The Homodimeric Hematopoietic Cytokine Receptor C-Mpl Requires Cross Phosphorylation in Order to Signal Cell Proliferation

Objectives: The three primary regulators of hematopoiesis, erythropoietin, granulocyte colony-stimulating factor and thrombopoietin, bind to homodimeric members of the cytokine receptor superfamily and utilize Janus Kinase (JAK) 2 to initiate signaling. Recently, mutations in JAK2, particularly JAK2V617F, were found to contribute to the pathogenesis of the myeloproliferative disorders. In vitro studies have determined that only homodimeric cytokine receptors can support JAK2-mediated cytokine hypersensitivity. As part of a strategy to identify novel approaches to inhibit mutant JAK2 function we tested whether the homodimeric receptors initiate signaling by JAK2 mediated receptor trans-phosphorylation, and whether JAK2V617F escapes this requirement. Methods: We introduced the engineered receptor Myr/FKBPF36V/c-Mplcyto into hematopoietic cell lines containing either wild-type JAK2 or JAK2V617F, a receptor designed to adopt either a monomeric or dimeric state depending on the absence or presence, respectively, of the chemical dimerizer AP20187. To evaluate the effects of receptor dimerization on the growth of wild-type and V617F mutant JAK2 cell lines we measured reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). If either of the cell lines were able to support cell proliferation in absence of receptor dimerization it would indicate that signaling is initiated by ipsilateral receptor phosphorylation, as opposed to the trans-phosphorylation employed by subfamilies of heterodimeric receptors, such as that for IL-2. Results: The Myr/FKBPF36V/c-Mplcyto receptor construct was subcloned into a retroviral vector, transduced into Baf3, Baf3/JAK2 and Baf3/JAK2V617F cells and spontaneous, IL-3- and AP20187-induced cell proliferation was assessed. Equal expression of the receptor construct in each cell line was confirmed by western blotting. Both Baf3/JAK2- and Baf3/JAK2V617F-derived cell lines transduced with wild type c-Mpl served as controls, and quantitative western blotting was used to verify that equal levels of the two receptor constructs were introduced into the cell lines. Growth factor dependence was confirmed in the control cell lines with both thrombopoietin and IL-3 and was confirmed with IL-3 in the experimental cell lines. There was an increased sensitivity to growth factors in the control cell line containing the JAK2 V617F mutant, consistent with a myeloproliferative phenotype. When Myr/FKBPF36V/Mplcyto was introduced into either Baf3/JAK2 or Baf3/JAK2V617F cells, the cells remained dependent on either IL-3 or AP20187, although maximal rates of cell growth were significantly greater in the Baf3/JAK2V617F/Myr/FKBPF36V/Mplcyto cells than in Baf3/JAK2/Myr/FKBPF36V/Mplcyto cells. The maximal rate of growth of Baf3/JAK2V617F/Myr/FKBPF36V/Mplcyto cells also significantly exceeded that of the Baf3 parental cell line. Furthermore, we found that in the absence of chemically induced dimerization neither Myr/FKBPF36V/c-Mplcyto/JAK2 nor Myr/FKBPF36V/c-Mplcyto JAK2V617F cells proliferated. Conclusions: These results argue that JAK2 induces signaling by trans-phosphorylation of the cytoplasmic domains of c-Mpl and that the kinase hyperactivity displayed by JAK2V617F cannot overcome this requirement. Therefore it may be possible to alter or inhibit trans-phosphorylation and attenuate JAK2V617F-mediated myeloproliferation.