Proc. Natl. Acad. Sci. USA Vol. 94, pp. 2350–2355, March 1997 Cell Biology Dissecting the thrombopoietin receptor: Functional elements of the Mpl cytoplasmic domain (tyrosine phosphorylationythrombopoietinysignal transductionyJAKySTAT) JONATHAN G. DRACHMAN* AND KENNETH KAUSHANSKY Division of Hematology, University of Washington Medical Center, Box 357710, Seattle, WA 98195 Communicated by Seymour Klebanoff, University of Washington School of Medicine, Seattle, WA, December 24, 1996 (received for review December 5, 1996) ABSTRACT Thrombopoietin (TPO) acts through its re- box2) that are conserved among most members of the hema- ceptor, Mpl, to stimulate the proliferation and maturation of topoietic cytokine receptor superfamily and are critical for all megakaryocytes and their progenitors. The Mpl cytoplasmic aspects of receptor function (6–8). Box1, identified by two domain controls this process through assembly of an active spatially conserved proline residues (PXXP), is located be- signaling complex using various receptor docking sites. In this tween cytoplasmic residues 17 and 20 of Mpl. Box2 is more report, eight carboxyl truncations of the 121-aa murine Mpl imprecisely defined by a region with increased serine and cytoplasmic domain were tested for the ability to support glutamic acid content that extends between residues 46 and 64 growth of a cytokine-dependent cell line (BayF3) and for their and by a proposed core motif between residues 52 and 61 (9). capacity to induce TPO-stimulated tyrosine phosphorylation Be´nit et al. (9) have demonstrated that deletion of either box1 of specific signaling proteins. Point mutations of the five or box2 abolished the transforming potential of v-mpl. Simi- tyrosine residues in the cytoplasmic domain of the receptor larly, Gurney et al. (10) have shown that loss of either the Mpl were subsequently used to confirm our conclusions. From box1 or box2 motifs blocks both activation of the Janus kinases these studies we demonstrate that: (i) TPO-induced prolifer- (JAKs) and cellular proliferation. ation is moderately reduced by truncation of as many as 53 The Mpl cytoplasmic region does not contain any recogniz- C-terminal amino acids of Mpl, including the sites of receptor able enzymatic motif, yet through the recruitment of nonre- tyrosine phosphorylation; (ii) truncationymutation of resi- dues 69–83 of the Mpl cytoplasmic domain enhances prolif- ceptor tyrosine kinases, it promotes rapid tyrosine phosphor- erative signaling, perhaps mediated by a decrease in receptor- ylation of numerous intracellular proteins after TPO stimula- driven cellular differentiation; (iii) Mpl can be phosphory- tion. In previous reports, we and others (11–21) have lated at either Y112 or Y117 but not at the three proximal demonstrated that TPO induces rapid tyrosine phosphoryla- cytoplasmic tyrosine residues (Y8, Y29, and Y78); (iv) Y112 of tion of Mpl, Shc, SHIP, JAK2, TYK2, STAT3, and STAT5. For Mpl is necessary for tyrosine phosphorylation of Shc and the JAKs and STAT family of latent transcription factors, Shc-associated p145 (SHIP); and (v) unlike STAT3, STAT5 is tyrosine phosphorylation is essential for activation. Mpl and partially phosphorylated in the absence of any tyrosine res- Shc acquire phosphotyrosine residues that function as poten- idues in the Mpl cytoplasmic domain. These studies identify tial binding sites for proteins that contain Src homology 2 subdomains of Mpl necessary for activation of several critical (SH2) or phosphotyrosine-binding domains. Differences signaling pathways and point to two potentially novel mech- within the SH2 domains of distinct molecules (e.g., STATs) anisms of TPO-induced signal transduction, an indirect path- direct binding to specific phosphotyrosine residues, depending way to STAT5 activation and a differentiation domain that on the adjacent amino acids (22–24). Analysis of the erythro- acts by limiting proliferation. poietin receptor has identified the individual tyrosine residues that recruit STAT5 to the signaling complex (25–27). The cloning of thrombopoietin (TPO) and its receptor, Mpl, The carboxyl portion of the Mpl cytoplasmic domain (res- has opened promising avenues toward understanding the idues 70–121) shares little homology with other cytokine molecular basis of megakaryocyte development. In the pres- receptors, whose length and primary sequence vary tremen- ence of TPO, multipotential hematopoietic cells undergo dously. Several reports have begun to explore the function of proliferation and maturation along the megakaryocyte lin- this receptor subdomain. Baumann et al. (28) showed that Mpl eage, characterized by nuclear endoreduplication, cytoplasmic does not contain a C-terminal box3 motif, which is required for expansion, display of lineage-restricted surface proteins, and lineage-specific gene expression in the G-CSF, gp130, and platelet production (1, 2). This programmed response is ini- leukemia inhibitory factor receptors (28, 29). Gurney et al. (10) tiated by the interaction of TPO and Mpl and is coordinated demonstrated that truncation of the final 20 aa of the Mpl by the cytoplasmic domain of the receptor. receptor abrogated Shc phosphorylation and c-fos mRNA Like the receptors for growth hormone, erythropoietin, and induction. Hill et al. (30) showed that a dominant-negative granulocyte colony-stimulating factor, Mpl is believed to be form of the Shc adapter protein blocked TPO-induced myeloid activated through homodimerization, independent of the three differentiation of 32D-mpl cells. Shc, which contains multiple receptor subunits shared by other cytokines [i.e., bc, gp130, and protein-interactive domains, is believed to be involved in interleukin (IL)-2 g chain; refs. 3–5 and unpublished results]. activation of the Ras pathway by simultaneously interacting The cytoplasmic domain of murine Mpl contains 121 aa, with several signaling molecules, such as Grb2, SOS, and the including two short, membrane-proximal motifs (box1 and recently cloned 145-kDa inositol phosphatase, SHIP (31–33). Finally, Porteu et al. (34) reported that amino acids 71–94 of The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviations: TPO, thrombopoietin; IL, interleukin; SH2, Src ho- mology 2; m, murine; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl Copyright q 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA tetrazolium bromide. 0027-8424y97y942350-6$2.00y0 *To whom reprint requests should be addressed. e-mail: drachman@ PNAS is available online at http:yywww.pnas.org. u.washington.edu. 2350 Downloaded by guest on September 28, 2021 Cell Biology: Drachman and Kaushansky Proc. Natl. Acad. Sci. USA 94 (1997) 2351 the Mpl signaling domain, just downstream of box2, were diphenyl tetrazolium bromide (MTT; Sigma) was added, at 1 required for megakaryocytic differentiation. mgyml, and incubated for 5 hr. The formazan granules were In the present study, eight truncation mutations of murine solubilized by adding 100 ml of MTT lysis buffer (40% Mpl were used to identify important regions for TPO-induced dimethylformamidey2% glacial acetic acidy20% sodium do- proliferation and signaling. Receptor point mutations of spe- decyl sulfatey0.215% concentrated hydrochloric acid) and cific tyrosine (Y) residues to phenylalanine (F) were then absorbance (570 nm-630 nm) was determined on an ELISA constructed and expressed in BayF3 cells to confirm that these plate reader. Each experiment was performed in triplicate. residues are critical docking sites for the SH2-containing Proliferation was expressed as a percentage of maximal mIL- signaling proteins under study. Our results reveal that Y112 is 3-induced growth. Statistical significance was tested for the critical for Shc and SHIP phosphorylation and that Y112 and combined results of several experiments using two-factor Y117 are both involved in Mpl and STAT3 phosphorylation. analysis of variance with replications. However, STAT5 is partially phosphorylated in response to Preparation of Cellular Extracts. Cells in logarithmic-phase TPO, even in cells bearing mutant receptors that contain no growth were washed and resuspended in serum-free, cytokine- cytoplasmic tyrosines, suggesting potentially novel mecha- free medium for 16 hr. Samples were either unstimulated or nisms of receptor-mediated signaling. In addition, deletion or exposed to TPO-containing medium (10–15 ngyml) for 10 mutation of residues 69–83, a region similar to the differen- min. Lysis was performed in a 1% Triton X-100 buffer, 100 ml tiation domain recognized by Porteu et al. (34), enhances per 107 cells as previously described (11). Final protein con- Mpl-induced proliferation. Together, these experiments fur- centrations were determined by modified Bradford assay ther our understanding of the signaling pathways employed by (Bio-Rad). Mpl and pose interesting questions about the molecular mech- Immunoprecipitations. Cellular extracts (750 mg) were in- anisms of TPO-induced megakaryocytopoiesis. cubated with appropriate antiserum (2 ml) or IgG (1 mg) for 2hrat48C. Immune complexes were collected for 1 hr with 20 MATERIALS AND METHODS ml of protein A-agarose (Santa Cruz Biotechnology). The beads were washed three times in lysis buffer and boiled in 30 Antibodies. Polyclonal Mpl antiserum was raised against the ml of denaturation buffer containing 2-mercaptoethanol and extracytoplasmic domain