Thrombopoietin induces an SH2-containing protein, CIS1, which binds to MP1: Involvement of the ubiquitin proteosome pathway

The interaction of thrombopoietin (TPO) with its receptor, Mpl, triggers growth and differentiation of megakaryocytes and their progenitors. The Mpl cytoplasmic domain controls this process through src homology 2 (SH2)- containing target molecules and their receptor docking sites. A novel cytokine inducible SH2-containing protein, CIS1, has been isolated. CIS1 is induced by interleukin-2 (IL-2), IL-3, GM-CSF, and erythropoietin (EPO), but not by IL-6, granulocyte colony-stimulating factor (G-CSF), or stem cell factor. To investigate the functional domains of Mpl for induction of CIS1, we examined FDCP-2 cell lines expressing seven carboxyl truncations of the human Mpl cytoplasmic domain. We found that the box1 and box2 regions of Mpl were necessary for induction of CIS1 after TPO stimulation. CIS1 was degraded very quickly and was found to be involved in the ubiquitin-proteosome pathway. A 4-hour depletion of TPO almost completely eliminated CIS1 protein; within 1 hour after TPO stimulation, CIS1 protein reappeared as 37- and 32- kDa proteins in the wild type Mpl-expressing FDCP-2 cells. Further, CIS1 was stably associated with tyrosine-phosphorylated Mpl. The SH2 domains of CIS1, constructed as glutathione S-transferase fusion protein, bound to activated Mpl in vitro. These results suggest that CIS1 may be an important signaling component downstream of Mpl and may regulate the proliferation and differentiation of hematopoietic cells.