title: Phosphorylated c-Mpl tyrosine 591 regulates thrombopoietin-induced signaling.
abstract: Thrombopoietin (TPO) is the primary regulator of platelet production, affecting cell survival, proliferation, and differentiation through binding to and stimulation of the cell surface receptor the cellular myeloproliferative leukemia virus oncogene (c-Mpl). Activating mutations in c-Mpl constitutively stimulate downstream signaling pathways, leading to aberrant hematopoiesis, and contribute to development of myeloproliferative neoplasms. Several studies have mapped the tyrosine residues within the cytoplasmic domain of c-Mpl that mediate these cellular signals; however, secondary signaling pathways are incompletely understood. In this study, we focused on c-Mpl tyrosine 591 (Y591). We found Y591 of wild-type c-Mpl to be phosphorylated in the presence of TPO. Additionally, eliminating Y591 phosphorylation by mutation to Phe resulted in decreased total receptor phosphorylation. Using a Src homology 2/phosphotyrosine-binding (SH2/PTB) domain binding microarray, we identified novel c-Mpl binding partners for phosphorylated Y591, including Src homology region 2 domain-containing phosphatase-1 (SHP-1), spleen tyrosine kinase (SYK) and Bruton's tyrosine kinase (BTK). The functional significance of binding partners was determined through small interfering RNA treatment of Ba/F3-Mpl cells, confirming that the increase in pERK1/2 resulting from removal of Y591 may be mediated by spleen tyrosine kinase. These findings identify a novel negative regulatory pathway that controls TPO-mediated signaling, advancing our understanding of the mechanisms required for successful maintenance of hematopoietic stem cells and megakaryocyte development.
authors.affiliation: Department of Medicine, Stony Brook University, Stony Brook, NY, USA.
authors.forename: Veena
authors.initials: V
authors.lastname: Sangkhae
authors.affiliation: Department of Hematology/Oncology, Eberhard Karls University Tübingen, Tübingen, Germany.
authors.forename: Sebastian Jonas
authors.initials: SJ
authors.lastname: Saur
authors.affiliation: Seattle Biomedical Research Institute, Seattle, WA, USA.
authors.forename: Alexis
authors.initials: A
authors.lastname: Kaushansky
authors.affiliation: Department of Medicine, Stony Brook University, Stony Brook, NY, USA.
authors.forename: Kenneth
authors.initials: K
authors.lastname: Kaushansky
authors.affiliation: Department of Hematology/Oncology, Eberhard Karls University Tübingen, Tübingen, Germany. Electronic address: ian.hitchcock@stonybrookmedicine.edu.
authors.forename: Ian Stuart
authors.initials: IS
authors.lastname: Hitchcock
mesh_terms: D000595:Amino Acid Sequence; D015153:Blotting, Western; D002460:Cell Line; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D053628:Receptors, Thrombopoietin / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics*; D013926:Thrombopoietin / Q000502:physiology*; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D053628:Receptors, Thrombopoietin; C082218:MPL protein, human; D014443:Tyrosine; D013926:Thrombopoietin; D048049:Extracellular Signal-Regulated MAP Kinases