UBC Theses and Dissertations
The role of SHIP in hematopoiesis Hughes, Michael R.
The SH2-containing inositol-5'-phosphatase, SHIP, has been shown, by comparing SHTP+/+ and -/- cells, to be. a critical negative regulator of the phosphatidylinositol (PI)-3- kinase (PI3K) pathway in many hematopoietic cell lineages. However, SHIP-/- mice are neither polycythemic nor profoundly anemic and we wanted to know why. Our studies revealed that SHIP-/- mice suffer from reticulocytosis and show an enhanced recovery from phenylhydrazine (PHz)-induced anemia accompanied by a more rapid accumulation of CFUE and late erythroid progenitors in the spleens of SHIP-/- mice. In addition, following PHz treatment, SHIP-/- plasma possesses higher erythroid growth-stimulating activity than SHIP+/+ plasma. Interestingly, however, erythropoietin (Epo) dose-response studies revealed little difference between SHIP+/+ and -/- progenitors and this may be because perturbed erythropoiesis in SHIP-/- mice is caused by extrinsic mechanisms. Although SHIP becomes tyrosine phosphorylated in response to multiple stimuli, the kinase(s) responsible has not been identified. Using phosphospecific antibodies we developed, we found that the two NPXYs within SHIP are the major sites of tyrosine phosphorylation in response to cytokines, growth factors, G protein-coupled receptor ligands, immunoreceptor ligands and osmotic stress. Moreover, using the Src family inhibitor, PP2 as well as cells from Lyn-/- mice, we found that the Src family is primarily responsible for SHIP'S tyrosine phosphorylation. Moreover, consistent with this being a direct effect, we found SHIP and Lyn associate in B-cells and this association increases with BCR + FcγRIIB aggregation. Interestingly, Lyn-SHIP association appears to be mediated via Lyn's SH3 and a previously unidentified PXXP motif proximal to SHIP'S SH2 domain. Lastly, we investigated the role of SHIP in human hematopoietic cells using small interfering RNA to silence SHIP expression in the erythroleukemic cell line, TF-1. We demonstrated complete and long-lasting knockdown of SHIP protein in these cells and this resulted in a more rapid phosphorylation of Akt, GSK3β and ERK1/2 in response to GMCSF. Furthermore, SHIP-deficient TF-1 cells survived better with low or no cytokines but displayed reduced proliferation at high cytokine concentrations. Further investigation into the enhanced survival of SHIP-knockdown TF-1 cells suggested that this was mediated in part by the PI3K-induced maintenance of the pro-survival Bcl-2 member, Mcl-1.
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