A structure-function analysis of serine/threonine phosphorylation of the thrombopoietin receptor, c-Mpl.
Thrombopoietin (TPO), the critical regulator of platelet production, acts by binding to its cell surface receptor, c-Mpl. Numerous studies have shown that TPO binding leads to JAK2 kinase activation and Tyr phosphorylation of c-Mpl and several intracellular signaling intermediates, events vital for the biological activity of the hormone. In contrast, ... virtually nothing is known of the role of Ser or Thr phosphorylation of c-Mpl. By using phosphoamino acid analysis we found that Ser residues of c-Mpl were constitutively phosphorylated in receptor-bearing cells, levels that were increased following exposure of cells to TPO. To identify which residues were modified, and to determine the functional consequences of their phosphorylation, we generated a series of Ser to Ala mutations of a truncated c-Mpl receptor (T69) capable of supporting TPO-induced cell growth. Of the eight Ser within T69 we found that at least four are phosphorylated in TPO-stimulated cells. The mutation of each of these residues alone had minimal effects on TPO-induced proliferation, but substitution of all of the phosphoserine residues with Ala reduced the capacity of the receptor to support cell growth by over 50%. Additionally, the Ser at cytoplasmic position 18 is not detectably phosphorylated. However, the mutation of Ser-18 to Ala nearly abrogates TPO-induced proliferation and co-precipitation of JAK2 with Mpl. This study provides the first systematic analysis of the role of Ser residues in c-Mpl signaling.
Mesh Terms:
Amino Acid Sequence, Animals, Cell Division, Cell Line, Enzyme Activation, Janus Kinase 2, Mice, Molecular Sequence Data, Mutagenesis, Site-Directed, Neoplasm Proteins, Peptide Mapping, Phosphopeptides, Phosphorylation, Phosphoserine, Precipitin Tests, Protein Binding, Protein-Tyrosine Kinases, Proto-Oncogene Proteins, Receptors, Cytokine, Receptors, Thrombopoietin, Recombinant Proteins, Signal Transduction, Structure-Activity Relationship, Threonine, Thrombopoietin
Amino Acid Sequence, Animals, Cell Division, Cell Line, Enzyme Activation, Janus Kinase 2, Mice, Molecular Sequence Data, Mutagenesis, Site-Directed, Neoplasm Proteins, Peptide Mapping, Phosphopeptides, Phosphorylation, Phosphoserine, Precipitin Tests, Protein Binding, Protein-Tyrosine Kinases, Proto-Oncogene Proteins, Receptors, Cytokine, Receptors, Thrombopoietin, Recombinant Proteins, Signal Transduction, Structure-Activity Relationship, Threonine, Thrombopoietin
J. Biol. Chem.
Date: Oct. 13, 2000
PubMed ID: 10918061
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