(Received for publication, December 6, 1994; and in revised form, January 9, 1995)
From the
c-Mpl is a member of the cytokine receptor superfamily,
expressed primarily on hematopoietic cells. Recently, the c-Mpl ligand
was cloned and found to have thrombopoietic activity. In this paper we
report that ligand binding induced tyrosine phosphorylation in BaF3
cells engineered to express the murine Mpl receptor (BaF3/mMpl).
Phosphorylation occurred within 1 min at cytokine concentrations
sufficient for proliferation of receptor-bearing cells. Using specific
antibodies for immunoprecipitation and Western blotting, several of
these phosphorylated proteins were identified. Shc and Jak2, known
cytokine signaling molecules, and the c-Mpl receptor were shown to be
major substrates for tyrosine phosphorylation. In contrast,
phospholipase C- and phosphatidylinositol 3-kinase displayed
little and no tyrosine phosphorylation, respectively, after
thrombopoietin stimulation. Co-immunoprecipitation studies demonstrated
that Jak2 became physically associated with c-Mpl relatively late in
the observed time course (20-60 min), significantly later than
tyrosine phosphorylation of Jak2 (1-5 min). These results suggest
that c-Mpl induces signal transduction pathways similar to those of
other known cytokines. Additionally, in light of its late physical
association with c-Mpl following ligand binding, Jak2 may not be the
initiating tyrosine kinase in the thrombopoietin-induced signaling
cascade.
Several years ago, the proto-oncogene v-mpl was cloned from the murine myeloproliferative leukemia
virus(1) . Shortly thereafter, a full-length cellular homolog, c-mpl, was cloned from both human (2) and
murine sources(3, 4) . Based on sequence homologies,
the encoded polypeptide was classified as a member of the cytokine
receptor superfamily(1, 2, 4) . This rapidly
growing group of type I transmembrane proteins is characterized by a
200-amino acid motif containing 4 conserved cysteines and a
Trp-Ser-X-Trp-Ser sequence near its carboxyl end and includes
the receptors for many interleukins, hematopoietic growth factors, and
several hormones(5) . Like the common -chain of IL-3, (
)IL-5, and granulocyte-monocyte colony stimulating factor
receptors, c-Mpl contains a duplication of the entire cytokine receptor
domain, the significance of which is not known(4) .
The intracytoplasmic domain of the murine Mpl receptor is 121 amino acids long and does not encode any recognized kinase domain, nucleotide binding site, or enzymatic motif(4) . Skoda et al.(3) have shown that this cytoplasmic domain is capable of transmitting a proliferative signal in a chimeric receptor construct. Like gp130 and other cytokine receptors, the membrane-proximal region of the c-Mpl cytoplasmic domain has a potential box 1/box 2 sequence, thought to be important for mitogenic signaling(6) . Experiments with truncated cytokine receptors have shown that this membrane-proximal region (50-60 amino acids) is necessary and sufficient to support proliferation of cytokine-dependent cell lines(3, 6, 7) . The carboxyl terminus of the intracytoplasmic domain seems to direct cytokine-specific differentiation(8) .
Recently, several groups, including our own, cloned the ligand for c-Mpl(9, 10, 11) . Both in vitro and in vivo studies indicate that it is the critical regulator of megakaryocyte proliferation and differentiation(12, 13) . On this basis, c-Mpl ligand has been termed thrombopoietin (TPO). The molecular events triggered when TPO binds to its receptor have not been previously described.
In the last few years, the central role of tyrosine phosphorylation in cytokine signal transduction has been elucidated. Although the cytokine receptors lack intrinsic kinase activity, ligand binding induces a rapid increase in cellular phosphotyrosine content(14) . Ligand/receptor interaction leads to aggregation of several subunits, forming the complete receptor complex. This complex activates intracellular tyrosine kinases, which are responsible for phosphorylation of various target molecules(8, 14) . The importance of this mechanism has been confirmed in cytokine-dependent cell lines; specific tyrosine kinase inhibitors block the proliferative response to growth factors(15) , while phosphatase inhibitors potentiate growth in the absence of cytokines(16) .
Detailed studies of the hematopoietic growth factor receptors indicate that a common set of signaling molecules are tyrosine-phosphorylated in response to ligand binding(14, 17, 18, 19) . These target proteins include various members of the Src (20, 21) and Janus (8, 22, 23, 24) tyrosine kinase families, mitogen-activated protein kinase(25) , Shc(18, 19) , and the cytokine receptors themselves(26, 27) .
Based on homology with other members of the cytokine receptor superfamily, thrombopoietin and c-Mpl were predicted to fit this model of tyrosine phosphorylation. In the studies presented here, we have begun to characterize the proliferative signal generated by the c-Mpl receptor. Although the phosphotyrosine pattern induced by TPO binding is complex, we have identified several target proteins and their temporal and spatial associations. Our results suggest that several of the signaling pathways described for other cytokine receptors are also important for megakaryocyte development.
Murine TPO was produced in baby hamster kidney cells engineered to secrete the new cytokine(9) . Cells were grown in serum-free medium, and the spent supernatant was used as a source of TPO. Activity for this reagent was determined by the ability to support growth of BaF3/mMpl cells in the absence of IL-3. A proliferation assay was employed to quantitate TPO based on reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (28) ; the dilution that produced 50% maximal proliferation was defined as 50 units/ml. The batch used for these experiments contained approximately 30 ng/ml and 50,000 units/ml.
Murine IL-3 (mIL-3) was produced by baby hamster kidney cells engineered to secrete this cytokine. The spent supernatant contained 1% fetal calf serum, and the activity was determined by MTT assay of BaF3 cells.
Antibodies were
obtained from the following sources: anti-phosphotyrosine (4G10),
anti-Jak2 (polyclonal sera), anti-PLC- (mixed monoclonal),
anti-Shc (purified IgG), anti-PI 3-K (antisera) were all purchased from
Upstate Biotechnology Inc. Antisera that recognizes c-Mpl was raised in
rabbits against the extracytoplasmic domain of the receptor. An
engineered soluble receptor, described previously(9) , was used
for immunization.
Figure 1:
TPO induces tyrosine phosphorylation in
BaF3 cells bearing the c-Mpl receptor. Parental BaF3 cells were
unstimulated (unstim) or exposed to TPO (750 units/ml) or
mIL-3 (5%) at 37 °C for 20 min. BaF3/mMpl cells were exposed to TPO
of varying concentrations (0-10,000 units/ml, as indicated) under
identical conditions. Cells were lysed and subjected to Western blot
analysis. The blot was probed with an -phosphotyrosine antibody
(4G10). Molecular size markers (kDa) are
shown.
In order to study the dose-response curve of tyrosine phosphorylation, BaF3/mMpl cells were exposed to increasing concentrations of TPO for 20 min. Increased phosphotyrosine incorporation was detected with only 10 units/ml and reached maximal levels with 1000 units/ml (Fig. 1). In MTT bioassays, 50 units/ml is the concentration required for half-maximal proliferation. Furthermore, normal megakaryocyte progenitor cells respond to similar concentrations of TPO(12) . Thus, tyrosine kinase activity was stimulated at concentrations that appear to have physiologic relevance.
By increasing the duration of exposure to TPO (37 °C) up to 60 min, we found that phosphotyrosine incorporation was stimulated at 1 min, peaked between 10 and 20 min, and diminished somewhat by 60 min (Fig. 2). This time course is very similar to that for IL-3 signaling (data not shown). The decreased phosphotyrosine content at 60 min, despite continued TPO exposure, presumably represents down-regulation of tyrosine kinases, activation of cellular phosphatases, or both.
Figure 2:
Time
course of TPO-induced tyrosine phosphorylation. BaF3/mMpl cells were
stimulated with TPO (750 units/ml) for 0-60 min, as indicated.
Cell lysates were analyzed by Western blot and probed with an
-phosphotyrosine antibody (4G10).
Figure 3:
Phosphorylation of signal transduction
molecules in response to TPO. BaF3/mMpl cells were either
unstimulated(-) or exposed to TPO (750 units/ml) for 20 min
(+). Cell lysates were immunoprecipitated and analyzed by Western
blot. TPO-induced phosphorylation was detected with an
-phosphotyrosine antibody (4G10). Results are shown for
immunoprecipitations performed with:
-PI 3-K antisera,
-PLC-
IgG,
-Shc IgG,
-Jak2 antisera, and
-Mpl
antisera. A separate sample was probed with the same antibody used for
precipitation to confirm the identity of the protein (data not shown).
The IgG band in each lane represents detection of the antibody used for
immunoprecipitation by the goat-anti-mouse antibody, coupled to
alkaline phosphatase.
Figure 4:
Co-immunoprecipitation of Jak2 and PI 3-K
with c-Mpl. BaF3/mMpl cells were stimulated with TPO (750 units/ml) for
0-60 min, as indicated. Cell lysates were immunoprecipitated (IP) with -Mpl,
-Jak2, or
-PI 3-K antisera, and
the precipitated proteins were analyzed by Western blotting. The blots
were probed with the indicated detecting antibody in order to compare
the temporal relationship of tyrosine phosphorylation with receptor
association.
We chose to study the role of
Jak2 in c-Mpl-induced signaling because of its well characterized
relationship with other hematopoietic cytokines. In the erythropoietin
and interferon- systems, Jak2 associates with receptors only after
ligand binding(38, 40) . Another group of receptors,
including prolactin and gp130-related receptors (IL-6, leukemia
inhibitory factor, oncostatin M), associate with Jak2 both before and
after ligand binding(24, 41, 42) . We
performed co-immunoprecipitation studies, which demonstrate that direct
interaction of c-Mpl and Jak2 was only detectable after 20 min of
ligand stimulation and increased at 60 min (Fig. 4A).
This rather delayed event is in marked contrast to the early
phosphorylation of Jak2, suggesting that Jak2 becomes phosphorylated
prior to binding the cytoplasmic domain of c-Mpl. However, several
caveats exist. First, weak, but physiologically important interactions
may not withstand the immunoprecipitation conditions. Second, a small
fraction of Jak2, below our limits of detection, might be associated
with c-Mpl before or immediately after TPO-binding.
This association/phosphorylation time course differs from that of the erythropoietin receptor (the only other system thus studied) in which Jak2's association with the receptor occurred in parallel with Jak2 phosphorylation(43) . These results suggest that another tyrosine kinase may first be activated by c-Mpl. An earlier kinase might then be responsible for Jak2 phosphorylation as part of a kinase cascade. Alternatively, Jak2 may be initially associated with a distinct receptor subunit and only later associates with c-Mpl, forming the complete thrombopoietin receptor. Additional studies will be necessary to identify the other kinase(s) activated by TPO.
In the studies described above, we have chosen to use BaF3/mMpl cells, rather than megakaryocytes, to study TPO-induced signal transduction. BaF3 cells, derived from early hematopoietic progenitors(44, 45) , appear to contain most of the signaling molecules described in other primary hematopoietic cells and cell lines. Many signaling studies have been performed in transfected BaF3 cells or similar cytokine-dependent hematopoietic cells, permitting direct comparison of cytokine responses. Furthermore, BaF3 cells engineered to express the erythropoietin receptor partially differentiate in response to erythropoietin(45) . Nevertheless, we plan to confirm our findings in normal megakaryocytes.
In summary, c-Mpl fits the general model of signal transduction developed for other members of the cytokine receptor superfamily. TPO binding leads to activation of tyrosine kinases, including Jak2, and results in increased phosphotyrosine content of known signaling molecules. It is likely that phosphorylated residues on the receptor provide binding sites for SH2-containing proteins. Despite these insights into TPOinduced signaling, many important questions remain. For example, how does c-Mpl activation specifically trigger megakaryocyte development when pathways common to other cytokines appear to be involved? Second, which pathways are involved in proliferation and which for differentiation? Third, are new signaling pathways yet to be identified in megakaryocytes? And fourth, does c-Mpl require additional subunits for signal transduction? Answers to these questions will only come from detailed study of purified megakaryocytes or their precursors, and from a better understanding of the signaling intermediates themselves.