl, which retained the box 1 and all distal
sequence motifs but lost JAK2 activation, retained its capacity to
support cellular proliferation in a TPO-dependent manner.
The explanation for the discrepancy of the findings is not immediately
clear, but our results add to the accumulating evidence that JAK2
activation is vital for hematopoiesis. Moreover, the structural basis
for the loss of JAK2 binding and TPO-induced activation in the S18A Mpl
mutant is not clear; whether the potential for hydrogen bonding through
Ser exceeds that of Ala at position 18 or whether a subtle tertiary
structural change was introduced by Ser to Ala substitution at this
position are uncertain.
The results of our Ser(P) mapping experiments might also shed some
light on the process of Ser phosphorylation in cytokine receptors. For
example, T69/S61A/S62A and T69/S64A/T65A cells lost the same
radiolabeled phosphopeptides in mapping experiments, suggesting that
phosphorylation of residues Ser-61, Ser-62, and/or Ser-64 is dependent
on one or two of the other Ser(P) residues. This result is similar to
that with Ser/Thr phosphorylation of the p55 TNF receptor, where
phosphorylation of Thr-236 and Ser-270 enable subsequent
phosphorylation of Ser-240 and Ser-244 (15). Similar hierarchical and
synergistic phosphorylation events were also reported for rhodopsin and
heat shock factor-1 (16, 17). Therefore, it is possible that
phosphorylation of Ser-61, Ser-62, or Ser-64 induces a conformational
change in the cytoplasmic domain of c-Mpl allowing other sites to
undergo phosphorylation. In the human GM-CSF receptor We do not yet know the Ser kinase responsible for phosphorylation of
c-Mpl. Liu et al. (24) preliminarily identified an H7
and staurosporine-sensitive 110-kDa Ser/Thr kinase constitutively associated with the IL-3 receptor. Interestingly we also observed an
~110-kDa polypeptide labeled with
[32P]orthophosphate and associated with c-Mpl and T69
(Figs. 2 and 5). Based on the sequence surrounding the sites of known
Ser phosphorylation of Mpl (i.e. SXX(D/E)), other
candidate kinases are casein kinase (CK) I and CKII. CKI is known to
phosphorylate the TNF receptor (25). In addition, if Ser-37 is
phosphorylated, it is immediately followed by Pro and hence represents
a site for a Pro-directed Ser/Thr kinase, such as c-Jun
NH2-terminal kinase, p38MAPK, and
p44/42MAPK. Although it is clear that
p44/p42MAPK is activated by TPO stimulation (9), the
activity of CKI and CKII following exposure to TPO has not yet been
studied. Further work will be necessary to establish the responsible
Ser kinases for c-Mpl phosphorylation.
In conclusion, we have identified a critical role for the Ser residue
at position 18 of Mpl in TPO-induced cell proliferation, and we have
mapped several sites of receptor Ser phosphorylation. The functional
role of these individual sites appears at least partially redundant for
cellular proliferation, and it remains possible that other physiologic
functions of Mpl, such as receptor internalization or cellular
differentiation, are also dependent on the modification of these sites.
Only further studies of the Mpl receptor will provide a better
understanding of the molecular basis of platelet production.
We appreciate the kind gift of rmTPO from Dr.
Akihiro Shimosaka and the anti-murine Mpl antibody from Dr. Takashi
Kato. We also appreciate the technical assistance for flow cytometry by Kathy Allen and our discussions with Dr. Kazuo Fujikawa on
phosphopeptide mapping.
*
This work was supported in part by Grants R01 CA31615 and
R01 DK49855 from the National Institutes of Health (to K. K.).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: Divisions of Hematology
and Cardiology, University of Washington School of Medicine, 1959 NE
Pacific St., Seattle, WA 98195. Tel.: 206-685-7868; Fax: 206-543-3560;
E-mail: kkaushan@u.washington.edu.
Published, JBC Papers in Press, July 28, 2000, DOI 10.1074/jbc.M005080200
The abbreviations used are:
TPO, thrombopoietin;
rmTPO, recombinant murine TPO;
Mpl, myeloproliferative leukemia virus
proto-oncogene product;
JAK, Janus kinase;
STAT, signal transducer and
activator of transcription;
MAPK, mitogen activated protein kinase;
PKC, protein kinase C;
IL, interleukin;
FCS, fetal calf serum;
MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide;
TNF, tumor necrosis factor;
EPO, erythropoietin;
GM-CSF, granulocyte-macrophage colony-stimulating factor;
CK, casein
kinase;
PAGE, polyacrylamide gel electrophoresis.
A Structure-Function Analysis of Serine/Threonine Phosphorylation
of the Thrombopoietin Receptor, c-Mpl*
,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The Mpl receptors utilized in this
study. Site-directed mutagenesis of the murine c-mpl
receptor gene followed by BaF3 cell transfection was used to generate
the receptor forms shown in this figure. The predicted sites of tryptic
digestion are shown by arrows.
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Fig. 2.
Phosphoamino acid analysis of thrombopoietin
receptor, c-Mpl, and its truncation mutant, T69. A,
murine BaF3 cells engineered to express c-Mpl (lanes 1 and
2) and T69 (lanes 3 and 4) were
labeled with [32P]orthophosphate. The cells were
incubated without (lanes 1 and 3) or with
(lanes 2 and 4) TPO for 10 min. The receptor was
immunoprecipitated with specific antibody and separated by SDS-PAGE
followed by transferring to polyvinylidene fluoride membrane. The blot
was exposed to film to detect 32P-labeled c-Mpl and T69
protein. B, the bands of c-Mpl and T69 from A
were cut out of the blot and subjected to hydrolysis with 6 N HCl, and phosphoamino acid analysis was performed. The
positions of serine (Ser), threonine (Thr), and tyrosine (Tyr) are
shown in the same figure.
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Fig. 3.
Phosphopeptide map of T69. T69 cells
were labeled with [32P]orthophosphate, and the
radiolabeled receptor was immunoprecipitated with specific antibody
before (A) and 10 min after (B) stimulation with
TPO. The immunoprecipitates were digested with trypsin and separated on
cellulose-coated glass plates for 2 h (the first dimension),
followed by the second dimension separation by thin layer
chromatography (TLC) for 5 h. Asterisks depict the
origin of chromatography. Each of the reproducibly detectable
phosphopeptides is indicated by a number.
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Fig. 4.
Phosphopeptide maps of Ser mutants. As
described in the legend to Fig. 3, phosphopeptide maps were obtained by
labeling the cells with [32P]orthophosphate and digestion
with trypsin. All of the cells were stimulated with TPO for 10 min for
the analysis. A, T69/S18A; B, T69/S37A/S39A;
C, T69/S46A/S53A; D, T69/S46A; E,
T69/S53A; F, T69/S61A/S62A/S64A/T65A; G,
T69/S61/S62A; and H, T69/S64A/T65A. Asterisks
depict the origin of separation.
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Fig. 5.
32P labeling of
T69/S37A/S39A/S46A/S61A/S62A/S64A/T65A cells. Equal numbers of
BaF3/T69 cells (lane 1) and
T69/S37A/S39A/S46A/S61A/S62A/S64A/T65A mutant cells (lane 2)
were labeled with [32P]orthophosphate, and the receptor
was immunoprecipitated 10 min after stimulation with TPO and subjected
to SDS-PAGE.
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Fig. 6.
Proliferation assay of serine mutants.
Several mutants were studied for proliferation with MTT assay. The
cells were incubated with sequential concentrations of TPO or IL-3 for
48 h, and the reduction of MTT was determined. The growth of each
mutant cell line was calculated based on the percentage of IL-3-induced
maximum growth. A, T69; B, T69/S18A;
C, T69/S37A/S39A; D, T69/S46A;
E, T69/S53A; F, T69/S61A/S62A; G,
T69/S64A/T65A; H, T69/S37A/S39A/S46A/S61A/S62A/S64A/T65A.
The abscissa shows the concentration of rmTPO (ng/ml),
and the ordinate displays the percentage of maximal
IL-3-induced growth. The results represent the mean (±S.D.) of
triplicate determination in a single representative experiment. All of
these experiments have been performed at least three times with
similar results.
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Fig. 7.
Activation and association of Jak2 tyrosine
kinase is diminished in T69/S18A and
T69/S37A/S39A/S46A/S61A/S62A/S64A/T65A cells. T69, T69/S18A, and
T69/S37A/S39A/S46A/S61A/S62A/S64A/T65A cells were incubated in
serum-free, cytokine-free medium for 14 h and stimulated with (+)
or without ( 
) TPO for 10 min. JAK2 kinase was immunoprecipitated
(IP) from the indicated cell lines with a specific antibody
and blotted with anti-phosphotyrosine antibody (A and
E). The same blots were stripped and reprobed with an
anti-JAK2 antibody (B and F). T69 and T69/S18A
proteins were immunoprecipitated with anti-Mpl antibody and blotted
with anti-JAK2 antibody (C). The same membrane was then
probed with anti-Mpl antibody to indicate the levels of protein loading
(D).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit and the interleukin (IL)-6 receptor subunit are
phosphorylated on Tyr, Ser, and Thr residues (19, 20). However, the
functional relevance of Ser and Thr phosphorylation of cytokine
receptors is poorly understood. To explore this aspect we used Ser to
Ala receptor mutants and assessed their phosphorylation and capacity to
support cell proliferation. We found that although alteration of any
single Ser residue (except Ser-18) failed to significantly impact the
capacity of Mpl to support cellular proliferation, alteration of all
sites of Ser(P) from the receptor substantially blunted BaF3 cell
growth at all concentrations of TPO tested (Fig. 6H).
Although we cannot rule out the possibility that the tertiary structure
of the cytoplasmic domain of Mpl might be subtly altered, leading to a
global reduction in function, it is also possible that only specific
signals are affected. Furthermore, we found the capacity of the mutant
receptor to stimulate JAK2 activation was reduced proportionately to
the diminished TPO-induced proliferation of these cells. Interestingly,
an observation reported by Sawyer and Penta (18) may relate to our
findings. These investigators found that only a highly modified form of
the EPO receptor, which was glycosylated and phosphorylated on Ser
residues, associated with JAK2.
subunit, it
was recently reported that Ser-538 binds to 14-3-3 
protein in
vivo and in vitro (23). As the 14-3-3 molecule is known
to bind to c-Raf and lead to MAPK activation, it is possible
phosphorylated Ser residues in c-Mpl also bind to 14-3-3 proteins
contributing to cellular proliferation and survival.
ACKNOWLEDGEMENTS
FOOTNOTES
Supported by National Institutes of Health Grant 1RO1HL64228 (to
B. G. and Dr. Marshall Corson).
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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