1. Induction of myeloproliferative disorder and myelofibrosis by thrombopoietin receptor W515 mutants is mediated by cytosolic tyrosine 112 of the receptor
by Christian Pecquet, Judith Staerk, Ronan Chaligné, Valerie Goss, Kimberly A. Lee, Xiaowu Zhang, John Rush, Joanne Van Hees, Hélène A. Poirel, Jean-Marie Scheiff, William Vainchenker, Stéphane Giraudier, Roberto D. Polakiewicz, and Stefan N. Constantinescu
Blood
Volume 115(5):
February 4, 2010
©2010 by American Society of Hematology

2. Myeloproliferative disorder induced in vivo by Δ5TpoR and TpoRW515A mutants.
Myeloproliferative disorder induced in vivo by Δ5TpoR and TpoRW515A mutants. (A) Sequencing of genomic DNA isolated from peripheral blood granulocytes of primary myelofibrosis patients. A W515L (TGG→TTG) mutation was identified in TpoR from patient 64. A W515A (TGG→GCG) mutation was detected in TpoR from patient 66. (B) Shown is the location of the amphipathic K(R)WQFP motif at the junction between the TpoR transmembrane (TM) and cytosolic domains. (C) Peripheral blood counts, taken 45 days after reconstitution with bone marrow cells expressing the indicated TpoR variants, demonstrated that Δ5TpoR and TpoRW515A induce a rapid myeloproliferative disease. (D) Hematocrit values (%) were determined 45 days for mice reconstituted with bone marrow cells expressing the indicated TpoR constructs.
Christian Pecquet et al. Blood 2010;115:
©2010 by American Society of Hematology

3. Histology of mice reconstituted with bone marrow–expressing TpoR variants.
Histology of mice reconstituted with bone marrow–expressing TpoR variants. Bone marrow cells were infected with empty vector pMEGIX, or pMEGIX viruses coding for wild-type TpoR (TpoRwt), Δ5TpoR, or TpoRW515A mutants. (A) Splenomegaly was detected in mice expressing the active TpoR mutants compared with control empty vector or TpoRwt mice. All mice present more than 95% GFP-positive cells at 3 weeks after reconstitution. (B) Histology of spleen sections of mice reconstituted with the indicated constructs. Spleen sections are stained with hematoxylin-eosin (HE; 3× or 40× magnification), silver stain (SL; 20× magnification), or trichrome (TB; 20× magnification). Spleen sections were performed 45 days after transplantation. Microscopic section of the spleen shows expansion of the red pulp (HE, 3×). The splenic enlargement is due mainly to extramedullary hematopoiesis (HE, 40×) as well as fibrosis (SL, 20× and TC, 20×) that occur in the splenic red pulp sinusoids. Megakaryocytes are prominent, but erythropoiesis and granulopoiesis are present as well in the splenic sinuses. Both Δ5TpoR and TpoRW515A induce some degree of early fibrosis with reticulin (see silver staining: SL, 20×) and collagen deposition stained in blue by the Masson trichrome (TC, 20×). Images were obtained using a Mirax Digital Slide System.
Christian Pecquet et al. Blood 2010;115:
©2010 by American Society of Hematology

4. Evidence that Y78 and Y112 of TpoR are phosphorylated in TpoRW515A cells selected for autonomous growth.
Evidence that Y78 and Y112 of TpoR are phosphorylated in TpoRW515A cells selected for autonomous growth. Phosphopeptides were prepared using PhosphoScan Kit (Cell Signaling Technology) after lysis of Ba/F3 TpoRW515A cells in urea buffer. Trypsin-digested lysates were immunoaffinity purified with pY-100 antibody, concentrated on reverse-phase microtips, and analyzed by mass spectrometry. (A) Mass spectrum representing phosphorylation of Y78 of TpoR (Y582, full murine TpoR numbering). (B) Mass spectrum representing phosphorylation of Y112 of TpoR (Y616, full murine TpoR numbering). Oxidized methionine is represented by M# and tyrosine phosphorylation, by Y*. Phosphorylated residues are denoted as y. (C) Schematic representation of TpoR with the location of the W515A mutation in the TpoR cytosolic juxtamembrane domain (red region) and the Y78 and Y112 in the cytosolic domain of the receptor.
Christian Pecquet et al. Blood 2010;115:
©2010 by American Society of Hematology

5. In vivo effects of Δ5TpoR and TpoRW515A where cytosolic tyrosine residues were substituted to phenylalanine.
In vivo effects of Δ5TpoR and TpoRW515A where cytosolic tyrosine residues were substituted to phenylalanine. (A) Peripheral blood counts of mice that received a transplant of bone marrow cells expressing the indicated Δ5TpoR (left) or TpoRW515 (right) mutants at day 30 after reconstitution. All mice present more than 95% GFP-positive cells at 3 weeks after reconstitution. (B) The Y112F mutation abrogates spleen myelofibrosis induced by Δ5TpoR or TpoRW515A at day 60 after reconstitution. Silver staining of spleen sections shows the presence of reticulin fibers in spleens from mice reconstituted with Δ5TpoR or TpoRW515A, but not in spleens from mice reconstituted with Δ5TpoR-Y112F, TpoRW515A-Y112F, or TpoRwt.
Christian Pecquet et al. Blood 2010;115:
©2010 by American Society of Hematology

6. Role of TpoR cytosolic residues Y78 and Y112 in the proliferation induced in Ba/F3 cells by TpoR signaling.
Role of TpoR cytosolic residues Y78 and Y112 in the proliferation induced in Ba/F3 cells by TpoR signaling. Proliferation of cells expressing either TpoRwt or the corresponding Y→F mutants, Δ5TpoR or the corresponding Y→F mutants (A), or TpoR W515A or the corresponding Y→F mutants (B) was examined after 4 days in the presence of 3 ng/mL Tpo. Shown are averages of triplicates ± SD of 1of 3 representative experiments. Proliferation of cells expressing Δ5TpoR (C) or TpoRW515A mutants (D) in the absence of any cytokine was examined after 8 days. Shown are averages of triplicates ± SD of 1 of 3 representative experiments. (E) Proliferation of Ba/F3 expressing either Δ5TpoR, TpoRW515A, TpoRW515K, or TpoRW515L in the absence of cytokine. Cell growth was measured using the Cell Titer Glo Kit (Promega). Shown are averages of absorbance units of triplicates ± SD obtained from 1 of 2 representative experiments. (F) The Y112F mutation inhibits proliferation induced in the absence of Tpo by TpoRW515L and TpoRW515K mutants. Shown are averages of cell counts performed in triplicates ± SD obtained from 1 of 2 representative experiments.
Christian Pecquet et al. Blood 2010;115:
©2010 by American Society of Hematology

7. Effects of Y78F and Y112F mutations on signaling pathways activated by TpoR.
Effects of Y78F and Y112F mutations on signaling pathways activated by TpoR. Ba/F3 cells retrovirally transduced with Δ5TpoR or TpoRW515A and mutants thereof (Y78F or Y112F) were sorted for similar expression levels of GFP and examined for protein expression (A), activation of JAK2 (B), and activation of several downstream signaling pathways (C). Detection was performed by Western blotting with anti–phospho-specific antibodies that recognize the major phosphorylation sites that are linked to activation of catalysis (for kinases and phosphatases) or transcription (for STAT proteins). JAK2 activation was assessed by Western blotting with anti–phospho-JAK2 Y1007, as phosphorylation of activation loop Y1007 is obligatory for activation of JAK2 signaling. The Y78F mutation enhanced JAK2 activation and signaling via TpoRwt and Δ5TpoR. The Y112F mutation did not alter JAK2 activation, but inhibited downstream signaling, especially by Δ5TpoR.
Christian Pecquet et al. Blood 2010;115:
©2010 by American Society of Hematology

8. Model for the roles of TpoR Y112 and Y78 in pathologic signaling by TpoR W515 mutants.
Model for the roles of TpoR Y112 and Y78 in pathologic signaling by TpoR W515 mutants. The wild-type TpoR (TpoRwt) contains a juxtamembrane domain K(R)WQFP that maintains the unliganded receptor inactive. TpoRwt is activated upon Tpo stimulation leading to conformational change of the receptor and to activation of major signaling pathways. In the absence of the K(R)WQFP juxtamembrane motif (Δ5TpoR), or in situations where the W515 of this motif is substituted by other residues (ie, W515A), the receptor assumes an active dimeric conformation, which activates JAK2 in a ligand-independent manner. Y78 plays a negative role in signaling through TpoRwt or the active TpoR. Mutation of Y78 into phenylalanine in the context of the activating W515 mutations led to strong pathologic signaling and myelofibrosis. In contrast, the TpoR-Y112F mutation decreases signaling of the receptor and abolishes the myeloproliferative and myelofibrosis phenotype.
Christian Pecquet et al. Blood 2010;115:
©2010 by American Society of Hematology