A structure-function analysis of serine/threonine phosphorylation of the thrombopoietin receptor, c-Mpl

Thrombopoietin (TPO), the critical regulator of platelet production, acts b y 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 k nown of the role of Ser or Thr phosphorylation of c-Mpl. By using phosphoam ino acid analysis we found that Ser residues of c-Mpl were constitutively p hosphorylated in receptor-bearing cells, levels that were increased followi ng exposure of cells to TPO. To identify which residues were modified, and to determine the functional consequences of their phosphorylation, we gener ated a series of Ser to Ala mutations of a truncated c-Mpl receptor (T69) c apable of supporting TPO-induced cell growth. Of the eight Ser within T69 w e 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 phosph orylated. However, the mutation of Ser-18 to Ala nearly abrogates TPO-induc ed proliferation and co-precipitation of JAK2 with Mpl. This study provides the first systematic analysis of the role of Ser residues in c-Mpl signali ng.