©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The c-Mpl Ligand (Thrombopoietin) Stimulates Tyrosine Phosphorylation of Jak2, Shc, and c-Mpl (*)

(Received for publication, December 6, 1994; and in revised form, January 9, 1995)

Jonathan G. Drachman (1)(§) James D. Griffin (2) Kenneth Kaushansky (1)

From the  (1)Department of Medicine, University of Washington, School of Medicine, Seattle, Washington 98195 and the (2)Division of Tumor Immunology, Dana Farber Cancer Institute, Harvard University, Boston, Massachusetts 02155

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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 beta-chain of IL-3, (^1)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.


MATERIALS AND METHODS

Reagents

BaF3 cells (kindly provided by Dr. Alan D'Andrea) were transfected with cDNA for murine c-mpl(9) . BaF3/mMpl cells were shown to express c-Mpl on their surface by flow cytometry and Western blotting, using an antiserum raised against a soluble form of murine Mpl (see below).

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.

Tissue Culture

BaF3 cells were grown in RPMI 1640 with 10% fetal calf serum, 2 mML-glutamine (Bio Whittaker), 100 units/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml amphotericin B (Bio Whittaker), and 0.2% mIL-3. BaF3/mMpl cells were grown in the same media plus 0.5 mg/ml synthetic hygromycin (G418, Life Technologies, Inc.) or in the presence of 150 units/ml recombinant TPO instead of mIL-3.

Cell Lysates

Cells were washed twice with RPMI and 0.5% bovine serum albumin, then resuspended in the same serum-free media at a concentration of 2 times 10^6/ml, and grown without exogenous growth factors overnight. TPO was added at a concentration of 0-10,000 units/ml and incubated at 37 °C for 1-60 min. Cells were then washed twice with phosphate-buffered saline at 4 °C. Cell pellets were resuspended at 1 times 10^7 cells/100 µl in lysis buffer containing 50 mM HEPES, pH 7.0, 1% Triton X-100, 1 mM sodium pyrophosphate, 1 mM sodium fluoride, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride (Sigma), 1 mM sodium vanadate, 10 µg/ml leupeptin (Sigma), and 10 µg/ml aprotinin (Sigma). After 20 min on ice, samples were spun in a microcentrifuge for 10 min at 12,300 times g, 4 °C. Supernatants were frozen (-70 °C) or used immediately for immunoprecipitation or polyacrylamide gel electrophoresis. In some cases, equal protein loading in all lanes was confirmed using the Bio-Rad protein assay/DC kit.

Immunoprecipitation

For each assay, 100 µl of cell lysate was diluted 1:5 with lysis buffer. alpha-Mpl antisera was diluted 1:250; all other purified immunoglobulins or antisera were diluted according to the supplier's guidelines for immunoprecipitation. After 1-2 h at 4 °C, immune complexes were precipitated by incubating with 50 µl of protein A-Sepharose beads (Pharmacia Biotech Inc.) for 1 h at 4 °C. Beads were washed three times in cold lysis buffer and then boiled in sample buffer with 1% beta-mercaptoethanol.

Western Blotting

Total cellular lysate from 4 times 10^6 cells (40 µl) or immunoprecipitation from 1 times 10^7 cells were boiled for 5 min in sample buffer (containing 62 mM Tris, pH 6.8; 1% sodium dodecyl sulfate (w/v), 10% glycerol (w/v), 1% beta-mercaptoethanol) and size-fractionated by electrophoresis through 7.5% polyacrylamide gels (29) . Protein was electrophoretically transferred to nitrocellulose sheets (Schleicher & Schuell, 0.45 µm) and blocked with 1% gelatin in Tris-buffered saline for 1 h. The detecting antibody was incubated at room temperature for 2-8 h at dilutions of 1:2000 (4G10, Jak2, PI 3-K, PLC-), 1:1000 (Mpl), or 1 µg/ml (Shc) in Tris-buffered saline + 0.5% Tween 20 (TBST). After extensive washing in TBST, blots were probed with either goat-anti-mouse or goat-anti-rabbit IgG coupled to alkaline phosphatase (1:3000 in TBST, Bio-Rad). Color development was carried out with p-nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate toluidine salt (Bio-Rad).


RESULTS AND DISCUSSION

TPO Stimulates Phosphorylation of Cellular Proteins

Cells expressing the c-Mpl receptor, but not the parental BaF3 cells, had a dramatic increase in tyrosine phosphorylation in response to TPO (Fig. 1). The most prominent phosphotyrosine proteins migrated at 52 kDa, a doublet at 95 kDa, several bands between 115 and 125 kDa, and 140 kDa; less intense bands were seen at 45, 65-75, and 110 kDa. Compared to maximal IL-3 stimulation, the overall phosphotyrosine pattern is quite similar. However, several differences were noted. TPO induced much greater phosphorylation of the 52-kDa band. Also, the bands at 65, 75, and 115-125 kDa are much more prominent after TPO stimulation. These observations suggest partial overlap of TPO and IL-3 signaling pathways.


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 alpha-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 alpha-phosphotyrosine antibody (4G10).



The Identification of TPO-responsive Signaling Intermediates

In order to identify intermediates in c-Mpl signal transduction, we used antibodies to several known signaling molecules in immunoprecipitation and Western blotting experiments. As shown in Fig. 3, TPO stimulation clearly induced phosphotyrosine incorporation of Jak2 (125 kDa), Shc (52- and 46-kDa forms), the Shc-associated 140-kDa protein, and c-Mpl (doublet at 95 kDa). Of these, Shc (52-kDa form) co-migrated with the most prominent band in total cellular lysates. In comparison, PLC had relatively little phosphotyrosine incorporation, and PI 3-K (85-kDa subunit) was not appreciably phosphorylated in response to TPO. The observation that alpha-Mpl antisera precipitated and recognized a double band suggests post-translational modification as a single receptor cDNA was used for constructing the cell line. In data not shown, we have demonstrated that these phosphotyrosine-containing proteins represent distinct bands in the whole cellular lysate. Immunoprecipitation with anti-Shc antiserum completely eliminated the intensely stained band at 52 kDa and a lighter band, corresponding to 45 kDa, and partially reduced the prominent band at 140 kDa. Immunoprecipitation of Jak2 eliminated one of several bands at approximately 125 kDa. In contrast, immunoprecipitation of Mpl only slightly reduced the intensity of bands at 95 kDa, suggesting that other phosphotyrosine-containing proteins co-migrate at this position.


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 alpha-phosphotyrosine antibody (4G10). Results are shown for immunoprecipitations performed with: alpha-PI 3-K antisera, alpha-PLC- IgG, alpha-Shc IgG, alpha-Jak2 antisera, and alpha-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.



Phosphorylation of c-Mpl

Stimulation of BaF3/mMpl cells with TPO induced rapid accumulation of phosphotyrosine in c-Mpl within 1 min (time course not shown). This finding has been demonstrated for many of the cytokine receptors, including the erythropoietin receptor(27) , IL-2 receptor beta chain(30) , and IL-3 receptor beta chain(7) . Phosphotyrosine residues act as docking sites for proteins containing Src homology domains (SH2), thus recruiting signaling molecules to the activated receptor complex(31) . However, receptor phosphorylation is not necessary for mitogenic signal transduction. Truncations of the IL-4, erythropoietin, and common beta-chain receptors, which eliminate all cytoplasmic tyrosine residues, can still induce proliferation(7, 26, 32) . Receptor phosphorylation may play a role in cytokine-specific differentiation or receptor down-regulation. The importance of c-Mpl-specific tyrosine phosphorylation needs to be further explored.

Phosphorylation of Shc

Shc and the Shc-associated 140 kDa protein (33) were phosphorylated within 1 min of TPO stimulation and achieved peak phosphotyrosine content between 10 and 20 min (time course not shown). Shc exists in several forms (46, 52, and 66 kDa) and contains both SH2 and SH3 domains(34) . Shc is thought to act as an adaptor protein, physically linking receptors with other signaling molecules, such as Grb2(35) . Cell lines engineered to constitutively overexpress Shc have a transformed phenotype, suggesting an important role in the proliferative response(34) . The role of these molecules in TPO-induced signaling remains to be explored.

Phosphatidylinositol 3-Kinase and Phospholipase C-

In response to TPO stimulation, PLC- acquired detectable phosphotyrosine content (Fig. 3). In contrast, the 85-kDa regulatory subunit of PI 3-K was not phosphorylated although several other phosphorylated bands were co-precipitated with PI 3-K (Fig. 3). For these molecules, enzymatic activation is not necessarily dependent on tyrosine phosphorylation(36, 37) . Thus, direct measurement of enzymatic activity will be required to assess the TPO-induced effects on PI 3-K and PLC-. In previous studies, the receptors for erythropoietin, IL-3, IL-4, IL-5, and stem cell factor associate with PI 3-K only after ligand-binding(36, 38) . In our co-immunoprecipitation studies, we found that c-Mpl associated with PI 3-K both before and after TPO stimulation (Fig. 4B). This suggests that c-Mpl/PI 3-K interaction occurs constitutively and is not altered by tyrosine phosphorylation of the receptor.


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 alpha-Mpl, alpha-Jak2, or alpha-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.



Jak2 Associates with c-Mpl

In response to TPO stimulation, Jak2 had increased phosphotyrosine content within 1 min and peaked at 5-10 min (Fig. 4A). Jak2 is a member of the Janus family of tyrosine kinases, which includes Jak1, Jak2, Jak3, and Tyk2(39) . These intracellular molecules are found in almost all cells and are activated by tyrosine phosphorylation. Jak kinases have been implicated in signal transduction by nearly all members of the cytokine receptor superfamily, as well as the related interferon receptors(8) . Studies with truncated receptors demonstrate that Jak phosphorylation is required for mitogenic signaling and appears to be physically linked via the box 1/box 2 membrane-proximal region(8) . Furthermore, various receptors associate with and activate different members of the Janus family(8, 24) .

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.


FOOTNOTES

*
This work was supported in part by the National Institutes of Health Grants R01DK43719 and CA31615. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of postdoctoral support (a molecular medicine training grant) from the National Institutes of Health. To whom correspondence should be addressed: University of Washington Medical Center, Division of Hematology, Mail Stop RM-10, Seattle, WA 98195. Tel.: 206-685-3051; Fax: 206-685-3062.

(^1)
The abbreviations used are: IL, interleukin; TPO, thrombopoietin; PI 3-K, phosphatidylinositol 3`-kinase; PLC-, phospholipase C-; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; TBST, Tris-buffered saline with 0.05% Tween 20; IgG, immunoglobulin; SH2, Src homology 2.


ACKNOWLEDGEMENTS

We thank Catherine Lofton-Day for preparation of the soluble c-Mpl receptor and Charlie Hart for raising rabbit antisera against the above receptor (Zymogenetics, Inc., Seattle, WA).


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