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Phosphorylated c-Mpl tyrosine 591 regulates thrombopoietin-induced signaling.

Authors
  • Sangkhae, Veena1
  • Saur, Sebastian Jonas2
  • Kaushansky, Alexis3
  • Kaushansky, Kenneth1
  • Hitchcock, Ian Stuart4
  • 1 Department of Medicine, Stony Brook University, Stony Brook, NY, USA.
  • 2 Department of Hematology/Oncology, Eberhard Karls University Tübingen, Tübingen, Germany. , (Germany)
  • 3 Seattle Biomedical Research Institute, Seattle, WA, USA.
  • 4 Department of Hematology/Oncology, Eberhard Karls University Tübingen, Tübingen, Germany. Electronic address: [email protected] , (Germany)
Type
Published Article
Journal
Experimental hematology
Publication Date
Jun 01, 2014
Volume
42
Issue
6
Identifiers
DOI: 10.1016/j.exphem.2014.02.007
PMID: 24607955
Source
Medline
License
Unknown

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.

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