Homodimeric class I cytokine receptors are assumed to exist as preformed dimers that are activated by ligand-induced conformational changes. We quantified the dimerization of three prototypic class I cytokine receptors in the plasma membrane of living cells by single-molecule fluorescence microscopy. Spatial and spatiotemporal correlation of individual receptor subunits showed ligand-induced dimerization and revealed that the associated Janus kinase 2 (JAK2) dimerizes through its pseudokinase domain. Oncogenic receptor and hyperactive JAK2 mutants promoted ligand-independent dimerization, highlighting the formation of receptor dimers as the switch responsible for signal activation. Atomistic modeling and molecular dynamics simulations based on a detailed energetic analysis of the interactions involved in dimerization yielded a mechanistic blueprint for homodimeric class I cytokine receptor activation and its dysregulation by individual mutations.

SummaryIn the two decades since its cloning, thrombopoietin (TPO) has emerged not only as a critical haematopoietic cytokine, but also serves as a great example of bench-to-bedside research. Thrombopoietin, produced by the liver, is the primary regulator of megakaryocyte progenitor expansion and differentiation. Additionally, as TPO is vital for the maintenance of haematopoietic stem cells, it can truly be described as a pan-haematopoietic cytokine. Since recombinant TPO became available, the molecular mechanisms of TPO function have been the subject of extensive research. Via its receptor, c-Mpl (also termed MPL), TPO activates a wide array of downstream signalling pathways, promoting cellular survival and proliferation. Due to its central, non-redundant role in haematopoiesis, alterations of both the hormone and its receptor contribute to human disease; congenital and acquired states of thrombocytosis and thrombocytopenia and aplastic anaemia as a result from dysregulated TPO expression or functional alterations of c-Mpl. With TPO mimetics now in clinical use, the story of this haematopoietic cytokine represents a great success for biomedical research.Keywords: thrombopoeitin, megakaryocytes, megakaryocytopoiesis, myeloproliferative disease. HistoryThe term erythropoietin (EPO) was first used in the literature in 1906 to describe the humoral substance responsible for erythropoiesis. At the time, blood platelets were barely distinguishable in the best microscopes, prompting their description with the pejorative phrase 'the dust of the blood'. However, the work of Carnot and of Wright and others in the early 20th century defined the critical role of blood platelets in coagulation and their origin from the marrow megakaryocyte, ultimately leading Kelemen and Tanos (1958) to coin the term thrombopoietin (TPO, also termed THPO) to describe the humoral substance responsible for platelet production.In the mid-1960s, several groups began attempting to purify TPO from the plasma of thrombocytopenic animals. These early efforts were severely handicapped by inconvenient and insensitive assays for the hormone, and the attempts failed to produce unequivocal proof of the existence of TPO. With the availability of in vitro megakaryocyte differentiation assays in the 1980s, additional purifications were attempted; however, while some claims were made of its biological activities, attempts to produce a cDNA for TPO, the sine qua non of the existence of a protein, also failed.Occasionally in science, a finding from one field, although by itself important, can have a catalytic effect on a seemingly unrelated area of research. The discovery and characterization of the murine myeloproliferative leukaemia virus (MPLV) had such an influence on the search for TPO. The virus causes an acute myeloproliferative syndrome in infected mice (Wendling et al, 1986). In 1990, the responsible oncogene (v-mpl, now termed Mpl) was cloned, and the protooncogene (c-mpl, also now termed Mpl) obtained 2 years later (Souyri et al, 1990;Vigon et al, 19...

Highlights d Single-cell-omics demonstrate megakaryocyte-biased hematopoiesis in myelofibrosis (MF) d Megakaryocyte progenitors (MkPs) show high expression of a fibrosis signature in MF d MkPs are heterogeneous in MF with aberrant metabolic and inflammatory signatures d Targeting aberrant surface G6B expression may selectively ablate the MF clone

Motile prostate cancer cell lines express vimentin. In tissue sections, the presence of vimentin positive tumour cells correlated positively to poorly differentiated cancers and the presence of bone metastases.

Significance The myeloproliferative neoplasms (MPNs) are a group of hematological malignancies characterized by increased numbers of myeloid blood cells, such as platelets, erythrocytes, and neutrophils. The main causes of illness and death in patients with MPNs are arterial and venous clotting and also, conversely, bleeding complications. However, the causes of these conditions are poorly understood. In this paper, we use a mouse model of MPNs to determine the cell types responsible for abnormal clotting in MPNs. We demonstrate that endothelial cells, the cell type that lines all blood vessels, have a significant role to play in MPN bleeding complications, potentially identifying a new cellular target for MPN therapies.

IntroductionFocal adhesion kinase (FAK) is an essential nonreceptor protein tyrosine kinase that is expressed ubiquitously and is conserved in mammals and lower eukaryotic organisms. [1][2][3][4] The principal FAK stimulus is integrin engagement (Guan and Shalloway 5 and reviewed in Parsons 6 ), although its direct interaction with, and activation via, platelet-derived and epidermal growth factor receptors suggests that it may also function downstream of other membrane-bound growth factor and cytokine receptors. 7 Autophosphorylation of FAK Tyr397 occurs rapidly following integrin activation, and the resulting phosphorylated residue acts as a docking site for the SH2 domains of Src-family kinases. 8,9 FAK catalytic activity is increased by subsequent Src-mediated phosphorylation of FAK residues Tyr576 and Tyr577, 10 followed by Tyr861 and Tyr925, which act as binding sites for the SH3 domain of p130CAS and the SH2 domain of the adaptor protein GRB2, respectively. 11,12 Tyr925, located in the focal adhesion targeting domain, also mediates interactions with integrin-associated proteins such as talin and paxillin, 13 thereby recruiting FAK to focal adhesion sites.Activated FAK modulates the activity of a broad range of downstream signaling proteins, including phosphoinositide 3-kinase (PI3-K) 14 and phospholipase C (PLC)-␥, 15 as well as a number of small GTPases such as Ras, Rac, and Rho (reviewed in 16 ). Extensive studies indicate that FAK is essential for normal cell migration. FAK-deficient cells migrate poorly in response to chemokines; form an increased number of prominent "immature" focal adhesions, apparently due to decreased focal adhesion turnover; and do not spread normally on extracellular matrices (ECMs). 17,18 Megakaryopoiesis and platelet production are tightly regulated by a number of growth factors and cytokines to maintain a normal number of circulating platelets. The principal regulator of megakaryopoiesis is thrombopoietin (Tpo), 19 although other fac 1 tors such as interleukin-3 and stem-cell factor work in synergy with Tpo during the earlier stages of megakaryocytic progenitor cell expansion. 20 It has become apparent that the microenvironments in which megakaryocytes function are also of critical importance to megakaryopoiesis. 21,22 Indeed, direct cell-cell and cell-ECM interactions have been demonstrated to influence megakaryocyte differentiation and proplatelet formation. [23][24][25] Although FAK is important in regulating cell spreading and migration in response to integrin-ECM interactions, the role of FAK in megakaryopoiesis remains unclear, partly because Fak deletion in mice is lethal at embryonic day 8.5, before the onset of significant definitive hematopoiesis. Although the role of proline-rich tyrosine kinase-2 (PYK2), which shares sequence homology and similar characteristics with FAK, has been characterized in megakaryocytes, 8,[26][27][28] its localization and dependence on intracellular calcium make the 2 proteins functionally different. 29 FAK activation in platelets require...

IntroductionThrombopoietin (Tpo) is critical for the maintenance of hematopoietic stem and progenitor cells and is also the primary regulator of megakaryocyte development. 1,2 The binding of Tpo to its receptor, c-Mpl, causes associated Janus kinase 2 (Jak2) activation, which in turn phosphorylates (activates) several downstream effectors including signal transducers and activators of transcription (STAT) 3 and 5, mitogen-activated protein kinase (MAPK), phosphotidylinositol-3-kinase (PI3-K), and protein kinase C (PKC). [3][4][5][6][7] Activation of these pathways promotes proliferation and survival in c-Mplexpressing cell lines and hematopoietic progenitor cells, in addition to megakaryocyte lineage differentiation and maturation. [8][9][10][11] It is critical that Tpo signal transduction is stringently controlled to prevent uncontrolled proliferation. Suppressors of cytokine signaling (SOCS) proteins, phosphatases, and negative regulators such FAK, Lnk, and Lyn have all been shown to down-modulate Tpo-induced signaling. [12][13][14][15] However, the most effective method of regulating Tpo signaling is by controlling expression of c-Mpl on the plasma membrane. Tpo-mediated c-Mpl endocytosis, recycling, and degradation are rapid mechanisms to control signaling longevity and represent a mechanism that regulates Tpo signaling without new protein expression.The principal mechanism of receptor-mediated endocytosis in eukaryotic cells is the clathrin-coated vesicle. 16 Soluble clathrin molecules self-assemble and are recruited to the plasma membrane, where they form lattice structures and interact with transmembrane receptors via adaptor proteins (APs), such as AP2, to form clathrin-coated pits. 17,18 These pits then further invaginate before finally budding from the membrane to form clathrin-coated vesicles. AP2 is a heterotetramer composed of ␣2, ␤2, 2, and 2 subunits. The ␣2 and ␤2 subunits localize AP2 to the membrane, recruit endocytic accessory proteins, and bind clathrin heavy chain. [19][20][21] Transmembrane proteins are associated with the AP2-clathrin complex via the 2 domain, which binds directly to cytoplasmic YXX⌽ (where X ϭ any amino acid and ⌽ ϭ bulky hydrophobic residue) and [DE]XXX-L[IL] motifs. 22,23 To ensure that AP2 specifically associates with membrane-bound proteins, phosphorylation of 156 Thr in the 2 subunit results in a conformational change in AP2, dramatically increasing the affinity of 2 for YXX⌽ motifs. 24,25 156 Thr is phosphorylated by adaptor-associated kinase 1 (AAK1), 26 the activity of which is maximized by its association with clathrin, 27,28 ensuring that AP2-cargo protein interactions are initiated only at the plasma membrane. In addition to being an endocytic signal, YXX⌽ motifs located between 6 to 9 amino acids from the transmembrane domain mediate targeting of cargo protein to the lysosome and lysosome-like organelles via interactions with AP3. [29][30][31][32] c Methods Chemicals and reagentsPharmacologic inhibitors JakI, LY294002, SU6656, and U0126 where all purchased f...

Regulation of growth factor and cytokine signaling is essential for maintaining physiologic numbers of circulating hematopoietic cells. Thrombopoietin (Tpo), acting through its receptor c-Mpl, is required for hematopoietic stem cell maintenance and megakaryopoiesis. Therefore, the negative regulation of Tpo signaling is critical in many aspects of hematopoiesis. In this study, we determine the mechanisms of c-Mpl degradation in the negative regulation of Tpo signaling. We found that, after Tpo stimulation, c-Mpl is degraded by both the lysosomal and proteasomal pathways and c-Mpl is rapidly ubiquitinated. Using site-directed mutagenesis, we were able to determine that c-Mpl is ubiquitinated on both of its intracellular lysine (K) residues (K 553 and K 573 ). By mutating these residues to arginine, ubiquitination and degradation were significantly reduced and caused hyperproliferation in cell lines expressing these mutated receptors. Using short interfering RNA and dominant negative overexpression, we also found that c-Cbl, which is activated by Tpo, acts as an E3 ubiquitin ligase in the ubiquitination of c-Mpl. Our findings identify a previously unknown negative regulatory pathway for Tpo signaling that may significantly impact our understanding of the mechanisms affecting the growth and differentiation of hematopoietic stem cells and megakaryocytes. (Blood. 2010;115: 1254-1263) IntroductionHematopoiesis is tightly regulated by several cytokines and growth factors to ensure that numbers of circulating blood cells remain constant under normal conditions. In many hematologic disorders, cytokine and growth factor signaling is dysfunctional, resulting in the overproduction or underproduction of 1 or more blood cell lineages. Thrombopoietin (Tpo) is a hematopoietic cytokine that, via its receptor c-Mpl, supports hematopoietic stem cell maintenance and proliferation and is the primary regulator of megakaryopoiesis. 1,2 Absence of Tpo signaling results in thrombocytopenia, reduced numbers of transplantable stem cells, and eventually aplastic anemia in humans. [3][4][5] Conversely, excessive Tpo signaling, usually due to mutations in c-Mpl or its secondary signaling proteins, results in hyperproliferation of numerous cell lineages, causing myeloproliferative syndromes. 6-8 Therefore, the control of Tpo-mediated signaling is critical in maintaining physiologic numbers of circulating blood cells.Protein phosphatases, suppressors of cytokine signaling (SOCS) proteins, and inhibitory intracellular mediators are all mechanisms that contribute to the negative regulation of cytokine signaling. [9][10][11][12] However, the process of receptor internalization and degradation is one of the quickest and most effective ways in which activated receptors are negatively regulated. We recently demonstrated a mechanism for Tpo-stimulated c-Mpl internalization, through the interaction of adaptor protein 2 with YRRL motifs located at Y 521 and Y 591 in the c-Mpl intracellular domain; elimination of these sites significantly reduced degrad...