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.
Divisions of Hematology and Cardiology, University of Washington School of Medicine, Seattle, Washington 98195, USA.
Y
Y
Miyakawa
J G
JG
Drachman
B
B
Gallis
A
A
Kaushansky
K
K
Kaushansky
D000595:Amino Acid Sequence; D000818:Animals; D002455:Cell Division; D002460:Cell Line; D004789:Enzyme Activation / Q000187:drug effects; D053614:Janus Kinase 2; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009363:Neoplasm Proteins; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000032:analysis / Q000737:chemistry; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism*; D011233:Precipitin Tests; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018121:Receptors, Cytokine; D053628:Receptors, Thrombopoietin; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D013912:Threonine / Q000378:metabolism*; D013926:Thrombopoietin / Q000494:pharmacology
D009363:Neoplasm Proteins; D010748:Phosphopeptides; D011518:Proto-Oncogene Proteins; D018121:Receptors, Cytokine; D053628:Receptors, Thrombopoietin; D011994:Recombinant Proteins; C082218:MPL protein, human; D010768:Phosphoserine; D013912:Threonine; D013926:Thrombopoietin; D011505:Protein-Tyrosine Kinases; C507924:JAK2 protein, human; C507926:Jak2 protein, mouse; D053614:Janus Kinase 2